401 Post-transcriptional Control of Stress Response

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401 Post-transcriptional Control of Stress Response mRNAs by a Zinc Finger Protein and AU-rich Elements Dorothea Droll, Igor Minia, Aditi Singh, Abeer Fadda, Christine Clayton Conference Paper · May 2012 CITATIONS

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RNA 2012 The 17th Annual Meeting of the RNA Society PROGRAM & ABSTRACTS

May 29 – June 2, 2012 Ann Arbor, Michigan, USA

Rachel Green, Johns Hopkins University School of Medicine/HHMI Nils Walter, University of Michigan, Ann Arbor Melissa Moore, University of Massachusetts Medical School/HHMI Gerhart Wagner, Uppsala University i

ACKNOWLEDGEMENTS Throughout the program listing, the numbers next to the titles refer to corresponding Oral or Poster numbers in the Abstract section of this book. These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication, and should be cited only with the consent of the author.

To encourage sharing of unpublished data at the RNA Society Meeting, taking of photographs and/or videos during scientific sessions (oral or posters), or of posters outside of session hours, is strictly prohibited. Violators of this policy may have their equipment confiscated (cameras, cell phones, etc.) and/or they may be asked to leave the conference and have their registration privileges revoked without reimbursement.

Front Cover

Structure of mitochondrial Proteinaceous RNase P 1 (PRORP1) from Arabidopsis thaliana with precursor tRNA substrate modeled in. Howard, Lim, Fierke, and Koutmos, manuscript in preparation. ii

MEETING SPONSORS

Biosearch Technologies www.biosearchtech.com

Toray Industries www.toray.com

PTC Therapeutics www.ptcbio.com

New England Biolabs www.neb.com

OSU Center for RNA Biology rna.osu.edu

National Science Foundation www.nsf.org

Michigan RNA Society no website

UM Dept of Biological Chemistry www.biochem.med.umich.edu

UM College of Literature, Science & the Arts www.lsa.umich.edu

UM Dept of Chemistry www.umich.edu/~michchem

University of Michigan www.umich.edu iii

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TABLE OF CONTENTS Meeting Sponsors.............................................................................................................................................iii Berry & Associates advertisement....................................................................................................................iv RNA Society Officers.......................................................................................................................................vi Invitation to Membership.................................................................................................................................vii Program – RNA 2012............................................................................................................................. viii – xi Additional Scheduled Events.................................................................................................................xii – xiv RNA Awards...........................................................................................................................................xv – xvi Abstract Listing..............................................................................................................................................xvii Oral Abstract Numbers 1 – 171..................................................................................................... Oral Sessions Poster Abstract Numbers 172 – 632............................................................................................ Poster Sessions Recent Poster Additions....................................................................................................................... 701 – 711 Author Index....................................................................................................................... Author Index 1 – 14 Keyword Index...................................................................................................................Keyword Index 1 – 5 The RNA Institute advertisement............................................................................................ Inside front cover University of Michigan Campus Map......................................................................................Inside back cover Abbreviated Schedule........................................................................................................... Outside back cover

v

The RNA Society Officers of the RNA Society FY 2012 President (2012) Douglas L. Black Howard Hughes Med Inst-UCLA Past President (2010) Manuel Ares, Jr. University of California, Santa Cruz

Director (’11/12) V. Narry Kim Seoul National University

Chief Executive Officer James McSwiggen McSwiggen & Associates

Director (’11/12) Scott Strobel Yale University

Chief Financial Officer James P. Bruzik Case Western Reserve University

Director (’12/13) Brenton Graveley Univ of Connecticut Hlth Ctr

Secretary/Treasurer (’12/14) Mary O’Connell MRC Human Genetics Unit, University of Edinburgh

Director (’12/13) Tracy Johnson Univ of California - San Diego

Director (’11/12) Kathy Collins University of California-Berkely

Director (’12/13) Mikiko Siomi University of Tokyo

RNA 2013 The 18th Annual Meeting of the RNA Society will be held in Davos, Switzerland from June 11-16, 2013, at the Congress Center Davos

2013 Organizers Frédéric Allain, ETH - Zurich Withold Filipowicz, Friedrich Miescher Inst Adrian Krainer, Cold Spring Harbor Laboratory Osamu Nureki, University of Tokyo Sarah Woodson, Johns Hopkins University 9650 Rockville Pike, Bethesda, Maryland, 20814-3998 Tel: 301-634-7120 vi

Fax: 301-634-7420

E-mail: [email protected]

Invitation to Membership The RNA Society was established in 1993 to facilitate sharing and dissemination of experimental results and emerging concepts in RNA research. The Society is an interdisciplinary, cohesive intellectual home for those interested in all aspects of RNA Science. We welcome new members from all disciplines and we look forward to sharing the new perspectives they bring to the Society.

Our members work in numerous areas of RNA science including but not limited to: RNAi and miRNA

Noncoding RNA

Ribosomes and Translation Regulation

Splicing Mechanisms

Splicing Regulation and Alternative Splicing

3’End Formation and Riboregulation of Development

RNA Turnover and Surveillance

RNA Transport and Localization

Integration of Nuclear Gene Expression Processes

RNP Biosynthesis and Function

RNA Regulation in Neurons and Specialized Cells

RNP Structure and RNA-Protein Interactions

RNA Structure and Folding

RNA Catalysis

RNA and Disease; Therapeutic Strategies

Heterochromatin Silencing

Viral RNA Mechanisms

Telomerases

Methods in RNA and RNP Research

Bioinformatics

Our members receive:

• Subscription to the Society journal, RNA (IF 6.051) with • 50% discount on page charges • 50% discount on first color figure charge (a savings of $225) • For those members who wish to have their articles completely open access immediately upon publication can do so at a reduced cost of $1500 (a $500 savings from non-member fee) • Reduced registration fees for the annual meeting of the Society (more than $100 saving) • The RNA Society Newsletter, a forum for disseminating information to members and discussing issues affecting the Society and RNA Science • Numerous opportunities for students and postdocs to become involved in the Society • The Directory of Members, available on the web and in print • Free job postings on the Society Employment and Careers website • Opportunities to request Travel Fellowship and Meeting Support for RNA-related meetings you are organizing These member savings more than offset the cost of a one-year membership in the Society. Two and three year memberships, as well as lifetime memberships, are now available through our online registration system with the added benefit of a discounted annual rate!

Take a moment to start or renew your membership using our online system at http://rnasociety.org/membership The RNA Society • 9650 Rockville Pike Bethesda, MD 20814-3998 Tel: 301-634-7120, Fax: 301-634-7099; E-mail: [email protected] vii

PROGRAM – RNA 2012 The 17th Annual Meeting of the RNA Society Ann Arbor, Michigan, USA May 29–June 2, 2012

Tuesday, May 29 14:00 – 22:00

Registration

Power Center (Lobby)

14:00 – 16:00

Campus Walking Tours (by prior reservation)

17:30 – 19:00 Barbecue Dinner

Departing from Power Center Lobby Ingalls Mall (outside/adjacent to Michigan League)

19:00 – 20:00

Reception and Dessert

Power Center (Lobby)

20:00 – 20:30

Welcome, Opening remarks, and brief Entertainment

Power Center

20:30 – 22:00

Opening Plenary Session Brenda Bass (Univ Utah) Olke Uhlenbeck (Northwestern Univ) Jonathan Weissman (UCSF)

Power Center

Wednesday, May 30 08:00 – 18:30

Registration

09:00 – 12:30

Session 1: RNA-protein interactions Keynote: Carol Fierke (Univ Michigan) Chair: Scott Blanchard (Cornell Univ)

12:30 – 14:00

Lunch

14:00 – 17:00

Concurrent Early Afternoon Sessions



Session 2A: Non-coding RNAs Keynote: Tom Gingeras (Cold Spring Harbor) Chair: Wade Winkler (Univ Maryland)



Session 2B: Ribosomes & translation Keynote: Harry Noller (UCSC) Chair: Kurt Fredrick (Ohio State Univ)

17:15 – 18:30

Concurrent Late Afternoon Sessions



Session 3A: RNA-protein architecture Chair: Hashim Al-Hashimi (Univ Michigan)



Session 3B: RNA-seq & computational structure prediction Chair: Alain Laederach (UNC Chapel Hill)

viii

Power Center (Lobby) Power Center

Michigan Union Power Center

Rackham Auditorium

Power Center Rackham Auditorium

18:30 – 20:00

Dinner

Michigan Union

18:30 – 20:30 Meetings Committee Meeting & Dinner (limit ~40) 19:30 – 20:30

Junior Scientists Social

20:00 – 22:30

Beer Hall and Poster Session 1

Michigan Union (Anderson Room D) Michigan Union (Patio) Michigan League (2nd Floor)

Poster Topic Bioinformatics Emerging & High-throughput Techniques for RNA RNA-Protein Interactions Mechanisms of RNA interference RNA and Epigenetics Riboregulation in Development Non-coding and Regulatory RNAs 3’ end processing Ribosomes and Translation Interconnections Between Gene Expression Processes

Abstracts 172 - 182 183 - 202 203 - 237 238 - 249 250 - 251 252 253 - 273 274 - 284 285 - 307 308 - 324

Sponsor: OSU Center for RNA Biology

Thursday, May 31 08:00 – 18:30

Registration

09:00 – 12:30

Session 4: RNA turnover Keynote: Joel Belasco (NYU) Chair: Oliver Mühlemann (Univ Bern)

12:30 – 14:00

Lunch

12:30 – 14:00 Mentor-Mentee Lunch 14:00 – 17:00

Concurrent Early Afternoon Sessions



Session 5A: Splicing mechanism Keynote: Kristen Lynch (U Penn) Chair: Tracy Johnson (UCSD)



Session 5B: RNA editing & modification Keynote: Jamie Williamson (Scripps Res Inst) Chair: Eric Phizicky (Univ Rochester)

17:15 – 18:30

Concurrent Late Afternoon Sessions



Session 6A: Aptamers Chair: Donald Burke (Univ Missouri)



Session 6B: Surveillance & decay Chair: Anita Hopper (Ohio State Univ)

Power Center (Lobby) Power Center

Michigan Union Michigan Union (Rogel Ballroom) (separate reservation required) Power Center

Rackham Auditorium

Power Center Rackham Auditorium

ix

18:30 – 20:00

Dinner

Michigan Union

18:30 – 20:30 RNA & Society Dinner Michigan Union (Rogel Ballroom) (limit 400, separate reservation required) Laurie Zoloth (Northwestern Univ) “Making Good: Ethical Issues in Synthetic Biology” 20:00 – 22:30

Beer Hall and Poster Session 2 Poster Topic RNP Structure, Function and Biosynthesis Splicing Mechanisms RNA Editing and Modification Translational Regulation RNA Turnover RNA structure and folding RNA Transport and Localization Viral RNAs

Michigan League (2nd Floor) Abstracts 325 - 335 336 - 363 364 - 379 380 - 393 394 - 417 418 - 456 457 - 463 464 - 478

Sponsor: PTC Therapeutics

Friday, June 1 08:00 – 17:00

Registration

09:00 – 12:30

Session 7: RNA & disease Keynote: Bruce Sullenger (Duke University Med Center) Chair: Andy Berglund (Univ Oregon)

12:30 – 14:00

Lunch

14:00 – 17:00

Concurrent Early Afternoon Sessions



Session 8A: RNA & RNP structure Keynote: Barbara Golden (Purdue Univ) Chair: Carl Correll (Rosalind Franklin Univ)



Session 8B: Small RNAs Keynote: Tom Tuschl (Rockefeller Univ) Chair: Javier Martinez (IMBA, Austrian Acad Sci)

Rackham Auditorium

17:30 – 19:30

Career Development Workshop

Rackham Auditorium

17:30 – 20:00

Board of Directors Meeting & Dinner

x

Power Center (Lobby) Power Center

Michigan Union Power Center

Michigan Union (Anderson Room D)

18:30 – 20:00

Dinner

Michigan Union

20:00 – 22:30

Beer Hall and Poster Session 3 Topic Small RNAs RNA Catalysis and Riboswitches Chemical and Synthetic Biology of RNA Splicing Regulation tRNA, snRNA, snoRNA, rRNA RNAs in Diseases Therapeutic RNAs Recent Poster Submissions

Michigan League (2nd Floor) Abstracts 479 - 501 502 - 530 531 - 535 536 - 576 577 - 603 604 - 623 624 - 632 701 - 711

Sponsor: UM Chemistry Department

Saturday, June 2 08:00 – 18:15

Registration

Power Center (Lobby)

09:00 – 12:30

Session 9: Interconnections & regulation Keynote: Doug Black (UCLA) Chair: Guillaume Chanfreau (UCLA)

12:30 – 14:00

Lunch

14:00 – 17:00

Concurrent Early Afternoon Sessions



Session 10A: Ribozymes & riboswitches Power Center Keynote: Adrian Ferré-D’Amaré (Nat’l Heart, Lung & Blood Inst) Chair: Peter Unrau (Simon Fraser Univ)



Session 10B: Function through sequence analysis Keynote: Fritz Roth (Univ Toronto) Chair: Mihaela Zavolan (Univ Basel)

Michigan Union

17:15 – 18:15 Awards Ceremony 18:30 – 19:00 Depart University of Michigan 19:00 – 23:00

Power Center

Rackham Auditorium

Power Center Transportation to the Henry Ford Museum by charter bus

Reception & Banquet The Henry Ford Museum Dinner, dancing and viewing of American and automotive history exhibits (separate reservation required)

Sunday, June 3 Conference concludes

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ADDITIONAL SCHEDULED EVENTS AT RNA 2012 Junior Scientists Pre-Conference Tour - Monday, May 28 1 PM-???; Meet at the Power Center • Open to all graduate students and post docs • No additional charge, no registration required

This is an informal gathering for graduate students and post docs to meet and socialize. It will be a great way to glimpse a part of Ann Arbor and catch up with colleagues before the meeting starts. The tour will start on campus then extend out to the Nichols Arboretum and Kerrytown, and finally finishing at Ashley’s bar and grill.

Campus Walking Tours - Tuesday, May 29 14:00-16:00 at the Power Center (Lobby) • Open to all attendees • No additional charge, but prior registration is required before May 1

Take a walking tour of the University of Michigan campus, enjoy the weather and meet fellow RNA 2012 attendees. These tours are scheduled before the official start of the meeting, so get here early and stretch your legs! The tours will be in groups of 20-30 people and will be hosted by the University of Michigan Office of Undergraduate Admissions; the same folks who give the tours to prospective students.

Meetings Committee Meeting - Wednesday, May 30 18:30-20:30 in the Michigan Union (Anderson Room D) • Open to the Meetings Committee, the Board of Directors, meeting sponsors, and (due to space constraints) a small number of additional observers

This meeting is where the venues for future RNA Society meetings are reviewed and selected. Any member of the RNA Society is welcome to attend, but due to space constraints one should request participation in advance by sending an email to the Meetings Committee Chair, David Lilley (d.m.j.lilley@ dundee.ac.uk).

Junior Scientists Social - Wednesday, May 30 19:30-20:30 in the Michigan Union (Patio) • Open to all graduate students and post docs • No additional charge, no registration required

The social is a nice setting to socialize with your fellow colleagues and talk some science over drinks.

Beer Hall and Poster Sessions – Wednesday-Friday, May 30-June 1 20:00-22:30 in the Michigan League (2nd Floor) • Open to all attendees • Traditional poster sessions with the addition of a beer hall

Wednesday sponsor: OSU Center for RNA Biology Thursday sponsor: PTC Therapeutics Friday sponsor: UM Chemistry Department and MI RNA Society

xii

Mentor/Mentee Lunch - Thursday, May 31 12:30-14:00 in the Michigan Union (Rogel Ballroom) • Open to all attendees • No additional charge, but registration is required before May 1

This lunch is an informal gathering that brings together 6-7 graduate students and post docs with one to two academic and industry mentors to answer student questions about careers. Topics include the pros and cons of academic vs industry careers, finding jobs, grant applications, and of course lots of interesting science. These lunches are fun for the mentors and hopefully fun and useful for the mentees as well. To the extent possible, mentors and mentees with common career and geographical objectives or experiences are grouped together.

RNA & Society Dinner – Thursday, May 31 (currently on “waitlist” basis only) 18:30-20:30 in the Michigan Union (Rogel Ballroom) • Open to all attendees • No additional charge, but registration is required due to space constraints Laurie Zoloth (Northwestern Univ) “Making Good: Ethical Issues in Synthetic Biology”

The RNA & Society dinners are an opportunity for RNA scientists to hear and to think about the impacts of science on society as well as the impacts of society on science.

Career Development Workshop – Friday, June 1 17:30-19:30 in Rackham Auditorium • Open to all attendees, but especially junior scientists

This is an opportunity for junior scientists to hear and discuss the issues around developing a career in science. The goal of this specific workshop is to demystify the interview process, and provide a forum in which young scientists can draw on the insights of established members of academia and industry.  This workshop should be particularly relevant to young members who will be interviewing for the next step in their careers. The workshop will feature a panel of speakers with diverse experiences. The focus will be on members who recently have been through the interview process, be it for a faculty position, tenure, or a job in industry.

Board of Directors Meeting – Friday, June 1 17:30-20:00 in the Michigan Union (Anderson Room D) • Open to the Board of Directors and (due to space constraints) a small number of additional observers

This is the business meeting of the RNA Society. Topics include an RNA journal update, results of the Meetings Committee deliberations, a report on finances and a vote on the next year’s budget, and new initiatives. Any member of the RNA Society is welcome to attend, but due to space constraints one should request participation in advance by sending an email to the CEO, Jim McSwiggen (mcswigj@ comcast.net).

xiii

Awards ceremony - Saturday, June 2 17:15-18:15 at the Power Center • Open to all attendees

This is our opportunity to honor the people who have made significant contributions to RNA science. This year’s awardees include: • Okle Uhlenbeck; RNA Society Lifetime Achievement Award • Brenda Peculis; RNA Society Service Award • RNA Society/Scaringe Award winners o Chenguang Gong o Tatjana Trcek Pulisic o Kotaro Nakanishi o Dipali Sashital • Poster prize winners

Conference Banquet - Saturday, June 2 18:30–19:00 Shuttles depart University of Michigan for The Henry Ford Museum 19:00–23:00 Banquet and Dance at The Henry Ford Museum • Open to all attendees • Registration required for planning purposes before May 15

The banquet will take place at the Ford Museum in Dearborn, Michigan. The museum is an American treasure, and we’ll have access to exhibits along with food, drink and dancing under an airplane suspended from the ceiling. This year, for the first time, we will have a scientist band playing for the dance. The band CTP from PTC Therapeutics will be playing a mixture of rock, blues, R&B. It will be great! We have chartered buses to transport attendees to the Ford Museum, which is located approximately 1/2 hour drive from Ann Arbor. Please let us know if you are attending the banquet, and whether you will ride the bus or provide your own transportation. Ford Museum offers plenty of free parking and maps will be provided for those who drive themselves. Guests are welcome! You may purchase additional banquet tickets for guests, or add back in if you originally opted out, by contacting registration@ rnasociety.org before May 15.

xiv

RNA 2012 AWARDS The RNA Society Lifetime Achievement Award The RNA Society Lifetime Achievement Award acknowledges the impact of an outstanding RNA scientist on the general scientific community. Each year beginning in 2003, the Board of Directors has selected the recipient who receives a lifetime membership in the RNA Society in recognition of their outstanding contributions. The award is presented at the Annual RNA Meeting where the recipient gives a special address to the RNA Society. Previous winners include Joan Steitz (2003), Harry Noller (2004), John Abelson (2005), Christine Guthrie (2006), Walter Keller (2007), Norm Pace (2008), Thomas Cech (2009), Fritz Eckstein (2010), and Witold Filipowicz (2011). Congratulations to Olke Uhlenbeck who is the winner of the 2012 RNA Society Lifetime Achievement Award..

The RNA Society Service Award The RNA Society Service Award is given in appreciation of outstanding service to the RNA community. The overall mission of the RNA Society is to facilitate sharing and dissemination of experimental results and emerging concepts in RNA research. Each year, the Board of Directors identifies the recipient of this award who has made exemplary contributions to these goals. Previous winners include Tim Nilsen (2003), Chris Greer (2004), Jean Beggs (2005), Olke Uhlenbeck (2006), Marvin Wickens (2007), Eric Westhof (2008), Anita Hopper (2009), Lynne Maquat (2010), and Evelyn Jabri (2011). Congratulations to Brenda Peculis who is the winner of the 2012 RNA Society Service Award.

The RNA Society/Scaringe Award The RNA Society/Scaringe Young Scientist Award was established to recognize the achievement of young scientists engaged in RNA research and to encourage them to pursue a career in the field of RNA. In 2004 and 2005, the RNA Society/Scaringe Award was made to the student author(s) of the best paper, as selected by the editors, published during the previous year in RNA. The winners of the 2004 and 2005 awards were Stefano Marzi and Ramesh Pillai, respectively. In 2006, this award was revamped and opened to all junior scientists (graduate students or postdoctoral fellows) from all regions of the world who have made a significant contribution to the broad area of RNA. The award is no longer restricted to authors who have published in the RNA journal. The award includes a cash prize and support for travel and registration costs for the awardee(s) to attend the annual RNA Society meeting. Previous graduate student winners include: Jeff Barrick (2006), Malte Beringer (2007), Qi Zhang (2008), Jeremey Wilusz (2009), John Calarco (2010), and Jasmine Perez (2011). Previous postdoctoral fellow winners include Megan Talkington (2006), Zefeng Wang (2007), Alexei Aravin (2008), Shobha Vasudevan (2009), Luciano Marraffini (2010), and Hani Zaher (2011). Congratulations to graduate students Chenguang Gong and Tatjana Trcek Pulisic, along with postdoctoral fellows Kotaro Nakanishi and Dipali Sashital, who are the winners of the 2012 RNA Society/Scaringe Award. xv

The ACS Chemical Biology Poster Prize ACS Chemical Biology is pleased to recognize junior scientists with a poster prize to be awarded at RNA 2012. The prize is for ‘innovative use of chemical biology applied to the study of RNA’, and consists of a free one-year online subscription to ACS Chemical Biology. All graduate students and postdoctoral fellows presenting posters at RNA 2012 are eligible.

The NSMB and NRMCB Poster Prizes Nature Structural & Molecular Biology (NSMB) is pleased to sponsor 3 poster prizes to be awarded at the 2012 RNA Society Meeting. The prizes, one in the area of molecular biology and biochemistry, one in genetics and development, and one in biophysics and structural biology, consist of a free one-year print and online subscription to NSMB. All graduate students and postdoctoral fellows presenting posters at the meeting are eligible.

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Nature Reviews Molecular Cell Biology (NRMCB) is pleased to sponsor a poster prize to be awarded at the 2012 RNA Society Meeting. The prize is for ‘innovation and interdisciplinary research,’ and consists of a free one-year print and online subscription to NRMCB. All graduate students and postdoctoral fellows presenting posters at the meeting are eligible.

The NIGMS 50th Anniversary Poster Prizes The National Institute of General Medical Sciences (NIGMS) is celebrating the 50th anniversary of the establishment of the institute in 1962. As part of that celebration, NIGMS will award two poster prizes in scientific areas that reflect the NIGMS mission—specifically, research that will increase understanding of life processes and lay the foundation for advances in disease diagnosis, treatment and prevention. The prizes will include travel awards to enable one graduate student and one postdoctoral fellow to attend and present a poster at the NIGMS 50th anniversary symposium in Bethesda, Maryland on October 17, 2012. The award will cover domestic airfare, lodging and meal expenses to enable the awardees to attend the half-day symposium.

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ABSTRACT LISTING (Note: Numbers refer to abstract numbers, not page numbers)

TUESDAY, MAY 29, 2012: 20:30 – 22:00 Opening Plenary Session - Power Center Abstracts 1 – 3 1

Cross-talk between dsRNA-mediated pathways: Is that my dsRNA or yours? Brenda Bass

2

tRNA Tuning in Translation Olke C. Uhlenbeck

3

Monitoring protein synthesis one codon at a time through ribosome profiling Jonathan Weissman

WEDNESDAY, MAY 30, 2012: 9:00 – 12:30 Keynote Speaker: Carol Fierke Session 1: RNA-protein interactions - Power Center Scott Blanchard, Chair Abstracts 4 – 16 4

Precursor tRNA Processing in Mitochondria and Chloroplasts Carol Fierke, Michael Howard, Wan Hsin Lim, Markos Koutmos

5

Insights into RNA Biology from a Mammalian Cell mRNA Interactome Alfredo Castello, Bernd Fischer, Katrin Eichelbaum, Rastislav Horos, Benedikt Beckmann, Claudia Strein, David Humphreys, Thomas Preiss, Lars Steinmetz, Jeroen Krijgsveld, Matthias Hentze

6

Searching for the Specificity of Non-specific RNA-binding Proteins Chaolin Zhang, Robert Darnell

7

High throughput sequencing kinetics (HTS-KIN) reveals hidden sequence determinants for an RNA-binding protein that binds substrates in a non-specific manner Ulf-Peter Guenther, Lindsay Yandek, Frank Campbell, David Anderson, Vernon Anderson, Eckhard Jankowsky, Michael Harris

8

Global Analysis of Yeast mRNPs Sarah Mitchell, Saumya Jain, Meipei She, Roy Parker

9

Higher Order mRNP Structure Revealed by the Cellular EJC Interactome Guramrit Singh, Alper Kucukural, Can Cenik, John Leszyk, Scott Shaffer, Zhiping Weng, Melissa Moore

10

CLIP-seq of the DEAD-box RNA Helicase eIF4AIII Reveals Transcriptome-wide Mapping of the Exon Junction Complex in Human Jerome Sauliere, Valentine Murigneux, Zhen Wang, Isabelle Barbosa, Hugues Roest Crollius, Herve Le Hir

11

Identification of targets of mRNA decay factors using RIP-seq reveals they target specific messages Jason Miller, Liye Zhang, Jennifer Kruk, B. Franklin Pugh, Joseph Reese

12

Allosteric inhibition of a stem cell RNA-binding protein by an intermediary metabolite Carina Clingman, Laura Deveau, Samantha Hay, Ryan Genga, Shivender Shandilya, Francesca Massi, Sean Ryder

13

Crystal structure of the eukaryotic RNA-induced silencing complex Kotaro Nakanishi, David Winberg, David Bartel, Dinshaw Patel

xvii

14

The Structural Basis for Binding and Unwinding of Duplex RNA by a DEAD-box Protein Anna Mallam, Mark Del Campo, Benjamin Gilman, David Sidote, Alan Lambowitz

15

DEAD-box Helicases Can Act as ATP-dependent RNA Clamps and as AMP Sensors Andrea Putnam, Fei Liu, Eckhard Jankowsky

16

Mechanism of foreign DNA selection in a bacterial adaptive immune system Dipali Sashital, Blake Wiedenheft, Jennifer Doudna

WEDNESDAY, MAY 30, 2012: 14:00 – 17:00 Keynote Speaker: Thomas Gingeras Concurrent Session 2A: Non-coding RNAs - Power Center Wade Winkler, Chair Abstracts 17 – 27 17

High resolution landscape of transcription in human cells Thomas Gingeras

18

Saccharomyces cerevisiae non-coding RNAs: bound to be broken Alex Tuck, David Tollervey

19

Non-coding RNA in transcriptional silencing Jordan Rowley, Qi Zheng, Brian Gregory, Andrzej Wierzbicki

20

Cancer-associated Long Noncoding RNA Regulates Cell Cycle Progression by Modulating the Expression of Oncogenic Transcription Factors Vidisha Tripathi, Xinying Zong, Zhen Shen, Ashish Lal, Supriya Prasanth, Kannanganattu Prasanth

21

LincRNA-p21 Suppresses Target mRNA translation Je-Hyun Yoon, Kotb Abdelmohsen, Subramanya Srikantan, Xiaoling Yang, Jennifer Martindale, Supriyo De, Maite Huarte, Ming Zhan, Kevin Becker, Myriam Gorospe

22

Architecture of Regulatory Long Non-coding RNAs Associated with Nuclear Receptor Biology Irina Novikova, Scott Hennelly, Karissa Sanbonmatsu

23

Telomerase RNA Biogenesis Involves Sequential Binding By Sm and Lsm Complexes Wen Tang, Peter Baumann

24

Saccharomyces cerevisiae Telomerase Activity is Exquisitely Sensitive to Subtle Perturbations of the TLC1 Pseudoknot 3’ Stem Fei Liu, Carla Theimer

25

The Microprocessor complex controls the activity of mammalian LINE-1 retrotransposons Sara Macias, Sara Heras, Mireya Plass, Eduardo Eyras, Jose Garcia-Perez, Javier Caceres

26

A Non-Coding RNA Produced by Arthropod-Borne Flaviviruses Inhibits the Cellular Exoribonuclease Xrn1 and Alters Host mRNA Stability Stephanie Moon, John Anderson, Carol Wilusz, Alexander Khromykh, Jeffrey Wilusz

27

Conservation of a Triple-Helix Forming RNA Stability Element in Noncoding and Genomic RNAs of Diverse Viruses Kazimierz Tycowski, Mei-Di Shu, Sumit Borah, Mary Shi, Joan Steitz

xviii

WEDNESDAY, MAY 30, 2012: 14:00 – 17:00 Keynote Speaker: Harry Noller Concurrent Session 2B: Ribosomes & translation - Rackham Auditorium Kurt Fredrick, Chair Abstracts 28 – 38 28

Some insights into the molecular mechanics of the ribosome Harry Noller, Jie Zhou, Laura Lancaster, Dmitri Ermolenko, Andrei Korostelev, John Paul Donohue

29

Nucleating 30S ribosome assembly through protein-RNA induced fit Sanjaya Abeysirigunawardena, Hajin Kim, Magan Mayerle, Taekjip Ha, Sarah Woodson

30

Real-Time Assembly Landscape of the Translation Initiation Machinery Pohl Milon, Riccardo Belardinelli, Cristina Maracci, Akanksha Goyal, Irena Andreeva, Liudmila Filonava, Marina Rodnina

31

Joining of 60S subunits and a translation-like cycle in 40S ribosome maturation Bethany Strunk, Megan Novak, Crystal Young, Katrin Karbstein

32

Evolutionary Divergence of Translation Quality Control Mechanisms During tRNA Charging Srujana Yadavalli, Noah Reynolds, Michael Ibba

33

EF-Tu Dynamics During Pretranslocation (PRE) Complex Formation Wei Liu, Chunlai Chen, Darius Kavaliauskas, Jared Schrader, Olke Uhlenbeck, Charlotte Knudsen, Yale Goldman, Barry Cooperman

34

Structural Studies of Streptomycin Resistant and Dependent Ribosomes Hasan Demirci, Steven Gregory, Frank Murphy, Gerwald Jogl, Albert Dahlberg

35

Role of Inter-subunit Bridges in Ribosomal Translocation Qi Liu, Kurt Fredrick

36

Single Molecule Visualization of Stalled Ribosomes: insight into the mechanism of ribosomal frameshifting Peiwu Qin, Dongmei Yu, Xiaobing Zou, Peter Cornish

37

HIV-1 Frameshift Efficiency is Determined Solely by the Stability of Base Pairs at the mRNA Entrance Tunnel of the Ribosome Kathryn Mouzakis, Andrew Lang, Kirk Vander Meulen, Samuel Butcher

38

‘Late’ Translation Arrest: An Unconventional Mode of Inhibition of Protein Synthesis by Macrolides Krishna Kannan, Nora Vazquez-Laslop, Alexander Mankin

WEDNESDAY, MAY 30, 2012: 17:15 – 18:30 Concurrent Session 3A: RNA-protein architecture - Power Center Hashim Al-Hashimi, Chair Abstracts 39 – 45 39

Characterization of the Single-stranded RNA Dynamical Ensemble Jun Feng, Katie Eichhorn, Hashim Al-Hashimi, Charles Brooks III

40

Pri-miR-17-92a Transcript Folds into a Tertiary Structure and Autoregulates its Processing Saikat Chakraborty, Shabana Mehtab, Anand Patwardhan, Yamuna Krishnan

41

Molecular Mimicry of Human tRNA by HIV-1 RNA Genome Facilitates Viral Replication Christopher Jones, Jenan Saadatmand, Erik Olson, Jeremy Fichtenbaum, Lawrence Kleiman, Karin Musier-Forsyth

42

Architectural Contributions of Genomic RNA to Retrotransposon Replication Katarzyna Purzycka, Michal Legiewicz, Emiko Matsuda, Linda Eizentstat, Sabrina Lusvarghi, Qing Huang, Jef Boeke, David Garfinkel, Stuart Le Grice

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43

YB-1 Binds to CAUC Motifs and Stimulates Exon Inclusion by Enhancing the Recruitment of U2AF to Weak Polypyrimidine Tracts Wenjuan Wei, Shirong Mu, Monika Heiner, Lijuan Cao, Jingyi Hui

44

Crystal Structure of Cwc2 Reveals a Novel Architecture of a Multipartite RNA-binding Protein Jana Schmitzova, Nicolas Rasche, Olexandr Dybkov, Katharina Kramer, Patrizia Fabrizio, Henning Urlaub, Reinhard Luehrmann, Vladimir Pena

45

The DEAD-box Protein Rok1 and Its Co-factor Rrp5 Catalyze Helix Formation During 40S Ribosome Assembly Crystal Young, Katrin Karbstein

WEDNESDAY, MAY 30, 2012: 17:15 – 18:30 Concurrent Session 3B: RNA-seq & computational structure prediction - Rackham Auditorium Alain Laederach, Chair Abstracts 46 – 52 46

Multilign: An Algorithm to Improve Prediction of Secondary Structures Conserved in Multiple RNA Sequences Zhenjiang Xu, David Mathews

47

Scanning Very Long Sequences for Suboptimal Structures Including Pseudoknots Using an Adjustable Window Size and Flexibility Wayne Dawson, Shingo Nakamura, Gota Kawai, Kiyoshi Asai

48

Deep sequencing Ribosome Protected mRNA Fragments from polysomes and monosomes isolated by size exclusion chromatography Scott Kuersten, Ramesh Vaidyanathan, Agnes Radek, Silvi Rouskin, Sajani Swami, Josh Dunn, Jonathan Weissman

49

Thermostable Group II Intron Reverse Transcriptase Fusion Proteins and their Applications in cDNA Synthesis and Next-Generation Sequencing Sabine Mohr, Eman Ghanem, Whitney Smith, Dennis Sheeter, Yidan Qin, Damon Polioudakis, Vishwanath Iyer, Scott Hunicke-Smith, Sajani Swamy, Scott Kuersten, Alan Lambowitz

50

RNA 3D Hub - a new online resource for RNA structural bioinformatics Anton Petrov, Craig Zirbel, Neocles Leontis

51

Encapsidated Viral RNA Structure Prediction Susan Schroeder, Samuel Bleckley, Jonathan Stone, Jui-wen Liu

52

Genesilico Web Servers for Predicting of RNA -Metal Ion and -Ligand Interactions Anna Philips, Kaja Milanowska, Grzegorz Lach, Michal Boniecki, Kristian Rother, Janusz Bujnicki

THURSDAY, MAY 31, 2012: 9:00 – 12:30 Keynote Speaker: Joel Belasco Session 4: RNA turnover - Power Center Oliver Mühlemann, Chair Abstracts 53 – 65 53

5’-Terminal Control of RNA Degradation Dan Luciano, Jamie Richards, Ping-kun Hsieh, Joel Belasco

54

Polyadenylation Helps Regulates Functional tRNA Levels in Escherichia coli Sidney Kushner, Bijoy Mohanty

55

Ribonucleases control RNA damage and protect cells against oxidative stress Zhongwei Li

56

Global Analysis Reveals Multiple Pathways for Unique Regulation of mRNA Decay in Induced Pluripotent Stem Cells Ashley Neff, Ju Youn Lee, Bin Tian, Jeffrey Wilusz, Carol Wilusz

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57

Mechanism of Processive and Cap-Stimulated mRNA Poly(A) Tail Degradation Niklas Henriksson, Per Nilsson, Magnus Lindell, Mikael Nissbeck, Samuel Flores, Anders Virtanen

58

Crystal Structure and Heterodimerization of the NOT Box Domains of human NOT2 and NOT3 Andreas Boland, Ying Chen, Lara Wohlbold, Praveen Bawankar, Oliver Weichenrieder, Elisa Izaurralde

59

Rrp47p Forms a Heterodimer with Yeast Nuclear Exosome Component Rrp6p and Stimulates its Activity Emil Dedic, Paulina Seweryn, Anette Jonstrup, Jan Jensen, Natalya Fedosova, Søren Hoffmann, Thomas Boesen, Ditlev Brodersen

60

Crystal Structure of a Yeast 11-Subunit Exosome Complex Bound to RNA Debora Makino, Marc Baumgärtner, Elena Conti

61

Human Staufen1 Dimerization via Swapping a Conserved Motif and a Degenerate Double-Stranded RNABinding Domain Augments UPF1 Binding and mRNA Decay Michael Gleghorn, Chenguang Gong, Clara Kielkopf, Lynne Maquat

62

Single Molecule Analysis of Nonsense-mediated mRNA Decay in Yeast Victor Serebrov, Nadia Amrani, Larry Friedman, Jeff Gelles, Melissa Moore, Allan Jacobson

63

A Large-Scale Survey Reveals Selective Ultra-Fast microRNA Turnover Rates Yanwen Guo, Yinghong Ma, Caihong Qiu, Jun Lu

64

A Primate Herpesvirus Promotes T-cell Activation via Degradation of microRNA-27 Eric Guo, Kasandra Riley, Joan Steitz

65

The Nuclear Poly(A) Binding Protein Promotes Polyadenylation-mediated Decay of Nuclear Transcripts in Human Cells Stefan Bresson, Nicholas Conrad

THURSDAY, MAY 31, 2012: 14:00 – 17:00 Keynote Speaker: Kristen Lynch Concurrent Session 5a: Splicing mechanism - Power Center Tracy Johnson, Chair Abstracts 66 – 76 66

Repression of Exon Splicing by an HnRNP Network that Alters Spliceosomal Interactions Upstream of the 5’ Splice Site Ni-ting Chiou, Ganesh Shankarling, Kristen Lynch

67

Group II Introns at Work: Intermediates of the Splicing Cycle Revealed by X-ray Crystallography Marco Marcia, Anna Pyle

68

Single Molecule pre-mRNA Splicing: Splice Site Juxtaposition During Spliceosome Assembly Mario Blanco, Ramya Krishnan, Matthew Kahlscheuer, John Abelson, Christine Guthrie, Nils Walter

69

A Role for the Stem-loop 4 of U1 snRNA in Splice Site Pairing Shalini Sharma, Somsakul Wongpalee, Douglas Black

70

Two unconventional RRMs of the RNA chaperone Prp24 have opposing effects on the U6 RNA internal stem-loop Ashley Richie, Elizabeth Curran, Kristie Andrews, Christine Treba, Samuel Butcher, David Brow

71

Cwc21p regulates branchsite usage in meiotic splicing Amit Gautam, Richard Grainger, David Barrass, Jean Beggs

72

Single Molecule Visualization of the DEAH-box ATPases Prp16p and Prp22p Interacting with Spliceosomes Eric Anderson, Aaron Hoskins, Larry Friedman, Jeff Gelles, Melissa Moore

73

Structural rearrangements within human spliceosomes following exon ligation Janine Ilagan, Robert Chalkley, Al Burlingame, Melissa Jurica

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74

Protein composition, morphology and disassembly mechanism of the intron-lariat spliceosome from S. cerevisiae Jean-Baptiste Fourmann, Jana Schmitzová, Berthold Kastner, Henning Christian, Henning Urlaub, Ralf Ficner, Patrizia Fabrizio, Reinhard Lührmann

75

Spliceosome Dynamics in the Catalytic Center Chi-Kang Tseng, Soo-Chen Cheng

76

Imaging pre-mRNA splicing in living cells with single-molecule sensitivity and high temporal resolution José Rino, Célia Carvalho, Tomas Kirchhausen, Maria Carmo-Fonseca

THURSDAY, MAY 31, 2012: 14:00 – 17:00 Keynote Speaker: Jamie Williamson Concurrent Session 5B: RNA editing & modification - Rackham Auditorium Eric Phizicky, Chair Abstracts 77 – 87 77

Assembly of Bacterial Ribosomes in Cells James R. Williamson

78

Mutations Within A Conserved loop Of Human ADAR2 Affect Base Flipping Of The Target Adenosine Ashani Kuttan, Brenda Bass

79

A Long-Range Tertiary Interaction Affects RNA Editing In Vivo Leila Rieder, Barry Hoopengardner, Lee Ann Smith, Robert Reenan

80

Rescue of neurodegeneration in Adar deficient flies is independent of RNA editing activity Simona Paro, Leeanne McGurk, Liam Keegan, Mary O’Connell

81

Developmental regulation of brain specific miRNAs by A-to-I RNA editing Ylva Ekdahl, Hossein Farahani, Mikaela Behm, Jens Lagergren, Marie Ohman

82

Maturation Of H/ACA Box SnoRNAs: PAPD5-Dependent Adenylation And PARN-Dependent Trimming Christiane Harnisch, Heike Berndt, Christiane Rammelt, Nadine Stöhr, Anne Zirkel, Juliane Dohm, Heinz Himmelbauer, Stefan Hüttelmaier, Elmar Wahle

83

Roquin Promotes Constitutive mRNA Deadenylation and Decay via an Abundant Stem-loop Recognition Element Kathrin Leppek, Johanna Schott, Sonja Reitter, Ming Hammond, Georg Stoecklin

84

Identification of human and Drosophila 5’-nucleotidases degrading 7-methyl guanosinemonophosphate Juliane Buschmann, Thomas Monecke, Bodo Moritz, Mandy Jeske, Thomas Rudolph, Ralf Ficner, Elmar Wahle

85

Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA Hardip Patel, Jeffrey Squires, Marco Nousch, Tennille Sibbritt, David Humphreys, Brian Parker, Catherine Suter, Thomas Preiss

86

Unexpected Complexity of Critical Methylation Reactions in the tRNA Anticodon Loop Michael Guy, Brandon Podyma, Eric Phizicky

87

Divergent Trans Editing Mechanisms used to Ensure Fidelity in Proline Codon Translation Karin Musier-Forsyth, Mom Das, Oscar Vargas-Rodriguez, Sandeep Kumar, Christopher Hadad

THURSDAY, MAY 31, 2012: 17:15 – 18:30 Concurrent Session 6A: Aptmers - Power Center Donald Burke, Chair Abstracts 88 – 94 88 xxii

No Target Left Behind: A Microfluidics Solution for Standardized Partitioning in Aptamer Selections Christina Birch, Han Wei Hou, Jongyoon Han, Jacquin Niles

89

New Aptamers, Neutral Networks, and Next-generation Sequencing: A Fresh Look at HIV Reverse Transcriptase Aptamers Mark Ditzler, Debojit Bose, Christopher Bottoms, Katherine Virkler, Andrew Sawyer, Angela Whatley, William Spollen, Scott Givan, Donald Burke

90

Aptamer Structure, Dynamics and Function as Investigated by Integrative Computational and Experimental Approaches Tianjiao Wang, Monica Lamm, Julie Hoy, Bruce Fulton, Mazdak Mina, Marit Nilsen-Hamilton

91

A Systematic Approach to Evolve Aptamers with New Specificities Muslum ILGU, Ragothaman Yennamalli, Megan Kleckler, Taner Sen, Monica Lamm, Marit Nilsen-Hamilton

92

Orthogonal Riboswitches As Tools For Controlling Gene Expression In Bacteria Christopher Robinson, Neil Dixon, John Duncan, Torsten Geerlings, Ming-Cheng Wu, Phillip Lowe, Jason Micklefield

93

Engineered mRNA Regulation Using an Inducible Protein-RNA Aptamer Interaction Brian Belmont, Stephen Goldfless, Jessica Liu, Jacquin Niles

94

Human adenosine aptamers Michael Vu, Nora Jameson, Stuart Masuda, Dana Lin, Rosa Larralde-Ridaura, Andrej Luptak

THURSDAY, MAY 31, 2012: 17:15 – 18:30 Concurrent Session 6B: Surveillance & decay - Rackham Auditorium Anita Hopper, Chair Abstracts 95 – 101 95

Global Analysis of the Nuclear Processing of Unspliced U12-type Introns by the Exosome Elina Niemela, Ger Pruijn, Mikko Frilander

96

tRNA Splicing Endonuclease: Investigating Cytoplasmic Function Unrelated to tRNA Splicing Nripesh Dhungel, Anita Hopper

97

Turnover of Pre-mRNA Splicing Intermediates by a Novel Debranching Enzyme Jay Hesselberth, Stephen Garrey, Masad Damha, Stanley Fields, Adam Kotolik

98

Role of TUT3 in the Initial Step of Histone mRNA Degradation Patrick Lackey, Michael Slevin, Shawn Lyons, William Marzluff

99

Air Proteins Control Differential TRAMP Substrate Specificity for Nuclear RNA Surveillance Karyn Schmidt, Zhenjiang Xu, David Mathews, J. Scott Butler

100 Human Pumilio proteins recruit CCR4-POP2 deadenylases to efficiently repress mRNAs Jamie Van Etten, Trista Schagat, Joel Hrit, Chase Weidmann, Justin Brumbaugh, Josh Coon, Aaron Goldstrohm 101 40S Subunit Dissociation and Proteasome-dependent RNA Degradation in Nonfunctional 25S rRNA Decay Makoto Kitabatake, Kotaro Fujii, Tokie Sakai, Tomoko Sakata, Mutsuhito Ohno

FRIDAY, JUNE 1, 2012: 9:00 – 12:30 Keynote Speaker: Bruce Sullenger Session 7: RNA & disease - Power Center Andy Berglund, Chair Abstracts 102 – 114 102 Driving Innovation by Forward and Reverse Translation with Aptamers Bruce Sullenger, Shahid Nimjee, Sabah Oney, Kristin Bompiani, Jaewoo Lee, Eda Holl, Shashank Jain, George Pitoc, Kam Leong 103 Decreased Transcription End Sites for SETX Knockdown in Motor Neuron Progenitors Matthew Hansen, Lulu Tsao, Nebiyou Samuel, Edgar Ibarra, Ebone Ingram, Tzu-Ying Chuang, Ben Greenberger, Rachel Moda, Rob Kulathinal, Miriam Bucheli xxiii

104 Extensive and dynamic heteroallelic expression and RNA editing in human blood cells using high-throughput RNA sequencing Jennifer Li-Pook-Than, Rui Chen, George Mias, Lihua Jiang, Hugo Lam, Hua Tang, Michael Snyder 105 Combinatorial Splicing Regulation by Muscleblind-like Proteins in Development and Disease Kuang-Yung Lee, Moyi Li, Mini Manchanda, Lily Shiue, Chris Chamberlain , Apoorva Mohan, Hannah Hong, Manuel Ares, Jr, Maurice Swanson 106 Structure Optimization of a Family of Compounds Identifies One Compound that Reverses the Hallmark Symptom of Myotonic Dystrophy in a Mouse Model and Reveals a Novel Mechanism of Action Leslie Coonrod, Masayuki Nakamori, Cameron Hilton, Micah Bodner, Michael Haley, Charles Thornton, Andrew Berglund 107 Loss-of-function Analysis of the ALS-associated Proteins Tdp-43 and Fus/Tls Reveals Global RNA Mis-regulation that is Conserved from Mouse to Humans Kasey Hutt, Magdalini Polymenidou, Clotilde Lagier-Tourenne, Anthony Vu, Stephanie Huelga, Sebastian Markmiller, Edward Wancewicz, Curt Mazur, Yalda Sedaghat, John Paul Donohue, Lily Shiue, C Frank Bennett, Don Cleveland, Gene Yeo 108 Structural basis for the regulation of the spliceosomal RNP remodeling enzyme, Brr2, by Prp8 and links to retinitis pigmentosa Sina Mozaffari Jovin, Traudy Wandersleben, Karine F. Santos, Markus C. Wahl, Reinhard Lührmann 109 Rescue of Hearing and Vestibular Function in Deaf Mice Using Antisense Oligonucleotides That Block a Cryptic Splice Site Francine Jodelka, Anthony Hinrich, Jennifer Lentz, Kate McCaffrey, Hamilton Farris, Matthew Spalitta, Dominik Duelli, Frank Rigo, Michelle Hastings 110 Mechanistic Defects of Mutant U4atac Minor Spliceosomal snRNAs in Primordial Dwarfism Faegheh Jafarifar, Rosemary Dietrich, Richard Padgett 111 Rescue of nonsense mutation containing mRNAs expression by amlexanox Sara Gonzalez-Hilarion, Jieshuang Jia, Nadège Debreuck, Fabrice Lejeune 112 Repeat associated Non-AUG initiated translation drives Polyglycine production and neurodegeneration in Fragile X Tremor Ataxia Syndrome Peter Todd, Seok Yoon Oh, Amy Krans, Michelle Frazer, Abigail Renoux, Fang He, Elan Louis, J. Paul Taylor, Henry Paulson 113 The AAA ATPases pontin and reptin in the R2TP complex remove SHQ1 from NAP57/dyskerin during biogenesis of H/ACA ribonucleoproteins Rosario Machado-Pinilla, Dominique Liger, Nicolas Leulliot, U. Thomas Meier 114 Effective inhibition of cytomegalovirus infection by external guide sequences in mice Xiaohong Jiang, Hao Gong, Yuan-Chuan Chen, Gia-Phong Vu, Phong Trang, Chen-Yu Zhang, Sangwei Lu, Fenyong Liu

FRIDAY, JUNE 1, 2012: 14:00 – 17:00 Keynote Speaker: Barbara Golden Concurrent Session 8A: RNA & RNP structure - Power Center Carl Correll, Chair Abstracts 115 – 125 115 An integrated picture of HDV ribozyme catalysis Barbara Golden, Ji Chen, Pallavi Thaplyal, Abir Ganguly, Sharon Hammes-Schiffer, Philip Bevilacqua 116 Structural Basis for Telomerase RNA Recognition and RNP Assembly by the Holoenzyme La Family Protein p65 Mahavir Singh, Zhonghua Wang, Bon-Kyung Koo, Anooj Patel, Duilio Cascio, Kathleen Collins, Juli Feigon 117 Crystal structure of the spliceosomal RNP remodeling enzyme, Brr2 Karine Santos, Sina Mozaffari Jovin, Gert Weber, Vladimir Pena, Reinhard Lührmann, Markus Wahl 118 The 2.7 Angstrom Crystal Structure of the 2’,5’ GIR1 Branching Ribozyme Mélanie Meyer, Eric Westhoff, Steinar Johansen, Henrik Nielsen, Benoît MASQUIDA xxiv

119 How The HIV Virus Selects Its Own mRNA For Export: The Topology OF The HIV-1 Rev Response Element, A molecular Beacon For Specificity And Coorperativity For Rev Binding Jinbu Wang, Xianyang Fang, Michelle Mitchell, Xiaobing Zuo, Yi Wang, Soenke Siefert, Randall Winnas, R. Andrew Byrd, Stuart LeGrice, Yun-Xing Wang 120 NMR structures of CPEB4 RRMs free and bound to RNA reveals an unexpected fold and mode of RNA recognition by two RRMs Tariq Afroz, Lenka Skrisovska, Frederic Allain 121 Nrd1 RRM binds RNAs via two distinct RNA-binding surfaces Veronika Bacikova, Karel Kubicek, Josef Pasulka, Fruzsina Hobor, Richard Stefl 122 Biochemical Analysis of Primary miRNA Structure Reveals an Extensive Capacity to Deform Near the Drosha Cut Site Kaycee Quarles, Debashish Sahu, Ellen Forsyth, Christopher Wostenberg, Scott Showalter 123 Differential Fine-Tuning of Ligand-Induced Folding in Single Transcriptional and Translational Riboswitches Krishna Suddala, Arlie Rinaldi, Jun Feng, Anthony Mustoe, Catherine Eichhorn, Hashim Al-Hashimi, Charles Brooks III, Nils Walter 124 SNitching Riboswitches; on the Structural Sensitivity of RNA to SNPs Justin Ritz, Joshua Martin, Alain Laederach 125 RNA Catalysis Through Compartmentalization Christopher Strulson, Rosalynn Molden, Christine Keating, Philip Bevilacqua

FRIDAY, JUNE 1, 2012: 14:00 – 17:00 Keynote Speaker: Thomas Tuschl Concurrent Session 8B: Small RNAs - Rackham Auditorium Javier Martinez, Chair Abstracts 126 – 136 126 Characterization of Regulatory Small RNAs and RNA-Binding Proteins Thomas Tuschl 127 Argonaute and RISC in the Mammalian Cell Nucleus Keith Gagnon, Roya Kalantari, David Corey 128 A novel autoregulatory loop involving Argonaute and let-7 regulates miRNA biogenesis Dimitrios Zisoulis, Zoya Kai, Roger Chang, Amy Pasquinelli 129 Intracellular Single Molecule High Resolution Localization and Counting of microRNAs Sethu Pitchiaya, John Androsavich, Nils Walter 130 Evolution and gene silencing capacity of Piwi-interacting RNAs Jessica Matts, Gung-wei Chirn, Christina Post, Charlotte Logan, Nelson Lau 131 Terminal A- and U-rich Motifs Inhibit Uridylation and Degradation of the 3’ end of the MALAT1 Noncoding RNA Jeremy Wilusz, Laura Lu, Phillip Sharp 132 A SigmaB Dependent Regulatory RNA Modulates Biofilm and Capsule Formation in Staphylococcus aureus Cedric Romilly, Claire Lays, Efthimia Lioliou, Florence Vincent, François Vandenesch, Pascale Romby, Sandrine Boisset, Tom Geissmann 133 A phylogenetically conserved hairpin switch controls 6S RNA transcriptional regulation by triggering sigma-70 release from bacterial polymerase Shyam Panchapakesan, Mariana Oviedo, Lindsay Shephard, Peter Unrau 134 Presence of Hfq Binding Site Facilitates Identification of Functionally Important mRNA Targets Martha Faner, Rebecca Swett, Amit Kumar, Cassandra Joiner, Andrew Feig 135 The Redox-Sensing Aconitase B Protein Act Against sRNA Distal Nucleolytic Cleavage Julie-Anna Benjamin, Marie-Claude Carrier, Eric Massé xxv

136 A Salmonella Small Non-Coding RNA Facilitates Bacterial Invasion and Intracellular Replication by Modulating the Expression of Virulence Factors Hao Gong, Gia-Phong Vu, Yong Bai, Elton Chan, Ruobin Wu, Edward Yang, Fenyong Liu, Sangwei Lu

SATURDAY, JUNE 2, 2012: 9:00 – 12:30 Keynote Speaker: Douglas Black Session 9: Interconnections & regulation - Power Center Guillaume Chanfreau, Chair Abstracts 137 – 149 137 The splicing regulator Rbfox2 is required for both cerebellar development and mature motor function Lauren Graham, Pratap Meera, Peter Stoilov, Lily Shiue, Janelle O’Brian, Miriam Meisler, Manuel Ares Jr., Thomas Otis, Douglas Black 138 The CBCAP Complex binds various Classes of RNAs and links the Cap to 3’-end Formation and RNA Export Marie Hallais, Frédéric Pontvianne, Daniela Lener, Marcello Clerici, Thierry Gostan, Marie-Cécile Robert, Stephen Cusack, Céline Verheggen, Edouard Bertrand 139 RNAi Keeps Atf1-Bound Stress Response Genes in Check at Nuclear Pores Katrina Woolcock, Rieka Stunnenberg, Dimos Gaidatzis, Hans-Rudolf Hotz, Stephan Emmerth, Pierre Barraud, Marc Bühler 140 Stalled Spliceosomes Trigger RNAi in Cryptococcus neoformans: A New Function for pre-mRNA Splicing in Genome Defense Phillip Dumesic, Prashanthi Natarajan, Changbin Chen, Anna Drinnenberg, Benjamin Schiller, James Moresco, James Thompson, John Yates III, Hiten Madhani 141 First Exon Length Controls Activating Histone Marks and Transcriptional Fidelity Nicole Bieberstein, Fernando Carrillo Oesterreich, Karla Neugebauer 142 Co-transcriptional Splicing Regulation During Environmental Stress in Budding Yeast Jaclyn Greimann, Megan Bergkessel, Christine Guthrie 143 The Heterogeneous Nuclear Ribonucleoprotein hnRNPM Accelerates EMT by Promoting Skipping of CD44 Variable Exons Yilin Xu, Chonghui Cheng 144 Ptbp2 Represses Adult-Specific Splicing To Regulate The Generation Of Neuronal Precursors In The Embryonic Brain Donny Licatalosi, Masato Yano, John Fak, Aldo Mele, Sarah Grabinski, Chaolin Zhang, Robert Darnell 145 Genome-wide Changes In Binding Of The RNA Processing Factor hnRNP L Are Induced In Response To Cell Signaling Pathways In Human Immune Cells Ganesh Shankarling, Brian Cole, Kristen Lynch 146 Genome-wide Analysis of Alternative Splicing in Drosophila Brenton Graveley, Angela Brooks, Ben Brown, Michael Duff, Gemma May, Robert Obar, Sara Olson, Peter Cherbas, Thom Kaufman, Steven Brenner, Tom Gingeras, Roger Hoskins, Brian Oliver, Spyros Artavanis-Tsakonas, Susan Celniker 147 Nuclear Envelope Budding Enables Large Ribonucleoprotein Particle Export During Synaptic Wnt Signaling Sean Speese, James Ashley, Vahbiz Jokhi, John Nunnari, Romina Barria, Yihang Li, Bulent Ataman, Alex Koon, Young-Tae Chang, Qian Li, Melissa Moore, Vivian Budnik 148 RanBP2/Nup358 Potentiates the Translation of a Subset of mRNAs Encoding Secretory Proteins Hui Zhang, Kohila Mahadevan, Abdalla Akef, Alexander Palazzo 149 Regulated Protein Synthesis Errors as a Potential, New Mechanism of Stress Response Thomas Jones, Chloe Weisberg, Tao Pan

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SATURDAY, JUNE 2, 2012: 14:00 – 17:00 Keynote Speaker: Adrian Ferré-D’Amaré Concurrent Session 10A: Ribozymes & riboswitches - Power Center Peter Unrau, Chair Abstracts 150 – 160 150 Ribozymes and Riboswitches: Structure, Function and Evolution Adrian Ferré-D’Amaré 151 Observation of Intrinsic Conformational Dynamics of TPP Riboswitch and Its Structural Transition Induced by TPP Binding Heesoo Uhm, Sungchul Hohng 152 Complete Thermodynamic and Kinetic Characterization of Riboswitch Functioning with a New ITC Methodology (kinITC) Philippe Dumas, Dominique Burnouf, Sondes Guedich, Eric Ennifar, Guillaume Bec, Mireille Baltzinger 153 Leader-Linker Interaction Abolishes Ligand Binding Cooperativity in Glycine Riboswitches Jingdong Ye, Eileen Sherman 154 New Mechanistic Insights into how Synthetic Riboswitches Regulate Translation Dennis Mishler, Justin Gallivan 155 Riboswitches Control mRNA Decay in Escherichia coli Marie-Pier Caron, Audrey Dubé, Laurène Bastet, Antony Lussier, Maxime Simoneau-Roy, Eric Massé, Daniel Lafontaine 156 A Thiamine-Utilizing Ribozyme Decarboxylates a Pyruvate-like Substrate Paul Cernak, Dipankar Sen 157 Alternative Splicing of a Group II Intron in Clostridium tetani Bonnie McNeil, Dawn Simon, Steven Zimmerly 158 Nucleobase-Mediated General Acid-Base Catalysis by the Hairpin Ribozyme Stephanie Kath-Schorr, Timothy Wilson, Nan-Sheng Li, Jun Lu, Joseph Piccirilli, David Lilley 159 The Hairpin Ribozyme Self-Cleavage Reaction Pathway Involving Deprotonated G8 as General Base and Protonated A38 as General Acid Seems to Be the Most Consistent with Experimental Data Vojtech Mlynsky, Pavel Banas, Nils Walter, Jiri Sponer, Michal Otyepka 160 Probing Metal Ion Binding Sites in the P4 Helix of Bacillus subtilis RNase P Yu Chen, Carol Fierke

SATURDAY, JUNE 2, 2012: 14:00 – 17:00 Keynote Speaker: Frederick (Fritz) Roth Concurrent Session 10B: Function through sequence analysis - Rackham Auditorium Mihaela Zavolan, Chair Abstracts 161 – 171 161 Interplay between 5’ UTR introns & nuclear mRNA export for secretory and mitochondrial genes Frederick Roth 162 Reconstruction of an Ancestral U1A/U2B”/SNF Family Protein Sandra Williams, Kathleen Hall 163 Inclusion of Large Internal Exons (>1 kb) in 5% of Human mRNAs Mohan Bolisetty, Karen Beemon 164 Deep Intron Elements Mediate Nested Splicing Events at Consecutive AG-dinucleotides to Regulate Alternative 3’ Splice Site Choice in Vertebrate 4.1 Genes Marilyn Parra, Thomas Gallagher, Sharon Amacher, Narla Mohandas, John Conboy xxvii

165 Unusual Patterns of RNA Processing in a microsporidian Parasite Revealed by RNA-seq Cameron Grisdale, Naomi Fast 166 Deep RNA-seq of Small RNAs Across 17 Diverse Archaea: New Frontiers in Gene Regulation Todd Lowe, David Bernick, Lauren Lui, Andrew Holmes, Patrick Dennis 167 A High-resolution CstF64-RNA Interaction Network Map Reveals Mechanisms For Poly(A) site Recognition And Novel Functions Chengguo Yao, Jacob Biesinger, Anke Busch, Xiaohui Xie, Yongsheng Shi 168 Regulation and evolution of alternative polyA sites in protein coding regions Zhe Ji, Mainul Hoque, Dinghai Zheng, Wenting Luo, Bei You, Wencheng Li, Bin Tian 169 U1 snRNP Determines mRNA Length and Regulates Isoform Expression Michael Berg, Larry Singh, Ihab Younis, Qiang Liu, Anna Maria Pinto, Daisuke Kaida, Zhenxi Zhang, Lili Wan, Gideon Dreyfuss 170 Two distinct tRNA Modifications Influenced the Evolution of Genome structure and Codon Usage Eva Novoa, Mariana Pavon-Eternod, Tao Pan, Lluís Ribas de Pouplana 171 Transcript Leader Annotation and Insight into Genes’ Translational Behavior Joshua Arribere, Wendy Gilbert

Poster Sessions WEDNESDAY, MAY 30, 2012: 20:00 – 22:30 Poster Session 1 - Michigan League (2nd Floor) Abstracts 172 – 324 172 NanoFolder: Multi-strand Secondary Structure Prediction and Sequence Design of RNA Nanoparticles Eckart Bindewald, Kirill Afonin, Luc Jaeger, Bruce Shapiro 173 CLIP-Based Prediction Of Mammalian MicroRNA Binding Sites Chaochun Liu, Bibekanand Mallick, Dang Long, William Rennie, Adam Wolenc, Charles Carmack, Ye Ding 174 Discovery of Novel ncRNA by Scanning Multiple Genome Alignments Yinghan Fu, David Mathews 175 Coordinated Dynamics of Cell Growth and Transcription David Gresham 176 Sharing and Archiving Nucleic Acid Structure Mapping data Philippe Rocca-Serra, Stanislav Bellaousov, Amanda Birmingham, Chunxia Chen, Pablo Cordero, Rhiju Das, Lauren Davis-Neulander, Caia Duncan, Matthew Halvorsen, Rob Knight, Neocles Leontis, David Mathews, Justin Ritz, Jesse Stombaugh, Kevin Weeks, Craig Zirbel, Alain Laederach 177 Identification of Allele-specific Alternative mRNA Processing via Transcriptome Sequencing Gang Li, Jae Hoon Bahn, Jae-Hyung Lee, Guangdun Peng, Zugen Chen, Stanley Nelson, Xinshu Xiao 178 R’N’B - a Database of RNA Metabolic Pathways Kaja Milanowska, Zuzanna Balcer, Anna Å ukasik, Katarzyna Mikolajczak, Marcin Skorupski, Kristian Rother, Janusz Bujnicki 179 Influenza A Virus Coding Regions Exhibit Host-specific Global Ordered RNA Structure Salvatore Priore, Walter Moss, Douglas Turner 180 Inference of Recurrent 3D RNA Motifs from Sequence James Roll, Craig Zirbel, Anton Petrov, Neocles Leontis 181 Abstract Withdrawn 182 Abstract Withdrawn xxviii

183 Rediscovery of the p53 transcriptome Mary Allen, Robin Dowell, Joaquin Espinosa 184 Deciphering the Logic of Bypassing Essential DExD/H-box Proteins Sean Shang-Lin Chang, Chung-Shu Yeh, Tien-Hsien Chang 185 Genome-wide Intron Mapping by Spliceosome Footprinting Weijun Chen, Hennady Shulha, Ami Ashar, Jing Yan, Charles Query, Nick Rhind, Zhiping Weng, Melissa Moore 186 Mass spectrometry-based quantitation of proteins in ribonucleoprotein complexes Romel Dator, Patrick Limbach 187 In vivo RNA Structural Mapping in the Model Plant Species Arabidopsis thaliana Chun Kit Kwok, Yiliang Ding, Philip Bevilacqua, Sarah Assmann 188 High-throughput Fluorescence Polarization Method for Studying Molecular Recognition of Inhibitors by Bacterial Ribonuclease P Xin Liu, Carol Fierke 189 High-throughput Screening of Chemical Libraries for the Discovery of RNA-binding Compounds Asako Murata, Yasue Harada, Takeo Fukuzumi, Shiori Umemoto, Seongwang Im, Masaki Hagihara, Kazuhiko Nakatani 190 Analysis of RNA Modification and Structural Rearrangement by Real Time Detection of Single Molecule Reverse Transcription Igor Vilfan, Yu-Chih Tsai, Tyson Clark, Jeffrey Wegener, Stephen Turner, Jonas Korlach, Qing Dai, Chengqi Yi, Tao Pan 191 Single Molecule Fluorescence with Nucleotide Analogues Eric Patrick, Elvin Alemán, Chamaree de Silva, Karin Musier-Forsyth, David Rueda 192 SeqZip - A Versatile Methodology For Analyzing Long RNAs Christian Roy, Phillip Zamore, Melissa Moore 193 Discovery of Small Molecular Inhibitors of Yeast Gene Expression Utilizing a High Throughput and Multiparameter Single-Cell Approach Matthew Sorenson, Ashwini Devkota, Eun Jeong Cho, Scott Stevens 194 Healthy and cancerous serum RNA profiling by the novel RNA extraction reagent and highly sensitive DNA chip “3D-Gene” Satoko Takizawa, Makiko Ichikawa, Hiroko Sudo, Yoji Ueda, Hideo Akiyama 195 Full Length cDNA Sequencing on the PacBio RS® Jason Underwood, Lawrence Lee, Tyson Clark, Michael Brown, Sara Olson, Brenton Graveley, Jonas Korlach, Kevin Travers 196 Sensitive and Selective Nucleic Acid Capture with Shielded Covalent Probes Jeffrey Vieregg, Hosea Nelson, Brian Stoltz, Niles Pierce 197 Development of a Fluorescence-based RNase P Assay Andrew Wallace, Lien Lai, Edward Behrman, Venkat Gopalan 198 High-Throughput Discovery of Novel Post-Transcriptional Regulatory Sequences Erin Wissink, Andrew Grimson 199 Rapid Pathogen Detection Using Novel RNA-Based Technologies Jacek Wower, Iwona Wower, Christian Zwieb 200 Abstract Withdrawn 201 Structure-function Analysis of Thermostable RNA Ligase - Engineering ATP Independent Enzyme. Alexander Zhelkovsky, Larry McReynolds 202 Genome-wide Characterization of Eukaryotic Transcriptomes Ting Ni, Han Wu, David Corcoran, Wenjing Yang, Uwe Ohler, Weiqun Peng, Jun Zhu

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203 Cap Independent Translation Control By 3’Untranslated Region (UTR) Elements Of Barley Yellow Dwarf Virus (BYDV) RNA Bidisha Banerjee, Sohani Das Sharma, Dixie Goss 204 characterization of the HDV RNA promoter required for recognition by RNA Polymerase Ii Yasnee Beeharry, Martin Pelchat 205 CELF1 Expression Patterns In Vertebrate Embryonic Development Yotam Blech-Hermoni, Samantha Stillwagon, Andrea Ladd 206 Selective Interaction of RNA Helicase A with 5’-Leader of Viral and Cellular Complex mRNAs: Components of the Core mRNP that Directs Progression through the Translation Cycle Sarah Fritz, Arnaz Ranji, Kathleen Boris-Lawrie 207 HITS-CLIP Reveals Points of Contact Between the SLBP and the Histone Stem-Loop Lionel Brooks 3rd, John Mahoney, Michael Whitfield 208 Binding of Mbl and MBNL to a Model RNA Jay Narasimhan, Danielle Cass 209 Isolation and Analysis of Peptides That Bind to Helix 69 of Bacterial 23S rRNA Moninderpal Kaur, Christine Chow 210 Differential Transcriptome Occupancy of hnRNP L upon T Cell Stimulation Revealed by Computational Genomics Brian Cole, Ganesh Shankarling, Kristen Lynch 211 Structural Studies on the NF90/NF45 Dimerisation Domain Complex Reveals an Evolutionary Relationship to RNA Modifying Enzymes Urszula Wolkowicz, Atlanta Cook 212 Deciphering the Molecular Determinants of the Complex Formed by the Immature microRNA let-7g and the Pluripotency Factor Lin28 Alexandre Desjardins, Ao Yang, Jonathan Bouvette, James Omichinski, Pascale Legault 213 RPL22 Targets p53: Ribosomal Protein Playing Outside The Ribosome Anne-Cecile Duc, Jason Stadanlink, Yong Zhang, David Wiest 214 A Novel Three-unit tRNA Splicing Endonuclease Found in Ultrasmall Archaea Kosuke Fujishima, Junichi Sugahara, Christopher Miller, Brett Baker, Massimo Di Giulio, Kanako Takesue, Asako Sato, Masaru Tomita, Jillian Banfield, Akio Kanai 215 Mutual Biochemical Modulation of eIF4G1 and the DEAD-box RNA Helicase Ded1p from Saccharomyces cerevisiae Zhaofeng Gao, Heath Bowers, Andrea Putman, Eckhard Jankowsky 216 Ionic strength analysis of a DEAD-Box protein reveals that the type of RNA can alter the ATP binding site Ivelitza Garcia, Michael Albring, Winnie Wong 217 Genome-wide Identification of Cellular RNA Targets of the DEAD-box Helicase Ded1p Ulf-Peter Guenther, Frank Tedesci, Akshay Tambe, Sarah Geisler, Jeffery Coller, Elizabeth Tran, Mark Adams, Eckhard Jankowsky 218 RNA binding and RNA remodeling activities of the Half-a-Tetratricopeptide (HAT) protein HCF107 underlie its effects on gene expression Kamel Hammani, William Cook, Alice Barkan 219 Recognition of Termination Signal of Non-coding RNAs by Nab3 Fruzsina Hobor, Odil Porrua-Fuerte, Roberto Pergoli, Karel Kubicek, Dominika Hrossova, Stepanka Vanacova, Domenico Libri, Richard Stefl 220 The Mechanism Of RNA Binding By The 27 kDa Trypanosoma brucei Pentatricopeptide Repeat Protein Pakoyo Kamba, Neil White, David Dickson, Charles Hoogstraten 221 Characterization of an Inhibitory Compound of Nematode Tandem Zinc Finger RNA-Binding Proteins Ebru Kaymak, Sean Ryder xxx

222 Investigation of Protein-RNA Interactions by UV Induced Cross-linking and Mass Spectrometry Katharina Kramer, Timo Sachsenberg, Oliver Kohlbacher, Henning Urlaub 223 Oligomeric Assembly of HIV-1 Rev on the Rev Response Element: Role of Cellular Cofactors Rajan Lamichhane, Rae Robertson-Anderson, Svitlana Berezhna, Edwin van der Schans , David Millar 224 Critical role of the RNA-binding protein Mex-3B in the control of phagocytosis and cell-cell adhesion Mailys Le Borgne, Nicolas Chartier, Marc billaud 225 Genome-wide analysis of RBFOX1 and RBFOX2-regulated exons extends their regulatory role to distal intronic elements Michael Lovci, Henry Marr, Justin Arnold, Marilyn Parra, Tiffany Liang, Sherry Gee, Joe Gray, Dana Ghanem, John Conboy, Gene Yeo 226 Identification of mRNAs and Novel non-coding RNAs Associated with Drosophila Sm Proteins Zhipeng Lu, A. Gregory Matera 227 Structural Basis of Lariat RNA Recognition by the Intron Debranching Enzyme, Dbr1 Eric Montemayor, Adam Katolik, Alexander Taylor, Jonathan Schuermann, Joshua Combs, Richard Johnson, Stephen Holloway, Masad Damha, Scott Stevens, P. John Hart 228 New Tools for the Enrichment and Detection of RNA- Protein Interactions Kay Opperman, Chris Etienne, Scott Meier, J. Schultz, Barbara Kaboord, Atul Deshpande 229 The HIV-2 Leader RNA Structure And Interactions With NCp8 Protein Katarzyna Pachulska-Wieczorek, Katarzyna Purzycka, Agnieszka Stefaniak, Ryszard Adamiak 230 Characterization of ΔN-Zfp36l2-mutant Associated with Early Embryonic Arrest and Female Infertility Silvia Ramos 231 Determinants and anti-determinants of substrate recognition by yeast RNAse III Kevin Roy, Guillaume Chanfreau 232 Mapping the Interaction Between E. coli tRNA and the Trans-Editing YbaK Protein Brianne Sanford, Maryanne Refaei, Mark Foster, Karin Musier-Forsyth 233 Amino Acid Signature Important for Modified tRNA Recognition: A Tool for Studying Modified RNA-Protein Interactions Jessica Spears, Caren Stark, Paul Agris 234 Post-translational Modifications of AUF1 during Erythroid Differentiation Regulate β-globin mRNA Expression Sebastiaan van Zalen, Grace Jeschke, Elizabeth Hexner, J. Eric Russell 235 LIN28 Interacts With GGAGA Motifs in Messenger RNA Melissa Wilbert, Stephanie Huelga, Anthony Vu, Thomas Stark, Katlin Massirer, Tiffany Liang, Stella Chen, Hilal Kazan, Quaid Morris, Gene Yeo 236 Depletion Of HuR Inhibits Proliferation Of Normal Breast Epithelial Cells In Parallel With Upregulation Of ΔNp63 Wensheng Yan, Yanhong Zhang, Xinbin Chen 237 Recruitment of Bromovirus Genomic RNA Replication Templates is 5’ Cap-Independent but RNA StructureInhibited Quansheng Yang, Brandi Gancarz, Paul Ahlquist 238 mirEX- comprehensive platform for for highthrougput analysis of Arabidopsis pri-miRNAs Jakub Dolata, Bielewicz Dawid, Zielezinski Andrzej, Alaba Sylwia, Szarzynska Bogna, Szczesniak Michal, Karlowski Wojciech, Jarmolowski Artur, Szweykowska-Kulinska Zofia 239 Restoration of protein function by miRNA interference – a tentative complementary mode of action for platinum based anticancer drugs? Alak Alshiekh, Sofi Elmroth 240 The Role of Argonaute in the Mammalian Nucleus Roya Kalantari, Keith Gagnon, David Corey

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241 Mechanisms of RNA Interference: siRNA Strand Selection and mRNA Target Cleavage at the Single Molecule Level Vishalakshi Krishnan, Sethuramasundaram Pitchiaya, Nils Walter 242 Substrate Recognition by Drosophila Dicer-2 and Loquacious-PD Sucharita Kundu, Xuecheng Ye, Philip Aruscavage, Qinghua Liu, Brenda Bass 243 dsRNA binding proteins alter human miRNA processing by Dicer Ho Young Lee, Jennifer Doudna 244 Argonaute-interacting GW protein directs transposon silencing in pathogenic fungus, Cryptococcus neoformans Prashanthi Natarajan, Phillip Dumesic, James Moresco, John Yates III, Hiten Madhani 245 Mechanistic Studies of Human RISC Loading Complex Function in RNA Interference Cameron Noland, Enbo Ma, Jennifer Doudna 246 Nuclear Localized Antisense Small RNAs with 5′-Polyphosphate Termini Regulate Long-term Transcriptional Gene Silencing in Entamoeba histolytica G3 Strain Hanbang zhang, Hussein Alramini, Vy Tran, Upinder Singh 247 Abstract Withdrawn 248 The Role of Dicer-dsRBD in Small Regulatory RNA Maturation Christopher Wostenberg, Kaycee Quarles, Scott Showalter 249 The RDE-10/RDE-11 complex triggers RNAi induced mRNA degradation by association with target mRNA in C. elegans Huan Yang, Ying Zhang, Jim Vallandingham, Hua Li, Laurence Florens, Ho Yi Mak 250 Involvement of Splicing Factors in Chromosome Segregation in Mammalian Cells Takashi Ideue, Kazuaki Tokunaga, Misato Morita, Kanako Nishimura, Madoka Chinen, Mistuyoshi Nakao, Tokio Tani 251 DNA Replication Facilitates Transcription of Epigenetically Silenced Genes Javier Peña-Diaz, Siv Hegre, Endre Anderssen, Per Aas, Robin Mjelle, Gregor Gilfillan, Robert Lyle, Finn Drabløs, Hans Krokan, Pål Sætrom 252 Abstract Withdrawn 253 Characterization of divergent transcripts from CpG island promoters in mouse embryonic stem cells Albert Almada, Ryan Flynn, Jesse Zamudio, Phillip Sharp 254 The Making of an OncomiR: Oncogenic microRNA-21 First Exhibits Unusually Reduced mRNA Binding and Silencing Activity in Healthy Mouse Liver John Androsavich, Nelson Chau, Balkrishen Bhat, Peter Linsley, Nils Walter 255 RNA controls the activation of the Aurora-B kinase to promote mitotic spindle assembly. Michael Blower, Ashwini Jambhekar 256 Tertiary Structure Of Pri-miR-17-92 Influences Its Processing Saikat Chakraborty, Shabana Mehtab, Anand Patwardhan, Yamuna Krishnan 257 snoRNAs Expression Units Are The Source Of New Non-Coding RNAs That Regulate Gene Expression Marina Falaleeva, Manli Shen, Pierre de la Grange, Eduardo Eyras, Justin Surface, Brian Rymond, Stefan Stamm 258 Biogenesis of Mammalian Mirtrons and Simtrons Mallory Havens, Ashley Reich, Dominik Duelli, Michelle Hastings 259 Mechanistic insights into crRNA biogenesis in type II and III CRISPR systems Martin Jinek, Michael Hauer, Ole Niewoehner, Jennifer Doudna 260 Identification And Characterization Of Breast Cancer-relevant Long Non-coding RNAs Katharina Kasack, Kristin Reiche, Inga Rye, Hege Russnes, Friedemann Horn, Anne-Lise Børresen-Dale, Jörg Hackermüller, Lars Baumbusch

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261 Novel class of transcripts in Arabidopsis xrn3 mutant of nuclear 5’-3 Michal Krzyszton, Monika Zakrzewska-Placzek, Joanna Kufel 262 Successive Tailing and Trimming of RISC-loaded miRNA by the 3’UTR-binding Protein HuR Sokol Lena, Lisa Young, Martin Hintersteiner, Jan Weiler, David Morrissey, Witold Filipowicz, Nicole Meisner-Kober 263 Novel Metabolite Analogs for Modulating Riboswitch Activity Christina Lünse, Magnus Schmidt, Valentin Wittmann, Fraser Scott, Colin Suckling, Günter Mayer 264 The Redox-Sensing Apo-Aconitase B Protein Act Against sRNA Distal Nucleolytic Cleavage Julie-Anna Benjamin, Marie-Claude Carrier, Eric Massé 265 Diversity of RNA-Based Ribosomal Protein Regulation in Different Bacterial Phyla Shermin Pei, Jon Anthony, Michelle Meyer 266 Splicing remodels let-7 primary miRNA for enhanced Drosha processing Vanessa Mondol, Amy Pasquinelli 267 Bacterial Regulatory RNAs in Competition for Binding to the Chaperone Protein Hfq Aleksandra Kaszynska, Agnieszka Hartwich, Anna Roszak, Mikolaj Olejniczak 268 Comparative analysis of miRNA expression patterns between Triops cancriformis (tadpole shrimp) and model species during development Kahori Takane, Yuka Hirose, Kiriko Hiraoka, Emiko Noro, Kosuke Fujishima, Masaru Tomita, Akio Kanai 269 Characterization of the sxRNA platform; A trans-acting RNA Switch Carla Theimer, Nakesha Smith, Sabarinath Jayaseelan, Francis Doyle, Paul Kutscha, Scott Tenenbaum 270 Multidimensional Regulation of and by LncRNAs: Non-Conservation and Networks Emily Wood, Leonard Lipovich 271 Genome-wide Analysis of Target Genes of Steroid Receptor RNA Activator - a Regulatory RNA Linghe Xi, Elaine Podell, David McKay, Thomas Cech 272 An Adenosine-rich Sequence is Crucial for the Integrator-dependent microRNA 3’ Processing in Herpesvirus saimiri Mingyi Xie, Diana Lenis, Joan Steitz 273 Discovery of Polymerase Binding Elements and their Functions Bob Zimmermann, Frederike von Pelchrzim, Jennifer Boots, Adam Weiss, Doris Chen, Marek Zywicki, Katarzyna Matylla-Kulinska, Renée Schroeder 274 3’-UTR G-quadruplexes Regule Gene Expression Throughout Alternative Polyadenylation Site Jean-Denis Beaudoin, Jean-Pierre Perreault 275 Post-Transcriptional Regulation of COX-2 Ashley Cornett, Carol Lutz 276 The Exo- and Endoribonuclease Activities of RNase BN Both Function In Vivo Tanmay Dutta, Arun Malhotra, Murray Deutscher 277 A Novel C-rich USE Globally Regulates mRNA 3’ Processing Xinjun Ji, Ji Wan, Melanie Vishnu, Yi Xing, Stephen Liebhaber 278 SR Protein-Regulated Polyadenylation of Rous Sarcoma Virus mRNA Stephen Hudson, Mark McNally 279 Analyzing Effect of La Mutants on Biogenesis of SRP RNA in S.cerevisiae Hatem Mohi El Din, Jeremy Brown 280 Replication Stress Linked Alternative Splicing Modulates the Activity of the RNA Binding Protein HBP/SLBP, a Key Factor in the Control of Histone Gene Expression Pamela Nicholson, Alexander Rattray, Berndt Mueller

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281 Non-Canonical 3’ End Formation Of Certain mRNAs In Drosophila S2 Cells Trinh Tat, Patricia Maroney, Timothy Nilsen 282 RNA-Dependent Control of Histone Synthesis by the Spinal Muscular Atrophy Protein Sarah Tisdale, Luciano Saieva, Francesco Lotti, George Mentis, Livio Pellizzoni 283 mRNAs Encoded by Linear Plasmids of the Yeast Kluyveromyces lactis are not Polyadenylated Vaclav Vopalensky, Martin Pospisek 284 Identification of Polyadenylation Mutations through a Novel Engineered Minigene Construct in Arabidopsis Jie wang, Q. Quinn Li 285 Multiple Resistance Mechanisms in a Multi-drug Producer Mashal Almutairi, Alexander Mankin 286 Reconstitution and characterization of in vitro translation system from Thermus thermophilus at high temperatures Ying Zhou, Haruichi Asahara, Eric Gaucher, Shaorong Chong 287 Probing the Function of the Truncated Insertion Domain in Rhodopsudomonas palustris Prolyl-tRNA Synthetase and Free Standing Homologs YbaK and ProX Jo Marie Bacusmo, Karin Musier-Forsyth 288 An RNA interference Screening Approach to Identify Factors Involved in 40S Ribosomal Subunit Biogenesis in Mammalian Cells Lukas Badertscher, Thomas Wild, Michael Stebler, Karol Kozak, Gábor Csúcs, Peter Horvath, Ulrike Kutay 289 Investigating the Physiological Role of Translation Factor LepA in E. coli Rohan Balakrishnan, Kurt Fredrick 290 Role of the Uncharacterized Factor Rbtf1 in Ribosome Synthesis Lukas Bammert, Ulrike Kutay 291 Structural Rearrangements and Antibiotic Targeting of Helix 69 in Bacterial Ribosomes Christine Chow, Yogo Sakakibara 292 Molecular Basis of Substrate Specificity and Mechanism of Catalysis by Bacterial Prolyl-tRNA Synthetase and YbaK Mom Das, Sandeep Kumar, Christopher Hadad, Karin Musier-Forsyth 293 Molecular Mechanism Of Translation Initiation In Barley Yellow Dwarf Virus (BYDV) Sohani Das Sharma, Jelena Kraft , W.Allen Miller, Dixie Goss 294 Antibiotics that Bind to the A site of the Large Ribosomal Subunit Can Induce mRNA Translocation Dmitri Ermolenko, Harry Noller 295 Single Molecule Dynamics of tRNA inside Ribosomes May Farhat, Philip Cunningham, David Rueda 296 Crystal structure of the ribosome in complex with the tRNA-like-domain of tmRNA and SmpB in the posttranslocation state Natalia Ivanova, Andrei Korostelev, John Paul Donohue, Jianyu Zhu, Harry Noller 297 Evidence That Domain Closure Influences Both Initial Selection And Proofreading Of Aminoacyl-tRNA By The Ribosome Sean McClory, Aishwarya Devaraj, Kurt Fredrick 298 Studying the Mechanism of Translation Termination Using Bulk Fluorescence Approaches Megan McDonald, Rachel Green 299 40S Maturation Requires Joining of 60S and a Subset of Translation Initiation Factors Megan Novak, Bethany Strunk, Katrin Karbstein 300 Unified Mechanism for Proofreading by Class I Aminoacyl-tRNA Synthetases John Perona, Ita Gruic-Sovulj, Nevena Cvetesic, Morana Dulic, Hari Bhaskaran xxxiv

301 Alternative Reading Frame Selection Mediated by a Viral tRNA-like Internal Ribosome Entry Site Qian Ren, Qing Wang, Andrew Firth, Mandy Chan, Joost Gouw, Marta Guarna, Leonard Foster, John Atkins, Eric Jan 302 Translation initiation of non-LTR retrotransposons by HDV-like self-cleaving ribozymes Dana Ruminski, Andrej Luptak 303 Residues in Different Parts of the tRNA Structure Collaborate in Establishing tRNA Performance, In Vivo Margaret Saks, Daniel Curtis, Devin Midura, Olke Uhlenbeck 304 Rbg1-Tma46 Dimer Structure Reveals New Functional Domains and Their Role in Polysome Recruitment Sandrea Francis, María-Eugenia Gas, Marie-Claire Daugeron, Jeronimo Bravo, Bertrand Séraphin 305 The Requirement for RPS25 Links IRES and Ribosomal Shunting Mechanisms of Initiating Ttranslation Marla Hertz, Dori Landry, Anne Willis, Guangxiang Luo, Sunnie Thompson 306 Peptidyl Transfer Center Dynamics Probed via Single Molecule FRET Ming Xiao, Yue Li, Mediha Altuntop, Yuhong Wang 307 Crystal structure of release factor RF3 trapped in the GTP state on a rotated conformation of the ribosome Harry Noller lab :, Jie Zhou, Laura Lancaster, Sergei Trakhanov, Harry Noller 308 Abstract Withdrawn 309 Molecular Characterization of Genome Unscrambling in the Ciliated Protozoan Oxytricha trifallax Leslie Beh, Laura Landweber 310 Comparison Between Developmental Stages of D. melanogaster and C. elegans with modENCODE RNA-Seq Data Jingyi Jessica Li, Haiyan Huang, Peter Bickel, Steven Brenner 311 Regulation of Splicing Factors by Alternative Splicing and NMD is Conserved Between Kingdoms Yet Evolutionarily Flexible Liana Lareau, Steven Brenner 312 The Role of the Canonical 50nt Rule in Targeting a Transcript for Nonsense-Mediated mRNA Decay in Human and in Fly Courtney French, Gang Wei, Angela Brooks, Steven Brenner 313 Transcriptome Analysis Reveals Extensive Alternative Splicing Coupled with Nonsense-Mediated mRNA Decay in a Human Cell Line Courtney French, Gang Wei, Angela Brooks, Steven Brenner 314 Btf and TRAP150 Localize to Transcription Sites and Affect the Cellular Distribution of mRNAs Sapna Varia, Divya Potabathula, Zhihui Deng, Athanasios Bubulya, Paula Bubulya 315 Characterization Of prp43 Alleles Provides Insights Into Pathway Specific Domains Jennifer Hennigan, Scott Stevens 316 Traf3 Alternative Splicing Regulates The Non-Canonical NFkB Pathway In T Cells Monika Regehr, Ilka Wilhelmi, Florian Heyd 317 Abstract Withdrawn 318 Transcriptional activation of PIK3R1 by PPARγ in adipocyte Yoon-Jin Kim, Sang Hoon Kim 319 Biochemical Characterization of Dbp2 at the Interface of RNA Export and lncRNA-dependent Transcription Regulation Wai Kit Ma, Sara Cloutier, Elizabeth Tran 320 Transcription elongation and mRNA processing are linked through ELL2 in lymphocyte Christine Milcarek, Michael Albring, Fortuna Arumemi, Creitekya Langer, Kyung Park 321 Inhibition of Co-transcriptional Splicing Affects RNA Polymerase II Elongation Kinetics Jarnail Singh, Richard Padgett xxxv

322 Functional Characterization of Polypyrimidine Tract-Binding Proteins from Arabidopsis Eva Stauffer, Andreas Wachter 323 A Phosphorylation-Dependent Signaling Pathway Links SLBP Ubiquitination To Histone mRNA Decay Nithya Krishnan, TuKiet Lam, Padriac Philbin, Donald Rempinski, Andrew Fritz, Kieran O’ Loughlin, Hans Minderman, Ronald Berezney, William Marzluff, Roopa Thapar 324 Investigating Novel Paradigms for RNA Binding Protein - miRNA Regulatory Interactions: A Study of the Poly(A) RNA Binding Protein, ZC3H14 Callie Wigington, Paula Vertino, Anita Corbett

THURSDAY, MAY 31, 2012: 20:00 – 22:30 Poster Session 2 - Michigan League (2nd Floor) Abstracts 325 – 478 325 The Exon Junction Complex is Restricted to a Specific Subset of Splice Junctions Nazmul Haque, Scot Harms, Malcolm Cook, Marco Blanchette 326 Stressed-out Adult Stem Cells? A Particular Class of Cytoplasmic mRNA Granules in Adipose-Derived Stem Cells. Alejandro Correa, Crisciele Kuligovski, Marco Stimamiglio, Axel Cofré, Bruno Dallagiovanna, Samuel Goldenberg 327 Abstract Withdrawn 328 Interactions of a Pop5/Rpp1 Heterodimer with the Catalytic Domain of Ribonuclease MRP Anna Perederina, Elena Khanova, Chao Quan, Igor Berezin, Olga Esakova, Andrey Krasilnikov 329 Non-canonical biogenesis of the catalytic subunit of the Drosophila RNase P- a tRNA processing enzyme Sathiya Manivannan, Lien Lai, Venkat Gopalan, Amanda Simcox 330 Mouse Cells Expressing Catalytically Inactive Dyskerin show Slow Growth and Unstable Ribosomal RNA Bai-Wei Gu, Jingping Ge, Jianmeng Fan, Monica Bessler, Philip Mason 331 Single Molecule Studies of Prp24-dependent U4/U6 di-snRNP Formation Margaret Rodgers, Ashley Richie , David Brow, Samuel Butcher, Aaron Hoskins 332 Novel Function for the Cajal body: Surveillance of SnRNP Assembly Ivan Novotny, Daniel Mateju, Martin Sveda, Zdenek Knejzlik, David Stanek 333 A Rapid Synergistic Approach for Determining RNA-Binding Domain Structure Rebecca Taurog, John Johnson, James Williamson, Blair Szymczyna 334 The Post-Transcriptional trans-Acting Regulator, TbZFP3, Coordinates Transmission-Stage Enriched mRNAs in Trypanosoma brucei Pegine Walrad, Paul Capewell, Katelyn Fenn, Keith Matthews 335 Roles of Ribosomal Proteins in Assembly of Yeast 60S Subunits In Vivo John Woolford, Jelena Jakovljevic, Michael Gamalinda, Jesus de la Cruz, Reyes Babiano, Philipp Milkereit, Jan Linneman, Uli Ohmayer 336 Secondary structure fold of the human U2-U6 snRNA complex. Ravichandra Bachu, Joerg Schlatterer, Michael Brenowitz, Nancy Greenbaum 337 Determining the molecular mechanism underlying splicing factor related Retinitis Pigmentosa Deepti Bellur, Jonathan Staley 338 Effects of Destabilizing the U4/U6 Complex Are Suppressed by a Mutation That Alters the U2/U6 Complex Jordan Burke, Dipali Sashital, David Brow, Samuel Butcher 339 Unravelling the alternative RNA splicing labyrinth with chemical biological tools Glenn Burley, Ian Eperon, Helen Lewis, Andrew Perrett, Rachel Dickinson 340 AU-rich Elements (AREs) As Intronic Enhancers: A Molecular Mechanism For The Lack Of Iterated AREs In Protein-coding Exonic Regions Durga Rao Chilakalapudi, Sandip Chorghade xxxvi

341 The Splicing Factor hnRNP K Inhibits G6PD RNA Splicing in Response to Changing Nutrient Availability Travis Cyphert, Lisa Salati 342 Identifying Small-Molecule Inhibitors of Human and Yeast pre-mRNA Splicing by High-throughput Screening Kerstin Effenberger, Walter Bray, Rhonda Perriman, Manuel Ares, Melissa Jurica 343 The Structure of ySad1 Suggests a Divergent Zinc Finger Ubiquitin Binding Domain Function. Haralambos Hadjivassiliou, Oren Rosenberg, Christine Guthrie 344 Zooming into the Splicing Cycle: Step by step Dissection of Pre-mRNA Dynamics Ramya Krishnan, Mario Blanco, Joshua Martin, Matthew Kahlscheuer, Alain Laederach, Christine Guthrie, John Abelson, Nils Walter 345 High Throughput Discovery of Factors Involved in Complex, Regulated Splicing Pathways in S. pombe Amy Larson 346 Ensemble and Single Molecule Characterization of Brr2 Helicase Activity on the U4/U6 snRNAs Sarah Ledoux, John Abelson, Haralambos Hadjivassiliou, Christine Guthrie 347 Compositional analysis of the pre-spliceosome intermediates associating with Prp5 protein Sujin Lee, Scott Stevens 348 The Role of Saccharomyces cerevisiae Prp5 in Splicing Fidelity Control Wen-Wei Liang, Soo-Chen Cheng 349 RNA-binding Protein GPKOW Interacts with Spliceosomal DExD/H-box Protein DHX16/hPRP2 and is Required for Pre-mRNA splicing Shengbing Zang, Ting-Yu Lin, Ren-Jang Lin 350 Identification of Critical Residues in the Pseudo-Helicase Domain of Brr2 Corina Maeder, William Boswell, Christine Guthrie 351 Role of U2 snRNA stem IIb in Spliceosome Function Alberto Moldon, Charles Query 352 Identification of an Early ATP-Independent Role for Prp28 in Spliceosome Assembly Argenta Price, Christine Guthrie 353 A Conserved Serine of HnRNP L Mediates Depolarization-Regulated Alternative Splicing of Potassium Channels Aleh Razanau, Guodong Liu, Yan Hai, Jiankun Yu, Muhammad Sohail, Vincent Lobo, Jiayou Chu, Sam Kung, Jiuyong Xie 354 Structural basis for regulation of Brr2 incorporation into U5 snRNP by the Aar2 protein Gert Weber, Vanessa Cristão, Flavia de L. Alves, Karine Santos, Nicole Holton, Juri Rappsilber, Jean Beggs, Markus Wahl 355 Branched Spliceosome Assembly Pathway Revealed by Single Molecule Microscopy Inna Shcherbakova, Aaron Hoskins, Larry Friedman, Victor Serebrov, Jeff Gelles, Melissa Moore 356 The U2AF35-related protein Urp contacts the 3’ splice site to promote U12-type intron splicing and the second step of U2-type intron splicing xuexiu zheng zheng, haihong shen 357 Role of a Unique RNA-RNA Long-distance Interaction in Spinal Muscular Atrophy Natalia Singh, Mariah Lawler, Daya Upreti, Ravindra Singh 358 Spliceosome Product Release and Disassembly is Dependent Upon the 3’-end of U6 snRNA Rebecca Toroney, Jonathan Staley 359 Backbone Oxygens in U6 snRNA Position a Catalytic Metal to Stabilize the Leaving Group During Exon Ligation Nicole Tuttle, Sebastian Fica, Thaddeus Novak, Qing Dai, Jun Lu, Nan-Sheng Li, Jonathan Staley, Joseph Piccirilli 360 Detecting Metal Ion Binding Pockets within the Group II Intron ai5γ in vivo Michael Wildauer, Christina Waldsich 361 Capturing DExD/H-box protein Prp28p in action Fu-lung Yeh, Tien-Hsien Chang xxxvii

362 Genome-wide in vivo RNA binding sites of key spliceosomal protein Prp8 identified using HITS-CLIP and CRAC Xueni Li, Wenzheng Zhang, Tao Xu, Jolene Ramsey, Jay Hesselberth, Rui Zhao 363 Self-Splicing of Group IIE and Group IIF Introns Katherine Zhou, Vivien Nagy, Anna Pyle 364 Reconstitution of the Sequential Pathway for 1-methyl Pseudouridine Formation in Archaeal tRNAs Kunal Chatterjee, Ian Blaby, Patrick Thiaville, Mrinmoyee Majumder, Henri Grosjean, Adam Yuan, Valerie de CrecyLagard, Ramesh Gupta 365 Alu-transposons Determine Site-selective A-to-I Editing Chammiran Daniel, Gilad Silberberg, Marie Ohman 366 The Rapid tRNA Decay Pathway Acts Broadly on Modification-Deficient Yeast Strains and Competes with Elongation Factor 1A for Substrates Joshua Dewe, Joseph Whipple, Irina Chernyakov, Laura Nemeth, Eric Phizicky 367 The Editing Deaminase is Required for Anticodon Loop Methylation of Threonyl-tRNA in Trypanosoma brucei Ian Fleming, Kirk Gaston, Zdenek Paris, Kady Krivos, Pat Limbach, Mary Anne Rubio, Juan Alfonzo 368 Structural Features of Archaeal Pus10 Proteins That Specify Pseudouridine Formation at tRNA Position 54 Archi Joardar, Sujata Jana, Priyatansh Gurha, Elisabeth Fitzek, Mrinmoyee Majumder, Kunal Chatterjee, Matt Geisler, Ramesh Gupta 369 Inverted Alu dsRNA Structures Alter Translation of Human mRNAs Independent of RNA Editing Claire Capshew, Kristen Dusenbury, Heather Hundley 370 Multiple Functions of Thg1 Family Enzymes in Mitochondria of Dictyostelium discoideum Yicheng Long, Maria Abad, Jane Jackman 371 Structural Features of Haloferax volcanii Cbf5 Protein Essential for In Vivo RNA-guided Pseudouridylation Mrinmoyee Majumder, Ramesh Gupta 372 3’-5’ Polymerization in tRNA Biogenesis Fuad Mohammad, Maria Abad, Yicheng Long, Jane Jackman 373 Down-regulation of tRNA mG Formation Affects Mitochondrial but not Cytoplasmic Protein Synthesis in Trypanosoma brucei Zdenek Paris, Eva Horakova, Mary Anne Rubio, Paul Sample, Ian Fleming, Julius Lukes, Juan Alfonzo 374 A Comprehensive Analysis of Developmental Expression Patterns of RNA Modifying Enzymes in Zebrafish Marion Pesch, Jana Pfeiffer, Erez Raz, Sebastian Leidel 375 Novel RNA Editing Events Necessary for Endonuclease Processing of tRNAs in Trypanosoma brucei Mary Anne Rubio, Juan Alfonzo, Chris Trotta 376 The Elongator Subcomplex Elp456 is a Hexameric RecA-like ATPase Sebastian Glatt, Juliette Létoquart, Céline Faux, Nicholas Taylor, Bertrand Séraphin, Christoph Müller 377 Substrate specificity of a multi-functional tRNA modification enzyme. William Swinehart, Jane Jackman 378 Genome-Wide Identification of RNA Editing Events in RNA-Seq Data Jae Hoon Bahn, Jae-Hyung Lee, Jason Ang, Xinshu Xiao 379 The Study of tRNA Modifications by Molecular Dynamics Xiaoju Zhang, David Mathews 380 Directed Translational Initiation by the Anticodon Mimicry Domain of a Viral Internal Ribosome Entry Site Hilda Au, Eric Jan 381 The Structure of Intact E. coli RelBE Suggests a Structural Basis for Conditional Cooperativity Andreas Bøggild, Nicholas Sofos, Ashley Easter, Lori Passmore, Ditlev Brodersen

xxxviii

382 Thriving under Stress: HIV-1 mRNAs Exploit Nuclear Cap-Binding Protein to Sustain Translation during Viral Impairment of eIF4E Activity Amit Sharma, Alan Cochrane, Kathleen Boris-Lawrie 383 Genome-wide Investigations of Translating mRNAs to Study the Cellular Functions of tRNA Nuclear-Cytoplasmic Dynamics in S. cerevisiae Hui-Yi Chu, Anita Hopper 384 Selection of Inhibitory Codon Combinations in Saccharomyces cerevisiae Kimberly Dean, Elizabeth Grayhack 385 Investigating the Targeting and Regulation of Ded1, a DEAD-box ATPase, in Returning Repressed mRNAs to Translation Angie Hilliker, Roy Parker 386 Post-transcriptional Regulation of Gene Expression by Khd1, Ccr4, and Pbp1 Kenji Irie, Yuichi Kimura, Xia Li, Tomoaki Mizuno 387 Characterization of Translational Regulation during Hypoxia in Human Colon Cancer HCT116 Cells Ming-Chih Lai, Shaw-Jenq Tsai, H. Sunny Sun 388 LARP1 Induces HeLa Cell Migration And Invasion By Activating Localised Protein Synthesis At The Cellular Leading Edge Manuela Mura, Normala Abd Latip, Theodora Michael, Jacqueline Fok, Thomas Hopkins, Francesco Mauri, Roberto Dina, Edward Curry, Sarah Blagden 389 Efficient Ribosomal Frameshifting on a Non-canonical Sequence in a Herpes Simplex Virus Mutant is Promoted by Non-stop mRNA Dongli Pan, Donald Coen 390 Physiological significance of tRNA thio-modification in protein translation Vanessa Rezgui, Kshitiz Tyagi, Namit Ranjan, Patrick Pedrioli, Matthias Peter 391 Ribosome Profiling of Caulobacter crescentus Development Jared Schrader, Gene-Wei Li, Jonathan Weissman, Lucy Shapiro 392 Biophysical Characterization Of The SLIP1-SLBP Complex Reveals New Insights Into The Role Of Oligomerization In Regulation Of Histone mRNAs Nitin Bansal, Minyou Zhang, Aishwarya Bhaskar, Patrick Itotia, EunHee Lee, Lyudmila Shlyakhtenko, Joseph Luft, TuKiet Lam, Andrew Fritz, Ronald Berezney, Yuri Lyubchenko, Walter Stafford, Roopa Thapar 393 Novel Activities of Drosophila Pumilio Reveal New Mechanisms of mRNA Regulation Chase Weidmann, Aaron Goldstrohm 394 Characterizing the disorder in Tristetraprolin and its contribution to RNA binding specificity Laura Andersh, Francesca Massi 395 High resolution analysis of the stable protein-associated RNA fraction in different nutrient starvation conditions in the yeast, Saccharomyces cerevisiae Rodoniki Athanasiadou, David Gresham 396 An eIF4E-Binding Protein Promotes mRNA Decapping and is Required for PUF Repression Nathan Blewett, Aaron Goldstrohm 397 Gene Expression Knock-Down by Forced Splicing-Dependent NMD Francesca Zammarchi, Gina Rocco, Sandra Vorlova, Clare LeFave, Luca Cartegni 398 Psp1p Interacts with Rbp1p to Mediate the Recruitment of Rbp1p to P-bodies Lin-Chun Chang, Ying-Chieh Chu, Li-Ting Jang, Fang-Jen S Lee 399 Implication that SMG5-PNRC2 is a more functionally dominant complex than SMG5-SMG7 under normal conditions: Cellular substrates targeted for nonsense-mediated mRNA decay have their own preference for Upf1-binding players Hana Cho, Sisu Han, Kyoung Mi Kim, Seung Gu Park, Sun Shim Choi, Yoon Ki Kim xxxix

400 The RNA Binding Protein Y14 Inhibits mRNA Decapping and Modulates Processing Body Formation Tzu-Wei Chuang, Wei-Lun Chang, Kuo-Ming Lee, Woan-Yuh Tarn 401 Post-transcriptional Control of Stress Response mRNAs by a Zinc Finger Protein and AU-rich Elements Dorothea Droll, Igor Minia, Aditi Singh, Abeer Fadda, Christine Clayton 402 The role of CNOT10 in the process of mRNA turnover Valentin Faerber, Esteban Erben, Abeer Fadda, Christine Clayton 403 A novel form of nonsense-mediated mRNA decay revealed by studies on COL10A1 mutations Yiwen Fang, Jacqueline Tan, Julian Mercer, Shireen Lamandé, John Bateman 404 Inhibition of Nonsense-mediated mRNA Decay by Retroviral RNA Elements Zhiyun Ge, Stacey Baker, J. Robert Hogg 405 EJC and NMD mutants in Cryptococcus neoformans Sara Gonzalez-Hilarion, Estelle Mogensen, Guilhem Janbon 406 Analysis of archaeal RNase E-like protein, FAU-1 in Pyrococcus furiosus Yoshiki Ikeda, Shinnosuke Murakami, Asako Sato, Masaru Tomita, Akio Kanai 407 Copper tolerance of Saccharomyces cerevisiae Nonsense-Mediated mRNA decay mutants Xuya Wang, Bessie Kebaara 408 Questioning the relevance of the interaction between Pab1 and eRF3 Marie Cerciat, Sylvain Roque, Isabelle Gaugue, Liliana Mora, Emmeline Huvelle, Miklos de Zamaroczy, Valerie HeurgueHamard, Stephanie Kervestin 409 Functional Analysis of Mtr4 as a Component of TRAMP Complex Yan Li, James Anderson 410 The Human La-related Protein 4 mRNA Contains a Functional AU-rich Element Sandy Mattijssen, Richard Maraia 411 Dissecting the Mammalian Nonsense-Mediated mRNA Decay Pathway by a Combined Tethering / Knockdown Approach Pamela Nicholson, Oliver Mühlemann 412 Biochemical Localization of mRNA Repression and Degradation Factors Susanne Brettschneider, Tracy Nissan 413 Degradation of rRNA in Bacteria Anton Paier, Tanel Tenson, Ülo Maiväli 414 Structural and Functional Analysis of the Rrp6-Rrp47 Interaction in RNA Degradation Processes Benjamin Schuch, Monika Feigenbutz, Claire Basquin, Phil Mitchell, Elena Conti 415 Regulation of CAF1-dependant deadenylation under stress conditions Sahil Sharma, Georg Stoecklin 416 D-foci - Sites for RNA Decay in Human Mitochondria Lukasz Borowski, Andrzej Dziembowski, Monika Hejnowicz, Piotr Stepien, Roman Szczesny 417 Proteasome-independent Ubiquitin Signaling in HuR-mediated mRNA Stability Control Hua-Lin Zhou, Hua Lou 418 Microsecond Timescale Molecular Dynamics SImulation of Ligand-Induced Strand Migration in an S-adenosyl methionine Riboswitch fareed aboul-ela, Wei Huang, Vamsi Boyapati, Joohyun Kim, Shantenu Jha 419 Highlighting Features of the B-Z Transition in DNA Heidi Alvey, Hashim Al-Hashimi 420 SAXS and ITC Analyses Reveal Important Role of the K-turn in Folding of Glycine Riboswitches Nathan Baird, Adrian Ferré-D’Amaré xl

421 Structural Similarities at the Active Sites of the VS and Hairpin Ribozymes Eric Bonneau, Genevieve Desjardins, Nicolas Girard, Jerome Boisbouvier, Pascale Legault 422 Correcting Pervasive Errors in RNA Crystallography Fang-Chieh Chou, Parin Sripakdeevong, Rhiju Das 423 Structure and folding of a rare kink turn with an A•A pair at the 2b•2n position Peter Daldrop, Kersten Schroeder, Scott McPhee, David M. J. J. Lilley 424 SHAPE Analysis of VSV RNA Transcipts Adam Davidson, Rebecca Alexander 425 Fragment-Based Drug Discovery: X-ray Structures of the TPP Riboswitch in Complex with Lead Compounds Katherine Deigan, Alison Smith, Chris Abell, Adrian Ferré-D’Amaré 426 Predicting RNA-RNA Interactions with Consideration for Competing Self Structure Laura DiChiacchio, David Mathews 427 Abstract Withdrawn 428 Previously Overlooked CoA Aptamers Revealed by High-Throughput Analysis Kyle Hill, Mark Ditzler, Collin Luebbert, Donald Burke 429 SHAPEclash: A Biochemical and Computational Approach for RNA Tertiary Structure Analysis and Refinement Philip Homan, Feng Ding, Nikolay V. Dokholyan, Kevin Weeks 430 Using a Non-Redundant Dataset of RNA Crystal Structures to understand RNA Structural Flexibility for Rational Design of RNA Molecules and RNP Complexes Swati Jain, Laura Murray, Jane Richardson, Bruce Donald 431 A Novel RNA Structural Motif with a Conserved Backbone Structure but non-Conserved Base Sequence Gary Kapral, Swati Jain, Jane Richardson, David Richardson 432 Model Building in RNA Structural Biology: The RCrane Approach for Crystallographic Applications Kevin Keating, Anna Marie Pyle 433 The Role of Salt Concentration and Magnesium Binding in HIV-1 Subtype-A and Subtype-B Kissing Loop Monomer Structures Taejin Kim, Bruce Shapiro 434 Abstract Withdrawn 435 The Hairpin Ribozyme, A Multiconformer Ribozyme? Matthew Marek, Berhanegebriel Assefa, Nils Walter 436 Restoring SNP Disrupted RNA Structural Ensembles with LNAs Joshua Martin, Justin Ritz, Lauren Neulander, Chetna Gopinath, Alain Laederach 437 Single-Molecule Studies of HIV-1 Dimerization Initiation Sequence Kissing Interaction and its Resolution to a Stable Extended Duplex Hansini Mundigala, Jonathan Michaux, Eric Ennifar, Andrew Feig, David Rueda 438 Solution NMR and X-ray Crystallographic Examination of RNA Plasticity Using and Aptamer for Ribosomal Protein S8 Edward Nikonowicz, James Donarski, Jiachen Wang, Yousif Shamoo 439 Transcriptional pausing coordinates folding of the aptamer domain and the expression platform of a riboswitch. George Perdrizet II, Irina Artsimovitch, Ran Furman, Tobin Sosnick, Tao Pan 440 Mutations in The UTRs of SERPINA1 Transcripts Are Involved in The Disease Associated Mechanisms Gabriela Phillips, Chetna Gopinath, Mat Halvorsen, Justin Ritz, Joshua Martin, Alain Laederach 441 Site-specific Crosslinking Using Cis-diamminedichloroplatinum (II) to Probe the Tertiary Structure of Complex RNAs Kory Plakos, Erich Chapman, Elaine Chase, Barbara Golden, Victoria DeRose xli

442 Structural Changes of a Group II Intron ID3 Stem Loop Associated with Binding of its Target Exon 1 Sequence Milena Popovic, Nancy Greenbaum 443 Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling Xiaojun Zhang, Peter Qin 444 Two Protein Cofactors Co-opt To Facilitate bI5 Intron Folding In Yeast Mitochondria Nora Sachsenmaier, Christina Waldsich 445 Dynamic Nature of pri-miRNA Revealed by Molecular Dynamics Simulations and Biochemical Methods Debashish Sahu, Kaycee Quarles, Scott Showalter 446 Two Retinoblastoma Associated SNVs in RB1 form a RiboSNitch Wes Sanders, Matt Halvorsen, Justin Ritz, Joshua Martin, Alain Laederach 447 Structural and Biochemical Studies of the dG Riboswitch Olga Pikovskaya, Anna Polonskaia, Dinsnaw Patel, Alexander Serganov 448 Non-Nearest Neighbor Dependence of Stability for Group III RNA Single Nucleotide Bulge Loops Martin Serra, Christina Hasson, Brandon Panaro, Dan Phillips, Michael McCann 449 Single Molecule Dynamics of Functional Spliceosomes Amanda Solem, Yu-Chih Tsai, Jonas Korlach, David Rueda 450 Abstract Withdrawn 451 Influence Of Ligand Binding On The Loop-Loop Interaction Of The Adenine Riboswitch Aptamer Patrick St-Pierre, Juan Penedo, Daniel Lafontaine 452 Antibodies as RNA crystallization chaperones Nikolai Suslov, Joseph Piccirilli 453 Characterization of Structural Changes Associated with HIV-1 Genomic RNA Dimerization Using Fluorescence, NMR, and SANS Andrea Szakal, John Marino, Susan Krueger 454 Single Molecule Studies of Prp24-Dependent Folding Dynamics of the U2/U6 Complex Chandani Warnasooriya, Zhuojun Guo, Samuel Butcher, David Brow, David Rueda 455 Orientational Information from FRET in the Structural Analysis of RNA Timothy Wilson, Stephanie Kath-Schorr, Jonathan Ouellet, Asif Iqbal, David Lilley 456 Towards Structural Insights Into Human Telomerase Georgeta Zemora, Samuel Coulbourn Flores, Christina Waldsich 457 mRNA Localization Mechanisms are Conserved in Trypanosomes Lysangela Alves, Arthur Oliveira, Samuel Goldenberg, Bruno Dallagiovanna 458 Association, Recruitment and Function of TREX Components Aly, THO and UAP56 are Interdependent Binkai Chi, Qingliang Wang, Guifen Wu, Min Shi, Lantian Wang, Hong Cheng 459 p180 Promotes the Ribosome-Independent Localization of a Subset of mRNA to the Endoplasmic Reticulum Xianying (Amy) Cui, Alexander Palazzo 460 Antibodies Specific for Active Spliceosomes Reveal the Global Extent and Subnuclear Location of co- and postTranscriptional Splicing Cyrille Girard, Cindy Will, Jianhe Peng, Evgeny Makarov, Berthold Kastner, Ira Lemm, Henning Urlaub, Klaus Hartmuth, Reinhard Lührmann 461 Regulation of tRNA nucleus-cytoplasm distribution by nutrient availability Rebecca Hurto, Anita Hopper 462 MicroRNA-Mediated Translational Repression of a Localized mRNA in Xenopus Oocytes Catherine Pratt, Kimberly Mowry xlii

463 The Dynamics of Splicing Factor Interactions with Active Genes in Living Cells Noa Neufeld, Yehuda Brody, Itamar Kanter, Shai Carmi, Eva-Maria Böhnlein, Karla Neugebauer, Yaron Shav-Tal 464 SRp20 Plays An Essential Role in the Regulation of HIV-1 RNA Processing Maria Calimano, Annie Mao, Raymond Wong, Alan Cochrane 465 Gammaherpesvirus 68 Exemplifies a Novel Biogenesis Pathway to Produce Biologically Functional miRNAs Kevin Diebel, Linda van Dyk 466 IFIT1 is an antiviral protein that recognises 5’-triphosphate RNA Andreas Pichlmair, Matthias Habjan, Caroline Lassnig, Cathleen Holze, Carol-Ann Eberle, Keiryn L Bennett, Jacques Colinge, Thomas Rülicke, Friedemann Weber, Mathias Müller, Giulio Superti-Furga 467 Effect of single nucleotide substitutions in Pepino mosaic virus genome on its virulence Beata Hasiow-Jaroszewska, Julia Byczyk, Henryk Pospieszny, Natasza Borodynko 468 A Non-Canonical Ribosomal Frameshift Signal in a Plant Viral RNA Alice Hui, Vijayapalani Paramasivan, Norma Wills, Betty Chung, Andrew Firth, John Atkins, W. Allen Miller 469 Structural Studies on the Panicum mosaic virus-like Cap-independent Translation Element: an Uncapped RNA That Tightly Binds the Cap-binding Protein eIF4E Jelena Kraft, Mariko Peterson, W. Allen Miller 470 Discovery of RNA Secondary Structure in Influenza Virus Walter Moss, Salvatore Priore, Lumbini Dela-Moss, Tian Jiang, Douglas Turner 471 In vivo Functional Selection and SELEX Reveal Flexible Length of the H2 Stem Loop in a Satellite RNA Allison Murawski, Tareq Azad, Johnathan Nieves, Holleh Tajalli, Nina Jean-Jacques, Biology 419 Students, Megan Young, Anne Simon, David Kushner 472 Involvement Of PSF In The Recognition Of An RNA Promoter Derived From The HDV RNA Genome Martin Pelchat 473 Comparison of SIVmac239 and HIV-1NL4-3 Genomic RNA Structures: Stabilization and Reformation of Structure Throughout Sequence Evolution Elizabeth Pollom, Kristen Dang, Elizabeth Potter, Robert Gorelick, Christina Burch, Kevin Weeks, Ronald Swanstrom 474 Novel microRNAs and Alternative mRNA Isoforms Arising During Human Cytomegalovirus Infection Thomas Stark, Brett Roberts, Justin Arnold, Deborah Spector, Gene Yeo 475 Physical Interactions Between eIF4G and the 3’ Cap-independent Translation Element of Barley yellow dwarf virus (BYDV) RNA Krzysztof Treder, Jelena Kraft, Zhaohui Wang, W. Allen Miller 476 Kinetic Analysis of the Double-stranded RNA Formation in Qβ RNA Replication. Kimihito Usui, Norikazu Ichihashi, Yasuaki Kazuta, Tomoaki Matsuura, Tetsuya Yomo 477 The Rous Sarcoma Virus RNA Stability Element Acts As an Insulator to Prevent Recognition of Unspliced Retroviral RNA by Host Cell Decay Machinery Johanna Withers, B. Lin Quek, Nicholas Ingolia, Karen Beemon 478 Requirement of Ded1p, a Conserved DExD/H-Box Translation Factor, in Yeast L-A Virus Replication Chung-Shu Yeh, Tien-Hsien Chang

FRIDAY, JUNE 1, 2012: 20:00 – 22:30 Poster Session 3 - Michigan League (2nd Floor) Abstracts 479 – 632 479 Self-cleavage Mechanism of the Hairpin Ribozyme Berhanegebriel Assefa, Matt marek, Nils Walter 480 Defective RNAs associated with Tomato black ring virus isolates collected from zucchini plants Beata Hasiow-Jaroszewska, Natalia Rymelska, Henryk Pospieszny, Natasza Borodynko xliii

481 Regulation of Drosha by Microprocessor-independent Viral miRNAs in Cells Latently Infected by Herpesvirus saimiri Demian Cazalla, Joan Steitz 482 Design and Synthesis of Small Molecules for RNA Internal Loop Takeo Fukuzumi, Asako Murata, Yasue Harada, Kazuhiko Nakatani 483 Phosphorylation Regulates MiRNA Biogenesis Kristina Herbert, Genaro Pimienta, Suzanne DeGregorio, Joan Steitz 484 A Role for AGO4 in the Male Mouse Germ Line Stephanie Hilz, Andrew Modzelewski, Rebecca Holmes, Andrew Grimson, Paula Cohen 485 Characterization of Stage-Specific Small RNAs during Development of Triops cancriformis (Tadpole Shrimp) Yuka Hirose, Kahori Takane, Emiko Noro, Kiriko Hiraoka, Masaru Tomita, Akio Kanai 486 Real-time Dynamics of RISC-mRNA Interaction Observed via Single-Molecule FRET Seung-Ryoung Jung, Eunji Kim, Ji-Joon Song, Sungchul Hohng 487 The Profile of snoRNA-derived MicroRNAs that Regulate Expression of Variant Surface Proteins in Giardia lamblia Wei Li, Ashesh Saraiya, Ching Wang 488 The mir-35-41 Family of MicroRNAs Regulates RNAi Sensitivity in C. elegans Katlin Massirer, Saida Perez, Vanessa Mondol, Amy Pasquinelli 489 Global miRNA profiling and Ago2 RIP-Chip identifies consistently deregulated miRNAs in neuronal adaptive and death responses induced by MPP+ Elena Miñones-Moyano, Birgit Kagerbauer, Michaela Beitzinger, Gunter Meister, Xavier Estivill, Eulalia Marti 490 Substrate Specificity In Small RNA Silencing Pathways Milijana Mirkovic-Hoesle, Klaus Foerstemann 491 Abstract Withdrawn 492 Characterization of Small RNAs by Using a Ribosome-enriched Fraction in Escherichia coli Shinnosuke Murakami, Yoshiki Ikeda, Emiko Noro, Masaru Tomita, Kenji Nakahigashi, Akio Kanai 493 Regulatory small RNAs generated from miRNA loops Katsutomo Okamura, Erik Ladewig, Eric Lai 494 Position of Hfq Distal Face Binding Site on RNA Controls the Rate of RNA Annealing Subrata Panja, Sarah Woodson 495 Complexity of murine cardiomyocyte miRNA biogenesis, sequence variant expression and function Jennifer Clancy, David Humphreys, Carly Hynes, Hardip Patel, Thomas Preiss 496 Transition of MicroRNA Function from Repression to Activation Depending on the Extent of Base Pairing with the Target Site Ashesh Saraiya, Ching Wang 497 Comparative Profiling of Prostate Epithelial and Stromal Cell MicroRNA Transcriptome by Deep Sequencing Girish Shukla, Guru Jagadeeswaran, Zheng Yun, Kavleen Sikand, Ramanjulu Sunker 498 Mapping Targets for sRNAs in Pathogenic E.coli Jai Tree, Sander Granneman, David Gally, David Tollervey 499 The Period protein homolog LIN-42 negatively regulates microRNA biogenesis in C. elegans Priscilla Van Wynsberghe, Emily Finnegan, Amy Pasquinelli 500 Comprehensive Analysis of Small RNAs in Oryzias latipes and Takifugu rubripes Chaninya Wongwarangkana, Ryota Terai, Kosuke Negoro, Masaki Akiba, Kazuhiro E. Fujimori, Shigeharu Kinoshita, Atsushi Shimizu, Kosuke Sakai, Sabine K. Kojima, Susumu Mitsuyama, Ikuya Kikuzato, Morimi Teruya, Maiko Nezuo, Shuichi Yano, Yukino Miwa, Yumi Imada, Yuki Sato, Tsukahara Masatoshi, Jun Kudoh, Takashi Hirano, Nobuyoshi Shimizu, Shugo Watabe, Shuichi Asakawa xliv

501 The General Role of Drosha Processing in the Regulation of microRNA Expression and Lessons Beyond microRNA Biogenesis Yong Feng, Xiaoxiao Zhang, Yan Zeng 502 The Butterfly Effect of Translationally-acting Riboswitches Laurène Bastet, Antony Lussier, Audrey Dubé, Daniel Lafontaine 503 A small kinase ribozyme with unusual dependence on pH and Cu for dual-site activity Elisa Biondi, Raghav Poudyal, Andrew Sawyer, Adam Maxwell, Donald Burke 504 Mechanism for Gene Control by a Natural Allosteric Group I Ribozyme Andy G. Y. Chen, Narasimhan Sudarsan, Ronald Breaker 505 Characterizing the Contribution of a Reverse G-U Wobble Pair to Metal Ion Catalysis in the HDV Ribozyme Ji Chen, Abir Ganguly, Zulaika Miswan, Sharon Hammes-Schiffer, Philip Bevilacqua, Barbara Golden 506 Efficiency and Fidelity of Splicing at the 3’-Splice Site of RmInt1 Group II Intron Isabel Chillon, Olga Fedorova, Francisco Martinez-Abarca, Anna Pyle, Nicolas Toro 507 The kinetics of the interaction between the btuB riboswitch and coenzymeB12 Pallavi Choudhary, Roland K. O. Sigel 508 Sequence-Specific Multicolor Imaging of Nucleic Acid Nanostructures Alexander Johnson-Buck, Jeanette Nangreave, Hao Yan, Nils Walter 509 Substrate Interaction And RNase P RNA Mediated Cleavage Leif Kirsebom, Shiying Wu, Guanzhong Mao, Abhishek Srivastava 510 Helix-length Compensation Studies for Cleavage of Alternate Substrates by the Neurospora VS Ribozyme Julie Lacroix-Labonté, Nicolas Girard, Sébastien Lemieux, Pascale Legault 511 SELEX Experiment Suggests that the Coenzyme-Dependent glmS Ribozyme Evolved from a Self-Cleaving Ribozyme Requiring Only Magnesium for Catalysis Matthew Lau, Adrian Ferré-D’Amaré 512 Structural Diversity in Riboswitches that Respond to Pre-Queuosine1 Jonathan Liang, Phillip McCown, Ronald Breaker 513 Thermodynamic characterization of Mg2+ function in the binding of tRNA to the T box riboswitch antiterminator Jia Liu, Jennifer Hines 514 Development of efficient methods to prepare natively folded RNA using photocleavable biotin-modified nucleotides Yiling Luo, Nadukkudy Eldho, Herman Sintim, Kwaku Dayie 515 The Diversity of Architectures in Lysine and TPP Riboswitches Phillip McCown, Adam Roth, Ronald Breaker 516 An In Vitro Assay for the Function of a Guanine Riboswitch Colin Nevins, Daniel Morse 517 In Vivo Evolution of Trans-Splicing Group I Intron Ribozymes Karen Olson, Gregory Dolan, Zhaleh Amini, Ulrich Müller 518 Characterization of the Trans Watson-Crick GU Base Pair Located in the Catalytic Core of The Antigenomic HDV Rbozyme. Dominique Lévesque, Cedric Reymond, Jean-Pierre Perreault 519 Targeted Modifications of Glucosamine-6-phosphate to Affect the Binding and Activation of the glmS Ribozyme Jeffrey Posakony, Adrian Ferré-D’Amaré 520 Differential Conformational Selection and Induced Fit of Structurally Similar Single Transcriptional and Translational Riboswitches Arlie Rinaldi, Krishna Suddala, Jun Feng, Anthony Mustoe, Charles Brooks III, Nils Walter xlv

521 Programming the highly structured HDV ribozyme into complex allostery with RNA oligonucleotides. Samuel Rouleau, Jean-Pierre Perreault 522 Mg-Dependent Folding of the btuB Riboswitch by Single Molecule FRET Studies Michelle F. Schaffer , Roland K.O. Sigel 523 A Newly Characterized Version of the Hepatitis Delta Virus Ribozyme Binds its Substrate Less Efficiently than Previous Versions Kamali Sripathi, Pavel Banáš, Wendy Tay, Jiſí Šponer, Michal Otyepka, Nils Walter 524 Exploring the Significance of a Water-Involving Hydrogen Bonding Network Within the Hairpin Ribozyme Wendy Tay, Nils Walter 525 Interaction between the Scissile Phosphate and a Putative Catalytic Metal Ion in the HDV Ribozyme Pallavi Thaplyal, Barbara Golden, Philip Bevilacqua 526 The Effects of Expression Platform Stability on Riboswitch Mediated Gene Expression Control Nakesha Smith, Shanelle Graham, Carla Theimer 527 Metal-Dependent Folding and Catalysis of a Native Hammerhead Ribozyme Luke Ward, Dan Morris, Matthew Hendricks, Victoria DeRose 528 The Hairpin Ribozyme: Metal Dependence to Tight Intermolecular Docking Neil White, Minako Sumita, Charles Hoogstraten 529 Effect of Spermidine on The T Box Riboswitch Antiterminator Model RNA Chunxi Zeng, Shu Zhou, Jennifer Hines 530 Base-pairing, Base-stacking, and Steric Occlusion Mediate Recognition of a Non-aminoacylated tRNA by a T-box Riboswitch Jinwei Zhang, Adrian Ferré-D’Amaré 531 Development of a New Fluorescent Toolbox for Imaging RNA in Live Cells Tucker Carrocci, Jacquelyn Turri, Aaron Hoskins 532 Preparation of Large RNAs by Splintless RNA Ligation Kevin Desai, Aaron Hoskins, Ronald Raines 533 Kinetic Analysis of Aptazyme-regulated Gene Expression in a Cell-free Translation System Shungo Kobori 534 Defining RNA-Platinum Adducts within the Eukaryotic Ribosome Maire Osborn, Jonathan White, Victoria DeRose 535 Use of Fluorescence Spectroscopy for High-Throughput Quantification of pKa Shifting in RNA Jennifer Wilcox, Philip Bevilacqua 536 HOW, an RNA Binding Protein, Regulates Alternative Splicing in D. melanogaster Nehemiah Alvarez, Malcolm Cook, Marco Blanchette 537 Pseudouridines induced during filamentous growth in yeast Anindita Basak, Charles Query 538 Splicing stimulates biogenesis of plant microRNAs Dawid Bielewicz, Lukasz Sobkowiak, Katarzyna Raczynska, Daniel Kierzkowski, Maria Kalyna, Andrea Barta, Franck Vazquez, Artur Jarmolowski, Zofia Szweykowska-Kulinska 539 A Subset of Introns is Abundant in poly-A mRNA Paul Boutz, Jesse Zamudio, Xuebing Wu, Phillip Sharp 540 Combinatorial Control of Alternative Splicing by SR Protein Regulatory Networks Todd Bradley, Malcolm Cook, Marco Blanchette

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541 Genome-wide Analysis of Splicing Regulation in Drosophila melanogaster by RNAi Depletion of 58 RNA Binding Proteins Angela Brooks, Gemma May, Li Yang, Michael Duff, Jane Landolin, Kenneth Wan, Jeremy Sandler, Susan Celniker, Brenton Graveley, Steven Brenner, Fly Transcriptome Group 542 Functional Analysis of the Conserved AU Di-nucleotides at the 5’-end of the U1 snRNA Jui-Hui Chen, Tien-Hsien Chang 543 SC35 and SF2/ASF Regulate Stress-Responsive Alternative Splicing of MDM2 Daniel Comiskey, Ravi Singh, Aixa Tapia-Santos, Dawn Chandler 544 Modifiers of SMN Splicing in Spinal Muscular Atrophy Catherine Dominguez, Thomas Bebee 545 Dissecting Minimal Domains Necessary for Alternative Splicing by Muscleblind-like Proteins Christopher Edge, Clare Gooding, Chris Smith 546 First splicing step in Saccharomyces cerevisiae requires Prp45 Ondrej Gahura, Zdenek Cit, Anna Valentova, Frantisek Puta, Petr Folk 547 De Novo Prediction of PTB Binding and Splicing Targets Reveals Unexpected Features of Its RNA Recognition and Function Areum Han, Peter Stoilov, Yu Zhou, Xiang-Dong Fu, Douglas Black 548 Identification and Characterization of RBM38 Regulated Alternative Splicing Events in Hematopoietic Cell Development Laurie Heinicke, Behnam Nabet, Russ Carstens 549 MDM2 Stress Responsive Splicing: An Intricate Interplay of Positive and Negative Elements and Splicing Regulatory Factors Aishwarya Jacob, Ravi Singh, Dawn Chandler 550 Contribution of Chromatin Marks to Alternative Splicing Regulation by Rbfox2 in Mouse Embryonic Stem Cells Mohini Jangi, Paul Boutz, Phillip Sharp 551 Regulated Use of Alternative Splice Sites During Stress in S.cerevisiae Tadashi Kawashima, Stephen Douglass, Guillaume Chanfreau 552 Understanding how PTB and NPTB Direct Different Splicing Outcomes Niroshika Keppetipola, Douglas Black 553 Mammalian Neuronal Development and Maturation Require the Splicing Regulator PTBP2 Qin Li, Chia-Ho Lin, Peter Stoilov, Lily Shiue, Manuel Ares Jr., Douglas Black 554 PTB and nPTB regulated splicing events during neural progenitor maintenance and motor neuron development. Anthony Linares, Douglas Black 555 Analysis of Site-Specific Phosphorylation Events and Their Influence on Splicing in Schizosaccharomyces pombe Michael Marvin, Jesse Lipp, Kevan Shokat, Christine Guthrie 556 Investigating the Function of Tissue-dependent Alternative Splicing in the Mammalian Circadian Clock Nicholas McGlincy, Inge van Bussel, Johanna Chesham, Jernej Ule, Michael Hastings 557 Evolution of alternative splicing regulation Joel McManus, Joseph Coolon, Michael Duff, Jodi Mains, Patricia Wittkopp, Brenton Graveley 558 Using a Drosophila genetic model to study crosstalk between chromatin and alternative splicing Michael Meers, A. Gregory Matera 559 Silent Effects of Splicing: Conservation of Splicing Signals in Coding Exons William Mueller, Klemens Hertel 560 Evolution and Functional Analysis of the Antisense Overlap Between mRNAs Encoding Two Mammalian Nuclear Receptors, TRα2 and Rev-erbα Stephen Munroe, Christopher Morales, Cynthia Aguilar, Paul Waters, Jennifer Graves xlvii

561 The Tumor Suppressor p53 Controls Alternative Splicing In Mammary Epithelial Cells Ryan Percifield, Daniel Myrphy, Peter Stoilov 562 Identifying and Characterizing the Mechanisms and Consequences of Nervous System Alternative Splicing Adam Norris, John Calarco 563 Loss of a Positive Regulator Within a Deep Intronic Sequence of SBP2 Contributes to a Genetic Disorder Eric Ottesen, Joonbae Seo, Senthilkumar Sivanesan, Natalia Singh, Ravindra Singh 564 Identification of New Splicing Inhibitors Andrea Pawellek, Stuart McElroy, Timur Samatov, Reinhard Luehrmann, Angus Lamond 565 A model in vitro system for co-transcriptional splicing YONG YU, Rita Das, Eric Folco, Robin Reed 566 Identification of epigenetic regulators of alternative pre-mRNA splicing Maayan Salton, Ty Voss, Tom Misteli 567 The role of Rbfox proteins during skeletal muscle differentiation Ravi Singh, Auinash Kalsotra, Chris Bland,Tomaž Curk, Jernej Ule, Liguo Wang, Wei Li, Thomas A. Cooper 568 Pyrvinium Pamoate Regulates Alternative Splicing Of The Serotonin Receptor 2C pre-mRNA By Changing RNA Structure Manli Shen, Peter Stoilov, Stefan Stamm 569 Increased Accumulation of Glucose-6-Phosphate Dehydrogenase mRNA Due to Enhanced Binding of the Splicing Factor SRSF3 In Response to Nutrients Amanda Suchanek, Callee Walsh, Travis Cyphert, Lisa Salati 570 HnRNPH/F, U1 snRNP and U11 snRNP co-operate to regulate the stability of the U11-48K pre-mRNA Bhupendra Verma, Janne Turunen, Mikko Frilander 571 Integrative Genome-wide Analysis Reveals Cooperative Regulation of Alternative Splicing by hnRNP Proteins Anthony Vu, Stephanie Huelga, Justin Arnold, Tiffany Liang, John Donohue, Lily Shiue, Shawn Hoon, Sydney Brenner, Manuel Ares Jr., John Taylor, Gene Yeo 572 EM Study of PTB-repressed Exon Complex Somsakul Wongpalee, Shalini Sharma, James Wohlschlegel, Hong Zhou, Douglas Black 573 A Conserved Alternative Splicing Event Leads to Differential Subcellular Localization of ESRP1 Yueqin Yang, Russ Carstens 574 Investigating the Regulation of PSF in Signal-induced T Cell Alternative Splicing Christopher Yarosh, James Lipchock, Kristen Lynch 575 Non-UGCAUG Elements Involved in Rbfox-mediated Alternative Splicing Regulation Yi Ying, Andrey Damianov, Chia-Ho Lin, Douglas Black 576 Structural and Biochemical Analysis of The Role of Phosphorylation of Splicing Factor 1 in Spliceosome Assembly Yun Zhang, Tobias Madl, Hyun-Seo Kang, Thomas Kern, Angela Krämer, Michael Sattler 577 Discovery of Pyrobaculum small RNA Families with a Subset of Pseudouridine Guide RNA Features David Bernick, Patrick Dennis, Matthias Höchsmann, Todd Lowe 578 Kinetics and Thermodynamics of tRNA Folding Monitored by Aminoacylation Hari Bhaskaran, John Perona 579 Small RNA Sequencing Reveals Modification Sites and Transcripts Antisense to Transfer RNAs in Archaea Patricia Chan, Todd Lowe 580 Kinetic Characterization of Escherichia coli MetRS Keng-Ming Chang, Rebecca Alexander 581 Cajal Body-Specific RNAs in Drosophila: New Members, New Targets? Svetlana Deryusheva, Joseph Gall xlviii

582 Post-Translational Addition of Amino Acids: tRNA Substrate Specificity Angela Fung, Richard Fahlman 583 nev-tRNA: A novel nematode-specific tRNA that decodes an alternative genetic code for leucine Kiyofumi Hamashima, Kosuke Fujishima, Takeshi Masuda, Junichi Sugahara, Masaru Tomita, Akio Kanai 584 Structure of Proteinaceous RNase P Provides Insight into Precursor-tRNA Processing in Mitochondria Michael Howard, Wan Hsin Lim, Markos Koutmos, Carol Fierke 585 The Mechanism of Bidirectional tRNA Nuclear-cytoplasmic Trafficking Hsiao-Yun Huang, Anita Hopper 586 Box C/D Guide RNA Containing a Typical K-loop is Non-Functional in Haloarchaea Sujata Jana, Archi Joardar, Ramesh Gupta 587 Defining The Division Of Labor Within The Human tRNA Ligase Complex Jennifer Jurkin, Johannes Popow, Stefan Weitzer, Karl Mechtler, Javier Martinez 588 Biochemical Characterization of Three RNA Ligases from the Hyperthermophilic Archaeon Pyrococcus furiosus Asako Sato, Takeshi Masuda, Masaru Tomita, Takashi Itoh, Akio Kanai 589 Impairment of Ribosomal RNA Modification Causes Developmental Defects in Zebrafish Sayomi Higa-Nakamine, Tamayo Uechi, Anirban Chakraborty, Takeo Suzuki, Tsutomu Suzuki, Naoya Kenmochi 590 A Role for Nuclear Import in tRNA Quality Control in Saccharomyces cerevisiae Emily Kramer, Anita Hopper 591 Pathways for 3’ End Processing of Yeast tRNAs Involve tRNase Z, Rex1 and Rrp6 Ewa Skowronek, Pawel Grzechnik, Bettina Späth, Anita Marchfelder, Joanna Kufel 592 Enzyme and Substrate Basis of Species-Specificity of tRNA N-Isopentenyl-A37 (iA37) Modification, Which Increases Codon Reading ~5-Fold in Yeast Tek Lamichhane, James Iben, Nathan Blewett, Amanda Crawford, Sandy Mattijssen, Phil Farabaugh, Richard Maraia 593 Evolution of tRNA Dependent Fidelity Mechanisms in Aminoacyl-tRNA Synthetases Susan Martinis, Michal Boniecki, Li Li, Andrés Palencia, Tiit Luuk, Zhi Li, Satish Nair, Stephen Cusack, Zan LutheySchulten 594 Kinetic investigation of nucleotide addition by a 3’-5’ polymerase Krishna Patel, Paul Yourik, Jane Jackman 595 Identification of a Novel Regulatory Factor of the Human RNA Ligase Johannes Popow, Alexander Schleiffer, Javier Martinez 596 In Vitro Characterization of Bacterial Thg1-like Proteins (TLPs): New Frontiers, Novel Avenues Bhalchandra Rao, Jane Jackman 597 Recognition of Guanosine by Dissimilar tRNA Methyltransferases Reiko Sakaguchi, Ya-Ming Hou 598 A Common Modification in an Unusual Place: Avoiding Conundrums in Mitochondrial Translation Paul Sample, Ludek Koreny, Kirk Gaston, Patrick Limbach, Julius Lukeš, Juan Alfonzo 599 Coupled Processing of S. cerevisiae sn/snoRNA 3’ and 5’ Termini Sylwia Szczepaniak, Dorota Adamska, Zaneta Matuszek, Pawel Grzechnik, Joanna Kufel 600 tRNA Transcription and tRNA Decay Are Coupled Tomasz Turowski, Iwona Karkusiewicz, Justyna Kowal, Magdalena Boguta 601 Early Pre-rRNA Processing in Human ITS1 uses both Different Processing Pathways and Nucleases from the Established Yeast Model Katherine Sloan, Sandy Mattijssen, David Tollervey, Ger Pruijn, Nick Watkins 602 The RNA kinase CLP1 is required for efficient tRNA splicing and regulates p53 activation in response to oxidative stress Stefan Weitzer, Toshikatsu Hanada, Barbara Mair, Josef Penninger, Javier Martinez xlix

603 A Genome-wide Analysis to Identify Novel Genes Involved in tRNA Metabolism and Subcellular Trafficking Jingyan Wu, Anita Hopper 604 DUX4 Induces Global Dysregulation of RNA Processing in Skeletal Muscle Stephen Tapscott, Zizhen Yao, Robert Bradley 605 Human microRNA Expression Profile in Amyotrophic Lateral Sclerosis: Role of microRNAs in the Regulation of Neurofilament Levels Danae Campos-Melo, Kathryn Volkening, Michael Strong 606 Hypoxia is a Modifier of SMN2 Splicing and Disease Severity in a Severe SMA Mouse Model Thomas Bebee, Catherine Dominguez, Somayeh Samadzadeh-Tarighat, Dawn Chandler 607 Splice Isoform Switching: A New Mechanism Controlling EMT and Breast Cancer Progression Rhonda Brown, Lauren Reinke, Yilin Xu, Marin Damerow, Denise Perez, Lewis Chodosh, Jing Yang, Chonghui Cheng 608 Rho Guanine Nucleotide Exchange Factor is a NFL mRNA Destabilizing Factor that Forms Cytoplasmic Inclusions in Amyotrophic Lateral Sclerosis Cristian Droppelmann, Brian Keller, Danae Campos-Melo, Kathryn Volkening, Michael Strong 609 Induced Pluripotent Stem Cells from Diamond Blackfan Anemia Patients Show Defects in Ribosome Biogenesis Jingping Ge, Loic Garcon, Marisa Apicella, Jason Mills, Paul Gadue, Deborah French, Mitch Weiss, Monica Bessler , Philip Mason 610 Noncoding Consequences of Disease Associated Mutations Set Against a Backdrop of Multiple Transcriptomic SNVs Matthew Halvorsen, Joshua Martin, Gabriela Phillips, Justin Ritz, Wes Sanders, Alain Laederach 611 Analysis of Novel NFL targeting MicroRNAs in Amyotrophic lateral Sclerosis (ALS) Muhammad Ishtiaq, Danae Campos Melo, Kathryn Volkening, Michael Strong 612 A BIM deletion polymorphism contributes to resistance against targeted cancer therapy by promoting splicing of non-apoptotic BIM variants Wen Chun Juan, King Pan Ng, Tun Kiat Ko, Axel Hillmer, Charles Chuah, Yijun Ruan, Xavier Roca, Sin Tiong Ong 613 Expanded CUG Repeat RNA reactivates the embryonic gene program in Myotonic dystrophy Auinash Kalsotra, Ravi Singh, Chad Creighton, Thomas Cooper 614 S6K1 alternative splicing modulates its oncogenic activity and regulates mTORC1 Vered Ben Hur, Polina Denichenco, Avraham Maimon, Adrian Krainer, Ben Davidson, Rotem Karni 615 Identification Of New Factors Associated With Translationally Repressed RNAs In Plasmodium Berghei Natalia Kozlova, Edwin Lasonder , António Mendes, Céline K. Carret, Ana M. Guerreiro, Gunnar Mair 616 Identification of altered MicroRNA expression in response to salmonella infection during early stage in intestinal epithelial cells Xingyin Liu, Jun Sun 617 A Drosophila Model of Spinal Muscular Atrophy Uncouples the snRNP Biogenesis Functions of Survival Motor Neuron from Locomotion and Viability Defects Kavita Praveen, Ying Wen, A. Gregory Matera 618 Role of double-stranded RNA-dependent protein kinase (PKR) in metabolic disease Takahisa Nakamura, Brenna Baccaro, Gökhan Hotamisligil 619 Aberrant mRNA processing, a major disease mechanism that precedes symptoms in a mouse model of Amyotrophic Lateral Sclerosis (ALS) Ramesh Narayanan, Marie Mangelsdorf, Robyn Wallace 620 A Novel Interplay Between the NMD Mechanism and ER Stress Response Under Normal Conditions and in Human Diseases Yifat Oren, Tamar Geiger, Miriam Manor, Matthias Mann, Batsheva Kerem 621 Novel Human Variation in MicroRNAs associated with Disease, Biomarkers, and Drug Metabolism. Renata Rawlings, Sarah Tishkoff l

622 Serum non-coding RNAs as Biomarkers for Osteoarthritis Progression After ACL Injury Maozhou Yang, Liang Zhang, Mark Hurtig, Lawrence White, Paul Marks, Gary Gibson 623 Identification of Cellular dsRNA Required for PKR Activation during Metabolic Stress Osama Youssef, Takahisa Nakamura, Gökhan Hotamisligil, Brenda Bass 624 DRAGins: Drug Binding Aptamers For Growing Intracellular Numbers Muslum Ilgu, Supipi Auwardt, Robert Feldges, Khalid Boushaba, Howard Levin, Marit Nilsen-Hamilton 625 Robust Suppression of HIV Replication by Intracellularly-expressed RNA Aptamers is Independent of Ribozyme Processing Margaret Lange, Angela Whatley, Tarun Sharma, Marc Johnson, Donald Burke 626 Therapeutic Application of Antisense Compounds to Target Pathogenic RTKs for Cancer Therapy Lee Spraggon, Sandra Vorlova, Gina Rocca, Luca Cartegni 627 Nanoparticle Delivery of MicroRNA Inhibition to Treat OncomiR-Addicted Lymphoma Tumors Christopher Cheng, Imran Babar, Mark Saltzman, Frank Slack 628 The Potential of Nanoparticles Composed of RNA-Bolaamphiphile Complexes as a Therapeutic siRNA Delivery Vehicle Taejin Kim, Kirill Afonin, Eliahu Heldman, Mathias Viard, Selene Sparks, Robert Blumenthal, Bruce Shapiro 629 Using Unlocked Nucleic Acid (UNA) Substitutions to Alter Strand Selection By the RNA Induced Silencing Complex Nicholas Snead, Julie Escamilla-Powers, Sabrina Shore, Natasha Paul, John Rossi, Anton McCaffrey 630 Gene Expression From Pseudouridine and 5-methylcytidine Modified Messenger RNAs Jiehua Zhou, Julie Escamilla-Powers, Anton P. McCaffrey, John Rossi 631 Therapeutic Application of RNA based Compounds to Target Pathogenic RTKs for Cancer Therapy Lee Spraggon, Sandra Vorlova, Gina Rocco, Luca Cartegni 632 Effective RNAi Therapeutics to Treat Chronic Hepatitis B Virus Infection Christine Wooddell, Matthias John, Markus Hossbach, Jochen Deckert, Philippe Hadwiger, Holly Hamilton, Qili Chu, Darren Wakefield, Daniel Sheik, Jason Klein, Kerstin Jahn-Hofmann, Alan McLachlan, Hans-Peter Vornlocher, David Rozema, David Lewis

LATE ADDITIONS Michigan League (2nd Floor) Abstracts 701 – 711 701 Molecular cloning and gene expresión of Fibrillarin in Phaseolus vulgaris Jesus Cortes, Josefina Huerta, A. Fco. Cabral, E. E. De Leon 702 Single-RNA FISH reveals that nonsense-mediated mRNA decay in human cells occurs in the cytoplasm near the nucleus Tatjana Trček Pulisic, Hanae Sato, Robert H. Singer, Lynne E. Maquat 703 A Direct Role for Quaking in Muscle-Specific Alternative Splicing W. Samuel Fagg, Megan Hall, Roland Nagel, Melissa Cline, Lily Shuie, Manuel Ares 704 Saccharomyces cerevisiae General Splicing Factor Required for the Stable U2 snRNP Binding to pre-RNAs Patricia Coltri, Carla Oliveira 705 Use of 19F-NMR to measure RNA structural dynamics Caijie Zhao, Matthew Devany, Nancy Greenbaum 706 Direct Interaction between RanBP2/Nup358 and ALREX-Promoting SSCR RNA Elements Hui Zhang, Serge Gueroussov, Kohila Mahadevan, Can Cenik, Frederick Roth, Alexander Palazzo, Alexander Palazzo 707 Characterization of guanine riboswitches for the development of selective analog inhibitors Anne-Marie Lamontagne, Jerome Mulhbacher, Daniel Lafontaine li

708 Kramers Rate Theory Describes the Viscosity Dependent Folding Kinetics of the Tetraloop-Receptor Motif Nicholas Dupuis, David Nesbitt 709 Conformational Selection of Initiation Factor 3 Signals Proper Substrate Selection During Translation Initiation Margaret Elvekrog, Ruben Gonzalez, Jr 710 Regulation of Ribosomal 70S Initiation Complex Stability during Translation Initiation Daniel MacDougall, Ruben Gonzalez 711 Cloning, Expression, and Purification of the S. cerevisiae Sub2 ATPase Yuliang Sun and Aaron A. Hoskins 712 The Development of Methods for the Site-Specific Fluorescent Labeling of Spliceosomal Proteins for use in Single-Molecule Studies Matthew Kahlscheuer, Ramya Krishnan, Mario Blanco, Nils Walter

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RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

1

Cross-talk between dsRNA-mediated pathways: Is that my dsRNA or yours?

2

tRNA Tuning in Translation

Brenda Bass University of Utah, Salt Lake City, (Utah), United States RNA strands form base-pairs, with themselves or another molecule, to create the basic unit of RNA secondary structure, the A-form double-stranded RNA (dsRNA) helix. Secondary structures of the most familiar RNA molecules, such as tRNA and rRNA, are comprised of multiple, non-contiguous, or “branched”, regions of dsRNA, and form the foundation for more complex 3-dimensional structures and non-canonical interactions. However, increasingly, it is clear that unbranched, rod-like dsRNA molecules, consisting of many, nearly contiguous base-pairs, play important roles in the RNA world. The precursors to small RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) are examples of unbranched, cellular dsRNA molecules. dsRNA binding proteins (dsRBPs) such as Drosha or Dicer bind and process these precursors to generate the small RNAs, which base-pair to mRNA to alter its expression. Another family of dsRBPs, the adenosine deaminases that act on RNA, or ADARs, deaminates adenosines in unbranched cellular dsRNA to create inosines. One function of these RNA editing enzymes is to alter codons in mRNAs so that one gene gives rise to many different protein isoforms, thus diversifying a proteome. Another class of dsRBPs is exemplified by PKR and RIG-I, which bind viral dsRNA to trigger a host response to pathogen. dsRBPs have unique challenges. Their functions mandate that they recognize many different sequences of dsRNA. In fact, the structure of the A-form helix makes sequence-specific contacts difficult, and dsRBPs bind any dsRNA they encounter, sometimes even the dsRNA of a different pathway. Existing literature offers examples of how different dsRNA-mediated pathways intersect and affect each other. After an introduction to the dsRNA world, and the functions of dsRBPs, examples of how dsRNA-mediated pathways co-exist, yet achieve specificity, will be discussed.

Olke C. Uhlenbeck Departments of Chemistry and Molecular Biosciences Northwestern University Each bacterial tRNA species possesses an idiosyncratic set of residues distributed throughout the molecule that distinguish it from tRNAs with different anticodons. We believe that these multivalent “tRNA consensus” sequences have primarily evolved to compensate for the intrinsically different physical properties of the esterified amino acid and the codon-anticodon interaction such that each tRNA can translate with similar rate and accuracy. I will first review data showing that three base pairs in the T stem adjust the affinity of tRNA to EF-Tu in a manner that compensates for the varying affinity of the esterified amino acid. As a result, each aa-tRNA binds tight enough to form a complex, but weak enough so it can subsequently release from the protein during decoding. Different bacteria often use different combinations of base pairs to achieve the characteristic affinity of a given tRNA species. A second set of experiments suggests that many of the remaining tRNA consensus residues have evolved to minimize misreading of near-cognate codons. Base pairs in the anticodon stem or base triples in the core of tRNAAla were mutated to residues present in tRNAAla of other bacteria (“consensus mutations”) or to residues never present in tRNAAla, but present in other tRNAs (“non-consensus mutations”). While all the mutations were fully active on cognate GCC codons, 13 of 16 non-consensus mutations showed misreading of GUC or ACC codons whereas all 6 consensus mutations tested were accurate. I will speculate on possible mechanisms how tRNA sequence can modulate decoding accuracy and propose why different tRNA species have evolved different consensus sequences. We believe that principles deduced from this classic system can be extended to other systems.

Opening Plenary Session Speaker

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

3

Monitoring protein synthesis one codon at a time through ribosome profiling

4

Precursor tRNA Processing in Mitochondria and Chloroplasts

Jonathan Weissman University of California-San Francisco/HHMI, San Francisco, (CA), USA The ability to sequence genomes has far outstripped approaches for deciphering the information they encode. We have developed a suite of techniques based on ribosome profiling (deep sequencing of ribosome protected fragments) that dramatically expand our ability to follow translation in vivo. I will present recent applications of our ribosome profiling approach including the following: (1) Development of ribosome profiling protocols for a wide variety of eukaryotic and prokaryotic organisms. (2) Uses of ribosome profiling to globally monitor when chaperones, targeting factors or processing enzymes engage nascent chains. (3) Deciphering the driving force and biological consequences underlying the choice of synonymous codons.

Carol Fierke1, Michael Howard1, Wan Hsin Lim1, Markos Koutmos2 1 Departments of Chemistry and Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, 2 Department of Biochemistry & Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814 Transfer tRNA in most organisms is transcribed as a precursor with additional sequences at both the 5’ and 3’ ends. In mitochondrial and chloroplast genomes the tRNA genes punctuate the RNA transcripts, making tRNA processing important for the maturation of other RNA species critical for organelle biogenesis. Maturation of the 5’ end oftRNA is catalyzed by ribonuclease P (RNase P) across all domains of life. Until recently, all known RNase P enzymes include a catalytic RNA component. However, RNaseP from human mitochondria and Arabidopsis thaliana chloroplast and mitochondria are composed solely of protein subunits and represent a new class of metallonucleases (PROteinacous RNase P (PRORP))1,2. Structural and biochemical studies of A. thaliana PRORP demonstrate that the enzyme contains three domains: (i) a metallonuclease domain that is a member of the PIN (PilT N-terminal) domain-like fold super-family and contains conserved aspartate and histidine side chains important for catalysis and metal binding; (ii) a pentatricopeptide repeat domain that enhances pre-tRNA affinity and orients the substrate for cleavage; and (iii) an intervening structural zinc site domain. These results provide insight into the evolution and catalytic mechanism of the newly discovered class of proteinaceous RNase P. This work is supported by NIH grant GM055387. 1. Holzmann J,Frank P, Löffler E, Bennett KL, Gerner C, Rossmanith W. (2008) RNase P without RNA: identification and functional reconstitution ofthe human mitochondrial tRNA processing enzyme. Cell 135, 462-536. 2. Gobert A,Gutmann B, Taschner A, Gössringer M, Holzmann J, Hartmann RK, Rossmanith W, Giegé P. (2010) A single Arabidopsis organellar protein has RNaseP activity. Nature structural &molecular biology 17, 740-744.

Opening Plenary Session Speaker & Session 1: Keynote

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

5

Insights into RNA Biology from a Mammalian Cell mRNA Interactome

6

Searching for the Specificity of Non-specific RNA-binding Proteins

Alfredo Castello1, Bernd Fischer1, Katrin Eichelbaum1, Rastislav Horos1, Benedikt Beckmann1, Claudia Strein1, David Humphreys2,3, Thomas Preiss2,3, Lars Steinmetz1, Jeroen Krijgsveld1, Matthias Hentze1,2 1 European Molecular Biology Laboratory (EMBL), Heidelberg, Germany, 2Victor Chang Cardiac Research Institute, Sydney, Australia, 3The John Curtin School of Medical Research, The Australian National University, Acton ACT, Australia RNA-binding proteins (RBPs) determine RNA fate from synthesis to decay. Employing two complementary protocols for covalent UV-crosslinking of RBPs to RNA, we have used a systematic, unbiased and comprehensive approach to define the mRNA interactome of proliferating human HeLa cells. Of the proteins captured with polyadenylated RNA, 860 are significantly enriched over negative controls shown by analysis of independent repeat experiments. Following validation, the in vivo HeLa mRNA interactome adds more than three hundred RBPs to those previously known. The described method is broadly applicable to study mRNA interactome composition and dynamics in varied biological settings. Our data shed new light on diverse aspects of RNA biology, including RBPs in disease, RNA-binding kinases and the potential existence of novel RNA-binding architectures. We also identify enzymes of intermediary metabolism that moonlight as RBPs in vivo, implicating these in the recently proposed REM (RNA/enzyme/metabolite) networks.

Chaolin Zhang, Robert Darnell Laboratory of Molecular Neuro-oncology, Howard Hughes Medical Institute, Rockefeller University, New York, NY, USA RNA-binding proteins (RBPs) interacting with their target transcripts are essential for multiple steps of RNA processing and gene expression regulation in mammals. However, many RBPs recognize very short and degenerate sequences, and it is unclear how the selectivity in target transcripts is derived. Part of this paradox is likely resolved by the fact that RBPs typically have multiple RNA binding domains or can multimerize to synergistically bind several motif sites clustered together, which can be further modulated by site accessibility through different RNA-secondary structures. Here we present a statistical machine learning approach based on a hidden Markov model (HMM) to integrate the number and spacing of individual motif sites, and their accessibility and cross-species conservation to predict functional RBP binding sites. This method takes advantage of a large number of in vivo RBP binding sites determined by high throughput sequencing of RNAs isolated by cross-linking and immunoprecipitation (HITS-CLIP) to estimate model parameters and evaluate performance. It was recently applied to predict YCAY (Y=C/U) clusters recognized by a neuron-specific RBP Nova at a genome-wide scale, which showed very reasonable accuracy and effectively improved prediction of Nova-regulated alternative exons. We have now performed systematic evaluation of the method, using multiple measures including concordance with independent microarray data and mutagenesis analysis of several mini-gene reporters. Subsets of Nova binding sites defined by CLIP data also show interesting differences from those predicted bioinformatically. In addition, we have now applied the method to other RBPs, demonstrating its general applicability to facilitate the inference of RNA-regulatory networks.

Session 1: RNA-protein interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

7 High throughput sequencing kinetics (HTS-KIN) reveals hidden sequence determinants for an RNA-binding protein that binds substrates in a non-specific manner

Ulf-Peter Guenther1, Lindsay Yandek2, Frank Campbell1, David Anderson3, Vernon Anderson2, Eckhard Jankowsky1, Michael Harris2 1 Center for RNA Molecular Biology and Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH, USA , 2Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, USA , 3Department of Decisions, Operations and Information Technologies, Robert R Smith School of Business, University of Maryland, College Park, MD, USA

Many RNA binding proteins accommodate a broad range of substrates that lack apparent sequence signatures in their binding sites. The biological importance of non-specific RNA binding modes is well recognized, but the molecular basis underlying an apparent lack of sequence specificity is not well understood. A key to advancing our understanding is achieving a comprehensive description of how sequence variation and binding affinity are correlated, given that the biological substrates identified for RNA binding proteins do not cover the entire sequence space of possible binding sites. Here, we describe a systematic and quantitative analysis of the entire sequence space for the pre-tRNA leader binding site of the protein subunit (C5) of the E. coli RNase P ribonucleoprotein. Mutation of pre-tRNA nucleotides contacted by C5 can alter the rate of processing by RNase P; however, genomically encoded leader sequences are highly variable. To define functional affinities for a complete set of binding site sequence variants, we generated a population of pre-tRNA in which the six nucleotides comprising the C5 binding site were randomized. This population was subjected to RNase P processing in vitro and changes in the distribution of individual variants were quantified over time by next generation sequencing. Using an internal competition method we calculated the apparent second order rate constant for processing by RNase P (V/K) for all 4096 leader sequence variants. This high throughput sequencing kinetics (HTS-KIN) approach revealed an over 50-fold difference in V/K for the entire spectrum of leader sequences. Strikingly, the distribution of rate constants for all C5 binding site sequence variants closely resembles the distribution of affinities for transcription factors. As seen for these highly specific DNA binding proteins, the optimal RNA sequences that interact with C5 show a clear sequence signature. However, few genomically encoded pre-tRNA leader sequences conform to the fastest reacting sequence motif. Our observations reveal hidden sequence determinants for RNA binding by the C5 protein, and show that its biological substrates are not optimized for the highest functional affinity. The data further raise the possibility that in at least a subset of RNA binding proteins, specific and non-specific binding modes are not fundamentally different, but rather represent distinct parts of a spectrum of affinity distributions. Finally, our results show that inherently specific RNA binding proteins can operate functionally in a non-specific manner.

8

Global Analysis of Yeast mRNPs

Sarah Mitchell, Saumya Jain, Meipei She, Roy Parker Howard Hughes Medical Institute, University of Arizona, Tucson, AZ, USA mRNA binding proteins regulate gene expression at the post-transcriptional level by controlling mRNA biogenesis, localization, translation and decay. A key goal is to identify the composition, diversity and function of individual mRNPs. To understand yeast mRNPs on a global level, we developed the ICLamP (In vivo Cross-Linking and mRNA Pull-down) procedure to identify the complete set of mRNA binding proteins in yeast. This procedure consists of crosslinking proteins to mRNA in vivo, purifying mRNPs by oligo(dT) pull-down under denaturing conditions and identifying bound proteins by mass spectrometry. ICLamP has identified 125 proteins that cross-link robustly to mRNA including 56 known mRNA binding proteins. In addition to identifying numerous new RNA binding proteins, our results reveal that multiple proteins known to bind rRNA and tRNA also function in mRNA control suggesting extensive cross talk between mRNA regulation and tRNA and rRNA function. We also identify several RNA or protein modification enzymes that crosslink to mRNA suggesting mRNA specific RNA and protein modification patterns exist. Localization studies of these mRNP proteins under growth and stress conditions demonstrate that mRNP components are highly dynamic with ~40% changing their subcellular location under stress. Interestingly, mRNP proteins accumulate in five distinct subcellular compartments during stress suggesting specific mRNAs are subject to different regulation under these conditions. CLIP studies on proteins that represent different subcellular compartments (e.g. P-bodies or stress granules) identified classes of mRNAs recruited to different mRNPs during stress, as well as revealing 5’ and 3’ position specific binding of some mRNP components. Taken together, these results define the yeast mRNP composition, reveal mRNA specific classes of mRNPs, and demonstrate the dynamics of the mRNP population during stress. We are building on these results to more completely define the mRNA-protein interaction map in yeast, its dynamics and functional consequences, and how this information can be used to develop a comprehensive model for mRNP biogenesis and function in eukaryotic cells.

Session 1: RNA-protein interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

9

Higher Order mRNP Structure Revealed by the Cellular EJC Interactome

Guramrit Singh1,2, Alper Kucukural1,2, Can Cenik1,2, John Leszyk2, Scott Shaffer2, Zhiping Weng2, Melissa Moore1,2 1 HHMI, 2UMass Medical School, Worcester, MA, 01605 As pre-mRNA splicing sculpts pre-mRNA transcripts into mature mRNAs by removing introns, it also greatly impacts the protein complement of the emerging mRNP. The exon junction complex (EJC), deposited upstream of exonexon junctions after splicing, is a major constituent of spliced mRNPs. We now report a comprehensive analysis of the endogenous human EJC protein and RNA interactomes. We confirm that the major “canonical” EJC occupancy site in vivo lies 24 nucleotides upstream of exon junctions. Surprisingly, however, endogenous EJCs predominantly exist as multimers in highly RNase-resistant megadalton complexes. Within these, EJCs interact with >70 proteins belonging to different classes, with SR and SR-like proteins being the most abundant. Remarkably, in the cellular EJC proteome, the SR proteins SRSF1, 3 and 7 are superstoichiometric to the EJC core factors. Furthermore, these large EJC/SR complexes protect from nucleases unexpectedly long RNA fragments that are enriched with EJC deposition sites and sequence motifs similar to the preferred binding sites of several SR and SR-like proteins. Over the last decade EJC and SR proteins have been found to display many similar functions (genomic stability, mRNA export, translation, mRNA surveillance). Our results strongly suggest that rather than being attributable to functional redundancies, the apparently parallel functions of EJC and SR proteins reflect their tight physical association. That is, these proteins may act cooperatively to stabilize their mutual association with spliced mRNPs and act together to perform these functions. Furthermore, within cellular spliced mRNPs, EJC multimerization and extensive interactions between EJCs and SR proteins lead to higher order structures that may act as “ribonucleosomes”. Functionally paralleling DNA nucleosomes, such structures may facilitate the packaging of mRNAs into highly compacted mRNPs tightly woven around proteinaceous cores consisting largely of EJCs and SR proteins. Thus, our results provide new insight into the structures of spliced mRNPs in vivo.

10 CLIP-seq of the DEAD-box RNA Helicase eIF4AIII Reveals Transcriptome-wide Mapping of the Exon Junction Complex in Human

Jerome Sauliere, Valentine Murigneux, Zhen Wang, Isabelle Barbosa, Hugues Roest Crollius, Herve Le Hir Institut de Biologie de l’Ecole Normale Superieure, CNRS UMR8197-INSERM U1024, Paris, France The multiprotein exon junction complex (EJC) is deposited onto mRNAs by the splicing machinery ~24 nucleotides upstream of exonic junctions. The EJC is a key effector of mRNAs metabolism, and influences pre-mRNA splicing as well as mRNA localization, translation and surveillance by NMD. Structurally, the EJC is organized around a tetrameric core complex composed of the DEAD-box RNA helicase eIF4AIII, MAGOH, Y14 and MLN51. eIF4AIII acts as an ATPdependent RNA clamp to ensure the tight and sequence-independent binding of the EJC core to the RNA. Despite a clear view of EJC structure and functions, very little is known about its assembly in vivo. To gain insights into transcriptome-wide EJC deposition in human cells, we used CLIP-seq (crosslinking and immunoprecipitation coupled to high-throughput sequencing) of eIF4AIII. After UV-crosslinking, RNAs bound directly to endogenous eIF4AIII were immunoprecipitated using specific antibodies. After stringent purification, the cDNA libraries were prepared and subjected to Illumina sequencing. CLIP-seq generated millions of uniquely mapped reads to the genome, and clusters of local high-density peaks were identified as the binding signature of eIF4AIII. Importantly, deep-sequencing of HeLa cell transcriptome allowed normalizing the intensity of eIF4AIII peaks by mRNA expression level. The distribution of eIF4AIII peaks concentrates in exons of spliced mRNAs compared to introns and intergenic regions. Peaks mark most exons indicating that a large proportion of spliced junctions are associated to eIF4AIII. Localization of the center of thousands of peaks reveal a strong enrichment of eIF4AIII at the canonical EJC position ~24 nt upstream of exon junctions. Moreover, distinct peaks are also distributed within exons. Surprinsigly, some of them also accumulate at a position ~30 nt downstream of spliced junctions. This unexpected splicing-dependent eIF4AIII signature strongly suggests the existence of a new EJC-like binding site. Comparison of the height of eIF4AIII peaks along some transcripts shows a striking heterogeneity supporting the notion that EJC can differentially mark spliced junctions in human. Current analyses of eIF4AIII crosslinking sites and the surrounding sequences are ongoing to determine what may influence EJC imprinting. In summary, our transcriptome-wide mapping of eIF4AIII by CLIP-seq provides unprecedented details on EJC assembly in human transcripts, opening the road to new investigations to better understand its role on gene expression in human. Session 1: RNA-protein interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

11 Identification of targets of mRNA decay factors using RIP-seq reveals they target specific messages

Jason Miller1,2, Liye Zhang1, Jennifer Kruk1,2, B. Franklin Pugh1, Joseph Reese1,2 The Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, University Park, PA, 16802, 2Center for RNA Molecular Biology, Department of Biochemistry and Molecular Biology, University Park, PA, 16802 Although a great deal of effort has been spent on uncovering how RNA binding proteins regulate mRNA decay and translation, the field still lacks a complete understanding of which mRNAs these proteins are recruited to on a transcriptome wide level. For example, do RNA decay factors preferentially target specific messages or do they perform only housekeeping functions? We undertook a cross-linking procedure, followed by deep RNA sequencing (RIP-seq), to identify RNA targets of Ccr4, Dhh1, and Puf5. All three of these factors are known to play a role in mRNA decay and translational repression, however they contribute to these processes in different ways. For example, Ccr4 is the main deadenylase in yeast, Puf5 binds mRNA targets to recruit Ccr4, and Dhh1 contributes to shuttling mRNAs between the translatable and non-translatable pool of mRNAs. Here, we determined the mRNAs bound to these proteins under normal growth conditions. We found that all three decay factors crosslink to specific mRNAs, rather than those of highest abundance or longest length. All three proteins preferentially cross-link to the 3’ ends of transcripts, however significant levels of cross-linking are also detected at the middle and the 5’ end among subsets of mRNAs. GO terms and overlap analysis suggest these proteins are recruited to mRNAs associated with osmotic stress resistance, transcription/chromatin regulators, nitrogen metabolism, and mitochondrial function. Interestingly, our analysis suggests that the recruitment of Ccr4 to its target is not sufficient to cause mRNA instability per se, suggesting a post-recruitment activation step of the deadenylase complex. Additionally, overlap analysis suggests an interesting connection between Dhh1p and Puf5p and the RNA export machinery. In summary, we detected protein-RNA interactions that were previously unidentified by non-cross linking methods, and analysis of these targets suggests mRNA decay factors may regulate specific groups of transcripts in addition to providing a “house keeping”function.

1

12

Allosteric inhibition of a stem cell RNA-binding protein by an intermediary metabolite

Carina Clingman, Laura Deveau, Samantha Hay, Ryan Genga, Shivender Shandilya, Francesca Massi, Sean Ryder University of Massachusetts Medical School, Worcester, MA, 01605, USA Imprecise regulation of gene expression can lead to a variety of developmental diseases and cancers. Musashi-1 (MSI1) is a stem cell RNA-binding protein that regulates the translation of mRNAs required for cell proliferation. MSI1 is overexpressed in a variety of epithelial and neural tumors. In order to identify inhibitors of MSI1 function, we developed a high throughput assay to screen for small molecules that disrupt MSI1 RNA-binding activity. In a pilot screen, we identified three inhibitors. The most potent specific inhibitor is the intermediary metabolite oleic acid. A second inhibitor is an agonist of the fatty acid responsive nuclear hormone receptor PPARα. The last inhibitor is aurintricarboxylic acid, a non-specific inhibitor of protein-nucleic acid interactions. Structure activity relationship studies revealed that MSI1 is specifically inhibited by 18-22 carbon cis ω-9 monounsaturated fatty acids. Mechanistic studies reveal that ω-9 fatty acids bind a hydrophobic pocket in RRM1 and induce a conformational change that precludes RNA binding. Metabolic control of post-transcriptional regulation has been widely documented in bacteria, but remains relatively unstudied in eukaryotes. We identify stearoyl-CoA desaturase-1 (SCD1) as a MSI1 target. SCD1 is the enzyme that converts stearic acid to oleic acid in mammals. MSI1 binds directly to the Scd1 3’UTR in vitro and in cells, and overexpression of MSI1 increases SCD1 levels. Together, our results show that MSI1 N-terminal RRM1 acts as a sensor that couples translation regulation to metabolite concentration, the first example of such activity observed for an RRM domain.

Session 1: RNA-protein interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

13

Crystal structure of the eukaryotic RNA-induced silencing complex

Kotaro Nakanishi1, David Winberg2, David Bartel2, Dinshaw Patel1 1 Memorial Sloan-Kettering Cancer Center, New York, USA, 2Howard Hughes Medical Institute, MIT Department of Biology, and Whitehead Institute, Cambridge, USA MicroRNAs and small interfering RNAs are incorporated into Argonaute to form the RNA-induced silencing complex (RISC). The assembly of RISC occurs in two steps: entry of a small-RNA duplex into Argonaute to form pre-RISC, and then loss of one of the two strands of the duplex to form mature RISC. The remaining RNA strand acts as a guide to recruit RISC to RNA targets based on sequence complementarity. If pairing between the guide RNA and the target RNA is extensive, Argonaute cleaves the target RNA. Here we report the 3.2 Å resolution crystal structure of the eukaryotic Argonaute protein from the budding yeast Kluyveromyces polysporus (KpAGO). When building the KpAGO model, we noticed electron density that was not explained by the protein and instead corresponded to RNA at the location expected for guide RNA. High-throughput sequencing and biochemical analysis showed that our recombinant KpAGO fortuitously copurified with thousands of different RNA fragments from the E. coli in which it had been expressed. When presented with their cognate targets, these fragments can direct RNA cleavage, thereby demonstrating their function as bona fide guide RNAs and indicating that we purified mature RISC with guide RNA positioned in the nucleic acid– binding channel. Strong electron density is observed for both the 5’ nucleotide and the RNA seed region (nucleotides 2–8, thought to nucleate pairing to the RNA target), which indicates that the seed regions of most (if not all) of the different guide RNAs must be oriented similarly. Electron density is not observed for RNA after nucleotide 9, which indicates that the 3’ regions of the RNAs are less consistently oriented. Protein contacts are made to nearly every 2’-OH and phosphate group of the seed region, while the bases of nucleotides 2 and 5–6 are positioned in a sequence-independent manner through interactions with N935 and a hydrophobic pocket, respectively. As a result, KpAGO tilts the bases of nucleotides 2–6 and presents their Watson–Crick faces to solvent while pre-organizing the sugar-phosphate backbone in a near A-form conformation. Structural comparison of KpAGO with the apo form of the MID-PIWI lobe of Neurospora crassa QDE-2 reveals correlated movements between the loops L1 and L2 and a eukaryote-specific helix-turn-helix segment. This suggests that a dramatic surface shape change occurs within the MID-PIWI lobe upon guide-RNA incorporation. We also identify a cluster of eukaryote-specific conserved insertion segments that fill a gap between the loop L1 and the PIWI domain, which shifts the N domain out of the way of the nucleic acid-binding channel. In contrast, the N domain of prokaryotic Argonautes is positioned at one end of the channel and thereby disrupts duplex formation beyond nucleotide 16. The resulting longer channel in eukaryotic Argonautes would enable guide–target base pairing to extend along the 3’-half of the guide RNA, which is consistent with the known contributions of pairing to this region of the guide.

14

The Structural Basis for Binding and Unwinding of Duplex RNA by a DEAD-box Protein

Anna Mallam, Mark Del Campo, Benjamin Gilman, David Sidote, Alan Lambowitz University of Texas at Austin, Austin, TX, USA DEAD-box proteins are ATP-dependent RNA helicases that are found in all domains of life and function in diverse cellular processes that require the remodeling of RNA and RNP substrates. All DEAD-box proteins contain a conserved ‘helicase core’ that consists of two RecA-like domains (domains 1 and 2), typically attached to additional domains that specialize the proteins for different functions. DEAD-box proteins differ from processive helicases in promoting RNAunwinding by localized strand separation rather than translocation through the duplex, enabling them to remodel RNAs and RNPs without globally disrupting RNA structure. This local strand separation is catalyzed by the helicase core, which undergoes conformational changes that couple cycles of ATP binding and hydrolysis to RNA binding and release. Here, we obtained X-ray crystal structures of the individual core domains of the yeast DEAD-box protein Mss116p in the presence and absence of bound substrates, including the first DEAD-box protein structures with bound duplex RNA. These structures together with biochemical and genetic analyses of Mss116p and other DEAD-box proteins indicate an RNA unwinding mechanism in which ATP binds initially to domain 1, while duplex RNA binds initially to domain 2. Core closure to form the ATPase active site causes domain 1 to distort the path of both strands of the bound RNA duplex, resulting in local strand separation. Domain 2 contains a binding pocket for double-stranded RNA that accommodates A-form but not B-form duplexes, providing a basis for RNA substrate specificity. Interactions between domain 2 and duplex RNA are at or near conserved DEAD-box protein motifs, one of which rearranges to help form the ATPase active site during core closure. Our results suggest a comprehensive structural model of how DEAD-box proteins bind and unwind RNA duplexes, which helps to explain many longstanding questions about DEAD-box protein function. These include the structural basis for the specificity of DEAD-box proteins for RNA duplexes, the cooperative tight binding of ATP and RNA, the requirement for ATP binding but not hydrolysis to promote RNA unwinding, protein cooperativity in unwinding RNA duplexes, and the ATP-independent strand annealing activity observed for some DEAD-box proteins. The model also provides new perspective for considering how the structurally related helicase cores of DEAD-box and other helicases could evolve to use different RNA-unwinding mechanisms. Session 1: RNA-protein interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

15

DEAD-box Helicases Can Act as ATP-dependent RNA Clamps and as AMP Sensors

16

Mechanism of foreign DNA selection in a bacterial adaptive immune system

Andrea Putnam, Fei Liu, Eckhard Jankowsky Case Western Reserve University, Cleveland, OH, USA DEAD-box RNA helicases have been shown to unwind and remodel RNAs and RNPs. All of these functions are based on the ability of DEAD-box helicases to couple ATP binding and hydrolysis to changes in RNA affinity, but how exactly RNA affinity is altered by nucleotide binding and hydrolysis is not well understood. Using various biochemical approaches, we have devised a quantitative framework for the coupling between ATP binding/hydrolysis and RNA affinity for the DEAD-box helicase Ded1p from S.cerevisiae. In addition to a general overview of the connection between ATP binding/hydrolysis to RNA binding, this framework reveals to two striking and unanticipated features of DEAD-box helicases. First, nucleotide analogs that mimic pre-hydrolysis and transition states of the ATP hydrolysis cycle form extremely long-lived complexes with RNA, with half-lives of t1/2 ~ 38 - 52 hrs. Further tests on other DEAD-box helicases show that the ability to form long-lived, ATP-dependent complexes on RNA is seen for all tested DEAD-box helicases. These observations show that the capacity to form ATP-dependent RNA clamps is a common feature of a broad range of DEAD-box proteins. Second, we find that AMP strongly decreases RNA affinity, even though the nucleotide is not a product of ATP hydrolysis by DEAD-box helicases. Adenosine and cyclic AMP have no effect. Most remarkably, functional binding constants for AMP are in the range of physiologically relevant AMP concentrations under stress conditions, such as glucose deprivation in yeast. Further tests of AMP binding with other DEAD-box helicases revealed that some, but not all were similarly inhibited by AMP. These findings suggest that a subset of DEAD-box proteins have the capacity to act as AMP sensors. Collectively, these findings expand the functional spectrum of DEAD-box helicases and raise the possibility that these ubiquitous enzymes have the potential to regulate RNA binding and remodeling in response to a common metabolite.

Dipali Sashital1, Blake Wiedenheft2, Jennifer Doudna2 1 The Scripps Research Insistute, La Jolla, CA, USA, 2University of California, Berkeley, CA, USA Effective immunity against infection requires detection and neutralization of foreign transgressors while avoiding reactions to self. Bacterial and archaeal CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) immune pathways use an RNA-guided complex to target invasive viral and plasmid DNA for destruction. Foreign “spacer” sequences are acquired and integrated between repetitive elements within CRISPR loci in the host genome. RNAs generated from these loci (crRNAs) can base pair with sequences in both the host CRISPR locus and foreign “protospacer” sequences in invading viruses or plasmids. A proto-spacer adjacent motif (PAM) is required to discriminate target sequences present in foreign genetic material from those found within the CRISPR locus [1], but the mechanism of non-self recognition is unknown. Here we show that CasA, the largest of 11 protein subunits of the Cascade complex in Escherichia coli, is required for non-self target binding and PAM recognition. Combining a 2.3 Å crystal structure of CasA with cryo-EM structures of Cascade [2], we have identified a loop containing a three amino acid motif that is required for viral defense at the levels of both Cascade assembly and DNA target recognition. The CasA loop directly contacts the target PAM sequence, implicating this protein in self versus non-self discrimination. Together, our data suggest a model for Cascade-mediated intracellular surveillance in which the CasA loop scans DNA for the short PAM sequence prior to proto-spacer binding, maximizing the efficiency with which the CRISPR machinery can identify and destroy invasive nucleic acids. 1. Marraffini, L. A. & Sontheimer, E. J. Nature 463, 568-571 (2010). 2. Wiedenheft, B. et al. Nature 477, 486-489 (2011).

Session 1: RNA-protein interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

17

High resolution landscape of transcription in human cells

18

Saccharomyces cerevisiae non-coding RNAs: bound to be broken

Thomas Gingeras Cold Spring Harbor Laboratory, NY, United States Steady state measurements of transcriptomes represent a snapshot of the sequence content and amounts of individual RNAs present in biological samples. As part of the ENCODE project, we have sought to both provide an accurate representation of a genome-wide catalogue of the human transcriptome and also to identify the sub-cellular context for distinct RNA classes. This goal was achieved by identifying and characterizing both previously annotated and novel RNAs that are enriched in either of the two major cellular sub-compartments (nucleus and cytosol) for all 15 cell lines studied and for one cell line, three additional sub-nuclear compartments. In addition, we sought to determine if identified transcripts are modified at their 5’ and 3’ termini (cap or polyadenylation) and to determine precursor-product relationships for many annotated and unannotated RNAs. Overall, a sampling of our result indicate that a total of 62.1% and 74.7% of the human genome were observed to be covered by either processed and primary transcripts, respectively with no cell line showing more than 56.7% of the union of the expressed transcriptome across all cells. A consequence of these high-resolution RNA mapping observations is that the intergenic regions of the human genome is shrinking in size (median length reduced by 3.6 fold ) having notable implications on the classic definition of a genic region. The range of expression for detected transcripts in each cell line was measured, and covers 6 orders of magnitude for protein coding, non-coding and novel intergenic/antisense genes (10-2 – 10-4 RPKM) in the polyadenylated fraction and 5 orders of magnitude (10-2 – 10-3 RPKM) in the non-polyadenylated fraction Isoform expression by genes was observed not follow a minimalistic strategy. While the number of expressed isoforms expressed in a cell type appears to increase with the number of associated annotated isoforms, the expressed number appears to plateau at about 10-12 expressed isoforms per gene per cell line. Finally, cell type-specific enhancers contain regulatory regions that are distinct from other transcriptional regulatory regions by the presence of specific types of RNA transcripts, chromatin marks and DNAse l hypersensitive sites. These and other results paint a picture of the human transcriptome that is complex but also indicate layers of regulation that have yet to be characterized.

Alex Tuck, David Tollervey Wellcome Trust Centre for Cell Biology, Edinburgh, UK Pervasive eukaryotic transcription produces antisense and intergenic transcripts termed “long non-coding RNAs” (lncRNAs). LncRNAs and mRNAs share many properties (5’ cap, poly(A) tail, transcribed by RNA Pol II) but the available data suggest their fates differ. The mRNAs are generally stable in the nucleus and exported to the cytoplasm for translation, whereas the lncRNAs tested are predominately degraded rapidly in the nucleus and can have distinct functions such as directing chromatin modifications. These differences may reflect systematic changes in patterns of associated RNA binding proteins. During their synthesis and maturation Pol II transcripts interact with a series of protein factors, and we hypothesized that somewhere in this pathway mRNAs and lncRNAs diverge to form distinct ribonucleoprotein particles. Factors binding mRNAs were therefore tested by CRAC (crosslinking and analysis of cDNAs) for association with lncRNAs. The export receptor Mex67 and cytoplasmic factors Xrn1 and Tif1 bound relatively few lncRNAs, suggesting that lncRNAs are mainly distinguished from mRNAs before assembly of export-competent RNPs. Conversely, the 3’-end processing factor Hrp1 bound numerous lncRNAs. Many of these also bound the nuclear exosome cofactor Trf4, and ~25% of Hrp1-bound fragments had short oligo(A) tails characteristic of decay. Together, these data suggest that besides its documented role in directing 3’-end processing factors to mRNA cleavage and polyadenylation sites, Hrp1 destabilises transcripts terminating at non-canonical sites (e.g. lncRNAs). This is reminiscent of Nrd1 and Nab3, termination factors promoting decay. We propose that lncRNA fate is predominantly determined upon completion of termination and 3’ end processing. Ongoing CRAC studies are identifying other participants in this decision. We are also investigating how lncRNA abundance is modulated via changes in transcription rate and surveillance activity. Following a nutrient shift, changes in lncRNA association with surveillance factors were detected by CRAC, and transcription rates analyzed by high-throughput sequencing of newly synthesized transcripts. LncRNA binding by the exosome cofactor Mtr4 was highly dynamic under conditions of nutrient shift, and an intriguing class of lncRNAs showed transient changes in Mtr4 association. This supports a role for surveillance in altering lncRNA expression, and identifies lncRNAs that may participate in rapid reprogramming of gene expression. Session 2A: Keynote & Session 2A: Non-coding RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

19

Non-coding RNA in transcriptional silencing

Jordan Rowley1, Qi Zheng2, Brian Gregory2, Andrzej Wierzbicki1 1 University of Michigan, Ann Arbor, MI, USA, 2University of Pennsylvania, Philadelphia, PA, USA Transcriptional gene silencing controls the activity of transposable elements and other repetitive sequences. It is mediated by small interfering RNAs which associate with Argonaute (AGO) proteins and determine the sequencespecificity of silencing. We have shown before that in Arabidopsis transcriptional silencing of several loci requires also a second class of RNA – long non-coding RNA (ncRNA) produced by RNA Polymerase V (Pol V). These ncRNAs interact with AGO4 and are required for AGO4 binding to chromatin suggesting that they recruit AGO4 to at least some of its targets in the genome. We now present ChIP-seq evidence that AGO4 binds to chromatin throughout the Arabidopsis genome with about a thousand high confidence binding sites. Surprisingly, AGO4 binding shows no enrichment on transposon rich centromeric regions, instead AGO4 preferentially targets promoters of protein-coding genes. Using a combination of genetic and genomic approaches we found that preferential AGO4 binding to gene promoters is mediated by long non-coding RNA produced by Pol V. Furthermore, lncRNA-dependent AGO4 binding directs DNA methylation to gene promoters. We found that a significant subset of genes targeted by AGO4 shows strong changes in expression levels in mutants lacking AGO4 or lncRNA production, therefore lncRNA-mediated AGO4 targeting to gene promoters may control gene expression. Overall, our data show that Pol V-produced lncRNA guides AGO4 to specific loci in the Arabidopsis genome, however controlling the activity of transposable elements does not seem to be the primary function of AGO4. We propose that AGO4 and the entire RNA-mediated transcriptional gene silencing pathway may be primarily involved in regulation of gene expression.

20 Cancer-associated Long Noncoding RNA Regulates Cell Cycle Progression by Modulating the Expression of Oncogenic Transcription Factors

Vidisha Tripathi1, Xinying Zong1, Zhen Shen1, Ashish Lal2, Supriya Prasanth1, Kannanganattu Prasanth1 1 University of Illinois at Urbana-Champaign, Urbana, Illinois, USA, 2National Cancer Institute, Bethesda, Washington DC, USA In eukaryotic cells, long ncRNAs (lncRNAs) play roles in crucial cellular processes and their aberrant expression is linked to several diseases. The cancer-associated MALAT1 lncRNA interacts with SR splicing proteins, regulates SR-protein activity and modulates alternative splicing. MALAT1 and SR proteins localize to nuclear speckles, a sub nuclear domain that is involved in pre-mRNA processing. MALAT1 and SR proteins facilitate the initial assembly of nuclear speckles in the interphase nucleus. MALAT1 displays cell cycle-regulated expression, with low levels during G1 and significantly elevated levels during G1/S and mitosis. MALAT1 regulates S-phase progression and mitosis, as MALAT1-depleted human primary cells show defects in G1 to S and G2 to M transition. Genome wide transcriptome analyses indicate that MALAT1 regulates the expression of several E2F-target genes, including the transcription and pre-mRNA processing of key oncogenic transcription factors that play crucial roles in cell cycle progression. Significant population of MALAT1-depleted cells undergoes senescence, with characteristic β-gal positive labeling and increased expression of senescence-associated genes. Our results indicate that MALAT1 acts like a “molecular sponge” and titrates the functional level of splicing proteins during cell cycle. This in turn influences the alternative splicing of specific premRNAs involved in cell cycle progression.

Session 2A: Non-coding RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

21

LincRNA-p21 Suppresses Target mRNA translation

Je-Hyun Yoon1, Kotb Abdelmohsen1, Subramanya Srikantan1, Xiaoling Yang1, Jennifer Martindale1, Supriyo De1, Maite Huarte2, Ming Zhan3, Kevin Becker1, Myriam Gorospe1 1 National Institute on Aging-Intramural Research Program, NIH, Baltimore, MD, US, 2Department of Oncology, CIMA, University of Navarra, 31008 Pamplona, Spain, 3Department of Systems Medicine and Bioengineering, The Methodist Hospital Research Institute, Houston, TX, US Mammalian long intergenic noncoding (linc)RNAs are best known for modulating transcription. We have identified a post-transcriptional function for lincRNA-p21 as a modulator of translation. Association of the RNA-binding protein HuR with lincRNA-p21 favored the recruitment of let-7/Ago2 to lincRNAp21, leading to lower lincRNA-p21 stability. Under reduced HuR levels, lincRNA-p21 accumulated, increasing its association with JunB and β-catenin mRNAs and selectively lowering their translation. With elevated HuR, lincRNA-p21 levels declined, which in turn derepressed JunB and β-catenin translation and increased the levels of the encoded proteins. We propose that HuR controls translation of a subset of target mRNAs by influencing the levels of lincRNA-p21. Our findings uncover a role for lincRNA as a posttranscriptional inhibitor of translation.

22 Architecture of Regulatory Long Non-coding RNAs Associated with Nuclear Receptor Biology

Irina Novikova, Scott Hennelly, Karissa Sanbonmatsu Los Alamos National Lab, Los Alamos, NM, USA While functional roles of some non-coding RNAs (lncRNAs) have been determined, the molecular mechanisms are not understood (1). Three basic mechanistic questions have yet to be answered: 1) Are lncRNAs highly structured or disordered? 2) Do they contain globular sub-domains or are they organized linearly in chains of stem-loops? 3) Does the RNA exist as an intermixed RNA–protein complex, or, is the structure dominated by RNA? So far, no biochemical characterization of their structures has been performed. Here, we report the first experimentally derived secondary structure of a human lncRNA, the steroid receptor RNA activator (SRA). The SRA RNA was one of the first human long RNAs discovered (0.87kB in length) and proven to act as noncoding transcript. It co-activates several human sex hormone receptors and directly binds a variety of proteins, suggesting the possible formation of a multicomponent RNA–protein complex. Our extensive experimental findings (SHAPE, in-line, DMS and RNase V1 probing) reveal that this lncRNA carries a complex structural organization, consisting of four domains, with a variety of unique secondary structure elements (2). Interestingly, alternatively spliced coding isoforms of SRA are also expressed to produce proteins, making the SRA gene a unique bifunctional system. We find that the coding isoform of SRA has a secondary structure similar to that of the noncoding form, with the exception of one subregion, suggesting that it may still function in a similar fashion to the noncoding transcript. Thus, in evolution, the gene needs to maintain not only the RNA structure, but also the amino acid reading frame. We also assess the co-evolution of the SRA gene at both the RNA structure and protein structure levels using comparative sequence analysis across vertebrates. We observe that the sub-domains evolve with different evolutionary rates. Rapid evolutionary stabilization of RNA structure, combined with frame-disrupting mutations in conserved regions, dominates the gene. We also report analogous structural investigations for other newly-discovered long noncoding RNAs associated with nuclear receptors. (1) Wapinski, O. and Chang, H.Y. (2011) Long noncoding RNAs and human disease. Trends Cell Biol., 21, 354–361. (2) Novikova, I.V., Hennelly, S.P. and Sanbonmatsu, K.Y. (2012) Structural architecture of the human long noncoding RNA, steroid receptor RNA activator. NAR, in press (Epub ahead of print is currently online). Session 2A: Non-coding RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

23

Telomerase RNA Biogenesis Involves Sequential Binding By Sm and Lsm Complexes

Wen Tang, Peter Baumann Stowers Institute for Medical Research, Kansas City, Kansas, USA In most eukaryotes, the progressive loss of chromosome-terminal DNA sequences is counteracted by the enzyme telomerase, a reverse transcriptase that uses part of an RNA subunit as template to synthesize telomeric repeats. Many cancer cells express high telomerase activity and mutations in telomerase subunits are associated with degenerative syndromes including dyskeratosis congenita and aplastic anaemia. The therapeutic value of altering telomerase activity thus provides ample impetus to study the biogenesis and regulation of this enzyme in human cells and model systems. We have previously identified a precursor of the fission yeast telomerase RNA subunit (TER1) and have demonstrated that the mature 3’ end is generated by the spliceosome in a single cleavage reaction akin to the first step of splicing. Directly upstream and partially overlapping with the spliceosomal cleavage site is a putative Sm protein binding site. Sm and Like-Sm (LSm) proteins belong to an ancient family of RNA binding proteins represented in all three domains of life. Members of this family form ring complexes on specific sets of target RNAs and play critical roles in their biogenesis, function and turnover. We now demonstrate that the canonical Sm ring and the Lsm2-8 complex sequentially associate with fission yeast TER1. The Sm ring binds to the TER1 precursor, stimulates spliceosomal cleavage and promotes the hypermethylation of the 5’ cap by Tgs1. Sm proteins are then replaced by the Lsm2-8 complex, which promotes the association with the catalytic subunit and protects the mature 3’ end of TER1 from exonucleolytic degradation. Our findings define the sequence of events that occur during telomerase biogenesis and characterize roles for Sm and Lsm complexes as well as for the methylase Tgs1.

24 Saccharomyces cerevisiae Telomerase Activity is Exquisitely Sensitive to Subtle Perturbations of the TLC1 Pseudoknot 3’ Stem

Fei Liu, Carla Theimer University at Albany, SUNY Functional characterization of the pseudoknot in the S. cerevisiae telomerase RNA (TLC1) demonstrated that tertiary structural interactions occur between loop 1 uridines and stem 2 Watson-Crick A-U pairs (1), as previously observed for the K. lactis and human telomerase RNA pseudoknots. In the same study, the contributions of backbone ribose groups in the pseudoknot to telomerase catalysis were investigated using 2’-OH (ribose) to 2’-H (deoxyribose) substitutions at several positions in the stem 2 helix , and it was proposed that one or more 2’-OH groups from the stem 2 sequences at or near the triple-helix participate in telomerase catalysis. More recently, 2’-O methylation sites were introduced into specific nucleotides within the same region and the 2’-O methylation of single backbone ribose sugars within the triple-helix region showed either increased or no effect on the in vivo and in vitro telomerase activity, while a single substitution adjacent to the triple helix resulted in substantial reduction of telomerase activity (2). From these studies it was proposed that, in the case of TLC1, 2’-OH substitutions in the triple helix cause changes in telomerase activity which correlate with the affinity of the substituted group for the C3’-endo sugar pucker and that the 2’-OH at position U809 contributes to telomerase activity. Based on investigations of the structural and thermodynamic properties of the TLC1 pseudoknot region, which provided a more detailed description of the secondary structure of the stem 2 helical region (3), including an additional base paired sequence, we examined the structural and thermodynamic perturbations of the 2’-O methyl and 2’-H substituted pseudoknots using UV-monitored thermal denaturation experiments, native gel electrophoresis, CD spectroscopy, and Nuclear Magnetic Resonance spectroscopy. These results demonstrate a correlation between A-form RNA geometry, thermodynamic stability, and telomerase activity in the triple helix substitutions and are consistent with the identification of the U809 2’-OH as a contributor to telomerase activity. (1) Qiao F, Cech TR. 2008. Triple-helix structure in telomerase RNA contributes to catalysis. Nat Struct Mol Biol 15: 634-640. (2) Huang C, Yu Y-T. 2010. Targeted 2’-O methylation at a nucleotide within the pseudoknot of telomerase RNA reduces telomerase activity in vivo. Mol Cell Biol 30:4368-4378. (3) Liu, F., Kim Y., Cruickshank, C., Theimer, C.A. 2012. Thermodynamic characterization of the Saccharomyces cerevisiae telomerase RNA pseudoknot domain in vitro. RNA in press. Session 2A: Non-coding RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

25

The Microprocessor complex controls the activity of mammalian LINE-1 retrotransposons

Sara Macias1, Sara Heras2, Mireya Plass3, Eduardo Eyras3, Jose Garcia-Perez2, Javier Caceres1 1 MRC Human Genetics Unit,Institute of Genetics and Molecular Medicine, University of Edinburgh , UK, 2 GENYO (Centre Pfizer-University of Granada-Junta de Andalucia of Genomics and Oncology); Granada, Spain, 3GRIB, Universitat Pompeu Fabra, Barcelona, Spain Long INterspersed Element-1 (LINE-1 or L1) retrotransposons comprise approximately 20% of the mammalian genome and ongoing LINE-1 retrotransposition events are a driving force in genetic diversity. However, little is known about how the host regulates the activity of LINE-1 elements. Although most L1s are retrotransposition defective, there are approximately 100 full-length Retrotransposition Competent L1s (RC-L1s) in an average human diploid genome. The activity of RC-L1s impacts the human genome in a myriad of ways, and the host actively represses their ongoing mobilization, as a defense mechanism. The Drosha-DGCR8 complex (Microprocessor) has a well characterized role in microRNA (miRNA) biogenesis. In order to identify RNA targets of the Microprocessor complex, we used high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) of DGCR8 in human 293T cells. Here, we show that the Microprocessor complex (Drosha/DGCR8), which is responsible for the generation of microRNAs, also recognizes the transcripts derived from several human active retrotransposons (LINE-1, Alu and SVA). We observed that CLIP reads distributed along the consensus sequence of a RC-L1, with main peaks located at the 5’end of the 6 Kb transcript, which is predicted to form a stable secondary structure. Expression analyses demonstrate that cells lacking a functional Microprocessor accumulate LINE-1 mRNA and the L1-encoded ORF1 proteins in both human and mouse cells. In addition, we show that structured regions of the LINE-1 mRNA can be cleaved in vitro by Drosha/DGCR8. Finally, we used a cell culture-based L1 retrotransposition assay to show that the Microprocessor negatively regulates L1 retrotranposition in vivo. Interestingly, this effect was partially abolished when the 5’UTR was removed from the engineered human L1. Altogether, these data reveal a new role for the Microprocessor as a post-transcriptional repressor of LINE-1 retrotransposition acting as a defender of human genome integrity against endogenous retrotransposons.

26 A Non-Coding RNA Produced by Arthropod-Borne Flaviviruses Inhibits the Cellular Exoribonuclease Xrn1 and Alters Host mRNA Stability

Stephanie Moon1, John Anderson1, Carol Wilusz1, Alexander Khromykh2, Jeffrey Wilusz1 Colorado State University, Fort Collins, CO, USA, 2The University of Queensland, Brisbane, Australia All arthropod-borne flaviviruses generate a non-coding RNA encompassing the viral 3’ untranslated region (UTR) that accumulates in the cell over the course of the infection. This short flavivirus RNA (sfRNA) is a unique decay product resulting from incomplete 5’ to 3’ exonucleolytic decay of the viral genome by the cellular enzyme Xrn1. The flavivirus 3’ UTR contains several structural elements that stall Xrn1 both in living cells and reconstituted systems. We hypothesized that formation of sfRNA by the stalling of Xrn1 might also repress the exonuclease as a non-competitive inhibitor. Using cytoplasmic extracts from human and mosquito cells as well as purified Xrn1 protein, we found that decay of a reporter RNA by Xrn1 was inhibited upon formation of sfRNA. Furthermore, several lines of evidence indicate that Xrn1 activity is also repressed during a flavivirus infection. Cells infected with Dengue virus or Kunjin virus were found to accumulate significant amounts of uncapped mRNAs, an intermediate in the 5’-to-3’ mRNA decay pathway that is normally rapidly degraded by Xrn1. Since Xrn1 repression would debilitate a major pathway of mRNA decay in cells, we next analyzed the relative stability of cellular mRNAs during flaviviral infections. Experiments to determine mRNA half-lives demonstrated that cellular mRNAs had increased stability in both Kunjin and Dengue virus infected 293T cells compared to uninfected cells. Importantly, a mutant Kunjin virus that cannot form sfRNA but replicates to normal levels in 293T cells failed to have any effect on host mRNA stability. Finally, we demonstrated that sfRNA formation was directly responsible for the stabilization of cellular mRNAs using a reporter construct containing the Dengue virus 3’ UTR in the absence of viral infection. Therefore we conclude that arthropod-borne flaviviruses incapacitate the host RNA decay factor Xrn1 during infection and dysregulate host mRNA stability as a result of sfRNA formation.

1

Session 2A: Non-coding RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

27 Conservation of a Triple-Helix Forming RNA Stability Element in Noncoding and Genomic RNAs of Diverse Viruses

Kazimierz Tycowski, Mei-Di Shu, Sumit Borah, Mary Shi, Joan Steitz Yale University, New Haven, CT, USA The long noncoding (lnc) PAN (polyadenylated nuclear) RNA from the human oncogenic gammaherpesvirus KSHV is required for the production of late viral proteins and thus infectious viral particles (1). Abundant expression of PAN RNA depends on a cis-element called the ENE (2). The ENE upregulates PAN RNA by inhibiting its rapid nuclear decay through triple helix formation with the poly(A) tail (3,4). Using structure-based bioinformatics, we identified six novel ENE-like elements in evolutionarily diverse viral genomes. Five are in double-stranded DNA viruses, including mammalian herpesviruses, insect polydnaviruses and a protist mimivirus. One is in an insect picorna-like positive-strand RNA virus, suggesting that the ENE can counteract cytoplasmic as well as nuclear RNA decay pathways. All ENE elements exhibit common features, not included in our bioinformatics selection, within the ENE structure itself. Moreover, all ENEs are located near either a canonical polyadenylation signal (AAUAAA) or a genomically-encoded poly(A) tract, indicating that all are functional RNA stability elements. Indeed, we demonstrated functionality of four of the new ENEs by increased accumulation of an intronless reporter transcript in human cells. Identification of these ENEs enabled the discovery of PAN RNA homologs in two additional gammaherpesviruses, RRV and EHV2, suggesting that PAN RNAs are widely expressed among gammaherpesviruses. Our findings demonstrate that search for structural elements can lead to rapid identification of lncRNAs, even in the absence of primary sequence conservation. 1. Borah, S., Darricarrere, N., Darnell, A., Myoung, J. & Steitz, J.A. (2011) PLoS Pathog. 7, e1002300. 2. Conrad, N.K. & Steitz, J.A. (2005) EMBO J. 24, 1831-1841. 3. Conrad, N.K., Mili, S., Marshall, E.L., Shu, M.D. & Steitz, J.A. (2006) Mol. Cell 24, 943-953. 4. Mitton-Fry, R.M., DeGregorio, S.J., Wang, J., Steitz, T.A. & Steitz, J.A. (2010) Science 330, 1244-1247.

28

Some insights into the molecular mechanics of the ribosome

Harry Noller1, Jie Zhou1, Laura Lancaster1, Dmitri Ermolenko2, Andrei Korostelev3, John Paul Donohue1 1 Center for Molecular Biology of RNA, University of California at Santa Cruz, Santa Cruz, (CA), USA, 2 Departments of Biochemistry and Biophysics, Univ. of Rochester Medical Center, Rochester, (NY), USA, 3 Departments of Biochemistry and Molecular Pharmacology, Univ. of Massachusetts Medical School, Worcester, (MA), USA In spite of much progress over more than five decades of research, many of the most fundamental questions concerning the molecular mechanism of protein synthesis remain unanswered. One such challenge is to understand the molecular basis of translocation, the EF-G-catalyzed, coupled movement of mRNA and tRNA through the ribosome. Translocation requires rapid and precise large-scale molecular movements (on the order of 20 to 70 Å) of tRNA from the A to P to E sites of the ribosome, which must be carried out while maintaining correct codon-anticodon pairing with the mRNA. It has long been supposed that this movement is coupled to dynamic changes in ribosome structure. The first steps were the discoveries of the involvement of hybrid-states intermediates and intersubunit rotation in the translocation process. More recently, structural and FRET studies in our lab and elsewhere have begun to suggest how tRNA movement is coupled to the molecular mechanics of the ribosome. Time-resolved FRET experiments allow direct observation of intersubunit movement in solution, in which rapid counterclockwise rotation of the 30S subunit from its classical state into its hybrid (rotated) state is followed by a slower, clockwise reversal of this motion. Parallel fluorescence-quenching experiments show that translocation of mRNA occurs during the second, clockwise rotational step. One puzzle is that, while intersubunit rotation kinetics can be fit to a single exponential, movement of mRNA is biphasic. Cate and co-workers have pointed out that a constriction between the head and platform of the 30S subunit creates a steric barrier to movement of tRNA between the P and E sites, and that this could be alleviated by movement of the head. Cryo-EM studies by Spahn and co-workers have identified a sub-population of EF-G-bound ribosomes in which such a rotation of the 30S head was observed. In the absence of an x-ray structure of EF-G bound to a rotated state of the ribosome, we solved the 3.3 Å crystal structure of the structurally-related release factor RF3 trapped in its GTP state bound to a rotated ribosome. In this complex, the body of the 30S subunit is rotated by 7° and the 30S head by 14°. These combined rotational movements result in displacement of elements of the P-tRNA binding site by 23 Å, corresponding almost exactly to the distance moved by P-tRNA in the 30S subunit during its translocation to the E site. If indeed rotational movement of the head is involved in authentic EF-G-catalyzed translocation, it may help to explain the biphasic behavior of mRNA movement. Session 2A: Non-coding RNAs & Session 2B: Keynote

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

29

Nucleating 30S ribosome assembly through protein-RNA induced fit

30

Real-Time Assembly Landscape of the Translation Initiation Machinery

Sanjaya Abeysirigunawardena1, Hajin Kim2,3, Magan Mayerle1, Taekjip Ha3, Sarah Woodson1 1 Johns Hopkins University, Baltimore, (Maryland), U.S.A., 2University of Illinois at Urbana-Champaign, (Illinois), USA, 3University of Illinois at Urbana-Champaign, Urbana, (Illinois), USA Binding of ribosomal proteins with the rRNA has been shown to stabilize productive assembly intermediates that lead to the formation of active ribosomes. S4 is an essential primary assembly protein that binds to the 5’-domain of the 16S rRNA and helps nucleate 30S assembly. Recent hydroxyl radical footprinting studies of the 16S 5’ domain showed that binding of primary assembly proteins generates two assembly intermediates, which can be distinguished by the orientation of 16S helix 3 with respect to helix 4. To follow the motion of helix 3 in real-time using FRET, the 5’-domain RNA and S4 protein were labeled with fluorescent dyes. Equilibrium single-molecule FRET (smFRET) measurements at high MgCl2 concentration revealed three stable conformations of the S4-RNA complex: a partially docked helix 3, an intermediate in which helix 3 is flipped away from S4, and a native-like complex with helix 3 stably docked under the base of helix 18. Real-time S4 binding experiments suggest an induced-fit mechanism in which S4 preferentially binds to RNA with partially docked helix 3 and progresses to a native-like structure through the flipped intermediate. Bulk fluorescence measurements indicate these rearrangements occur after very fast initial binding of S4 to the RNA. Proteins S17, S16 and S20 that bind to the core region of the 16S 5’-domain influence assembly by biasing the population of S4 complexes toward certain assembly intermediates. Equilibrium titrations show that protein S16 favors the nativelike S4-RNA complex due to its ability to stabilize stacking interactions of helices 15 and 17. Surprisingly, protein S17 stabilizes the flipped intermediate, in agreement with hydroxyl radical footprinting experiments. The results illustrate how individual proteins drive assembly by biasing dynamic fluctuations of the rRNA.

Pohl Milon, Riccardo Belardinelli, Cristina Maracci, Akanksha Goyal, Irena Andreeva, Liudmila Filonava, Marina Rodnina Max Planck Institute for Biophysical Chemistry, Goettingen, Germany Translation initiation is a crucial step of protein synthesis which largely defines how the cellular composition of the transcriptome is converted to the proteome and controls the response and adaptation to environmental stimuli. In bacteria, translation regulation can account for differences in gene expression ranging over three orders of magnitude among genes. Although diverse mechanisms of mRNA loading to the ribosome have being characterized, the timing of events during assembly of translation initiation complexes and their compositional dynamics are not known. Here, we use pre-steady state analysis to study the real-time mechanism of 30S pre-initiation complex (PIC) assembly, its transition to the 30S and 70S initiation complexes (IC) and the co-translational loading of following ribosomes onto the same mRNA. Our results suggest that although the components of the initiation machinery can bind to the 30S ribosome stochastically, there exists a kinetically favored pathway: IF3 --> IF2 --> IF1 --> fMet-tRNAfMet. mRNA can bind at any time with velocities largely depending on the secondary structure of the translation initiation region (TIR). The 30S PIC rearranges to the 30S IC upon start codon recognition, resulting in the binding stabilization of IF1, IF2, mRNA and fMet-tRNAfMet while IF3 is destabilized. Subsequently, the 30S IC is rapidly joined by the large ribosomal subunit, triggering a non-linear array of reactions which ultimately leads to the elongation-capable 70S IC. Our data indicate that regulation of mRNA selection is achieved at several checkpoints, from initial docking of structured mRNAs to the 30S PIC to kinetic partitioning during 70S IC formation. Furthermore, our results propose mechanisms by which the leading 70S IC influences the loading of subsequent ribosomes onto the same mRNA, further shaping the protein outcome by regulating the number of ribosomes per polysome.

Session 2B: Ribosomes & translation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

31

Joining of 60S subunits and a translation-like cycle in 40S ribosome maturation

32

Evolutionary Divergence of Translation Quality Control Mechanisms During tRNA Charging

Bethany Strunk1, Megan Novak2, Crystal Young1, Katrin Karbstein1 1 Scripps Florida, Jupiter, (Florida), USA, 2Furman University, Greenville, (South Carolina), USA Assembly factors prevent premature translation initiation on small (40S) ribosomal subunit assembly intermediates by blocking ligand binding. However, it is unclear how assembly factors are displaced from maturing 40S ribosomes, if or how maturing subunits are assessed for fidelity, and what prevents premature translation initiation once assembly factors dissociate. We show that maturation involves a translation-like cycle whereby the translation factor eIF5B promotes joining of large (60S) subunits with pre-40S subunits to give 80S-like complexes, which are subsequently disassembled by the termination factor Rli1. The assembly factors Tsr1 and Rio2 block the mRNA channel and initiator tRNA binding sites and thus 80S-like complexes lack mRNA or initiator tRNA. After Tsr1 and Rio2 dissociate from 80S-like complexes Rli1-directed displacement of 60S subunits allows for translation initiation. This cycle thus provides a functional test of 60S subunit binding and the GTPase site before ribosomes enter the translating pool, and establishes a new paradigm for understanding ribosome maturation.

Srujana Yadavalli, Noah Reynolds, Michael Ibba Ohio State University, Columbus, Ohio, USA Mistranslation can potentially follow two events during protein synthesis: production of non-cognate amino acid:tRNA pairs by aminoacyl-tRNA synthetases (aaRSs) and inaccurate selection of aminoacyl-tRNAs (aa-tRNAs) by the ribosome. Many aaRSs actively edit non-cognate amino acids, but editing mechanisms are not evolutionarily conserved and their physiological significance remains unclear. To address the connection between aaRSs and mistranslation, the evolutionary divergence of tyrosine editing by phenylalanyl-tRNA synthetase (PheRS) was used as a model. Certain PheRSs are naturally error-prone, most notably a Mycoplasma example that displayed a low level of specificity consistent with elevated mistranslation of the proteome. Mycoplasma PheRS was found to lack canonical editing activity, relying instead on discrimination against the non-cognate amino acid transition state for quality control. This mechanism of discrimination is inadequate for organisms where translation is more accurate, as Mycoplasma PheRS failed to support Escherichia coli growth. However, minor changes in the defunct editing domain of the Mycoplasma enzyme were sufficient to restore E. coli growth, indicating that translational accuracy is an evolutionarily adaptable trait. These findings indicate a mechanism by which aaRSs facilitate adaption to changes in cellular physiology by altering the accuracy of translational of certain codons, which may prove advantageous for growth under different environmental conditions.

Session 2B: Ribosomes & translation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

33

EF-Tu Dynamics During Pretranslocation (PRE) Complex Formation

34

Structural Studies of Streptomycin Resistant and Dependent Ribosomes

Wei Liu1, Chunlai Chen2, Darius Kavaliauskas1,2,5, Jared Schrader3,4, Olke Uhlenbeck3, Charlotte Knudsen5, Yale Goldman2, Barry Cooperman1 1 Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA, 2Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, PA, USA, 3Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA, 4Current address, School of Medicine, Stanford University, Stanford, CA, USA, 5 Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark During the first elongation cycle of protein synthesis, aminoacyl-tRNA (aa-tRNA) having an anticodon cognate to the codon triplet at the ribosome decoding center binds to the 70S initiation complex (70SIC) via a ternary complex (TC), consisting of aa-tRNA.EF-Tu.GTP. Such binding places the aa-tRNA in the A/T site of the ribosome. aa-tRNA is next accommodated into the A-site, with concomitant GTP hydrolysis, rapid peptide bond formation and dissociation of EF-Tu·GDP and Pi from the ribosome, resulting in PRE complex formation. The initial binding of cognate TC to the ribosome in the A/T state places EF-Tu in close proximity to ribosomal protein L11, while maintaining EF-Tu interaction with aa-tRNA. Fluorescent labels placed on L11, EF-Tu and aa-tRNA form an equilateral triangle within the ribosomal complex formed prior to accommodation, with distances between each other that are appropriate for fluorescence energy transfer studies (FRET). Here we combine time-dependent ensemble and single-molecule FRET measurements between these labels on rapid mixing of 70SIC with TC with parallel ensemble studies measuring the rates of Pi release and of accommodation. Our results indicate that for wt-tRNA, Pi release, EF-Tu separation from L11 and aa-tRNA accommodation all proceed prior to EF-Tu release from aa-tRNA and the ribosome. Furthermore, EF-Tu release can occur prior to the EF-Tu conformational change that is a consequence of GTP hydrolysis, or either concomitant with or following such conformational change. The rates of both EF-Tu separation from L11 and aa-tRNA accommodation can be fine-tuned by modifying the EF-Tu:aa-tRNA interface via mutation of tRNA, providing direct support for an earlier suggestion that the tRNA affinity towards EF-Tu determines the rate of aa-tRNA accommodation. These results form an important part of our overall effort to obtain a detailed understanding of the dynamics of EF-Tu participation in tRNA selection and accommodation during PRE complex formation.

Hasan Demirci1, Steven Gregory1, Frank Murphy2, Gerwald Jogl1, Albert Dahlberg1 1 Brown University, Providence, (RI), USA, 2Argonne National Laboratory, Argonne, (IL), USA The 30S subunit undergoes a global conformational shift from an open to a closed form upon codon recognition. Our recent structure of wt-30S subunits bound to the drug streptomycin reveals that streptomycin affects the equilibrium of this transition, inducing an alternate state that differs from that induced by cognate codon recognition. We have recently obtained crystal structures of five different streptomycin-resistant mutant 30S ribosomal subunits. The base substitutions map in the central pseudoknot of 16S rRNA, which forms part of the streptomycin binding pocket. Our data reveal that mutations at the central pseudoknot perturb the conformation of the neighboring structure and rearrange the position of the backbone directly contacting streptomycin. The precise mechanism of streptomycin dependence is not well understood. In order to explore the structural effects of dependence phenotypes, we determined the crystal structures of 30S subunits bearing a streptomycin dependence mutation in the helix 18 pseudoknot of 16S rRNA. This pseudoknot stabilizes the position of G530, a residue central to codon recognition. The G524U mutant subunits crystallize in a novel monoclinic form that diffracts to beyond 4.5 angstrom resolution. We further investigated the streptomycin dependence caused by ribosomal protein S12 mutations. We determined the structures of two additional streptomycin dependent mutants, P90W and P90L, in a new crystal form that is different from both the wild type and G524U mutant. These new crystal forms diffracted beyond 3.5 angstrom resolution and showed remarkable rearrangement of the decoding site. Strikingly, co-crystallization of G524U, P90W and P90L mutant subunits with streptomycin produces the tetragonal crystal form in space group P41212 previously observed with wild-type subunits. Thus, the crystallization behavior of these mutants mirrors their phenotype. In summary, streptomycin dependent 30S subunits exhibit a unique conformational deformation in the decoding site and also explain why these mutant ribosomes are non-functional in the absence of streptomycin.

Session 2B: Ribosomes & translation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

35

Role of Inter-subunit Bridges in Ribosomal Translocation

Qi Liu, Kurt Fredrick The Ohio State University, Columbus, OH, United States During each elongation cycle of translation, the ribosomal complex moves 3 nucleotides along the mRNA, a process termed translocation and catalyzed by Elongation Factor-G (EF-G). Work from Wintermeyer and coworkers has shown that the rate of translocation is limited by a conformational change of the ribosome, termed unlocking. Despite its importance in protein synthesis, the nature of the unlocked state and the molecular mechanism of its formation remain unknown. Previous structural and biochemical studies suggest that interactions between the subunits may be altered during unlocking. To systematically investigate the roles of individual inter-subunit bridges in translocation, we targeted most bridges by mutagenesis and characterized the effects on both EF-G-catalyzed forward and spontaneous reverse translocation. Mutations that disrupt bridge B1a and B4 showed significant acceleration of both forward and reverse translocation. These bridges are predicted to constrain 30S head swiveling and inter-subunit rotation respectively. These data provide evidence that both 30S head swiveling and inter-subunit rotation are part of the rate-limiting step of translocation.

36 Single Molecule Visualization of Stalled Ribosomes: insight into the mechanism of ribosomal frameshifting

Peiwu Qin1, Dongmei Yu2, Xiaobing Zou3, Peter Cornish1,2 Department of Biochemistry, University of Missouri, Columbia, MO, USA, 2Department of Biological Engineering, University of Missouri, Columbia, MO, USA, 3X-ray Science Division, Argonne National Laboratory, Argonne, IL, USA During protein synthesis, mRNA and tRNAs are moved through the ribosome by the process of translocation. The small diameter of the mRNA entrance tunnel only permits linear mRNA to pass through. However, there are structured elements within mRNA that present a barrier for translocation that must be unwound. The ribosome has been shown to unwind RNA in the absence of additional factors, but the mechanism remains unclear. Here, we show using single molecule Förster resonance energy transfer and small angle X-ray scattering experiments a novel global conformational state of the ribosome in the presence of the RNA structures. RNA mutants have been created to further confirm the new conformational state. This previously unobserved conformational state provides structural insight into the helicase activity of the ribosome and has important implications for understanding the mechanism of ribosomal frameshifting.

1

Session 2B: Ribosomes & translation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

37 HIV-1 Frameshift Efficiency is Determined Solely by the Stability of Base Pairs at the mRNA Entrance Tunnel of the Ribosome

Kathryn Mouzakis, Andrew Lang, Kirk Vander Meulen, Samuel Butcher University of Wisconsin Madison, Madison, WI, USA A -1 programmed ribosomal frameshift (PRF) in human immunodeficiency virus type 1 (HIV-1) is required for translation of its enzymatic proteins. The efficiency of the frameshift (FS) event determines the ratio of structural to enzymatic proteins produced. The FS site consists of two cis-acting RNA elements: a heptanucleotide slippery sequence (UUUUUUA) followed by a downstream RNA structure. Despite extensive characterization of the HIV-1 FS site, the mechanism of FS stimulation is unclear. FS efficiency may be influenced by multiple factors, including RNA thermodynamic and mechanical stability and potential structural interactions with the translational machinery. We have investigated the role of the HIV-1 RNA structure and thermodynamics in frameshifting using mutagenesis and in vitro frameshift assays. First, we analyzed frameshifting efficiency of the genomic HIV frameshift site structure that forms into a three-helix junction secondary structure (1), and compared this efficiency to frameshifting induced by a construct containing only the stable downstream stem-loop (2). We found no significant difference between the two, and conclude that the minimal stemloop is sufficient to promote efficient frameshifting. Next, a set of mutant stem-loops were designed to specifically dissect the determinants of FS efficiency within the stem-loop. Contrary to previously published studies (3), we find no correlation between frameshift efficiency and overall stem-loop thermodynamic stability. Instead, there is an extremely strong correlation (R2 = 0.96) between FS efficiency and the stability of the 3 base pairs at the stem-loop base. Furthermore, changes in linker length between the slippery site and the stem-loop alter FS efficiency in a predictable manner that correlates with the stability of base pairs exactly 9 nt from the slippery site. When considering the direction of force required for translocation (4), these data are consistent with a model in which frameshifting is determined simply by the mechanical stability of 3 base pairs encountered at the mRNA entrance tunnel during frameshifting. Once translation has progressed beyond these 3 base pairs, the register of the reading frame is set; therefore, the stability of other base pairs in the stem-loop has little effect on overall frameshift efficiency. 1. J. M. Watts et al. 2009. Nature, 460, 711; 2. D. W. Staple, S.E. Butcher. 2005. J. Mol. Biol. 349, 1011; 3. L. Bidou et al., 1997 RNA 3, 1153; 4. X. Qu et al. 2011. Nature, 475, 118

38 ‘Late’ Translation Arrest: An Unconventional Mode of Inhibition of Protein Synthesis by Macrolides

Krishna Kannan, Nora Vazquez-Laslop, Alexander Mankin University of Illinois at Chicago, Chicago, Illinois, USA Macrolide antibiotics, like erythromycin, prevent translation by binding in the ribosomal exit tunnel and obstructing the egress of nascent polypeptides. It is assumed that the synthesis of all bacterial proteins is blocked by macrolides. In contrast to this long-standing paradigm, we found that macrolides differentially affect the synthesis of polypeptides: while many proteins are inhibited, a subset of proteins is actively synthesized in the presence of the drugs. Some ‘resistant’ proteins are synthesized by the antibiotic-bound ribosome: the N-terminal sequence of such proteins allows the nascent peptide to thread through the exit tunnel obstructed by the bound macrolide molecule. Depending on the amino acid sequence, the synthesis of the protein can be successfully completed, or it can be arrested at later stages. Specific nascent peptide sequences can lead to a ‘late’ translation arrest through the formation of stalled ribosome complex carrying bound antibiotic and a long nascent peptide. Both new phenomena, the nascent peptide threading through the antibioticobstructed tunnel and the late translation arrest depend on the sequence of the nascent peptide and the structure of the antibiotic bound in the tunnel. These findings open new ways for the development of superior antibiotics and regulating translation by small molecules bound in the exit tunnel of the ribosome.

Session 2B: Ribosomes & translation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

39

Characterization of the Single-stranded RNA Dynamical Ensemble

40

Pri-miR-17-92a Transcript Folds into a Tertiary Structure and Autoregulates its Processing

Jun Feng, Katie Eichhorn, Hashim Al-Hashimi, Charles Brooks III University of Michigan, Ann Arbor, MI, USA Single-stranded RNAs (ssRNAs) are ubiquitous RNA elements that serve diverse functional roles. However, little is known about the structural and dynamical information of ssRNAs. Molecular dynamics (MD) simulations offer an invaluable tool that is able to characterize the molecular structure at the atomic detail. With the joint approach from nuclear magnetic resonance (NMR), we have previously studied the solution dynamics of the 12-nt ssRNA derived from the prequeuosine riboswitch and unveiled structural complicity in such a small ssRNA. While MD simulation is able to capture the key features of the ssRNA observed by NMR, a few discrepancies remain, particularly regarding the dynamical information provided by residual dipolar couplings (RDCs). The free tumbling of ssRNA in solution prevents us from decoupling the internal and overall motions in order to quantitatively access the structure ensemble generated by the MD simulations. Therefore, in this study we use domain elongation strategy to anchor the ssRNA on RNA double strands that predominantly define the molecular orientation. Detailed comparisons are made between the NMR and MD reported dynamics of the elongated ssRNA. We also apply an ensemble selection method which utilizes a Monte Carlo simulated annealing procedure to select the conformers from the MD trajectory pool. Based on the RDCs, a ssRNA dynamical ensemble is constructed and examined to further uncover the solution dynamics and structural characteristics of the ssRNA. This approach generally applies to the conformational selection of proteins and RNAs.

Saikat Chakraborty, Shabana Mehtab, Anand Patwardhan, Yamuna Krishnan National Centre for Biological Sciences MicroRNAs control gene expression by either by RNA transcript degradation or translational repression. Expressions of miRNAs are highly regulated in tissues, disruption of which leads to disease. But how this regulation is achieved and maintained is still largely unknown. MiRNAs that reside on clustered or polycistronic transcripts represent a more complex case where individual miRNAs from a cluster are processed with different efficiencies despite being co-transcribed. To shed light on the regulatory mechanisms that might be operating in these cases we considered the long polycistronic primary miRNA transcript pri-miR-17-92a that contains six miRNAs with diverse function (1). The six miRNA domains on this cluster are differentially processed to produce varying amounts of resultant mature miRNAs in different tissues (2,3). How this is achieved is not known. We show using various biochemical and biophysical methods coupled with mutational studies that pri-miR-17-92a adopts a specific three dimensional architecture which poses a kinetic barrier to its own processing (4). This tertiary structure could create suboptimal protein recognition sites on the pri-miRNA cluster due to higher order structure formation. References: 1. Mendell, J.T. 2008. miRiad roles for the miR-17-92a cluster in development and disease. Cell 133: 217-222. 2. Tang, G.Q., Maxwell, E.S. 2008. Xenopus microRNA genes are predominantly located within introns and are differentially expressed in adult frog tissues via post-transcriptional regulation. Genome Res. 18: 104-112. 3. Thomson, J.M., Newman, M., Parker, J.S., Morin-Kensicki, E.M., Wright, T., Hammond, S.M. 2006. Extensive post-transcriptional regulation of microRNAs and its implications for cancer. Genes Dev. 20: 2202-2207. 4. Chakraborty, S., Mehtab, S., Patwardhan, A. R., Krishnan, Y. 2012. Pri-miR-17-92a transcript folds into a tertiary structure and autoregulates its processing. RNA (in press)

Session 3A: RNA-protein architecture

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

41

Molecular Mimicry of Human tRNALys by HIV-1 RNA Genome Facilitates Viral Replication

42

Architectural Contributions of Genomic RNA to Retrotransposon Replication

Christopher Jones2, Jenan Saadatmand1, Erik Olson2,3, Jeremy Fichtenbaum2, Lawrence Kleiman1, Karin MusierForsyth2 1 Lady Davis Institute for Medical Research, McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada, 2Department of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University, Columbus, OH, 3Department of Chemistry, Carleton College, Northfield, MN A retrovirus, human immunodeficiency virus type 1 (HIV-1) has a single-stranded RNA genome that is reverse transcribed into cDNA during infection of target cells. The primer for initiating reverse transcription in HIV-1 is cellular tRNALys3, which is selectively packaged into HIV-1 through a specific interaction between the major tRNALys-binding protein, human lysyl-tRNA synthetase (hLysRS), and the viral proteins Gag and GagPol. Annealing of the tRNA primer onto the complementary 18-nt primer-binding site (PBS) in the ~9.4 kB viral RNA is mediated by the nucleocapsid domain of Gag. However, the mechanism by which tRNALys3 is targeted to the PBS and released from hLysRS prior to annealing is unknown. Here we show that hLysRS specifically binds to a tRNA-like element (TLE) in the 5’ untranslated region (UTR) of the HIV-1 genome, which mimics the anticodon loop of tRNALys and is located proximal to the PBS. Mutation of the U-rich sequence within the TLE attenuates binding of hLysRS in vitro and reduces the amount of annealed tRNALys3 in virions. The TLE determines the specificity of LysRS binding and is part of a larger LysRS binding domain in the viral RNA that includes elements of the psi packaging signal. Although our functional studies suggest that HIV-1 uses molecular mimicry of tRNALys to increase the efficiency of tRNALys3 annealing to viral RNA, the tertiary structure of this part of the HIV-1 genome is unknown. Using small-angle X-ray scattering, we have begun to study the tertiary structure of the 5’ UTR of the HIV-1 genome to determine if it indeed mimics the canonical tRNA fold. This research was supported by Ohio State University and by a fellowship to CPJ from the OSU Center for RNA Biology.

Katarzyna Purzycka1, Michal Legiewicz1, Emiko Matsuda2, Linda Eizentstat2, Sabrina Lusvarghi1, Qing Huang3, Jef Boeke3, David Garfinkel2, Stuart Le Grice1 1 RT Biochemistry Section, HIV Drug Resistance Program, NCI-Frederick, Maryland 21702, 2Department of Biochemistry and Molecular Biology, The University of Georgia, Athens, Georgia 30602, 3Department of Molecular Biology and Genetics and the High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Ty1 is a long terminal repeat (LTR)-containing retrotransposon of Saccharomyces cerevisiae. While this class of retroelements is structurally and evolutionarily related to vertebrate retroviruses, there are many important differences between the two elements. While both elements contain LTRs and replicate using a multifunctional reverse transcriptase, LTR-containing retrotransposons lack an envelope gene, which restricts them to an intracellular life cycle. Another differentiating aspect of LTR-retrotransposon replication is the structural diversity of minus-strand initiation complexes among and within retrotransposon families. In Ty1, for example, minus strand DNA synthesis initiates from a host derived tRNA that is partially hybridized to a bipartite primer binding site comprised of non-contiguous nucleotides, and the entire complex appears to be stabilized by long-range interactions between the 5’ and 3’ termini of the Ty1 genome. This structural organization is distinct from both retroviruses and other LTR retrotransposons, and is more complex than has previously been shown. In order to gain a deeper understanding of cis-acting elements in the Ty1 (+) RNA genome that control reverse transcription, we applied chemoenzymatic probing to viral RNA/tRNA complexes assembled in vitro as well as within purified Ty1 virus-like particles (VLPs). A combined structural, functional and genetic analysis allowed us to characterize previously unknown determinants of Ty1 replication, including extensive intra- and intermolecular tertiary interactions crucial for dimerization, protein binding and packaging. In addition, we investigated a unique mechanism of copy number control in which antisense Ty1 RNA is incorporated into VLPs (1). We found this mechanism particularly intriguing since, during the course of evolution, S.cerevisiae has lost conserved RNAi pathways for silencing endogenous transposons (2). The structural basis for antisense RNA-mediated copy number control will be presented. (1) E. Matsuda and D.J. Garfinkel, 2009, Posttrnaslational interference of Ty1 retrotransposition by antisense RNAs., PNAS 106(37) 15657-62 (2) I.A. Drinnenberg, G.R. Fink and D.P. Bartel, 2011, Compatibility with killer explains the rise of RNAi-deficient fungi., Science 333 1592 Session 3A: RNA-protein architecture

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

43 YB-1 Binds to CAUC Motifs and Stimulates Exon Inclusion by Enhancing the Recruitment of U2AF to Weak Polypyrimidine Tracts

Wenjuan Wei, Shirong Mu, Monika Heiner, Lijuan Cao, Jingyi Hui State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China The human Y box-binding protein 1 (YB-1) is a member of the evolutionarily conserved nucleic acid binding protein family, which exhibits multiple functions in DNA repair, transcription, splicing, mRNA stability, and translation. It is mainly localized in the cytoplasm, but is highly expressed in the nucleus of tumors, particularly in breast cancer cells. A number of studies indicated its role in malignant transformation. Recently, several lines of evidence indicate that YB-1 is a spliceosome-associated protein and is involved in alternative splicing, but the underlying mechanism has remained elusive. In this study, we defined both CAUC and CACC as high-affinity binding motifs for YB-1 by SELEX and demonstrated that these newly defined motifs function as splicing enhancers. Interestingly, on the endogenous CD44 gene, YB-1 appears to mediate a network interaction to activate exon v5 inclusion via multiple CAUC motifs in both the alternative exon and its upstream polypyrimidine tract. We provide evidence that YB-1 activates splicing by facilitating the recruitment of U2AF65 to weak polypyrimidine tracts through protein-protein interactions. Together, these findings suggest a vital role of YB-1 in activating a subset of weak 3’ splice sites in mammalian cells. Currently, we are undertaking a genome-wide search for specific RNA targets of YB-1 in cancer cells by RNA-Seq.

44 Crystal Structure of Cwc2 Reveals a Novel Architecture of a Multipartite RNA-binding Protein

Jana Schmitzova, Nicolas Rasche, Olexandr Dybkov, Katharina Kramer, Patrizia Fabrizio, Henning Urlaub, Reinhard Luehrmann, Vladimir Pena Max Planck Institute for Biophysical Chemistry, Goettingen, Germany The yeast splicing factor Cwc2 contacts several catalytically important RNA elements in the active spliceosome, suggesting that Cwc2 is involved in determining their spatial arrangement at the spliceosome’s catalytic center. We have determined the crystal structure of the Cwc2 functional core, revealing how a previously uncharacterized Torus domain, an RNA recognition motif (RRM) and a zinc-finger (ZnF) are tightly integrated in a compact folding unit. The ZnF plays a pivotal role in the architecture of the whole assembly. UV-induced crosslinking of Cwc2-U6snRNA allowed the identification by mass spectrometry of six RNA-contacting sites: four in or close to the RRM domain, one in the ZnF and one on a protruding element connecting the ZnF and RRM domains. The three distinct regions contacting RNA are connected by a contiguous and conserved positively-charged surface, suggesting an expanded interface for RNA accommodation. Cwc2 mutations confirmed that the connector element plays a crucial role in splicing. We conclude that Cwc2 acts as a multipartite RNA binding platform to bring RNA elements of the spliceosome’s catalytic centre into an active conformation.

Session 3A: RNA-protein architecture

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

45 The DEAD-box Protein Rok1 and Its Co-factor Rrp5 Catalyze Helix Formation During 40S Ribosome Assembly

Crystal Young, Katrin Karbstein The Scripps Research Institute, Jupiter, FL, USA DEAD-box proteins are involved in every aspect of RNA metabolism and are found in all kingdoms of life. While often referred to as RNA helicases, their described biochemical activities additionally include protein displacement from RNAs, ATP-dependent RNA binding and RNA annealing. In the simple eukaryote Saccharomyces cerevisiae, 25 DEAD-box proteins have been identified; their cellular roles include ribosome biogenesis, pre-mRNA splicing, mRNA export and decay and translation initiation. In agreement with DEAD-box proteins containing a highly conserved helicase core, in vitro analyses of DEAD-box proteins indicate that, in all cases except one, they lack substrate specificity. This finding, however, contradicts the observation that DEAD-box proteins have non-redundant functions and therefore high specificity in vivo. Consequently, it is believed that helicase co-factors may increase the specificity of DEAD-box proteins by specifically binding individual RNA sequences, thereby contributing to their unique and generally non-overlapping roles in various biological processes. Surprisingly, there is no strong experimental evidence for this simple hypothesis. Here, we show that Rok1, an assembly factor involved in 18S rRNA maturation, is a unique DEAD-box protein in that it preferentially binds double-stranded RNA over single-stranded RNA and has annealing but no unwinding activity. Excitingly, the presence of the C-terminus of Rok1’s binding partner Rrp5, an RNA-binding protein, greatly enhances this annealing activity in an RNA-sequence specific manner. Furthermore, the RNA duplex that is preferentially annealed is part of an inhibitory duplex in the pre-rRNA that serves to regulate the final cleavage step in 18S rRNA maturation. These biochemical results suggest that Rok1 and Rrp5 promote formation of this inhibitory duplex during rDNA transcription; preliminary in vivo structure probing experiments support this requirement of Rok1. In addition to exemplifying the first in vivo role of helicase annealing activity, these results also serve as an example of a co-factor increasing the specificity of a DEAD-box protein.

46 Multilign: An Algorithm to Improve Prediction of Secondary Structures Conserved in Multiple RNA Sequences

Zhenjiang Xu, David Mathews Dept. of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, US With discovery of diverse roles for RNA, its centrality in cellular functions has become increasingly apparent. Structure prediction is an attractive tool to study RNA function-structure relationship with high speed and low cost. A new algorithm, called Multilign, is presented to find the lowest free energy RNA secondary structure common to multiple sequences. Multilign is based on Dynalign, which is a program that simultaneously aligns and folds two sequences to find the lowest free energy conserved structure. For Multilign, Dynalign is used to progressively construct a conserved structure profile from multiple pair-wise calculations, with one sequence used in all pair-wise calculations. A base pair is predicted only if it is contained in the set of low free energy structures predicted by all Dynalign calculations. In this way, Multilign is able to improve prediction accuracy by keeping the genuine base pairs and excluding competing false base pairs. Multilign predicts secondary structures of multiple sequences with computation complexity linear in the number of sequences. Multilign was tested on extensive datasets of tRNA, 5S rRNA, Signal Recognition Particle RNA, RNase P RNA, and small subunit rRNA and its prediction accuracy is among the best of available algorithms. The results show Multilign can run on long sequences (>1,500 nt) and an arbitrarily large number of sequences. The algorithm is implemented in ANSI C++ and will be available as part of the RNAstructure package at: http://rna.urmc.rochester.edu/.

Session 3A: RNA-protein architecture & Session 3B: RNA-seq & computational structure prediction

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

47 Scanning Very Long Sequences for Suboptimal Structures Including Pseudoknots Using an Adjustable Window Size and Flexibility

Wayne Dawson1, Shingo Nakamura2, Gota Kawai3, Kiyoshi Asai4,1 Dept of Comp Bio, Fac Frontier Sci, Univ Tokyo, Kashiwa, Chiba, Jpn, 2Pharm. Res. Div., Takeda Pharm. Co. Ltd; Business Dev. - Biologics, Catalent Pharma Solutions, Minato-ku, Tokyo, Jpn., 3Chiba Inst. Tech., Narashino, Chiba, Jpn, 4CBRC, AIST, Koto-ku, Tokyo, Jpn In recent work, we reported that there is a physical basis behind the apparent size of the domains in known RNA structures (JNAI, in press). For free energy based computational approaches, this means that a legitimate cutoff can be used in scanning for RNA structure without concern about loss of information. We have been developing an RNA structure prediction program that computes suboptimal structures including pseudoknots and have recently upgraded the program to evaluate these suboptimal structures with a sliding window. The approach allows the user to specify particular points in an RNA sequence where the window size should change or the flexibility (as measured by the Kuhn length) should be changed. By limiting the window size, it is possible to compute much longer sequences than is feasible without this option, and in principle, diverse regions can be calculated independently in parallel. Moreover, this relaxes some of the constraints on the way natural selection introduces mutations in RNA because the impact can only extend over a physically limited range. In general, mutations in this model appear to be less deleterious to RNA structures because the method aims at the maximum entropy in folded RNA structures. 1

48 Deep sequencing Ribosome Protected mRNA Fragments from polysomes and monosomes isolated by size exclusion chromatography

Scott Kuersten1, Ramesh Vaidyanathan1, Agnes Radek1, Silvi Rouskin2, Sajani Swami3, Josh Dunn2, Jonathan Weissman2 1 Epicentre, Madison, WI, USA, 2UCSF, San Francisco, CA, USA, 3Illumina, Hayward, CA, USA RNA abundance measured either by microarrays or high-throughput sequencing reflect protein levels for transcripts that are not subjected to translation control. However, it is known that a large proportion of mRNAs are regulated at some stage of their biogenesis and only some of these regulatory steps are known to affect transcript levels in the cell. The isolation of translating ribosomes (polysomes and monosomes) and analysis of the associated mRNAs provide a better measure of translation rates to estimate the copies of the synthesized protein. Furthermore, comparison of total mRNA to the Ribosome Protected mRNA Fragments (RPF) can provide quantitative analysis of the translationally active verses repressed pools of transcripts present at a particular time or condition. Current methods to isolate polysomes and monosomes rely on ultracentrifugation using either a sucrose gradient or a cushion. While the sucrose cushion avoids the need for more specialized gradient fractionation step, it still requires access to an ultracentrifuge and several hours of centrifugation. We have investigated size exclusion chromatography as an alternate to ultracentrifugation to isolate polysomes. As reported in the literature, we found polysomes in the void volume of commonly used size exclusion resins. The size-exclusion method is simpler and rapid and because of polysome exclusion it does not require any special equipment. We are investigating the use of size-excluded polysomes for ribosome profiling using the method of Ingolia et al [Science 324, 218-223 (2009)] to monitor the protein production in cells and define the proteome. Furthermore, we have designed, developed and commercialized a library prep kits to convert ribosome protected RNA fragments into samples compatible with sequencing on Illumina’s instruments and will present data comparing classic sucrose gradient/cushion samples with gel filtration spin column sample preparations.

Session 3B: RNA-seq & computational structure prediction

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

49 Thermostable Group II Intron Reverse Transcriptase Fusion Proteins and their Applications in cDNA Synthesis and Next-Generation Sequencing

Sabine Mohr1, Eman Ghanem1, Whitney Smith1, Dennis Sheeter1, Yidan Qin1, Damon Polioudakis1, Vishwanath Iyer1, Scott Hunicke-Smith1, Sajani Swamy2, Scott Kuersten3, Alan Lambowitz1 1 UT Austin, Austin, TX, USA, 2Illumina Inc, Hayward, CA, USA, 3Epicentre, Madison, WI, USA Reverse transcriptases (RTs) are used in research and biotechnology for a variety of applications that require cDNA synthesis, including qRT-PCR, transcriptome and miRNA profiling, RNA-structure mapping and footprinting, and the cloning and sequencing of protein-bound RNA fragments in HITS-CLIP and related procedures. However, retroviral RTs, which are currently used for these methods, suffer from low processivity and fidelity. Another large superfamily of RTs encoded by mobile group II introns and other non-LTR-retroelements exists in nature, but have been difficult to express and purify with high activity in large quantities. Some of these enzymes are present in thermophiles and potentially have high thermostability, a useful property that enables reverse transcription at high temperatures that melt RNA secondary structure. We have now developed general methods for the high-level expression of thermostable and other group II intron-encoded RTs as fusion proteins with a rigidly linked, non-cleavable solubility tag. We find that these group II intron RT fusion proteins have higher processivity, fidelity, and thermostability than retroviral RTs, and we demonstrate their advantages for a variety of applications, including qRT-PCR, capillary electrophoresis, and next-generation RNA sequencing (RNA-seq). In RNA-seq, the higher processivity of the thermostable group II intron RTs makes it possible to obtain relatively uniform 5’- to 3’-end read coverage of human mRNAs from preparations of whole-cell RNAs using an annealed oligo(dT) primer. Thus, these enzymes enable RNA-seq to be done directly on whole-cell RNAs with minimal manipulation compared to current methods employing retroviral RTs, which require rRNA depletion/poly(A) selection, RNA fragmentation, and random priming to achieve uniformity. Additionally, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base pairing to the 3’ ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without using RNA ligase. This novel templateswitching method enables facile and less biased cloning of non-polyadenylated RNAs, such as miRNAs or protein-bound RNA fragments in procedures like HITS-CLIP. Our findings demonstrate inherent and unique advantages of group II intron RTs for cDNA synthesis, with potentially wide applications in RNA research and other areas.

50

RNA 3D Hub - a new online resource for RNA structural bioinformatics

Anton Petrov, Craig Zirbel, Neocles Leontis Bowling Green State University, Bowling Green, (OH), USA Many internal and hairpin loops, which are usually drawn as unstructured in RNA secondary structure diagrams, in fact, form sophisticated structural motifs stabilized by non-Watson-Crick interactions. These RNA 3D motifs are often recurrent, occurring in various types of RNA molecules, and are essential for many biological functions and RNA folding. It is desirable to create a comprehensive collection of RNA 3D motifs in order to advance the RNA 2D and 3D structure prediction and ncRNA discovery methods, to interpret mutations that affect ncRNAs, and to guide experimental functional studies. To this end, a new online resource called RNA 3D Hub (http://rna.bgsu.edu/rna3dhub) has been developed. It houses RNA 3D motifs extracted from all RNA 3D structures and the RNA 3D Motif Atlas, a representative collection of RNA 3D motifs. In addition, RNA 3D Hub hosts non-redundant sets of RNA-containing 3D structures and structural annotations of various pairwise interactions found in 3D structures, such as basepairing and stacking. Unique and stable ids are assigned to all motifs, to all motif instances, and to all non-redundant equivalence classes of structure files. RNA 3D Hub is updated automatically on a regular schedule, and a versioning system is implemented to provide independent access to data snapshots. Both human and computer friendly interfaces for data access are provided. RNA 3D Motif Atlas is built upon a new RNA 3D motif clustering approach, which is based on exhaustive all-against-all geometric FR3D searches using each motif in turn as a query. The search results are checked for incompatible structural features and organized in a graph where the nodes are formed by motif instances connected by edges if the corresponding motif instances matched each other during FR3D searches. The motifs are identified as maximum cliques in this graph. RNA 3D Hub and RNA 3D Motif Atlas lay the groundwork for further research into RNA 3D motif prediction starting from sequence and provide useful online resources for the scientific community worldwide.

Session 3B: RNA-seq & computational structure prediction

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

51

Encapsidated Viral RNA Structure Prediction

52

Genesilico Web Servers for Predicting of RNA -Metal Ion and -Ligand Interactions

Susan Schroeder, Samuel Bleckley, Jonathan Stone, Jui-wen Liu University of Oklahoma, Norman, OK, USA The diverse landscape of RNA conformational space includes many canyons and crevices distant from the lowest minimum free energy valley that remain unexplored by traditional RNA structure prediction methods. Viral genomic RNA adopts many conformations during its life cycle as the genome is replicated, translated, and encapsidated, which poses many interesting RNA folding challenges. Multiple solutions to the genome packaging problem could be an evolutionary advantage for viruses. In such cases, an ensemble of structures that share favorable global features best represents the RNA fold. New computational tools, Crumple and Sliding Windows and Assembly, are presented to evaluate and explore the possible secondary structures for encapsidated Satellite Tobacco Mosaic Virus RNA and MS2 bacteriophage RNA. The Crumple algorithm provides a method to compute completely and efficiently all possible secondary structures for a given RNA sequence without consideration of thermodynamic parameters. Traditional free energy minimization methods do not consider stabilizing RNA tertiary interactions, RNA-protein interactions, or the possibility that kinetics rather than thermodynamics determines the functional structure. Crumpling an RNA sequence, like crumpling a piece of paper, is a fast and indiscriminate way of folding. Efficient parallel computing and effective experimental filters make the complete enumeration of all possible structures for an RNA sequence a reasonable approach. Experimental filters from chemical or enzymatic probing, phylogenic covariation, SELEX, crystallography, or cryoelectron microscopy can reduce the conformational space without overlooking structures that may be stabilized by tertiary and quaternary interactions. The minimum number and length of helices has a significant effect on reducing conformational space. The combined effect of all filters reduces the possible number of structures for an Alfalfa Mosaic Virus protein binding sequence from over 50 million structures to a set of 91 structures. The advantage of the Crumple, Sliding Windows, and Assembly approach is to modulate the influence of thermodynamic parameters on RNA structure prediction. The predictions from this new approach can test hypotheses and models of viral assembly and guide construction of complete three-dimensional models of virus particles.

Anna Philips1,2, Kaja Milanowska1, Grzegorz Lach2, Michal Boniecki2, Kristian Rother1,2, Janusz Bujnicki2,1 1 Adam Mickiewicz University, Poznan, Poland , 2International Institute of Molecular and Cell Biology, Warsaw, Poland Interactions of RNA with other molecules such as metal ions or ligands play crucial roles in many biological processes. Mono- and divalent cations drive proper folding of RNA and stabilize its secondary and tertiary structures. Ions also act as essential cofactors in many reactions catalyzed by RNAs. The hammerhead ribozyme, group I and group II introns as well as ribonuclease P (RNaseP) ribozymes are examples of RNA that need divalent ions to cleave phosphodiester bonds. Moreover, many RNA molecules are essential elements of cellular or viral physiology, and they serve as targets for small-molecule ligands that may act as drugs. For example, the majority of antibiotics target the ribosome, and in particular active sites composed of ribosomal RNAs. We have developed novel bioinformatics tools for predicting RNA interactions with metal ions and small molecules. MetalionRNA determines sites around a user-specified RNA 3D structure, where Mg2+, Na+, and K+ cations are most likely to bind. LigandRNA scores and ranks user-defined RNA-ligand complexes (e.g. poses obtained by ligand docking methods). Both methods employ a grid-based algorithm and a knowledge-based potential derived from metal ion and ligand binding sites from experimentally solved PDB structures. Our methods can be used to assist X-ray crystallographic structure determination, e.g. by identifying tentative metal ion sites to be further validated during structure refinement. It can also be used in a fully predictive mode to identify or to design drugs targeting RNA (e.g. novel antibiotics targetting bacteria ribosomes or novel inhibitors affecting riboswitch structure and function) or to propose metal positions for structural models that typically lack coordinates of ions, e.g. RNA structures determined by nuclear magnetic resonance (NMR) spectroscopy or theoretical models. The MetalionRNA and LigandRNA programs are available free of charge as web servers at http://genesilico.pl/ Acknowledgements: This work has been supported by the Foundation for Polish Science (FNP, grant TEAM/20094/2). A.P. was supported by MNiSW (GDWB-04/2011).

Session 3B: RNA-seq & computational structure prediction

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

53

5’-Terminal Control of RNA Degradation

54

Polyadenylation Helps Regulates Functional tRNA Levels in Escherichia coli

Dan Luciano, Jamie Richards, Ping-kun Hsieh, Joel Belasco New York University School of Medicine, New York, NY, USA In bacteria, the lifetimes of mRNAs can differ by more than an order of magnitude, with corresponding effects on gene expression. Our recent findings have shown that bacterial mRNA decay is often triggered by a non-nucleolytic event that precedes ribonuclease attack and marks transcripts for rapid turnover: the conversion of the 5’ terminus from a triphosphate to a monophosphate by the RNA pyrophosphohydrolase RppH. In Escherichia coli, this modification creates better substrates for the endonuclease RNase E, whose cleavage activity is greatly enhanced when the RNA 5’ end is monophosphorylated, whereas in Bacillus subtilis it triggers 5’-exonucleolytic degradation by RNase J. Many but not all E. coli transcripts are targeted for degradation by this mechanism. An investigation of the basis for this selectivity has begun to identify the features of transcripts that determine the pathway by which they are degraded and to provide insights into what limits the rate of RppH-dependent RNA degradation.

Sidney Kushner, Bijoy Mohanty University of Georgia, Athens, GA USA We will present data that demonstrates a new regulatory mechanism for tRNA processing in Escherichia coli whereby RNase T and RNase PH, the two primary 3’ → 5’ exonucleases involved in the final step of 3’ end maturation, compete with poly(A) polymerase I (PAP I) for tRNA precursors in wild-type cells. In the absence of both RNase T and RNase PH, there is a >30-fold increase of PAP I-dependent poly(A) tails that are 2-fold decrease in growth rate. Only 7 out of 86 tRNAs are not regulated by this mechanism and are also not substrates for RNase T, RNase PH or PAP I. Surprisingly, neither PNPase nor RNase II, the primary enzymes in E. coli that degrade poly(A) tails, has any effect on tRNA poly(A) tail length. Our data also suggest that the polyadenylation of tRNAs by PAP I likely proceeds in a distributive fashion unlike what is observed with mRNAs where the average poly(A) tail length is between 10-30 nt. Furthermore, additional experiments have shown that the toxicity associated with increased levels of PAP I results from a drastic reduction in the intracellular levels of functional tRNAs. This work was supported in part by research grants from the National Institute of General Medical Sciences (GM57220 and GM81554) to S.R.K.

Session 4: Keynote & Session 4: RNA turnover

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

55

Ribonucleases control RNA damage and protect cells against oxidative stress

Zhongwei Li Florida Atlantic University, Boca Raton, FL, USA Nucleic acid damaging agents cause damages to both DNA and RNA. Emerging evidences suggest that RNA damage is detrimental to cells and is strongly implicated in various degenerative diseases. We have shown greater oxidation damage to RNA than to DNA under both normal and oxidative stress (OS) conditions. Upon treatment of Escherichia coli cells with hydrogen peroxide, the level of the oxidative lesion 8-hydroxyguanine (8-oxo-G) rise in RNA within minutes. After removal of the oxidant, 8-oxo-G levels quickly drop in total RNA, demonstrating the existence of efficient surveillance mechanisms that can eliminate 8-oxo-G RNA rapidly. We have shown that degradation may play a major role in eliminating oxidatively-damaged RNA in Escherichia coli and in cultured human cells. The 3’ to 5’ exoribonuclease polynucleotide phosphorylase (PNPase) is able to bind 8-oxo-G RNA with high affinity. In the absence of PNPase, E. coli cells become hypersensitive to OS challenge, accompanied by a sharp increase of 8-oxo-G in RNA but not in DNA. Importantly, mutant strains lacking PNPase are also impaired in eliminating 8-oxo-G RNA after removal of oxidant. Several other RNases and proteins also play a role in reducing oxidized RNA and protecting E. coli cells against OS. The human PNPase homolog, hPNPase, is localized mainly in mitochondria. Increased expression of hPNPase rendered HeLa cell more resistant to hydrogen peroxide treatment and lowered 8-oxo-G in RNA. Knock-down of hPNPase resulted in higher sensitivity to oxidants and increased 8-oxo-G in RNA. Our results strongly suggested that RNA damage can be deleterious to cells and organisms, and specific mechanisms are employed to remove damaged RNA species.

56 Global Analysis Reveals Multiple Pathways for Unique Regulation of mRNA Decay in Induced Pluripotent Stem Cells

Ashley Neff1, Ju Youn Lee2, Bin Tian3, Jeffrey Wilusz1, Carol Wilusz1 Colorado State University, Fort Collins, (CO), USA, 2New York University, New York, (NY), USA, 3UMDNJ - New Jersey Medical School, Newark, (NJ), USA Pluripotency is a unique state in which cells can self-renew indefinitely but also retain the ability to differentiate into other cell types upon receipt of extracellular cues. Although it is clear that stem cells have a distinct transcriptional program, little is known about how alterations in post-transcriptional mechanisms, such as mRNA turnover, contribute to the achievement and maintenance of pluripotency. Here we have assessed the rates of decay for the majority of mRNAs expressed in induced pluripotent stem (iPS) cells and the fully differentiated fibroblasts (HFFs) they were derived from. Comparison of decay rates for 5,481 mRNAs expressed in both cell types led to the discovery of three independent regulatory mechanisms that allow coordinated turnover of specific groups of mRNAs. One mechanism results in increased stability of many replication-dependent histone mRNAs in iPS cells. A second pathway stabilizes a large set of C2H2type zinc finger protein mRNAs, potentially through reduced levels of miRNAs that target them. Finally, a group of transcripts bearing 3’UTR C-rich sequence elements, many of which encode transcription factors, are significantly less stable in iPS cells. Intriguingly, two poly(C) binding proteins that recognize this type of element, PCBP3 and PCBP4, are reciprocally expressed in iPS and HFF cells. PCBP4 is a tumor suppressor whose overexpression leads to apoptosis and cell cycle arrest, consistent with our findings that it is expressed more highly in HFFs. Overall, our results highlight the importance of post-transcriptional control in pluripotent cells and identify miRNAs and RNA-binding proteins whose activity may coordinately control expression of a wide range of genes in iPS cells. This work was funded by an NIH R01 award (GM072481) and ARRA supplement, as well as a Colorado State University CVMBS College Research Council Award to JW. The Tian lab was funded by an NIH R01 award (GM084089) to BT.

1

Session 4: RNA turnover

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

57

Mechanism of Processive and Cap-Stimulated mRNA Poly(A) Tail Degradation

58

Crystal Structure and Heterodimerization of the NOT Box Domains of human NOT2 and NOT3

Niklas Henriksson, Per Nilsson, Magnus Lindell, Mikael Nissbeck, Samuel Flores, Anders Virtanen Uppsala University, Uppsala, Sweden Poly(A)-specific ribonuclease (PARN)is an oligomeric, processive and cap-interacting exoribonuclease that efficiently degrades mRNA poly(A) tails. PARN is a key regulator of AU-rich element (ARE) containing mRNAs encoding proto-oncogenes and cytokines. Here, we propose a mechanistic framework for PARN action. The model has been experimentally tested and is supported by structural evidence. In the model the RNA recognition motif (RRM) of PARN pushes the poly(A) tail into the active site, which simultaneously pulls the substrate into the hydrolytic site where the movement of a catalytically essential histidine residue is coordinated with the pushing of the RRM. The model provides a mechanistic explanation for how the cap-structure of the mRNA can stimulate poly(A) tail hydrolysis. We have also investigated the functional significance of divalent metal ions in the active site and revealed that divalent metal ions are required for both hydrolysis and substrate translocation in the active site. Our data imply that three divalent metal ions are required for proper action. Two ions participate in hydrolysis while the third plays a key role during translocation. The generality of this proposal in relationship to other processive enzymes participating in cleavage or formation of phosphodiester bonds will be discussed.

Andreas Boland, Ying Chen, Lara Wohlbold, Praveen Bawankar, Oliver Weichenrieder, Elisa Izaurralde Max Planck Institute for Developmental Biology, Tübingen, Germany The CCR4-NOT complex plays a crucial role in post-transcriptional mRNA regulation in eukaryotic cells. It catalyzes the removal of mRNA poly(A) tails thereby repressing translation and committing mRNAs to decay. The complex consists of a catalytic module comprising two deadenylases (POP2/CAF1 and CCR4) and the NOT module comprising minimally the NOT1, NOT2 and NOT3 subunits. NOT2 and NOT3 are related proteins that both contain a highly conserved C-terminal domain termed the NOT-box. Crystal structures are available for the deadenylase POP2 and the nuclease domain of CCR4, but lacking for the minimal NOT module. Furthermore it is still unclear how the NOT proteins contribute to the activity of the deadenylase complex in vivo. Here we show that the NOT-box is a heterodimerization domain mediating the assembly of the NOT2-NOT3 subcomplex. We have solved the crystal structures of the human NOT2 and NOT3 NOT-boxes at 2.4Å and 2.5Å resolution, respectively. The NOT box consists of a four-stranded C-terminal open β-barrel as well as N-terminally located α-helices, which are required for heterodimerization. Furthermore, we defined the domains of NOT1 required for the interaction with the NOT2-NOT3 subcomplex as well as with the catalytic subunit POP2/CAF1. We have investigated the effects of mutations that specifically disrupt interactions of these complex components in deadenylation assays in vivo. Our findings shed light on the assembly of the CCR4-NOT complex and provide the basis for dissecting the role of the NOT subunits in mRNA deadenylation.

Session 4: RNA turnover

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

59 Rrp47p Forms a Heterodimer with Yeast Nuclear Exosome Component Rrp6p and Stimulates its Activity

Emil Dedic1, Paulina Seweryn1, Anette Jonstrup1, Jan Jensen2, Natalya Fedosova3, Søren Hoffmann4, Thomas Boesen5, Ditlev Brodersen1 1 Centre for mRNP Biogenesis and Metabolism, Aarhus University, Denmark, 2Department of Molecular Biology and Genetics, Aarhus University, Denmark, 3Department of Biomedicine, Aarhus University, Denmark, 4Institute for Storage Ring Facilities, Aarhus University, Denmark, 5Centre for Membrane Pumps in Cells and Disease PUMPKIN, Aarhus University, Denmark S. cerevisiae Rrp6p is an RNase D-type 3’-5’ exonuclease that functions as a nuclear-specific cofactor of the RNA exosome. During processing of stable RNAs in the yeast nucleus, Rrp6p associates with Rrp47p/Lrp1p, a protein that has been shown to form multimers and bind double-stranded RNA in vitro. We present that Rrp47p associates with Rrp6p into a 1:1 heterodimeric complex with an overall elongated shape consistent with binding of Rrp47p at the top of the PMC2NT domain of Rrp6p, opposite of the HRDC domain. We also show that complex formation leads to structural rearrangements that potentially could affect the activity of the nuclease and that both proteins form multimers in absence of their binding partner. We further demonstrate that Rrp47p stimulates the exonucleolytic activity of Rrp6p on both single-stranded and double-stranded RNA substrates without significantly altering the affinity towards structured RNA. The results support a model in which Rrp47p does not specifically recruit structured RNAs to the exosome, but rather acts as a general allosteric activator of Rrp6p.

60

Crystal Structure of a Yeast 11-Subunit Exosome Complex Bound to RNA

Debora Makino, Marc Baumgärtner, Elena Conti Max-Planck Institute of Biochemistry, Martinsried, Germany The exosome is a 3’-to-5’ ribonuclease responsible for the degradation and processing of a wide range of RNA substrates in eukaryotic cells. The conserved core of the exosome present in both the nucleus and the cytoplasm of all eukaryotes studied to date is a complex of ~400kDa comprised of 10 different proteins (Exo-10). Only one subunit of Exo-10, Rrp44, harbors ribonuclease activity (Liu et al., 2006; Dziembowski et al., 2007). Yet, the catalytically inactive subunits of Exo-10 are all essential for viability in S. cerevisiae (Allmang et al., 1999). The catalytically inert subunits of the eukaryotic exosome have a barrel-like structure similar to that of the archaeal exosome and bacterial PNPase, despite lacking the phosphorolytic activity of these complexes (Liu et al., 2006; Lorentzen et al., 2005; Buttner et al., 2005; Symmons et al., 2000). Structural studies have also shown that Rrp44 contains an N-terminal PIN domain that tightly binds to one of the exosome subunits (Bonneau et al., 2009) and an RNase II-like domain that binds a 9-nucleotidelong RNA (Lorentzen et al., 2008). In biochemical assays, however, Exo-10 recognizes a 31-33 nucleotide-long RNA (Bonneau et al., 2009). These data, together with mutational analysis, suggested that RNA reaches the exoribonuclease site of Exo-10 by threading through the 9- subunit barrel structure, reminiscent of the archaeal complexes. To visualize how RNA binds within the eukaryotic exosome and is poised for degradation, we have determined the 2.8 Å resolution crystal structure of Exo-10 in complex with the interacting region of Rrp6 (the eleventh subunit of the nuclear exosome in yeast) and RNA. The structure shows how RNA is threaded through the entire complex. It also shows how the 5’ end of the RNA is unwound by the exosome and how the 3’ end is positioned to the active site via a large conformational change in Rrp44.

Session 4: RNA turnover

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

61 Human Staufen1 Dimerization via Swapping a Conserved Motif and a Degenerate DoubleStranded RNA-Binding Domain Augments UPF1 Binding and mRNA Decay

Michael Gleghorn, Chenguang Gong, Clara Kielkopf, Lynne Maquat Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY, USA Staufen1-mediated mRNA decay (SMD) contributes to many important biological processes, including myogenesis and wound healing. SMD occurs when translation terminates sufficiently upstream of a Staufen1 (STAU1)-binding site (SBS) that is located in the 3’ UTR of an mRNA and bound STAU1 recruits the ATP-dependent RNA helicase UPF1. Human (h)STAU1 contains four regions having varying degrees of similarity to the canonical double-stranded (ds) RNA-binding domain (RBD). RBD3 and RBD4 are known to bind dsRNA, but ‘RBD’2 and ‘RBD’5 do not, and the latter two have been reported to be important regions for the oligomerization of hSTAU1 on mRNA. Our work demonstrates that hSTAU1 dimerization is critical for efficient SMD. We have identified and solved at 1.7 Å resolution the X-ray crystal structure of a new motif, which we name the Staufenswapping motif (SSM), together with ‘RBD’5. An SSM is present in all examined vertebrate paralogs of hSTAU1. It consists of two-helices connected by a flexible linker N-terminal to ‘RBD’5 that recognizes ‘RBD’5 of an adjacent hSTAU1 via domainswapping. Gel-filtration and analytical ultracentrifugation analyses demonstrated that SSM-’RBD’5 forms a dimer in vitro. In cells, two different ‘RBD’5 domains alone are insufficient for dimerization, but the inclusion of an SSM in just one of two molecules is sufficient for dimerization. The X-ray crystal structure illustrates that: 1) while ‘RBD’5 is a degenerate RBD, it still adopts the typical α-β-β-β-α topology of a true RBD but with critical differences that render it incapable of binding dsRNA, and 2) the two α-helices of SSM form a hydrophobic core with the two α-helices of ‘RBD’5 that is positioned by two polar interactions. SSM and ‘RBD’5 appear to have co-evolved residues important for their interaction. In cells, SSM-’RBD’5-mediated dimerization is critical for efficient SMD triggered by SBSs formed either in trans (i.e., formed by base-pairing between a 3’ UTR Alu element and the partially complementary Alu element of a long-noncoding (½sbs) RNA or in cis (i.e., formed by an intramolecular 3’ UTR stem-apex structure). We can impair full-length hSTAU1 dimerization in vivo by expressing tagged ‘RBD’5 alone. Remarkably, impairment decreases the affinity of hSTAU1 for the SMD factor hUPF1. Since the affinity of hSTAU1 for RNA is more than 4-orders of magnitude higher than the affinity of hSTAU1 for another hSTAU1, we propose that hSTAU1 dimerization occurs on SBSs. Thus, SMD is regulated in vivo by SBS length, hSTAU1 concentration-dependent SSM-mediated dimerization, and the recruitment of hUPF1.

62

Single Molecule Analysis of Nonsense-mediated mRNA Decay in Yeast

Victor Serebrov1, Nadia Amrani2, Larry Friedman3, Jeff Gelles3, Melissa Moore1,4, Allan Jacobson2 1 Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA, 2Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, USA, 3Department of Biochemistry, Brandeis University, Waltham, MA, USA, 4Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA, USA Nonsense mediated decay (NMD) is a conserved mRNA quality control and regulatory mechanism that specifically recognizes premature termination of translation and triggers accelerated degradation of the aberrant mRNA. Many details of the mechanism of NMD remain unclear. Here, we use multi-color co-localization single molecule spectroscopy (CoSMoS)1,2 in conjunction with a SNAP- and CLIP-tag labeling system3 to investigate the biochemical details of NMD in S.cerevisiae.  In CoSMoS, spectrally distinct fluorescent dyes are placed on different proteins and visualized during a reaction. Complex formation by the proteins is then quantitatively monitored using the co-localization of their distinguishable fluorescence emissions. We created a collection of yeast strains expressing C-terminal SNAP or CLIP-tagged alleles of the genes encoding the NMD factors (UPF1, UPF2/NMD2, and UPF3), release factors, and several other translation factors, thereby allowing for highly specific labeling of these proteins with fluorescent dyes in translation-competent yeast extracts. The mutually orthogonal nature of SNAP and CLIP tags permits simultaneous labeling of any combination of two of the tagged proteins with different color dyes in the same extract, allowing us to detect concerted binding events that involve multiple factors. In addition, we utilize a fluorescent probe displacement assay that takes advantage of the helicase (strand displacement) activity of the elongating ribosome and helps delineate its positioning with respect to termination events. CoSMoS experiments with mRNAs containing either a premature or a normal terminator confirm preferential association of the NMD factors with nonsense-containing mRNAs, and afford the first direct observations of molecular events underlying NMD. 1. Friedman LJ, Chung J, Gelles J. Biophys J. (2006) 91(3), 1023-31; 2. Hoskins AA, Friedman L.J., Gallagher S.S., Crawford D.J., Anderson E.G.,Wombacher R., Ramirez N., Cornish V.W., Gelles J., Moore M.J. (2011) Science 331(6022), 1289-95. 3. New England Biolabs.  Session 4: RNA turnover

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

63

A Large-Scale Survey Reveals Selective Ultra-Fast microRNA Turnover Rates

64

A Primate Herpesvirus Promotes T-cell Activation via Degradation of microRNA-27

Yanwen Guo1, Yinghong Ma2, Caihong Qiu2, Jun Lu3 1 Department of Genetics, Yale University School of Medicine, New Haven, CT, USA, 2Yale Stem Cell Center and Cancer Center, New Haven, CT, USA, 3Department of Genetics, Yale University School of Medicine, Yale Stem Cell Center and Cancer Center, Yale Center for RNA Science and Medicine, New Haven, CT, USA Despite the importance of microRNAs (miRNAs) in diverse biological settings, limited information exists on the extent and how miRNAs are actively turned over in mammalian cells. Although it is commonly believed that many miRNAs are highly stable, studies that systematically characterize miRNA stability are rare and performed on limited cell types. Through a large-scale study on miRNA turnover rates in mammalian cells, we report here that a select group of Ago-bound miRNAs display cell-type-independent ultra-short half-lives, whereas the majority of miRNAs are long-lived. We followed the expression of 528 endogenous miRNAs in 8 human and mouse cancer and normal cell types treated with transcription inhibitors, generating a total of 218 expression profiles. A select group of the previously categorized miRNA* species displays ultra-short half-lives of less than 1 hour in both cancer and normal cells. In contrast, the vast majority of detectable miRNAs, including other miRNA* species, possess turnover rates comparable to or slower than ribosomal RNAs. These short-lived miRNAs are exemplified by miR-222-5p, whereas miR-222-3p, produced from the same hairpin precursor, is highly stable. Using a tet-off inducible system that controls the exogenous production of both miR-222-5p and miR-222-3p, we found that this fast turnover is an intrinsic property of miR-222-5p, and is independent of genome-wide transcription inhibition. Furthermore, miR-222-5p is bound to Ago proteins at comparable fractions to either miR-222-3p or other long-lived miRNA species, arguing against the possibility of our measuring the pre-RISCloading transient miRNA duplexes. Interestingly, we did not find evidence that extracellular secretion is a major contributor to this short half-life, strongly suggesting that an active intra-cellular mechanism is responsible for its rapid turnover. Our results highlight that differential turnover rate can be a significant contributor to the unequal strand presence for miRNAs produced from the same hairpins in mammalian cells. Our data also support a model that two distinctive pools of Ago-bound miRNAs co-exist in cells, which possess vastly different turnover rates.

Eric Guo, Kasandra Riley, Joan Steitz Yale University, New Haven, CT, USA Herpesvirus saimiri (HVS) is a T-lymphotropic herpesvirus that causes aggressive T-cell lymphomas and leukemias in New World primates. During latent infection, HVS expresses seven U-rich small non-coding RNAs, called the HSURs. It was shown by our lab that HSUR1 base-pairs with the host miR-27 family of microRNAs (miRNAs), leading to their degradation (1). We hypothesize that miR-27 plays an important role in the transformation of infected marmoset T cells and/or host immune evasion. To identify miR-27 target genes in HVS-infected T cells, we applied the HITS-CLIP (High-throughput Sequencing of RNA after Crosslinking Immunoprecipitation) method (2). Current efforts are underway to validate novel targets of miR-27 obtained by HITS-CLIP in the infected T cells and test their influence on transformation and immune regulation. Our results show that miR-27 targets mRNAs of several genes in the T-cell Receptor (TCR) signaling pathway. By degrading miR-27, HVS should upregulate the cellular mRNA targets of this miRNA. This is consistent with previous findings where genes that are hallmarks of T-cell activation are selectively upregulated by HSUR1 and/or 2 in virally transformed T cells (3). Since increased levels of TCR signaling promote activation and proliferation of T cells, our study suggests that HVS hijacks the host TCR signaling pathway to ensure the survival of infected T cells. 1. Cazalla D, Yario T, Steitz JA. 2010. Down-regulation of a host microRNA by a Herpesvirus saimiri noncoding RNA. Science. 328(5985):1563-6. 2. Chi SW, Zang JB, Mele A, Darnell RB. 2009. Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature. 460(7254): 479-86 3. Cook HL, Lytle JR, Mischo HE, Li MJ, Rossi JJ, Silva DP, Desrosiers RC, Steitz JA. 2005. Small nuclear RNAs encoded by Herpesvirus saimiri upregulate the expression of genes linked to T cell activation in virally transformed T cells. Curr Biol. 15(10):974-9. Travel award was provided by the Yale Center for RNA Science and Medicine Session 4: RNA turnover

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

65 The Nuclear Poly(A) Binding Protein Promotes Polyadenylation-mediated Decay of Nuclear Transcripts in Human Cells

Stefan Bresson, Nicholas Conrad UT Southwesterm, Dallas, Texas, United States Messenger RNA abundance is regulated by balancing both transcription and decay rates. While cytoplasmic mRNA decay pathways are fairly well characterized in human cells, less is known about nuclear decay pathways. We are using the viral polyadenylated nuclear (PAN) RNA as a model transcript to investigate RNA decay in human nuclei. Here we show that PAN RNA is degraded by a polyadenylation-dependent decay pathway involving the nuclear poly(A)-binding protein (PABPN1), the poly(A) polymerases PAPα and PAPγ and the exosome. We show that nonadenylated versions of PAN RNA are more stable in cells and a poly(A) tail increases decay rates in nuclear extract supporting a destabilizing role for the poly(A) tail in nuclear RNA decay. RNAi-mediated knockdown of PABPN1 dramatically stabilizes PAN RNA as does co-depletion of the highly related PAPα and PAPγ, or the nuclear exosome components hDis3 and hRrp6. Interestingly, newly transcribed PAN RNA still has a poly(A) tail upon PABPN1 or PAPα/γ depletion, but it is significantly shorter than the poly(A) tail in the control knockdown, consistent with the idea that hyperadenylation by PABPN1 and PAPα/γ leads to PAN RNA decay. Further supporting this model, we show that hyperadenylated PAN RNA accumulates upon knockdown of exosome subunits. Importantly, this decay pathway is not restricted to PAN RNA. An intronless β-globin reporter mRNA is subject to PABPN1-mediated decay, but an analogous spliced β-globin transcript is not. This observation suggests that the decay pathway may have a role in the quality control of unspliced, and presumably inefficiently exported, transcripts. Preliminary analysis of endogenous cellular mRNAs has revealed that a large fraction of newly made transcripts is subject to PABPN1-mediated hyperadenylation. We propose that nuclear RNAs are subject to a PABPN1-mediated hyperadenylation-dependent decay pathway that may be involved in both RNA quality control and gene regulation.

66 Repression of Exon Splicing by an HnRNP Network that Alters Spliceosomal Interactions Upstream of the 5’ Splice Site

Ni-ting Chiou, Ganesh Shankarling, Kristen Lynch University of Pennsylvania, Philadelphia, (PA), USA The mammalian spliceosome is comprised of the U1, U2, U4, U5, and U6 snRNPs, which assemble sequentially on pre-mRNA to catalyze intron removal and exon joining. Importantly, control of spliceosome assembly allows for alternative pre-mRNA splicing, an essential and ubiquitous means of regulating gene expression. The majority of wellcharacterized examples of alternative splicing involve regulation of the earliest steps in spliceosome assembly. However, an increasing number of studies have demonstrated the potential for regulation also at later steps in the assembly process. Interestingly, recent studies have suggested that kinetic traps in the spliceosome assembly pathway may not only reduce splicing efficiency but also alter the choice of competing splice sites. As a model for dissecting mechanisms of splicing control, our laboratory has been investigating the mechanism by which hnRNP L represses one of its best characterized target exons, CD45 exon 4. Previously, we have shown that hnRNP L represses CD45 exon 4 after the ATP-dependent addition of the U1 and U2 snRNPs. We now demonstrate that the U4/U5/U6 tri-snRNP is also efficiently recruited to exon 4 in the presence of hnRNP L, but is not stably assembled on the substrate. Through purification and analysis of spliceosomal intermediate complexes we demonstrate that hnRNP L recruits hnRNP A1 to a weak cognate binding site immediately upstream of the 5’ splice site. Together, hnRNP L and A1 induce aberrant contacts between the 5’ splice site-bound U1 snRNA and neighboring exonic sequences. This altered conformation of U1 inhibits appropriate U1 displacement, as well as recruitment of the nineteen complex (NTC), thereby precluding splicing catalysis. Our results here demonstrate that conformational inhibition of splicing is a naturally occurring mechanism for controlling alternative splicing decisions, consistent with the kinetic equilibrium model. Furthermore, our data reveal what may be a widespread mechanism to regulate use of weak 5’ splice sites in a cell type or condition-specific manner through recruitment of hnRNP A1. Finally, our study provides direct evidence of NTC regulation as a mechanism for splicing repression, adding to repertoire of transitions within spliceosome assembly that are bona fide control points in the regulation of gene expression. Session 4: RNA turnover & Session 5A: Keynote

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

67

Group II Introns at Work: Intermediates of the Splicing Cycle Revealed by X-ray Crystallography

68

Single Molecule pre-mRNA Splicing: Splice Site Juxtaposition During Spliceosome Assembly

Marco Marcia1, Anna Pyle1,2 1 Molecular Cellular and Developmental Biology Department, Yale University, New Haven, CT, USA, 06511, 2 Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA Group II introns are ubiquitous self-splicing ribozymes possessing a common ancestor with the eukaryotic spliceosome. The structures of Oceanobacillus iheyensis group II intron previously determined by our group set a milestone for understanding the complex architecture of this ribozyme, but its detailed splicing mechanism was still uncharacterized. Here we report a set of novel crystal structures describing for the first time the intron at intermediate stages of its catalytic cycle. First, we describe the mechanism of the first step of splicing by hydrolysis, presenting the structures of the intron in the pre- and post-catalytic states. Second, we provide structural and functional evidence for a reversible conformational change that may favor the intron’s rearrangement between the first and the second steps of splicing. Third, we report the first structure of the intron in an exon-free state at the end of the splicing cycle and thus we can propose an explanation for its high reactivity as a retrotransposable element. Our new structures show that group II introns possess pronounced differences from other self-splicing ribozymes, i.e. group I introns, but a striking similarity with protein endonucleases, i.e. BamHI. They elegantly reveal how closely RNA can mimic proteins in forming efficient catalytic sites. Furthermore, they provide the first complete and unambiguous view of the group II intron active site, thereby allowing us to draw concrete structural comparisons with the spliceosome. Such comparisons offer unpredicted new insights into the mechanism of nuclear pre-mRNA maturation in eukaryotes. This project was supported by the National Institute of Health (RO1GM50313). Prof. Pyle is a Howard Hughes Medical Institute Investigator. Data were collected at the NE-CAT beamlines 24-ID-C/E, Advanced Photon Source (APS), Argonne, IL.

Mario Blanco1, Ramya Krishnan1, Matthew Kahlscheuer1, John Abelson2, Christine Guthrie2, Nils Walter1 1 University of Michigan, Ann Arbor, MI, USA, 2University of California, San Francisco, USA The spliceosome is the ribonucleoprotein (RNP) complex that catalyzes the removal of intervening sequences (introns) from coding regions (exons) in eukaryotic pre-mRNAs. The ordered assembly of the spliceosome makes it a compositionally dynamic system where protein and RNA components are shuttled in and out in a highly regulated manner. Catalytic activation of the spliceosome requires a dynamic set of ATP dependent RNA-RNA and RNA-protein interactions. The pre-mRNA substrate which acts as both the scaffold for assembly and is a key component of the chemical steps after activation must be recognized and positioned properly to ensure splicing efficiency and fidelity. To dissect the kinetic and conformational requirements for pre-mRNA positioning during spliceosome assembly we developed an in vitro single molecule FRET (smFRET) splicing assay in which the position of conserved intronic sequences have been tracked in real-time throughout splicing. Previously characterized mutations in either the pre-mRNA or splicing components allow us to stall the splicing process at defined steps. Through a combination of spliceosome complex enrichment, advanced data analysis techniques and single molecule splicing confirmation assays we can dissect the role of spliceosome components in regulating the juxtaposition of splice sites at various assembly steps.

Session 5A: Splicing mechanism

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

69

A Role for the Stem-loop 4 of U1 snRNA in Splice Site Pairing

Shalini Sharma2, Somsakul Wongpalee1, Douglas Black2,3 1 MBI, 2HHMI, 3MIMG, UCLA, Los Angeles, (CA), USA Critical steps in the assembly of spliceosome include the association of the U1 snRNP with the 5’ splice site and of the U2 snRNP with the branch point/3’ splice site to form the prespliceosomal A complex. Interactions that pair the two splice sites within the prespliceosome are decisive in committing the reaction to a particular splicing choice. These choices must occur with a high degree of specificity and fidelity for the appropriate expression of functional mRNAs. Candidate factors for mediating contact between the 5’ and 3’ splice site complexes include the U2 snRNP associated DEAD box protein Prp5 and SR related proteins. However, it is likely that additional interactions occur. Using a U1 snRNP suppression/complementation assay, we show that the stem-loop 4 of U1 snRNP (U1-SL4) is essential for splicing. Results show that the G-C rich region at the base of the stem is important. Using a combination of Stable Isotopic Labeling of Amino Acids in Culture (SILAC), biotin/Neutravidin affinity pull down, and mass spectrometry we find that U1-SL4 interacts with proteins of the SF3a complex, which is a component of the U2 snRNP. Addition of free SL4 to the splicing reaction inhibits splicing and blocks complex assembly at the transition from E to A complex, indicating a role for this region of U1 snRNA in splice site pairing and commitment to splicing.

70 Two unconventional RRMs of the RNA chaperone Prp24 have opposing effects on the U6 RNA internal stem-loop Ashley Richie, Elizabeth Curran, Kristie Andrews, Christine Treba, Samuel Butcher, David Brow University of Wisconsin, Madison, WI, USA

U6 RNA pairs with U2 RNA in the core of the spliceosome, where the two RNAs scaffold the pre-mRNA for the catalysis of splicing. In the U2/U6 complex, the U6 internal stem-loop (ISL) is thought to play a direct role in catalysis. After splicing, the U2/U6 complex is disrupted and U6 pairs with U4 RNA, which maintains U6 in an inactive conformation and shuttles it to the next intron via the U4/U5/U6 tri-snRNP. The conversion of U2/U6 to U4/U6 requires unwinding the U6 ISL, so that it can pair with U4 RNA to form U4/U6 Stem II. To investigate the mechanism of the conformational changes of U6 RNA during the splicing cycle, we previously introduced a stabilizing mutation that converts an A-C mismatch at the base of the U6 ISL to a G-C pair (A62G). This mutation results in cold-sensitive (cs) growth defect (1). To provide insight into the nature of the cs defect, we selected 95 independent spontaneous cold-resistant revertants of U6-A62G. A third of these have intragenic suppressor mutations in the U6-A62G allele (1). Another third have extragenic suppressor mutations in the PRP24 gene, which encodes a U6 RNA-binding protein with four RNA recognition motifs (RRMs). PRP24 was previously identified by Shannon and Guthrie in a selection for suppressors of a cs mutation in U4 RNA that destabilizes U4/U6 Stem II (2). The suppressor loci in the remaining third of our revertant strains are as yet unidentified. Our U6-A62G-suppressor mutations in PRP24 identify 30 different single amino acid substitutions that compensate for stabilization of the ISL. Most of the substitutions are in RRM3 or the linker between RRMs 3 and 4, and cluster in the solvent-exposed surface of the beta-sheet face and adjoining loops of RRM3. These substitutions suggest that RRM3 binds and stabilizes the U6 ISL, an unusual activity for an RRM but consistent with our chemical shift perturbation studies (3). Disruption of this stabilizing interaction by the suppressor substitutions is expected to offset ISL stabilization by the U6-A62G mutation. Ten of the suppressor substitutions are in RRM4 or its flanking alpha-helices, which we have collectively named an occluded RRM (oRRM), since the flanking alpha-helices bind and occlude the canonical RNA-binding surface of RRM4. We previously showed that isolated oRRM4 unwinds the U6 ISL in vitro (3). RRM3 added in trans inhibits unwinding of the ISL by oRRM4, consistent with stabilization of the ISL by RRM3. Furthermore, while wild-type oRRM4 is incapable of unwinding the U6-A62G ISL in vitro, the suppressor mutant oRRM4-S350R does unwind the U6-A62G ISL under the same conditions. Thus, S350R is a gain-of-function mutation. Intriguingly, in our structural model the side chain of Prp24-S350 is adjacent to the A62-C85 pair at the base of the U6 ISL, so the guanidinium group introduced by the S350R substitution may make a favorable contact with U6-C85 to promote unwinding of the ISL. 1) Fortner DM, Troy RG, Brow DA. (1994) Genes Dev 8:221-233. 2) Shannon KW, Guthrie C. (1991) Genes Dev 5:773-785. 3) Martin-Tumasz S, Richie AC, Clos AJ 2nd, Brow DA, Butcher SE. (2011) Nucl Acids Res 39:7837-7847.

Session 5A: Splicing mechanism

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

71

Cwc21p regulates branchsite usage in meiotic splicing

Amit Gautam, Richard Grainger, David Barrass, Jean Beggs Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK Yeast Cwc21p is one of twelve Bact complex proteins that associate with spliceosomes shortly before the first step of splicing catalysis. Cwc21p interacts directly with the U5 snRNP proteins Prp8p and Snu114p, as does its human orthologue, the SR protein SRm300/SRRM2 (Grainger, Barrass et al. 2009). We will present evidence for genetic interactions between CWC21 and critical residues in the U2, U5 and U6 snRNAs that suggests a role for Cwc21p in stabilising RNA interactions in the catalytic centre of the spliceosome. Cwc21p is not essential for yeast cell viability; however, we found that Cwc21p is required for an early step in sporulation. By screening intron-containing genes that are expressed in meiosis, we discovered that Cwc21p is required for splicing HRB1 transcripts. We found that HRB1 is also required during meiosis. The HRB1 intron contains an unusual branchsite sequence, TACTAATG, which when changed to the consensus branchsite sequence restores sporulation in the absence of Cwc21p. Therefore, we propose that Cwc21p promotes the expression of HRB1 during an early stage of meiosis by stabilising its pre-mRNA in the catalytic centre of spliceosome. Our study demonstrates an essential and novel function for Cwc21p during meiosis. Significantly, the corresponding C.elegans protein rsr-2, has been shown to cause severe abnormalities in germ line cells, causing sterility and embryonic lethality (Longman, McGarvey et al. 2001; Ceron, Rual et al. 2007), suggesting a role in development. Ceron, J., J. F. Rual, et al. (2007). «Large-scale RNAi screens identify novel genes that interact with the C. elegans retinoblastoma pathway as well as splicing-related components with synMuv B activity.» BMC Dev Biol 7: 30. Grainger, R. J., J. D. Barrass, et al. (2009). “Physical and genetic interactions of yeast Cwc21p, an ortholog of human SRm300/SRRM2, suggest a role at the catalytic center of the spliceosome.” RNA 15(12): 2161-73. Longman, D., T. McGarvey, et al. (2001). “Multiple interactions between SRm160 and SR family proteins in enhancerdependent splicing and development of C. elegans.” Curr Biol 11(24): 1923-33.

72 Single Molecule Visualization of the DEAH-box ATPases Prp16p and Prp22p Interacting with Spliceosomes

Eric Anderson1,4, Aaron Hoskins2, Larry Friedman3, Jeff Gelles3, Melissa Moore1,4 1 UMass Medical School, Worcester, (Massachusetts), USA, 2University of Wisconsin, Madison, (WI), USA, 3Brandeis University, Waltham, (MA), USA, 4Howard Hughes Medical Institute Pre-mRNA splicing is catalyzed by a vast and dynamic RNP complex called the spliceosome. The chemistry and fidelity of splicing are dependent upon a series of structural rearrangements mediated by trans-acting splicing factors. One key class of these factors is the DEAH-box ATPase subfamily, whose members couple ATP hydrolysis to RNP structural changes. This subfamily is typified by Prp16p, which promotes transition between the first and second step catalytic conformations of the spliceosome, and Prp22p, which facilitates spliced exon release. Both proteins have also been implicated as kinetic proofreaders that facilitate discard of aberrantly assembled splicesomes. While these roles are well documented, very little is known regarding the comings and goings of these ATPases relative to the rest of the splicing machinery. By combining yeast genetics, chemical biology and Colocalization Single Molecule Spectroscopy (CoSMoS), we have developed a toolkit for analyzing pre-mRNA splicing by single-molecule fluorescence (Hoskins, et al., Science 2011). Using these methodologies, we find that Prp22p’s interactions with the spliceosome are highly dynamic and reversible, with only one Prp22 protein molecule binding at a time. By simultaneously monitoring Prp22p and individual splicing subcomplexes, we can place Prp22p signals in context relative to specific steps in spliceosome assembly. Experiments in which both U5 snRNP and Prp22p are labeled reveal that, consistent with current models of the spliceosome cycle derived from ensemble studies, U5 recruitment precedes Prp22p binding. Co-localized Prp22p and U5 signals often disappear simultaneously, or in the order U5->Prp22p, suggesting that Prp22p may remain briefly bound to spliced exons after the remainder of the spliceosome departs. We are now working to frame Prp22 binding events relative to the binding of other trans-acting factors (e.g. Prp16) and discard pathways for aberrant spliceosomes.

Session 5A: Splicing mechanism

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

73

Structural rearrangements within human spliceosomes following exon ligation

Janine Ilagan1, Robert Chalkley2, Al Burlingame2, Melissa Jurica1 1 University of California, Santa Cruz, Santa Cruz Ca USA, 2University of California, San Francisco, San Francisco Ca USA Pre-mRNA splicing is catalyzed by a large, highly dynamic macromolecular machine called the spliceosome. Understanding the RNA/RNA and RNA/protein interactions that position the pre-mRNA substrate in the spliceosome for the two catalytic steps of splicing is key to determining the molecular mechanisms of this dynamic complex. Previously, it was shown in yeast that an extended 3’ exon is required for the DEAD/H-box protein Prp22 to promote mRNA release. We also find that in human splicing extracts a shortened 3’ exon blocks cleaved lariat intron and mRNA release to trap the spliceosome after second step chemistry. We term this complex as post-catalytic (PC). In comparison to C complex, which is blocked after first step chemistry, we find specific changes in interactions with the RNA substrate. Although protections to DNA oligo-mediated RNase H cleavage remain constant to the last 30 nt of the 5’ exon, a new crosslink forms to a ~30 kD protein in PC complex 2 nucleotides upstream of the 5’ splice site. In the 3’ exon, we see additional protection extending approximately 9 nt from the exon-exon junction in PC that is not present in C complex. In addition, 1 nt downstream of the 3’ splice site we see a much stronger crosslink to a ~250 kD protein in PC complex. These data indicate that between the two complexes, structural rearrangements exist for the substrate RNA. In order to examine differences in protein association, we used single reaction monitoring (SRM) mass spectrometry to quantitatively compare PC and C complex components. Although relative amounts of most proteins appear unchanged, we detect enrichment for SF3B proteins in PC complex, which we confirmed by western analysis. In contrast, we see loss of an RNA-dependent ATPase, DHX35. These differences likely reflect additional conformation changes in the spliceosome that occur with exon ligation. We are now using electron microscopy to explore structural differences between C and PC complex.

74 Protein composition, morphology and disassembly mechanism of the intron-lariat spliceosome from S. cerevisiae

Jean-Baptiste Fourmann1, Jana Schmitzová1, Berthold Kastner1, Henning Christian2, Henning Urlaub1, Ralf Ficner2, Patrizia Fabrizio1, Reinhard Lührmann1 1 Max-Planck-Institute of Biophysical Chemistry, Goettingen, Germany, 2Institute for Microbiology and Genetics Georg-August-University, Goettingen, Germany During each round of splicing, the spliceosome undergoes a cascade of major structural rearrangements that are driven by RNA helicases. While remodeling of the spliceosome during catalytic activation and the catalytic phase has been well studied, the protein composition and the disassembly mechanism of the post-splicing intron-lariat spliceosome (ILS) are poorly understood. We established an in vitro system which recapitulates both catalytic steps of S. cerevisiae splicing with purified components. We use the thermosensitive yeast strain prp2-1 to purify BactΔPrp2 spliceosomes via MS2 affinity-selection. The addition of Prp2, Spp2 and Cwc25 to the BactΔPrp2 complex leads to step 1 of splicing and the formation of the C complex. Step 2 is promoted by adding Prp16, Slu7 and Prp18, yielding the post-splicing complex. This system was now used to study the spliceosome disassembly process. Addition of Prp22 to matrix-bound post-splicing complexes promotes their disassembly into mRNA and a ~35S ILS that is released from the matrix. For the first time the protein composition of the ILS was characterized by mass spectrometry (MS) and its morphology by electron microscopy (EM). EM revealed that, like the C complex, the ILS exhibits a maximal length of ~40 nm but it has a thinner, elongated appearance. By adding the DEAH box NTPase Prp43 plus Ntr1 and Ntr2 to the purified ILS in solution, we show that it dissociates into the intron-lariat, the U2 and U5 snRNPs and the U6 snRNA, as analyzed by gradient centrifugation under physiological salt. MS and immunoprecipitation revealed that all known U2 proteins, including the SF3a/b complexes, as well as the U5 proteins Prp8, Brr2 and Snu114, remain stably bound to their respective snRNAs after disassembly. Remarkably, the NTC-complex proteins were associated with both the free intron-lariat and U2 snRNP. The fate of the NTC-related proteins, including Cwc2, is currently under investigation. It was suggested (Small et al., Mol Cell 06) that Prp43 collaborates with the ATPase Brr2 and the GTPase Snu114 during the disassembly of the ILS. We show that disassembly also occurs efficiently in the presence of UTP, consistent with the broad rNTP specificity of Prp43. As Brr2 is strictly ATP-specific, this suggests that in our purified system disassembly of the ISL is solely due to the action of Prp43 and that it is a highly cooperative event. Thus, our data shed light into the Prp43-mediated spliceosome disassembly process. Session 5A: Splicing mechanism

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

75

Spliceosome Dynamics in the Catalytic Center

Chi-Kang Tseng, Soo-Chen Cheng Academia Sinica, Taipei, Taiwan The spliceosome is a large and dynamic ribonucleoprotein complex, which undergoes structural remodeling throughout the entire splicing pathway. We have previously shown that the affinity-purified spliceosome, when incubated under proper conditions, can catalyze reverse splicing in both steps: reverse of the second reaction (R2) to yield splicing intermediates, and reverse of the first reaction (R1) to yield pre-mRNA. The post-catalytic spliceosome can also catalyze the hydrolytic spliced-exon reopening reaction (SER), cleaving the ligated exons at the splice junction to yield free exon 1 and exon 2. When arrested at the pre-Prp16 stage to accumulate splicing intermediates, the spliceosome can also catalyze the hydrolytic debranching reaction to yield linear intron-exon 2. All of these reactions are highly efficient without needing extra factors or ATP, suggesting that after the spliceosome is fully assembled, a large conformational rearrangement is not required to promote different catalytic reactions. Recently, we have further found that the binding of antibodies to the N-terminus or C-terminus of the component situated at the catalytic center can also direct the spliceosome to catalyze different reactions. These include antibodies to step one factor Yju2 and Cwc25, and step two factor Slu7. These results strongly suggest that various reactions catalyzed by the spliceosome are directed by different conformations of the spliceosome, which can be modulated by ionic environments of the spliceosome or by altering the structure of spliceosomal components, which bind at the catalytic center.

76 Imaging pre-mRNA splicing in living cells with single-molecule sensitivity and high temporal resolution

José Rino1, Célia Carvalho1, Tomas Kirchhausen2, Maria Carmo-Fonseca1 1 Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Portugal, 2Harvard Medical School, Boston, Massachusetts, USA How long does it take for an intron to be excised from a pre-mRNA molecule? Several studies using either electron microscopy of fixed samples or ensemble approaches, inferred splicing rates of less than 2 minutes in the cell nucleus. Here, we visualize splicing in real-time in living cells. We combined genomic integration of a single beta-globin gene in human cells, intron labelling with the MS2 technique and spinning disk confocal microscopy to directly image the kinetics of intron excision from pre-mRNA. The fluorescence intensity associated a single transcription site, which appears as a diffraction-limited object, was quantified as a function of time. Increments in the fluorescence signal result from de novo transcription of MS2-binding sites, and its disappearance reflects intron excision. These fluorescence intensity fluctuations were used to determine the intron lifetime. We also determined the number of introns present at each individual transcription site at any given time point based on the number of GFP molecules bound to intronic MS2-stem loops. The results reveal that the introns are excised within 20-30 seconds after synthesis and are immediately degraded in close proximity to the transcription site.

Session 5A: Splicing mechanism

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

77

Assembly of Bacterial Ribosomes in Cells

James R. Williamson Departments of Molecular Biology and Chemistry; The Skaggs Institute for Chemical Biology; The Scripps Research Institute, La Jolla, CA 92037 The bacterial 70S ribosome is the macromolecular machine responsible for synthesis of all cellular proteins. Biogenesis of ribosomes, which is composed of three large RNA molecules and over fifty small ribosomal proteins, accounts for about one third of the energy budget of a rapidly growing cell. The process of ribosome assembly must be efficient and coordinated, and the stoichiometric production of all of the components is tightly regulated permitting growth under a wide range of conditions. Efficient in vitro assembly reactions for the 30S and 50S ribosomal subunits have mapped the thermodynamic dependencies for ribosomal protein association, resulting in the Nomura and Nierhaus assembly maps, respectively. In our laboratory, we have been developing techniques to allow us to measure the kinetics of assembly in vitro. We have developed an isotope pulse chase assay, using 15N-labeled ribosomal proteins, that measures the rates of binding of all 20 small subunit ribosomal proteins to 16S rRNA simultaneously. The method takes advantage of quantitative analysis of 14N and 15N proteins using mass spectrometry. We have used this assay to measure the rates of binding of proteins under a variety of conditions, and in the presence of ribosome assembly factors. Most recently, we have adapted our quantitative mass spectrometry approach to monitor ribosome biogenesis directly in rapidly growing bacterial cells. A pulse of 15N medium into a culture labels the newly synthesized ribosomal proteins and assembly intermediates more rapidly than fully assembly ribosomes. Quantitation of the flux of the isotope label reveals kinetic information on the biogenesis process. We can identify a range of assembly intermediates for the 30S and 50S subunits, and quantitatively analyze the ribosomal protein content of the intermediates. In addition, we can perform proteomic studies to identify the presence of assembly cofactors associated with these intermediates. We are exploring the role of these cofactors in ribosome assembly using deletion strains and overexpression approaches. Information from all of the in vitro and cell-based approaches is being synthesized into a mechanistic framework for studying ribosome assembly.

78 Mutations Within A Conserved loop Of Human ADAR2 Affect Base Flipping Of The Target Adenosine

Ashani Kuttan, Brenda Bass University of Utah Adenosine deaminases that act on RNA (ADARs) bind double-stranded RNA (dsRNA) and deaminate adenosines to create inosines. The extent an adenosine is edited depends on its sequence context. Human ADAR2 (hADAR2) has 5’ and 3’ neighbor preferences (Eggington et al., 2011), but it is not known which amino acids mediate these preferences. We adapted a previously reported screen in yeast (Pokharel and Beal, 2006) to identify mutations in hADAR2 catalytic domain that allow editing of an adenosine within a disfavored triplet, GAC; a favored triplet, UAG, served as positive control. Hairpin substrates containing the triplets were based on the R/G editing site of glutamate receptor B. We identified seven positives that could edit GAC more than wildtype (WT) hADAR2, and a negative, T490A, that did not edit GAC and edited UAG poorly. We will present data for two mutants, E488Q and V493T, which showed maximum editing of GAC in vivo, and also for T490A; all three mutants were on a highly conserved loop close to the modeled-in adenosine. Gel shift assays confirmed that binding affinities of WT and mutants were similar with all hairpins, indicating that discrimination was not derived from differences in binding affinities. We also determined catalytic rates and performed base-flipping experiments by substituting the target adenosine with the fluorescent base analog 2-aminopurine (2-AP). In all cases, observed activity correlated with base-flipping, as measured by fluorescence intensity (FI). For example, WT hADAR2 readily deaminated the UAG substrate (k2 ~ 0.9/min), and showed an increase in FI of ~ 4.6 when mixed with a U-2AP-G substrate. T490A showed a lower FI (~3.9) for this substrate, correlating with its reduced activity. Similarly, the WT protein deaminated the GAC substrate poorly (k2 ~ 0.00044/min), with a lower FI (~ 1.9), when mixed with the G-2AP-C substrate. The E488Q mutant showed a dramatic increase in catalytic rate for the UAG substrate compared to WT (k2 ~ 7.3/min) and a corresponding increase in FI (~ 9.8) when mixed with the U-2AP-G substrate. E488Q also had enhanced activity for the GAC substrate (k2 ~ 0.026/min, FI ~ 2.6) compared to WT. Our data provide the first information on the residues important for base flipping, and point to a conserved loop as key. Unexpectedly, our data suggest that hADAR2’s preference for UAG over GAC is derived from differences in baseflipping, rather than direct recognition of the 5’ neighbor. Session 5B: Keynote & Session 5B: RNA editing & modification

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

79

A Long-Range Tertiary Interaction Affects RNA Editing In Vivo

80

Rescue of neurodegeneration in Adar deficient flies is independent of RNA editing activity

Leila Rieder1, Barry Hoopengardner2, Lee Ann Smith3, Robert Reenan1 1 Brown University, Providence, RI, USA, 2Benedictine University, Lisle, IL, USA, 3Central Connecticut State University, New Britain, CT, USA The human genome encodes for nine sodium channels, while that of Drosophila contains only one—paralytic (para). Nevertheless, transcriptional diversity is achieved via para alternative splicing and RNA editing to generate >2 million potential isoforms. Because changes in paralytic activity have dramatic behavioral and neuronal effects in the fly, Drosophila is an ideal system for studying the biological effects of post-transcriptional RNA processing. Adenosine-to-inosine RNA editing occurs in para constitutive exon 19 at three closely positioned sites, around which complex and conserved secondary, and even tertiary, structures are predicted to form in the pre-mRNA. We have elected to use homologous recombination to introduce numerous engineered mutations into the endogenous paralytic locus to test various structural predictions. In this way we can subtly alter the endogenous gene sequence and pre-mRNA structure and then carefully assess effects on RNA processing. Our structural mutations confirm the necessity for expected editing site secondary structure, as documented for other editing sites in numerous phyla. In addition, we also demonstrate a structural element that appears to act at a distance to modulate editing effectiveness via interactions with RNA splicing. Furthermore, we discovered through in vivo mutation/ countermutation that the formation of a complex conserved tertiary loop structure is absolutely required for one particular adenosine deamination in paralytic. Our results suggest a novel model for editing substrate recognition, which does not rely on the standard connectivity within a dsRNA region.

Simona Paro, Leeanne McGurk, Liam Keegan, Mary O’Connell MRC Human Genetics Unit Drosophila have a single Adar gene that converts adenosine to inosine in dsRNA. Flies deficient for this RNA editing enzyme have reduced viable; do not mate, defective locomotion and develop age-related neurodegeneration. We want to elucidate why lacking ADAR causes age-related neurodegeneration. Vacuoles are present in the brain of the complete null Adar5G1. In mutants these vacuoles are visible by day 30 in the mushroom body calyses which is involved in olfactory associative learning and memory. We find that Adar5G1 have defects in autophagy which is a pathway involved in the lysosomal degradation of cytoplasmic organelles and protein aggregates. Autophagy has been linked to human neurodegenerative disorders such as Alzheimers Disease. In a separate genetic screen for hemizygous DrosDel deletions that suppressed the reduced viability in Adar5G1 at eclosion, we find that a reduction in Tor gene dosage rescues viability. Tor is a kinase that activates translation and inhibits autophagy under well-fed conditions. Reducing Tor activity increases autophagy. We demonstrate that the rescue of Adar5G1 viability by reduced Tor dosage is due to increase autophagy. We also observe increased autophagy in the large fat cells in mutant larvae compared to wildtype larvae by staining with Lysotracker which detects acidic lysosomes. Surprisingly a P-element insertion in Tor that is viable in an Adar5G1 background rescues not only neurodegeneration, but also locomotion and viability. Therefore despite having 972 editing sites in Drosophila, an increase in autophagy alone without any editing, substantially suppresses all Adar mutant phenotypes. In addition we have found that an inactive Adar transgene rescues neurodegeneration but not locomotion. Therefore the presence of an ADAR protein but not editing activity rescues neurodegeneration.

Session 5B: RNA editing & modification

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

81

Developmental regulation of brain specific miRNAs by A-to-I RNA editing

Ylva Ekdahl1, Hossein Farahani2, Mikaela Behm1, Jens Lagergren2, Marie Ohman1 1 Stockholm University, Stockholm, Sweden, 2Royal Institute of Technology (KTH), Stockholm, Sweden. Adenosine to inosine (A-to-I) RNA editing targets double-stranded RNA stem loop structures in the mammalian brain. We have previously shown that A-to-I editing of most transcripts coding for genes involved in neurotransmission are regulated during brain development, with low levels of editing during embryogenesis and a gradual increase until adulthood. Here we have analyzed the frequency of editing in miRNAs during brain development. Editing of miRNA can either lead to alterations in target recognition, if it occurs within the crucial seed sequence, or inability of the miRNA to be further processed by Drosha or Dicer. The recent development of powerful high throughput sequencing methods has opened novel possibilities to detect low levels of editing events and to analyze short RNA sequences with the size of mature miRNAs. We show that most of the miRNAs previously known to be edited follow an increasing trend of editing throughout development with very low levels of editing in the embryo. Using RNA-Seq we also identify novel sites of editing in miRNAs. We have found that one miRNA cluster (miR379-410) is particularly targeted for regulation by editing during mouse brain maturation. In neurons, transcription of this miR379-410 cluster that contains over 50 miRNAs, is induced by the transcription factor Mef2 in a synaptic activity-dependent manner. We show that several miRNAs within this cluster are inefficiently edited or not edited at all during mouse embryogenesis, while they are highly edited in the adult brain. One example is the +6 site of miR-376b where 10% of the sequences are edited at embryonic day 15 while more than 75% of all sequences are edited in the adult brain. The edited site is situated within the seed sequence that interacts with the target mRNA. Since inosine is interpreted as guanosine (G) by the cellular machineries, the effect of A-to-I editing is a functional A to G change. A perfect match between the seed sequence of the miRNA and its target mRNA is essential. Therefore, 75% of the miR-376b sequences in the adult brain no longer recognize the same predicted targets as the non-edited form. A potential target for the non-edited miR-376b is the Pum2 mRNA, a translational repressor that inhibits dendritic outgrowth during dentritogenesis. We discovered two novel substrates for A-to-I editing (miR-381 and miR-134) that have been shown to target Pum2 in their unedited forms. Both of these are also expressed from the miR379-410 cluster. Editing of miR-381 is low during embryogenesis but increases during brain development, in a similar manner as miR-376b, so that the majority of the miRNAs are edited in the seed sequence in the adult brain. Taken together, our data imply that RNA editing regulates the miRNA repertoire during brain maturation and thereby controls dendritogenesis. Our results raise the possibility that miRNA editing regulates other fundamental processes involved in neuronal development.

82 Maturation Of H/ACA Box SnoRNAs: PAPD5-Dependent Adenylation And PARN-Dependent Trimming

Christiane Harnisch1, Heike Berndt1,3, Christiane Rammelt1, Nadine Stöhr2, Anne Zirkel2, Juliane Dohm4, Heinz Himmelbauer4, Stefan Hüttelmaier2, Elmar Wahle1 1 Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Halle, Germany, 2 Section for Molecular Cell Biology, Department of Medicine, Martin Luther University Halle-Wittenberg, Halle, Germany, 3Institute of Biology, Martin Luther University Halle-Wittenberg, Halle, Germany, 4Centre for Genomic Regulation (CRG) and UPF, Barcelona, Spain The poly(A)-specific ribonuclease, PARN, is a homodimeric 3’ exonuclease that prefers poly(A) as a substrate and is moderately stimulated by a 5’ cap on the RNA. In Xenopus, PARN has been identified as the main deadenylase regulating maternal mRNAs in oocytes. In human somatic cells, PARN is localized to the nuclei and concentrated in nucleoli and Cajal bodies, whereas mRNA deadenylation typically takes place in the cytoplasm. Using PARN knock down in combination with microarray and qRT-PCR analysis, we were able to identify various putative PARN targets, including small nucleolar RNAs and small Cajal body RNAs (snoRNAs and scaRNAs). These noncoding RNAs are subdivided into the H/ACA box and C/D box type. PARN knock-down causes the accumulation of oligoadenylated processing intermediates of H/ACA box RNAs, which is in agreement with the nucleolar localization of PARN. Oligo(A) tails are attached by the non-canonical poly(A) polymerase PAPD5 to a short stub of intron sequences remaining beyond the mature 3’ end of the H/ACA box snoRNAs. We suggest that deadenylation by PARN is coupled to clean 3’ end trimming. RRP6, which is responsible for final 3’end trimming of yeast snoRNAs, plays a minor role in H/ACA box snoRNA processing in human cell.

Session 5B: RNA editing & modification

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

83 Roquin Promotes Constitutive mRNA Deadenylation and Decay via an Abundant Stem-loop Recognition Element

Kathrin Leppek1, Johanna Schott1, Sonja Reitter1, Ming Hammond2, Georg Stoecklin1 1 German Cancer Research Centre (DKFZ), Heidelberg, Germany, 2Departments of Chemistry and of Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA, USA Tumor necrosis factor alpha (TNFα) is the most potent pro-inflammatory cytokine of the mammalian organism. Numerous posttranscriptional mechanisms control the expression of this potentially harmful cytokine, including the constitutive decay element (CDE) in the 3’UTR. The CDE mediates rapid mRNA degradation of TNFα mRNA independently of the well characterized AU-rich element. We now demonstrate that the CDE is a 17 nt long sequence that does not serve as a microRNA binding site. Rather, structural probing and mutagenesis provide evidence that it forms a short RNA stem-loop in its active conformation. In order to identify CDE-binding effector proteins, we optimized a protocol for RNP affinity purification by developing S1m, an improved streptavidin-binding RNA aptamer. Following RNP purification, proteins associated with the CDE were identified by mass spectrometry. Thereby, we found the CCCHtype zinc and RING finger proteins Roquin (Rc3h1) and Roquin2 (Rc3h2) to be CDE-binding proteins. By RNA-IP, we confirmed that Roquin and Roquin2 specifically bind to the TNFα CDE. Overexpression and knockdown analyses further showed that Roquin and Roquin2 are required for the degradation of CDE-containing mRNAs. Using a bioinformatics approach, we identified 56 highly conserved putative CDEs in the mouse transcriptome. ICOS mRNA, a known target of Roquin, also contains a functional CDE. In macrophages, we could verify that Roquin associates with the CDE-containing mRNAs encoding TNFα, Nfkbid, Nfkbiz and Ier3, but not with control mRNAs. This suggests that Roquin targets numerous transcripts including several that encode immune response regulators. Finally, we provide evidence that Roquin and Roquin2 localize in processing-bodies and promote mRNA deadenylation by recruiting the Caf1-Not deadenylase complex. This study identifies Roquin and Roquin2 as adaptor proteins that cause rapid mRNA deadenylation through the CDE, a novel class of abundant 3’UTR stem-loop recognition elements.

84 Identification of human and Drosophila 5’-nucleotidases degrading 7-methyl guanosinemonophosphate

Juliane Buschmann1, Thomas Monecke2, Bodo Moritz1, Mandy Jeske1, Thomas Rudolph3, Ralf Ficner2, Elmar Wahle1 Institute of Biochemistry and Biotechnology, Martin Luther University Halle/Wittenberg, Halle 06120, Germany, 2 Institute of Microbiology and Genetics, GZMB, Georg August University Göttingen, Göttingen 37077, Germany, 3 Institute of Biology, Martin Luther University Halle/Wittenberg, Halle 06099, Germany Turnover of RNA constantly releases mononucleotides, the majority of which is presumably recycled to NTPs for new RNA synthesis. However, mRNA decay also releases the 7-methyl G cap, incorporation of which into new RNA is not desirable. While the mRNA cap can be released by two different types of enzymes, the universal product of both pathways has been proposed to be m7GMP. We have discovered a 5’ nucleotidase that cleaves the nucleotide to m7guanosine and orthophosphate. Both the enzyme from Drosophila and its orthologue from H. sapiens have been characterized. Whereas both nucleotidases prefer m7GMP, two other human nucleotidases tested do not accept the methylated nucleotide. We propose that the m7G-specific enzymes serve in the removal of m7GMP to prevent its incorporation into RNA or, after potential conversion to the deoxynucleotide, DNA. Crystal structure analysis of a product complex shows that the nucleotidase binds the base in a hydrophobic cage, similar to other m7G binding proteins. The structure of a complex with a transition state analog is consistent with the two step reaction mechanism, via a phospho-aspartate intermediate, known from similar enzymes. Flies homozygous for a deletion of the gene encoding the m7G-specific nucleotidase are viable and fertile. More subtle phenotypes are being investigated.

1

Session 5B: RNA editing & modification

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

85

Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA

86

Unexpected Complexity of Critical Methylation Reactions in the tRNA Anticodon Loop

Hardip Patel1, Jeffrey Squires2, Marco Nousch2, Tennille Sibbritt1, David Humphreys2, Brian Parker1, Catherine Suter2, Thomas Preiss1 1 ANU, Canberra, ACT, Australia, 2VCCRI, Sydney, NSW, Australia The modified base 5-methylcytosine (m(5)C) is well studied in DNA, but investigations of its prevalence in cellular RNA have been largely confined to tRNA and rRNA. In animals, the two m(5)C methyltransferases NSUN2 and TRDMT1 are known to modify specific tRNAs and have roles in the control of cell growth and differentiation. To map modified cytosine sites across a human transcriptome, we coupled bisulfite conversion of cellular RNA with next-generation sequencing. We confirmed 21 of the 28 previously known m(5)C sites in human tRNAs and identified 234 novel tRNA candidate sites, mostly in anticipated structural positions. Surprisingly, we discovered 10,275 sites in mRNAs and other non-coding RNAs. We observed that distribution of modified cytosines between RNA types was not random; within mRNAs they were enriched in the untranslated regions and near Argonaute binding regions. We also identified five new sites modified by NSUN2, broadening its known substrate range to another tRNA, the RPPH1 subunit of RNase P and two mRNAs. Our data demonstrates the widespread presence of modified cytosines throughout coding and non-coding sequences in a transcriptome, suggesting a broader role of this modification in the post-transcriptional control of cellular RNA function. Squires et al. Nucleic Acids Res. 2012 PMID: 22344696

Michael Guy, Brandon Podyma, Eric Phizicky University of Rochester, Rochester, (NY), USA Post-transcriptional modification of the tRNA anticodon loop is critical for proper translation and growth. In yeast, 2’-O-methylation of residues 32 and 34 (Nm32 and Nm34) occurs on tRNAPhe, tRNATrp and tRNALeu(UAA), and requires Trm7 (Pintard, L., et al., 2002, EMBO J. 21:1811-1820). trm7-Δ mutants have a severe growth defect, but the cause of this defect is not known. It seems likely that Trm7 and its modifications are important throughout eukaryotes, since Nm32 and Nm34 are found widely in eukaryotes, since Trm7 is also highly conserved, and since mutations in the putative human Trm7 ortholog FTSJ1 are associated with non-syndromic X-linked mental retardation. We report here that the growth defect of trm7-Δ mutants is due to loss of methylation of tRNAPhe, because overexpression of this tRNA, but not other known Trm7 substrates, suppresses slow growth. Unexpectedly, we find that Trm7 requires a conserved, uncharacterized ORF for Nm32 modification, and a conserved WD40 repeat protein for Nm34 modification. Trm7 appears to form separate complexes with each protein cofactor. We also show that the slow growth of trm7-Δ mutants is due to lack of both Cm32 and Gm34, because the individual mutants that affect only Nm32 or Nm34 are each healthy, whereas the double mutant phenocopies a trm7-Δ mutant. However, we present genetic evidence demonstrating that each modification has a distinct role in the cell. We also demonstrate that FTSJ1 is the human ortholog of Trm7, because its expression suppresses the slow growth of trm7-Δ mutants. Remarkably, our results demonstrate that FTSJ1 recruits the yeast cofactor for formation of Cm32 on tRNAPhe, and that Trm7 can recruit the corresponding human ortholog. These results suggest that similar Trm7 interacting proteins are conserved in higher eukaryotes to direct formation of Nm32 or Nm34.

Session 5B: RNA editing & modification

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

87

Divergent Trans Editing Mechanisms used to Ensure Fidelity in Proline Codon Translation

Karin Musier-Forsyth, Mom Das, Oscar Vargas-Rodriguez, Sandeep Kumar, Christopher Hadad Ohio State University, Columbus, Ohio, USA The fidelity of protein translation relies on accurate amino acid discrimination by aminoacyl-tRNA synthetases. For example, prolyl-tRNA synthetase (ProRS) must discriminate amino acids with similar (Cys) or smaller (Ala) molecular volumes from cognate Pro. Due to the high rate of misactivation of these amino acids, the evolution of ProRSs has led to the addition of editing activities that help to prevent the misincorporation of Ala at Pro codons. In contrast, Cys, which is the same molecular volume as Pro, is not cleared by these mechanisms. We have previously shown that the Cys paradox is solved by the freestanding YbaK protein, which clears Cys-tRNAPro in trans. YbaK is one member of a “YbaK superfamily” of homologous domains that are believed to possess tRNA editing function. Thus, a “triple-sieve” editing mechanism has evolved in many bacteria, in which the aminoacylation active site of ProRS rejects larger amino acids but allows activation of Ala, Cys, and Pro. The second sieve (a distinct domain of ProRS known as INS) acts in cis to edit smaller Ala-tRNAPro, whereas the third sieve (YbaK) acts in trans to edit Cys-tRNAPro. Using biochemical and computational approaches, we have probed the substrate specificity and mechanisms of these homologous editing domains. Whereas the INS domain catalyzes hydrolysis of Ala-tRNA via a steric exclusion-based sieve whose specificity can be re-engineered by mutagenesis, the YbaK editing reaction is carried out via substrate-assisted sulfhydryl side-chain chemistry and thus represents a novel chemical sieve. Although this triple sieve model of editing applies to many bacteria, certain bacterial ProRSs lack a full-length INS, raising the question of how fidelity is maintained in these organisms. Caulobacter crescentus (Cc) is a bacterium that possesses a ProRS with a truncated INS, but encodes YbaK and PrdX, another member of the YbaK superfamily. We explored the function of the truncated INS and the catalytic activities of Cc YbaK and PrdX. Our results show that Cc ProRS can mis-acylate tRNAPro with both Ala and Cys. The severely truncated INS lacks hydrolytic activity, but its function appears to be replaced by PrdX, which acts in trans to deacylate Ala-tRNAPro. As expected, Cc YbaK is responsible for the editing of Cys-tRNAPro. Thus, C. crescentus is an example of an organism that exclusively employs trans editing domains for maintaining the fidelity of proline codon translation. Moreover, whereas INS lacks specific tRNA recognition capability, which is provided by the ProRS anticodon domain, PrdX recognizes specific elements in the tRNA acceptor stem and also collaborates with elongation factor Tu to prevent Ala-tRNAAla deacylation. The evolution of acceptor stem recognition and the discovery of an alternative triplesieve mechanism for editing highlights the diversity of approaches used by bacteria to ensure translational fidelity.

88 No Target Left Behind: A Microfluidics Solution for Standardized Partitioning in Aptamer Selections

Christina Birch, Han Wei Hou, Jongyoon Han, Jacquin Niles Massachusetts Institute of Technology, Cambridge, MA, USA Since its introduction in 1990, the systematic evolution of ligands by exponential enrichment (SELEX) strategy has been used to generate RNA and DNA aptamers with high target affinity and specificity to a wide variety of small molecule, protein, and whole-cell targets. These aptamers are generally obtained after many rounds of laborious, time-intensive selection. Arguably the most important phase of any SELEX round is the partitioning of target-binding aptamers from a background of 1014-1015 non-interacting sequences. The partitioning method can determine the success or failure of the selection and governs how quickly a desired solution is attained. Despite all of the variations and modifications made to the basic underlying SELEX protocol, successful aptamer selection is still considered an art. The most significant advances to the field have incorporated modern technologies, such as capillary electrophoresis, deep sequencing, magnetic microfluidics, and automated manipulation, with the goal of improving the reliability of the selection process. A major limitation of these methods is their inability to provide a highly efficient, standardized partitioning that does not require modification of the target’s natural state. Whole-cell selections, in particular, have a sparse toolkit of biocompatible library partitioning techniques. By applying the unique physical properties and conveniences of microfluidic devices to the demands of whole-cell selections, we aim to augment the SELEX practitioner’s ability to efficiently and reliably generate aptamers to challenging unmodified targets. To this end, we have designed an easy-to-use inertial microfluidic device capable of separating unmodified whole cells and surface-bound aptamers from nonbinding sequences in solution. We validated the device by constructing a novel cell-surface protein and aptamer system that enabled us to quantify aptamer recovery and partitioning efficiency. Our inertial microfluidic device is able to discriminate cell-surface binding aptamers from a nonbinding background library with reproducibly high efficiency competitive with the best current strategies. We present this standardized partitioning method as an inexpensive, simple-to-use and robust tool that can be tailored for SELEX against a wide array of molecular and cellular targets.

Session 5B: RNA editing & modification & Session 6A: Aptamers

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

89 New Aptamers, Neutral Networks, and Next-generation Sequencing: A Fresh Look at HIV Reverse Transcriptase Aptamers

Mark Ditzler1, Debojit Bose1,2, Christopher Bottoms3, Katherine Virkler1, Andrew Sawyer4, Angela Whatley1, William Spollen3, Scott Givan3, Donald Burke1,4 1 Molecular Microbiology and Immunology, University of Missouri, Columbia, MO, USA, 2Proteomics and Structural Biology Unit, Institute of Genomics and Integrative Biology, CSIR, Delhi, India, 3Informatics Research Core Facility, University of Missouri, Columbia, MO, USA, 4Department of Biochemistry, University of Missouri, Columbia, MO, USA Systematic Evolution of Ligands through Exponential Enrichment (SELEX) generates nucleic acid populations that are enriched for specified functions. While standard cloning-based sequencing methods have proven successful in extracting valuable information from these populations, the small size of the data sets places significant limitations on their ability to fully describe the enriched populations. High-throughput sequencing provides an opportunity to identify important features of these populations that are lost in the noise of low-throughput sequencing. Here, we use high-throughput comparative sequence analysis to evaluate RNA populations selected to bind HIV-1 reverse transcriptase (RT). The populations are enriched in RNAs of independent lineages that converge on shared motifs, as well as RNAs with nearly identical sequences that share common ancestry; we exploit both of these features to predict the secondary structures of enriched RNAs, the RNAs’ minimal structural requirements for binding, and the nature of the RNA-protein complexes. The statistical power afforded by sequencing these populations in depth reveals how the populations respond to changes in selection pressure. Monitoring population dynamics while applying increasingly stringent selection pressure enables direct comparison of the relative binding characteristics of the RNAs within the population and the identification of RNA inhibitors of RT that are more potent than those identified previously. A detailed evaluation of diversity within the converged motifs reveals structural and functional details that are otherwise obscured by simple consensus descriptions. Structural and functional predictions derived from this high-throughput sequence analysis are supported biochemically, thus validating our high-throughput comparative sequence analysis pipeline for evaluating SELEX populations.

90 Aptamer Structure, Dynamics and Function as Investigated by Integrative Computational and Experimental Approaches

Tianjiao Wang, Monica Lamm, Julie Hoy, Bruce Fulton, Mazdak Mina, Marit Nilsen-Hamilton Iowa State University With the view that both structure and dynamics are essential elements of aptamer function, we are taking an integrated computational and experimental approach to understanding the interaction of aptamers and their ligands, which includes molecular dynamics (MD) simulations and biochemical and biophysical analyses. Using the malachite green aptamer (MGA) as a model system, we demonstrate the effectiveness of this approach by explaining the structural basis of the ability of the MGA to protect its ligand from oxidation. The observation that chemical reactivity can be controlled by an aptamer has potential in controlling metabolism and sensing metabolites in vivo. We also show that the MGA undergoes a global conformational change upon binding its ligand. This prediction from atomistic simulations was confirmed experimentally and reveals a mechanism by which this aptamer can convey a structural change upon binding its ligand that can be applied in developing engineered RNA constructs for sensors and other applications. The results of these studies demonstrate how an investigative approach that integrates computational and experimental studies is an effective means of revealing atomistic details of aptamer structure, dynamics and function

Session 6A: Aptamers

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

91

A Systematic Approach to Evolve Aptamers with New Specificities

92

Orthogonal Riboswitches As Tools For Controlling Gene Expression In Bacteria

Muslum ILGU1, Ragothaman Yennamalli2, Megan Kleckler1, Taner Sen1, Monica Lamm1, Marit Nilsen-Hamilton1 1 Iowa State University, 2University of Wisconsin Aptamers are single-stranded nucleic acids with high affinities and specificities for the targets against which they are selected. Both features, along with an ability to be integrated into a large variety of sensors, make possible a wide-range of aptamer applications. However, changing aptamer specificity and/or affinity generally requires additional rounds of selection. To eliminate this secondary selection, we are exploring a novel systematic approach in which we combined wet-lab experiments with molecular docking and molecular dynamics (MD) simulations to “evolve” aptamers with altered properties. The initial studies were performed with the neomycin aptamer and 11 aminoglycoside ligands. With the exception of one ligand (ribostamycin), the energy scores obtained using docking analysis were in good agreement with experimental values obtained by isothermal titration calorimetry (ITC). Parallel, molecular dynamics (MD) simulations of the neomycin aptamer without its ligand using GROMACS showed a mobile structure consistent with the ability of this aptamer to interact with a wide range of ligands. From molecular docking and MD simulations, we identified the neomycin aptamer residues that might contribute to its ligand selectivity and designed a series of new aptamers accordingly. Through this systematic approach, we have obtained a variety of aminoglycoside aptamers with different selectivities and specificities. We believe that this approach can be applied to develop other aptamers of desired specificity and affinity.

Christopher Robinson, Neil Dixon, John Duncan, Torsten Geerlings, Ming-Cheng Wu, Phillip Lowe, Jason Micklefield School of Chemistry and Manchester Interdisciplinary Biocentre, The University of Manchester, Manchester, UK. Small-molecule inducible gene expression systems have many applications in functional genomics, recombinant protein expression, synthetic biology and metabolic engineering. However, our ability to independently control the expression of multiple genes has been hampered by the lack of tunable expression systems which are compatible within the same cell. Riboswitches are relatively simple, protein-free, small-molecule dependent genetic switches that are attractive targets for re-engineering in this context. Using a combination of chemical genetics and genetic selection, we have developed new mRNA regulatory elements based on the add A-riboswitch from Vibrio vulnificus. These mutant ON-riboswitches control protein expression through a translational mechanism, and are activated by specific synthetic small-molecules, whilst no longer responding to natural intracellular metabolites. The orthogonal selectivity of these riboswitches is demonstrated both in vivo, through gene expression assays, and in vitro, using isothermal titration calorimetry and X-ray crystallography. The mutually-orthogonal riboswitches can be deployed in the same bacterial cell to independently control co-expression of multiple genes in a dose-dependent manner; allowing for convenient access to highly dynamic expression landscapes and desirable protein stoichiometries. Following on from this initial success, we have subsequently developed orthogonal OFF-riboswitches, based on the transcriptionally acting PreQ1 riboswitch from the queC gene of Bacillus subtilis. This approach could be further adapted to re-engineer a wide range of natural riboswitches in both prokaryotes and eukaryotes, promising an unprecedented array of new small-molecule responsive genetic switches.

Session 6A: Aptamers

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

93

Engineered mRNA Regulation Using an Inducible Protein-RNA Aptamer Interaction

94

Human adenosine aptamers

Brian Belmont, Stephen Goldfless, Jessica Liu, Jacquin Niles Massachusetts Institute of Technology, Cambridge, MA, USA The importance and pervasiveness of naturally occurring regulation of RNA function in biology is increasingly being recognized. A common regulatory mechanism uses inducible protein-RNA interactions to shape diverse aspects of cellular RNA fate. Recapitulating this using a novel set of protein-RNA interactions is appealing given the potential to subsequently modulate RNA biology in a manner decoupled from normal cellular physiology. We describe a ligandresponsive protein-RNA interaction module that can be used to target a specific RNA for subsequent regulation. Using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, RNA aptamers binding to the bacterial Tet Repressor protein (TetR) with low- to sub- nanomolar affinities were identified. This interaction is reversibly controlled by tetracycline in a manner analogous to the interaction of TetR with its cognate DNA operator. Aptamer minimization and mutational analyses support a functional role for conserved sequence and structural motifs in TetR binding. We illustrate the utility of this chemically-inducible RNA–protein interaction to directly regulate translation and RNA subcellular localization in the model eukaryote, Saccharomyces cerevisiae. By genetically encoding TetRbinding RNA elements into the 5’-untranslated region (5’-UTR) of a given mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs. In endogenous and synthetic 5’-UTR contexts, this modular system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from non-functional RNA–TetR interactions. We also demonstrate engineering this TetR-aptamer module to regulate subcellular mRNA localization. This is efficiently achieved by fusing TetR to proteins natively involved in localizing endogenous transcripts, and genetically encoding TetR-binding RNA aptamers into the target transcript. Using this platform, we achieve tetracycline-regulated enhancement of target transcript subcellular localization. We also systematically examine some rules for successfully forward engineering this RNA localization system. Altogether, these results define and validate an inducible protein-RNA interaction module that incorporates desirable aspects of a ubiquitous mechanism for regulating RNA function in Nature and that can be used as a foundation for functionally and reversibly controlling multiple fates of RNA in cells.

Michael Vu, Nora Jameson, Stuart Masuda, Dana Lin, Rosa Larralde-Ridaura, Andrej Luptak University of California, Irvine Aptamers are structured macromolecules in vitro selected to bind molecular targets and in nature form the ligandbinding domains of riboswitches. Adenosine aptamers of a single structural family were discovered several times in random libraries but not in genomic sequences. We used two unbiased methods, structure-based bioinformatics and human genome-based in vitro selection, to identify three human aptamers that form the same adenosine-binding structure. Two of the aptamers map to introns of annotated genes, RAB3C and FGD3, which code for a GTPase and a guanine nucleotide exchange factor, respectively. The RAB3C aptamer binds ATP with dissociation constants about ten times lower than physiological ATP concentration, while the minimal FGD3 aptamer binds ATP only co-transcriptionally. Our data suggest that these human aptamers may be ATP-sensing modules of kinetically controlled riboswitches.

Session 6A: Aptamers

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

95

Global Analysis of the Nuclear Processing of Unspliced U12-type Introns by the Exosome

96

tRNA Splicing Endonuclease: Investigating Cytoplasmic Function Unrelated to tRNA Splicing

Elina Niemela1, Ger Pruijn2, Mikko Frilander1 1 University of Helsinki, Helsinki, Finland, 2Radboud University Nijmegen, Nijmegen, The Netherlands U12-type introns have earlier been shown to splice at a slower rate compared to the major U2-dependent pathway. This suggests a rate-limiting regulatory function for the minor spliceosome in the nuclear processing of transcripts containing U12type introns (Patel et al. 2002). In support to this model, an elevated level of unspliced U12-type introns have been detected in the steady-state RNA populations in various organisms (Patel et al. 2002; Pessa et al. 2006; Pessa et al. 2010) However, both the mechanism of slower splicing kinetics and the fate of mRNAs containing unspliced U12-type introns is unknown. Here we have analyzed globally the inactivation of the exosome by knockdown of either the Rrp41 or Dis3 subunits using SOLiD RNA sequencing technology. We hypothesized that pre-mRNAs containing unspliced U12-type introns are degraded by the exosome complex in the nucleus. Following the knockdown we fractionated the the cells to nuclear and cytoplasmic pools and subjected the isolated total RNA preparations to deep sequencing. The sequencing libraries were constructed from RNA pools depleted of ribosomal RNAs to allow sequencing of nonpolyadenylated and noncoding RNAs. We obtained on average 30 (cytoplasmic) to 120 (nuclear) million mapped cellular compartment-specific reads per sample allowing the detection of unspliced pre-mRNAs. Consistent with our hypothesis, we found that exosome inactivation stabilizes unspliced U12-type introns in the nuclear fraction, while U2-type introns in the same genes are less affected. We observe that the bulk effect on intron retention levels and gene expression changes correlate between the two knockdowns, but we also detect effects that are gene specific to either Rrp41 or Dis3 knockdown. This suggests that the individual subunits of the exosome may have overlapping but nonidentical target pools. Patel AA, McCarthy M, Steitz JA. (2002) The splicing of U12-type introns can be a rate-limiting step in gene expression. EMBO J. 21:3804-15. Pessa HK, Ruokolainen A, Frilander MJ. (2006) The abundance of the spliceosomal snRNPs is not limiting the splicing of U12-type introns. RNA. 12:1883-92. Pessa, H.K.J., Greco, D., Kvist, J., Wahlström, G., Heino, T.I., Auvinen, P., and Frilander, M.J. (2010). Gene Expression Profiling of U12-Type Spliceosome Mutant Drosophila Reveals Widespread Changes in Metabolic Pathways. PLoS ONE 5, e13215. Nripesh Dhungel, Anita Hopper The Ohio State University, Columbus, (OH), USA Pre-tRNA splicing is an essential process in all eukaryotes. In yeast and vertebrates the enzyme catalyzing intron removal from pre-tRNA is an essential heterotetrameric complex (SEN complex). Sen2 and Sen34 are the catalytic subunits that catalyze 5’ and 3’ cleavage, respectively, whereas the functions of the other two subunits, Sen15 and Sen54 are not very well understood. Although the SEN complex is conserved, the subcellular location where pre-tRNA splicing occurs is not. In yeast, the SEN complex is located at the cytoplasmic surface of mitochondria, whereas in vertebrates pre-tRNA splicing is nuclear. To understand the differences in localization of the SEN complex, we devised a two-prong strategy. First (Dhungel & Hopper, 2012), we engineered yeast that express nuclear tRNA SEN, essentially mimicking the vertebrate cell biology of tRNA splicing and demonstrate that all three steps of pre-tRNA splicing, as well as tRNA nuclear export and aminoacylation occur efficiently when the SEN complex is nuclear. However, nuclear pre-tRNA splicing fails to complement growth defects of cells with defective mitochondrial-located splicing, suggesting that the yeast SEN complex surprisingly serves a novel and essential function in the cytoplasm that is unrelated to tRNA splicing. Furthermore, our studies show that the novel function requires all four SEN complex subunits and the catalytic core. A subset of pre-rRNAs accumulates when the SEN complex is restricted to the nucleus, indicating that the SEN complex moonlights in rRNA processing and may or may not be the essential cytoplasmic function. Second, we investigated interactions and assembly of the heterotetrameric subunits in vivo in order to understand the differences in location of the tRNA SEN between yeast and vertebrates. We utilized a co-transformation strategy to investigate subunit interactions. To assess the ability of subunits to interact with and translocate each other, one subunit N-terminally tagged with NLS and C-terminally tagged with mCherry localizing to the nucleus was co-transformed with another subunit C-terminally tagged with GFP localizing to the endogenous location at mitochondria. The studies confirm known subunit interactions in addition to demonstrating a novel Sen15-Sen15 dimer in vivo. Since the dimer is not a part of the tRNA splicing heterotetramer and Sen15 is not a catalytic subunit for tRNA splicing, the dimer may also have other roles unrelated to tRNA splicing. Thus, our findings collectively suggest that selection for the subcellular distribution of the SEN complex and SEN subunits may reside not in canonical, but rather in novel activities. Session 6B: Surveillance & decay

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

97

Turnover of Pre-mRNA Splicing Intermediates by a Novel Debranching Enzyme

98

Role of TUT3 in the Initial Step of Histone mRNA Degradation

Jay Hesselberth1, Stephen Garrey1, Masad Damha2, Stanley Fields3,4, Adam Kotolik2 1 University of Colorado School of Medicine, Aurora, CO, 2McGill University, Montreal, Canada, 3University of Washington, Seattle, WA, USA, 4Howard Hughes Medical Institute Branched RNAs formed during pre-mRNA splicing are degraded via specialized pathways. We have identified and characterized a novel component of the spliceosome, Drn1/Ygr093w, in the budding yeast S.cerevisiae. Drn1 and its orthologs are conserved through humans, and have previously been identified in spliceosomal subcomplexes. Drn1 is homologous to the lariat debranching enzyme Dbr1, and displays 2’-5’ phosphodiesterase activity in vitro against synthetic branched and lariat substrates. Mutational analysis of Drn1 indicates that the Dbr1-like domain is dispensable for debranching in vitro and in vivo, whereas conserved residues in the C-terminal CwfJ domains are required for debranching activity. Drn1 is required for the turnover of a subset of pre-mRNA splicing intermediates in vivo, and co-purifies with branched RNA intermediates from in vitro splicing extracts. We have performed CLIP-seq analysis of Drn1 and Dbr1, and found both common and distinct targets of these two debranching enzymes in vivo. These studies define Drn1 as a conserved, previously unidentified debranching activity present during pre-mRNA splicing.

Patrick Lackey, Michael Slevin, Shawn Lyons, William Marzluff University of North Carolina, Chapel Hill, NC, USA Replication-dependent histone mRNAs end in a 3’ stem-loop (SL) rather than a poly (A) tail. This unique 3’ end is the cis-element responsible for the bulk of cell cycle regulation of the histone mRNA. It binds directly to a single protein, the stem-loop binding protein (SLBP), and the SL/SLBP complex interacts with a novel set of factors to regulate histone mRNA metabolism, functioning in the synthesis, translation and degradation of histone mRNA. SLBP binds to the stem-loop on nascent pre-mRNA and is required for all of the major events in the histone mRNA life cycle. Histone mRNAs are rapidly degraded at the end of S-phase and when DNA replication is inhibited in S-phase cells. Two unique aspects of this rapid degradation are the requirement for Upf1 and the addition of an oligo(U) tail to the 3’ end of the histone mRNA. Degradation occurs both 5’ to 3’ and 3’ to 5’, and the oligo(U) tail provides a binding site for the Lsm1-7 complex, which in turn provides a binding site for the recruitment of decapping and other decay factors. We have detected two distinct oligouridylation steps in histone mRNA degradation. The first is oligouridylation of the 3’ end of the mRNA, triggered by inhibition of DNA replication. We also found additional uridylated degradation intermediates (a result of partial degradation of the mRNA 3’ to 5’). These intermediates are capped, suggesting that a subset of histone mRNA molecules is degraded completely 3’ to 5’. The enzyme that adds the oligo(U) tail has not been definitively identified but previous work by our lab suggests that a non-canonical poly(A) polymerase(s), which includes enzymes known as terminal uridylyltransferases (TUTases), may be responsible for this oligouridylation. Knockdown of TUT3 slows the degradation of histone mRNAwhen DNA replication is inhibited1. We have found that TUT3 binds directly to SLBP in vitro. A region of TUT3 near the C-terminus of the protein, clearly distinct from the presumed catalytic domain, interacts with SLBP, consistent with the possibility that TUT3 is responsible for the initial oligouridylation at the 3’ end of the mRNA. It is possible that additional TUTase(s) are involved in the oligouridylation of the3’ to 5’ degradation intermediates. 1. T. E. Mullen and W. F. Marzluff, Genes Dev. 22, 50 (2008).

Session 6B: Surveillance & decay

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

99

Air Proteins Control Differential TRAMP Substrate Specificity for Nuclear RNA Surveillance

100

Human Pumilio proteins recruit CCR4-POP2 deadenylases to efficiently repress mRNAs

Karyn Schmidt, Zhenjiang Xu, David Mathews, J. Scott Butler University of Rochester, Rochester, (NY), USA RNA surveillance systems function at critical steps during the formation and function of RNA molecules in all organisms. The RNA exosome plays a central role in RNA surveillance by processing and degrading RNA molecules in the nucleus and cytoplasm of eukaryotic cells. The exosome functions as a complex of proteins composed of a 9-member core and two ribonucleases. The identity of the molecular determinants of exosome RNA substrate specificity remains an important unsolved aspect of RNA surveillance. In the nucleus of Saccharomyces cerevisiae, TRAMP complexes recognize and polyadenylate RNAs, which enhances RNA degradation by the exosome and may contribute to its specificity. TRAMPs contain either of two putative RNA binding proteins, Air1 or Air2. Previous studies suggested that these proteins function interchangeably in targeting the poly(A)-polymerase activity of TRAMPs to RNAs. To better define the specificities of the TRAMP complexes, we used phenotypic analysis and RNA deep-sequencing technology to measure differences in global RNA polyadenylation in air mutants, revealing specific requirements for each Air protein in the regulation of the levels of non-coding and coding RNAs. Air2 functions in the regulation of transcripts encoding proteins involved in carbon metabolism and iron transport, and is specifically required for turnover of many snoRNAs. Loss of Air1, however, results in plasmid inheritance defects of the endogenous 2-micron plasmid. These findings reveal differential functions for Air proteins in RNA metabolism and indicate that they control the substrate specificity of the RNA exosome.

Jamie Van Etten3, Trista Schagat3,1, Joel Hrit3, Chase Weidmann3, Justin Brumbaugh2, Josh Coon2, Aaron Goldstrohm3 1 Promega Corporation, Madison, WI USA, 2Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA, 3Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA PUF proteins are a conserved family of eukaryotic RNA binding proteins that regulate specific mRNAs to control diverse biological processes including stem cell proliferation, fertility and memory formation. PUFs repress protein expression from their target mRNAs. The mechanism of PUF repression remains unclear, especially for human PUFs. Humans possess two related PUF proteins, PUM1 and PUM2, which exhibit identical RNA binding specificities. Here we report new insights into their regulatory activities and mechanisms of action. We developed cell-based functional assays to measure sequence specific repression by PUM1 and PUM2. Both PUM1 and PUM2 act redundantly to robustly inhibit translation and promote mRNA degradation. We engineered each PUM to bind to a new mRNA sequence and demonstrate efficient repression by each individual PUM. Purified PUM1 and PUM2 complexes were analyzed by mass spectrometry, resulting in identification of subunits of the CCR4-NOT (CNOT) complex. The CNOT complex contains multiple enzymes related to CCR4 and POP2 that catalyze mRNA deadenylation - the 3’ exonucleolytic degradation of poly-Adenosine tails. We confirmed the interaction of PUMs with the CNOT deadenylase subunits by co-immunoprecipitation. These interactions are not dependent on RNA, indicating that they are mediated by protein contacts. Indeed, we demonstrate direct interactions between recombinant PUMs and CNOT deadenylases in vitro. To demonstrate the importance of CNOT deadenylases for PUM repression, we utilize three approaches: First, dominant negative mutants of CNOT7 and CNOT8 specifically reduce PUM repression. Second, RNA interference depletion of the deadenylases alleviates PUM repression. Third, the poly (A) tail is shown to be necessary for maximal PUM repression. These findings highlight an evolutionarily conserved mechanism of PUF mediated repression via direct recruitment of the CCR4-POP2-NOT deadenylase complex by PUFs to target mRNAs, leading to translational inhibition and accelerated mRNA degradation.

Session 6B: Surveillance & decay

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

101 40S Subunit Dissociation and Proteasome-dependent RNA Degradation in Nonfunctional 25S rRNA Decay

Makoto Kitabatake, Kotaro Fujii, Tokie Sakai, Tomoko Sakata, Mutsuhito Ohno Institute for Virus Research, Kyoto, Japan Eukaryotic cells have quality control systems that eliminate nonfunctional rRNAs with deleterious mutations (nonfunctional rRNA decay, NRD). We have previously reported that 25S NRD requires an E3 ubiquitin ligase complex, which is involved in ribosomal ubiquitination. However, the degradation process of nonfunctional ribosomes has remained unknown. Here, using genetic screening, we identified two ubiquitin-binding complexes, the Cdc48–Npl4–Ufd1 complex (Cdc48 complex) and the proteasome, as the factors involved in 25S NRD. We show that the nonfunctional 60S subunit is dissociated from the 40S subunit in a Cdc48-complex-dependent manner, before it is attacked by the proteasome. When we examined the nonfunctional 60S subunits that accumulated under proteasome-depleted conditions, the majority of mutant 25S rRNAs retained their full length at a single-nucleotide resolution. This indicates that the proteasome is an essential factor triggering rRNA degradation. We further showed that ribosomal ubiquitination can be stimulated solely by the suppression of the proteasome, suggesting that ubiquitin–proteasome-dependent RNA degradation occurs in broader situations, including in general rRNA turnover.

102

Driving Innovation by Forward and Reverse Translation with Aptamers

Bruce Sullenger, Shahid Nimjee, Sabah Oney, Kristin Bompiani, Jaewoo Lee, Eda Holl, Shashank Jain, George Pitoc, Kam Leong Department of Surgery, Duke University Medical Center, Durham, (NC), 27720 Thrombosis remains the major cause of death and disability in the western world. We will describe our recent efforts to develop RNA aptamers to control thrombosis by targeting coagulation factors and platelet proteins. We will present recent preclinical and clinical studies evaluating the ability of aptamers targeting individual or combinations of coagulation factors. We have observed that combinations of aptamers that target multiple steps in the coagulation cascade are extremely potent inhibitors of clotting. We will also present recent work demonstrating that aptamers targeting VWF are potent antithrombotic agents in mammalian models of stroke. However as expected from studies on VWF knock out mice and humans lacking VWF, such VWF aptamers also engender significant bleeding when animals are surgically challenged. To control such bleeding in the surgical setting, we also will describe the development of two different antidote molecules that counteract aptamer activity. Furthermore, we will discuss results indicating that one of these types of antidotes, nucleic acid scavengers, have intrinsic anti-thrombotic activity themselves as well as anti-inflammatory properties. Thus we believe that nucleic acid scavengers may represent novel therapeutic agents to combat thrombotic and inflammatory diseases caused by release of nucleic acids into the extracellular space.

Session 6B: Surveillance & decay & Session 7: Keynote

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

103

Decreased Transcription End Sites for SETX Knockdown in Motor Neuron Progenitors

Matthew Hansen1, Lulu Tsao2, Nebiyou Samuel2, Edgar Ibarra2, Ebone Ingram2, Tzu-Ying Chuang3, Ben Greenberger2, Rachel Moda2, Rob Kulathinal1, Miriam Bucheli4 1 Temple University, Philadelpha, PH, 2Harvard University, Cambridge, MA, 3University of Virginia, 4San Francisco University of Quito Two neurodegenerative disorders result from mutations in the Senataxin (SETX) gene, an autosomal recessive Ataxia oculomotor apraxia type 2 and a dominant juvenile form of Amyotrophic Lateral Sclerosis. SETX, a DEAxQ-box DNA/ RNA helicase and the human ortholog of yeast Sen1, is involved in transcription termination and RNA maturation. Depletion of SETX causes an RNA polymerase II-dependent transcription termination defect described as “readthrough”. Further transcriptional characterization recently revealed a specific role for SETX concerning “pause site”-dependent termination. The helicase activity of SETX was shown to be essential for the resolution of RNA/DNA hybrid structures (R-loops) that form behind elongating RNA polymerase II (RNAPII) at pause sites located downstream of polyA signals. Exonuclease Xrn2 degradation is required in the termination mechanism and is thus dependent on the unwinding activity of SETX. Sen1 functions in the termination of non-coding RNAPII transcripts, snRNAs and small nuclear (sno) RNAS. Currently, the genomic targets of SETX are unknown. We performed a transcriptome characterization using a murine system of stem cells differentiated in a lineage-committed pathway to motor neurons. To identify those genomic targets specific to SETX, we used a gene trap ES cell line harboring an insertion mutation in the SETX gene. We show that transcription end sites (TES) in the SETX knockdown cell lines from different stages of the differentiation are significantly reduced in comparison to the controls. Additional analysis focuses on genes specifically expressed in motor neurons and are further marked with a decrease in TES. Particularly in neurons and cell lineage differentiation, proper 3’end transcription may be critical for the production of functional mRNAs. Our data provides evidence that SETX plays an important role in the regulation of TES in neuron-specific genes.

104 Extensive and dynamic heteroallelic expression and RNA editing in human blood cells using high-throughput RNA sequencing

Jennifer Li-Pook-Than, Rui Chen, George Mias, Lihua Jiang, Hugo Lam, Hua Tang, Michael Snyder Stanford University The plethora of information that we gained from high-throughput RNA sequencing (RNAseq) was integrated with both genomic and proteomic (mass-spectrometry) data of peripheral blood mononuclear cells (PBMCs) in an individual (54 year old male). We obtained an average of 123 million mapped transcriptomic reads for over 20 time points (including 2 infection states) collected over a year (>400 days). This study focused on allele specific gene expression (ASE), as well as the phenomena of RNA editing. RNA editing is a post-transcriptional event, usually from a deamination process: cytidine to uridine (C-to-U) or adenosine to inosine (A-to-I) conversion. To correctly identify RNA variants, we developed the RIT(E)2-seq pipeline (RNA Identifier Tool for Expression and Editing) which compared RNA information with the respective individual’s genome (~3.7 million called SNPs).The typical gamut of edits (100,000s) that were first predicted are whittled down to a high confidence range (~2000) as high instances of false positives are removed; namely from intrinsic platform errors, and misalignment to multigene-regions and pseudogenes. Unusual edited sites and modified nucleotides were found, suggesting a novel amination process. Subsets of these were corroborated with digital droplet PCR and respective proteomic data. This analysis was part of an integrative Personal Omics Profiling (iPOP) proof-ofprincipal project whereby, for the first time, a fully comprehensive genotype-to-phenotype whole-omics profiling analysis (genome, transcriptome, proteome, metabolome) was performed. This iPOP approach showed the value of studying the complex transcriptome isoforms (differentiating over time at the allelic and editing expression level) which correlated variants with phenotype, captured health state and risk of an individual, as well as gave insights into human diversity studies.

Session 7: RNA & disease

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

105

Combinatorial Splicing Regulation by Muscleblind-like Proteins in Development and Disease

Kuang-Yung Lee1,2, Moyi Li1, Mini Manchanda1, Lily Shiue3, Chris Chamberlain 4, Apoorva Mohan1, Hannah Hong1, Manuel Ares, Jr3, Maurice Swanson1 1 University of Florida, Gainesville, (FL), USA, 2Chang-Gung Memorial Hospital, Keelung, Taiwan, 3University of California, Santa Cruz, (CA), USA , 4University of Minnesota, (MN), USA The muscleblind-like (MBNL) proteins regulate the alternative splicing of hundreds of genes during development and loss of MBNL splicing activity is a key pathogenic feature underlying the neuromuscular disease myotonic dystrophy (DM). Since the MBNL gene family consists of three paralogs, which vary in temporal and spatial tissue expression patterns, we have proposed that the multi-systemic disease manifestations of DM result from combinatorial loss of MBNL proteins. To test this hypothesis, we have generated conditional Mbnl1, Mbnl2 and Mbnl3 knockout (KO) mice and studied the effects of single and multiple Mbnl knockouts on embryonic and postnatal development. Splicing microarrays, RNA-seq and HITS-CLIP analyses demonstrate that the Mbnl1 and Mbnl2 proteins regulate hundreds of splicing events in skeletal muscle and the brain, respectively, via the recognition of a similar RNA core sequence motif, YGCY. While Mbnl1 KOs develop DM-associated muscle and eye pathologies, loss of Mbnl2 expression does not significantly affect muscle function but instead has a profound impact on the brain with spatial learning/memory deficits on a hippocampaldependent task, a decrease in NMDAR synaptic transmission, impaired hippocampal synaptic plasticity and REM sleep abnormalities. Mbnl1-/-; Mbnl2-/- double KOs are embryonic lethal but Mbnl1-/-; Mbnl2+/- are viable and show enhanced pathological phenotypes in skeletal and cardiac muscle suggesting that Mbnl2 partially compensates for Mbnl1 loss in these tissues. The molecular basis of this compensatory function will be discussed.

106 Structure Optimization of a Family of Compounds Identifies One Compound that Reverses the Hallmark Symptom of Myotonic Dystrophy in a Mouse Model and Reveals a Novel Mechanism of Action

Leslie Coonrod1, Masayuki Nakamori2, Cameron Hilton1, Micah Bodner1, Michael Haley1, Charles Thornton2, Andrew Berglund1 1 University of Oregon, Eugene, OR, USA, 2University of Rochester, Rochester, NY, USA Myotonic dystrophy (DM) is one of the most common forms of muscular dystrophy, characterized by its hallmark symptom myotonia. DM is an autosomal dominant disease caused by a toxic gain of function RNA. The toxic RNA is produced from expanded non-coding CTG/CCTG repeats, and these CUG/CCUG repeats sequester various RNA binding proteins. The Muscleblind-like (MBNL) family of RNA binding proteins are sequestered to the expanded CUG/CCUG repeats. The MBNL proteins are regulators of alternative splicing, and their sequestration has been linked with missplicing events in DM. Previously in a limited screen of known nucleic acid binding molecules we identified pentamidine as compound able to rescue splicing defects associated with DM. In this study, analysis of a series of methylene linker variants of pentamidine revealed that heptamidine can reverse splicing defects in a DM1 tissue culture model at lower doses compared to pentamidine and rescues myotonia in a myotonic dystrophy mouse model. We had original proposed that pentamidine worked by releasing the sequestered MBNL proteins from the CUG repeats in the DM tissue culture and mouse model. Our recent data suggests that pentamidine and its analogues function through other mechanisms. We have found in tissue culture that pentamidine significantly reduces the level of CUG repeat RNA but not the level of controls RNAs. Our current working model is that pentamidine inhibits transcription elongation through repeat DNA. The generality of our results for other CTG and CAG repeat diseases such as Huntington’s disease will be discussed.

Session 7: RNA & disease

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

107 Loss-of-function Analysis of the ALS-associated Proteins Tdp-43 and Fus/Tls Reveals Global RNA Mis-regulation that is Conserved from Mouse to Humans

Kasey Hutt1, Magdalini Polymenidou1, Clotilde Lagier-Tourenne1, Anthony Vu1, Stephanie Huelga1, Sebastian Markmiller1, Edward Wancewicz2, Curt Mazur2, Yalda Sedaghat2, John Paul Donohue3, Lily Shiue3, C Frank Bennett2, Don Cleveland1, Gene Yeo1 1 University of California San Diego, La Jolla, California, USA, 2ISIS Pharmaceuticals, Carlsbad, California, USA, 3 University of California Santa Cruz, Santa Cruz, California, USA TDP-43 and FUS/TLS are RNA-binding proteins that have both recently been identified as major causative factors in a myriad of neurodegenerative diseases, including Amyolotrophic Lateral Sclerosis and Frontotemporal Lobar Dementia. Typically found in the nucleus, mislocalization of TDP-43 and FUS/TLS to form cytoplasmic inclusions is a hallmark of ALS-diseased motor neurons. To understand the pathology caused by loss of TDP-43 and FUS/TLS from the nucleus and reveal their normal functions, it is imperative to identify the functional RNA targets of both TDP-43 and FUS/TLS in the central nervous system. As a follow-up to our recently published TDP-43 targets in mice brain, here, we have performed genome-wide and computational studies to discover the functional RNA targets of FUS/TLS in vivo mouse brains. Importantly, we compared the functional RNA targets of both TDP-43 and FUS/TLS to elucidate if common pathways are evoked in the disease. CLIP-seq of FUS/TLS in mouse and human brains showed widespread binding across many genes, with FUS/ TLS being enriched on distal intronic regions. We have discovered that FUS/TLS bind to a distinct, degenerate sequence motif, and while FUS/TLS is often located in regions distinct from TDP-43 occupancy, it targets a largely overlapping set of genes. Depletion of FUS/TLS in mouse striata revealed a drastic down-regulation of many neuronal genes that contain strikingly long-introns, reminiscent of the functional targets of TDP-43. Splicing-sensitive microarray analysis also revealed that a quarter of alternative splicing events are common targets of both proteins. Furthermore, nearly 95% of the overlapping splicing events changed in the same direction when either TDP-43 or FUS/TLS are depleted. To demonstrate the these targets are regulated across evolution, we validated a subset using a lentiviral-mediated depletion of human FUS/TLS in human neural progenitor cells and neurons differentiated from pluripotent stem cells, and also in a zebrafish model using morpholinos to deplete the FUS/TLS ortholog. Taken altogether, our results show a strong connection of RNA regulation between these two proteins, some of which are relevant to human neurodegenerative diseases.

108 Structural basis for the regulation of the spliceosomal RNP remodeling enzyme, Brr2, by Prp8 and links to retinitis pigmentosa

Sina Mozaffari Jovin1, Traudy Wandersleben2, Karine F. Santos2, Markus C. Wahl2, Reinhard Lührmann1 1 Max-Planck-Institute of Biophysical Chemistry, Department of Cellular Biochemistry, Am Fassberg 11, D-37077 Göttingen, Germany, 2Freie Universität Berlin, Laboratory of Structural Biochemistry, Takustrasse 6, D-14195 Berlin, Germany

Brr2 is a DExD/H-box protein and a unique representative of the Ski2-like subfamily of helicase superfamily 2 (SF2) in the spliceosome. Many SF2 helicases show low processivity, poor RNA unwinding activity and no specificity towards RNA substrates in vitro. Hence, most of these helicases require accessory proteins for their specific and/or optimal RNA/RNP remodeling function to unfold in vivo. Within the assembled precatalytic spliceosome, Brr2 is known to function during the activation process by catalyzing U4 snRNP displacement, which leads to a major compositional and structural rearrangement in the spliceosome. The RNPase activity of Brr2 is thought to be tightly regulated through a combinatorial function of the key scaffolding protein of the spliceosome, Prp8, and the EF-G-like GTPase Snu114. Previously, it has been shown that a C-terminal region of Prp8, encompassing consecutive RNase H-like and Jab1/MPN-like domains stimulates Brr2 helicase activity, while reducing its ATPase activity [1]. The first domain is known to interact with conserved RNA elements in the catalytic core of the spliceosome, and the latter is a ubiquitin-interacting domain. We dissected these domains and showed that the Jab1/MPN domain of Prp8 but not the RNase H-like domain stably interacts with Brr2 and serves as the main Brr2 helicase cofactor. Biochemical analyses allowed us to clarify different aspects of the regulatory mechanism of this Prp8 domain on Brr2-catalyzed ATP hydrolysis and U4/U6 unwinding. Several mutations in an unstructured tail of the Prp8 Jab1/MPN domain are linked to a severe type of autosomal dominant retinitis pigmentosa (adRP13) [2]. We have investigated the consequences of these adRP-linked mutations on Prp8-Brr2 interaction as well as on the enzymatic activities of Brr2, showing in which way they lead to dysregulation of Brr2. We have also solved the crystal structure of a large portion of Brr2, containing both helicase cassettes, in complex with the Prp8 Jab1/MPN domain, providing a firm molecular basis for our biochemical findings. Together, our results reveal novel molecular mechanisms for a versatile regulation of Brr2 activity by Prp8. [1] Maeder C, Kutach AK, Guthrie C. ATP-dependent unwinding of U4/U6 snRNAs by the Brr2 helicase requires the C terminus of Prp8. Nat Struct Mol Biol. 2009; 16(1):42-8. [2] McKie AB, et al. Mutations in the pre-mRNA splicing factor gene PRPC8 in autosomal dominant retinitis pigmentosa (RP13). Hum Mol Genet. 2001; 10(15):1555-62. Session 7: RNA & disease

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

109 Rescue of Hearing and Vestibular Function in Deaf Mice Using Antisense Oligonucleotides That Block a Cryptic Splice Site

Francine Jodelka1, Anthony Hinrich1, Jennifer Lentz2, Kate McCaffrey1, Hamilton Farris2, Matthew Spalitta2, Dominik Duelli3, Frank Rigo4, Michelle Hastings1 1 Department of Cell Biology and Anatomy, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA, 2Neuroscience Center and Dept. of Otorhinolaryngology, LSU Health Sciences Center, New Orleans, LA, USA, 3Department of Cellular and Molecular Pharmacology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA, 4Isis Pharmaceuticals, Carlsbad, CA, USA Hereditary deafness is often mediated by the developmental failure or degeneration of cochlear hair cells. There are no cell-based treatments for deafness, and until now, it was not known whether such congenital failures could be mitigated by therapeutic intervention. Here we show that hearing and vestibular function can be rescued in a mouse model of human hereditary deafness. Antisense oligonucleotides (ASOs) were used to correct defective pre-mRNA splicing of transcripts from the mutated USH1C.216G>A gene, which causes human Usher syndrome, the leading genetic cause of combined deafness and blindness. Mice homozygous for the c.216G>A mutation are completely deaf, have severe vestibular dysfunction and develop retinitis pigmentosa. A single systemic treatment of ASOs to neonate mice partially corrected Ush1c splicing, increased protein expression in the cochlea and retina, and protected cochlear hair cells from degeneration. Remarkably, ASO-treatment rescued hearing to normal levels, as measured by auditory-evoked brain stem response analysis. Vestibular dysfunction was also completely eliminated in ASO-treated mice. The effects of ASO treatment on hearing and balance have lasted more than six months. Our results indicate the therapeutic potential of ASOs in the treatment of deafness and demonstrate that congenital deafness can be overcome by treatment early in development to correct gene expression.

110

Mechanistic Defects of Mutant U4atac Minor Spliceosomal snRNAs in Primordial Dwarfism

Faegheh Jafarifar, Rosemary Dietrich, Richard Padgett Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA We have investigated the biological defects in U4atac small nuclear RNA (snRNA) function due to mutations that cause Microcephalic Osteodysplastic Primordial Dwarfism Type 1 (MOPD1) or Taybi-Linder Syndrome. This is an autosomal recessive developmental disorder characterized by extreme intrauterine growth retardation, brain and bone malformations and unexplained postnatal death. The gene encoding U4atac snRNA, a component of U12-dependent (minor) spliceosome, is point-mutated in individuals with MOPD1. The splicing activity of MOPD1 minor spliceosomes is reduced by greater than 90% and the steady state levels of U12-dependent introns in MOPD1 cells increase by almost two fold. The biochemical function of mutant U4atac snRNAs is analyzed in this study. 1) We measured the stability of U4atac snRNA carrying each of seven known MOPD1 U4atac point mutations using qRT-PCR. Our results showed that the MOPD1 mutations have little effect on the stability of U4atac snRNAs. 2) We have derived and functionally characterized induced pluripotent stem (iPS) cell lines from MOPD1 patient cells carrying the U4atac 51G>A mutation. These cells and control iPS cells were used to determine the effect of the 51G>A U4atac mutation on the distribution of U4atac-containing snRNP complexes. The U4atac snRNP can exist as a mono-snRNP, a di-snRNP with U6atac or a tri-snRNP with U6atac and U5. Whole cell extracts from normal and patient iPS cells were prepared, the snRNP complexes were separated on glycerol gradients and the snRNP profiles were analyzed. Our data showed that the majority of U4atac snRNAs was found in the U4tac/U6atac di-snRNP in both normal and patient cells. However, the amount of U4atac/U6atac.U5 tri-snRNP was severely reduced in patient cells compared to normal cells. Thus patient cells appear to be defective in the formation of tri-snRNP complexes, leading to the inhibition of U12-dependent splicing in these cells. 3) Most MOPD1 mutations are clustered in the 5’ stemloop structure of U4atac snRNA, which is the binding site of the 15.5K protein and the hPrp31 protein. The ordered binding of these proteins is necessary for the formation of active di- and tri-snRNPs. Using a gel shift assay to study the effect of these mutations on the binding of the 5’ stem-loop to these proteins, we showed that the 51G>A mutation reduces binding of the 15.5K protein by about 10 fold. These results suggest that the splicing defects in MOPD1 are due to the failure of the mutant U4atac snRNA to form the correct RNA-protein complexes required for productive U12-dependent splicing. MOPD1 is the first disease to be directly associated with defects in a spliceosomal snRNA. Session 7: RNA & disease

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

111

Rescue of nonsense mutation containing mRNAs expression by amlexanox

Sara Gonzalez-Hilarion1, Jieshuang Jia3,2, Nadège Debreuck2, Fabrice Lejeune4,2 1 Institut Pasteur, Paris, France, 2Institut Pasteur, Lille, France, 3Université Lille 2, Lille, France , 4Inserm - IFR 142, Lille, France 11% of the mutations found in the human mutation database are nonsense mutations (Mort et al., 2008). mRNA harboring a nonsense mutation are often degraded by a mechanism called nonsense-mediated mRNA decay (NMD). Several strategies have been developed to rescue the expression of a gene harboring a nonsense mutation such as nonsense mutation containing exon skipping, readthrough of nonsense mutation or NMD inhibition. Using a screening system that mimics NMD, we identified amlexanox as a molecule capable of increasing the amount of nonsense mutation containing mRNAs and also capable of stimulating nonsense mutation readthrough. In our study, we used 3 cell lines derived from patient cells harboring a nonsense mutation in P53, dystrophin or CFTR gene leading to cancer, Duchenne muscular dystrophy or cystic fibrosis, respectively. We showed that incubation with amlexanox promotes in these cells an increase in the amount of nonsense mutation mRNAs and also the synthesis of the full length proteins from the nonsense mutation containing mRNAs. The functionality of the newly synthesized full length proteins was assessed and allowed us to conclude that amlexanox is able to correct the presence of a nonsense mutation. Additionally, amlexanox showed a better efficacy in the rescue of the expression of genes harboring nonsense mutations than ataluren (PTC124) or G418 at least in our models. Finally, the in vivo study of amlexanox on 2 different mouse models confirms the capacity of amlexanox at correcting nonsense mutation. Amlexanox is a drug on the market for more than 20 years and uses for athma and aphteous ulceria. The new property of amlexanox at rescuing the expression of genes with nonsense mutations could represent a new hope in the treatment of genetic diseases caused by nonsense mutation. Indeed, amlexanox might become in the future another example of drug used for personalized medicine. Mort, M., Ivanov, D., Cooper, D.N., and Chuzhanova, N.A. (2008). A meta-analysis of nonsense mutations causing human genetic disease. Hum Mutat 29, 1037-1047.

112 Repeat associated Non-AUG initiated translation drives Polyglycine production and neurodegeneration in Fragile X Tremor Ataxia Syndrome

Peter Todd1, Seok Yoon Oh1, Amy Krans1, Michelle Frazer1, Abigail Renoux1, Fang He1, Elan Louis2, J. Paul Taylor3, Henry Paulson1 1 University of Michigan, Ann Arbor, MI 48109, 2Columbia University, New York, New York, USA, 3St Judes Medical Center, Memphis, TN, USA FragileX-associated tremor ataxia syndrome (FXTAS) results from a CGG repeat expansion in the 5’UTR of the FMR1 gene. This repeat is thought to elicit toxicity as RNA, yet disease brains demonstrate ubiquitin-positive intranuclear neuronal inclusions, a pathologic hallmark of protein-mediated neurodegeneration. We explain this paradox by demonstrating that the FXTAS repeat expansion triggers translation of a polyglycine-containing protein via a novel non-AUG dependent initiation mechanism. This cryptic protein accumulates in ubiquitin-positive inclusions in Drosophila, cell culture, a CGG knock-in mouse model and FXTAS human disease brain. In cell-based experiments, AUG-independent translation initiates just proximal to the repeat structure, at repeat sizes ranging from normal (25) to pathogenic (90), with nuclear inclusion formation occurring only with larger repeat sizes. We propose a model of FXTAS pathogenesis in which a CGG repeat mRNA hairpin promotes aberrant translation initiation of an upstream open reading frame, driving production of a polyglycine-containing protein that contributes to neurodegeneration.

Session 7: RNA & disease

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

113 The AAA ATPases pontin and reptin in the R2TP complex remove SHQ1 from NAP57/dyskerin during biogenesis of H/ACA ribonucleoproteins

Rosario Machado-Pinilla1, Dominique Liger2, Nicolas Leulliot2, U. Thomas Meier1 1 Albert Einstein College of Medicine, Bronx, New York, 2Laboratoire de Cristallographie et RMN Biologiques, UMR CNRS 8015, Université Paris Descartes, Paris, France The AAA ATPases pontin and reptin, true to their name (ATPases associated with diverse cellular activities), function in a vast array of cellular processes including chromatin remodeling, DNA damage repair, transcriptional regulation, and assembly of macromolecular complexes, such as RNA polymerase II, phosphatidylinositol 3-kinase-related kinases, mRNA surveillance complexes, and small nucleolar ribonucleoproteins (snoRNPs). However, the molecular mechanism for none of these AAA ATPase-mediated activities is known. We now document that, during the biogenesis of the H/ ACA class of snoRNPs that includes telomerase, the assembly factor SHQ1 holds the pseudouridine synthase NAP57/ dyskerin in a viselike grip, and that pontin and reptin (as components of the R2TP complex) are required to pry SHQ1 from NAP57. Surprisingly, neither ATP depletion nor addition impacts the R2TP-mediated release reaction indicating that ATPases pontin and reptin function in an ATP-independent manner. The NAP57 domain captured by SHQ1 harbors most mutations underlying the X-linked bone marrow failure syndrome dyskeratosis congenita implicating the interface between the two proteins as a target of this inherited disease.

114

Effective inhibition of cytomegalovirus infection by external guide sequences in mice

Xiaohong Jiang1, Hao Gong1, Yuan-Chuan Chen1, Gia-Phong Vu1, Phong Trang1, Chen-Yu Zhang2, Sangwei Lu1, Fenyong Liu1 1 University of California-Berkeley, Berkeley, USA, 2Nanjing University, Nanjing, P.R. China Ribonuclease P (RNase P) complexed with external guide sequence (EGS) represents a novel nucleic acid-based gene interference approach for modulation of gene expression. Compared to other strategies, such as RNA interference, the EGS-based technology is unique because a custom-designed EGS molecule can hybridize with any mRNA and recruit intracellular RNase P for specific degradation of the target mRNA. It has not been reported whether the EGS-based technology can modulate gene expression in mice. In this study, a functional EGS was constructed to target the mRNA encoding the protease (mPR) of murine cytomegalovirus (MCMV), which is essential for viral replication. Furthermore, a novel attenuated strain of Salmonella was generated for gene delivery of EGS in cultured cells and in mice. Efficient expression of EGS was observed in cultured cells treated with the generated Salmonella vector carrying constructs with the EGS expression cassette. Moreover, a significant reduction in mPR expression and viral growth was found in MCMV-infected cells treated with Salmonella carrying the construct with the functional EGS sequence. When MCMVinfected mice were orally treated with Salmonella carrying EGS expression cassettes, viral gene expression and growth in various organs of these animals were reduced and animal survival significantly improved. Our study provides the first direct evidence to suggest that EGS RNAs, when expressed following Salmonella-mediated gene transfer, effectively inhibit viral gene expression and infection in mice. Furthermore, these results demonstrate the feasibility of developing Salmonella-mediated delivery of EGS as a novel approach for treatment of viral diseases in vivo.

Session 7: RNA & disease

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

115

An integrated picture of HDV ribozyme catalysis

Barbara Golden1, Ji Chen1, Pallavi Thaplyal2, Abir Ganguly2, Sharon Hammes-Schiffer2, Philip Bevilacqua2 1 Purdue University, West Lafayette, IN, USA, 2Penn State University, University Park, PA, USA The hepatitis delta virus (HDV) ribozyme and HDV-like ribozymes are widely dispersed in nature. These RNAs are small nucleolytic ribozymes that self-cleave to generate products with a 2’,3’-cyclic phosphates and free 5’-hydroxyls. Structural, spectroscopic and mechanistic analyses have demonstrated that the HDV ribozyme active site contains a cytosine with a perturbed pKa that can serve as a general acid, protonating the leaving group in the cleavage reaction. There is significant biochemical evidence that a Mg2+ ion participates directly in catalysis and a recent crystal structure of the HDV ribozyme revealed that there is a metal-binding pocket in the HDV ribozyme active site. Modeling of the cleavage site dinucleotide into the structure suggested that this metal ion can interact directly with the scissile phosphate and the nucleophile. In this manner, the Mg2+ ion is well positioned to serve as a Lewis acid. This metal ion can participate in catalysis by facilitating deprotonation of the nucleophile and stabilizing the conformation of the cleavage site for in-line attack of the nucleophile at the scissile phosphate. This catalytic strategy had previously been observed only in much larger ribozymes, including group I and group II introns, RNase P, and, possibly, the spliceosome. Thus, in contrast to most large and small ribozymes, the HDV ribozyme uses two distinct catalytic strategies, general acid and Lewis acid catalysis, in its cleavage reaction. Continuing studies will help elucidate the contributions of the active site metal ion in HDV ribozyme catalysis.

116 Structural Basis for Telomerase RNA Recognition and RNP Assembly by the Holoenzyme La Family Protein p65

Mahavir Singh1, Zhonghua Wang1, Bon-Kyung Koo1, Anooj Patel1, Duilio Cascio1, Kathleen Collins2, Juli Feigon1 1 University of California, Los Angeles, CA, USA, 2University of California, Berkeley, CA, USA Telomerase is a ribonucleoprotein complex essential for maintenance of telomere DNA at linear chromosome ends. The catalytic core of Tetrahymena telomerase comprises a ternary complex of telomerase RNA (TER), telomerase reverse transcriptase (TERT), and the essential La family protein p65. The unique C-terminal domain of p65 was shown to be essential and sufficient for the hierarchal assembly of TERT with TER. Here we present the structures of p65 C-terminal domain free and in complex with stem IV of TER. The structures reveal that RNA recognition is achieved by a novel combination of single- and double-strand RNA binding, which induces a large bend in TER. The structure has several unusual features including a long internal loop and an unstructured C-terminal extension that gets structured in the complex and is necessary for hierarchical assembly of TERT with p65-TER. This work provides the first structural insight into biogenesis and assembly of TER with a telomerase-specific protein. Additionally, our studies define a structurally homologous domain in genuine La and LARP7 proteins and suggest a general mode of RNA binding for biogenesis of their diverse RNA targets.

Session 8A: Keynote & Session 8A: RNA & RNP structure

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

117

Crystal structure of the spliceosomal RNP remodeling enzyme, Brr2

118

The 2.7 Angstrom Crystal Structure of the 2’,5’ GIR1 Branching Ribozyme

Karine Santos1, Sina Mozaffari Jovin2, Gert Weber1, Vladimir Pena2, Reinhard Lührmann2, Markus Wahl1 1 Freie Universitaet Berlin, Berlin, Germany, 2Max-Planck-Institute of Biophysical Chemistry, Goettingen, Germany Splicing entails the removal of non-coding intervening sequences from eukaryotic pre-mRNA and the ligation of the neighboring coding regions and is carried out by the spliceosome, a large and dynamic RNA-protein (RNP) enzyme [1]. While many macromolecular machines require elaborate assembly mechanisms to be put to work at the right place and time, the spliceosome takes this principle to an extreme in that none of its subunits comprises a preformed active site for the splicing reactions. Instead an active spliceosome only evolves on the substrate by a stepwise assembly that is driven by members of the DExD/H-box family of ATPases/RNA helicases [2]. Major conformational and compositional RNP remodeling is required to convert an initially inactive complex into an active spliceosome. The Brr2 protein is the key player in this catalytic activation process. It stands out both architecturally and functionally among all other spliceosomal helicases. Brr2 belongs to a unique group of nucleic acid helicases, whose members are exceptionally large (ca. 250 kDa in the case of Brr2) and which contain two expanded helicase units fused in tandem. Until now there is no detailed structure and very little mechanistic information available on this class of enzymes. Furthermore, unlike other spliceosomal helicases, Brr2 is preassembled with one of its substrates, the U4/U6 di-snRNP, before incorporation into the spliceosome. Subsequently, it is stably associated with the spliceosome and is used at least twice during a splicing cycle. Thus, it has to be reliably switched on and off multiple times during spliceosome assembly, splicing catalysis and spliceosome disassembly. Presently, it is unclear how the unusual architecture of Brr2 meets these specific functional and regulatory requirements. We have determined the crystal structure of an active, 200 kDa portion of Brr2, showing that its two helicase units intimately interact with each other. The C-terminal unit, while inactive in ATP hydrolysis and RNA duplex unwinding, strongly stimulates the N-terminal helicase. Using structure-guided mutagenesis, we delineated communication lines between the cassettes required for this modulation. Our results reveal how Brr2 is not merely optimized for helicase activity but rather for versatile regulation on various levels and suggest new, additional functions for at least some of the many spliceosomal proteins that interact at the C-terminal helicase unit of Brr2. References [1] Wahl, M. C., Will, C. L., Lührmann, R. (2009) The spliceosome: design principles of a dynamic RNP machine Cell 136, 701-718. [2] Staley, J. P., Guthrie, C. (1998) Mechanical devices of the spliceosome: motors, clocks, springs, and things Cell 92, 315-26. Mélanie Meyer1, Eric Westhoff1, Steinar Johansen2, Henrik Nielsen3, Benoît Masquida1 1 Institut de Biologie Moléculaire et Cellulaire-CNRS, Strasbourg, France, 2University of Tromsø, Tromø, Norway, 3 University of Copenhagen, Copenhagen, Denmark The 2’,5’ GIR1 branching ribozyme belongs to the complex twin-ribozyme intron of the SSU RNA precursor from the myxomycete Didymium iridis. This intron consists of a conventional group I splicing ribozyme GIR2 containing in its P2 domain the GIR1 branching ribozyme followed by a homing endonuclease (HE) mRNA. In connection with D. iridis life cycle, three different intron processing pathways are observed. The first one consists in regular splicing catalysed by GIR2 followed by GIR1 2’,5’ branching leading to capping by an unusual three nucleotides lariat of the HE mRNA which can then be released. The co-transcriptional folding of GIR1 leads to an inactive state in order to allow sequential actions of the two ribozymes that is essential for the correct rRNA processing. The switch between the GIR1 active and the inactive states seems to be regulated by its peripheral domain P2P2.1 which alternatively forms a hairpin HEG P1 disturbing the formation of the active site or a three-way junction. Noteworthy, HEG P1 is present in the 5’ UTR of the mature HE mRNA immediately downstream the lariat cap. In the second pathway, the GIR2 ribozyme yields a full-length circle intron and unligated exons. GIR1 is not active in this pathway. In the third pathway, induced by starvation, GIR1 branching occurs without any prior GIR2 activity. The resulting 7.5 kb RNA product seems to be stored as a precursor of the HE mRNA. We now have a 2.7 Å resolution crystal structure of a full-length (192 nucleotides) construct of the DiGIR1 ribozyme. The MAD experimental map is readily interpretable and reveals most of the GIR1 architectural features.

Session 8A: RNA & RNP structure

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

119 How The HIV Virus Selects Its Own mRNA For Export: The Topology OF The HIV-1 Rev Response Element, A molecular Beacon For Specificity And Coorperativity For Rev Binding

Jinbu Wang1, Xianyang Fang1, Michelle Mitchell1, Xiaobing Zuo1,2, Yi Wang1, Soenke Siefert2, Randall Winnas2, R. Andrew Byrd1, Stuart LeGrice1, Yun-Xing Wang1 1 National Cancer Institute, Frederick, Maryland, USA, 2The Argonne National Laboratory, Argonne, IL, USA Viruses always find ways to circumvent regulatory systems in order to propagate within host cells. Retrovirus HIV genomes contain multiple splicing sites. Unspliced RNAs normally are not exported out of the nucleus in host cells. In order to overcome this problem, the virus uses the regulator of virion (Rev) to bind cooperatively and specifically to the Rev response element (RRE), which is located in an intron in the viral env gene region. The Rev–RRE complex can then ‘hitchhike’ on Crm1 (Xpol1), a host exporting complex, to sneak out of the nucleus and release viral RNA in the cytoplasm for viral replication and packaging. However, the topology of RRE that enables such a specific and cooperative interaction is not known, despite more than two decades of efforts. We have determined the first three-dimensional (3D) topology of the 232-nt RRE using small-angle X-ray scattering (SAXS). RRE has a unique, extended structure and folds into an uncanny shape like a ‘scarlet letter A’. Dissecting experiments, modeling and SAXS-WAXS-restrained ensemble calculations reveal that two specific binding sites, stem loops IIB and IA, are located at the two ‘legs’ of the ‘A’, facing each other by a separation of 50-60 Å, a distance span similar that between the two RRE binding sites in the Rev dimer crystal structures. This duet of binding sites serve as a nucleation sites and exerts a topological constraint, and, together with its unique, extended shape, constitutes the basis for the specificity, cooperativity, and multimerization of the Rev– RRE interaction. The RRE topology settles the decades-long important question about the structural basis of how the seemly promiscuous Rev and its cognate RNA partner mutually select each other, not host RNAs, to export. The RRE structure may also lead to designing a new class of drugs that target this duet of the key nucleation sites.

120 NMR structures of CPEB4 RRMs free and bound to RNA reveals an unexpected fold and mode of RNA recognition by two RRMs

Tariq Afroz, Lenka Skrisovska, Frederic Allain ETH Zurich Cytoplasmic polyadenylation is one of the mechanisms of controlling mRNA translation and is regulated by CPEB, a highly conserved sequence specific RNA binding protein that binds to cytoplasmic polyadenylation element (CPE) present in 3’-UTRs of mRNAs. By regulating mRNA translation, CPEB influences gametogenesis, early development, synaptic plasticity and cellular senescence. The best-characterized mechanism of cytoplasmic polyadenylation is the one occurring during oocyte maturation and is centered around CPEB1, the founding member of the family. However, it has been recently shown that CPEB1 is replaced by CPEB4 during the late stages of meiotic progression in oocytes. In order to better understand this temporal regulation mediated by CPEB proteins, we solved the solution structure of the two carboxy-terminal RNA Recognition Motifs (RRM12) of human CPEB4 in the free form and in complex with the CPE RNA. The structure of free RRM12 of CPEB4 reveals unusual features in both RRM domains. RRM1 adopts a novel topology with two additional anti-parallel β-strands compared to the canonical RRM. These additional strands, extending the β-sheet surface have implications in RNA binding. RRM1 and RRM2 interact via the inter-domain linker, bringing them in close proximity with a defined orientation. The structure of RRM12 in complex with the CPE RNA highlights the importance of these inter-domain interactions that position the two RRM’s in a unique way exposing a hydrophobic RNA binding groove. Four nucleotides are recognized by RRM1 while one nucleotide is recognized by RRM2. The directionality of RNA binding is very different from other previously reported RRM-RNA complexes. These structural features imply a novel mode of RNA recognition distinct from other RRM-RNA complexes. It also explains the sequence specificity in RNA recognition for CPEB4 and other members of the CPEB proteins at the molecular level.

Session 8A: RNA & RNP structure

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

121

Nrd1 RRM binds RNAs via two distinct RNA-binding surfaces

Veronika Bacikova, Karel Kubicek, Josef Pasulka, Fruzsina Hobor, Richard Stefl CEITEC - Central European Institute of Technology, Masaryk University, Czech Republic In Saccharomyces cerevisiae, Nrd1-dependent termination pathway play important role in the control of pervasive transcription and processing of non-coding RNAs such as sn- and sno-RNAs. The termination and processing is dependent on the Nrd1 complex that consists of two RNA-binding proteins Nrd1 (nuclear pre-mRNA down-regulation) and Nab3 (nuclear polyadenylated RNA-binding) and Sen1 helicase. Nrd1 and Nab3 proteins cooperatively recognize specific termination elements within nascent RNA, GUAA/G and UCUUG, respectively. Due to cooperativity between both proteins, the affinity of Nrd1-Nab3 heterodimer to RNA is significantly higher compared to the affinities of isolated subunits. Interestingly, some transcripts do not require GUAA/G motif for sufficient transcription termination in vivo, suggesting that there may be other termination elements recognized by Nrd1. Here we aimed to identify alternative Nrd1 binding sites. To this end we used fluorescence anisotropy (FA) measurement, nuclear magnetic resonance (NMR) spectroscopy, and fenotypic analyses in yeast to study RNA binding properties of Nrd1 RRM and its mutants. Our FA data showed that not only GUAA but also several other G-rich and AU-rich motifs are able to bind Nrd1 with affinity in a low micromolar range. NMR analysis of interaction between Nrd1 RRM and these RNA sequences revealed two distinct binding regions that are specific for AU-rich and G-rich sequences. These results highlight Nrd1 RRM as a unique RNA-binding motif which is able to recognize various RNA sequences via different binding sites within one domain, a feature that was not observed before for RNA-recognition motifs.

122 Biochemical Analysis of Primary miRNA Structure Reveals an Extensive Capacity to Deform Near the Drosha Cut Site

Kaycee Quarles, Debashish Sahu, Ellen Forsyth, Christopher Wostenberg, Scott Showalter Pennsylvania State University, University Park, (PA), US Since their discovery in 1996, microRNAs (miRNAs) have been shown to play a critical role in several developmental and differentiation processes in the human body, in addition to participation in various cancerous and disease states. Yet very little is known about the maturation process of primary miRNAs at the molecular or atomic level, and the role its secondary structure plays in targeting it to the Microprocessor complex for maturation is only qualitatively defined. Moreover, the lack of experimentally-derived primary miRNA structures impedes the potential understanding of the structure-function relationship between the primary miRNA and the Microprocessor components DGCR8 and Drosha. In this study, SHAPE chemistry and ribonuclease structure mapping were used to determine the secondary structures of a panel of primary miRNAs. These structure mapping techniques indicate that a majority of the small helical imperfections located in the RNA stem—such as single nucleotide bulges and mismatches—are not as severely disruptive to the A-form helix as originally predicted by standard computational methods. Using these biochemical constraints as input into the MC-Pipeline, an ensemble of 3-dimensional structures was generated that depicts the elongated nature of the primary miRNA stem and the lack of deformability from the smaller mismatches. However, the structure ensembles do feature extensive deformability in the stem adjacent to the Drosha cut site nearest the single-stranded RNA: double-stranded RNA junction that may provide a recognition site for the Microprocessor. In addition, a second site enriched in deformation is found approximately one turn of A-form helix from the Drosha cut site opposite the single-stranded RNA: doublestranded RNA junction. The structure models generated herein support the hypothesis that periodic deformations created by non-canonical structure elements, in combination with the necessary single-stranded RNA: double-stranded RNA junction, identify the correct Drosha cut site. Using these RNA ensembles, this study aims to distinguish the catalytic effects of Drosha from the binding abilities of DGCR8 caused by structure differences of various native and mutated primary miRNAs.

Session 8A: RNA & RNP structure

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

123 Differential Fine-Tuning of Ligand-Induced Folding in Single Transcriptional and Translational Riboswitches

Krishna Suddala1, Arlie Rinaldi1, Jun Feng1, Anthony Mustoe1, Catherine Eichhorn1, Hashim Al-Hashimi1, Charles Brooks III1,2, Nils Walter1 1 University of Michigan, Ann Arbor, (MI), USA, 2University of California, San Diego, (CA), USA The family of transcriptional and translational preQ1 riboswitches contains some of the smallest metabolite sensing aptamer domains found in nature. Crystal and NMR structures of the transcriptional Bacillus subtilis (Bsu) and translational Thermoanaerobacter tengcongensis (Tte) preQ1 riboswitch aptamers show them to be structurally similar pseudoknots, yet their ligand-induced folding has been proposed to be distinct. Here, we use single molecule fluorescence resonance energy transfer to show that both riboswitches adopt two distinct conformations, similarly utilizing a pre-folded state to sense ligand. Contrary to previous suggestions, both riboswitches exist primarily in this pre-folded state in the absence of ligand, in near-physiological buffer. Together with atomistic simulations, we show that the main distinction between the two riboswitches lies in their relative tendency to fold into the ligand-bound state by conformational selection (Bsu) or induced fit (Tte). Finally, we demonstrate that mutation of a single nucleotide distal from ligand binding site predictably switches their properties. K.C.S and A.J.R have contributed equally to this work.

124

SNitching Riboswitches; on the Structural Sensitivity of RNA to SNPs

Justin Ritz, Joshua Martin, Alain Laederach University of North Carolina, Chapel Hill, NC, USA The structure of RiboNucleic Acid (RNA) has the potential to be altered by a Single Nucleotide Polymorphism (SNP). Disease-associated SNPs mapping to non-coding regions of the genome that are transcribed into RiboNucleic Acid (RNA) can potentially affect cellular regulation (and cause disease) by altering the structure of the transcript. We performed a large-scale meta-analysis of Selective 2’-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) data, which probes the structure of RNA. We found that several single point mutations exist that significantly disrupt RNA secondary structure in the five transcripts we analyzed. Thus, every RNA that is transcribed has the potential to be a “RiboSNitch;” where a SNP causes a large conformational change that alters regulatory function. Predicting the SNPs that will have the largest effect on RNA structure remains a contemporary computational challenge. We therefore benchmarked the most popular RNA structure prediction algorithms for their ability to identify mutations that maximally affect structure. We also evaluated metrics for evaluating the effect on structure. Although no single algorithm/metric combination dramatically out performed the others, small differences in AUC (Area Under the Curve) values reveal that certain approaches do provide better agreement with experiment. The experimental data we analyzed nonetheless show that multiple single point mutations exist in all five RNA transcripts that significantly disrupt structure in agreement with the predictions.

Session 8A: RNA & RNP structure

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

125

RNA Catalysis Through Compartmentalization

126

Characterization of Regulatory Small RNAs and RNA-Binding Proteins

Christopher Strulson1, Rosalynn Molden1,2, Christine Keating1, Philip Bevilacqua1 1 The Pennsylvania State University, University Park, PA, USA, 2Princeton University, Princeton NJ, USA Compartmentalization of RNA is essential for many cellular functions such as RNP processing and assembly, gene silencing, and transcription control, and may have played a pivotal role in the emergence of life. Local concentration impacts numerous biomolecular functions; however, effects of cellular compartmentalization on RNA function are largely unknown. One process that could be affected by compartmentalization is catalysis, since effective concentrations of enzyme and substrate are increased. To mimic intracellular compartmentalization and crowding, we partitioned RNA in an aqueous two-phase system (ATPS) and then tested the kinetics of phosphodiester bond cleavage in several two-piece hammerhead ribozymes. The ATPS, which consists of PEG8kDa and dextran10kDa, enriched the concentration of RNA up to 3,000-fold in the dextran-rich phase. Under sub-saturating single-turnover enzyme conditions, this compartmentalization increased the rate of RNA catalysis in a fashion that was roughly proportional to the relative volumes of the two phases, and up to approximately 70-fold overall. Observation of enhanced catalysis by simply compartmentalizing RNA supports the importance of compartmentalization in the attainment of function in an RNA World.

Thomas Tuschl The Rockefeller University Our laboratory studies how small-RNA-containing ribonucleoprotein complexes (RNPs) and RNA-binding proteins (RBPs) regulate messenger RNAs (mRNAs) in human cells. We develop experimental approaches to record small RNA expression profiles in order to assess deregulation in normal and disease conditions. We also define the targeting sites and underlying RNA recognition elements (RREs) of RBPs and RNPs and examine regulation of mRNA stability or RNA processing upon binding. The identification of posttranscriptional regulatory networks will increase our understanding of the molecular causes of disease and may lead to the rational design of new therapeutic agents.

Session 8A: RNA & RNP structure & Session 8B: Keynote

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

127

Argonaute and RISC in the Mammalian Cell Nucleus

128

A novel autoregulatory loop involving Argonaute and let-7 regulates miRNA biogenesis

Keith Gagnon, Roya Kalantari, David Corey UT Southwestern Medical Center, Dallas, TX, USA Our lab has demonstrated that small RNAs complementary to nuclear-localized RNAs can regulate transcription and splicing in human cells. These small RNAs bind AGO2 protein and guide it to complementary target RNAs. AGO2 protein is known to associate with other RNA interference (RNAi) factors, assemble into RNA-induced silencing complexes (RISC), and cause cleavage of target RNAs. However, we often fail to observe cleavage of nuclear RNA targets in our studies. While AGO2 and RISC have been well-characterized in the cytoplasm, their activity and significance inside mammalian somatic cell nuclei is unknown. We have detected the RNAi factors AGO2, Dicer, TRBP, and GW182/TNRC6A in nuclear extracts of human T47D cells. Co-immunoprecipitation has demonstrated that these nuclear RNAi factors can interact. These results suggest that AGO2 RISC complexes are in the nucleus. Using in vitro AGO2 cleavage assays with cell extracts, we have discovered that AGO2 activity is conditional. Addition of siRNA to nuclear extracts does not cause cleavage of target RNA, whereas cleavage in cytoplasmic extracts is robust. Testing of nuclear AGO2 siRNA binding showed that duplex RNA loading is significantly impaired. Roles for nuclear AGO proteins in regulation and genome integrity have been shown in other organisms like yeast, drosophila and tetrahymena. However, mechanisms for nuclear AGO function and biological relevance have been missing in mammals. Our findings suggest that a nuclear-specific regulation of AGO2 inside mammalian cells that restricts catalytic activity or the pool of potential RNA targets. They also suggest roles for nuclear-localized AGO2 that are independent of catalytic activity.

Dimitrios Zisoulis1, Zoya Kai1, Roger Chang2, Amy Pasquinelli1 1 University of California at San Diego, La Jolla CA, USA, 2Stockholm University, Stockholm, Sweden MicroRNAs (miRNAs), comprise a class of small RNA molecules that postranscriptionally regulate the expression of protein-coding genes in multiple biological processes. Mature miRNAs are derived from long primary transcripts via a series of processing events: miRNAs are transcribed as long primary transcripts (pri-miRNA) that undergo Drosha processing producing short hairpin precursor miRNAs (pre-miRNAs); pre-miRNAs are subsequently cleaved by Dicer and the resulting mature miRNAs are loaded onto the Argonaute protein. Mature miRNAs guide Argonaute-containing miRNA induced silencing complexes (miRISC) to specific target sequences in protein-coding mRNAs via imperfect base-pairing interactions and this association results in mRNA degradation and/or translational repression. Here we show that not only mRNA transcripts, but also non-coding transcripts are bound and regulated by Argonaute. Genome-wide analysis of interactions between the Argonaute Like Gene 1 (ALG-1) and target transcripts in C. elegans revealed an Argonaute-binding site located close to the 3’ end of the primary let-7transcript. We confirmed that ALG-1 physically associates with let-7 primary transcripts and that the ALG-1-binding site is essential for this interaction by RNA immunoprecipitation (RIP) assays. This interaction is mediated by mature let-7 miRNA via a conserved complementary site in its own primary transcript. Our data support a model whereby the association of Argonaute/let-7 with the primary let-7 transcript promotes miRNA maturation, thus creating a positive feedback loop. Furthermore,we demonstrate that Argonaute binds primary let-7 transcripts in human cells, suggesting that this new function of the miRNA pathway is conserved across species. Our studies reveal a novel role for Argonaute in promoting the autoregulation of let-7 biogenesis setting a new paradigm for controlling miRNA expression.

Session 8B: Small RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

129

Intracellular Single Molecule High Resolution Localization and Counting of microRNAs

130

Evolution and gene silencing capacity of Piwi-interacting RNAs

Sethu Pitchiaya1,3, John Androsavich2, Nils Walter1,3 1 Single Molecule Analysis in Real-Time (SMART) Center, University of Michigan, Ann Arbor, Michigan, USA, 2 Program in Chemical Biology, University of Michigan, Ann Arbor, Michigan, USA, 3Department of Chemistry, Single Molecule Analysis Group, University of Michigan, Ann Arbor, Michigan, USA microRNAs (miRNAs) associate with components of the RNA induced silencing complex (RISC) to assemble on messenger RNA (mRNA) targets and regulate protein expression in higher eukaryotes. While standard ensemble assays, including intracellular fluorescence microscopy, have revealed a wealth of information, the mechanism of gene repression by miRNAs is still debated. We have therefore developed an incisive tool to investigate the localization, mobility and assembly of miRNAs inside living (and fixed) human cells using single particle tracking and stepwise photobleaching at 30 nm spatial accuracy and 100 ms time resolution in pursuit of their still elusive mode of action. Upon microinjection, fluorophore labeled miRNAs were visualized as well isolated, diffraction limited particles that diffused with a wide range of diffusion coefficients (~0.7 - 0.0001 μm2/s) and demonstrated multifarious diffusive patterns (Brownian, corralled and directed) in living HeLa cells. These diffusion coefficients were distributed within (at least) two distinct Gaussian populations and were indicative of miRNA-bound mRNAs and processing bodies (PBs) respectively, functional intermediates of miRNA mediated gene regulation. Stepwise photobleaching of fluorescent probes in fixed cells revealed that the largest fraction of these particles contained single fluorescent miRNA molecules. A still significant fraction of particles, however, contained multiple labeled miRNA molecules, strongly invoking the formation of higher-order miRNA complexes either assembled on mRNA targets or associated with PBs. Time dependent changes in diffusion and assembly of miRNA particles were also observed, suggesting the existence of two kinetic processes. We are currently using multicolor single molecule imaging to colocalize and co-track miRNAs with their corresponding target mRNAs and PB proteins in an effort to map the dynamic interaction network of the RNA silencing pathway.

Jessica Matts, Gung-wei Chirn, Christina Post, Charlotte Logan, Nelson Lau Brandeis University, Waltham, MA, USA RNA interference (RNAi) is a natural gene silencing pathway in animals which utilizes small regulatory RNAs. One of these systems, the Piwi-interacting RNAs (piRNAs), operates in animal germ cells to repress selfish genetic parasites like transposons and perhaps directs the regulation of maternal transcripts. To gain a better understanding of animal health that is impacted by changes in piRNAs and the PIWI pathway, we wish to dissect molecular mechanisms of piRNA biogenesis and the regulation of mRNAs targeted by piRNAs and PIWI proteins. One of our approaches is to pinpoint mechanisms governing piRNA evolution and biogenesis through an evolutionary examination of genic piRNAs. Building upon our earlier discovery that many piRNAs are made from the 3’ UnTranslated Region (3’UTR) of messenger RNAs, we wish to discover patterns which might inform on precursor selection and piRNA accumulation through an evolutionary and bioinformatics approach. To accomplish this, we have been deeply sequencing new libraries with genic piRNA from cohorts of rodents and fruit flies. Our data suggests that despite a disconnect in piRNA biogenesis from homologous transcripts between flies and rodents, the conservation of genic piRNA biogensis is much stronger within rodent and Drosophilid cohorts. Surprisingly, we discovered several cases of differential genic piRNA biogenesis patterns for abundant piRNA precursors within both rodent and Drosophilid cohorts. For example, the Traffic Jam gene generates tremendous piRNAs in D. melanogaster and D. erecta, but few piRNAs in D. yakuba and D.virilis despite active gene transcription. We are analyzing these specific cases to discern evolutionary rates and motif signatures that may inform on biogenesis patterns. Although many piRNAs are presumed to silence transposons, the majority of metazoan piRNAs do not appear to target transposons; and the targets of these piRNAs are completely unknown. To gain insight into how piRNAs and PIWI proteins molecularly recognize target transcripts and to formally test the regulatory potential of PIWI/piRNA complexes, we tested classical reporter gene assays in insect cell lines that endogenously express PIWI and AGO proteins and piRNAs, endo-siRNAs, and miRNAs. This allowed us to compare one form of gene regulation potential between PIWI/piRNA complexes, AGO/siRNA complexes, and AGO/miRNA complexes. In agreement with published reports, we can observe canonically robust silencing of reporter genes with perfectly complementary binding sites against miRNAs and siRNAs, however, gene silencing potential was surprisingly modest for reporters directed against piRNAs. Nevertheless, we did observe with certain forms of reporter genes that gene silencing is the operational function of piRNAs, and propose a revised model for how meaningful piRNA targets may be considered. Session 8B: Small RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

131 Terminal A- and U-rich Motifs Inhibit Uridylation and Degradation of the 3’ end of the MALAT1 Noncoding RNA

Jeremy Wilusz, Laura Lu, Phillip Sharp Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA The MALAT1 locus is commonly misregulated in many human cancers and produces two noncoding RNAs via an unusual 3’ end processing mechanism. Although transcribed by RNA polymerase II, MALAT1 is not cleaved at its 3’ end by the cleavage and polyadenylation machinery but instead by the tRNA processing factor RNase P. Cleavage by RNase P simultaneously generates the mature 3’ end of the ~6.7-kb MALAT1 noncoding RNA and the 5’ end of a small 61-nt tRNA-like transcript known as mascRNA (MALAT1-associated small cytoplasmic RNA). The mechanism by which the 3’ end of the long MALAT1 noncoding RNA is stabilized despite the lack of a canonical poly(A) tail as well as the biological function of mascRNA are, however, unknown. To address these issues, we have developed a novel expression plasmid that accurately recapitulates MALAT1 3’ end processing in vivo. Placing a 174-nt fragment from the 3’ end of the MALAT1 locus (which includes the mascRNA tRNA-like structure) downstream of the GFP open reading frame is sufficient for RNase P cleavage of the CMV promoter driven transcript in HeLa cells. In addition to generating mascRNA, this plasmid generates a mature GFP transcript that is stable and, rather surprisingly, efficiently translated in vivo despite lacking a canonical poly(A) tail. We have identified a short genomically encoded A-rich tract immediately upstream of the MALAT1 RNase P cleavage site, which may perform many of the functions of a canonical poly(A) tail. Using extensive mutagenesis, we have determined that base pairing between this short A-rich sequence and upstream U-rich motifs is critical for stability of the long RNA in vivo. Subtle mutations to these sequences cause the GFP transcript to be efficiently degraded. Using 3’ RACE, we find short poly(U) tails added to the 3’ ends of the mutant transcripts, implicating uridylation in the degradation process. As this expression plasmid also allows efficient over-expression of mascRNA in vivo, we are exploring the effect of modulating mascRNA levels on cellular physiology as well as characterizing the mechanisms by which mutant tRNAs are degraded in vivo (such as CCACCA addition by the CCA-adding enzyme). In summary, by studying the 3’ end of MALAT1, we are gaining important new insights into how long transcripts that lack poly(A) tails are stabilized, translated, and function.

132 A SigmaB Dependent Regulatory RNA Modulates Biofilm and Capsule Formation in Staphylococcus aureus

Cedric Romilly1, Claire Lays2, Efthimia Lioliou1, Florence Vincent2, François Vandenesch2, Pascale Romby1, Sandrine Boisset2, Tom Geissmann2 1 Institut de Biologie Moléculaire et Cellulaire CNRS UPR9002-ARN, Strasbourg F-67084, France, 2INSERM U851, Lyon F-69008, France Université de Lyon, Université Lyon1, Lyon, F-69008, France Hospices Civils de Lyon, Centre National de Référence des Staphylocoques, Bron F-69500, France Staphylococcus aureus is a remarkable versatile pathogen responsible of 30% of nosocomial infections, with dramatic outbreaks in immuno-compromised patients. The pathogenicity of the bacteria results from the production of a plethora of virulence factors. The expression of these factors is temporally regulated by complex networks including transcriptional regulators, two-component systems, and regulatory RNAs. Here, we have characterized the function of RsaA [1]. This 138 nucleotides non-coding RNA (ncRNA) is expressed upon stationary growth phase and is under Sigma B control, a transcriptional regulator involved in stress adaptation [2]. To investigate the function of RsaA, we used a differential proteomic analysis combined with prediction of stable RsaA/mRNA duplexes. This strategy led to the identification of two direct target mRNAs, mgrA and yabJ-spoVG. MgrA is a global transcriptional factor affecting multiple genes involved in virulence, biofilm and antibiotic resistance [3] while SpoVG is involved in cap operon regulation and affect capsule formation, an adaptive mechanism against phagocytosis [4]. Using a combination of in vitro and in vivo approaches, we have validated mgrA as a direct target of RsaA. In vitro, RsaA uses a C-rich motif to form an extended duplex with the Shine and Dalgarno sequence of the mRNA. A second interaction involves a loop-loop interaction with a hairpin located in the coding region. Binding of RsaA prevents the ribosome binding to inhibit the translation of mgrA mRNA. In vivo, we showed that RsaA mutants showed a delay in early biofilm formation. Moreover, RsaA deleted strain produces less biofilm. Furthermore, we showed that rsaA inactivation enhances the capsule production. These observations were confirmed in several genetic backgrounds. More surprisingly, deletion of rsaA causes some defects in the internalization of the bacteria by macrophages but the phenotype was different in various genetic background such as S. aureus Newman and Becker strains. We are presently analyzing the importance of RsaA in pathogenesis and host-pathogen interaction. 1. Geissmann T et al. (2009) Nucleic Acids Res.; 2. Bischoff M. et al (2004) J. Bacteriol.; 3. Luong TT et al. (2006) J. Bacteriol. 4. Meier S et al. (2007) Infect. Immun. Session 8B: Small RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

133 A phylogenetically conserved hairpin switch controls 6S RNA transcriptional regulation by triggering sigma-70 release from bacterial polymerase Shyam Panchapakesan, Mariana Oviedo, Lindsay Shephard, Peter Unrau SFU, Burnaby, (BC), Canada

The 6S RNA binds to RNA Polymerase holoenzyme (core:σ70) in stationery phase and suppresses transcription in low nutrient conditions. Release from this transcriptional inhibition is achieved by the synthesis of a short product RNA (pRNA) when nutrients are abundant (Wassarman et al., Science 2006). Recent work has shown that 6S release is a complex multi-step process that involves changes in binding interaction between the 6S RNA and the holoenzyme complex as a function of pRNA synthesis (Beckmann et al., EMBO J. 2012, Shephard et al., RNA 2010). We demonstrate here that a highly conserved hairpin switch in the gamma-proteobacteria serves not only to dramatically enhance the rate of 6S release but also triggers the ejection of σ70 from the holoenzyme complex prior to the release of the 6S:pRNA from core polymerase. Nuclease mapping indicates that the 6S:pRNA complex released from E. coli polymerase contains a hairpin structure not found in the initial 6S RNA. This hairpin is formed when pRNA synthesis opens a highly conserved downstream helix, allowing the top strand of the duplex to pair with the conserved -10 region of the 6S (located in the top strand of the open ‘transcription’ bubble). Since we previously demonstrated that mutating the -10 sequence alters both 6S binding and 6S release rate (Shephard et al., RNA 2010) this implicates the hairpin, which would have the capability to strip away protein-RNA interactions in the -10 region, in the 6S release process. Indeed mutating the 6S downstream sequence to preclude hairpin formation dramatically slows 6S release, while maintaining full initial binding. Unexpectedly, native gel analysis showed that these mutants accumulate a new release intermediate that lacks σ70. Critically, nucleotide feeding experiments using wild type 6S revealed that a core:6S:pRNA intermediate lacking σ70 is transiently populated during normal 6S release and that this sigma-less intermediate very rapidly depopulates by additional pRNA synthesis to core plus 6S:pRNA when NTPs are present. This surprising finding matches well to a complementary in vitro selection study where we isolated three characteristic types of 6S release defective mutant. The first precluded all pRNA synthesis, the second involved extensive template slippage, while the third involved the synthesis of unusually long pRNAs that where typically associated with defects in 6S hairpin formation. Reinforcing the importance of rapid 6S release rate in normal transcriptional regulation, a release defective 6S expressed in E. coli remained fully bound to holoenzyme in high nutrient conditions and led to a profound suppression of bacterial growth. This hairpin dependent mechanism has important implications for understanding global transcriptional regulation in the gammaproteobacteria. Coupling the ejection of σ70 with pRNA synthesis provides an elegant mechanism to explain the rapid destabilization of the entire 6S:holoenzyme complex. Even in low nutrient conditions the abrupt conformational change induced by the formation of the hairpin is expected to trigger complex destabilization making it possible to efficiently reassemble core polymerase with alternative sigma factors / specific regulatory factors as environmental conditions demand.

134

Presence of Hfq Binding Site Facilitates Identification of Functionally Important mRNA Targets

Martha Faner1, Rebecca Swett1, Amit Kumar1, Cassandra Joiner2, Andrew Feig1 1 Wayne State University, Detroit, (MI), USA, 2Madonna University, Livonia, (MI), USA The ability of bacteria to respond to environmental conditions is crucial for their survival. One adaptive mechanism they use to regulate gene expression involves sRNAs and the RNA binding protein, Hfq. These trans-sRNAs are complementary to the mRNA that they regulate and form base pairs with the help of Hfq to either up or down regulate translation of the mRNA. Because trans-sRNAs act by imperfect base pairing they often regulate multiple mRNAs, forming a web of regulatory activities that occur in response to the environment of the bacterium. Annotation of sRNAs in bacteria has been extensive but the number of known mRNA targets is lacking. Recently we have developed a novel approach to mRNA target identification based on the presence of Hfq binding motifs in the 5’UTRs of regulated messages. This motif has been observed in several mRNAs (fhlA, rpoS, and glmS) previously, has the sequence (ARN)2-4, and is found within or surrounded by a highly structured region of the 5’UTR. We have shown that all mRNAs known to be regulated by trans-sRNAs contain such a motif but the motif is present in many more messages - approximately 20% of E.coli transcriptional units. This finding suggests that many more mRNAs may be regulated than previously recognized. We asked whether the mRNAs that have an Hfq binding motif are bona fide targets of trans-sRNAs. We will present data showing that the predicted targets interact with sRNA partners in an Hfq dependent manner in vitro. These data show that the presence of an ARN motif leads to identification of previously unidentified sRNA-mRNA pairs. Using in vivo reporter studies we have validated several of these regulatory circuits. The discovery of many new mRNA targets demonstrates an increased complexity of the sRNA regulatory network in bacteria. More importantly, the ability to predict new mRNA targets by bioinformatics provides significant benefits for genomes beyond E. coli where less is known about the biology of regulatory RNAs and provides a means to rapidly elucidate these pathways.

Session 8B: Small RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

135

The Redox-Sensing Aconitase B Protein Act Against sRNA Distal Nucleolytic Cleavage

Julie-Anna Benjamin, Marie-Claude Carrier, Eric Massé Université de Sherbrooke Post-transcriptional regulation by bacterial small RNAs (sRNAs) typically occurs through the pairing of a sRNA to the translational initiation region of a target mRNA, allowing the sRNA to directly compete with initiating 30S ribosomes and thus to inhibits translation initiation. This is often followed by a rapid degradation of the target mRNA through the recruitment of the RNA degradosome. Here, we report the first example of repressed sRNA-induced degradation, which depends on cellular conditions. The well-known RyhB sRNA which regulates Fe-homeostasis target and quickly degrade acnB mRNA when expressed in Fe-rich media. However, in Fe-poor conditions, RyhB is expressed and rapidly induces degradation of its targets, but not acnB mRNA anymore. In condition of Fe starvation, it is known that the enzymatic Holo-aconitase B protein switch in its Apo-aconitase B mRNA binding protein state. Also, in this metabolic state, Apo-aconitase B seems to stabilise acnB mRNA, possibly by binding to the 3’-UTR of its own mRNA. Our data indicate that RyhB is still able to bind acnB mRNA in Fe starvation and block translation. Also, we propose that the stabilisation of acnB mRNA could result from protection against RNase E-mediated cleavage after RyhB sRNA pairing. This novel mechanism demonstrates a new level of regulation of sRNA-induced mRNA degradation, which depends on the metabolic state of the enzyme/ RNA-binding protein Aconitase B.
90%) leader-linker duplex involving leader nucleotides upstream of the previously reported consensus glycine riboswitch sequences. In more than 50% of the glycine riboswitches, the leader-linker interaction forms a kink-turn motif. Characterization of three glycine ribsowitches showed that the leader-linker interaction improved the glycine binding affinities by 4.5 to 86 fold. In-line probing and native gel assays with two aptamers in trans suggested synergistic action between glycine binding and inter-aptamer interaction during global folding of the glycine riboswitch. Mutational analysis showed that there appeared to be no ligand binding cooperativity in the glycine riboswitch when the leader-linker interaction is present, and the previously measured cooperativity is simply an artifact of a truncated construct missing the leader sequence.

Dennis Mishler, Justin Gallivan Emory University, Atlanta, GA, USA Riboswitches are RNA sequence elements that control gene expression through ligand-dependent structural changes. In the past decade numerous natural riboswitches have been discovered, and a variety of synthetic riboswitches have been engineered. Synthetic riboswitches are derived from aptamers discovered using established in vitro selection techniques, and can in principle, be used to sense nearly any molecule that can bind to an RNA. Thus, synthetic riboswitches are attractive tools for controlling gene expression in a small molecule-dependent fashion to create novel phenotypes. Our lab has engineered a number of riboswitches that regulate translation initiation in the presence of a small molecule ligand. Previous studies suggested that these riboswitches operate under equilibrium conditions, in which addition of the ligand perturbs the equilibrium between translationally-active and translationally-inactive conformations in the absence of active transcription. Here, we present new data suggesting that when transcription is active, there is a substantial kinetic contribution to the mechanistic picture. We performed our studies in an E. coli S30 extract that is capable of transcribing DNA templates, and translating mRNAs into protein. When an mRNA template encoding a synthetic riboswitch in the 5’ UTR of our luciferase reporter gene was added to the extract (in the absence of a DNA template), we observed modest ligand-dependent increases in the production of luciferase. This observation is consistent with previous data obtained from intact E. coli cells in which active transcription was arrested using the antibiotic rifampicin. However, when a DNA template encoding a synthetic riboswitch in the 5’ UTR of the luciferase reporter gene was added to the extract, the performance of the riboswitches (fold-activation; expression levels relative to controls) improved significantly when ligand was present during active transcription, but not if ligand was added after transcription was halted with rifampicin. These new results suggest that while there is an equilibrium component to the mechanisms of action of these synthetic riboswitches, there is a kinetic component that appears to be the dominant contributor when transcription is active. Our results suggest that synthetic riboswitches derived from aptamers that bind their ligands quickly may perform better than those derived from aptamers that display slow ligand-association kinetics. Session 10A: Ribozymes & riboswitches

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

155

Riboswitches Control mRNA Decay in Escherichia coli

156

A Thiamine-Utilizing Ribozyme Decarboxylates a Pyruvate-like Substrate

Marie-Pier Caron, Audrey Dubé, Laurène Bastet, Antony Lussier, Maxime Simoneau-Roy, Eric Massé, Daniel Lafontaine Université de Sherbrooke, Sherbrooke, (Québec), Canada Riboswitches are genetic control elements that specifically recognize cellular metabolites and that modulate gene expression at various levels such as transcription, translation and splicing. The lysine riboswitch was first characterized in Bacillus subtilis, where it was shown that lysine binding to the riboswitch induces transcription attenuation of lysC (1). However, in contrast to B. subtilis, sequence alignments predicted that the Escherichia coli lysC riboswitch could control gene expression at the translational level by modulating ribosome access to the ribosome binding site (RBS)(2). In this model, lysine binding induces the riboswitch to sequester the RBS into a stem-loop structure, thereby inhibiting translation initiation. We have studied the in vivo regulation mechanism of the E. coli lysC riboswitch. As expected from earlier predictions, our results are consistent with the riboswitch modulating translation initiation as a function of lysine. In addition, we observed that lysine binding to the riboswitch also targets RNase E cleavage to the riboswitch domain, significantly reducing lysC mRNA level. RNase E cleavage sites were mapped within the riboswitch expression platform, which become accessible to RNase E uniquely when the riboswitch is bound to lysine. The conformation of the lysine-free riboswitch prevents mRNA cleavage thereby allowing lysC mRNA accumulation and translation. We also found that both regulatory activity can be uncoupled, directing the riboswitch to regulate either at the level of translation initiation or mRNA decay. Interestingly, we have obtained results indicating that the TPP-dependent E. coli thiC riboswitch also relies on RNase E activity for regulation of the thiCEFSGH operon. Thus, our results suggest that both lysC and thiC riboswitches directly inactivate the translational process through mRNA endonucleolytic cleavages, consistent with both riboswitches employing a nucleolytic repression mechanism to achieve genetic regulation (3). This is in contrast to other riboswitches, such as thiM and btuB, for which we have observed that the mRNA decay is tightly coupled to the inhibition of translation initiation. Our study provides the first strong experimental evidence indicating that riboswitches may control mRNA decay upon ligand binding, thus expanding the known regulatory functions of riboswitch. (1) Grundy et al, PNAS 2003 100:12057; Sudarsan et al, Genes & Dev 2003 17:2688. (2) Rodionov et al, NAR 2003 31:6748. (3) Dreyfus, Prog Mol Biol. 2009 85:423

Paul Cernak, Dipankar Sen Simon Fraser University, British Columbia, Canada Thiamine (vitamin B1) is a cofactor central to modern metabolism and is used by variety of protein enzymes to catalyze a series of difficult metabolic reactions, including the decarboxylation of α-keto acids such as pyruvate. Along with other vitamins, thiamine has been hypothesized to be a relic of the RNA World, and a likely participant in RNAmediated primordial metabolism. We have been searching for empirical evidence for the capability of RNA to utilize thiamine, comparably to modern protein enzymes, for the manipulation of α-keto acids. To this end, we carried out in vitro selection for catalytic RNAs that decarboxylate a pyruvate-based suicide substrate (SS, appended to the 5’ ends of all RNAs within the random pool), and used thiamine attached to biotin (TB) to enrich for catalytic RNAs. Following 13 rounds of selection, clone thi4-13 was characterized in depth for its ability to decarboxylate the α-keto acid SS. Mass spectral analysis of the stable guanosine adduct obtained by a limit digest of the RNA of clone thi4-13 yielded a product with the precise mass as well as atomic composition of the expected product: Guanosine-SS-TB, minus CO2. This discovery of a catalytic RNA that utilizes a vitamin cofactor, thiamine, to catalyze chemistry associated with pyruvate decarboxylase and related protein enzymes, is a milestone towards affirming the hypothesis that modern metabolic pathways still incorporate vestiges of RNA-orchestrated catalytic processes.

Session 10A: Ribozymes & riboswitches

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

157

Alternative Splicing of a Group II Intron in Clostridium tetani

158

Nucleobase-Mediated General Acid-Base Catalysis by the Hairpin Ribozyme

Bonnie McNeil1, Dawn Simon2, Steven Zimmerly1 1 University of Calgary, Calgary, AB, Canada, 2University of Nebraska, Kearney, Nebraska, USA Group II introns are large ribozymes that are thought to be ancestors of spliceosomal introns and the spliceosome. While spliceosomal introns frequently undergo alternative splicing to produce several products per gene, this property has not been reported for group II introns. Here we report a naturally-occurring intron that produces four alternatively spliced RNAs in vivo. The intron was detected in a bioinformatic search for bacterial group II introns that lack intronencoded proteins (IEPs). IEPs are normally required for both splicing and mobility of their host introns. The genome of Clostridium tetani was found to contain such a candidate group II intron that lacks an IEP, but contains all six standard ribozyme structural domains (D1-D6); however, three additional copies of domains 4b-6 were observed in the downstream 10 kb region. This raised the possibility that the downstream intron fragments could be alternatively spliced utilizing domains 1-4a of the upstream intron copy. RT-PCR assays of RNA isolated from C.tetani detected ligated exon sequences corresponding to all four predicted 3’ intron ends, thus confirming the hypothesis. The four spliced mRNAs contained accurate, in-frame ligation of the upstream surface layer protein ORF to the four downstream ORFs, each of which contains putative surface layer and/or protease domains. Unexpectedly, the 5’ splice site did not occur at the typical intron boundary sequence (5’GUGYG), but eight nucleotides upstream. Use of the shifted 5’ splice-site results in the excision of the 5’ exon’s stop codon, allowing for the fusion of 5’ and 3’ exon ORFs. In vitro self-splicing assays showed that the intron self-splices with high efficiency and that the shifted 5’ splice site is an intrinsic property of the ribozyme, being conferred by an altered IBS1-EBS1 pairing and a novel sequence insertion in the EBS1 loop. Further mutagenesis experiments confirmed several novel features of the intron’s secondary structure that contribute to the adapted role in alternative splicing. Quantitative RT-PCR assays were used to determine the relative levels of the four splicing forms in C. tetani cells subjected to various growth or stress conditions. These experiments did not detect significant changes in alternative splicing or in splicing ratios under conditions tested, and also revealed that unspliced transcript was the most abundant RNA. Further experiments are underway addressing the significance of alternative splicing in C. tetani.

Stephanie Kath-Schorr1, Timothy Wilson1, Nan-Sheng Li2, Jun Lu2, Joseph Piccirilli2, David Lilley1 1 University of Dundee, Dundee, UK, 2The University of Chicago, Chicago, USA The role of general acid-base catalysis mediated by nucleobases in the hairpin ribozyme has been the subject of significant debate. There is considerable evidence indicating that the cleavage reaction is subject to general base catalysis by G8 and general acid catalysis by A38, closely similar to the proposed catalytic mechanism for the VS ribozyme. Alternative viewpoints have it that the nucleobases provide catalysis only by electrostatic stabilization of the transition state or that A38 alone participates in catalysis. We critically examined the role of A38 and G8 in the hairpin ribozyme by means of 5’-phosphorothiolate (5’-PS) substitution of the scissile phosphate. Sulfur is a far better leaving group than oxygen, and will therefore no longer require protonation by a general acid. If a particular nucleobase provides rate acceleration by protonation of the leaving group such that catalysis is impaired upon substitution of that nucleotide, then the rate of cleavage should be restored by the 5’-PS substitution for that variant. We find that the activity of an A38Purine ribozyme is indeed restored by 5’-PS substitution of the substrate. This strongly suggests that A38 acts as general acid. The rate of cleavage of the 5’-PS substrate by the A38Purine ribozyme rises log-linearly with pH, but reaches a plateau at neutral pH for a ribozyme with a G8DAP and an A38Purine substitution, consistent with general base catalysis by G8 and DAP8 respectively. 5’-PS substitution data are consistent with general acid-base catalysis by A38 and G8. However, the data show that other processes provide some contribution to the overall rate enhancement. These processes are likely to include stabilization of the active conformation and transition state stabilization. T. J. Wilson, A. C. McLeod and D. M. J. Lilley. A guanine nucleobase important for catalysis by the VS ribozyme. EMBO J. 26, 2489-2500 (2007). T. J. Wilson, N.-S. Li, J. Lu, J. K. Frederiksen, J. A. Piccirilli and D. M. J. Lilley. Nucleobase-mediated general acidbase catalysis in the Varkud satellite ribozyme. Proc. Natl. Acad. Sci. USA 107, 11751–11756 (2010). T. J. Wilson and D. M. J. Lilley. Do the hairpin and VS ribozymes share a common catalytic mechanism based on general acid-base catalysis? A critical assessment of available experimental data. RNA 17, 213-221 (2011). Session 10A: Ribozymes & riboswitches

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

159 The Hairpin Ribozyme Self-Cleavage Reaction Pathway Involving Deprotonated G8 as General Base and Protonated A38 as General Acid Seems to Be the Most Consistent with Experimental Data

Vojtech Mlynsky1, Pavel Banas1, Nils Walter2, Jiri Sponer3, Michal Otyepka1 1 Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Palacky University Olomouc, Czech Republic, 2Department of Chemistry, Single Molecule Analysis Group, University of Michigan, Ann Arbor, MI, USA, 3Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic The hairpin ribozyme is a member of the group of small ribozymes, achieving the self-cleavage and ligation without direct participation of metal ions.[1] Experiments identified two catalytically active residues guanine 8 (G8) and adenine 38 (A38), but their exact roles remain still elusive.[2] We carried out all atom molecular dynamics (MD) simulations in explicit solvent on 50-500 ns time scales in order to compare impact of various protonation states of G8 and A38 on the active site geometry. We observed that the geometries with the canonical G8 and protonated A38H+ agreed well with the crystal structures, while those bearing deprotonated G8- and canonical A38 gradually perturbed the active site.[3] It is worth noting, that the active site geometry with G8- was transiently stable on ps time scale.[4] The MD simulations generated geometries of potential reactive states, which we further analyzed by hybrid quantum-mechanical/molecular mechanical (QM/MM) method.[5] We calculated energies along the reaction pathways and indentified activation barriers and the rate limiting steps. We found three possible reaction scenarios having activation barriers in a good agreement with those derived from experiments (20-21 kcal/mol).[6] One scenario was (i) general base (deprotonated G8-)/general acid (protonated A38H+) mechanism with activation barrier of 20.4 kcal/mol, (ii) two mechanisms involved proton shuttle via the nonbridging oxygen in the presence of canonical G8 together with A38 or A38H+ forms (20.5 or 21.0 kcal/mol, respectively), and (iii) the combined proton shuttle/ general acid mechanism with canonical G8 and protonated A38H+ (21.0 kcal/mol). In all cases, the initial nucleophile attack of the A-1(2’-OH) group on the scissile phosphate represented the rate-limiting step along the reaction paths. The protonated A38H+ decreased the activation barrier of the exocyclic cleavage step by 7.7 kcal/mol.[4] We suggest that RNA self-cleavage may benefit from several microscopic pathways which are energetically comparable. Among those pathways, the reaction mechanism with deprotonated G8- as general base and protonated A38H+ as general acid is the most consistent with the experimentally measured kinetic pH profiles. This work was supported by the Grant Agency of the Czech Republic (grants 203/09/H046, P208/11/1822, P208/12/1878 and P301/11/P558), by Student Project PrF_2011_020 of Palacky University. This work was also supported by the Operational Program Research and Development for Innovations - European Social Funds (CZ.1.05/2.1.00/03.0058 and CZ.1.07/2.3.00/20.0058). NIH grant 2R01 GM062357 to NGW is also gratefully acknowledged. References: M. J. Fedor, Annu. Rev. Biophys. 38, 271-299 (2009); T. J. Wilson and D.M. Lilley, RNA 17, 213-221 (2011); V. Mlynsky, P. Banas, D. Hollas, K. Reblova, N. G. Walter, J. Sponer and M. Otyepka, J. Phys. Chem. B 114, 6642-6652 (2010); V. Mlynsky, P. Banas, N. G. Walter, J. Sponer and M. Otyepka, J. Phys. Chem. B 115, 6642-6652 (2011); P. Banas, P. Jurecka, N. G. Walter, J. Sponer and M. Otyepka, Methods 49, 202-216 (2009); M. J. Fedor, J. Mol. Biol. 297, 269-291 (2000).

160

Probing Metal Ion Binding Sites in the P4 Helix of Bacillus subtilis RNase P

Yu Chen, Carol Fierke University of Michigan, Ann Arbor, MI, USA

The endoribonuclease P, RNase P, is responsible for catalyzing the 5’-end maturation of precursor tRNAs1. Like many large ribozymes, divalent ions stabilize the folded structure and enhance catalytic function of RNase P2. P4 helix, the most highly conserved region in PRNA, is essential for RNase P activity and has been suggested to contain catalytic and/or cocatalytic metal ion binding sites3; 4. NMR spectroscopy of a P4 stem-loop mimic revealed an inner-sphere metal interaction with residues corresponding to G378 and G379 in the P4 helix of Bacillus subtilis RNase P5. Modeling of this site suggests that the base O6 and N7 of G378/G379 form inner and outer sphere interactions with a metal ion respectively5. To evaluate whether a metal ion binds to the same site in full-length PRNA and to investigate the function of the metal at this site, 2-aminopurine and N7-deaza guanine are specifically substituted for guanine in the P4 helix. These substitutions decrease the substrate binding affinity at low concentration of metal ion but not at high concentration. Single turnover kinetics show that 2-aminopurine and N7-deaza guanine substitutions have no effect on the rate of the conformational change step in the RNase P · pre-tRNA complex. However, the 2-aminopurine substitution at G379 decreases the cleavage rate constant by 3-fold in Ca2+ and 8-fold in Mg2+, demonstrating the importance of this site for catalytic activity, without altering the K1/2 for Mg2+-dependent activation of cleavage. These data indicate that O6 of G379 is important both for stabilizing pre-tRNA affinity in a metal-dependent fashion and for enhancing catalytic activity. The magnitude of these effects are smaller than expected for an inner-sphere metal ion interaction with O6 of G379 but are consistent with outer-sphere metal ion coordination. 1. Harris, M. E. & Christian, E. L. (2003). Recent insights into the structure and function of the ribonucleoprotein enzyme ribonuclease P. Current Opinion in Structural Biology 13, 325-333. 2. Smith, J. K., Hsieh, J. & Fierke, C. A. (2007). Importance of RNA-protein interactions in bacterial ribonuclease P structure and catalysis. Biopolymers 87, 329-338. 3. Christian, E. L., Kaye, N. M. & Harris, M. E. (2000). Helix P4 is a divalent metal ion binding site in the conserved core of the ribonuclease P ribozyme. RNA 6, 511-519. 4. Crary, S. M., Kurz, J. C. & Fierke, C. A. (2002). Specific phosphorothioate substitutions probe the active site of Specific phosphorothioate substitutions probe the active site of Bacillus subtilis ribonuclease P. RNA 8, 933-947. 5. Koutmou, K. S., Casiano-Negroni, A., Getz, M. M., Pazicni, S., Andrews, A. J., Penner-Hahn, J. E., Al-Hashimi, H. M. & Fierke, C. A. (2010). NMR and XAS reveal an inner-sphere metal binding site in the P4 helix of the metallo-ribozyme ribonuclease P. Proceedings of the National Academy of Sciences 107, 2479-2484. Session 10A: Ribozymes & riboswitches

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

161 Interplay between 5' UTR introns & nuclear mRNA export for secretory and mitochondrial genes

Frederick Roth Harvard Medical School, Boston, (MA), United States In higher eukaryotes, mRNAs are exported from the nucleus via factors deposited near the 5' end of the transcript during splicing. The signal sequence coding region (SSCR) has been shown to support an alternative type of mRNA export (ALREX) that does not depend on splicing. However, the vast majority of SSCR-containing genes also have introns, so the potential interplay between splicing-dependent export and ALREX remains unclear. I will describe evidence that introns in the 5' untranslated region (5'UTR) interfere with ALREX, and that SSCRs from 5'UTR-intron-lacking (5UI-) genes promote mRNA export, while SSCRs from 5'UTR-intron-containing (5UI+) genes do not. Unexpectedly, 5'UTR introns are also depleted among genes with a mitochondrial-targeting sequence coding region (MSCR). This led to the discovery that MSCRs from 5UI- genes contain ALREX-like nucleotide signatures and promote non-canonical mRNA export in vivo. Finally, I will describe a machine learning method to predict which transcripts are capable of using the ALREX pathway. Our results suggest that many human genes, including a substantial subset of those encoding secretory and mitochondrial proteins, share a common capacity for non-canonical mRNA export.

162

Reconstruction of an Ancestral U1A/U2B”/SNF Family Protein

Sandra Williams, Kathleen Hall Washington University Medical School, St Louis, MO USA Using newly developed bioinformatics methods (1) and the now extensive database of protein sequences, we have constructed a new phylogenetic tree of U1A/U2B”/SNF proteins. A family tree was first proposed by Polycarpou-Schwartz in 1996 (2), but new data and new bioinformatics methods provide a more robust model. Our new tree shows that U1A and U2B” duplicated late in vertebrate evolution, leading to our speculation that they are evolving to fulfill unique functions. The predicted last common ancestor protein of all bilaterans is a single protein with two RRMs that resembles Drosophila SNF. We have constructed the gene for this Ur-bilateran protein (which we call Urb) and over-expressed it in E coli. Its RRM1 is thermodynamically rather unstable, but it does specifically bind RNA. We will describe Urb RNA binding profiles and compare them to U1A, U2B”, and SNF. 1. Harms MJ, Thornton JW. 2010 Curr Opin Struct Biol. 20:360-6. 2. Polycarpou-Schwartz et al., 1996. RNA 2:11-23.

Session 10B: Keynote & Session 10B: Function through sequence analysis

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

163

Inclusion of Large Internal Exons (>1 kb) in 5% of Human mRNAs

Mohan Bolisetty, Karen Beemon Johns Hopkins University, Baltimore, MD, USA Human internal exons have an average size of 147 nts; 98.5% are As an initial test of SeqZip, we assessed the proposed influence of EDA exon choice on subsequent splicing of the IIICS exon in the mouse fn1 gene. These two exons are separated by ~6 kb in the genomic sequence (815 nt in the mRNA), a region that encodes 6 constitutively included exons. In contrast to previous findings, our data indicate that the respective percent inclusions of EDA exon and IIICS exon are the same when these exons are analyzed individually or together. This suggests that there is little or no influence of the 5’ EDA exon on subsequent splicing decisions at the 3’ IIICS exon. n>In summary, we have developed a quantitative methodology, which does not require reverse transcriptase, but maintains connectivity and evaluates the integrity of distant intramolecular RNA sequences. In addition to examination of alternative splicing, we are currently utilizing SeqZip to investigate other long RNAs, including mammalian piRNA precursor transcripts and viral genomic RNA. Poster Session 1: Emerging & High-throughput Techniques for RNA

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

193 Discovery of Small Molecular Inhibitors of Yeast Gene Expression Utilizing a High Throughput and Multiparameter Single-Cell Approach

Matthew Sorenson1, Ashwini Devkota1,2, Eun Jeong Cho1,2, Scott Stevens1 1 University of Texas at Austin, 2The Texas Institute for Drug and Diagnostics Development The processes involved in gene expression have each been individually studied in model organisms for decades. These efforts have been strongly aided by the use of reporter genes that are sensitive to defects in a certain process. In recent years, it has become increasingly evident that gene expression processes in eukaryotes involve communication and coordination between many complex independent macromolecular machines. For example several laboratories have described connections between histone modification, transcription and pre-mRNA splicing which ensures efficient cotranscriptional intron removal. We have previously described the development of a versatile gene expression reporter for budding yeast amenable to high-throughput flow cytometry. Cells harboring the reporter generate green and red fluorescence from spliced and unspliced transcripts respectively. Our reporter exhibits a unique signature for defects in many gene expression processes including transcription, pre-mRNA splicing, mRNA export and mRNA decay. Although we have identified genetic determinates of gene expression, the identification of specific inhibitors would not only will aid those researching the target pathway, but also provide novel targets and compounds for future molecular therapies. To address these needs, we have adapted our reporter assay to perform high-throughput screening for small molecular inhibitors of specific gene expression processes using an inducible reporter. In our pilot study, we screened hundreds of small molecules that have a history of use in human clinical trials. By means of plate reader and flow cytometry analysis, we have identified many small molecules that affect gene expression and cell growth. For example, some compounds result in increased green to red fluorescence ratio, while others results in a decreased ratio. Additionally, by virtue of the single-cell nature of our assay, we have identified molecules that increase cell-to-cell variation in reporter expression. We are currently identifying patterns between our primary hit compounds and their effects on our gene expression reporter.

194 Healthy and cancerous serum RNA profiling by the novel RNA extraction reagent and highly sensitive DNA chip “3D-Gene”

Satoko Takizawa, Makiko Ichikawa, Hiroko Sudo, Yoji Ueda, Hideo Akiyama Toray Industries, Inc. Kamakura, Japan Proteins, metabolites and DNA are already known as components of serum or plasma biomarkers, however RNA has not been a strong biomarker candidate because of its instability. Exosomes that are small vesicles secreted by various cells are recently reported to play important roles in intercellular communications by transferring proteins, DNA and also RNA to distant cells through circulatory system. Surprisingly, the exosomal RNA in serum preserves its integrity and thus holds a potential to be a new blood biomarkers. In this report, we show the exhaustive analysis of miRNA and mRNA in serum by DNA chip for the highly purified RNA extracted with a novel reagent. Serum contains various types of nucleic acids, mainly small RNA, mRNA, and also short DNA fragment. We suppose that the contamination of DNA to the extracted RNA often causes a discrepancy between the DNA chip analysis and qRT-PCR validation. The novel reagent was able to extract RNA from serum without contamination of short DNA fragment, resulting in better RNA quantification and decreasing DNA-related noise outputs. Using this novel RNA extraction reagent and the highly sensitive DNA chip “3D-Gene”, we analyzed healthy and cancerous serum miRNA profiles. Over 500 miRNAs are detected in healthy and cancerous sera reproducibly, and some miRNAs were detected specifically in cancerous sera, such as breast, gastric, cervix cancer.

Poster Session 1: Emerging & High-throughput Techniques for RNA

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

195

Full Length cDNA Sequencing on the PacBio RS®

196

Sensitive and Selective Nucleic Acid Capture with Shielded Covalent Probes

Jason Underwood1, Lawrence Lee1, Tyson Clark1, Michael Brown1, Sara Olson2, Brenton Graveley2, Jonas Korlach1, Kevin Travers1 1 Pacific Biosciences, Menlo Park, CA USA, 2University of Connecticut Health Center, Farmington, CT USA cDNA sequencing from fully intact RNA molecules is required to build a comprehensive catalog of alternative isoforms present in an organism. Eukaryotic transcripts can display rich patterns of alternative events at diverse positions along the body of a transcript including numerous transcription start sites, internal splice site choices, and cleavage/ polyadenylation sites, as well as more subtle changes from RNA editing. It is likely that some aspects of co-regulation amongst these globally distant events remain undetected since both microarray or short read sequencing often assay short portions of the transcript and then stitch together an average of these observations. To address this gap, we applied the long readlength capabilities of the PacBio® RS to sequence full-length cDNAs. We sequenced libraries generated from budding yeast, the breast cancer line MCF7 and human cerebellum tissue and demonstrate that full length transcript sequences are highly enriched in the sequencing data. We also coupled the full length cDNA approach with normalization methods and the Agilent SureSelect platform to detect splicing patterns of full length human kinome transcripts expressed in these cell types. To further understand how highly complex splice site choices are interconnected, we also generated an amplicon library that assesses the splicing patterns of mRNAs encoding the extracellular domain of the Drosophila DSCAM axon guidance receptor.

Jeffrey Vieregg, Hosea Nelson, Brian Stoltz, Niles Pierce California Institute of Technology, Pasadena, CA USA Nucleic acid probes are used for diverse applications in vitro, in situ, and in vivo. In any setting, their power is limited by imperfect selectivity (binding of undesired targets) and incomplete affinity (binding is reversible and not all desired targets are bound). These limitations stem from reliance on base pairing to both reject off-targets and retain desired targets. To address this selectivity/affinity tradeoff, shielded covalent probes achieve selectivity via conformational change and durable capture via covalent crosslinking of the target with a photoactive nucleoside analog. In vitro assays show that mismatches are efficiently rejected and desired targets are durably captured. For probes designed to reject two-nucleotide mismatches, desired targets are captured nearly quantitatively. Single-nucleotide mismatches are discriminated near the thermodynamic limit. The probes operate isothermally and crosslinking activation is rapid with low-cost light sources. If desired, the crosslinks can be reversed to release the target after capture, suggesting the potential for isolating RNAs from complex biological mixtures. We envision a wide array of applications.

Poster Session 1: Emerging & High-throughput Techniques for RNA

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

197

Development of a Fluorescence-based RNase P Assay

198

High-Throughput Discovery of Novel Post-Transcriptional Regulatory Sequences

Andrew Wallace, Lien Lai, Edward Behrman, Venkat Gopalan Ohio State University, Columbus, Ohio, USA The endoribonuclease RNase P participates in tRNA biogenesis by removing the 5’-leader of precursor-tRNAs (pre-tRNAs). A possible relic of the RNA world, RNase P uses an RNA catalyst, associated with a different number of essential protein cofactors in the three domains of life, to catalyze this site-specific phosphodiester hydrolysis. In addition to being a paradigm for studies of protein-aided RNA catalysis, there is some interest in exploiting structural differences between the bacterial and eukaryotic variants to develop novel antibacterial agents. Facile high-throughput assays would aid the latter objective. Since its discovery four decades ago, RNase P activity has typically been assessed by monitoring the cleavage of internally or terminally 32P-labeled pre-tRNAs. Following denaturing polyacrylamide gel electrophoresis to resolve the substrate and products, the extent of cleavage can be quantitated through use of a phosphorimager. Fluor-labeled pre-tRNAs provide an attractive non-radioisotopic alternative,1 while retaining a high sensitivity and offering the potential for high-throughput assays. With some modifications of the method described by Paredes and Das,2 who demonstrated the copper(I)-mediated alkyne-azide cycloaddition as a bioconjugation strategy for RNAs, we have now used this ligand-free “click” chemistry to generate at low cost 5’-fluor-labeled pre-tRNAs. An in vitro transcription primed with 5’-deoxy-5’-azidoguanosine yielded a 5’-azide-bearing pre-tRNA, which was then coupled to a homemade alkyne-bearing carboxyfluorescein derivative. Our experiments revealed that two such economically prepared fluor-labeled pre-tRNAs are cleaved under multiple-turnover conditions accurately and efficiently by in vitro reconstituted Escherichia coli RNase P. We will present results from our ongoing studies that seek to compare RNase P activities calculated using these fluor-labeled or the more commonly used radiolabeled pre-tRNAs. Since capillary electrophoresis has been successfully employed for separating the substrate and products in an RNase P reaction,3 we are also exploring the use of an automated DNA sequencer with our fluor-labeled pre-tRNAs. 1. J. Hsieh, K. S. Koutmou, D. Rueda, M. Koutmos, N. G. Walter and C. A. Fierke, J. Mol. Biol., 2010, 400, 38-51. 2. E. Paredes and S. R. Das, ChemBioChem, 2011, 12, 125-131. 3. M. Lazard and T. Meinnel, Biochemistry, 1998, 37, 6041-6049.

Erin Wissink, Andrew Grimson Cornell University, Ithaca, NY, USA Post-transcriptional gene regulation contributes to the control of protein production for each mRNA produced in a cell. These events, while necessary for the correct expression of many genes, are not well understood. Regulatory elements in genes, often within the 3’ untranslated region (3’UTR) of the mRNA, bind to trans-acting factors to regulate an mRNA’s nuclear export, stability, and translation efficiency, which in turn affects the amount of protein made from each transcript. Some examples are known, but many cis-regulatory elements in post-transcriptional biology are likely still unidentified. In order to discover novel regulatory elements, we have tested many thousands of short sequences for their ability to regulate gene expression within 3’UTRs. To do so, we inserted random 8-nucleotide sequences into a 3’UTR attached to a GFP reporter, then integrated that collection of reporters into the genome of human cultured cells. The GFP intensity of each cell indicated the effect of the random sequence on gene expression. To discover which sequences changed GFP intensity, we used flow cytometry to isolate cells with high and low GFP intensity, then used next-generation sequencing to determine which random sequences were over-represented in those pools. Sequences enriched in sorted populations were validated in an orthogonal reporter assay. Using this assay, we can screen large numbers of possible regulatory sequences and find robust effectors of gene expression. Doing so will improve our ability to understand post-transcriptional regulation on a genome-wide scale.

Poster Session 1: Emerging & High-throughput Techniques for RNA

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

199

Rapid Pathogen Detection Using Novel RNA-Based Technologies

200

Abstract Withdrawn

Jacek Wower1, Iwona Wower1, Christian Zwieb2 1 Auburn University, Auburn, AL, USA, 2University of Texas Health Science Center at San Antonio, San Antonio, TX, USA We exploit properties of nucleic acids to fabricate flexible programmable biosensors for the rapid detection of RNAcontaining pathogens. First, we amplify the target RNA into its complementary RNA (cRNA) using isothermal RNA amplification (IRA). The cRNA is a synthetic RNA molecule that does not exist in nature and thus, when analyzed, will not suffer from contamination by naturally occurring living organism. As a result, a high level of specificity is achievable. The detection of the cRNAs requires their hybridization to a receptor as well as base pairing of the cRNA with various types of enhancers. DNA-linked gold nanoparticles (DNA-Au-NPs) can serve as both a receptor and an enhancer module. The requirement for two independent hybridization reactions permits manipulation of the sensor to optimize RNA target detection. The goal of this work is to synthesize receptor-cRNA-enhancer modules for incorporation into RNA sensors and evaluate their effectiveness for the detection of bacteria. These sensors will target tmRNA, an RNA molecule that is present in all bacteria. Because tmRNA is absent in eukaryotic cells, the possibility that RNAs derived from humans, animals and plants will interfere with the detection process is minimal. Our preliminary experiments demonstrated that 10-17 M cRNAs could be detected using DNA-Au-NPs as both a receptor and an enhancer. Given that each actively dividing E. coli cell contains 3,000 tmRNA molecules, and a two-minute-long IRA reaction produces thousands of cRNA copies, our findings indicate that the cRNA-based sensor will able to detect a single cell of E. coli. To increase sensitivity of detection we use as an enhancer RNA networks composed of multiple copies of an aptamer that binds Malachite Green (MG). When bound to its RNA aptamer, MG emits strong fluorescence that can be readily monitored by minimally trained personnel. Our technologies are expected to have significant impact in food agriculture, medicine, research and national security.

Poster Session 1: Emerging & High-throughput Techniques for RNA

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

201 Structure-function Analysis of Thermostable RNA Ligase - Engineering ATP Independent Enzyme.

Alexander Zhelkovsky, Larry McReynolds New England Biolabs, Ipswich, MA, USA RNA ligases are essential reagents for many methods in molecular biology including NextGen RNA sequencing. To prevent ligation of RNA to itself, ATP independent mutant ligases defective in self-adenylation are often used in combination with activated pre-adenylated linkers. Ligation bias caused by the secondary structure of the singlestranded substrates or linkers as well as reverse de-adenylation of pre-adenylated substrates are the major concerns in RNA ligation. To overcome these problems we performed mutational analysis of the archaeal thermophilic RNA ligase from Methanobacterium thermoautotrophicum. We identified amino acids essential for ATP binding and reactivity but dispensable for phosphodiester bond formation with 5’ pre-adenylated donor substrate. The motif V lysine mutant (K246A) showed reduced activity in the first two steps of ligation reaction. The mutant is fully active in ligation with pre-adenylated substrates but also in reverse step 2, deadenylation. Alanine substitution of the catalytic lysine of motif I (K97A) abolished activity in the first two steps of the ligation reaction, but preserved wild type ligation activity in step 3. The activity of the K97A mutant is similar with either pre-adenylated RNA or single-stranded DNA (ssDNA) as donor substrates but we observed two-fold preference for RNA as an acceptor substrate compared to ssDNA with an identical sequence. In contrast, truncated T4 RNA ligase 2 is significantly more active using pre-adenylated RNA as a donor versus pre-adenylated ssDNA, and the T4 RNA ligases are inefficient in ligation using ssDNA acceptors. The K97A mutant described here can be used for 3’ RNA ligation with pre-adenylated linker at650C to reduce secondary structure bias, and for ssDNA ligation in 5’ ligation independent cloning experiments.

202

Genome-wide Characterization of Eukaryotic Transcriptomes

Ting Ni1, Han Wu1, David Corcoran2, Wenjing Yang3, Uwe Ohler2, Weiqun Peng3, Jun Zhu1 1 National Heart Lung Blood Institute,Bethesda, MD, 20892, 2Duke University Medical Center, Durham, NC 27705, 3George Washington University, Washington, DC 20052 Emerging sequencing technology has provided unprecedented throughput for monitoring transcriptome complexity. In particular, alternative regulations in multiple steps of mRNA biogenesis, including but not limited to transcriptional initiation, pre-mRNA splicing and polyadenylation, have been shown to serve as major contributors for variant transcript production. One fundamental question is how genetic and epigenetic codes are wired in the genome to control tissue and/or cell-specific gene expression. We have developed several sequencing-based technologies to investigate differential promoter usage, antisense transcription and alternative 3’-endformation of eukaryotic transcriptomes. Our early work showed that Drosophila promoters, similar to that of mammalian ones, exhibit “peak” and “broad” initiation patterns. Further analyses demonstrated that core promoter motifs as well as their location preferences, in together with local chromatin structure, play important roles in promoter choices. In addition, a compendium of genomics data has also been collected to investigate the connectivity and feedback regulation of transcription initiation/elongation and alternative polyadenylation at the systems level. Taken together, integration of genomics and epigenomics information is expected to shed light on the complexity and regulation of eukaryotic transcriptomes

Poster Session 1: Emerging & High-throughput Techniques for RNA

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

203 Cap Independent Translation Control By 3’Untranslated Region (UTR) Elements Of Barley Yellow Dwarf Virus (BYDV) RNA

Bidisha Banerjee, Sohani Das Sharma, Dixie Goss Department of Chemistry and Biochemistry, Hunter College and the Graduate Center of the City University of New York, New York, NY 10065, USA BYDV belongs to luteoviridae family which represents pervasive groups of economically important plant viruses. BYDV can severely limit food grain production causing yield losses resulting in food scarcity. Many plant viruses including BYDV lack a 5’ cap and 3’poly A tail and are translated by cap-independent translation (CIT) mechanism, an alternate means employed to circumvent host defense mechanisms. 5’ UTR of some viruses e.g. Tobacco Etch Virus contain an internal ribosome entry site (IRES) which obviate the need for a 5’ cap by direct recruiting of the ribosome without upstream scanning. The BYDV translation element (BTE) located in 3’ UTR of mRNA stimulates CIT initiation at the 5’upstream AUG via base-pairing (kissing stem loop interaction) of BTE to a complementary loop, SL-D placed within the 5’-UTR of viral genomic RNA. In addition to recruiting ribosomes or translation factors, the 3’ BTE also transfers them to the 5’ end of the viral RNA where translation initiates. This mechanism is being investigated by studying the role of eukaryotic initiation factors (eIFs) eIF4F, in the translation initiation complex formation using the biophysical techniques fluorescence anisotropy and stopped flow kinetics. Protein-RNA complexes formed during the translation process are dynamic and were studied both as an equilibrium reaction and a dynamic process. Wild type BTE and its mutants with in-vitro translation efficiencies from 5-164 % were studied. eIF4F binding to BTE and its mutants gives equilibrium binding constant values (Kd) which are a measure of relative complex stability. We show 1) translation efficiencies correlated well with eIF4F binding with one exception; 2) Kinetics and interaction of other eIFs contribute; 3) BTE-eIF4F interaction is both enthalpically (52%) and entropically (47%) favorable with approximately 5 % more enthalpic contribution to ΔG° at 25°C. This quantitative information along with the stability measurements and binding-release kinetics determines the nature of interactions and yields unique information on the sequential assembly of the initiation complex and it’s binding to viral mRNA.

204

characterization of the HDV RNA promoter required for recognition by RNA Polymerase Ii

Yasnee Beeharry, Martin Pelchat University of Ottawa, Ottawa, Ontario, Canada The hepatitis D virus (HDV), the smallest known RNA virus to infect animal, is composed of a circular singlestranded RNA of 1700 nucleotides that folds into a rod-like structure. HDV uses the human RNA Polymerase II (RNAP II) for its replication, therefore using RNAP II as an RNA dependent RNA polymerase, without any DNA intermediate. The alignment of 750 000 sequences from a whole viral population, obtained by deep-sequencing showed that the HDV promoter region was extremely conserved. The alignment of sequences from natural HDV isolates showed that there was a high-level of covariation, strongly suggesting that it is the RNA promoter structure that is responsible of the interaction with RNAP II. Altogether, this work allowed to construct a model of the features required for the recognition of an HDV RNA by RNAP II. This work will help to define the use of the unexplored function of the RNAP II to act as an RNA dependent RNA polymerase and will contribute to better understand HDV replication.

Poster Session 1: RNA-Protein Interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

205

CELF1 Expression Patterns In Vertebrate Embryonic Development

Yotam Blech-Hermoni1,2, Samantha Stillwagon2, Andrea Ladd2 1 Case Western Reserve University, Cleveland, (OH), USA, 2Lerner Research Institute - Cleveland Clinic, Cleveland, (OH), USA BACKGROUND: CELF (CUG-BP, Elav-Like Family) proteins are multifunctional RNA-binding proteins. CELF1 in the nucleus regulates alternative splicing, while CELF1 in the cytoplasm has been shown to regulate mRNA deadenylation, stability, and translation. CELF1 has been shown to be post-transcriptionally regulated postnatally, while prenatal regulation has not been investigated. In heart and skeletal muscle, CELF1 protein levels are high during embryonic development, drop precipitously prior to birth, and remain low in adult tissue. Work on CELF1 has focused primarily on striated muscle and brain during late-fetal and adult stages and in tissues associated with myotonic dystrophy pathology. Investigations of CELF1 in earlier development have focused on the role of the protein in the processing of maternal transcripts in Xenopus oocytes, but the expression of CELF1 in the normally developing embryo has been largely neglected. AIM: The aims of this work are to characterize the spatial and temporal patterns of CELF1 transcript and protein expression during vertebrate embryogenesis, and to evaluate evolutionary conservation by comparing expression patterns in chicken and mouse. METHODS: The distribution of CELF1 transcripts in the developing embryo was analyzed by in-situ hybridization and transcript levels in different tissues during development were compared using real-time PCR. The tissue distribution of CELF1 protein was investigated by immunofluorescence, while protein levels (as well as the subcellular distribution of the protein) were compared in the different tissues during development by western blot. FINDINGS: CELF1 is expressed from very early stages of embryonic development, with high expression in the nervous system and in muscle. The subcellular distribution of CELF1 protein is tissue-specific: it is predominantly found in the nucleus in the myocardium and in other muscle cells, while it is predominantly found in the cytoplasm in the nervous system. In the developing heart, protein levels are transiently increased during early stages and then drop precipitously before birth. These findings suggest that CELF1 may be regulating targets in different tissues by largely different mechanisms.

206 Selective Interaction of RNA Helicase A with 5’-Leader of Viral and Cellular Complex mRNAs: Components of the Core mRNP that Directs Progression through the Translation Cycle

Sarah Fritz, Arnaz Ranji, Kathleen Boris-Lawrie Ohio State University RNA helicases are multi-domain RNA binding proteins involved in all aspects of RNA biology and replication of viruses. DHX9/RNA helicase A (RHA) is necessary for translation of selected complex mRNAs. RHA specifically interacts with retroviral and junD mRNA and recruits them to polysomes. The specific RNA-protein interaction requires: 1) the RHA-responsive post-transcriptional control element (PCE) in the 5’-untranslated region; 2) RHA’s amino-terminal doublestranded RNA-binding domains (dsRBDs). Conserved basic residues interact with the cognate PCE RNA and tether the conserved helicase domain to the structured RNA, stimulating ATPase-dependent ribonucleoprotein (RNP) rearrangement and protein production. RHA mutations within the conserved dsRBDs and/or DEIH helicase core fail to complement PCE activity following downregulation of endogenous RHA, and remain stalled within 40S ribosomal fractions in mRNA polysomal profiles. Carboxyl-terminal mutants become stalled on polysomes, implicating termination activity. Given the role of RHA in translation and data from mass spectrometry, we hypothesized that these defects are explained by a loss of functional interaction with the translation initiation factor PABP and additional translational regulatory proteins. Glutathione-S-transferase (GST) pull-down assay and IP in cultured cells validated that RHA associates with PABP and HuR (human antigen R). The interaction is conferred through RHA’s N-terminal and C–terminal residues. RHA associates with translation release factor eRF3 (eukaryotic release factor 3) and the transcriptional/translational regulatory protein YB-1 (Y-box binding protein 1), and the interaction is conferred by RHA’s central domains. These newly discovered cellular binding partners of RHA modulate stability and control of translation initiation, elongation, and termination, suggesting that RHA functions as the core mRNP protein that directs progression through the translation cycle. Results posit that RHA coordinates the association of the mRNA stability proteins HuR and YB-1 with target transcripts to protect against mRNA degradation and to prevent compact mRNA folding, respectively. And RHA facilitates the appropriate interaction between PABP and eRF3 to enable translation termination and translation re-initiation. Our proteomics, IP and functional analysis have determined RHA domains that are necessary for translation control of retroviral and junD mRNAs coordinate the association with PABP and 3 new cofactors of RHA. Poster Session 1: RNA-Protein Interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

207

HITS-CLIP Reveals Points of Contact Between the SLBP and the Histone Stem-Loop

208

Binding of Mbl and MBNL to a Model RNA

Lionel Brooks 3rd, John Mahoney, Michael Whitfield Dartmouth Medical School, Hanover, NH, USA The replication-dependent histone mRNAs are cell cycle-regulated primarily by post-transcriptional mechanisms. The histone 3’ stem-loop is bound by the SLBP and is necessary and sufficient to confer regulation on a heterologous gene. We developed a bioinformatics analysis pipeline specifically for the analysis of HITS-CLIP data which conducts data pre-processing, identifies regions with significant sequence coverage, plots nuclease cleavage sites and identifies putative crosslink points by mapping indels in the short sequence reads. We performed SLBP RNA-binding protein IPs (RIP) without crosslinking followed by Solexa sequencing (RIPseq) of the bound targets and find that the most enriched genes are exclusively histone genes. In order to identify MNase resistant SLBP RNP complexes, we performed cross-linked RNA binding protein immunoprecipitation (HITS-CLIP) using either anti-SLBP or a non-specific IgG. We examined both read coverage and MNase cleavage site distribution by selecting enriched messages from different size complexes and clustering the coverage vector (reads/base) across each histone mRNA. We find these coverage vectors form three distinct groups with each mRNA mapping to a single group. One group has reads mapping primarily to the stem-loop, while two other groups of mRNAs show enrichment of two different MNase resistant complexes. These two complexes likely represent terminating ribosomes captured on the messages in the CLIP experiment. In order to delineate the boundaries of these complexes, we mapped the MNase cleavage sites from the sequencing data. We find MNase cleavage sites flanking the stem-loop sequence but no evidence of cleavage within the stem-loop. This suggests that the SLBP completely protects the stem-loop from MNase cleavage. In order to identify the point of contact between SLBP and the stem-loop, we examined our sequence reads for indels resulting from the UV induced crosslink. We find evidence for crosslinking to at least one of the four uridines of the loop. Examination of three naturally occurring histone stem-loop variants with a cytosine in the second position of the loop show significantly reduced crosslinking and fewer mapped reads. A small number of indels are recovered at the first uridine of the loop. Consistent with prior NMR results, these data suggest the SLBP directly contacts the loop, primarily crosslinking to the first or second 5’ nt in the loop. Supported by NIH grant 5R01HG004499 to MLW.

Jay Narasimhan, Danielle Cass Reed College Alternative splicing has been shown to be an integral part of gene regulation. One protein factor that has been implicated in regulating alternative splicing is Muscleblind (MBNL), a protein also associated with the disease Myotonic Dystrophy (DM). Patients with DM misregulate MBNL by sequestering it away from its role in alternative splicing through binding of a toxic RNA element. This disease state results in misspliced mRNAs that result in some of the physical features of DM such as cardiac problems and insulin resistance. The family of Muscleblind proteins was first discovered in drosophila and it has since been shown that Mbl is the Drosophila homologue of MBNL. Due to their homogeneity, it is interesting to note that the domain structure of MBNL is not conserved in Mbl. MBNL consists of four zinc fingers while Mbl consists of only two zinc finger domains. This study compares these zinc finger domains using a fluorescence anisotropy assay.

Poster Session 1: RNA-Protein Interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

209

Isolation and Analysis of Peptides That Bind to Helix 69 of Bacterial 23S rRNA

Moninderpal Kaur, Christine Chow Wayne State University, Detroit, MI, USA The goal of this study was to identify compounds that bind to bacterial 23S rRNA in an effort to discover new RNA-binding motifs with potential therapeutic applications. The helix 69 region was chosen as a target because of its important structural and functional roles in translation. Peptides binding to helix 69 of E. coli 23S rRNA were selected from a 7mer phage display library. Several peptide sequences dominated the phage pool after four rounds of selection. One peptide contained amino acid residues similar to those found in a region of the ribosome recycling factor that is known to make contact with helix 69. To evaluate the relative binding affinities of the selected peptides with helix 69, fluorescent bead assays were carried out with immobilized peptides. The highest binding peptide in the screening assay was then further quantified with respect to helix 69 affinity by using a more sensitive method, namely electrospray ionization mass spectroscopy (ESI MS). The apparent dissociation constant obtained for helix 69 and this peptide was in the low micromolar range. This value is comparable to that of aminoglycoside antibiotics binding to the A-site RNA of 16S rRNA. The ESI MS binding experiments were also carried out with variants of helix 69, in order to identify critical components for favorable interactions. These experiments revealed that the presence of modified nucleotides, specifically pseudouridine and 3-methylpseudouridine, impact peptide binding to helix 69. In addition, the effect of pH on complex formation between helix 69 and peptide was studied, in which there was a three-fold difference of the apparent dissociation constant for the 1:1 complex of RNA and peptide, indicating that either protonation of the RNA or the peptide structure influenced the binding interaction. Finally, specificity of the peptide for bacterial helix 69 was tested by employing related RNAs such as human helix 69 and unrelated RNAs such as helix 31 and the A-site of 16S rRNA. The peptide displayed at least a three-fold reduced affinity for all of the RNAs tested relative to the parent helix 69 RNA, suggesting that the selected peptide has features that are suitable for it to be developed further as a potential lead compound for novel antimicrobials.

210 Differential Transcriptome Occupancy of hnRNP L upon T Cell Stimulation Revealed by Computational Genomics

Brian Cole, Ganesh Shankarling, Kristen Lynch Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA T cell stimulation results in global changes in gene expression and splicing. The abundant nuclear splicing regulatory protein hnRNP L is required for the excision of three exons from the CD45 phosphatase, a hallmark of T cell activation. Previous work by our laboratory has demonstrated the regulation of alternative splicing by hnRNP L in response to T cell stimulation, yet the mechanism by which signals translate to alternative pre-mRNA processing remains unknown. To investigate the global role of hnRNP L in the transition from resting to activated transcriptomes, we examined the RNA binding pattern of hnRNP L before and after T cell stimulation with crosslinking and immunoprecipitation followed by sequencing (CLIP-Seq). We sequenced over 440 million reads from resting and stimulated T cells in biological triplicate, both for a Jurkat-derived line and primary human CD4+ T cells. Approximately 80% of these reads mapped within RefSeq genes. Analysis of these genic reads resulted in discovery of almost 50,000 binding sites in pre-mRNAs with replicate support, with over 95% of binding sites discovered in introns. Canonical CA-rich and CA-repeat elements were the most frequent motifs found in hnRNP L binding sites, in agreement with previous in vitro SELEX data. We discovered binding sites in the two best-characterized hnRNP L targets, its own transcript and that of CD45. Our data also provide evidence for over 5,000 additional transcripts bound by hnRNP L, including several other splicing regulatory proteins, suggesting a network of splicing factors regulated by hnRNP L. This analysis revealed a profound redistribution of hnRNP L on target transcripts between resting and stimulated T cells, which is not explained by changes in gene expression or sequence features within binding sites. Studies are currently underway to determine the molecular basis of the differential transcriptome occupancy of hnRNP L before and after T cell stimulation. Specifically, we are investigating if this is due to post-translational modification-induced alterations in binding specificity or cooperative binding with other proteins. We are also directly analyzing the expression of individual genes to determine if differential occupancy of hnRNP L throughout the transcriptome confers signal-induced changes in the RNA processing of specific target genes. Poster Session 1: RNA-Protein Interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

211 Structural Studies on the NF90/NF45 Dimerisation Domain Complex Reveals an Evolutionary Relationship to RNA Modifying Enzymes

Urszula Wolkowicz, Atlanta Cook Wellcome Trust centre for Cell Biology, University of Edinburgh, Edinburgh, Scotland Nuclear factors NF90 and NF45 form a complex involved in a variety of cellular processes. NF90 contains two dsRNA binding domains and interacts with a variety of cellular RNAs, affecting gene expression at transcriptional and post-transcriptional levels. In addition, this complex participates in the life cycle of several viruses, including Hepatitis C virus and Dengue virus, by direct interaction with viral RNA. NF90 and NF45 dimerise via their common “DZF” domain (domain associated with zinc fingers) that currently has no known function. We present the 1.9 Å crystal structure of the NF90/NF45 dimerisation domain. The DZF domain shows a remarkable similarity to a family of RNA modifying enzymes, but has lost critical catalytic residues. We further discuss the ligand binding properties of NF45 and its role in a variety of related cellular complexes.

212 Deciphering the Molecular Determinants of the Complex Formed by the Immature microRNA let-7g and the Pluripotency Factor Lin28

Alexandre Desjardins, Ao Yang, Jonathan Bouvette, James Omichinski, Pascale Legault Universite de Montreal, Montreal (Qc) Canada Lin28 is a highly conserved protein comprising a unique combination of RNA-binding motifs, an N-terminal coldshock domain and a C-terminal region containing two retroviral-type CCHC zinc-binding domains. An important function of Lin28 is to inhibit the biogenesis of the let-7 family of microRNAs through a direct interaction with let-7 precursors. Here, we systematically characterize the determinants of the interaction between Lin28 and pre-let-7g by investigating the effect of protein and RNA mutations on in vitro binding. We determine that Lin28 binds with high affinity to the extended loop of pre-let-7g and that its C-terminal domain contributes predominantly to the affinity of this interaction. In addition, we uncover remarkable similarities between this C-terminal domain of Lin28 and the NCp7 protein of HIV-1, not only in terms of primary structure but also in their modes of RNA binding. This NCp7-like domain of Lin28 recognizes a G-rich bulge within the extended loop of pre-let-7g, which is adjacent to one of the Dicer cleavage sites. We hypothesize that the NCp7-like domain initiates RNA binding and partially unfolds the RNA. This partial unfolding would then enable multiple copies of Lin28 to bind different sites present in the extended loop of pre-let-7g and protect the RNA from cleavage by the pre-microRNA processing enzyme Dicer.

Poster Session 1: RNA-Protein Interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

213

RPL22 Targets p53: Ribosomal Protein Playing Outside The Ribosome

214

A Novel Three-unit tRNA Splicing Endonuclease Found in Ultrasmall Archaea

Anne-Cecile Duc, Jason Stadanlink, Yong Zhang, David Wiest Fox Chase Cancer Center Ribosomal proteins are increasingly found to play roles beyond their involvement in protein synthesis within the ribosome. Previously data from our lab indicated that the loss of Ribosomal Protein Large Subunit 22 (RPL22) selectively interfered with T-cell development in a lineage-specific manner. Unlike the loss of most other ribosomal proteins, which causes widespread deleterious effects, RPL22 deletion in mice specifically inhibits T-cell commitment to the α/β, but not the γ/δ lineage. Deletion of tumor suppressor p53 along with L22 rescues development of α/β lineage T cells, demonstrating that the block in T cell development in RPL22-deficient mice is p53 dependent. The focus of this research is the interaction of RPL22, an RNA-binding protein, with the mRNA of p53 and its effect on p53 translation regulation. The protein/RNA interactions were investigated using RNase protection assays. The role in translation regulation was studied by in vitro translation assays. The insight about the interaction of RPL22 with p53 mRNA will provide us with an understanding towards RPL22 extra-ribosomal role(s) in controlling the expression of the p53 tumor suppressor.

Kosuke Fujishima1,2, Junichi Sugahara2, Christopher Miller3, Brett Baker4, Massimo Di Giulio5, Kanako Takesue2, Asako Sato2, Masaru Tomita2, Jillian Banfield6, Akio Kanai2 1 NASA Ames Research Center, Moffett Field, CA, USA, 2Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan, 3Department of Earth and Planetary Science, University of California, Berkeley, CA, USA, 4Dept. of Geological Sciences, University of Michigan, Ann Arbor, MI, USA, 5Laboratory for Molecular Evolution, Institute of Genetics and Biophysics-ABT, CNR, Naples, Napoli, Italy, 6Department of Environmental Science, Policy, and Management, University of California, Berkeley, CA, USA In Archaea and Eukaryotes, some precursor transfer RNAs (Pre-tRNAs) are intervened by short introns, permuted or disrupted into multiple fragments that require the help of RNA splicing enzyme during the maturation step. Previously, three types of archaeal tRNA splicing endonucleases (α4, α2 and (αβ)2) and a single type of eukaryotic splicing endonuclease (αβγδ) have been reported. Despite the differences in the subunit composition and substrate specificity, they all share a common functional four-unit architecture to interact with and cleave the RNA substrate, which was thought to be a universally conserved trait. Here we report a first example of three-unit splicing endonuclease in the Archaeal Richmond Mine acidophilic nanoorganisms (ARMAN), an ultrasmall Archaea with a diameter of minimum 200 nm that is uniquely branched within the Archaeal tree. The three-unit endonuclease found specifically in ARMAN-1 and ARMAN-2, has undergone dynamic gene rearrangement: a duplication of thecatalytic α subunit, and fusion with a structural β subunit that was once encoded as a separate gene. This enzyme function as a homodimer and thus form an irregular six-unit architecture (defined as a fourth type of archaeal splicing endonuclease: ε2 type). Surprisingly, the ε2 endonuclease is capable of cleaving both strict and relaxed bulge-helix-bulge motif in vitro, which was previously known as a crenarchaeal (αβ)2 endonuclease-specific feature. This enzymatic property explains why only the tRNA genes found in ARMAN-2 are highly disrupted by intron at various positions. Accordingly, coevolution of tRNA genes and their processing enzymes will be discussed along the course of invention of ε2 splicing endonuclease and the gain of tRNA introns.

Poster Session 1: RNA-Protein Interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

215 Mutual Biochemical Modulation of eIF4G1 and the DEAD-box RNA Helicase Ded1p from Saccharomyces cerevisiae

Zhaofeng Gao, Heath Bowers, Andrea Putman, Eckhard Jankowsky Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, OH The DEAD-box RNA helicase Saccharomyces cerevisiae both activates and represses translation initiation through formation and resolution of an eIF4F-mRNA complex. In this complex, Ded1p directly interacts with eIF4G1. Here, we investigate how Ded1p and eIF4G1/eIF4E modulate their biochemical activities. We show that Ded1p increases the affinity of eIF4G1/eIF4E for RNA. Intriguingly, Ded1p allows eIF4G1 to form complexes with short RNAs that eIF4G1 alone cannot bind. While eIF4G1 does not affect the RNA-stimulated ATP hydrolysis by Ded1p, eIF4G1 inhibits the unwinding activity of Ded1p. This inhibition is caused by interference of eIF4G1with the oligomerization of Ded1p that is needed for optimal unwinding activity. Detailed analysis of the inhibitory effects of eIF4G1 on Ded1p unwinding reveals that eIF4G1 undergoes the strongest interaction with Ded1p bound to RNA, although an interaction between the proteins is seen also in the absence of RNA and ATP. Collectively, our results reveal complex mutual modulation of the biochemical activities of both eIF4G1 and Ded1p. In turn, the interaction between the two proteins is modulated by RNA binding of Ded1p.

216 Ionic strength analysis of a DEAD-Box protein reveals that the type of RNA can alter the ATP binding site

Ivelitza Garcia, Michael Albring, Winnie Wong Allegheny College, Meadville, PA, USA DEAD-box proteins are found in many organisms and are implicated in most RNA metabolic pathways. Classically, these motor proteins are defined as enzymes that couple RNA duplex unwinding with ATP hydrolysis through protein conformational changes. Studies have shown that DEAD-box proteins are not limited to RNA unwinding and can modulate RNA-protein as well as protein-protein interactions [1]. One example is the S. cerevisiae ribosome biogenesis pathway in which rRNA maturation requires at least 14 DEAD-box proteins, each with a unique functional role. The specific RNA target of these ribosomal proteins has yet to be elucidated since the kinetic and thermodynamic properties are insignificantly altered when comparing a wide range of RNA substrates. In contrast, the bacterial DEAD-box protein DbpA requires hairpin 92 (H92) within domain 5 of the 50S rRNA to stimulate optimal hydrolysis activity. RNA substrates that contained H92 as well as various segments of domain 5 altered the observed activity of the protein [2]. Thus, the architecture of the RNA potentially affects the interactions within the RNA and ATP binding sites for DEAD-box proteins. To explore the RNA effects on the network of interactions within DEADbox proteins, the monovalent ionic strength dependence of binding and ATP hydrolysis was examined for Dbp3p (S. cerevisiae ribosome biogenesis DEAD-box proteins) in the presence of various RNAs. All RNAs selected for this analysis facilitated similar catalytic behavior at low ionic strength. The rate of ATP hydrolysis was insensitive to increasing ionic strengths for U-rich RNAs. The binding of ATP in the presence of U-rich RNAs became progressively weaker with increasing monovalent salt. In accordance to the counterion condensation theory, U-rich RNAs facilitates a single monovalent ion displacement upon ATP binding. In contrast, the stimulation of ATPase activity with other RNA substrates resulted in a greater dependency on the concentration of monovalent salt in Dbp3p. ATP hydrolysis gradually decreased while the ATP binding affinity remained unchanged as a function of increasing ionic strength. The binding of ATP did not result in an ion displacement event in this case. The displacement of an ion for certain RNAs indicates that interactions within the ATP binding site can been altered. This comparative examination suggests that the architecture of RNAs fine-tunes the intermolecular interactions of DEAD-box proteins. [1] Cordin, O. , et al. Gene. 267, 17-37 (2006). [2] Tsu, C.A., et al. RNA 7,702–709 (2001). Poster Session 1: RNA-Protein Interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

217

Genome-wide Identification of Cellular RNA Targets of the DEAD-box Helicase Ded1p

Ulf-Peter Guenther1, Frank Tedesci1, Akshay Tambe1, Sarah Geisler1, Jeffery Coller1, Elizabeth Tran2, Mark Adams1, Eckhard Jankowsky1 1 Case Western Reserve University, Cleveland, OH, USA, 2Purdue University, West Lafayette, IN, USA DEAD-box RNA helicases use ATP to bind and remodel RNA and RNP complexes. In vitro, most DEAD-box proteins display little sequence specificity, but in the cell many of these enzymes are thought to target specific RNAs at specific regions. Although most eukaryotic DEAD-box proteins have been implicated in defined cellular processes, it is often not known which RNAs are targeted and where targeted RNAs are bound. The absence of this critical information greatly complicates the understanding of biological functions of DEAD-box proteins on a molecular scale. Here, we define binding sites in RNA targets of the DEAD-box helicase Ded1p from Saccharomyces cerevisae. We combined in vivo crosslinking of genomically encoded, histidine-biotin (HB) affinity tagged Ded1p with protein purification under denaturing conditions, followed by Next Generation Sequencing of crosslinked RNAs. Ded1p has been implicated in various cellular processes, including translation initiation and ribosome biogenesis. Accordingly, we find that Ded1p binds (pre-) ribosomal RNA and a large cross-section of expressed mRNAs. The number of mRNA sequence reads correlates broadly with the mRNA expression level, consistent with a role of Ded1p as general translation initiation factor. Bindings sites of Ded1p are distributed along the entire length of mRNAs, and many are found in the ORF. A major binding site of Ded1p in most mRNAs is seen slightly downstream of the initiation codon. The binding sites do not have an apparent sequence signature, but appear to favor proximity to potential RNA secondary structure elements such as hairpins. This preference correlates with higher affinity of Ded1p for hairpins, compared to non-structured RNAs in vitro. We are currently determining functional implications of Ded1p binding to specific sites on mRNAs.

218 RNA binding and RNA remodeling activities of the Half-a-Tetratricopeptide (HAT) protein HCF107 underlie its effects on gene expression

Kamel Hammani1, William Cook2, Alice Barkan1 University of Oregon, Eugene, OR, USA, 2Midwestern State University, Wichita Falls, TX, USA The Half-a-Tetratricopeptide Repeat (HAT) motif is a helical repeat motif related to the tetratricopeptide repeat (TPR), a degenerate 34 amino acid motif that forms a pair of antiparallel alpha-helices. TPR motifs are typically found in tandem arrays, which stack to form a broad surface that binds protein ligands. The HAT motif has been functionally linked to RNA due to its presence exclusively in complexes that influence RNA metabolism, including rRNA biogenesis, RNA splicing and polyadenylation. This functional association with RNA suggested that HAT repeat tracts might bind RNA. However, RNA binding activity has not been reported for any HAT repeat tract, and recent literature has emphasized a protein-binding role. HCF107 is a nucleus-encoded HAT protein that localizes to the chloroplasts of land plants. Mutations in Arabidopsis HCF107 cause defects in the translation and stabilization of mRNAs derived from the chloroplast psbH gene. In this study, we demonstrated that HCF107 binds single-stranded RNA with specificity for sequences at the 5’ end of the processed psbH mRNA isoforms that require HCF107 for their accumulation. In addition, we show that bound HCF107 (i) blocks a 5-3 exonuclease in vitro, accounting for its ability to stabilize psbH RNA in vivo; and (ii) remodels local RNA structure in a manner that can account for its ability to enhance psbH translation. We suggest that the RNA binding, RNA protection, and RNA remodeling activities observed for HCF107 are likely to be general features of long HAT repeat tracts, and that these properties are likely to contribute to the functions of HAT domains found in other ribonucleoprotein complexes in the nuclear-cytosolic compartment. Reference: Hammani K, Cook BW, Barkan A. RNA binding and RNA remodeling activities of the Half-aTetratricopeptide (HAT) protein HCF107 underlie its effects on gene expression. in press. 1

Poster Session 1: RNA-Protein Interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

219

Recognition of Termination Signal of Non-coding RNAs by Nab3

Fruzsina Hobor1, Odil Porrua-Fuerte2, Roberto Pergoli1, Karel Kubicek1, Dominika Hrossova1, Stepanka Vanacova1, Domenico Libri2, Richard Stefl1 1 CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic, 2Centre de Génétique Moléculaire,CNRS-UPR3404, Gif sur Yvette, France Non-coding RNAs transcribed by RNA polymerase II are processed by the poly(A)-independent termination pathway, that requires the Nrd1 complex. The Nrd1 complex consists of the RNA binding proteins, Nab3 and Nrd1, and the putative RNA helicase Sen1. Nrd1 and Nab3 form a stable heterodimer and each of them binds specific termination signals in the nascent transcript, initiating the termination and subsequent processing or degradation of these transcripts. Here we used a combination of structural, biochemical, and genetic methods to decipher the rules that govern the recognition of Nab3 termination element and shed light on the role of these factors in the assembly of Nrd1-dependent termination machinery. We show that the high-affinity RNA binding is based on a cooperativity between Nrd1 and Nab3. We demonstrate that a minimal heterodimer containing only the dimerization domains and the RNA recognition motifs (RRM) shows the same RNA-binding affinity as the full-length Nrd1-Nab3 heterodimer. Artificial selection and genome wide analysis of Nrd1/Nab3-dependent termination signals in yeast, suggested that the Nab3 binding site is longer than previously reported. Indeed, we show that this extended termination signal increases the binding affinity of the Nrd1-Nab3 complex and the isolated Nab3 with the RNA. Interestingly, the RRM of Nab3 is not sufficient for the recognition of the extended Nab3-terminator but also requires the presence of a distal α-helical element of Nab3. We present the structural basis, for how this novel element can effect the RNA binding affinity and substrate selectivity of a classical RRM, via an induced fit mechanism. Mutations either in residues at the binding surface of Nab3, or in the extended Nab3 binding site strongly decrease the binding affinity and impair Nrd1-Nab3 dependent termination, suggesting that the interactions in the studied complex are important for proper transcription termination of non-coding RNAs.

220 The Mechanism Of RNA Binding By The 27 kDa Trypanosoma brucei Pentatricopeptide Repeat Protein

Pakoyo Kamba, Neil White, David Dickson, Charles Hoogstraten Michigan State University, East Lansing, Michigan, USA. Infection by Trypanosoma brucei (T. brucei) is fatal if untreated, yet available drugs have high toxicity and inadequate efficacy. Developing new treatments requires elucidation of novel drug targets in the parasite which are absent in humans. Among these are the pentatricopeptide repeat (PPR) proteins, a group of organellar RNA binding proteins characterized by tandem repeats of 35 amino acids. These proteins are highly conserved in trypanosomatids, and their downregulation causes severe phenotypes in T. brucei, implying functions that are essential to the parasite. The manner in which PPR proteins interact with RNA is however unknown, mainly because structure-function studies are hindered by difficulties in heterologous protein production. In an effort to further understanding of these proteins, we have targeted the smallest T. brucei PPR protein with a molecular mass of 27 kDa (hence PPR27) and five PPR motifs. Soluble recombinant protein production was achieved after fusion to maltose binding protein (MBP). Trypanosomal PPR proteins are found to be highly alpha-helical, in agreement with modeling based on the related tetratricopeptide (TPR) peptide-binding motif. We have used in vitro selection and binding assays with homopolymeric RNA and DNA to understand the ligand specificity of PPR27. We have also used various deletion mutants lacking one or two PPR motifs to investigate the contribution of each PPR motif to RNA binding. We are now using nuclear magnetic resonance (NMR) spectroscopy to gain insight into the amino acid residues involved in RNA contacts

Poster Session 1: RNA-Protein Interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

221 Characterization of an Inhibitory Compound of Nematode Tandem Zinc Finger RNA-Binding Proteins

Ebru Kaymak, Sean Ryder UMASS Medical School, Worcester, (MA), USA Post-transcriptional regulatory mechanisms guide early development of C. elegans embryogenesis. RNA-binding proteins regulate maternally supplied mRNAs to establish body axes and define cell fate. Understanding RNAbinding protein function and mechanism will define how maternal mRNA regulation contributes to patterning during embryogenesis. Functional studies of maternal RNA-binding proteins are complicated by embryonic lethality and sterility phenotypes. Small molecule inhibitors could provide a useful tool to dissect post-transcriptional regulatory mechanisms and the roles of RNA-binding proteins during development. To identify small molecule inhibitors, we adapted fluorescence polarization assays to high-throughput screening format for three C.elegans RNA-binding proteins: MEX-5, POS-1 and MEX-3. MEX-5 and POS-1 are tandem zinc finger (TZF) proteins required for embryonic axis polarization, and MEX-3 is a tandem KH domain protein required for anterior cell fate specification in the embryo. All three assays were screened using the 30,000-compound Chembridge library. Following confirmatory tests, eight compounds were found to inhibit both MEX-5 and POS-1, but not MEX-3. No inhibitors of MEX-3 were identified. Compound UMW-CE showed 5-fold selectivity for POS-1 than MEX-5. The inhibition constant is not affected by increasing Zn+2 concentration, suggesting that the compound does not chelate zinc. DTT reverses inhibition of POS-1, but TCEP does not. We hypothesize that oxidized UMW-CE reacts with cysteine sulfhydryls required to coordinate zinc, and that DTT blocks inhibition by competition and by reversing conjugation. Future experiments will test the mechanism of inhibition, and address the basis of specificity. A reversible, covalent inhibitor could be useful to study TZF protein function in worm extracts.

222 Investigation of Protein-RNA Interactions by UV Induced Cross-linking and Mass Spectrometry

Katharina Kramer1, Timo Sachsenberg2, Oliver Kohlbacher2, Henning Urlaub1 MPI for Biophysical Chemistry, Goettingen, Germany, 2University of Tuebingen, Tuebingen, Germany UV-induced protein–RNA cross-linking is a common method for studying protein–RNA interactions. After excitation of a nucleic acid base by UV light, a novel covalent bond can form to an amino acid residue in the base’s close spatial proximity. Mass spectrometric analysis of such cross-links has become a powerful tool for studying protein–RNA contact sites and for identifying proteins, peptides and even amino acids that interact directly with RNA; novel RNA-binding motifs have also been identified. However, the low cross-linking yield and the analysis of mass spectrometry data still present various challenges. Enrichment based on titanium dioxide is used to enhance the proportion of cross-linked species in the excess of noncross-linked material. Despite this, cross-linked peptide–RNA oligonucleotide heteroconjugates are of very low abundance and require highly sensitive analytical methods for their detection and identification. Recent advances in the speed, sensitivity and accuracy of mass spectrometers greatly aid the unambiguous identification of heteroconjugates. We have developed a novel data analysis approach in which an algorithm subtracts the theoretical masses of all possible nucleotide combinations from the experimental precursor mass. A database search of the modified precursor masses together with the unaltered fragment information identifies the cross-linked peptide. Thus, this approach allows automated searching for cross-link candidates and greatly reduces the analysis time. This is especially important, as state-of-the-art mass spectrometers produce large amounts of data, at a rate that makes manual evaluation nearly impossible. We have successfully applied our data analysis approach to a number of small ribonucleoprotein complexes, namely spliceosomal U1 snRNP and a model complex for ASH1-mRNA transport. The number of identified cross-linked heteroconjugates was greatly increased compared with earlier approaches, and the greatly improved data quality reduced ambiguity. Currently, we are combining improved instrumental and analytical aspects to investigate protein–RNA contacts in more extended systems. We are establishing a workflow in which mRNA is isolated from nuclear or cellular extract by immunoprecipitation or by affinity purification of RNA-binding proteins. In combination with UV cross-linking, we expect to identify contact sites of abundant RNA-binding proteins at the peptide or even amino-acid level.

1

Poster Session 1: RNA-Protein Interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

223

Oligomeric Assembly of HIV-1 Rev on the Rev Response Element: Role of Cellular Cofactors

Rajan Lamichhane, Rae Robertson-Anderson, Svitlana Berezhna, Edwin van der Schans , David Millar The Scripps Research Institute, La Jolla, (CA), USA Rev, a key regulatory protein of HIV-1, activates nuclear export of unspliced and partially spliced viral mRNAs, which encode the viral genome and the genes encoding viral structural proteins, respectively. Initially, a single Rev monomer binds to a highly conserved region, stem IIB located on the Rev Response Element (RRE) of viral mRNA. Following this nucleation step, additional Rev monomers are recruited to the RRE through a combination of RNA-protein and proteinprotein interactions, resulting in the formation of a functional nuclear export complex. In addition, several cellular proteins, such as the DEAD box helicases DDX1 and DDX3 are known to be required for efficient Rev function in vivo, although their precise role is unknown. In this study, single-molecule total internal reflection fluorescence (smTIRF) microscopy was used to visualize oligomeric assembly of Rev on the RRE with single monomer resolution. Binding of up to eight fluorescently labeled Rev monomers to a single immobilized RRE molecule was observed in real-time as discrete jumps in fluorescence intensity, and the event frequencies and corresponding binding and dissociation rates for the different Rev-RRE stoichiometries were determined. The smTIRF assay was used to study Rev-RRE assembly in the presence of DDX1, DDX3 and other cellular proteins. The presence of DDX1 promotes oligomeric assembly by accelerating the first few Rev monomer binding steps, suggesting that DDX1 acts as a chaperone of Rev. The smTIRFmeasurements are being extended to a multi-color format, in order to directly visualize the colocalization of Rev and selected cellular proteins on the same immobilized RRE molecules. These measurements are revealing the precise timing of various protein binding events during ribonucleoprotein assembly. Supported by NIH grant P50 GM082545.

224 Critical role of the RNA-binding protein Mex-3B in the control of phagocytosis and cell-cell adhesion

Mailys Le Borgne, Nicolas Chartier, Marc billaud Institut Albert Bonniot grenoble france We have previously reported the identification of a family of four mammalian RNA-binding proteins homologous to Mex-3, a post-transcriptional regulator that specifies blastomeres identity in the early Caenorhabditis elegans embryo. Here, we report that male mice with a targeted disruption of one of these four genes, called Mex-3B, are hypofertile and present a progressive obstruction of seminiferous tubules resulting in a decreased sperm output. The defect is intrinsic to somatic Sertoli cells that fail to recruit junctional proteins : N-cadherin, occludin and connexin-43 at the peripheral membrane, thereby destabilizing Sertoli-Sertoli junctional complexes that form the blood-testis barrier. In addition, phagocytic properties of Sertoli cells are impaired, thus impeding the clearance of residual bodies released during spermatid maturation. Exploration of the underlying mechanisms revealed that Mex-3B is a key regulator of the small GTPase-protein Rap1 that controls cell adhesion and phagocytosis. Thus, these findings unveil a key role for Mex-3B in controlling activation of Rap1 that regulates Sertoli cell physiology during spermatogenesis.

Poster Session 1: RNA-Protein Interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

225 Genome-wide analysis of RBFOX1 and RBFOX2-regulated exons extends their regulatory role to distal intronic elements

Michael Lovci1, Henry Marr2, Justin Arnold1, Marilyn Parra2, Tiffany Liang1, Sherry Gee2, Joe Gray2, Dana Ghanem2, John Conboy2, Gene Yeo1 1 UC San Diego, La Jolla, California, 2Lawrence Berkeley National Labs Mechanisms of post-transcriptional RNA processing and RNA metabolism such as alternative pre-mRNA splicing (AS) are strongly regulated according to cell-type and developmental stage by RNA binding proteins (RBPs) which interact with coding or non-coding regulatory elements. Using genome-wide cross-linking and immunoprecipitation followed by sequencing methods (CLIP-seq), we had previously shown that the RBFOX2 protein binds in proximal intronic regions to control AS in embryonic stem cells. Here, in addition to confirming RBFOX-family members’ role in proximal intronic regions, we extend their function to include the regulation of AS via distal (>500nt) sites within intron regions. Comparative genomics approaches identify that these distal sites are highly conserved across evolution and identify several more in the genome. We demonstrate that one of these distal splicing control elements enhances the splicing of an exon within the ENAH gene in cultured cells using minigene splicing reporters, and in vivo using splicing enhancer-blocking antisense vivo-morpholinos in live mice. Our genome-wide analysis reveals that distal conserved splicing control elements can be found downstream of breast cancer subtype-specific exons, and exons mis-regulated in myotonic dystrophy, frontotemporal dementia and genes implicated in autism. The prevalence of RBFOX distal regulatory elements, especially with in light of their proximity to several disease-associated exons, suggests that a vitally important mechanism of gene regulation that has, until now, gone under-studied. Understanding alternative splicing networks in normal development and in disease will require consideration of proximal and distal motifs whose combined activities determine splicing outcomes.

226 Identification of mRNAs and Novel non-coding RNAs Associated with Drosophila Sm Proteins

Zhipeng Lu, A. Gregory Matera University of North Carolina, Chapel Hill, NC, USA Sm proteins are a family of highly conserved RNA-binding proteins present in all three domains of life. In eukaryotes, Sm proteins bind small nuclear RNAs (snRNAs) to form snRNPs, which are basic components of the pre-mRNA splicing machinery. However, little is known about other functions of Sm proteins in eukaryotic cells, given their divergent roles in regulating mRNA stability in bacteria and archaea. We developed an experimental strategy to identify RNAs associated with Sm proteins by massively parallel sequencing of RNA immunopurified from RNA-Sm-protein complexes, and statistical methods to find RNAs that are enriched in the immunoprecipitates. Using this approach, we uncovered a subset of mRNAs and novel unannotated non-coding RNAs that are associated with Sm proteins. One novel Sm-class snRNA is highly conserved in five of the twelve sequenced Drosophilid species, and is unrelated to any of the spliceosomal snRNAs at the sequence level. We also developed a new approach to identify the terminal stem-loops of non-coding RNAs based on their propensity to self-prime during preparation of the cDNA sequencing library.

Poster Session 1: RNA-Protein Interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

227

Structural Basis of Lariat RNA Recognition by the Intron Debranching Enzyme, Dbr1

228

New Tools for the Enrichment and Detection of RNA- Protein Interactions

Eric Montemayor1, Adam Katolik2, Alexander Taylor1, Jonathan Schuermann3, Joshua Combs4, Richard Johnson2, Stephen Holloway1, Masad Damha2, Scott Stevens4, P. John Hart1 1 Univ. of Texas Health Sci. Center, San Antonio, TX, USA, 2McGill University, Montreal, Canada, 3Cornell University, Ithaca, NY, USA, 4University of Texas, Austin, TX, USA The spliceosome removes introns from pre-mRNA in the form of a lariat that contains an unusual 2’,5’-phosphodiester linkage. This linkage must be hydrolyzed by the intron debranching enzyme (Dbr1) before a spliced intron can be metabolized or processed into essential cellular factors such as snoRNA and miRNA. Dbr1 is also involved in the propagation of retrotransposons and HIV-1, ostensibly through a transient 2’,5’-phosphodiester linkage that has been proposed to facilitate the strand-transfer reaction of reverse transcription. Despite extensive biochemical characterization over several decades, the exact enzyme mechanism and structural basis of lariat RNA recognition by Dbr1 has remained elusive. Here, we describe the first structures of Dbr1, in complex with several RNA compounds that mimic the branchpoint structure in lariat RNA. The structures demonstrate how Dbr1’s catalytic machinery is compatible with the 2’,5’-phosphodiester linkage and not the far more abundant 3’,5’-phosphodiester linkage. A combination of cell-based functional assays, in vitro activity assays, inductively coupled plasma mass spectrometry (ICP-MS) and X-ray anomalous diffraction methods were used to derive an enzyme mechanism for Dbr1. The proposed mechanism is novel in that it involves a dinuclear metal-binding center with functional alternation between single and double metal ion configurations. These findings provide a framework for understanding the role of Dbr1 in retrotransposon and retrovirus replication, and draw further attention to the potential evolutionary relationship between retrotransposons, retroviruses and pre-mRNA splicing.

Kay Opperman, Chris Etienne, Scott Meier, J. Schultz, Barbara Kaboord, Atul Deshpande Thermo Fisher Scientific, Rockford, (IL), USA Currently, enrichment and detection of RNA-protein interactions are limited by inefficient enrichment and release of the RNA-protein complex without disruption the interaction. Our approach to improve the efficiency of enrichment has been to utilize the RNA as the bait for the RNA-protein complex. The RNA is 3’- end labeled with a modified cytidine 3’, 5’ bisphosphate containing a spacer arm with an affinity handle using T4 RNA ligase. We have evaluated both desthiobiotin and biotin as affinity handles to determine the best attachment and elution of the complex. Several different spacer lengths and compositions were assessed to maximize accessibility of the RNA to the protein once attached to the bead without compromising secondary structure. The efficiency of the enrichment of the RNA-protein complex was tested using known RNA:protein interactions, including miRNA:Argonaute 2, poly A RNA:PolyA Binding Protein, and SNRNPA/U1 RNA. The endogenous RNA binding proteins were obtained from cell lysates or expressed in human in vitro translated cell lysates. Non-specific binding was determined by incubation of lysate with beads only, or with an unrelated labeled RNA. Elution with biotin allowed for more flexibility for further downstream applications, such as mass spectrometry. Our results using control systems suggest that our enrichment method is able to efficiently capture and detect RNA binding proteins with labeled RNA as bait. Interestingly, we were also able to enrich additional proteins in the binding complex, as evidenced by the isolation higher molecular weight complexes of miRNA:Argonaute detected by Western blot.

Poster Session 1: RNA-Protein Interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

229

The HIV-2 Leader RNA Structure And Interactions With NCp8 Protein

Katarzyna Pachulska-Wieczorek, Katarzyna Purzycka, Agnieszka Stefaniak, Ryszard Adamiak Institute of Bioorganic Chemistry Polish Academy of Science, Poznan, Poland Retroviruses selectively encapsidate a dimeric RNA genome assembled from two identical positive sense strands interacting near their 5’-ends. The signals important for the HIV genome dimerization and encapsidation are located in the untranslated leader RNA region (5’UTR) that contains highly structured domains which play various regulatory roles in the viral replication. The Gag and Gag-derived nucleocapsid (NC) proteins are considered important for dimerization, encapsidation and reverse transcription of retroviral RNA. The functions of these proteins are correlated with their ability to act as nucleic acids chaperones. The interaction sites of Gag and NC proteins within the viral RNA are not definitely determined, especially in the case of HIV-2. Furthermore, different dimerization and encapsidation mechanisms were proposed for HIV-1 and HIV-2. We have recently determined in vitro secondary structure of the HIV-2 leader RNA captured as a loose dimer. Two contact interfaces in the loose dimer form were identified within the SL1 and TAR motifs. We showed that both SL1 and TAR as isolated domains are bound by recombinant HIV-2 NC protein (NCp8) with high affinity, contrary to the hairpins downstream of SL1 (1). Foot-printing of the SL1/NCp8 complex indicates that the major binding site maps to the SL1 upper stem. The relatively tight binding of NCp8 to the TAR domain is especially interesting in view of the data indicating the involvement of the TAR hairpin III in the formation the HIV-2 leader RNA loose dimer. Nucleic acid-chaperoning activities of recombinant NCp8 protein and chemically synthesised NCp8 peptide were examined. We report that, much as NCp7, NCp8 has potent nucleic acid-chaperoning activities in the standard DNA annealing, DNA and RNA strand exchange and RNA ribozyme cleavage assays. 1. Purzycka,K.J., Pachulska-Wieczorek,K. and Adamiak,R.W. (2011) The in vitro loose dimer structure and rearrangements of the HIV-2 leader RNA. Nucleic Acids Res., 39, 7234-7248

230 Characterization of ΔN-Zfp36l2-mutant Associated with Early Embryonic Arrest and Female Infertility

Silvia Ramos University of North Carolina, Chapel Hill, NC, USA The zinc finger protein 36-like 2, Zfp36l2/TIS11D, has been implicated in mouse female infertility, since an N-terminus truncation mutation (ΔN-Zfp36l2) leads to two-cell stage arrest of embryos derived from the homozygous mutant female gamete. Zfp36l2 is a member of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins that can bind to transcripts containing AU-rich elements (ARE), resulting in deadenylation and destabilization of these transcripts. I show here that the mouse Zfp36l2 is composed of two exons and a single intron, encoding a polypeptide of 484 amino acids. I observed that ΔN-Zfp36l2 protein is similar to both wild-type Zfp36l2 and TTP (Zfp36) in that it shuttles between the cytoplasm and nucleus, binds to RNAs containing AREs, and promotes deadenylation of a model ARE-transcript in a cell-based co-transfection assay. Surprisingly, in contrast to TTP, Zfp36l2 mRNA and protein were rapidly down-regulated upon LPS exposure in bone marrow-derived macrophages. The ΔN-Zfp36l2 protein was substantially more resistant to stimulus-induced down-regulation than the WT. I postulate that the embryonic arrest linked to the ΔN-Zfp36l2 truncation might be related to its resistance to stimulus-induced down-regulation.

Poster Session 1: RNA-Protein Interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

231

Determinants and anti-determinants of substrate recognition by yeast RNAse III

232

Mapping the Interaction Between E. coli tRNAPro and the Trans-Editing YbaK Protein

Kevin Roy, Guillaume Chanfreau UCLA Double-stranded RNA binding domains (dsRBDs) participate in a wide variety of cellular processes and constitute the second most abundant RNA binding domain. Many dsRBDs bind only to their specific double-stranded RNA (dsRNA) substrates in vivo. In general, the molecular basis for the substrate specificity of dsRBDs is poorly understood. In budding yeast, the major RNAse III Rnt1p contains a dsRBD which recognizes hairpins capped by A/uGNN or AAGU tetraloops, which adopt a conserved fold upon dsRBD interaction for structure-specific recognition of these two classes of tetraloops. Previous solution structures have shown that a single conserved binding mode is used by the Rnt1p dsRBD for all its substrates. The structural data suggest a model whereby Rnt1p dsRBD scans dsRNA in a weakly bound conformation until specific substrate elements are recognized, triggering a conformational change into the tightly bound state. While the most of the binding specificity appears to be achieved through the interactions of the dsRBD alpha helix 1 with the tetraloop minor groove, other residues including A395 of the dsRBD contact the stem at a highly conserved AU base pair 11 nucleotides from the tetraloop. Mutation of the AU to a GC but not other base pairs resulted in a mild processing defect in vivo for the snR47 precursor, a model Rnt1p substrate. This suggests that the GC may function as an antideterminant, but also calls into question the basis for the high enrichment of AU at that position in Rnt1p substrates. We are currently investigating the role of this AU base pair in other Rnt1p substrates, as well as the role of A395 in substrate specific recognition.

Brianne Sanford1, Maryanne Refaei2, Mark Foster1,2, Karin Musier-Forsyth1,2 Deparment of Chemistry and Biochemistry, Center for RNA Biology, 2Ohio State Biochemistry Program, The Ohio State University, Columbus, OH, USA Prolyl-tRNA synthetases (ProRS) commonly misactivate alanine and cysteine, which are similar in size to cognate proline, and this error must be corrected to avoid mis-incorporation during protein synthesis. E. coli ProRS uses a socalled “triple-sieve” editing mechanism wherein an editing domain within ProRS (INS) edits Ala-tRNAPro in cis and a free-standing protein, YbaK, edits mischarged Cys-tRNAPro in trans. Previous fluorescence anisotropy binding studies showed that YbaK and ProRS interact in vitro, and chemical cross-linking studies support the formation of a ProRSYbaK-tRNAPro ternary complex1. Recent work inspecting these interactions through a split-GFP assay demonstrated that YbaK and ProRS also interact in vivo (Byung Ran So, PhD Thesis, 2010). Although these studies indicate that YbaK, ProRS, and tRNAPro interact as a ternary complex, there is currently no high resolution structural information on this interaction. The present work is focused on defining the interaction between ProRS, tRNAPro, and YbaK at the molecular level. Two-dimensional 1H-15N NMR correlation spectra of 15N-enriched YbaK have been obtained in the absence and presence of tRNAPro. Preliminary studies reveal a subset of peaks perturbed upon tRNAPro binding. In addition to the NMR experiments, cross-linking studies are being conducted to characterize the binding of tRNA to both YbaK and ProRS. The NMR and cross-linking data will be combined with computational docking and on-going Förster resonance energy transfer studies to yield the first three-dimensional model of this editing complex. Reference: 1.An, S. and K. Musier-Forsyth, Cys-tRNA(Pro) editing by Haemophilus influenzae YbaK via a novel synthetaseYbaK-tRNA ternary complex. J Biol Chem, 2005. 280(41): p. 34465-72. 1

Poster Session 1: RNA-Protein Interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

233 Amino Acid Signature Important for Modified tRNA Recognition: A Tool for Studying Modified RNA-Protein Interactions

Jessica Spears1,2, Caren Stark1,2, Paul Agris1,2 University at Albany-SUNY, Albany, (New York), USA, 2The RNA Institute, SUNY Albany, (New York), USA HumanLys3UUU is the nearly exclusive primer for HIV replication via reverse transcription; the HIV-1 nucleocapsid protein, NCp7, facilitates the tRNALys3 use by binding to and remodeling the tRNA structure. Human tRNALys3 is posttranscriptionally modified, but until recently the importance of those modifications in tRNA recognition by NCp7 was unknown. Recent studies show that modifications such as mcm5s2U34 and t6A37 in the anticodon stem loop (ASL) are

1

critical in the recognition of human tRNALys3UUU by NCp7 (compare Kd = 0.28 ± 0.03 μ M vs. 2.30 ± 0.62 μ M for unmodified ASLLys3). Furthermore, 15 amino acid peptide mimics also preferentially recognize these same modifications and could inhibit binding by NCp7. Using these NCp7 peptide mimics in a combination of in silico and in vitro studies, we have expanded these initial studies to determine which amino acids are important for modified tRNA recognition. Evolutionary algorithms and AMBER simulations were used to optimize the peptide’s amino acid sequence in silico while a combination of fluorescence, band shift (EMSA) and circular dichroism assays were used to verify the in silico results and elucidate an amino acid signature (R-W-Q/N-H-x2-F-x3-W-R-x2-G, where x can be most amino acids) for modified tRNA recognition and binding. The benefit of this amino acid signature is two-fold. First, this signature can be applied to further studies of HIV inhibition via optimized peptide competition with NCp7. Secondly and perhaps more importantly, this amino acid signature can be used as a tool to study other modified RNA-protein interactions specifically those between tRNAs and their modification enzymes and/or synthetases.

234 Post-translational Modifications of AUF1 during Erythroid Differentiation Regulate β-globin mRNA Expression

Sebastiaan van Zalen, Grace Jeschke, Elizabeth Hexner, J. Eric Russell Perelman School of Medicine University of Pennsylvania, Philadelphia PA, USA We recently demonstrated that two RNA-binding proteins, AUF1 and YB1, assemble a cytoplasm-restricted mRNP on the 3’UTR of β-globin mRNA that regulates its level in erythroid cells. Although both AUF1 and YB1 are ubiquitously expressed, their β-globin mRNA-specific regulatory properties are restricted to terminally differentiating erythroid cells that accumulate the highly stable β-globin mRNA. Based upon these observations, we reasoned that the erythroid- and differentiation stage-restricted assembly of AUF1 and YB1 into the regulatory mRNP reflects post-transcriptional or translational changes in their structures. Our analyses of AUF1 focus on two of its four alternatively spliced isoforms that are expressed in the erythroid cytoplasm. A GST-AUF1 fusion isoform that retains exon 7 (AUF1p45) fails to bind the β-globin 3’UTR, while an otherwise identical isoform that excludes exon 7 (AUF1p40) binds the 3’UTR with high efficiency. These results, which implicate the functional importance of AUF1 exon 7 exclusion, were subsequently validated in erythroid K562 cells using an AUF1 isotype-specific siRNA knockdown strategy. In these experiments, specific depletion of AUF1p40 resulted in a two-fold decrease in β-globin mRNA, while knock-down of AUF1p45 had no effect. The isotype-specific characteristics of AUF1 appear to be part of a constitutive erythropoietic regulatory program, as erythroid-induced hematopoietic stem cells display increased AUF1p40 and decreased AUF1p45 protein expression, without corresponding changes in the levels of their encoding mRNAs, suggesting functionally relevant changes in AUF1 mRNA translation and/or AUF1 isoform protein stability. We also noted that AUF-1 exon 7 encodes an unusually high number of phosphorylation-capable residues. In vitro analyses demonstrate that the β-globin mRNA-binding activity of exon 7-retained AUF1p45 could be restored by prior dephosphorylation, suggesting that post-translational modifications of AUF1 alter its mRNA-binding specificities. Current experiments are designed to identify the relevant phosphorylated amino acids. Collectively, our analyses indicate that the β-globin mRNA-regulating specificity of AUF1 is determined by post-translational events. In addition, the isoform-specific increase of AUF1 suggests a more extensive role for AUF1p40 in post-transcriptional processes that occur in erythroid terminal differentiation, during an interval when gene transcription is undergoing global silencing. Poster Session 1: RNA-Protein Interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

235

LIN28 Interacts With GGAGA Motifs in Messenger RNA

Melissa Wilbert1, Stephanie Huelga1, Anthony Vu1, Thomas Stark1, Katlin Massirer1, Tiffany Liang1, Stella Chen1, Hilal Kazan2, Quaid Morris2, Gene Yeo1 1 UCSD, La Jolla, CA, USA, 2Banting and Best Department of Medical Research, Toronto, Canada LIN28 is a conserved RNA binding protein implicated in pluripotency, reprogramming and oncogenesis. Previously shown to act primarily by repressing let-7 microRNAs, here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Using cross-linking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28 binding sites in a quarter of human genes. These sites revealed that LIN28 binds to GGAGA sequences enriched within predicted loop structures in mRNAs, reminiscent of its interaction with let-7. Among LIN28 targets, we found that LIN28 positively regulates its own mRNA levels, and affects the abundance of splicing regulators. Splicingsensitive microarrays confirmed that LIN28 expression causes widespread alternative splicing changes. These findings show that LIN28 contributes to cellular homeostasis via direct mRNA interactions, alterations of which may promote cancer and differentiation.

236 Depletion Of HuR Inhibits Proliferation Of Normal Breast Epithelial Cells In Parallel With Upregulation Of ΔNp63

Wensheng Yan, Yanhong Zhang, Xinbin Chen University of California at Davis, Davis, (CA), USA HuR, a critical RNA binding protein, has been suggested to function as a tumor maintenance gene in breast cancer, permissive for tumor growth, more aggressiveness and poor prognosis. However, the cellular function and molecular mechanism of this protein remains largely unknown in normal breast epithelial cells. Here, we showed that in MCF-10A breast epithelial cells, HuR knockdown inhibits cell proliferation and enhances premature senescence. In addition, we found that in three dimensional cultures, MCF-10A cells with HuR knockdown can not develop normal acinar architectures with hollow lumen, instead of forming acini with cell-filled lumen. Furthermore, we found that depletion of endogenous HuR increases ΔNp63, but decreases wild-type p53, expression in MCF-10A cells. Consistent with this, we identified two U-rich elements in the 3’ UTR of p63 mRNA, which HuR can specifically bind to. Furthermore, we found that HuR knockdown enhances translation of ΔNp63, but without affecting ΔNp63 mRNA turnover. Together, our data suggested that HuR maintains proliferation of MCF-10A breast epithelial cells via a refined control on ΔNp63 expression.

Poster Session 1: RNA-Protein Interactions

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

237 Recruitment of Bromovirus Genomic RNA Replication Templates is 5’ Cap-Independent but RNA Structure-Inhibited

Quansheng Yang, Brandi Gancarz, Paul Ahlquist University of Wisconsin Upon infection, positive-strand genomic RNAs must first be translated to produce RNA replication factors, which then selectively recruit the same genomic RNAs from translation to membrane-bound viral RNA replication complexes. To further define the relationship between genomic RNA translation and replication, we studied brome mosaic virus (BMV). BMV genomic RNA3 is recruited into vesicular RNA replication compartments on perinuclear ER membranes by the multifunctional BMV 1a methyltransferase/helicase, which recognizes an internal recruitment element (RE) in RNA3. 1a-directed recruitment of RNA3 to replication was unaffected when translation elongation was blocked by frameshift mutations in the 3a open reading frame, or when translation initiation was blocked by an oligo(G) insertion in the 5’-untranslated region, which prevents 40S ribosome scanning for the initiation codon. Similarly, while a 5’ m7G cap is critical for RNA3 translation, uncapped RNA3 initiated RNA replication with efficiency similar to capped RNA in cells of both yeast and barley, a natural BMV host. In addition, we showed that 1a recruits uncapped as well as capped RNA3 to membrane-associated BMV RNA replication complexes. Strikingly, despite the above results, 1a-mediated RNA3 recruitment to replication sites was blocked by inserting a stemloop at or near the RNA3 5’ end. Thus, 1a-mediated RNA3 recruitment for RNA replication was highly sensitive to the state of the RNA3 5’ end, even though RNA3 translation initiation was not required and the only specific cis-acting signal required is the internal RE recruiting element, located 1 kb downstream of the RNA3 5’ end. Inserting the same moderately stable stemloop at internal sites in RNA3 did not block RNA3 recruitment, but internal insertion of more highly stable stemloops did inhibit recruitment. As expected, all mutations inhibiting 1a-mediated RNA3 recruitment similarly blocked RNA3 replication when BMV RNA-dependent RNA polymerase was also expressed. These and other results suggest that RNA3 recruitment into BMV RNA replication vesicles, which requires an active 1a helicase domain, may involve 1a-mediated directional translocation of RNA3 into the vesicular RNA replication compartment.

238

mirEX- comprehensive platform for for highthrougput analysis of Arabidopsis pri-miRNAs

Jakub Dolata, Bielewicz Dawid, Zielezinski Andrzej, Alaba Sylwia, Szarzynska Bogna, Szczesniak Michal, Karlowski Wojciech, Jarmolowski Artur, Szweykowska-Kulinska Zofia Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland MicroRNAs are key post-transcriptional regulators of gene expression in eucariots. In Arabidopsis thaliana miRNA genes that encode the same miRNA species are grouped in gene families. MicroRNA genes that belong to the same family, although identical or almost identical as mature microRNAs, differ considerably in the gene organization and sequence. We designed gene-specific primers for 291 microRNA A.thaliana genes (miRBase release 18) to simultaneously monitor pri-miRNA expression pattern of individual genes using quantitative real-time PCR. Our analyses so far cover seven plant developmental stages. This database aims to be useful to anyone investigating the role of microRNAs in shaping plant development, organ formation and response to different biotic and abiotic stresses.

Poster Session 1: RNA-Protein Interactions & Mechanisms of RNA interference

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

239 Restoration of protein function by miRNA interference – a tentative complementary mode of action for platinum based anticancer drugs?

Alak Alshiekh, Sofi Elmroth Biochemistry and Structural Biology, Dept Chemistry, Lund University, Lund, Sweden During the past decade, the class of non-coding RNAs has been established as a group of nucleic acids with ability to fine-tune protein production. Most attention so far has been paid to the family of miRNAs which suppress protein production by interaction with the 3’-UTRs of protein mRNA. The suppressive function relies on interaction between individual miRNAs and the RISC-complex – an interaction that result in arrest of protein translation after location of the target 3’-UTR sequence using the miRNA as a guide. This pathway is also shared by siRNAs. Not too surprisingly perhaps, dysregulation of miRNA levels is today known to be associated with many common diseases, e.g. cancer, diabetes and inflammation. At present, miRNAs are getting established as therapeutic targets, with the strategy of targeting disease related miRNAs as the preferred mode of action.(1) Clinical treatment of cancers typically involve several treatment regimens where protein targeting drugs are combined with so called “alkylating drugs”, i.e. compounds with high affinity for nucleic acids. In the latter case, the group of platinum based drugs has been particularly successful. Their mode of action involves DNA-binding with down-stream effects on both replication and transcription.(2) Studies by us and others have shown that also RNAs can be targeted by this type of drugs.(3-5) In the Elmroth lab, work has therefore been initiated with the aim of investigating the sensitivity of the mi- and siRNA-controlled translational machinery towards drug exposure. We have previously shown that platination is compatible with cellular uptake and repressive function.(6, 7) In addition, metalation of non-seed regions, i.e. bases outside of the region involved in target recognition with the mRNA, was recently reported to fine tune silencing capacity.(8) In the present study we for the first time turn our attention to effects arising from interaction with the seed region, i.e. the bases directly involved target recognition. The here observed restoration of protein production resulting from platination will be discussed in light of tentative miRNA targets with proapoptotic- and tumor suppressor function. SELECTED KEY REFERENCES (1) GAMBARI, R., FABBRI, E., BORGATTI, M. et al. (2011) Biochemical Pharmacology, 82, 1416-1429. (2) TODD, R. C. & LIPPARD, S. J. (2010) Chemistry & Biology, 17, 1334-1343. (3) HAGERLOF, M.,PAPSAI, P., CHOW, C. S. & ELMROTH, S. K. C. (2006) JBIC, 11, 974-990. (4) RIJAL, K. & CHOW, C. S. (2009) Chemical Communications, 107-109. (5) HOSTETTER, A. A.,OSBORN, M. F. & DEROSE, V. J. (2012) ACS Chemical Biology, 7, 218-225. (6) HAGERLOF, M., HEDMAN, H. & ELMROTH, S. K. C. (2007) BBRC, 361, 14-19. (7) SNYGG, A. S. & ELMROTH, S. K. C. (2009) BBRC, 379, 186-190. (8) HEDMAN, H. K., KIRPEKAR, F. & ELMROTH, S. K. C. (2011) JACS, 133, 11977-11984.

240

The Role of Argonaute in the Mammalian Nucleus

Roya Kalantari, Keith Gagnon, David Corey UT Southwestern Medical Center, Dallas, (TX), USA RNA interference (RNAi) is a system that has been largely studied and defined by its ability to affect gene expression and translation in the cytoplasm. However, Argonaute (AGO) proteins, which are the major catalytic component of the RNA-induced silencing complex (RISC), have been found to be involved in nuclear roles outside of the canonical RNAi pathway. Within non-mammalian systems such as yeast and plants, AGOs have been shown to be involved in functions such as DNA methylation and heterochromatin formation. Our lab has utilized systems involving small RNAs to target nuclear events such as transcription and splicing in human cells. In the case of transcription, we have shown that small RNAs are capable of targeting long non-coding RNAs (lncRNAs) along both the promoter and past the 3’ end of genes in order to control gene expression. We have also demonstrated that targeting of small RNAs to introns and exons of pre-mRNA can robustly alter the splicing pattern. Within these systems, we have found that AGO proteins are recruited by the small RNAs to the nuclear target. However, the protein-protein interactions and mechanisms involved remain unclear. Identification and understanding of the interactions of AGO proteins in the nucleus is essential for comprehension of the mechanisms by which these proteins act. Through immunoprecipitation and mass spectrometry techniques, we have identified potential novel interacting factors of AGO. These candidates range in function from DNA binding, to transcription, and splicing. In order to verify the list of possible candidates, methods of sample preparation such as SILAC and I-DIRT are used, which have been shown to be reliable quantitative proteomic approaches in a range of studies. The interactions of these novel factors with AGO are confirmed using candidate-directed CoIPs. The results suggest that nuclear AGO proteins are involved in several protein complexes that function outside of the RNAi pathway within mammalian systems.

Poster Session 1: Mechanisms of RNA interference

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

241 Mechanisms of RNA Interference: siRNA Strand Selection and mRNA Target Cleavage at the Single Molecule Level

Vishalakshi Krishnan, Sethuramasundaram Pitchiaya, Nils Walter University of Michigan, Ann Arbor, MI, USA RNA interference (RNAi) is a conserved gene regulatory mechanism wherein exogenous long dsRNAs are cleaved (diced) into ~21-28 nt long siRNA duplexes with characteristic 5’-phosphate and 3’-dinucleotide termini that are subsequently loaded into the effector RNA Induced Silencing Complex (RISC). The mature duplex siRNA is then unwound and activated in a strand-specific and ATP-dependent manner. Upon recognition of a target mRNA, the siRNA guides RISC to initiate sequence specific mRNA cleavage (slicing) by the Argonaut (Ago) protein, thus inhibiting the production of a specific protein. It is thought that during strand selection of the siRNA duplex the RNA strand with the thermodynamically less stable 5’ end is preferentially loaded into RISC as the guide strand. In the absence of such differential thermodynamics, the RNA strand with its 3’ end bound to Dicer has been shown to be preferentially incorporated as the guide strand, thus making it equally likely for either strand of the siRNA duplex to affect RNAi. It is not known, however, whether strand selection depends on the presence of the target mRNA and whether there is a bias in selection of a strand when its complementary target is present. To address these questions, single molecule techniques are employed here to visualize the binding of siRNA-containing complexes onto fluorescently labeled target mRNAs as well as to monitor target cleavage in HeLa cytosolic extract with dicing and splicing activity in vitro. We directly observe mRNA target cleavage at the single molecule level upon assembly of specific siRNA-associated complexes with the mRNA, but not in the presence of non-specific siRNAs. Our study is illuminating the basic mechanism of siRNA strand selection followed by specific target mRNA cleavage.

242

Substrate Recognition by Drosophila Dicer-2 and Loquacious-PD

Sucharita Kundu1, Xuecheng Ye2, Philip Aruscavage1, Qinghua Liu2, Brenda Bass1 1 University of Utah, Salt Lake City, Utah, USA, 2University of Texas Southwestern Medical Center, Dallas, Texas, USA Dicer is one of the essential players in the RNA interference pathway and cleaves long double-stranded RNA (dsRNA) into small interfering RNA (siRNA) or microRNA (miRNA) products. Delineating how the various domains of Dicer coordinate and regulate efficient cleavage of dsRNA with different termini is crucial for a complete understanding of dsRNA-mediated pathways of gene expression. We have studied the binding affinity of Drosophila Dicer-2 with dsRNA substrates that have blunt and 3’-overhanging termini. Using gel shift assays, we find that Dicer-2 is able to differentiate between blunt-ended and 3’-overhanging substrates in the presence of a slowly hydrolysable analog of ATP, ATPγS. Studies with a dsRNA binding protein (dsRBP) partner Loquacious-PD (Loqs-PD) suggest that Loqs-PD enhances the affinity of Dicer-2 for its dsRNA substrate. Furthermore, Loqs-PD uses a cooperative mode of binding to bind to dsRNA substrates.

Poster Session 1: Mechanisms of RNA interference

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

243

dsRNA binding proteins alter human miRNA processing by Dicer

Ho Young Lee, Jennifer Doudna UC Berkeley, CA, United States Small non-coding RNAs such as miRNAs and siRNAs play central roles in controlling gene expression in human cells. Sequencing data show that many miRNAs are produced at different levels and as multiple isoforms, but the regulation and functional significance of these RNAs are largely unknown. Here we show that human dsRNA binding proteins (dsRBPs), TRBP and PACT, affect Dicer processing by changing kinetics, substrate specificities and iso-miRNA (isomiR) formation. As the enzyme responsible for processing most miRNAs/siRNAs in the eukaryotic cytoplasm, Dicer must recognize a variety of dsRNA substrates as well as various proteins that are part of the RNA interference (RNAi) machinery. In particular, Dicer binds to TRBP as well as PACT and also interacts with proteins in the Argonaute (Ago) family of endonucleases. Recently it was shown that Dicer processes different kinds of dsRNA substrates at rates that differ by ~100-fold, and that TRBP can enhance the processing rates of these substrates. Furthermore, mutation or truncation of TRBP has been observed in several cancers in which altered miRNA levels are also detected. These data suggest a possible regulatory role of dsRBPs in miRNA/siRNA biogenesis. Our recent results show that TRBP changes the rates of pre-miRNA cleavage in an RNA-structure-specific manner. In addition, TRBP can trigger the generation of isomiRs that are longer than the canonical sequence by one nucleotide. We show that this change in miRNA processing site can alter guide strand selection, resulting in preferential silencing of a different mRNA target. In contrast, PACT binds to Dicer but does not induce changes in pre-miRNA processing rates or product identity as TRBP. The distinct effects of TRBP and PACT suggest that human Dicer functions differently depending on its dsRBP partner.

244 Argonaute-interacting GW protein directs transposon silencing in pathogenic fungus, Cryptococcus neoformans

Prashanthi Natarajan1, Phillip Dumesic1, James Moresco2, John Yates III2, Hiten Madhani1 UCSF, San Francisco, CA, USA, 2The Scripps Research Institute, La Jolla, CA, USA Argonaute containing RNA silencing complexes (RSC) play a central role in small RNA mediated genome defense in a wide range of organisms from plants to humans. Proteins containing Glycine-tryptophan (GW) Argonaute-binding motifs have been shown to be essential components of RSC, especially in executing translation repression and deadenylation of plant and animal microRNA targets. So far, their study has been confined to only a few systems. Here, we report the identification of a GW-protein, Gwo1, in the basidiomycete fungus Cryptococcus neoformans that interacts with the only Argonaute protein in this organism. We have characterized this association to be specific through biochemical experiments. Localization of Gwo1 reveals distinct cytoplasmic foci, which overlap with mRNA processing bodies (P-bodies). This pattern bears striking resemblance to the well- known mammalian miRNA effector protein, GW182. Gwo1 associates with transposon derived small RNAs and their cognate mRNAs, which further strengthens its similarity to previously reported GW proteins. The increase in transposonactivity that results upon the deletion of Gwo1 shows the functional importanceof this protein in Cryptococcusgenome defense. These findings expand the repertoire of GW proteins and provide a genetically tractable model organism for future research. 1

Poster Session 1: Mechanisms of RNA interference

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

245

Mechanistic Studies of Human RISC Loading Complex Function in RNA Interference

Cameron Noland, Enbo Ma, Jennifer Doudna UC Berkeley RNA interference (RNAi) is a conserved mechanism of post-transcriptional gene silencing in which single-stranded guide RNAs base pair with cognate mRNAs, directing their endonucleolytic cleavage or translational repression by protein complexes termed RNA-Induced Silencing Complexes (RISCs). RNAi is initiated by long dsRNAs or hairpin RNAs, which are processed by the RNase III enzyme Dicer to yield short 21-23 nt dsRNAs, termed short-interfering RNAS (siRNAs) or microRNAs (miRNAs). Following dicing, siRNAs are loaded onto the RISC-associated endonuclease Ago2 such that one strand (the “passenger” strand) is cleaved and removed. The remaining “guide” strand then targets complementary mRNAs for silencing. The transfer of a nascent siRNA or miRNA to Ago2 involves the RISC Loading Complex (RLC), which is composed minimally of Dicer, Ago2, and the dsRNA binding protein (dsRBP) TRBP. Another dsRBP called PACT has also been implicated in this process. One substantial grey area in the study of RNAi in humans lies in our understanding of the individual contributions of each RLC component to optimal guide strand selection and target cleavage. Using in vitro reconstituted RLC complexes containing TRBP or PACT, we sought to shed light on this issue.

246 Nuclear Localized Antisense Small RNAs with 5′-Polyphosphate Termini Regulate Longterm Transcriptional Gene Silencing in Entamoeba histolytica G3 Strain

Hanbang zhang, Hussein Alramini, Vy Tran, Upinder Singh Stanford Uni., Stanford, (CA), USA In the deep-branching eukaryotic parasite Entamoeba histolytica, transcriptional gene silencing (TGS) of the Amoebapore A gene (Ap-A) in the G3 strain has been reported with subsequent development of this parasite strain for gene silencing. However, the mechanisms underlying this gene silencing approach are poorly understood. We report that antisense small RNAs (sRNAs) specific to the silenced Ap-A gene can be identified in G3 parasites. Furthermore, when additional genes are silenced in the G3 strain, antisense sRNAs to the newly silenced genes can also be detected. Characterization of these sRNAs demonstrates that they are ~27nt in size, have 5’-poly-phosphate termini, and persist even after removal of the silencing plasmid. Immuno-fluorescence analysis (IFA) and fluorescence in situ hybridization (FISH) show that both the Argonaute protein EhAGO2-2 and antisense sRNAs to the silenced genes are localized to the parasite nucleus. Furthermore, α-EhAGO2-2 immunoprecipitation (IP) confirmed the direct association of the antisense sRNAs with EhAGO2-2. Finally, chromatin immuno-precipitation (CHIP) assays demonstrate that the loci of the silenced genes are enriched for histone H3 and EhAGO2-2 indicating that both chromatin modification and the RNA-induced transcriptional silencing complex are involved in permanent gene silencing in G3 parasites. Our data demonstrate that G3-based gene silencing in E. histolytica is mediated by an siRNA pathway, which utilizes antisense 5’-polyphosphate sRNAs. To our knowledge, this is the first study to show that 5’-polyphosphate antisense sRNAs can mediate TGS, and is the first example of RNAi- mediated TGS in protozoan parasites.

Poster Session 1: Mechanisms of RNA interference

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

247

Abstract Withdrawn

248

The Role of Dicer-dsRBD in Small Regulatory RNA Maturation

Christopher Wostenberg, Kaycee Quarles, Scott Showalter The Pennsylvania State University One of the most exciting recent developments in RNA biology has been the discovery of small non-coding RNAs that effect gene expression through the RNA interference (RNAi) mechanism. Recent work has focused on developing RNAi based therapeutics due to the important roles RNAi plays in the cell including cellular defense, development and homeostasis. Two major classes of RNAs involved in RNAi are small-interfering RNA (siRNA) and micoRNA (miRNA). siRNAs are derived either endogenously from repetitive sequences or exogenously from viral RNAs, whereas miRNAs are only endogenously transcribed. Even through the starting points of siRNA and miRNA production are different both are processed by the RNase III enzyme Dicer prior to forming the RNA-induced silencing complex. RNase III enzymes contain a dsRNA binding domain (dsRBD), with only a few exceptions known to date. Most dsRBDs bind A-form dsRNA in a non-sequence specific way through interactions between positively charged residues and the negatively charged phosphate backbone. The crystal structure of Giardia intestinalis Dicer, which lacks a dsRBD, showed that the PAZ domain recognizes the 2nt 3’ overhang, a feature of precursor miRNAs and siRNAs, in order to position the catalytic domain sites. This model for Dicer binding fails to encompass the complexity of Dicer in higher eukaryotes since Giardia Dicer lacks additional domains. Human Dicer-dsRBD, containing part of the RNase IIIb domain, inhibits full length Dicer from binding dsRNA. Additionally, a construct of human Dicer lacking the dsRBD binds dsRNA, but the initial cleavage rate is reduced. Together, this data shows that Dicer-dsRBD is competent to bind RNA, but the precise role it plays in the mechanism of binding and cleavage is underdetermined. To further explore the role of human Dicer-dsRBD, we expressed it in isolation and determined its binding affinity to various RNAs modeling both pre-miRNA and pre-siRNA. In this study, we show that Dicer-dsRBD is able to discriminate between miRNA and siRNA precursors based on the presence of a terminal loop, which is observed in pre-miRNAs and not pre-siRNAs. In addition, NMR spin relaxation and MD simulations provide an overview of the role dynamics contribute to binding. We compare this current study with our previous studies of the dsRBDs from Drosha, another RNase III enzyme involved in miRNA processing, and its cofactor DGCR8, to give an overall mechanistic view of dsRBD binding of dsRNA Poster Session 1: Mechanisms of RNA interference

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

249 The RDE-10/RDE-11 complex triggers RNAi induced mRNA degradation by association with target mRNA in C. elegans

Huan Yang1,2, Ying Zhang1, Jim Vallandingham1, Hua Li1, Laurence Florens1, Ho Yi Mak1,2 1 Stowers Institute for Medical Research, Kansas City, MO, USA, 2The University of Kansas Medical Center, Kansas City, KS, USA The molecular mechanisms for target mRNA degradation in C. elegans undergoing RNA interference (RNAi) are not fully understood. Using a combination of genetic, proteomic and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. The RDE-10/RDE-11 complex acts in parallel of nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a 5-fold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and micro-RNA biogenesis is normal. Therefore, the RDE10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans.

250

Involvement of Splicing Factors in Chromosome Segregation in Mammalian Cells

Takashi Ideue1, Kazuaki Tokunaga2, Misato Morita1, Kanako Nishimura1, Madoka Chinen1, Mistuyoshi Nakao2, Tokio Tani1 1 Department of Biological Sciences, Graduate School of Science and Technology, 2Institute of Molecular Embryology and Genetics, Department of Medical Cell Biology, Kumamoto University, Japan. Proper chromosome segregation is one of essential steps for cell division. Kinetochore assembled on the centromere plays an important role in the chromosome segregation. It has been known that heterochromatin formation is prerequisite for the assembly of the kinetochore at the centromere. However, the mechanism of the heterochromatin formation at the centromere is largely unknown in mammalian cells. In fission yeast, the RNA interference (RNAi) system induces the formation of centromeric heterochromatin. In that process, non-coding RNAs transcribed from the centromere are processed to siRNAs that are required for the heterochromatin formation. We found that prp14-1, the fission yeast splicing mutant, yields the lagging chromosomes generated by abnormal chromosome segregation. In that mutant, unprocessed centromeric ncRNAs accumulated abnormally. Several lines of experiments demonstrated that Prp14p is involved in the RNAi-mediated formation of centromeric heterochromatin in fission yeast. To examine whether involvement of Prp14p and other splicing factors in the formation of centromeric heterochromatin is conserved in mammalian cells, we knocked down human homologue of Prp14p (hPrp16p) and other splicing factors using RNAi in HeLacells. We found that knockdown of hPrp16p generates the nucleus with abnormal morphology called as a grape-shape phenotype. To clarify the process of generating this phenotype, we carried out the time-lapse observation of knockdown cells. As a result, we found that hPrp16p knockdown cells are defective in chromosome segregation at a mitotic phase. RT-PCR analysis showed that the knockdown of hPrp16p does not cause significant splicing defects, suggesting that the grape-shape phenotypeis not a secondary effect of the splicing inhibition. We will discuss the possibility that several splicing factors are directly involved in chromosome segregation in mammalian cells.

Poster Session 1: Mechanisms of RNA interference & RNA and Epigenetics

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

251

DNA Replication Facilitates Transcription of Epigenetically Silenced Genes

252

Abstract Withdrawn

Javier Peña-Diaz2,1, Siv Hegre2,3, Endre Anderssen2, Per Aas2, Robin Mjelle2, Gregor Gilfillan4, Robert Lyle4, Finn Drabløs2, Hans Krokan2, Pål Sætrom2,5 1 Institute of Molecular Cancer Research (IMCR), University of Zurich, Zurich, Switzerland, 2Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway, 3St. Olavs Hospital, Trondheim, Norway, 4Department of Medical Genetics and Norwegian Sequencing Centre, Oslo University Hospital, Oslo, Norway, 5Department of Computer and Information Science, Norwegian University of Science and Technology, Trondheim, Norway During the somatic cell cycle, DNA and epigenetic modifications in DNA and histones are copied to daughter cells. DNA replication timing is tightly regulated and linked to GC content, chromatin structure, and gene transcription, but how maintenance of histone modifications relates to replication timing and transcription is less understood. Here, we identify an association between replication and expression of epigenetically silenced human genes. Specifically, we identify genes that have cell cycle dependent expression in HaCaT keratinocytes and HeLa cells and show that genes expressed during DNA replication are enriched with the Polycomb silencing mark histone H3 lysine 27 trimethylation (H3K27me3). Consistent with being epigenetically silenced in other cell cycle phases, these genes have generally lower expression than have other cell cycle genes. Moreover, the gene expression is related to replication timing, as genes expressed during G1/S transition and early S phase generally have higher GC content and are replicated earlier than genes expressed during late S phase. These results indicate widespread replication-dependent expression in mammals and suggest a general role for replication-dependent transcription in modulating Polycomb-mediated epigenetic silencing in humans.

Poster Session 1: RNA and Epigenetics & Riboregulation in Development

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

253 Characterization of divergent transcripts from CpG island promoters in mouse embryonic stem cells

Albert Almada1, Ryan Flynn2, Jesse Zamudio1, Phillip Sharp1 Massachusetts Institute of Technology, Cambridge, MA, U.S.A, 2Stanford School of Medicine, Stanford, CA, U.S.A RNA Polymerase II (RNAPII) transcription is a highly regulated process controlling cell type and state. We previously found that RNAPII transcribes low abundant, small (19-25 nts), antisense RNA upstream of most transcription start sites (TSSs) in mouse embryonic stem cells (mESCs) (Seila, 2008). Surprisingly, RNAPII only productively elongates in the protein-coding sense direction from these divergent promoters. These data suggest that control of RNAPII elongation may be a major point of transcriptional regulation and that mechanisms controlling this process may dictate whether a stable RNA molecule is synthesized. Although multiple studies have identified distinct RNA species from mammalian promoters, the precise mapping of RNAs produced from divergent CpG island promoters has not been described. Therefore, we investigated RNAPII divergent transcription through a detailed biochemical analysis of the upstream antisense RNAs (uaRNAs) from four divergent CpG island promoters in mESCs. We find that uaRNAs have distinct capped 5’ termini and heterogeneous 3’ ends ranging in size from 40-1100 nts. uaRNAs are short-lived with average half-lives of 18 minutes and are present at 1-4 copies per cell. We further show that the steady-state levels of uaRNAs, at least in part, are controlled by the RNA exosome. These uaRNAs are probably initiation products since their capped termini correlate with peaks of paused RNAPII. Knockdown of the pausing factor complexes NELF or DSIF results in an increase in the levels of uaRNAs. P-TEFb controls the release from pausing through its phosphorylation of the RNAPII-CTD at Ser2, NELF, and DSIF. Treatment with a P-TEFb inhibitor, flavopiridol, results in decreases in uaRNA and nascent mRNA transcripts with similar kinetics. Further, we find that induction of the Isg20l1 divergent promoter reveals equivalent increases in transcriptional activity in the sense and antisense directions. These data suggest that control of productive elongation at divergent promoters must occur after P-TEFb recruitment. Preliminary data demonstrates that uaRNAs can be spliced and polyadenylated at the upstream antisense polyadenylation signal sequence (PAS). We will explore the contribution of splicing signals to the abundance and stability of these RNAs.

1

254 The Making of an OncomiR: Oncogenic microRNA-21 First Exhibits Unusually Reduced mRNA Binding and Silencing Activity in Healthy Mouse Liver

John Androsavich1, Nelson Chau2, Balkrishen Bhat2, Peter Linsley2, Nils Walter1 University of Michigan, Ann Arbor, MI, USA, 2Regulus Therapeutics, San Diego, CA, USA MicroRNAs (miRNAs) are strongly implicated in cancer and other diseases, yet the mechanisms underlying their aberrant activities and contributions to pathogenesis are convoluted by a lack of knowledge concerning their physiological roles under healthy cellular conditions. miR-21 is a ubiquitous, highly abundant, and stress-responsive miRNA linked to several diseases including cancer, fibrosis and inflammation. We present here that pharmacological inhibition or genetic deletion of miR-21 in healthy mouse liver has little impact on regulation of canonical seed-matched mRNAs, and only a limited number of genes enriched in stress response pathways. These surprisingly weak and selective regulatory effects contrast with those of other abundant liver miRNAs such as miR-122 and let-7. Moreover, miR-21 shows greatly reduced binding to polysome-associated target mRNAs compared to miR-122 and let-7. Bioinformatic analysis suggests that reduced thermodynamic stability of seed pairing and target binding may contribute to this deficiency of miR-21. Significantly, these trends are reversed in human cervical carcinoma (HeLa) cells, where miR-21 shows enhanced target binding within polysomes and triggers strong degradative activity towards target mRNAs. Taken together, our results suggest that under normal cellular conditions miR-21 operates below a threshold required for binding and silencing most of its targets, while in diseased and stressed cells, miR-21 surpasses this threshold, resulting in a gain-of-function enabled by enhanced association with polysome-associated mRNA. Our findings also underscore that RNA silencing can be highly altered between healthy tissues and transformed cell lines.

1

Poster Session 1: Non-coding and Regulatory RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

255

RNA controls the activation of the Aurora-B kinase to promote mitotic spindle assembly.

256

Tertiary Structure Of Pri-miR-17-92 Influences Its Processing

Michael Blower, Ashwini Jambhekar Harvard Medical School, Boston, (MA), United States Recent studies have shown that a large portion of most eukaryotic genomes is transcribed into RNA with little protein coding capacity. However, very little is known about the function of these transcribed ncRNAs. Work from several groups has shown that RNA functions in a translation-independent manner to promote microtubule assembly during meiosis and mitosis, yet it was not known how RNA functioned to promote spindle assembly. We have found that the proteinkinase Aurora-b is present in a complex containing RNAs and RNA binding proteins. Furthermore, we found that RNA is a structural component of the Aurora-b complex, which is required for proper complex assembly and localization. RNA is also required for activation of Aurora-b kinase activity during mitosis and meiosis. Interestingly, inhibition of the downstream Aurora-b target protein MCAK rescues the majority of spindle assembly defects observed after RNase treatment, demonstrating that RNA acts as a structural component of the Aurora-b complex to promote mitosic spindle assembly. Aurora-b associates with a wide variety of both protein coding and noncoding RNAs, suggesting that mRNAs can serve a binary function both as protein coding messages and as structural components of a protein complexs. Identification of the Aurora-b complex as a RNP will provide insight into the many uses of RNA within the cell.

Saikat Chakraborty, Shabana Mehtab, Anand Patwardhan, Yamuna Krishnan NCBS, Bangalore, India MicroRNAs are 21-22 nucleotide small RNAs, that control gene expression by either by RNA transcript degradation or translational repression (1). Mature microRNA concentrations are currently thought mainly to be regulated at the transcriptional level (2). However evidence of intense regulation at the post transcriptional level has been recently obtained where mechanisms have been found that facilitate or inhibit targeted processing of mature miRNA from its precursors (3). Pri-miR-17-92 cluster is a well accepted model system for asymmetric processing of co-transcribed miRNAs. This polycistronic miRNA cluster contains six miRNAs with diverse function and is differentially processed to produce varying amounts of resultant mature miRNAs in different tissues (4). We considered the hypothesis that tertiary structure of this transcript could result in asymmetric processing at the very initial stages. Phylogenetic analysis indicated possible higher order structure formation by this intronic cluster. Following this, we have shown using SHAPE analysis, hydroxyl radical foot printing as well as bulk biophysical methods that pri-miR-17-92 adopts a specific three dimensional architecture with solvent inaccessible regions in an Mg2+ dependent manner. Using a fast acting reagent, benzoyl cyanide, we could elucidate the secondary structure of the whole cluster using SHAPE analysis. Further DMS probing of the cluster yielded the Hoogsteen base pairing sites in the pri-miRNA that mediated tertiary contacts. We have shown that tertiary structure adoption by pri-miR-17-92 cluster poses a kinetic barrier to its own processing (5). The secondary structural map not only provides a means to arrive at the tertiary structure but also indicates how protein binding could modulate processing in a context dependent manner. Reference:

1. Ambros, V. 2004.The functions of animal microRNAs. Nature 431: 350-355. 2. Saini, H.K., Griffiths-Jones, S., Enright, A.J. 2007. Genomic analysis of human miRNA transcripts. Proc. Natl. Acad. Sci USA 104: 17719-17724. 3. Filipowicz, W., Bhattacharyya, S.N., Sonenberg, N. 2008. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Genet. 9: 102-114. 4. Tang, G.Q., Maxwell, E.S. 2008. Xenopus microRNA genes are predominantly located within introns and are differentially expressed in adult frog tissues via post-transcriptional regulation. Genome Res. 18: 104-112. 5. Chakraborty, S., Mehtab, S., Patwardhan, A. and Krishnan, Y. 2012. Pri-miR-17-92a transcript folds into a tertiary structure and autoregulates it’s processing. RNA (in press). Poster Session 1: Non-coding and Regulatory RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

257 snoRNAs Expression Units Are The Source Of New Non-Coding RNAs That Regulate Gene Expression

Marina Falaleeva1, Manli Shen1, Pierre de la Grange2, Eduardo Eyras3, Justin Surface1, Brian Rymond1, Stefan Stamm1 1 University of Kentucky, Lexington, KY, USA, 2GenoSplice Technology, Paris, France, 3Universitat Pompeu Fabra, Barcelona, Spain The Prader-Willi syndrome is the most frequent genetic cause for obesity and type II diabetes. It is caused by the loss of expression of non-coding RNAs, HBII-52 and HBII-85 (SNORD 115 and 116) that resemble C/D box snoRNAs. HBII-52 promotes inclusion of an alternative exon of the serotonin receptor 2C pre-mRNA, which generates an appetite suppressing form of the receptor, and controls alternative splicing of several other pre-mRNAs. Originally HBII-52 and HBII-85 expression units were reported to form canonical C/D box snoRNAs, but they generate mainly smaller RNAs that we termed psnoRNAs for processed snoRNAs. psnoRNAs associate with hnRNPs and not with canonical C/D box snoRNP-proteins. High-throughput sequencing data show that most snoRNAs expressing units give rise to metabolically stable psnoRNAs, indicating the existence of a large, new class of non-coding nuclear RNAs. To determine psnoRNAs target genes, we performed genome-wide array analyses using RNA from transfected cell lines and defined brain regions from Prader-Willi patients. In addition to changes in alternative splicing, we detected an influence of psnoRNAs on the total abundance of multiple mRNAs. MBII-52 and MBII-85 act synergistically on several genes. The target genes function mainly in fat/energy metabolism, cell-cell interactions, and signal transduction, which is consistent with a psnoRNA role in generating a metabolic syndrome. Using a bioinformatics approach, we identified short binding sites of psnoRNAs in target genes. Their locations partially overlapped with known epigenetic modifications, suggesting that psnoRNAs act on some genes via an epigenetic mechanism. Surprisingly, human psnoRNAs are processed correctly when expressed in a heterologous intron in yeast, which allows determining their processing using a genetic approach. psnoRNAs are processed similar to canonical C/D box snoRNAs. They are dependent on splicing of the hosting intron, further trimming by exonucleases and additional action of exosome complex components. Furthermore, psnoRNAs production is independent from the miRNA machinery. The psnoRNAs concentration is physiologically important, as it is changed after eight hours of food withdrawal in the mouse brain. This shows that nutrients can control MBII-52 and MBII-85 concentration, which influences the expression patterns of a large number of genes, showing a physiological function of this new form of non-coding nuclear RNAs.

258

Biogenesis of Mammalian Mirtrons and Simtrons

Mallory Havens2, Ashley Reich1, Dominik Duelli2, Michelle Hastings2 1 Lake Forest College, Lake Forest, IL, USA, 2Rosalind Franklin University, North Chicago, IL, USA Canonical microRNA biogenesis requires the Microprocessor components, Drosha and DGCR8, to generate precursormiRNA, and Dicer to form mature miRNA. The Microprocessor is not required for processing of some miRNAs, including mirtrons, in which spliceosome-excised introns are direct Dicer substrates. Here, we demonstrate that although some predicted human mirtrons are splicing-dependent, as expected, miR-1225 and miR-1228 are produced in the absence of splicing. Knockout celllines and knockdown experiments indicate that biogenesis of these splicing-independent mirtron-like miRNAs, termed ‘simtrons’, does not require the canonical miRNA biogenesis components, DGCR8, Dicer, Exportin-5 or Argonaute 2. However, simtron biogenesis was reduced by expression of a dominant negative form of Drosha. Simtrons interact with Drosha and are processed in vitro in a Drosha-dependent manner. Both simtrons and mirtrons function in the silencing of target transcripts as demonstrated by luciferase reporter assays and simtron association with the Argonaute proteins of the RISC complex. Our findings reveal a non-canonical miRNA biogenesis pathway that can produce functional regulatory RNAs.

Poster Session 1: Non-coding and Regulatory RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

259

Mechanistic insights into crRNA biogenesis in type II and III CRISPR systems

Martin Jinek, Michael Hauer, Ole Niewoehner, Jennifer Doudna University of California, Berkeley, USA

CRISPR loci (clusters of regular interspaced short palindromic elements), found in numerous bacteria and archaea, constitute an RNA-based adaptive immune system that targets invasive genetic elements such as bacteriophages and plasmids (1, 2). These loci are composed of arrays of direct sequence repeats specific to the host organism, interspaced with unique non-repetitive sequences (referred to as spacers) that derive from previously encountered genetic invaders. Transcription of CRISPR loci yields long precursor RNAs that are processed to generate libraries of short RNAs (crRNAs), each containing the sequence corresponding to a single spacer. crRNAs in turn act as sequence-specific guides that recognize invading nucleic acids through base complementarity and target them for destruction. The enzymatic activities underlying spacer acquisition, crRNA biogenesis, and targeting are provided by CRISPR-associated (cas) genes. Comparative genomics studies have identified three major types of CRISPR systems (I-III), each including a distinct set of 4-10 cas genes associated with a particular CRISPR locus (3). We previously showed how CRISPR-specific endonucleases found in type I CRISPR systems specifically recognize and cleave repeat sequences within precursor CRISPR transcripts (4, 5). The cas proteins Csy4 (from Pseudomonas aeruginosa) and Cse3 (from E. coli) utilize a unique combination of sequence-specific and shape-specific RNA recognition mechanisms to achieve high affinity binding to stem loops structures formed by the CRISPR repeat sequence. Our current work is focused on the molecular mechanisms of crRNA biogenesis in type II and III CRISPR systems. In type III systems, precursor transcripts are processed by endonucleases of the Cas6 family. Cas6 proteins from different species recognize RNA substrates that either contain stable hairpins or are devoid of secondary structures. Type II systems encode a trans-acting RNA (tracrRNA) that base pairs with the repeat sequence in the precrRNA transcript. The resulting duplex is recognized and cleaved by the host enzyme RNase III, a dsRNA-specific endonuclease, in the presence of the cas protein Csn1/Cas9 (6). Our poster will highlight our recent progress towards understanding crRNA biogenesis in these systems at the biochemical and structural levels. 1. B. Wiedenheft, S. H. Sternberg, J. A. Doudna, Nature 482, 331–338 (2012). 2. L. A. Marraffini, E. J. Sontheimer, Nat Rev Genet 11, 181–190 (2010). 3. K. S. Makarova et al., Nat Rev Microbiol (2011). 4. R. E. Haurwitz, M. Jínek, B. Wiedenheft, K. Zhou, J. A. Doudna, Science 329, 1355–1358 (2010). 5. D. G. Sashital, M. Jínek, J. A. Doudna, Nat Struct Mol Biol (2011). 6. E. Deltcheva et al., Nature 471, 602–607 (2011).

260

Identification And Characterization Of Breast Cancer-relevant Long Non-coding RNAs

Katharina Kasack1,2, Kristin Reiche3,2, Inga Rye4, Hege Russnes4, Friedemann Horn5,2, Anne-Lise Børresen-Dale4,6, Jörg Hackermüller3,7, Lars Baumbusch4 1 LIFE Research Center for Civilization Diseases, University of Leipzig, Germany, 2Fraunhofer Institute for Cell Therapy and Immunology, AG RNomics, Leipzig, Germany, 3Helmholtz Centre for Environmental Research â¿¿ UFZ, Young Investigators Group Bioinformatics and Transcriptomics, Leipzig, Germany, 4Department of Genetics, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Oslo, Norway, 5University of Leipzig, Dept. of Molecular Immunology, Leipzig, Germany, 6K.G. Jebsen Center for Breast Cancer Research, Instiute for Clinical Medicine, University of Oslo, Oslo, Norway, 7University of Leipzig, Dept. of Computer Science, Bioinformatics Group, Leipzig, Germany Long non-coding RNAs (lncRNAs) have recently come into focus of research as new players in complex diseases like cancer. LncRNAs are predominantly expressed in a highly controlled, cell type and state specific manner and it has been shown that those transcripts are frequently aberrantly expressed in a variety of human cancers. However, the biological functions of the vast majority remain unclear. Breast cancer is one of the most frequent types of cancer worldwide. Despite advances in the diagnosis and therapy of this disease, mortality remains high. One reason is the heterogeneity of this disease, based on obvious structural features like morphology and structural organization as well as the diversity on the molecular level. The class of lncRNAs might give access to heterogeneity on the molecular level as a potential role in oncogenic and tumor suppressive pathways as well as in epigenetic processes has been shown. In this study we used the nONCOchip - a custom microarray interrogating experimentally identified ncRNAs regulated by oncogenes, tumorsuppressors and cyclines, all human RefSeq mRNAs and predicted ncRNAs from databases - to explore lncRNA expression in 26 breast carcinoma samples representing five clinically relevant tumor mRNA expression subclasses as well as normal breast tissue from breast reduction operations. We detect a large number of lncRNAs being differentially expressed between breast tumors and normal breast samples. Differential expression could be validated using quantitative PCR. Using a breast cancer cell line model the subcellular localization of selected lncRNAs was analyzed by RNA fluorescence in-situ hybridization (RNA-FISH). Subsequent functional studies will give insights into the role of those lncRNAs in breast carcinoma and might help to better understand the complexity of this disease. Poster Session 1: Non-coding and Regulatory RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

261

Novel class of transcripts in Arabidopsis xrn3 mutant of nuclear 5’-3

Michal Krzyszton, Monika Zakrzewska-Placzek, Joanna Kufel Institute of Genetics and Biotechnology University of Warsaw, Warsaw, Poland Arabidopsis thaliana two nuclear 5’-3’ exonucleases AtXRN2/3 are homologs of Xrn2/Rat1, which is involved in degradation and processing of several classes of nuclear RNAs and in transcription termination of RNA polymerases I and II. We have shown that AtXRN2/3 contribute to polyadenylation-mediated nuclear quality control for rRNA precursors and excised spacer fragments. Here, we report that in xrn3 mutants several mRNAs and pri-miRNAs are significantly upregulated, without the effect on their stability. Readthrough transcripts detected upstream and downstream of these genes indicate that this may result from defects in polymerase termination. Chromatin immunoprecipitation confirmed increased PolII occupancy in these regions, pointing to a role of AtXRN3 nuclease in the torpedo mechanism of termination. In addition, in xrn3 mutants we observe strong accumulation of intergenic transcripts, named XATs (XRN3-associated transcripts), which are transcribed by PolII and belong to both classes, polyadenylated or lacking the poly(A) tail. These ncRNAs start and end in promoters, coding sequences and downstream of genes, and often contain fragments of coding regions. Screening for accumulation of XATs in other RNA metabolism mutants suggests they depend only on AtXRN3. It is possible that in plants these aberrant RNAs may activate RNAi,, considering that plant XRN2/3 act as endogenous silencing suppressors, but we were not able to detect changes in DNA methylation or histon modifications in these regions. We can propose two models for XATs biogenesis, that they represent pervasive transcripts normally degraded by AtXRN3 or that they arise as a result of AtXRN3 involvement in transcription termination.

262 Successive Tailing and Trimming of RISC-loaded miRNA by the 3’UTR-binding Protein HuR

Sokol Lena2,3, Lisa Young1, Martin Hintersteiner2, Jan Weiler2, David Morrissey1, Witold Filipowicz3, Nicole MeisnerKober2 1 Novartis Institutes for Biomedical Research, Cambridge, MA, USA, 2Novartis Institutes for Biomedical Research, Basel, Switzerland, 3The Friedrich Miescher Institute for Biomedical Reserach, Basel, Switzerland HuR is a central protein in posttranscriptional regulation by AU-rich elements (AREs). It controls expression of a wide variety of transiently expressed ARE-containing mRNA encoding proteins involved in cellular processes as diverse as proliferation, differentiation, metabolic or immune responses. On a molecular level, it can regulate its target mRNAs by promoting nuclear export, protection from degradation, translational activation, or a combination thereof. Despite many years of research on AREs, it is still not entirely clear how HuR mediates its effects at a molecular mechanistic level. More recently, Bhatthacharyya et al demonstrated that miR-122 mediated translational repression of CAT-1 mRNA can be reversed by HuR upon its binding to the target mRNA. Here we show that HuR can modify the 3’end of RISC loaded miRNAs using its recently identified enzymatic activity associated with RRM3 . More specifically, we show that the nucleotide transferase activity of the HuR RRM3 catalyzes polyA-tailing of miRNAs preferentially containing a pentanucleotide GU-rich motif at their 3’end. In addition, we demonstrate that HuR also has another previously unnoted enzymatic, 3’-> 5’exonucleolytic, activity which resides in a protein pocket distinct from that of the adenylate transferase. By interplay of the three activities – ARE binding, transferase, and 3’->5’exonuclease – HuR promotes tailing and trimming of the RISC-loaded as well as free miRNA substrates. These observations are potentially related to recent findings by Ameres et al. on the target directed tailing and trimming of miRNAs in Drosophila. Based on our data, we propose a model according to which HuR, when recruited to a miRNA-repressed mRNA via its ARE binding domains RRM1&2, can relieve the repression by: (1) contacting, via RRM3, the relatively accessible 3’ terminus of the RISC-loaded miRNA; (2) adding a homopolymeric stretch of adenosines to extend into the more distant exonuclease pocket in RRM1&2 which is bound to the ARE; and, finally (3) trimming of the tailed miRNA by the 3’->5’ exonucleolytic HuR activity which eventually results in degradation of miRNA and removal of RISC from the silenced mRNA. These data demonstrate a molecular mechanism for interplay between mRNA 3’UTR-binding proteins and miRNAs on the message. In light of accumulating evidence for a connection between ARE binding proteins and miRNAs, our findings may have more far reaching implications for how the 3’UTR-binding trans-acting factors mediate their regulatory function. Poster Session 1: Non-coding and Regulatory RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

263

Novel Metabolite Analogs for Modulating Riboswitch Activity

264

The Redox-Sensing Apo-Aconitase B Protein Act Against sRNA Distal Nucleolytic Cleavage

Christina Lünse1, Magnus Schmidt2, Valentin Wittmann2, Fraser Scott3, Colin Suckling3, Günter Mayer1 1 LIMES Institute, Bonn, Germany, 2University of Konstanz, Konstanz, Germany, 3University of Strathclyde, Glasgow, UK Riboswitches are RNA elements mostly found in the 5’ UTR of bacterial mRNA. Consisting of a metabolite-binding aptamer domain and an expression domain, which inhibits transcription or translation initiation, they are involved in the regulation of up to 2-4% of all bacterial genes. The glmS riboswitch is a catalytically active RNA (a ribozyme) that regulates gene expression by recognition of glucosamine-6-phosphate (GlcN6P), subsequent self-cleavage, and degradation of glmS mRNA. This ribozyme tightly regulates the expression of the GLMS enzyme which catalyses the synthesis of GlcN6P, a precursor of bacterial cell wall biosynthesis. In H. influenza and other pathogenic bacteria thi-box ribowitches have been shown to be involved in the regulation of essential genes. Interference with the orderly expression of those genes may enable control of bacterial growth, thus making riboswitches a promising new target for developing antibiotics. Therefore, we sought to identify modulators for these riboswitches based on structural analogy to their natural ligand. We designed and synthesized small libraries of metabolite analogs and investigated them regarding their activation of the glmS ribozyme of S. aureus or the E.coli thiM riboswitch. While the glmS ribozyme was activated by a carba-sugar in comparable levels to the natural metabolite GlcN6P, the thi-box riboswitch is modulated by a novel thiamine analog in vivo. Moreover, activation by the thiamine analog was shown to be more effective than by pyrithiamin, a known artificial thi-box activator. Investigating riboswitches and their artificial regulation does not only enable their exploration as new antibacterial target, but may also allow encountering the increasing appearance of multi-resistant pathogenic strains, which seriously threaten our ability to control many microbial pathogens today.

Julie-Anna Benjamin, Marie-Claude Carrier, Eric Massé Université Sherbrooke Post-transcriptional regulation by bacterial small RNAs (sRNAs) typically occurs through the pairing of a sRNA to the translational initiation region of a target mRNA, allowing the sRNA to directly compete with initiating 30S ribosomes and thus to inhibits translation initiation. This is often followed by a rapid degradation of the target mRNA through the recruitment of the RNA degradosome. Here, we report the first example of repressed sRNA-induced degradation, which depends on cellular conditions. The well-known RyhB sRNA which regulates Fe-homeostasis target and quickly degrade acnB mRNA when expressed in Fe-rich media. However, in Fe-poor conditions, RyhB is expressed and rapidly induces degradation of its targets, but not acnB mRNA anymore. In condition of Fe starvation, it is known that the enzymatic Holo-aconitase B protein switch in its Apo-aconitase B mRNA binding protein state. Also, in this metabolic state, Apo-aconitase B seems to stabilise acnB mRNA, possibly by binding to the 3’-UTR of its own mRNA. Our data indicate that RyhB is still able to bind acnB mRNA in Fe starvation and block translation. Also, we propose that the stabilisation of acnB mRNA could result from protection against RNase E-mediated cleavage after RyhB sRNA pairing. This novel mechanism demonstrates a new level of regulation of sRNA-induced mRNA degradation, which depends on the metabolic state of the enzyme/ RNA-binding protein Aconitase B.

Poster Session 1: Non-coding and Regulatory RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

265

Diversity of RNA-Based Ribosomal Protein Regulation in Different Bacterial Phyla

266

Splicing remodels let-7 primary miRNA for enhanced Drosha processing

Shermin Pei, Jon Anthony, Michelle Meyer Boston College The biosynthesis of over half of the ribosomal protein genes in E. coli is controlled by more than 10 distinct RNA regulatory structures. Each structure is present within the mRNA and inhibits translation or transcription in response one of the ribosomal proteins whose synthesis is regulated. Detailed mechanistic studies have been performed for many of these autogenous regulatory elements. However, most of this work was completed prior to the age of comparative genomics and the extent to which the RNA structures are conserved across different phyla of bacteria remains largely unexplored, and Rfam alignments are available for fewer than half of the RNAs. In order to systematically assess the phylogenetic distribution of RNA elements regulating ribosomal protein biosynthesis in E. coli, we obtained from Rfam, or constructed based on available literature, sequence and secondary structure alignments for each RNA. These alignments were used in combination with Infernal (INFERence of RNA ALignment) to identify homologous examples in other bacterial genomes. From this analysis it is clear that most of the RNA mechanisms present in E. coli are not widely distributed, but rather are narrowly restricted to a few orders of gammaproteobacteria. Furthermore, de novo ncRNA searches have identified a plethora of distinct RNA structures associated with ribosomal protein operons across different bacterial phyla. This suggests that while RNA-based autogenous regulation of ribosomal protein synthesis is common, that the RNA structures responsible for this regulation are likely to be exceedingly diverse in different bacterial phyla.

Vanessa Mondol, Amy Pasquinelli Division of Biological Sciences, UC San Diego, La Jolla, CA, USA MiRNAs are important post-transcriptional regulators involved in a multitude of pathways and various human diseases. Many miRNAs exhibit distinct temporal and spatial expression patterns through mechanisms that are not fully understood. My overall aim is to define the regulatory mechanisms that contribute to the precise temporal production of let-7 in a developing organism. Specifically, I am focused on understanding the role of trans-splicing in let-7 biogenesis and determining whether structural rearrangements due to splicing can serve as a general post-transcriptional mechanism of miRNA regulation. In C. elegans, a trans-splicing event occurs in ~70% of transcribed mRNAs. A 22 nt spliced leader sequence (SL1 or SL2) donated by a snRNP is appended to the first exon of an mRNA. Trans-splicing is thought to aid translation, so it was surprising when our lab discovered SL1-spliced pri-let-7. Interestingly, alleles of let-7 that contain deletions, which include or are near the splice site, produce none or very little mature let-7. This splicing event is hypothesized to allow secondary structure rearrangements that permit Drosha processing. I am currently working to confirm secondary structure predictions of the unspliced and SL1 trans-spliced pri-let-7 transcripts using RNase structure assays. In addition, I am characterizing transgenic worms with mutations predicted to disrupt splicing or folding in order to better analyze the importance of this event. It has yet to be determined if other miRNAs in C. elegans are transspliced as well. I have identified potential splice sites within 500 nt upstream of the annotated precursor sequence for 8 miRNAs and am in the process of testing for evidence of trans-spliced forms. Results from this study will give more insight into the purpose of SL1 trans-splicing and the importance of secondary structure in miRNA biogenesis. Although SL-mediated trans-splicing is not found in most vertebrates, the primary transcripts of several human let-7 genes are part of spliced transcripts. My work shows that the genomic sequence does not necessarily serve as the miRNA primary transcript and suggests that splicing in general could be a mechanism for remodeling some miRNA primary transcripts to enhance their recognition by Drosha.

Poster Session 1: Non-coding and Regulatory RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

267

Bacterial Regulatory RNAs in Competition for Binding to the Chaperone Protein Hfq

Aleksandra Kaszynska, Agnieszka Hartwich, Anna Roszak, Mikolaj Olejniczak Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland The bacterial chaperone protein Hfq is required for the regulation of translation by trans-encoded small RNAs (sRNAs). This homohexameric, ring-shaped protein binds sRNAs and mRNAs, and facilitates their pairing, which in turn affects the ribosome access to mRNAs or their stability. Recent data showed that the concentration of Hfq in cells can be limiting for translation regulation. Moreover, it was observed that this may lead to competition among sRNAs for access to Hfq in vivo. Here, several structurally different E.coli sRNAs were selected to test if sRNAs differ in their binding to Hfq and competition for this protein. The thermodynamic and kinetic properties of their binding to the Hfq protein were compared using a high-throughput filter binding assays. The analysis of the data suggested the presence of intermediate steps in the pathway of sRNA association to Hfq. Interestingly, the results showed that Hfq bound different sRNAs with similar affinities and kinetics. However, the sRNAs differed in their ability to outcompete other sRNAs from the complex with Hfq. This suggests a hierarchy of sRNAs in their competition for this protein. Overall, the differences in the competition performance of the individual sRNAs, which have been observed here, could serve to modulate their access to Hfq. Such tuning of translation regulation could be important for the flexible bacterial cell adaptation to changing environmental conditions.

268 Comparative analysis of miRNA expression patterns between Triops cancriformis (tadpole shrimp) and model species during development

Kahori Takane2,1, Yuka Hirose2,1, Kiriko Hiraoka2, Emiko Noro2, Kosuke Fujishima2,3, Masaru Tomita2,1, Akio Kanai2,1 1 Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa 252-8520, Japan, 2Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan, 3NASA Ames Research Center, Moffett field, CA 94043 microRNAs (miRNAs) are approximately 22 nucleotide non-coding RNAs that regulate gene expression at the posttranscriptional level. miRNAs play an important role in the cellular processes such as development and cell differentiation. While many miRNAs are identified and analyzed in various model species, there are few reports of miRNAs in nontraditional model species. Here, we focused on Triops cancriformis which changes their morphology dramatically during their early larval developmental stages. We hypothesized that miRNA expression also dramatically changes in accordance with the morphological changes. We first constructed small RNA libraries from six different stages of T. cancriformis development (egg, 1st to 4th instar larvae and adult). Deep sequencing analysis of these libraries resulted approximately 46 million reads ranging from 12 to 45 nucleotides in length. Bioinformatics analysis have shown 101 conserved miRNAs in T. cancriformis by using known miRNA sequences registered in miRBase. Among these miRNA candidates, the expression of eight T. cancriformis miRNAs in adult stage were confirmed by northern blotting analysis. We also analyzed the miRNA expression patterns of 101 miRNAs in the six developmental stages and found that approximately half of these miRNAs are strongly expressed in one of the six stages (stage-specific expression). We also compared the miRNA expression patterns with that of D. melanogaster reported from previous studies, and found that 4 candidates (tcf-miR-87, tcf-miR-100, tcf-miR-125, tcf-let-7) showed conserved expression profiles. Whereas, eight T. cancriformis miRNA candidates showed different patterns, (e.g., T. cancriformis tcf-miR-79 is strongly expressed in 3rd instar larvae stage, D. melanogaster dme-miR-79 is constantly expressed from egg to adult stages. The inconsistency in the expression profile of conserved miRNAs suggest that although these miRNAs possess similar sequences, they may play a different role depending on the species. Thus, we believe that this study will provide a new insight into the relationships between dynamic morphological changes and the miRNA expression pattern during the developmental stages. Poster Session 1: Non-coding and Regulatory RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

269

Characterization of the sxRNA platform; A trans-acting RNA Switch

Carla Theimer1, Nakesha Smith1, Sabarinath Jayaseelan2, Francis Doyle2, Paul Kutscha2, Scott Tenenbaum2 1 University at Albany, SUNY, 2College of Nanoscale Science and Engineering, Nanobioscience Constellation, University at Albany, SUNY We are characterizing a novel RNA-based nano-switch technology termed structurally interacting RNAs (sxRNAs), which utilize post-transcriptional gene regulation as a reporter of non-coding RNA expression, such as miRNA. Unlike other reporter systems that decrease gene expression upon miRNA induction, our system results in an increase in reporter gene expression upon miRNA induction. The sxRNA platform is based on transacting RNA complexes that can enhance or disrupt RNA binding protein (RBP) interactions. RBPs frequently bind to a stem-loop structure within the non-coding portion of the mRNA, such as the downstream-untranslated region of a message. Some RBPs increase the translation of an mRNA by an order of magnitude when they bind. It is possible to modify the mRNA to modulate translation of the reporter gene by controlling the binding of the RBP. This is accomplished by strategically altering the natural stem-loop structural target of an RBP. We have demonstrated that we can custom design a sxRNA in which the natural RBP-binding structure is altered so that it only correctly forms when a second RNA, such as a miRNA, binds in trans and stabilizes it, by base-pairing to the flanking region. In this study, we have biophysically characterized in vitro a switch that demonstrated increased binding (by as much as 5X) for the histone stem-loop binding protein (HSLBP). These biophysical studies examine the interactions of this sxRNA platform and how that interaction can be modulated to alter protein binding.

270

Multidimensional Regulation of and by LncRNAs: Non-Conservation and Networks

Emily Wood, Leonard Lipovich Wayne State University School of Medicine, Detroit, (MI), USA Regulatory network interactions between long non-coding RNAs (lncRNAs) and protein-coding genes exploit multiple mechanisms, a subset of which is based on sharing genomic space. ‘Chains’ of lncRNA and protein-coding mRNAs connected by sense-antisense (SAS) overlaps and bi-directional promoters (BDPs) allow coordinated regulation. LncRNAs may also regulate targets at other loci through epigenetic and post-transcriptional mechanisms. These targets can include transcription factors (TFs) and epigenetic modulators (EMs). We term the resulting multidimensional regulatory space of lncRNAs CUBE: Convergent-divergent Ubiquity of Bi-directional Expression. The literature suggests that lncRNA interspecies conservation is limited. To test the hypothesis that CUBE describes non-conserved lncRNA-dependent networks, we designed and implemented three discovery pipelines: two bioinformatic (one automated, one manual) and one experimental, to identify chains in the human genome that contain both non-conserved lncRNAs and TF/EM genes. We identified primate specific (PS) lncRNAs from GENCODE annotated datasets, as well as from BLAT searches. We used PANTHER to highlight TF/EM genes in chains. We found 192 lncRNA-containing chains: 9 with PS lncRNAs, and 65 with both lncRNAs and TFs/EMs, and 118 others. 35 of the 192 (~18%) showed co-expression of all chain members in at least one FANTOM5 cell line. One four-gene chain contained the EM PCGF1, the homeobox TF LBX2, and two non-conserved lncRNAs. This chain unites a TF gene with an EM gene through a non-conserved lncRNA which overlaps both. All members of this chain were co-expressed in FANTOM5 HEPG2 and THP-1 cell lines, indicating CUBE potential. In our experimental pipeline, we used our custom microarray of 5,586 cDNA-supported lncRNAs to identify 127 estrogen-responsive lncRNAs in MCF7 cells, including 3 GENCODE PS lncRNAs. We identified 4 chains which contain estrogen-responsive lncRNAs and TF/EMs. These chains were predominantly composed of cell cycle regulators, apoptosis and proliferation effectors, as well as an EM (HHATL), suggesting that gene chains may participate in multidimensional disease networks. We expect lncRNAs at CUBE interfaces to perform non-conserved network regulation. CUBE exposes lncRNA-anchored differences between species, highlighting the need to examine gene structure, not just sequence, in comparative genomics. Our results will help to validate CUBE and fit non-conserved lncRNAs into regulatory networks. Poster Session 1: Non-coding and Regulatory RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

271 RNA

Genome-wide Analysis of Target Genes of Steroid Receptor RNA Activator - a Regulatory

Linghe Xi, Elaine Podell, David McKay, Thomas Cech University of Colorado at Boulder, Boulder, Colorado, USA Steroid Receptor RNA Activator (SRA) is the first identified bifunctional RNA which can function as an mRNA as well as a regulatory RNA. Both SRA and the protein it encodes (SRA protein, SRAP) act as coregulators of a wide-range of transcription factors which control important developmental and metabolic processes [1]. SRA and SRAP have also been reported to play significant roles in tumorigenesis. Knocking down SRA in breast cancer cells remarkably decreases invasiveness [2]. In order to provide genome-wide identification of target genes directly regulated by SRA in breast cancer cells, we are performing RNA-seq and ChIRP-seq [3] experiments on MDA-MB-231 cells upon treatment with siRNA directed against SRA or control siRNA. The siRNA directed against SRA achieved ~80% knockdown in wholecell RNA and ~70% knockdown in nuclear RNA. The long-term goal of this study is to reveal the regulatory network of SRA and to lay the foundation for investigations into its functional mechanism. [1] Reviewed by Colley & Leedman (2011), Biochimie 93, 1966-1972, doi:10.1016/j.biochi. 2] Foulds et al. (2010), Mol Endocrinol 24(5):1090–1105. 3] Chu et al. (2011), Molecular Cell, doi:10.1016/j.molcel.2011.08.027.

272 An Adenosine-rich Sequence is Crucial for the Integrator-dependent microRNA 3’ Processing in Herpesvirus saimiri

Mingyi Xie1, Diana Lenis2, Joan Steitz1 1 Department of Molecular Biophysics and Biochemistry, Yale University, Howard Hughs Medical Institute, New Haven, CT, U.S., 2Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, U.S. Herpesvirus saimiri (HVS) causes T-cell leukemias and lymphomas in New World primates and transforms human T cells in vitro. In latently infected T cells, the viral microRNAs (miRNA) are co-transcribed with small nuclear RNAs (snRNA) immediately upstream. More specifically, each of the HVS precursor (pre-) miRNA hairpins is located downstream of the 3’ processing signal (3’ box) of a viral snRNA: HSUR (Herpesvirus saimiri U RNA). These are the first snRNA-miRNA chimeras to be identified. Intriguingly, the Integrator complex cleaves to create the 3’ end of a HSUR and the 5’ end of the pre-miRNA hairpin, thereby bypassing canonical Drosha/DGCR8 cleavage (1). The mechanism of HVS pre-miRNA 3’ end formation is unknown. We recently discovered that a conserved adenosine track, termed HVS miRNA 3’ box, downstream of the HVS premiRNA hairpin is important for viral miRNA biogenesis. Mutational analysis revealed that the essential elements of this sequence are the consecutive adenosine residues. Interestingly, the miRNA 3’ box resembles adenosine-rich snRNA 3’ box, which is essential for 3’ end processing of the HSURs. In vivo knockdown and rescue experiments confirmed that the 3’ end processing of HVS pre-miRNA, like its 5’ end processing, also relies on the Integrator complex. Therefore, HVS has evolved a unique sequence to hijack the host Integrator processing mechanism to generate its own miRNAs. References: 1. Cazalla, D., Xie, M., and Steitz, J. A. (2011) Mol Cell 43, 982-992

Poster Session 1: Non-coding and Regulatory RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

273

Discovery of Polymerase Binding Elements and their Functions

274 Site

3’-UTR G-quadruplexes Regule Gene Expression Throughout Alternative Polyadenylation

Bob Zimmermann1, Frederike von Pelchrzim1, Jennifer Boots1, Adam Weiss1, Doris Chen1, Marek Zywicki2, Katarzyna Matylla-Kulinska1, Renée Schroeder1 1 Max F. Perutz Laboratories, 2University of Innsbruck Growing evidence that most, if not all, the genome is transcribed into RNA suggests that a plethora remain to be discovered. However, approaches for the discovery of functional, non-coding RNA requires a high expression level when the RNA is isolated from a cell or a significant or conserved structure. We employ an in vitro expression level bias-free approach, Genomic SELEX, to discover RNAs encoded in the human genome that bind RNA Polymerase II (PolII). We developed an algorithm to discover the minimal required sequence for binding based on high-throughput Genomic SELEX data. The most enriched repeat element from the experiment, ACRO satellites, have not been discussed extensively in the literature. The elements, when inserted serially into a template inhibit the PolII transcription of their own RNA. Many more of these sequences remain to be explored, and we envision that these elements confer riboregulation of gene expression.

Jean-Denis Beaudoin, Jean-Pierre Perreault Groupe ARN/RNA group, Département de biochimie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, QC, J1H 5N4, Canada ([email protected]) Given that greater than 90% of the human genome is expressed, it is logical to assume that post-transcriptional regulatory mechanisms must be the primary mean for controlling the flow of information from the mRNA to the protein. Guanine-rich nucleic acid sequences can fold into non-canonical, four stranded helical structures called G-quadruplexes. This report describes a robust approach that includes in silico, in vitro, as well as in cellulo experiments to evaluate the presence of G-quadruplex structures in human 3’-UTRs. Bioinformatic analysis revealed an enrichment of G-quadruplexes in 3’-UTRs. Subsequently, two potential G-quadruplex sequences located either in the 3’-UTR of the low density lipoprotein receptor-related protein 5 (LRP5) gene or fragile X mental retardation autosomal homolog 1 (FXR1) gene were initially investigated. Their ability to fold into a G-quadruplex structure in vitro was evaluated using both circular dichroism and thermal denaturation (two classical approaches) as well as in-line probing (a new method for this purpose). Results showed that the 3’-UTR of both the LRP5 and FXR1 genes includes a G-quadruplex structure. Moreover, each of these G-quadruplex structures increases by 2-fold the gene expression of a reporter gene by stimulating the polyadenylation of its mRNA throughout an alternative site located downstream of the canonical site of their corresponding 3’-UTR. Sequence analysis, site directed mutagenesis, miRNA regulation network analysis and G-quadruplex ligand experiments were performed to define rules governing this phenomenon. In light of these results, we suggest that 3’-UTR G-quadruplexes can regulate alternative polyadenylation sites, leading to the expression of shorter transcripts, and can interfer with the miRNA regulatory network of a specific mRNA.

Poster Session 1: Non-coding and Regulatory RNAs & 3’ end processing

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

275

Post-Transcriptional Regulation of COX-2

276

The Exo- and Endoribonuclease Activities of RNase BN Both Function In Vivo

Ashley Cornett, Carol Lutz UMDNJ-New Jersey Medical School and the Graduate School of Biomedical Sciences, Newark, NJ The oxidative conversion of arachidonic acid to prostaglandin H2 is carried out by a set of two enzymes termed cyclooxygenases, abbreviated as COX. COX-1 is constitutively expressed in normal tissues, while COX-2 is transiently induced from external stimuli, such as pro-inflammatory cytokines. COX-2 is also overexpressed in numerous cancers. We show that COX-2 protein expression is constitutive in a lung cancer cell line, A549, but not expressed in a normal bronchial cell line, Beas2B. Previous work from our lab has shown that COX-2 has two polyadenylation signals present in its 3’UTR that can potentially be utilized. Alternative polyadenylation is a post-transcriptional mechanism by which mRNAs can produce variable 3’ untranslated region (UTR) lengths is through usage of alternative poly(A) sites. Our RNAse H-coupled RT-PCR data indicate that both COX-2 mRNA isoforms, resulting from usage of two different poly(A) sites, are transcribed in A549 lung cancer cells. Another means of post-transcriptional regulation is mediated through microRNA repression. We have Real-Time qPCR data and microarray data that show decreased expression of miR-146a in lung cancer cells as compared to normal lung cells. miR-146a is predicted to target the COX-2 3’UTR. We speculate that many post-transcriptional mechanisms work in concert to regulate COX-2 expression, which may explain the employment of an alternative poly(A) signal.

Tanmay Dutta, Arun Malhotra, Murray Deutscher Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, 1011, N.W. 15th Street, Miami, Fl-33136 Escherichia coli RNase BN, a member of the RNase Z family of endoribonucleases, was initially discovered as a ribonuclease required for the maturation of phage T4 tRNA precursors. Studies with synthetic phage tRNA precursors showed that RNase BN could remove a single 3’-terminal nucleotide suggesting that it is an exoribonuclease. More recently, we showed that RNase BN actually has both endo- and exoribonuclease activity on synthetic RNA substrates and tRNA precursors in vitro, and that under conditions more closely resembling those thought to occur in vivo, the enzyme functions primarily as an endoribonuclease to generate mature tRNA. Comparison of the known X-ray structures of E. coli RNase BN and Bacillus subtilis RNase Z, which is an endoribonuclease, revealed that RNase BN has a narrower and more rigid exit channel than RNase Z. To determine whether this structural difference might be responsible for its exoribonuclease activity, we generated a mutant RNase BN in which a proline residue within a loop in the exit channel was converted to the corresponding glycine residue present in RNase Z, thus widening the channel. The resulting mutant RNase BN was essentially devoid of exoribonuclease activity in vitro. Substitution of the mutant rbn gene for wild type rbn in the E. coli chromosome revealed that the exoribonuclease activity of RNase BN was not required for maturation of phage T4 tRNA precursors, a specific function of this RNase. On the other hand, removal of the exoribonuclease activity in a cell lacking other processing RNases led to slowed growth and affected maturation of multiple host tRNA precursors. These findings provide a structural basis for the ability of RNase BN to act as both an exo- and an endoribonuclease and demonstrate that both activities are capable of functioning in vivo.

Poster Session 1: 3’ end processing

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

277

A Novel C-rich USE Globally Regulates mRNA 3’ Processing

Xinjun Ji1, Ji Wan2, Melanie Vishnu1, Yi Xing3, Stephen Liebhaber4 1 Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA, 2Interdepartmental Graduate Program in Genetics, University of Iowa, Iowa City, Iowa, USA, 3Interdepartmental Graduate Program in Genetics, Departments of Internal Medicine, Biostatistics and Biomedical Engineering, University of Iowa, Iowa City, Iowa, USA, 4Departments of Genetics and Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA Objective: The present study was designed to assess the role of the C-rich aCP RNP complex as a global enhancer of mRNA 3’ processing. Background: Post-transcriptional controls are regulated by sequence-specific RNA-protein interactions. Prior studies have revealed that the KH-domain protein, aCP, binds to a 3'UTR C-rich motif of ha-globin transcript and enhances the efficiency of 3’ processing (Ji, et al, EMBOJ, 2011). Based on this finding, and on the knowledge that aCPs can bind to a large number of mRNA targets, we hypothesized that aCP RNP complexes function as genome-wide regulators of 3' processing and alternative polyA site utilization (APA). Approach: K562 cells were transiently co-depleted of the two major aCP isoforms (aCP1 and aCP2) by siRNA transfection. The impact of aCP depletion on polyadenylation site utilizaiton was evaluated by massively parallel 3' terminal sequencing (Direct RNA Sequencing (DRS, Helicos)) of mRNAs from depleted and control cells. Results: Bioinformatic analysis of the DRS data sets revealed 490 significant alterations in PA site utilization specifically linked to the aCP depletion (FDR< 0.05); of these, 208 were simple APA events (two sites competing within the terminal exon) and 239 were splicing-associated APA events (coupled with 3' and 5' splice site choices). A subset of this data was validated by targeted real-time PCR. Gene ontology (Go) analysis indicated that aCP impacted on many important cellular functions. The motif search revealed that APA events triggered by depletion of aCPs correlated strongly with presence of C-rich sequences. In particular, we found C-rich sequences 30-40 nt 5' to polyA signal (AAUAAA) that were repressed upon aCP depletion. In the case of directly competing polyA sites within a terminal exon, we observed that the enhanced use of a 'distal' polyA site after the aCP depletion is accompanied by the presence of a C-rich motif 5' to the competing 'proximal' polyA sites. Reciprocally, an increase in use of the 'proximal' polyA site utilization upon aCP depletion was linked to the presence of a C-rich motif 5' to the distal polyA signal. This observed linkage of APA patterns with positioning of C-rich motifs is consistent with the known C-rich binding specificity of aCP and an enhancing activity of the aCPs complex on 3' processing. Conclusion: We conclude that the aCP RNP complex constitutes a novel C-rich USE. This complex enhances mRNA 3’ end processing, can modulate the relative utilization of competing polyA addition sites, and in this manner constitutes a significant global determinant of APA controls.

278

SR Protein-Regulated Polyadenylation of Rous Sarcoma Virus mRNA

Stephen Hudson, Mark McNally Medical College of Wisconsin, Milwaukee, WI, USA Polyadenylation is an important post-transcriptional modification of host messenger RNAs (mRNA), and expression of transcripts derived from viruses often requires this modification. Despite a suboptimal polyadenylation site, ~85% of Rous sarcomavirus (RSV) transcripts are polyadenylated in vivo. Previous work showed that SR protein binding to two far upstream elements, the negative regulator of splicing (NRS) and the envelope (Env) splicing enhancer, are required for optimal RSV polyadenylation. The NRS is an RNA processing control element that blocks splicing by acting as a pseudo 5’ ss that interacts non-productively with the 3’ ss. The NRS promotes polyadenylation by juxtaposing SR proteinbinding sites within the NRS to those at the Env enhancer, and this synergy is required to promote polyadenylation to the distant downstream pA site. Presumably, a threshold level of SR proteins, generated by the juxapositioning of the two elements, is required for long-range stimulation of RSV polyadenylation. To test the threshold hypothesis, we replaced the Env enhancer with an increasing number of SRSF1 SELEX-derived binding sites. Deletion of the Env enhancer lowered polyadenylation efficiency, and addition of one or three SELEX sites had no effect, but five sites restored efficient polyadenylation. Another prediction is that the requirement for both the NRS and the Env enhancer would be lost if the distance to the polyA site was decreased. Both elements were still required for optimal polyadenylation when the distance between the enhancer and polyA site was decreased from ~4,200 nt to ~1,400 nt; the results of decreasing the distance further will be reported. Based on a report that the polyadenylation factor CFIm interacts with SR proteins and our data that SR proteins promote polyadenylation in RSV, we hypothesized that SR proteins recruit CFIm to the RSV polyA site, which lacks a strong upstream element. Placing SELEX-derived binding sites for SRSF1 (SF2/ASF) and SRSF7 (9G8) upstream of the poorly used RSV polyA site increased the crosslinking of the 25-kDa subunit of CFIm, as judged by cross-linking – IP from HeLa nuclear extract. The results of similar experiments with purified proteins will be reported. These observations support the model where a threshold level of SR protein binding, via juxtaposition of SR protein-binding sites within NRS and Env enhancer, is required to mediate-long distance stimulation of polyadenylation by recruiting or stabilizing CFImto the weak RSV polyA site. Poster Session 1: 3’ end processing

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

279

Analyzing Effect of La Mutants on Biogenesis of SRP RNA in S.cerevisiae

Hatem Mohi El Din, Jeremy Brown Institute for cell and molecular biosciences, Newcastle University, UK Biogenesis of ribonucleoproteins is process that is tightly controlled. Many factors are involved in this process including the TRAMP (trf4/air2/Mtr4) complex, the nuclear exosome complex, other exonucleases such as the Rex1 exonuclease, as well as the RNA binding protein La. scR1, the RNA component of the signalrecognition particle in S.cerevisiae is a RNA Polymerase III  transcript and, as such, is bound by La. While La is bound to the RNA it protects it from exonucleolytic digestion. As La requires a 3’UUU-OH tail to bind, this provides a first check point in the qualitycontrol of scR1 (or any Pol III transcript). La comprises 3 domains;an N-terminal La motif (LM), a RNA recognition motif (RRM) and a disordered C-terminus. The LM and RRM are involved in binding the UUU-OH tail of the RNA transcript. Recently the C-terminus has been shown to be important for La function as when deleted La could no longer support the folding of aberrant tRNAs. While there are data that show the effect of the absence of La on various RNA levels there is no detailed investigation into the effect of La mutants on 3’ end integrity of RNAs in S.cerevisiae. We show here that certain mutations in the LM, RRM as well as C-terminus abolish the ability of La to protect scR1 from adenylation/ exonuclease digestion. We also show that the TRAMP complex as well as the exonucleolytic activity of the exosome complex are critical for correct scR1 maturation as when either is mutated scR1 can no longer form wild type 3’end.

280 Replication Stress Linked Alternative Splicing Modulates the Activity of the RNA Binding Protein HBP/SLBP, a Key Factor in the Control of Histone Gene Expression

Pamela Nicholson1, Alexander Rattray2, Berndt Mueller2 Departement für Chemie und Biochemie, Universität Bern, Bern, Switzerland , 2School of Medical Sciences, University of Aberdeen, Aberdeen, Scotland, UK Packaging of DNA with proteins is a feature of life. In eukaryotes, histone proteins H2A, H2B, H3, H4 and H1 are key components of chromatin and condense DNA in the cell nucleus. During S phase, chromatin is unwrapped and the DNA is replicated and then re-packaged into chromatin. This re-packing requires the production of new histone proteins which are provided by the expression of replication-dependent histone genes in animals. The histone RNA binding protein HBP/SLBP coordinates the expression of replication-dependent histone genes during the cell cycle. HBP/SLBP binds to a conserved RNA hairpin present in the 3’ untranslated region of all histone mRNAs and participates in nuclear histone RNA processing, histone mRNA translation and stability control. We have previously described an alternative HBP/SLBP splice variant that has reduced translation activity in Xenopus oocytes (1). We have identified additional alternative HBP/SLBP mRNA splice variants that lack exons coding for sequences that are important for HBP/SLBP function. The alternatively spliced transcripts accumulate when cells are exposed to replication stress, and disappear when replication stress is relieved. This suggests that HBP/SLBP mRNA splicing is influenced by cell physiology and linked to DNA replication, possibly by checkpoint signalling. We are currently investigating the function of the protein isoforms encoded by the alternatively spliced transcripts and we will report the outcome of this investigation. (1) Gorgoni et al., (2005). The Stem-Loop Binding Protein controlling histone gene expression stimulates translation at an early step in the initiation pathway. RNA 11, 1030.

1

Poster Session 1: 3’ end processing

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

281

Non-Canonical 3’ End Formation Of Certain mRNAs In Drosophila S2 Cells

282

RNA-Dependent Control of Histone Synthesis by the Spinal Muscular Atrophy Protein

Trinh Tat, Patricia Maroney, Timothy Nilsen Case Western Reserve University, Cleveland, OH, USA With the exception of histone mRNAs, 3’end formation of all mRNAs in higher eukaryotes is thought to occur via cleavage and polyadenylation. To address the role of deadenylation in miRNA-mediated gene regulation, we developed a sensitive method to determine polyA tail length. The method uses yeast polyA polymerase to add G and I residues to the existing 3’end of mRNAs. cDNA synthesis using a dC primer followed by PCR and gel electrophoresis reveals the length of the polyA tail.We used this method to analyze polyA tail length of a number of mRNAs in Drosophila S2 cells. While several mRNAs displayed the expected heterogeneous tail, two mRNAs, those encoding Hid andReaper appeared to be cleaved but not polyadenylated. This phenomenon was observed when the 3’UTR of Hid or Reaper were appended to luciferase reporter constructs. Further analysis revealed that “aberrant” 3’end formation did not require miRNA-mediated activity; both Hid andReaper are miRNA targets. We then focused on the Reaper 3’UTR. Systematic deletion and substitution analyses demonstrated that three cis-acting sequences, one located 187-83bases upstream of an AAUAAA hexanucleotide, the hexanucleotide itself and bases73-115 downstream of the hexanucleotide were necessary and sufficient to cause aberrant 3’end formation. Interestingly, the position of the first element relative to the hexanucleotide is important. We are currently attempting to refine the mapping of cis-actingelements as a prelude to seeking trans-actingfactors that presumably interact with these elements.

Sarah Tisdale, Luciano Saieva, Francesco Lotti, George Mentis, Livio Pellizzoni Columbia University, New York, NY, USA Histones are highly conserved nuclear proteins that facilitate the ordered packing of DNA into chromatin and play a central role in the regulation of genome function. The metazoan core histones H2A, H2B, H3, H4 and the linker histone H1 are members of a replication-dependent family of histone genes, whose mRNAs lack introns and poly(A) tails. The only processing event occuring on histone precursor mRNA is a single 3’-end endonucleolytic cleavage, which is dependent on U7 snRNP function and required for proper regulation of histone gene expression. Here we investigated a role for the survival motor neuron (SMN) protein in U7 snRNP biogenesis and function both in vitro and in vivo. We found that SMN deficiency disrupts U7 snRNP assembly leading to a strong decrease in the levels of mature U7 snRNP and accumulation of U7 snRNA precursors in mammalian cells. Analysis of the functional consequences of SMNdependent U7 snRNP reduction highlighted severe alterations in the normal 3’-end processing of histone mRNAs as well as changes in histone protein expression. These defects occur early and increase in a time-dependent manner upon depletion of SMN, indicating that they are direct and specific downstream effects of loss of SMN function. Importantly, SMN-dependent changes in 3’-end processing of histone mRNAs are observed in a mouse model of spinal muscular atrophy (SMA), a motor neuron disease caused by a deficiency in SMN. These findings demonstrate an essential role for SMN in U7 snRNP biogenesis and proper histone mRNA 3’-end formation. They also reveal disruption of a novel SMN-dependent RNA pathway in SMA.

Poster Session 1: 3’ end processing

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

283 mRNAs Encoded by Linear Plasmids of the Yeast Kluyveromyces lactis are not Polyadenylated

Vaclav Vopalensky, Martin Pospisek Charles University, Prague, Czech Republic Linear plasmids were found in a number of yeast species belonging to nine genera. The genetic organization of yeast linear plasmids appears to be quite uniform with most thoroughly studied plasmids pGKL1 and pGKL2 from the yeast Kluyveromyces lactis. pGKL plasmids are peculiar in many respects since both plasmids are cytoplasmically localized, linear with distinct proteins covalently linked to terminal inverted repeats. Here we present molecular and functional analyses of plasmid specific mRNAs UTRs, 5’ and 3’ respectively. First, we present a molecular analysis of a putative capping enzyme encoded by K2ORF3. We produced K2Orf3p as GSTtagged fusion protein in E. coli expression system, purified K2Orf3p by affinity chromatography and successfully tested for guanylyltransferase activity. Surprisingly, we were not able to detect any N7-methyltransferase activities of any of the purified K2Orf3p protein. Second, we show that pGKL specific mRNAs do not bind to the cap-binding translation initiation factor 4E from S. cerevisiae in vitro while cellular mRNAs do. This result is further supported by our finding that killer toxin, naturally encoded by pGKL plasmids, is translated by cap-independent pathway, while control killer toxin gene artificially expressed under the control of the strong Pol II driven promoter is translated by cap-dependent pathway. And last but not least, we analyzed 3’ UTRs of the plasmid specific mRNAs and reveal that these mRNAs are not polyadenylated. Possible regulation elements of the 3’ ends naturally occurring at pGKL-specific mRNAs will be discussed.

284 Identification of Polyadenylation Mutations through a Novel Engineered Minigene Construct in Arabidopsis

Jie wang, Q. Quinn Li Miami University, Oxford, (OH), USA Messenger RNA (mRNA) polyadenylation plays an essential role in eukaryotic cell gene expression and regulation. Mutational studies of genes encoding polyadenylation protein factors contributed significantly to the understanding of the regulation mechanisms. However, most of such mutations cause drastic effects or lethal on these essential polyadenylation factors. Thus there are only few homozygous mutants suitable for genetic analysis in plants. Taking advantage of rich genetic resources in the model plant Arabidopsis, we intend to generate and screen for hypomorphic mutants of any genes involved in mRNA polyadenylation. This project is about testing of suitable minigene constructs that contain a selection marker, the expression of which will indicate a lack of functional polyadenylation system thus it is possible for screening new polyadenylation mutations. To create and screen for new polyadenylation mutations, we constructed minigene containing an inducible promoter that drives the expression of two selection markers, GFP (green fluorescence protein) and RNAi-PDS (phytoene desaturase gene), and a strong polyadenylation signal from CaMV (cauliflower mosaic virus) terminator that is inserted in between GFP and RANi-PDS. A knock-down of PDS will cause white lesions on leaves. In desired poly(A) factor mutants, upon induction of the promoter, the CaMV poly(A) site would not be used effectively, thus producing RNAi-PDS and lesion. The latter will be used as a mutant selection marker. This minigene has been transformed into wild-type Arabidopsis and the homozygous transgene will be subjected to EMS mutagenesis. The M3 generation will be screened for target mutations. After getting polyadenylation mutations, we will further identify the mutated genes that may have an impact on polyadenylation.

Poster Session 1: 3’ end processing

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

285

Multiple Resistance Mechanisms in a Multi-drug Producer

Mashal Almutairi, Alexander Mankin Center for Pharmaceutical Biotechnology, University of Illinois at Chicago Bacteria of the genera Streptomyces produce a wide array of antibiotics. The producers avoid suicide by harboring resistance genes. One of the Streptomyces venezuelae strains produces two related macrolide antibiotics, methymycin and pikromycin. Two putative resistance genes, pikR1 and pikR2 are associated with the macrolide biosynthetic gene cluster that encode fairly similar enzymes homologous to Erm RNA methyltransferases that modify a unique nucleotide, A2058, in the ribosome at the macrolide-binding site. We are trying to understand why the producer would carry two similar resistance enzymes. In an attempt to answer this question, we investigated the mechanisms that regulate the expression of pikR genes and identify the rRNA residues modified by these enzymes in the ribosome. We found that both pikR genes are preceded by short leader peptides – a feature often associated with inducible resistance genes. To test whether pikR genes are inducible by macrolides, a plasmid-based reporter construct was engineered in which the leader regions of pikR1 or pikR2 control the expression of the downstream lacZgene. With the use of these reporters, we found that the expression of pikR2 was induced by a pikromycin-like antibiotic RU56006, as well as by erythromycin and telithromycin, showing that pikR2 expression is inducible by 14-member ring macrolides. However, PikR1 was not induced indicating that either different antibiotic induce it or its expression is regulated by another mechanism. Primer extension analysis showed that PikR2 dimethylates A2058 in the 23S rRNA, however, PikR1 either modifies a different nucleotide or monomethylates A2058. We are currently attempting to define the target site and modification type of PikR1 and characterize its inducibility. The study of such resistance mechanisms in the producers can improve our understanding of similar mechanisms in the clinical pathogens and provide important insights in the biology of antibiotic production.

286 Reconstitution and characterization of in vitro translation system from Thermus thermophilus at high temperatures

Ying Zhou1, Haruichi Asahara1, Eric Gaucher2, Shaorong Chong1 1 New England Biolabs, Inc., Ipswich, MA, USA, 2Georgia Institute of Technology, Atlanta, GA, USA Thermus thermophilus is a thermophilic model organism that is distantly related to the mesophilic model organism Escherichia coli. We reconstituted the translation of T. thermophilus in vitro from purified ribosomes, tRNAs, and 33 recombinant proteins (translation factors, aminoacyl-tRNA synthetases and energy regeneration enzymes). This reconstituted system was fully functional, capable of translating natural mRNA into active full-length proteins at temperatures up to 65°C and with yields up to 60 μg/ml. Surprisingly, the protein synthesis also occurred at 37°C, a temperature well below the minimal growth temperature of 47°C for T. thermophilus. The reconstituted T. thermophilus translation system required the presence of a polyamine, with tetraamine being most effective, for translation at both high and low temperatures. Using such a defined in vitro system, we demonstrated the functional compatibility of key translation components between T. thermophilus and E. coli, and the functional conservation of a number of resurrected ancient elongation factors in the translation reactions. This work sets the stage for future experiments that apply abundant structural information of T. thermophilus to biochemical characterization of translation.

Poster Session 1: Ribosomes and Translation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

287 Probing the Function of the Truncated Insertion Domain in Rhodopsudomonas palustris Prolyl-tRNA Synthetase and Free Standing Homologs YbaK and ProX

Jo Marie Bacusmo, Karin Musier-Forsyth Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus OH Similarities in isosteric properties of amino acids cause misactivation by aminoacyl-tRNA synthetases. In the case of prolyl-tRNA synthetases (ProRS), Ala and Cys are misactivated and charged onto cognate tRNAPro. Thus, editing mechanisms have evolved to ensure fidelity of Pro codon translation. In many bacterial systems including Escherichia coli (Ec), a triple-sieve editing mechanism is employed, which consists of the ProRS active site that discriminates amino acids based largely on volume and size, the ProRS editing domain (INS) that hydrolyzes Ala-tRNAPro, and a free-standing homolog called YbaK that clears Cys-tRNAPro via unique sulfhydryl side-chain chemistry. The gram-negative purple non-sulfur bacterium Rhodopseudomonas palustris (Rp) genome encodes a ProRS containing a truncated insertion domain instead of the full-length INS, in addition to two free-standing homologs, YbaK and ProX. Here, we elucidate the function of the mini-INS and the catalytic activities of Rp YbaK and ProX. Rp ProRS activates Cys and Pro with similar catalytic efficiency as Ec ProRS, whereas Ala activation is ~25-fold reduced relative to the Ec enzyme. Rp ProRS shows “pre-transfer” editing activity against the Ala adenylate that is comparable to that of Ec ProRS. However, Rp ProRS mischarges Ala onto tRNAPro. Therefore, despite the lower overall activation of Ala and the relatively robust pre-transfer editing activity, we hypothesize that “post-transfer” editing of Ala-tRNAPro is required in the Rp system. We show that the mini-INS lacks hydrolytic activity, but appears to be critical for Rp ProRS structure. As expected, Rp YbaK possesses robust Cys-tRNAPro editing activity. The function of the free-standing ProRS editing domain homolog ProX has not been reported for any species. Rp ProX has been cloned, purified and to date we have demonstrated that it lacks Ala-tRNAPro and Cys-tRNAPro editing function in vitro. Studies to elucidate ProX substrate specificity in vitro and in vivo are underway.

288 An RNA interference Screening Approach to Identify Factors Involved in 40S Ribosomal Subunit Biogenesis in Mammalian Cells

Lukas Badertscher1, Thomas Wild2, Michael Stebler3, Karol Kozak3, Gábor Csúcs3, Peter Horvath3, Ulrike Kutay1 1 Institute of Biochemistry, ETH Zurich, Zurich, Switzerland, 2Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany, 3Light Microscopy Centre, ETH Zurich, Zurich, Switzerland The synthesis of proteins in cells is performed by ribosomes, macromolecular complexes consisting of about 80 proteins and 4 distinct rRNAs. Mature ribosomes are made up of two subunits. In vertebrates, the 60S subunit contains three rRNA chains and approximately 46 ribosomal proteins. In contrast, the 40S subunit contains a single rRNA and about 32 ribosomal proteins. In eukaryotes, ribosome biogenesis is a highly compartmentalized process. It starts in the nucleolus by transcription of pre-rRNA, which undergoes several processing, modification and protein assembly steps leading to a 90S pre-ribosome. Following cleavage of the 90S particle into pre-40S and pre-60S ribosomal subunits, both particles mature separately in the nucleus and are finally exported to the cytoplasm, where they join to form translational competent ribosomes. The biogenesis pathway is assisted by more than 150 non-ribosomal proteins, known as transacting factors. Most of our current knowledge on ribosomal biogenesis derives from studies in yeast, however little is known about the process of ribosomal subunit assembly in mammalian cells. We used a screening approach relying on the RNAi technology to identify factors required for 40S ribosomal biogenesis in human cells. Our assays were image-based and a cell line was used that carries a fluorescently tagged ribosomal protein of the small subunit. Thereby, defects at different steps along the ribosomal biogenesis pathway could be detected. We identified about 350 factors with a potential role in ribosome biogenesis, including the gene products of a number of uncharacterized human ORFs. We will report on the molecular and functional characterization of these factors, some of which play a direct role in the nuclear assembly of ribosomal subunits.

Poster Session 1: Ribosomes and Translation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

289

Investigating the Physiological Role of Translation Factor LepA in E. coli

290

Role of the Uncharacterized Factor Rbtf1 in Ribosome Synthesis

Rohan Balakrishnan, Kurt Fredrick The Ohio State University, Columbus, Ohio, USA LepA is a paralog of EF-G, conserved in all bacteria,mitochondria, and chloroplasts. Despite this high degree of conservation, strains of E. coli lacking LepA have no obvious phenotype. LepA interacts with elongating ribosomes invivo and may be involved in membrane protein biogenesis, based on studiesof Guf1, the mitochondrial homolog of yeast. However, its role in bacteria remains elusive. To address this question, we screened for mutations synthetically lethal in the absence of LepA in E. coli. The marked null allele ΔlepA::cat was moved by conjugation into each strain of the Keio collection, an ordered set of single-gene deletion strains, and those crosses failing to yield transconjugants were sought. Two Hfr ΔlepA::cat donor strains, which differed in the site of F insertion, were used in the screen. While we did not find any mutations that were synthetically lethal with ΔlepA::cat, we found several mutations that were more deleterious in combination with ΔlepA::cat. These data and their potential relevance to LepA function will be discussed.

Lukas Bammert, Ulrike Kutay ETH Zurich, Zurich, Switzerland Ribosomes play a central role in protein synthesis and thereby directly drive cellular growth. Production of these huge macromolecular structures is one of the most energy consuming cellular processes. Ribosome biogenesis includes transcription of rRNA, expression of ribosomal proteins and the activity of ribosomal maturation factors, which are processing rRNA and assembling ribosomal particles. In yeast, it has been shown that ribosome production is tightly regulated in respect to environmental and intracellular signals. In human cells, the regulatory network upstream of ribosome synthesis appears even more complex; yet little is known as to how the various nutrient and growth factor induced signaling pathways cooperate to regulate ribosome synthesis. We use a combination of image-based and biochemical assays to monitor rRNA transcription, expression of ribosomal proteins and maturation of ribosomal precursors in human cells. To identify the cellular repertoire of factors involved in 40S subunit biogenesis, we have recently performed a genome-wide siRNA screen. The screen identified a number of candidate proteins that control ribosome synthesis, including the uncharacterized, putative transcription factor Rbtf1. We show that Rbtf1 is essential for both 40S and 60S subunit production. Rbtf1 is a nucleoplasmic protein, which is not required for rRNA transcription by RNA polymerase I, but for nuclear maturation of ribosomal subunits. Based on these data, we are currently testing whether Rbtf1 plays a direct role in subunit assembly, or influences ribosome synthesis by its function as a transcription factor.

Poster Session 1: Ribosomes and Translation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

291

Structural Rearrangements and Antibiotic Targeting of Helix 69 in Bacterial Ribosomes

Christine Chow, Yogo Sakakibara Wayne State University, Detroit, MI, USA As part of the central core domain of bacterial ribosomes, helix 69 of 23S rRNA in the 50S subunit participates in an important intersubunit bridge called B2a and contacts several translation factors and tRNAs. Defining the solution conformational states and roles of highly conserved nucleotides, including the modified nucleotide pseudouridine, in this domain is essential for a complete understanding of helix 69 function. A combined approach of chemical synthesis and biophysical characterizations (e.g., UV melting, circular dichroism, and NMR spectroscopy) on model RNA systems, and chemical probing (with dimethylsulfate and diethylpyrocarbonate) and SHAPE analysis (with N-methylisatonic anhydride) on both wild-type E. coli and pseudouridine-deficient 70S ribosomes, has revealed local structural rearrangements of helix 69 that can be modulated by changing the solution conditions (e.g., pH, temperature, and magnesium ion concentrations), level of modification, and subunit association. These data provide insight into the functional roles of helix 69 dynamics and regulation of these conformational states by pseudouridine modification, and also reveal the suitability of helix 69 as a novel antibiotic target site.

292 Molecular Basis of Substrate Specificity and Mechanism of Catalysis by Bacterial ProlyltRNA Synthetase and YbaK

Mom Das, Sandeep Kumar, Christopher Hadad, Karin Musier-Forsyth The Ohio State University, Columbus, OH, USA Prolyl-tRNAsynthetases (ProRSs) mischarge cognate tRNAPro with the smaller noncognate amino acid Ala, and with Cys, which is of similar size as Pro. Fidelity in translation is partly ensured by a discrete editing active site (INS domain) that is appended to most bacterial ProRS aminoacylation cores. INS functions to selectively hydrolyze mischarged AlatRNAPro but fails to hydrolyze Cys-tRNAPro, which is edited by a homologous free-standing domain, YbaK, via a novel mechanism involving substrate sulfhydryl side chain chemistry. On the other hand, computational docking and mutagenesis studies of E. coli (Ec) ProRS INS bound to 5’-CCA-Ala revealed that the methyl side chain of the substrate Ala binds in a well-defined and tunable hydrophobic pocket. Site-specific mutation of several active site residues including the highly conserved Ile263 led to a significant loss in Ala-tRNAPro hydrolysis, and altering the size of the pocket modulated the substrate specificity of the INS domain. This is consistent with a size-exclusion based mechanism and general watermediated hydrolysis. In contrast, manipulating the size of the active-site pocket of YbaK, whereby we changed the Gly30 residue, which aligns with Ile263 in INS, to a larger Ile or Val fails to alter the substrate specificity. To further understand the mechanism of catalysis by E. coli ProRS INS, we have combined hybrid QM/MM calculations with biochemical assays to probe the role of various substrate functional groups and putative active site residues in catalysis. Our results suggest that hydrolysis by INS may not require active participation of protein side chains, but is mediated by a nucleophilic water molecule activated by the substrate 2’-OH of A76 of tRNAPro.We also propose a role for the Gly261 backbone carbonyl in proton shuttling. Consistent with this mechanism, substitution of the 2’-OH of the substrate or deletion of Gly261 leads to complete loss of hydrolytic activity. Ongoing studies are also aimed at investigating tRNA substrate specificity of YbaK in vitro. Results to date indicate that although YbaK deacylates Cys attached to a variety of tRNA frameworks, the efficiency of deacylation appears to be modulated by the acceptor stem sequence.

Poster Session 1: Ribosomes and Translation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

293

Molecular Mechanism Of Translation Initiation In Barley Yellow Dwarf Virus (BYDV)

Sohani Das Sharma1, Jelena Kraft 2, W.Allen Miller2, Dixie Goss1 1 Hunter College and the Graduate Center ,City University of New York,New York,NY,USA., 2Iowa State University , Ames, IA, USA. Protein synthesis of several positive-sense RNA viruses follows cap-independent initiation using either an internal ribosomal entry site (IRES) or cap independent translation element (CITE), which are located mostly in the 5’ untranslated region (UTR) of viral mRNA. BYDV, which lacks both a cap and poly(A) tail has a CITE (called BTE) present in its 3’UTR, that helps in efficient translation initiation at the 5’ proximal AUG. The mechanism of ribosome recruitment during this 3’UTR (CITE) dependent BYDV translation remains unknown. We are investigating the assembly of translation factors and detailed molecular mechanism of ribosome binding events during the initiation step in BYDV protein synthesis. Using fluorescence anisotropy and gel mobility shift assays we demonstrated that initiation factor eIF4F binds to the 3’UTR with a very high affinity (Kd ~30±8nM) and has extremely low affinity for any other part of mRNA. Our results also show that ribosome (40S) has moderate binding affinity to the 3’ UTR (Kd~600nM) but binding increases nearly six fold (Kd~100nM) in the presence of the eIF4F-4B-4A-ATP helicase complex. Our preliminary observations indicate a novel virus translation mechanism where the 3’UTR acts as a translational signal hub. In order to identify the structural element that interacts with initiation factors or ribosome we probed the secondary structure of both 3’ and5’ UTR using Selective 2’-hydroxylacylation analyzed by primer extension (SHAPE) technique. These results identified a very stable secondary structure for both 5’and 3’ UTRs. We are now characterizing specific binding site of ribosome to BYDV mRNA using primer extension inhibition (toe-printing) assay. Using RNA-RNA (3’-5’ UTR) gel shift assay and translation studies with different mutations between 3’ and 5’ UTR of BYDV it was showed that there is a long distance RNA-RNA tertiary interaction between 3’ and 5’UTR.We hypothesize that this long distance kissing stem loop interaction might help in delivery of the translation machinery from the 3’UTR to the 5’ end of RNA to start translation. In summary, our study helps us get insight into understanding the ribosome recruitment pathway in BYDV translation.

294 Antibiotics that Bind to the A site of the Large Ribosomal Subunit Can Induce mRNA Translocation

Dmitri Ermolenko1, Harry Noller2 1 University of Rochester, Rochester, NY, USA, 2University of California, Santa Cruz, CA, USA In the absence of elongation factor EF-G, ribosomes undergo spontaneous fluctuation between the pre-translocation (classical) and intermediate (hybrid) states of translocation. These fluctuations do not result in productive mRNA translocation. Extending previous findings1> that the peptidyl transferase inhibitor sparsomycin induces translocation, we identify additional A-site binding antibiotics that can trigger mRNA translocation. We also show that the antibiotics, which bind in the peptidyl transferase cavity, but do not occupy the A site, fail to induce mRNA translocation. Our results support models which propose that the ribosome is a Brownian ratchet machine, whose intrinsic dynamics can be rectified into unidirectional translocation by ligand binding. 1. Fredrick, K. & Noller, H.F. Catalysis of ribosomal translocation by sparsomycin. Science 300, 1159-62 (2003).

Poster Session 1: Ribosomes and Translation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

295

Single Molecule Dynamics of tRNA inside Ribosomes

May Farhat, Philip Cunningham, David Rueda Wayne State University, Detroit, MI, USA The ribosome is a large macromolecular machine that matches the codon-anticodon in the decoding center (DC) and catalyzes peptide bond formation between the aminoacylated tRNA and the growing polypeptide chain in the peptidyl transferase center (PTC). Protein elongation is a dynamic process during which tRNA traverses the ribosomal binding sites along a path of approximately 100 Å that requires large structural rearrangements of the tRNA and ribosome, as well as the GTPase elongation factors EF-Tu, and EF-G. Cryo-EM, and single-molecule studies have suggested that the small ribosomal subunit rotates counterclockwise to the large ribosomal subunit in the rotated or hybrid state. Prior to translocation, deacylated tRNA bound to the P site fluctuates spontaneously between classical (P/P) and hybrid (P/E) states in the pretranslocation (PRE) complex. During translocation, A/A and P/P tRNAs pass through the hybrid intermediate A/P and P/E states within a solvent-accessible channel formed by the interface of large and small subunits. Upon release of EF-G, the ribosome returns to the nonrotated or classical conformation and the tRNAs move to the P/P and E/E sites. Here, we have used single molecule Protein Induced Fluorescence Enhancement (smPIFE) to study the dynamics of tRNA inside the ribosome. Our data reveal changes in the Cy3-labeled tRNA fluorescence intensity caused by changes in the fluorophore environment. We show that these changes are sensitive to the magnesium concentration and the presence of antibiotics. We also observe two populations of tRNA in the ribosome; a static one, in which the ribosome may be in the locked state, and a dynamic one, in which the tRNA switches between different sub-states at constant rates, consistent with spontaneous ribosomal ratcheting.

296 Crystal structure of the ribosome in complex with the tRNA-like-domain of tmRNA and SmpB in the post-translocation state

Natalia Ivanova1, Andrei Korostelev2, John Paul Donohue1, Jianyu Zhu1,3, Harry Noller1 Center for Molecular Biology of RNA and Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, CA 95064, USA, 2Biochemistry & Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655, USA, 3Cocrystal Discovery, Inc., Mountain View, CA 94043, USA Bacterial ribosomes stalled on problematic mRNAs during translation can be rescued by transfer-messenger RNA (tmRNA) and its helper – small protein B (SmpB) by the process of trans-translation. The mechanism of trans-translation and the interactions of tmRNA and SmpB with the ribosome have been the subjects of numerous biochemical and structural studies. Although much has been learned, detailed structural information concerning the 70S ribosome:tmRNA:SmpB posttranslocation complexes is lacking. In this work, we present the crystal structure of a complex of Thermus thermophilus ribosomes containing mRNA, deacylated tRNAfMet bound to the P site and the tRNA-like domain (TLD) of tmRNA with SmpB bound in the ribosomal E site. The 4 Å resolution structure represents a state following the second step of translocation of the t-RNA-like domain of tmRNA. The shape and orientation of the TLD:SmpB complex mimic that of a long-variable-arm tRNA bound in the ribosomal E site. The contacts made by the acceptor arm and CCA end of the TLD with the ribosome closely mimic the ones formed by canonical tRNAs.

1

Poster Session 1: Ribosomes and Translation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

297 Evidence That Domain Closure Influences Both Initial Selection And Proofreading Of Aminoacyl-tRNA By The Ribosome

Sean McClory, Aishwarya Devaraj, Kurt Fredrick Ohio State University, Columbus, OH, USA The ribosome controls the accuracy of aminoacyl(aa)-tRNA selection during two phases of decoding, initial selection and proofreading. During initial selection, aa-tRNAs delivered to the ribosome by EF-Tu·GTP compete for binding to the ribosomal A site. Cognate aa-tRNAs bind to the A site with high affinity and stimulate GTP hydrolysis by EF-Tu, while near- and non-cognate aa-tRNAs bind less tightly and fail to promote rapid GTP hydrolysis. In the proofreading phase, cognate aa-tRNAs are efficiently accommodated into the 50S A site, whereas near-cognate aa-tRNAs are most often rejected. While decoding is essential for the correct translation of the genetic code, details of the molecular mechanism involved remain unclear. We have recently isolated a large number of mutations in the 16S rRNA that impair the ribosome’s ability to correctly select aa-tRNA, providing us tools to study the role of distinct regions of the ribosome in the decoding process. Most of the mutations are located along the periphery of the 30S shoulder domain and stimulate GTP hydrolysis by EF-Tu for both cognate and near-cognate aa-tRNA, implicating shoulder domain rotation in the mechanism of GTPase activation. Several other mutations were localized in or near the 30S A site and also stimulate the GTP hydrolysis step. Interestingly, we found that those mutations predicted to promote shoulder rotation all impair ribosomal proofreading, whereas mutations of the A-site nucleotide C1054 do not. These data suggest that movement of the shoulder domain regulates steps during both phases of decoding.

298

Studying the Mechanism of Translation Termination Using Bulk Fluorescence Approaches

Megan McDonald, Rachel Green Johns Hopkins School of Medicine, Baltimore, MD, USA Translation termination is signaled in all organisms by one of three stop codons in the ribosomal decoding site. Instead of being recognized by aminoacylated-tRNAs, these stop codons are decoded by proteins known as Class I Release Factors (RFs). When a class I RF recognizes a stop codon, it catalyzes the hydrolysis reaction that releases the completed protein chain from the P-site tRNA on the ribosome. Class II Release Factor RF3 (a translational GTPase) then interacts with the complex to accelerate dissociation of the class I RF, and potentially to facilitate ratcheting of the ribosomal subunits. While there is considerable evidence to indicate that RFs function in many ways analogously to tRNAs, a detailed mechanistic understanding of this critical process in translation is lacking. We are measuring the thermodynamic and kinetic parameters of the interaction of RF1 with stop and near-stop codon-programmed ribosome complexes using stopped flow bulk fluorescent approaches. We are additionally following the interaction of RF3 with RF1:ribosome complexes and are defining the nucleotide dependence of this interaction. Through these studies, we hope to provide a thermodynamic and kinetic framework for deciphering ribosome function during this critical step of translation. Our current results are consistent with previous biochemical and structural work in the field1,2, but can now assign rate constants to various crucial steps. A synopsis of this ongoing work will be presented. 1. Zavialov AV, Buckingham RH, Ehrenberg M. 2001. A posttermination ribosomal complex is the guanine nucleotide exchange factor for peptide release factor RF3. Cell 107: 115-124. 2. Zhou J, Lancaster L, Trakhanov S, Noller HF. 2012. Crystal structure of release factor RF3 trapped in the GTP state on a rotated conformation of the ribosome. RNA 18: 230-240.

Poster Session 1: Ribosomes and Translation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

299

40S Maturation Requires Joining of 60S and a Subset of Translation Initiation Factors

300

Unified Mechanism for Proofreading by Class I Aminoacyl-tRNA Synthetases

Megan Novak2,1, Bethany Strunk1, Katrin Karbstein1 1 The Scripps Research Institute, Jupiter, FL, USA, 2Furman University, Greenville, SC, USA Ribosome biogenesis is a complex procedure in which rRNA precursors are transcribed and processed into mature rRNA, and associate with ribosomal proteins to form two distinct ribosomal subunits called the 40S (small) and 60S (large) subunits. The maturation process also requires assembly factors (AFs) to facilitate processing, modifications, and folding; in Saccharomyces cerevisiae the assembly process requires the cooperation of ~200 such factors. However, it is unclear how AFs are displaced from maturing 40S ribosomes, if or how maturing subunits are assessed for fidelity, and what prevents premature translation initiation once AFs dissociate. Here we show maturation involves a translation-like cycle, in which the translation factor eIF5B promotes joining of large (60S) subunits with pre-40S subunits to give 80S-like complexes, which are subsequently disassembled by the termination factor Rli1. The AFs Tsr1 and Rio2, which block spontaneous binding of 60S subunits and initiator tRNA, respectively, dissociate from 80S-like complexes. As 80S-like complexes lack mRNA or initiator tRNA, translation initiation requires Rli1-directed displacement of 60S subunits. This cycle thus provides a functional test of 60S subunit binding and the GTPase site before ribosomes enter the translating pool, and establishes a new paradigm for understanding ribosome maturation.

John Perona1, Ita Gruic-Sovulj2, Nevena Cvetesic2, Morana Dulic2, Hari Bhaskaran1 1 Portland State University, Portland OR 97207, 2University of Zagreb, Zagreb, Croatia Steady-state and transient kinetic studies of the synthetic and editing activities of E. coli isoleucyl-, valyl and leucyltRNA synthetases provide a comprehensive kinetic and thermodynamic framework for hydrolytic proofreading. Among these three class I tRNA synthetases possessing a dedicated CP1 post-transfer editing domain, LeuRS and ValRS rely primarily on post-transfer editing, while IleRS is distinct in retaining a distinct tRNA-dependent pre-transfer editing activity to clear noncognate amino acids before misacylation. Rigorous evaluation of various proposed models for pretransfer editing demonstrate that it is an enzyme-catalyzed activity residing in the synthetic site of the enzyme, and that kinetic partitioning between hydrolysis of noncognate aminoacyl adenylate and transfer to tRNA controls the relative dependence on pre- versus post-transfer editing pathways. Kinetic proofreading does not play a significant role in editing by any of the three enzymes. Thus, the synthetic active site of editing tRNA synthetases possesses both synthetic and editing activities, leading to a fundamental reconceptualization of the classic double sieve mechanism. Implications for the evolution of editing mechanisms, and contrasts with the proofreading activities of DNA polymerases will also be presented.

Poster Session 1: Ribosomes and Translation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

301 Site

Alternative Reading Frame Selection Mediated by a Viral tRNA-like Internal Ribosome Entry

302

Translation initiation of non-LTR retrotransposons by HDV-like self-cleaving ribozymes

Qian Ren1, Qing Wang1, Andrew Firth2, Mandy Chan1, Joost Gouw1, Marta Guarna1, Leonard Foster1, John Atkins3, Eric Jan1 1 University of British Columbia, Vancouver, BC, CA, 2University of Cambridge, Cambridge, U.K., 3University of Utah, Salt Lake City, UT, USA Dicistroviruses are linear positive-strand ssRNA viruses, containing two distinct internal ribosome entry sites (IRES) that direct translation of two open reading frames (ORFs) encoding the viral non-structural and structural proteins. The intergenic region (IGR) IRES utilizes an unusual mechanism, involving mimicry of a P-site tRNA, to directly assemble 80S ribosomes and initiate translation at a specific non-AUG A-site codon. Bioinformatic analyses have revealed that there is a hidden ORF (ORFx) in the +1 frame overlapping the structural polyprotein ORF starting downstream of the intergenic region (IGR) IRES region. Using the honey bee virus Israeli acute paralysis virus as a model, we show that the IGR IRES can mediate translation in the 0 and +1 reading frames to produce two distinct protein products by using extensive mutagenesis and mass spectrometric analysis. Furthermore, an ORFx peptide is detected using multiple reaction monitoring mass spectrometry in virus-infected honey bees, strongly suggesting that +1 frame translation occurs in vivo. We are further exploring using mutagenesis and structure probing analysis to elucidate the essential nucleotide(s) within the tRNA-mimicry PKI region for +1 frame translation. In summary, we have identified a novel viral strategy in which a tRNA-mimicking domain of the IGR IRES shifts the ribosome in the +1 frame to increase coding capacity.

Dana Ruminski, Andrej Luptak UC Irvine, Irvine, CA, USA Many non-long terminal repeat (non-LTR) retrotransposons lack internal promoters and are co-transcribed with their host genes. These transcripts need to be liberated before inserting into new loci. Using structure-based bioinformatics, we have recently shown that several classes of retrotransposons in phyla spanning arthropods, nematodes, and chordates utilize self-cleaving ribozymes of the hepatitis delta virus (HDV) family for processing their 5’ termini. Ribozyme-terminated retrotransposons include rDNA-specific R2, R4 and R6; telomere-specific SART; and Baggins and RTE elements. The R2 and R6 ribozymes were recently shown to promote translation initiation of downstream open reading frames in translation reactions in vitro with rabbit reticulocyte lysate and they also demonstrate in vivo activity in Drosophila S2 cell transfections. Translation initiation now extends to other retrotransposon-associated ribozymes including those that do not insert site-specifically. Novel roles, such as those presented here, highlight the biological importance of self-cleaving ribozymes as well as functional RNAs in general.

Poster Session 1: Ribosomes and Translation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

303 Residues in Different Parts of the tRNA Structure Collaborate in Establishing tRNA Performance, In Vivo

Margaret Saks, Daniel Curtis, Devin Midura, Olke Uhlenbeck Northwestern University, Evanston, (IL), USA In vivo studies, using an E. coli tRNA knockout strain, revealed that certain base pair substitutions in the tRNAThrUGU T stem are lethal by virtue of their deleterious effect on protein synthesis (Saks et al. 2011 RNA 17:1038-1047). Unexpectedly, although T-stem mutations can substantially influence the affinity with which tRNA binds to EF-Tu, a defect in ternary complex formation does not entirely account for the growth inhibition. Indeed, many of the lethal substitutions had only a negligible effect on protein binding. These observations raised the possibility that tRNA performance depends on a proper association between the sequence of the T stem and residues in other parts of the tRNA structure. To test this idea, genes corresponding to inactive parental tRNAs were subjected to error-prone PCR and expressed in an E. coli strain lacking a functional copy of the essential tRNAThrUGU gene. Plasmid DNA was purified from the viable colonies and sequenced to identify compensatory mutations. Of particular interest are the cases where the effect of a deleterious T-stem sequence is ameliorated by a single point mutation in an entirely different region of the tRNA. The compensatory mutations were individually introduced into wild type E. coli tRNAThrUGU as well as several inactive parental tRNAs and the translation efficiency of each second-generation variant was determined using β-galactosidase assays. Surprisingly, these assays revealed that the T stem sequence-context profoundly influences the extent to which individual second-site mutations improve translation efficiency. Investigations are in progress to elucidate the structural and mechanistic basis for these observations.

304 Rbg1-Tma46 Dimer Structure Reveals New Functional Domains and Their Role in Polysome Recruitment

Sandrea Francis1, María-Eugenia Gas2, Marie-Claire Daugeron3, Jeronimo Bravo1, Bertrand Séraphin2 1 Instituto de Biomedicina de Valencia (IBV-CSIC), Valencia, Spain , 2IGBMC, Illkirch, France, 3CGM, Gif/ Yvette, France DRG proteins are highly conserved GTPases that associate with DFRP proteins. The resulting complexes have recently been shown to participate in eukaryotic translation. The structure of the Rbg1 GTPase, a yeast DRG protein, in complex with the C-terminal region of its DFRP partner, Tma46, was solved by X-ray diffraction. These data reveal that DRG proteins are multimodular factors with three additional globular domains, HTH, S5D2L and TGS, packing against the GTPase platform. Surprisingly, one of these new domains is inserted in the middle of the GTPase sequence. In contrast, the region of Tma46 interacting with Rbg1 adopts an extended conformation typical of intrinsically unstructured proteins that contacts the GTPase and TGS domains. Functional analyses demonstrate that the various domains of Rbg1, as well as Tma46, modulate the GTPase activity of Rbg1 and contribute to the function of this complex in vivo. Dissecting the role of the different domains revealed that one of the Rbg1 domains is essential for the recruitment of this factor in polysomes, supporting further the implication of these conserved factors in translation.

Poster Session 1: Ribosomes and Translation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

305 The Requirement for RPS25 Links IRES and Ribosomal Shunting Mechanisms of Initiating Ttranslation

Marla Hertz1, Dori Landry1, Anne Willis2, Guangxiang Luo3, Sunnie Thompson1 University of Alabama at Birmingham, Birmingham, AL, USA, 2University of Leicester, Leicester, UK, 3 University of Kentucky, Lexington, KY, USA During cellular stress or viral infection cap-dependent translation is inhibited and translation of mRNAs occurs by alternate mechanisms of initiation. mRNAs that contain an IRES (internal ribosome entry site) can recruit a ribosome internally to the mRNA. We previously demonstrated that transient knockdown of ribosomal protein S25 (RPS25) specifically inhibited IRES-mediated translation by Dicistroviridae and hepatitis C viral IRESs. To further examine the role of RPS25 in translation, we generated a stable human cell line with RPS25 knocked down. These cells display little to no defect in cell growth or cap-dependent translation. However, translation initiation of a number of viral and cellular IRESs was impaired. Some IRESs were severely inhibited when RPS25 was knocked down, while others were only modestly decreased suggesting that there are at least two mechanisms of IRES-mediated translation. Interestingly, knock down of RPS25 also demonstrated a significant inhibition of ribosomal shunting by the adenovirus tripartite leader. We furthermore demonstrate that knock down of RPS25 resulted in a decrease in amplification for viruses that use either IRES or ribosomal shunting. Importantly, RPS25 knockdown had no effect on the replication of herpes simplex virus 1, which relies solely on cap-dependent translation. This demonstrates that cells deficient in RPS25 are not impaired in viral amplification or cap-dependent translation. These results suggest that IRESs and shunting, two structurally, functionally and biologically distinct mechanisms of translation initiation, both require an RPS25-dependent step in initiation. We present a model that can explain the common mechanism shared between these two disparate methods of translation initiation. 1

306

Peptidyl Transfer Center Dynamics Probed via Single Molecule FRET

Ming Xiao, Yue Li, Mediha Altuntop, Yuhong Wang University of houston, Houston, TX, USA During protein biosynthesis, the ribosome slides along the mRNA precisely 3 nucleotides for each amino acid incorporated. Biochemical and structural studies revealed that ribosome dynamics changed from flexible to more static after the transition of pre- to post-translocation configurations, which referred as ribosome “unlocking”. During the elongation cycles, the ribosome oscillates between “unlocked” and “locked” dynamics while repeatedly transits between pre- and post-translocation conformations. The bacterial ribosome was found to incorporate L27 within 10 Å away from the A- and P-site tRNAs. However, this protein is dispensable. By labeling the L27 and tRNA with FRET paired dyes, we have developed a novel platform to study the catalytic center of the ribosome via single molecule FRET microscopy, without compromising the ribosome competence. We have found that the ribosome hybrid state fluctuation is hierarchically regulated (1) and persistent even when the ribosome ratcheting was inhibited (2). Prolonged tracking of the pre-translocation complexes indicates that multiple native and active subpopulations co-exist in the same ribosome. These native conformations exchange to each other spontaneously and translocations are observed from each of them. However, the real time tracking of translocation in the presence of EF-G shows that the ribosome prefers one specific tRNA configuration (0.2 FRET state in our experiments) prior to translocation, regardless of the subpopulation the ribosome resides. We propose that EF-G binding biases the ribosome into one specific configuration for the optimal translocation processes. However, this preferred configuration is not the putative A/P-P/E state, but rather the A/A-P/E state. Consistent with ribosome locking, we do not observe much fluctuation in the post-translocated ribosomes. 1. Altuntop, M. E., C. T. Ly, and Y. Wang. 2010. A single molecule study of ribosome’s hierarchic dynamics at the peptidyl transferase center. Biophys J 99:1-8. 2. Ly, C. T., M. E. Altuntop, and Y. Wang. 2010. A single molecule study of Viomycin’s inhibition mechanism on ribosome translocation. Biochemistry epub ahead of print.

Poster Session 1: Ribosomes and Translation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

307 Crystal structure of release factor RF3 trapped in the GTP state on a rotated conformation of the ribosome

Harry Noller lab:1, Jie Zhou1, Laura Lancaster1, Sergei Trakhanov2, Harry Noller1 UC. Santa Cruz, Santa Cruz, CA, U.S.A., 2Max Planck Institute for Biophysical Chemistry, Göttingen, Germany The class II release factor RF3 is a GTPase related to elongation factor EF-G, which catalyzes release of class I release factors RF1 and RF2 from the ribosome after termination of protein synthesis. The 3.3 Å crystal structure of the RF3·GDPNP·ribosome complex provides a high-resolution description of interactions and structural rearrangements that occur when binding of this translational GTPase induces large-scale rotational movements in the ribosome. RF3 induces a 7° rotation of the body and 14° rotation of the head of the 30S ribosomal subunit, and itself undergoes interand intradomain conformational rearrangements. We suggest that ordering of critical elements of switch loop I and the P loop, which help to form the GTPase catalytic site, are caused by interactions between the G domain of RF3 and the sarcin–ricin loop of 23S rRNA. The rotational movements in the ribosome induced by RF3, and its distinctly different binding orientation to the sarcin–ricin loop of 23S rRNA, raise interesting implications for the mechanism of action of EF-G in translocation

1

308

Abstract Withdrawn

Poster Session 1: Ribosomes and Translation & Interconnections Between Gene Expression Processes

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

309 Molecular Characterization of Genome Unscrambling in the Ciliated Protozoan Oxytricha trifallax

Leslie Beh, Laura Landweber Department of Ecology and Evolutionary Biology, Princeton University The pond-dwelling protozoan Oxytricha trifallax is an emerging model system for the study of epigenetic regulatory mechanisms. Previous studies have largely focused on the ciliates Paramecium and Tetrahymena (class: oligohymenophorea), placing Oxytricha (class: spirotrichea) as a promising phylogenetic outgroup for comparative analyses. Like other ciliates, O. trifallax possesses a heterochromatin-rich germline micronucleus (MIC), and a transcriptionally active somatic macronucleus (MAC). During the sexual cycle of O. trifallax, a new MAC terminally differentiates from its zygotic nuclear precursor. Massive genome rearrangements occur during MAC development, entailing the elimination of ~95% of the precursor genome, and the reordering of macronuclear destined segments (MDSs) to form gene-sized nanochromosomes. Currently, the molecular mechanisms underlying global genome remodeling in the developing MAC remain unclear. To address this, we have developed fractionation methods to isolate the developing MAC from sexual progeny. This purification protocol will facilitate downstream analysis of the developing MAC through genome sequencing, transcriptional profiling, and mass spectrometry. Such studies could shed light on the temporal series of molecular events underlying genome rearrangements in O. trifallax, and facilitate comparative analysis of analogous pathways in both ciliate and non-ciliate systems.

310 Comparison Between Developmental Stages of D. melanogaster and C. elegans with modENCODE RNA-Seq Data

Jingyi Jessica Li, Haiyan Huang, Peter Bickel, Steven Brenner University of California, Berkeley, CA, USA We are undertaking a comparison of the developmental time courses of the model organisms, D. melanogaster and C. elegans, seeking commonalities in orthologous genes’ transcription. The availability of RNA-Seq data of different developmental stages of the two organisms in modENCODE enables a transcriptome-wide comparison study. Our current approach centers on identifying genes specific to each D.melanogaster and C. elegans developmental stage. These stagespecificgenes are selected as those highly expressed at that stage but lowly expressed at a few other stages in the time course. In this study, we have employed the set of one-to-one orthologs in TreeFam to link genes of the two organisms. To test the dependence between a pair of D. melanogaster and C. elegans stages, we use an overlap statistic, which is the number of one-to-one orthologous gene pairs specific to both stages. Under the null hypothesis that the two stages are independent, a p-value can be calculated exactly for the observed value of the overlap statistic. We find that temporally adjacent stages in each of fly and worm development time courses show common stage-specific genes, supporting the validity of this approach. We also find other connections, such as female fly adults showing similar stage-specific genes as fly embryos. Most important, when comparing fly with worm, we find a strong colinearity of their developmental time courses from early embryos to late larvae. Another parallel collinear pattern between fly white prepupae through adults and worm late embryos through adults is observed. Small p-values are also observed between fly early embryos and worm adults, and between fly female adults and worm adults. Our results are the first findings regarding the comparison between D. melanogaster and C. elegans mRNA RNA-seq time courses. Those stage-specific genes overlapped between D. melanogaster and C. elegans are currently being studied; some having known biological functions have been verified to play similar roles in both organisms, and their mapping in this study may help inform their functions in the development of D. melanogaster and C. elegans.

Poster Session 1: Interconnections Between Gene Expression Processes

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

311 Regulation of Splicing Factors by Alternative Splicing and NMD is Conserved Between Kingdoms Yet Evolutionarily Flexible

Liana Lareau1,2, Steven Brenner1 1 University of California, Berkeley, CA, USA, 2Currently at: Stanford University School of Medicine, Stanford, CA, USA Ultraconserved elements, unusually long regions of perfect sequence identity, are involved in regulating the expression of numerous RNA-binding proteins including SR splicing factors. Regulation occurs via alternative splicing of these regions to yield mRNAs that are degraded by nonsense-mediated mRNA decay (NMD), a process termed unproductive splicing (Lareau et al., 2007; Ni et al., 2007). As all human SR genes are affected by alternative splicing and NMD, one might expect this regulation to have originated in an early SR gene and persisted as duplications expanded the SR family. But in fact, unproductive splicing of most human SR genes arose independently (Lareau et al., 2007). This paradox led us to investigate the origin and proliferation of unproductive splicing in SR genes. We demonstrate that unproductive splicing of the splicing factor SRSF5 (SRp40) is conserved between animals and fungi; this is the first example of alternative splicing conserved between kingdoms, yet its effect is to trigger mRNA degradation. As the gene duplicated, the ancient unproductive splicing was lost in paralogs, and distinct, functionally equivalent splicing evolved rapidly and repeatedly to take its place. SR genes have consistently employed unproductive splicing, and while it is exceptionally conserved in some of these genes, turnover in specific events among paralogs shows flexible means to the same regulatory end.

312 The Role of the Canonical 50nt Rule in Targeting a Transcript for Nonsense-Mediated mRNA Decay in Human and in Fly

Courtney French1, Gang Wei1,2, Angela Brooks1,3, Steven Brenner1 1 University of California, Berkeley, CA, USA, 2Currently at: Fudan University, Shanghai, China, 3Currently at: Broad Institute of MIT and Harvard, Cambridge, MA, USA Nonsense-mediated mRNA decay (NMD) is an RNA surveillance system that degrades isoforms containing a premature termination codon. This pathway is conserved throughout eukaryotes and protects against the production of harmful truncated proteins. Additionally, NMD coupled with alternative splicing is a mechanism of post-transcriptional gene regulation that affects the mRNA levels of thousands of genes in human. The canonical model of defining a premature termination codon in mammals is the 50nt rule: a termination codon more than 50 nucleotides upstream of an exon-exon junction is recognized as premature and triggers degradation by NMD. In addition to mammals, there is evidence that this rule holds in Arabidopsis but not in other eukaryotes such as Drosophila. There is also evidence that a longer 3’UTR triggers NMD in plants, flies, and mammals. Details of these mechanisms and their prevalence remain unclear in many species. The importance of each mechanism appears to vary between species, and it is currently unclear which is the major mechanism at work in human cells. We have performed RNA-Seq analysis on human cells where NMD has been inhibited via knockdown of UPF1, a critical protein in the degradation pathway. We assembled isoforms and quantified their abundance with the Cufflinks software suite. By comparing the isoform abundance in cells with inhibited NMD to that in normal cells, we are able to determine which isoforms were being degraded by NMD. We see that isoforms with a premature termination codon according to the 50nt rule are more than twice as likely to be degraded by NMD (70% of 50nt rule isoforms are at reduced abundance when NMD is active compared to 30% of normal isoforms). In contrast, we found very little correlation between the likelihood of degradation by NMD and 3’UTR length in the absence of a 50nt rule premature termination codon. We also performed the analogous RNA-seq experiment in a Drosophila cell line. Overall, we found that hundreds of alternative isoforms are degraded by NMD in the fly. Unexpectedly, we did not find a clear correlation between 3’UTR length and likelihood of degradation in fly.

Poster Session 1: Interconnections Between Gene Expression Processes

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

313 Transcriptome Analysis Reveals Extensive Alternative Splicing Coupled with NonsenseMediated mRNA Decay in a Human Cell Line

Courtney French1, Gang Wei1,2, Angela Brooks1,3, Steven Brenner1 1 University of California, Berkeley, CA, USA, 2Currently at: Fudan University, Shanghai, China, 3Currently at: Broad Institute of MIT and Harvard, Cambridge, MA, USA Many alternatively spliced isoforms contain a premature termination codon that targets them for degradation by the nonsense-mediated mRNA decay (NMD) RNA surveillance system. Some such unproductive splicing events have a regulatory function, whereby alternative splicing and NMD act together to impact protein expression. Previous studies to identify NMD targets have been predominantly constrained to examining only those isoforms inferred from cells where NMD is active and the NMD targets are expected to be present at very low abundance. To effectively survey the targets of NMD genome-wide, we performed RNA-Seq on human tissue culture cells in which NMD had been inhibited via knockdown of UPF1, a critical protein in the degradation pathway. Using the Cufflinks software suite to assemble novel isoforms, quantify isoform-level abundance, and determine differential expression, we found 22,000 different isoforms from 10,600 genes that were moderately or highly expressed. We defined NMD targets as those isoforms that have a premature termination codon according to the canonical 50nt rule in mammals and significantly increased in abundance (>1.5 fold) when NMD was inhibited. Altogether, we report a strict set of 2,443 isoforms (11%), produced by 1,924 genes (18%), as putative NMD targets. Our results verify the NMD degradation of previously inferred unproductive isoforms for the SR proteins and hnRNPs. Moreover, we discovered thousands of previously uncharacterized putative targets of NMD. NMD-targeted isoforms were derived from genes involved in many functional categories, and were significantly enriched for RNA splicing factors. This indicates that auto-regulation of splicing factors through NMD and more general NMD-related regulation is more widespread than previously inferred and affects diverse biological processes. We found a significant enrichment in genes targeted by NMD of ultraconserved elements, predominantly for genes that function in RNA processing. Of these, 22 have an ultraconserved element overlapping a cassette exon that, when included, triggers NMD, indicating a potential role for ultraconserved elements in regulation of alternative splicing coupled with NMD. Our findings demonstrate that gene expression regulation through NMD is more widespread than previously inferred and this important level of posttranscriptional gene regulation impacts a large number of functionally distinct proteins and processes.

314 Btf and TRAP150 Localize to Transcription Sites and Affect the Cellular Distribution of mRNAs

Sapna Varia, Divya Potabathula, Zhihui Deng, Athanasios Bubulya, Paula Bubulya Wright State University, Dayton, (OH), USA Transcription of protein-coding genes is coordinated with pre-mRNA processing as well as mRNP assembly and export in mammalian cell nuclei. Btf (Bcl-2 associated transcription factor) and TRAP150 (Thyroid Hormone Receptor Associated Protein of 150 kDa or THRAP3) are serine-arginine-rich (SR) proteins that have 39% sequence identity and 66% sequence similarity; however it is not clear if the functions of these two proteins completely overlap. Btf and TRAP150 were previously reported to associate with synthetic affinity-purified in vitro spliced mRNPs, and also as a part of the spliceosome complex, suggesting they are involved in pre-mRNA processing. We used an in situ approach to show that both Btf and TRAP150 are recruited to a constitutively active beta-tropomyosin reporter minigene locus in HeLa cells as well as to the U2OS 2-6-3 inducible reporter gene locus in its transcriptionally active but not inactive state. Upon inhibition of RNA polymerase II, Btf and TRAP150 were absent from the locus, indicating their presence at the locus requires transcription. At the activated locus, both Btf and TRAP150 showed some overlap with reporter RNA and other pre-mRNA processing factors, but showed the most extensive overlap with the exon junction complex protein Magoh. Intriguingly, RNA-FISH with fluorescently tagged oligo-dT probes showed an increase in cytoplasmic polyadenylated RNA in HeLa cells specifically following Btf depletion but not TRAP150 depletion. qRT-PCR revealed an increase of beta-tropomyosin minigene reporter transcripts in the cytoplasm in Btf depleted cells. Our data suggests that modulation of Btf and TRAP150 at transcription sites affects processing and nuclear/cytoplasmic distribution of mRNAs.

Poster Session 1: Interconnections Between Gene Expression Processes

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

315

Characterization Of prp43 Alleles Provides Insights Into Pathway Specific Domains

316

Traf3 Alternative Splicing Regulates The Non-Canonical NFkB Pathway In T Cells

Jennifer Hennigan, Scott Stevens University of Texas, Austin, Texas, USA The essential DEAH box helicase Prp43p is involved in both ribosome biogenesis and spliceosome disassembly in S. cerevisiae. In splicing, Prp43p removes the intron from the post-splicing complex, a mechanism dependent upon an interaction with the G patch protein Ntr1p (Tsai et al. 2005, Tanaka et al. 2006). In ribosome biogenesis, Prp43p binds to multiple regions of the yeast pre-rRNA, suggesting a potential for more than one function in this process. Its loss contributes to disrupted distribution of multiple snoRNAs on the pre-ribosome (Bohnsack et al. 2009). Prp43p also appears to be crucial in remodeling around the D-site for proper cleavage of 20S, and this function is supported by another cofactor, Pfa1p (Pertschy et al. 2009, Lebaron et al. 2009). To further understand pathway specificity and potential regulation of Prp43p, we identified conditional alleles using a random PCR mutagenesis screen targeting the C-terminus of Prp43p. Interestingly, we have identified prp43 alleles with a previously uncharacterized splicing defect as detected by both U3 primer extensions and native snRNP gel analysis. Several prp43 alleles display an increase in di-snRNP levels and a decrease in tri-snRNP levels, indicative of a U5 recycling defect seen in the absence of Ntr1p (Boon et al. 2006). Other mutants display varying rRNA defects despite their relatively close proximity in three dimensional space when mapped on the crystal structure (He et al. 2010). Two of three alleles within the winged helix domain result in 35S pre-rRNA accumulation, while the third rapidly downregulates all ribosome precursors. To correlate the processing defects seen in each allele to cofactors, we have determined the extent of association of prp43p mutants with each G patch protein by coimmunoprecipiation. Understanding Prp43p specificity in these processes in yeast will be a key step towards discerning the molecular mechanism of recruiting other RNA helicases to their substrates, many of which remain enigmatic.

Monika Regehr, Ilka Wilhelmi, Florian Heyd Marburg University, Germany The non-canonical NFkB (ncNFkB) pathway controls the expression of several chemokines involved in formation and maintenance of secondary lymphoid organs and thus plays a pivotal role in adaptive immunity. In addition, it regulates survival of naive B cells and its misregulation contributes to malignant transformation. Activity of the ncNFkB pathway depends on the presence of its upstream activating kinase NIK. One of the proteins crucially involved in negatively regulating the abundance of NIK is Traf3. It induces formation of a NIK-Traf3-Traf2-cIAP complex, in which NIK is constitutively targeted for degradation by cIAP-mediated ubiquitylation to keep NIK expression and ncNFkB signaling at low basal levels. Signaling through several transmembrane receptors, e.g. in B cells, leads to Traf3 degradation which disrupts this complex allowing NIK accumulation and ncNFkB activity. Here we show that Traf3 alternative splicing in T cells generates a Traf3 isoform that, in contrast to the full length protein, activates ncNFkB signaling. This Traf3 splicing switch is cell type specific, as it is observed upon PMA-activation of human Jsl1 T cells but not in B cell lines. Overexpression of the shorter Traf3 variant in resting cells induces the ncNFkB pathway as does PMA treatment, providing a direct link between Traf3 alternative splicing and ncNFkB signaling. These data were confirmed by Morpholino-mediated manipulation of Traf3 isoform expression: a Traf3 splicing switch in resting cells is sufficient to activate ncNFkB signaling as assessed by increased formation of the active NFkB2 protein, reporter gene assays and increased expression of endogenous ncNFkB targets. To further support our results we have analyzed the effect of Traf3 alternative splicing on NIK expression and formation of the NIK-Traf3-Traf2-cIAP complex. Using immunoprecipitation as well as gel filtration analysis we show that the presence of the shorter Traf3 variant disrupts this complex and allows the accumulation of NIK to initiate ncNFkB signaling thus providing a mechanistic basis for our observations. Finally, we show that Traf3 exon skipping also correlates with increased activity of the ncNFkB pathway in primary T cells, confirming the in vivo relevance of our data. Taken together, we provide a detailed analysis of the functional outcome of Traf3 alternative splicing which reveals a role in regulating the ncNFkB pathway in T cells with implications for T cell activation and survival. Poster Session 1: Interconnections Between Gene Expression Processes

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

317

Abstract Withdrawn

318

Transcriptional activation of PIK3R1 by PPARγ in adipocyte

Yoon-Jin Kim, Sang Hoon Kim KyungHee University, Seoul, Republic of Korea Phosphatidylinositol 3-kinase plays an important role in the metabolic actions of insulin, and is required for adipogenesis. The PIK3R1/p85 α regulatory subunit is a critical component of the PI3K signaling pathway. Activation of PPARγ by rosiglitazone can induce the expression of PIK3R1. However, the transcriptional regulation of PIK3R1 in adipocytes remains unknown. In the present study, we investigated whether the mouse PIK3R1 is a target of PPARγ which is known as a key regulator for adipogenesis. The expression level of PIK3R1 in 3T3-L1 was increased after the induction of adipocyte differentiation and was also induced by overexpression of PPARγ. Two putative peroxisome proliferator response elements (PPREs) in PIK3R1 promoter were identified as PPARγ binding sites. By chromatin immunoprecipitation assay, we observed that PPARγ interacts to the PPREs of PIK3R1 promoter. In addition, luciferase reporter assays were showed that the -1183/-1161 and -573/-551 region of the PIK3R1 promoter contains essential elements for binding PPARγ. Taken together, these results suggest that PPARγ is essential for transcriptional activity of PIK3R1 during adipogenesis.

Poster Session 1: Interconnections Between Gene Expression Processes

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

319 Biochemical Characterization of Dbp2 at the Interface of RNA Export and lncRNAdependent Transcription Regulation

Wai Kit Ma1, Sara Cloutier1, Elizabeth Tran1,2 1 Department of Biochemistry, Purdue University, West Lafayette, (IN), USA, 2Purdue Cancer Center, Purdue University, West Lafayette, (IN), USA Regulation of gene expression is necessary for cell survival and function. In eukaryotes, there are many complex and highly coupled mechanisms that involve proteins to regulate gene expression at the level of transcription. In addition, long non-protein coding RNAs (lncRNAs) have also been shown to regulate transcription of protein coding genes. It is well understood that dynamic rearrangement of RNA structures, reassortment of RNA-binding proteins, and disassembly of RNA:protein complexes is central to post-transcriptional gene expression steps. However, the precise role of RNA:protein dynamics in lncRNA-mediated events has not been fully investigated. One class of enzymes that plays a critical role in RNA biology are the DEAD-box RNA helicases which utilize ATP hydrolysis to promote localized changes to RNA structure. Our laboratory has recently found that the DEAD-box protein Dbp2 in Saccharomyces cerevisiae required for resetting the transcriptional state of genes in response to overlapping lncRNA synthesis. Using series of biochemical and molecular techniques, we now show that Dbp2 prefers specific RNA substrates for stimulation of enzymatic activity. We also report that Dbp2 interacts directly with the nuclear RNA-binding protein Yra1. Yra1 functions as an adaptor protein for the mRNA export receptor, Mex67, to facilitate mRNA transport. We will provide evidence that will test the model that Dbp2 antagonizes the role of lncRNAs by promoting clearing from genomic loci via the mRNA transport pathway.

320

Transcription elongation and mRNA processing are linked through ELL2 in lymphocyte

Christine Milcarek, Michael Albring, Fortuna Arumemi, Creitekya Langer, Kyung Park University of Pittsburgh, Pittsburgh, PA, USA High levels of ELL2, a transcription elongation factor, are responsible for Igh mRNA being both alternatively processed to the secretory-specific form and for its increased abundance in plasma cells versus B cells. This leads to high levels of secreted Ig mRNA and protein and a robust immune response in plasma cells [Nat Imm 10:1102 (2009)]. Histone H3K4 di- methyl and H3K4 and H3K79 tri-methylation patterns are associated with actively transcribing genes. On the Igh gene, knocking out ELL2 with siRNA decreased those activating histone modifications. Associations with CstF-64 (the cleavage/ polyadenylation factor) and pTEFb (a ubiquitous elongation factor) were also decreased by ELL2 siRNA addition. Chromatin IP studies show that ELL2 is associated with RNA polymerase II on Igh and essential regulatory plasma cell genes including blimp-1 and IRF4. Using luciferase transcription assays we show that ELL2 is able to increase transcription from its own and the blimp-1, and IRF-4 promoters. Elongation is linked to increased mRNA production and the use of a promoter proximal poly(A) site in the IRF-4 gene. Studies on the role of ELL2 in Ig secretion and IRF4 expression using conditional knockout mice indicate a role for ELL2 in Igh mRNA production in vivo. Therefore, transcription elongation, RNA processing/ polyadenylation, and histone modifications are interdependent processes important for Igh alternative mRNA expression. This work was supported by NSF# MCB-0842725 (to C.M.)

Poster Session 1: Interconnections Between Gene Expression Processes

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

321

Inhibition of Co-transcriptional Splicing Affects RNA Polymerase II Elongation Kinetics

322

Functional Characterization of Polypyrimidine Tract-Binding Proteins from Arabidopsis

Jarnail Singh, Richard Padgett Cleveland Clinic Transcription and pre-mRNA processing are tightly coupled in the mammalian system. Consequently most of the introns are removed from the pre-mRNA by co-transcriptional splicing during transcription elongation by RNA polymerase II. It is therefore presumed that there may be crosstalk between transcription and splicing machinery and modulation of one might affect the other. A popular model suggests that there is coupling of transcription and splicing through the C-terminal domain of RNA Pol II. In support of this model, a number of studies have shown that the modulation of the transcription elongation rate by RNA Pol II can affect alternative splicing events in cells. However, the effect of modulating the rate or extent of splicing on the RNA Pol II elongation rate has been less well studied. Here we measured the in vivo rates of transcription elongation in human HEK 293 cells to show that inhibition of co-transcriptional splicing reduces the rate of transcription. This effect is most pronounced in exon-rich regions of genes with little or no effect on the transcription elongation rate observed within large introns. These data support the idea that transcription and splicing are coupled during mammalian gene expression.

Eva Stauffer, Andreas Wachter University of Tübingen, Tübingen, Germany One in vertebrates well-studied member of the heterogeneous ribonucleoprotein family is the Polypyrimidine tractbinding protein (PTB), which binds to CU-rich motifs in target precursor mRNAs and thereby can influence splice site selection. In mammals, PTBs were demonstrated to regulate not only splicing, but also other steps in mRNA metabolism, including transport and translation of mRNAs. Our previous analysis of three PTB homologues from Arabidopsis thaliana (AtPTBs) provided first evidence that this group of proteins can regulate alternative splicing in plants. Furthermore, AtPTBs were found to be able to regulate gene activity in a splicing-independent manner, which, in combination with splicing control, serves as basis for auto- and cross-regulation of their expression. Given that AtPTBs can repress gene activity in a splicing-independent manner and that they are not only localized in the nucleus and cytosol, but are also present in processing bodies, we suggest that plant PTB homologues might have a function in regulating mRNA translation. To further address this question, we perform polysomal profiling of mutant Arabidopsis lines with altered levels of the three AtPTBs. As previous experiments indicated that the accumulation of AtPTB proteins inhibits the expression of AtPTB-based reporter constructs, we expect to observe altered distributions of AtPTB mRNAs between mono- and polysomal fractions from AtPTB misexpression plants. Furthermore, members of the hnRNP family have been shown to be subject to posttranslational modifications, which can have important implications for their functioning. Our current analyses aim to identify sites of posttranslational modifications in the AtPTBs and to unravel their functional implications. Preliminary data support the occurrence of arginine dimethylation for at least one of the three AtPTBs and investigation of motif mutants indicate a role of those residues in PTB functions. In summary, our work aims at elucidating the intricate functions of PTBs in plant mRNA metabolism as well as providing novel insight into the posttranscriptional, regulatory mechanisms of this multifaceted protein family.

Poster Session 1: Interconnections Between Gene Expression Processes

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

323 A Phosphorylation-Dependent Signaling Pathway Links SLBP Ubiquitination To Histone mRNA Decay

Nithya Krishnan1, TuKiet Lam2, Padriac Philbin3, Donald Rempinski1, Andrew Fritz3, Kieran O’ Loughlin4, Hans Minderman4, Ronald Berezney3, William Marzluff5, Roopa Thapar1,3 1 Hauptman Woodward Medical Research Institute, Buffalo, NY 14203, USA, 2WM Keck Foundation Biotechnology Resource Laboratory, Yale University, New Haven CT 06511, USA , 3State University of New York at Buffalo, Buffalo, NY, USA, 4Department of Flow and Image Cytometry, Roswell Park Cancer Institute, Buffalo, NY 14263, USA, 5Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA Stem-Loop Binding Protein (SLBP) is a histone mRNA specific processing factor and the SLBP-histone mRNA complex functions as a coordinate unit in all steps of histone mRNA metabolism. SLBP is the only protein whose expression during S-phase is temporally correlated to that of histone mRNAs. SLBP and histone mRNAs are degraded at the end of S-phase. SLBP is degraded by the ubiquitin-proteasome pathway and SLBP degradation is triggered by phosphorylation of two threonines in the SLBP N-terminus which is catalyzed by Cyclin A/Cdk1 and CK2 kinases. Not much is known about the mechanism of SLBP ubiquitination and if the timing of SLBP degradation and decay of the histone mRNA may be linked by a common signaling pathway. Using high-resolution FT-ICR and LC MS-MS mass spectrometry, we have mapped 23 phosphoryation sites in human SLBP. Six of these phosphorylation sites occur in SP and TP sequences which are substrate recognition sites for the prolyl isomerase Pin1. Using NMR spectroscopy we determined that Pin1 binds the Thr171-Pro172 sequence in HPKTPNK sequence in the SLBP RNA binding domain in a phosphorylation dependent manner in vitro. We also crystallized Pin1 with phosphopeptides corresponding to this region. To test the hypothesis that SLBP is a substrate for Pin1 in vivo, we examined the effects of Pin1 knockdown on SLBP protein stability, ubiquitination, cell cycle regulation, cellular localization, and its interaction with histone mRNA. Chemical inhibition of Pin1 or down-regulation of Pin1 by siRNA increases the protein stability of SLBP as well as the mRNA stability of all five core histone mRNAs. Pin1 inhibition increased accumulation of ubiquitinated SLBP and augmented its localization in the nucleus, the site of SLBP degradation. Intriguingly, Pin1 acts along with the protein phosphatase PP2A to dissociate SLBP from the histone mRNA hairpin. These data suggest that Pin1 and PP2A act to remodel the histone mRNP and work to intersect two major pathways of gene expression at the end of S-phase, namely the degradation of SLBP by the ubiquitin proteasome system and the exosome-mediated degradation of the histone mRNA.

324 Investigating Novel Paradigms for RNA Binding Protein - miRNA Regulatory Interactions: A Study of the Poly(A) RNA Binding Protein, ZC3H14

Callie Wigington1, Paula Vertino2, Anita Corbett1 Emory University School of Medicine, Atlanta, GA, USA, 2Emory University Winship Cancer Institute, Atlanta, GA, USA Dysregulation of gene expression contributes to numerous disease states and there is increasing appreciation of posttranscriptional mechanisms that are involved in gene expression. These post-transcriptional events are tightly interconnected and are mediated by a myriad of RNA-binding proteins (RNA-BPs) as well as regulatory RNAs such as miRNAs. We are interested in gaining insight into the overall picture of post-transcriptional processing and understanding how RNA-BPs integrate with miRNAs to control gene expression. Our recent efforts have focused on understanding the role that the novel zinc finger poly(A) RNA-BP, ZC3H14, plays in regulating target mRNA transcripts. Although the molecular function of ZC3H14 is unknown, the budding yeast counterpart, Nab2, is required for proper control of poly(A) tail length and mRNA export from the nucleus, consistent with a critical role for ZC3H14 in post-transcriptional regulation. A previous study of Nab2 revealed an interaction between Nab2 and Pub1 (yeast ortholog of HuR) that influences the stability of AU-containing mRNA transcripts. Recent data demonstrate that HuR can participate in dynamic interplay with miRNAs on AU-containing transcripts to impact expression of target mRNAs. These intriguing studies led us to investigate a potential relationship between ZC3H14 and HuR that may include a role for miRNAs. In a genome-wide analysis of transcripts differentially expressed upon knockdown of either ZC3H14 or HuR, we identified putative consensus elements located within the 3’ UTR of candidate mRNA targets of ZC3H14 as well as a significant overlap between candidate upregulated ZC3H14 and HuR targets. These data suggest that ZC3H14, like HuR, has specific mRNA targets, and that ZC3H14 and HuR may have a functional relationship. In an effort to delineate the mechanism by which ZC3H14 regulates candidate target mRNAs identified and validated in the genome-wide analysis, we have selected one intriguing target, programmed cell death 4 (PDCD4), for further analysis. PDCD4 is not only one of the common upregulated targets that contains the consensus 3’ UTR element, but also a well-defined target of miR-21. We have confirmed that the steady-state level of PDCD4 mRNA and protein increases significantly upon siRNA-mediated knockdown of either ZC3H14 or HuR. Additionally, we are able to demonstrate that PDCD4 is a novel target of HuR by RNA-IP analysis. These studies provide a platform for understanding the interplay of RNA-BPs and miRNAs in regulating target mRNA transcripts. 1

Poster Session 1: Interconnections Between Gene Expression Processes

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

325

The Exon Junction Complex is Restricted to a Specific Subset of Splice Junctions

Nazmul Haque, Scot Harms, Malcolm Cook, Marco Blanchette Stowers Institute for Medical Research, Kansas City, MO, US The Exon Junction Complex (EJC) is assembled at spliced exon-exon junctions by the spliceosome, and functions as an integrator of cell signals during the mRNA lifecycle. EJC assembly is a selective process– occurring at specific junctions in intron containing 3’ UTRs. As a first step toward understanding the basis of this selectivity, we have comprehensively characterized the locations of EJC complexes assembled on a transcriptome in Drosophila S2 cells. We found that at least 92% of intron-containing transcripts are stably associated with EJCs. When individual exon-exon junctions – from transcripts not associated with EJCs – were tested in a heterologous context, we found that they could sustain EJC assembly. This suggested that most, if not all, intron-containing transcripts associate with at least one EJC at some point during their lifecycle. When we examined binding within transcripts, we found that only 61% of exonexon junctions are stably bound by an EJC – corresponding to 51% of multi-intron transcripts with EJC-free junctions that can not be explained by EJC removal by the pioneer round of translation. Finally, we have uncovered classes of functionally related transcripts associated with different EJC intermediates. Our results show that EJC is restricted to a subset of spliced junctions. Moreover, these observations suggest that EJC assembly is a highly regulated process involving multiple layers of control.

326 Stressed-out Adult Stem Cells? A Particular Class of Cytoplasmic mRNA Granules in Adipose-Derived Stem Cells.

Alejandro Correa, Crisciele Kuligovski, Marco Stimamiglio, Axel Cofré, Bruno Dallagiovanna, Samuel Goldenberg Inst. Carlos Chagas, Curitiba, Brazil The potential of adult stem cells for use in tissue regeneration has been demonstrated by in vitro, preclinical and clinical approaches. Adipose tissue-derived stem cells (ASCs) are easy to obtain and at high frequency (0.5%) in human adipose tissue. ASCs are considered a potentially useful tool for cellular therapy, and even as a carrier for gene therapies. However, little is known about the regulation of gene expression in adult stem cell such as ASCs. Posttranscriptional processes play a key role in the regulation of eukaryotic gene expression. Cellular mRNAs associate with proteins in messenger ribonucleoprotein (mRNP) complexes, to control mRNA translation, stability and distribution. Two types of cytoplasmic mRNA granules have been characterized in detail in eukaryotic cells: P-bodies (PBs) and stress granules (SGs). We investigated the presence and possible function of cytoplasmic mRNA granules in human adipose-derived stem cells (ASCs), by analyzing a stress granule (SG)-specific protein, TIA1/TIAR, and a P-body (PB)-specific component, DCP1. P-bodies were present in ASCs and resembled those in other mammalian cells. By contrast, TIA-1/TIAR-containing granules were unusually abundant in non stressed ASCs and also contained phosphorylated eIF2α but not RPS7. TIA granules were dependent on ASCs mRNA, most of which was present in these granules, and highly stringent stress conditions resulted in the disassembly of most TIA granules. ASCs containing TIA granules displayed weak translation activity and were more resistant to stress conditions than non-stem cells. Moreover, all these characteristics seem to be associated with the differentiation state of ASCs, at least during adipogenesis. Financial Support, CNPq, Fiocruz.

Poster Session 2: RNP Structure, Function and Biosynthesis

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

327

Abstract Withdrawn

328

Interactions of a Pop5/Rpp1 Heterodimer with the Catalytic Domain of Ribonuclease MRP

Anna Perederina, Elena Khanova, Chao Quan, Igor Berezin, Olga Esakova, Andrey Krasilnikov Penn State Uninersity, University Park, PA, USA Ribonuclease (RNase) P is an essential RNA-based enzyme found in all three domains of life. In the eukaryotes, the RNase P lineage has split into two, giving rise to a closely related enzyme RNase MRP, which has a similar composition and structural organization but a distinct specificity. Bacterial RNase P consists of a large catalytic RNA and a small protein component that is essential for the enzyme’s activity under physiological conditions. Archaeal RNase P is more complex, and in addition to the catalytic RNA component (which is similar to the one in bacteria) contains 4-5 proteins. Yeast RNases P/MRP have an apparent catalytic RNA component resembling that of bacterial and archaeal RNases P, plus 9-10 essential proteins, several of which are homologues of archaeal RNase P proteins. The region of RNase P RNA that is involved in interactions with the single protein component in bacterial RNase P is phylogenetically conserved throughout the three domains of life; however, archaeal RNase P and eukaryotic RNases P/MRP do not have protein components showing any degree of homology to the bacterial protein. Here we show that Pop5, a protein component of yeast RNases P/MRP, forms a heterodimer with the RNase P/MRP protein Rpp1 and interacts with the region of the RNase MRP RNA that corresponds to the part of bacterial RNase P RNA involved in interactions with the bacterial RNase P protein. Phylogenetic and structural analysis of the bacterial RNase P protein and yeast Pop5 show that while these two proteins are structurally distinct and apparently evolutionarily unrelated, the known RNA-binding interface of the bacterial protein has an apparent counterpart in yeast Pop5 where some of the distinctly connected secondary structure elements form similar RNA-binding surfaces. These results support the idea that in RNase MRP (and, by inference, in eukaryotic RNase P as well) the protein component Pop5, acting in a heterodimeric complex with Rpp1, performs a role similar to that of the single protein component in bacterial RNase P.

Poster Session 2: RNP Structure, Function and Biosynthesis

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

329 Non-canonical biogenesis of the catalytic subunit of the Drosophila RNase P- a tRNA processing enzyme

Sathiya Manivannan, Lien Lai, Venkat Gopalan, Amanda Simcox The Ohio State University, Columbus (Ohio), USA In all three domains of life, RNase P is a ribonucleoprotein (RNP) complex that catalyzes removal of the 5’- leader in precursor tRNAs (pre-tRNAs). The RNase P RNA (RPR) is the catalytic subunit responsible for this Mg2+-dependent endonucleolytic cleavage. In silico searches have identified the genes that code for the RPR in several eukaryotes including members of Drosophilidae. Interestingly, the locus of the putative RPR in Drosophila lies within the second intron of the protein coding gene CG1746 that has no apparent functional association with RNase P. The RPR gene is in the same orientation as CG1746 and lacks a typical pol III promoter and terminator in its proximal flanking regions; this observation contrasts with Homo sapiens, Saccharomyces cerevisiae, Caenorhabditis elegans and Danio rerio RPRs that are generated by pol III transcription. The unusual gene structure of Drosophila RPR motivated us to examine if this RPR is generated by a novel biogenesis mechanism involving (i) splicing of the CG1746 mRNA, and (ii) subsequent processing of the excised intron. Towards uncovering and establishing such a novel RPR biogenesis mechanism, we first sought to prove that the annotated gene indeed codes for RNase P. We tested the authenticity of the intronic RNase P RNA (iRPR) through biochemical characterization of the native RNase P holoenzyme isolated from Drosophila cultured cells. Partially purified RNase P, obtained after tandem anion and cation exchange chromatography of a crude lysate, cleaved a pre-tRNAGly substrate accurately compared to in vitro reconstituted Escherichia coli RNase P. Fractions with RNase P activity also contained iRPR as determined by RT-PCR, showing that iRPR co-eluted with the holoenzyme. Moreover, addition of an RNA oligonucleotide, complementary to the iRPR, to the assay reaction inhibited the activity of Drosophila RNase P. Since this evidence supports iRPR as the bona fide Drosophila RPR, we are testing several possible models for its biogenesis. As a first step, we are using a red fluorescent protein (RFP)-based reporter system that carries the second intron of CG1746 to determine if splicing is required for biogenesis of the iRPR and to investigate if all the necessary cis elements are present in the intron. We will present results from these studies designed to elucidate how the mature iRPR is generated from its intronic location.

330 Mouse Cells Expressing Catalytically Inactive Dyskerin show Slow Growth and Unstable Ribosomal RNA

Bai-Wei Gu, Jingping Ge, Jianmeng Fan, Monica Bessler, Philip Mason Children’s Hospital of Philadelphia, Philadelphia, PA, USA Pseudouridine is the most abundant modified nucleotide in ribosomal RNA but its role in ribosome biogenesis and translation is not clear. Specific uridine residues are converted to pseudouridine by H/ACA snoRNPs, the protein dyskerin being the active pseudouridine synthase. In vertebrates dyskerin is also present in telomerase RNA, where it is involved in assembly and stability of the telomerase complex. Thus a highly conserved complex has been co-opted in vertebrates for a different function. It is intriguing to consider whether the pseudouridine synthase activity of dyskerin is required for telomerase function, though at the moment there is no evidence that this is the case. To probe the role of pseudouridine in telomerase and ribosomal RNA of mouse embryo fibroblasts we have replaced the wild type dyskerin gene, Dkc1, with a gene encoding a catalytically inactive molecule, Dkc1D125A. The resulting protein is very unstable but nevertheless supports slow growth and ribosome biogenesis. In Dkc1D125A cells ribosomal RNA precursors are abnormally processed and mature ribosomal RNA is very unstable. Telomerase activity is greatly reduced in Dkc1D125A cells which show increased rates of telomere shortening compared with MEFs containing wild-type dyskerin. However it is not clear if this is due to the low amounts of dyskerin or to the lack of pseudouridine synthase activity. Ribosomal RNA processing is abnormal in Dkc1D125A cells according to the pattern of accumulation of precursors. Ribosomal RNA synthesis is also slightly delayed in the mutant cells. The biggest difference is in the stability of the mature cytoplasmic rRNA molecules. Using a pulse chase experiments very little labeled RNA is present in these cells 20 hours after the labeling whereas in WT cells at the same time there is no detectable loss of label. We conclude that pseudouridine is essential for the longevity of rRNA, most likely by stabilizing its functional conformation.

Poster Session 2: RNP Structure, Function and Biosynthesis

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

331

Single Molecule Studies of Prp24-dependent U4/U6 di-snRNP Formation

332

Novel Function for the Cajal body: Surveillance of SnRNP Assembly

Margaret Rodgers, Ashley Richie , David Brow, Samuel Butcher, Aaron Hoskins University of Wisconsin-Madison, Madison, WI, USA Assembly of large macromolecular machines like the spliceosome involves intricate recycling and regulatory pathways. During spliceosome assembly, 5 small nuclear ribonucleoproteins (the U1 and U2 snRNPs and the U4/U6.U5 tri-snRNP) associate with a pre-mRNA. However, only three of them remain during catalysis (U2, U5, and U6). Thus, recycling of the U4 snRNP to regenerate a U4/U6.U5 tri-snRNP must occur prior to each subsequent round of splicing. The U4 and U6 small nuclear ribonucleoproteins (snRNPs) are first associated with one another in a di-snRNP before joining with U5. The U4 and U6 snRNAs must anneal to form two intermolecular helices, a process which is greatly accelerated by the splicing factor Prp24. Despite the essential role Prp24 plays in eukaryotic gene expression, the kinetic pathway for annealing of U4/U6 is unknown. We have shown that we can prepare and anneal fluorescently labeled U4 and U6 RNAs in an in vitro model system. Utilizing this model system, we will study the mechanism of Prp24 dependent disnRNP formation by single molecule fluorescence resonance energy transfer (smFRET). Our experiments will analyze intramolecular FRET within each snRNA as well as intermolecular FRET between U4 and U6 in the presence and absence of Prp24. In addition, we are developing methods for fluorescently labeling and isolating endogenous Prp24 and U4 and U6 snRNPs from yeast. Using a combination of smFRET and Colocalization Single Molecule Spectroscopy (FRETCosSMoS), we will be able to examine binding events occurring during the annealing reaction and probe the influence of other snRNP components (e.g., the Lsm ring of U6) on Prp24-promoted U4/U6 di-snRNP formation with high spatial and temporal resolution. The combined results from these experiments will significantly extend our understanding of U4/U6 di-snRNP formation in vitro and in vivo.

Ivan Novotny1, Daniel Mateju1, Martin Sveda2, Zdenek Knejzlik2, David Stanek1 1 Institute of Molecular Genetics ASCR, Prague, Czech Republic, 2Institute of Chemical Technology, Prague, Czech Republic Assembly of spliceosomal snRNPs and their incorporation into the spliceosome has been well described. However, the fate of misfolded snRNPs and the molecular system that would prevent their integration into the splicing machinery has not been identified. Here, we provide evidence that incompletely assembled snRNPs are sequestered in a nuclear structure called the Cajal body. We interfered with different stages of snRNP assembly (e.g. U5 snRNP maturation or tri-snRNP formation) and in all cases observed retention of immature snRNP complexes in Cajal bodies. Moreover, inhibition of tri-snRNP assembly induced formation of Cajal bodies in the cell line that normally lacked visible Cajal bodies. Cells lacking coilin and thus devoid of functional Cajal bodies were not able to sequester immature snRNPs and were more sensitive to inhibition of the snRNP assembly pathway than cells containing Cajal bodies. Next, we identified the protein that specifically targets tri-snRNP components to Cajal bodies. Depletion of this protein reduced retention of incomplete snRNPs in Cajal bodies. The protein interacted with the Cajal body marker coilin and individual tri-snRNP components but not with complete tri-snRNP, which provided a mechanistic explanation how immature snRNP complexes were identified and sequestered to Cajal bodies.

Poster Session 2: RNP Structure, Function and Biosynthesis

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

333

A Rapid Synergistic Approach for Determining RNA-Binding Domain Structure

Rebecca Taurog1, John Johnson1, James Williamson1, Blair Szymczyna2 1 The Scripps Research Institute, La Jolla, CA, USA, 2Western Michigan University, Kalamazoo, MI, USA NMR spectroscopy and X-ray crystallography are currently the two most widely applied methods for the determination of macromolecular structure at high resolution, but both techniques still suffer from time-consuming bottlenecks. Recently, significant advances in the algorithms for de novo prediction of protein structure have been made, but only in a few, favorable cases do the predicted models agree well with experimentally determined structures. A combination of these methods, which takes advantage of the most accessible aspects of each structural technique, can dramatically accelerate the structure determination process. This effective, overall strategy for rapidly determining the molecular structure of proteins through the synergistic combination of NMR spectroscopy, de novo structure prediction and X-ray crystallography will be presented. My laboratory is exploiting this combinatorial approach and other biophysical techniques to investigate the structure and function of domains involved in the alternative splicing of RNA and RNA virus structure and replication.

334 The Post-Transcriptional trans-Acting Regulator, TbZFP3, Coordinates Transmission-Stage Enriched mRNAs in Trypanosoma brucei

Pegine Walrad, Paul Capewell, Katelyn Fenn, Keith Matthews Centre for Immunity, Infection and Evolution, University of Edinburgh Post-transcriptional gene regulation is essential to eukaryotic development. This is particularly emphasized in trypanosome parasites where genes are co-transcribed in polycistronic arrays but not necessarily co-regulated. The small CCCH protein, TbZFP3, has been identified as a trans-acting post-transcriptional regulator of Procyclin surface antigen expression in Trypanosoma brucei. To investigate the wider role of TbZFP3 in parasite transmission, a global analysis of associating transcripts was carried out. Examination of selected transcripts revealed their increased abundance through mRNA stabilization upon TbZFP3 ectopic overexpression, dependent upon the integrity of the CCCH zinc finger domain. Reporter assays demonstrated that this regulation was mediated through 3’UTR sequences for a subset of target transcripts. Global developmental expression profiling of the cohort of TbZFP3-selected transcripts revealed their significant enrichment in transmissible forms of the parasite. Immunofluorescent assays demonstrate that TbZFP3 colocalizes with P bodies in starvation granules, and with a subset of procyclin transcripts in transmissible stage parasites. This analysis of the specific mRNAs selected by the TbZFP3mRNP provides evidence for a developmental regulon with the potential to stabilize and coordinate genes important in parasite transmission.

Poster Session 2: RNP Structure, Function and Biosynthesis

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

335

Roles of Ribosomal Proteins in Assembly of Yeast 60S Subunits In Vivo

336

Secondary structure fold of the human U2-U6 snRNA complex.

John Woolford1, Jelena Jakovljevic1, Michael Gamalinda1, Jesus de la Cruz3, Reyes Babiano3, Philipp Milkereit2, Jan Linneman2, Uli Ohmayer2 1 Carnegie Mellon University, Pittsburgh,PA USA, 2Universitat Regensburg, Regensburg,Germany, 3 Universidad de Sevilla, Sevilla, Spain Ribosome assembly involves alternating steps of folding and processing of pre-rRNA in concert with binding of ribosomal proteins. To systematically investigate the roles of ribosomal proteins in assembly of the 60S subunit, we are depleting each protein and assaying pre-rRNA folding, processing, and turnover, and association of ribosomal proteins and assembly factors with pre-ribosomes. Phenotypes fall into three general classes: apparent blocks in early, middle, or late steps in pre-rRNA processing, as indicated by increased amounts of “upstream” pre-rRNAs and decreased amounts of “downstream” pre-rRNAs. These phenotypes correlate with the location of the ribosomal proteins in mature 60S subunits. Those proteins important for early steps are associated primarily with domains I or II of rRNA in a belt around the equator on the solvent-exposed surface of the subunit. Those important for middle steps are located mostly near the “bottom” third of the subunit. The proteins important for late steps of processing and nuclear export are located near the “top” and on the subunit interface. More detailed investigation of two proteins in the first phenotypic class, L7 and L8, reveal that in their absence nearby ribosomal proteins in domains I or II cannot assemble nor can six assembly factors previously implicated in processing of 27SA3 pre-rRNA. Consequently, pre-ribosomes are degraded before they can complete processing of 27SA3 pre-rRNA. We hypothesize that L7 and L8 are important to establish RNP structures within nascent ribosomes necessary for stable assembly of domains I and II, prior to removal of ITS1 sequences from 27SA3 pre-rRNA. Investigation of three proteins from the second class, L17, L35, and L37, reveal that in their absence the two assembly factors Nsa2 and Nog2 required for processing of 27SB pre-rRNA cannot assemble. The structure of the ITS2 spacer that is removed in this step does not appear to be affected. Nevertheless, these abortive assembly intermediates are turned over, although more slowly than those in the first phenotypic class.

Ravichandra Bachu1, Joerg Schlatterer2, Michael Brenowitz2, Nancy Greenbaum1 1 The Graduate Center and Hunter College, CUNY, New York, NY, USA, 2Albert Einsterin College of Medicine, Bronx, NY, USA The complex formed between U2 and U6 small nuclear (sn)RNAs is implicated in a critical role in precursor messenger (pre-m) RNA splicing. Several lines of evidence from other investigators suggest that the U2-U6 complex undergoes conformational rearrangement between the two steps of splicing. In vivo studies on yeast U2-U6 snRNA complex identified a three-way junction structure as catalytically important. In vitro structural studies on the same complex in its protein free state have shown that the complex can form two different secondary structure folds: one model with a four-way junction and another with a three-way junction depending upon the sequences and Mg2+ ion concentration. However, mutational studies on human U2-U6 snRNA complex identified the four-way junction structure as catalytically important. Because of the differences in sequence and variable resistance to the mutations of the yeast and human U2-U6 snRNA complex, it is possible that the human and yeast U2-U6 snRNA complexes have different lowest energy structures in the proteinfree state. In this work, we used enzymatic structure probing to evaluate the secondary structural fold of protein-free human U2-U6 snRNA complex. Cleavage patterns resulting from probing reactions were consistent with formation of four stem regions surrounding the junction, therefore favoring the four-helix junction model consistent with the in vivo studies on human U2-U6 snRNA complex. Upon addition of up to 100 mM Mg2+, a slight increase in cleavage by enzymes specific for both single-stranded and double-stranded regions was observed at the junction region, suggesting that this region is becoming more accessible to the enzymes. Analytical ultracentrifugation experiments revealed a small amount of compaction of the tertiary structure in the presence of Mg2+. Therefore, it appears that Mg2+ may contribute to tertiary structure. These results act as a good starting point to characterize further, the effects of spliceosomal proteins on the conformation of human U2-U6 snRNA complex.

Poster Session 2: RNP Structure, Function and Biosynthesis & Splicing Mechanisms

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

337 Determining the molecular mechanism underlying splicing factor related Retinitis Pigmentosa

Deepti Bellur, Jonathan Staley The University of Chicago, Chicago (IL), USA Retinitis pigmentosa (RP) is one of the most common inherited causes of blindness. While many RP genes are specific to the eye, mutations causing the autosomal dominant form of RP (adRP) are also found in genes involved in the ubiquitous pre-mRNA splicing process. Previous studies suggested that RP mutations may cause a defect in U4/U6.U5 triple snRNP assembly. However, our studies and those of others have shown that several RP factors in the triple snRNP mediate and/or regulate unwinding of U4/U6 snRNAs, a key transition during spliceosome activation, suggesting that adRP may instead or additionally derive from a defect in U4/U6 unwinding. We recently published an analysis of two missense mutations in the SNRNP200 gene co-segregating with the RP phenotype in affected families. The SNRNP200 gene encodes human Brr2, the U4/U6.U5 snRNP component that catalyzes U4/U6 unwinding. The altered residues, S1087L and R1090L, reside in the “ratchet” helix of the first Sec63 domain implicated in the directionality and processivity of nucleic acid unwinding. We observed marked defects in U4/ U6 unwinding in budding yeast for the analogous mutations (N1104L and R1107L). Additionally, the R1107L (R1090L in human) mutation, which leads to a much earlier onset of RP, leads to an accumulation of U1 and U4 snRNAs, implicating a delay in spliceosome activation as a result of this mutation. Thus, the severity of the RP disease phenotype correlated with the severity of the unwinding defect for each of these Brr2 mutations. Furthermore, adRP mutations have been identified in other essential splicing factors that are also part of the triple snRNP - Prp8, Prp6 and Prp31 - of which Prp6 and Prp31 dissociate upon spliceosome activation. The linkage of these triple snRNP factors to adRP suggests that the mechanism of pathogenesis for splicing-factor related RP may derive from a defect in hBrr2-dependent U4/U6 RNA unwinding and a consequent defect in spliceosome activation. We are currently investigating additional RP-causing mutations in Brr2 and Prp8 to test this hypothesis.

338 Effects of Destabilizing the U4/U6 Complex Are Suppressed by a Mutation That Alters the U2/U6 Complex Jordan Burke, Dipali Sashital, David Brow, Samuel Butcher University of Wisconsin, Madison, WI, USA

Interactions between the U2, U4 and U6 snRNA molecules are central to spliceosome assembly and catalysis. U6 snRNA forms mutually exclusive interactions with U4 and U2 snRNA that are essential to spliceosome function. A mutation in yeast U4 RNA, U4G14C, that weakens an intermolecular base pair in U4/U6 Stem II results in cold-sensitive growth and destabilizes the U4/U6 complex in vitro (1) . This phenotype is suppressed by U6-A91G, which surprisingly maps far from the disrupted U4/U6 helix and does not alter the stability of the isolated U4-G14C/U6 complex (1) . Thus, U6-A91G suppresses the U4/U6 pairing defect by an indirect mechanism. We have found that the U6-A91G mutation destabilizes the U6 internal stem loop (ISL) in the context of the U2/U6 complex. An 83-nt RNA that contains U2/U6 intermolecular Helices I and II as well as the U6 ISL exhibits base-pairing in all three regions as determined by NMR spectroscopy. However, the presence of the A91G mutation in this RNA results in disruption of the U6 ISL due to formation of an extended U2/U6 Helix II. Formation of the U6 ISL in the U2/U6-A91G construct can be rescued by introducing a stabilizing mutation in the U6 ISL, U6-A62G. In the absence of the A91G mutation, the A62G mutation results in an extended U6 ISL and decreased levels of U4/U6 complex in vivo (2) . We show that the cold-sensitive growth defect of a yeast strain containing the U6A62G mutation is suppressed by the U6-A91G mutation. The U6-A62G/A91G strain also contains in vivo levels of U4/U6 complex more similar to wild type. Combination of U6-A62G with either of the U4/U6 destabilizing mutations, U4-G14C (1) or G14U (3), is synthetic lethal; however, this phenotype can be suppressed through incorporation of U6-A91G. Surprisingly, these triple mutants do not contain detectable levels of U4/U6 complex in deproteinized total cellular RNA, despite being viable at all temperatures tested. We are currently testing if these strains are able to bypass U4/U6 complex formation entirely. Given that the U6 ISL is mutually exclusive with U4/U6 stem II, our results suggest that U6-A91G may act as a suppressor by restoring the equilibrium between U4/U6 and U2/U6 pairing. The equilibrium may be mediated through U2/U6 Helix II, which forms simultaneously with U4/U6 in human cell extracts (4). This mechanism could explain suppression of U4-G14C cold-sensitivity by U6A91G. Additionally, stabilization of U2/U6 Helix II may bypass the need for stable association of the U4/U6 complex. 1. Shannon, K.W. and Guthrie, C. (1991) Genes Dev. 2. Vidaver, R.M., et al. (1999) Genetics. 3. McManus, C.J., et al. (2007) RNA. 4. Wassarman, D.A. and Steitz, J.A. (1992) Science. Poster Session 2: Splicing Mechanisms

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

339

Unravelling the alternative RNA splicing labyrinth with chemical biological tools

Glenn Burley1, Ian Eperon2, Helen Lewis2, Andrew Perrett2, Rachel Dickinson2 1 University of Strathclyde, 2University of Leicester Pre-mRNA splicing is one of the most important determinants of gene expression. Almost all protein-coding genes contain introns, which affect transcription and export of the mRNA. Most such genes express multiple spliced isoforms of mRNA, about 7 on average, which can differ in their coding potential or stability. Splicing therefore determines in part the level of expression and, above all, determines which mRNA and protein sequences are made. Our research programme aims to understand the mechanistic aspects of splicesite selection in pre-mRNA splicing by developing biophysical as well as chemical biological tools to manipulate key interactions within this process. I will present progress towards this goal by highlighting newly developed molecular tools – both small molecule as well as oligonucleotide-based strategies - in which we have developed disentangle processes leading to splicesite selection. References 1 Owen, N. etal. Design principles for bifunctional targeted oligonucleotide enhancersof splicing. Nucleic Acids Res. 39, 7194-7208, doi:10.1093/nar/gkr152(2011). 2 Cherny, D. et al. Stoichiometry of a regulatory splicing complex revealed bysingle-molecule analyses. Embo Journal29, 2161-2172,doi:10.1038/emboj.2010.103 (2010).

340 AU-rich Elements (AREs) As Intronic Enhancers: A Molecular Mechanism For The Lack Of Iterated AREs In Protein-coding Exonic Regions

Durga Rao Chilakalapudi, Sandip Chorghade Indian Institute of Science, Bangalore, India Since the demonstration of AREs as important modulators of mRNA stability in 1986, a large body of evidence has accumulated on the importance of AREs as determinants of mRNA degradation in a stimulus and cell type-specific manner. AREs are commonly found in the 3’UTRs just upstreanm of the polyadenylation site of mammalian mRNAs, but not observed in protein coding exonic regions. However, the molecular basis for their presence primarily in the 3’ UTR near to the polyadenylation signal in the last exon but not in the protein coding exonic regions remained unexplained to date. During studies on expression of PDGF-B from an expression vector, it was observed that the PDGF-B ORF derived from the cDNA was re-spliced at 4/5 exon junction with the downstream splice acceptor present in the vector. Investigations revealed that a single nonameric AU-rich sequence as well as SR-protein binding sites present in the 66-nt region of the 1.7 kb 3’ UTR present in the construct just downstream of the terminator codon significantly contributed to the re-splicing event. By replacing the 66-nt 3’UTR region present downstream of the translational termination codon of PDGF-B ORF, with the well studied iterated AREs, present in the 3’ UTRs of GM-CSF, c-fos, c-JUN, TNF-a mRNAs, we demonstrate that AREs function as intronic enhacers by activating cryptic splice sites leading to their elimination in the mature mRNA. Different AREs exhibited differences in their ability to function as introninc enhacers. Employing several methods, we further demonstrate specific interactions among AU-BPs and splicing factors. Differential interactions between AUBPs and splicing factors suggest a likely link between pre-mRNA splicing, mRNA transport and mRNA degradation. These studies provide a new direction towards understanding the role of AREs and AU-BPs in alternative splicing and the relative abundance of mRNAs containing AREs of different sequence contexts and the proteins derived from them.

Poster Session 2: Splicing Mechanisms

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

341 The Splicing Factor hnRNP K Inhibits G6PD RNA Splicing in Response to Changing Nutrient Availability

Travis Cyphert, Lisa Salati West Virginia University Splicing of nascent RNA transcripts is a newly described target of nutrient regulation. Glucose-6-phosphate dehydrogenase (G6PD) is a lipogenic gene that has been a paradigm for analyzing the mechanisms for nutrient regulation of splicing. Expression of G6PD mRNA increases 15 to 17 fold during refeeding and is inhibited 80-90% by starvation and/or the addition of polyunsaturated fat to the diet. Starvation and dietary polyunsaturated fatty acids alter pre-mRNA splicing by decreasing the rate of intron removal, leading to intron retention and degradation of the pre-mRNA. A regulatory element within exon 12 of the G6PD pre-mRNA that contains both an ESS and an ESE mediates these changes in splicing efficiency. SR proteins, like SRSF3, along with hnRNP K and L bind to this regulatory region. HnRNP binding is enhanced during conditions that lead to intron retention in vivo. To study the function of these hnRNPs, HepG2 cells that stably express a G6PD reporter containing exon 12 and downstream intron 12 and exon 13 were used for loss of function and gain of function experiments. SiRNA depletion of hnRNP K in HepG2 cells results in an increase in splicing of the RNA reporter while siRNA mediated depletion of hnRNP L had no effect on reporter splicing. Over-expression of hnRNP K results not only in a decrease of reporter expression but also a decrease in the splicing of the reporter. Alternatively, hnRNP K depletion in HepG2 cells that express a G6PD reporter lacking exon 12 resulted in no change in reporter splicing or expression. RNA immunoprecipitation (RIP) has identified that hnRNP K binds to exon 12 of the reporter as well as in the endogenous G6PD mRNA. Binding of hnRNP K to exon 12 was next evaluated by RIP in the livers of mice that had been starved versus starved and then refed. HnRNP K bound 23-fold more to exon 12 of G6PD in livers from mice that were starved than mice that were fed a high carbohydrate diet. Together, this data suggests hnRNP K and not hnRNP L is part of the mechanism of splicing inhibition during changing nutrient availability. (Supported by a grant from NIDDK, DK 046897)

342 Identifying Small-Molecule Inhibitors of Human and Yeast pre-mRNA Splicing by Highthroughput Screening

Kerstin Effenberger, Walter Bray, Rhonda Perriman, Manuel Ares, Melissa Jurica University of California, Santa Cruz, USA The spliceosome, the complex macromolecular machine responsible for pre-mRNA splicing, is highly dynamic and constantly exchanges and rearranges RNA-RNA, RNA-protein, and protein-protein interactions. Currently, there are few methods available to accumulate homogenous population of intermediate splicing complex, a prerequisite for further biochemical, functional, and structural analysis of spliceosomes. To discover new tools to accumulate currently unavailable spliceosome intermediates, we developed a high-throughput assay that identifies splicing inhibitors within large compounds libraries. Our assay examines in vitro splicing reactions with HeLa nuclear extracts in 384-well plates in the presences of candidate inhibitors. We use quantitative RT-PCR to measure the amount of spliced mRNA produced and identify splicing inhibitors by the resulting decrease of splicing efficiency. In a pilot screen, we examined a library of 3,000 bioactive compounds for small molecules that inhibit splicing. We identified three compounds that inhibit splicing in a dose-dependent manner with an IC50 in the low M range. The three inhibitors are structurally unrelated and stall spliceosome assembly at different stages. We find that two of the inhibitors also inhibit splicing chemistry and complex formation in S. cerevisiae extracts. Currently, we are further characterizing the splicing complexes stalled in presence of the inhibitors in both human and yeast systems. Additionally, we are finding that derivatives of the compounds are providing information about the structure-activity relationship of the inhibitors. For example, one compound with a naphthazarin moiety appears to function, in part, through sulfhydryl oxidation that does not affect spliceosome assembly but impairs the 2nd step of splicing chemistry. The splicing inhibitors that we have identified and will continue to identify in expanded screens of small molecule and natural product libraries hold great potential as tools for building a mechanistic understanding of the spliceosome. Furthermore, they promise to aid in deciphering the roles of the splicing machinery in cell physiology and gene expression in vivo. Also, because other inhibitors of splicing have shown cytotoxicity against various cancer cell lines and have in vivo anti-tumor effects, our new inhibitors represent leads for developing additional chemotherapeutics. Poster Session 2: Splicing Mechanisms

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

343 The Structure of ySad1 Suggests a Divergent Zinc Finger Ubiquitin Binding Domain Function. Haralambos Hadjivassiliou, Oren Rosenberg, Christine Guthrie University of California, San Francisco, USA.

Ubiquitin functions additively with the GDP bound state of Snu114 to regulate U4/U6 unwinding in the tri-snRNP (1). Snu114 has strong genetic interactions with Sad1, an essential splicing factor with roles in snRNP assembly and tri-snRNP joining to the spliceosome (2, 3, 4). Sad1 is composed of two domains, a zinc finger ubiquitin binding domain (ZnF UBP) and an inactive ubiquitin specific protease (iUSP) domain. How these domains function with ubiquitin and other spliceosomal factors is unknown. We used splicing microarrays to analyze Sad1 function in vivo and found widespread accumulation of pre-mRNAs, demonstrating Sad1’s universal role in splicing. To gain structural insights into Sad1 function, we determined the crystal structure of full-length Sad1 at 1.8A resolution. We find that the iUSP domain forms the characteristic ubiquitin-binding pocket, with the finger, palm, and thumb features found in the USP family of deubiquitinases. However, in place of an active site cysteine, there is an aspartate residue, and while recombinant Sad1 is active in in vitro splicing assays, it does not show deubiquitinase activity in vitro. The ZnF UBP domain of Sad1 shares high structural similarly to other ZnF UBPs except that the high affinity ubiquitin binding motif appears to be degenerate. Additionally, we have been unable to detect an interaction between recombinant Sad1 and ubiquitin using a variety of biophysical techniques. ZnF UBP domains that have lost their ability to recognize ubiquitin have been found in similar domain topologies with other USPs (5), but it is harder to explain the lack of detectable ubiquitin binding by the iUSP domain, whose ubiquitin binding site appears intact. In the case of the similar but active USP, UBP8, the ZnF UBP domain has evolved to serve as a protein scaffold to recruit activators for its neighboring USP domain (6). Similarly, it is possible that the iUSP domain of Sad1 could recognize ubiquitin and the spliceosome, but that this will be dependent upon ZnF UBP interactions either with activating factors or spliceosomal recruitment factors. Our current efforts focus on identifying the proteins that interact with the ZnF UBP domain and understanding how Sad1’s iUSP domain may interact with a spliceosomally linked ubiquitin, or ubiquitin like substrate. 1. Bellare P, et al. 2008. 2. Brenner TJ, et al. 2005. 3. Lygerou Z, et al. 1999. 4. Makarova OV, et al. 2001. 5. Bonnet J, et al. 2008. 6. Samara NL, et al. 2010.

344

Zooming into the Splicing Cycle: Step by step Dissection of Pre-mRNA Dynamics

Ramya Krishnan1, Mario Blanco1, Joshua Martin2, Matthew Kahlscheuer1, Alain Laederach2, Christine Guthrie3, John Abelson3, Nils Walter1 1 University of Michigan, 2University of North Carolina, Chapel Hill, USA, 3University of California, SF, USA The formation of a mature messenger RNA (mRNA) requires an orchestrated, stepwise assembly of the spliceosome on the pre-mRNA in order to splice the exons and remove the intron. This process, termed splicing, requires about 80 proteins and 5 snRNAs in Saccharomyces cerevisiae. Although the conformational dynamics of the pre-mRNA serves as the signature of this process, and is likely exploited at each step during the assembly, lack of suitable tools has limited its detailed study. We have used single-molecule fluorescence resonance energy transfer (smFRET) to dissect the real-time dynamics of the pre-mRNA at each step of spliceosome assembly by using biochemical methods to stall the splicing at various stages. Using fluorescent labels on the exons and/or introns of the short yeast pre-mRNA Ubc4, we are able to comprehensively measure ATP- and splicing-signal dependent conformational transition kinetics for individual premRNA molecules. We have used sequence alignment and clustering algorithms to help determine the series of FRET states that are unique and enriched at the different steps along the pathway. Additionally, we have focused on the first step of splicing by affinity purifying the catalytically active B* spliceosome assembled on Ubc4 with fluorescent labels at the 5’SS and branch site. Preliminary smFRET analysis of this complex reveals conformational remodeling of the spliceosome when chased to a functional C complex with the stepwise addition of three recombinant proteins, Prp2, Spp2 and Cwc25. Analysis of such a conformationally and compositionally well defined complex allows us to precisely dissect the splicing cycle, and to identify new transitions and rate-limiting steps of the process.

Poster Session 2: Splicing Mechanisms

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

345 High Throughput Discovery of Factors Involved in Complex, Regulated Splicing Pathways in S. pombe

Amy Larson Cornell University, Ithaca, NY, USA Here we describe the development and implementation of a high throughput reverse genetic screen in S. pombe to identify novel factors in the splicing pathway, as well as characterize roles of known factors. Our approach utilizes a library of ~3000 S. pombe strains, each containing a deletion of a single gene. Using robotic methods similar to those recently described for S. cerevisiae (Albulescu etal., PLoS Genetics 2012, in press), we automate cell growth, RNA extraction, cDNA synthesis, then perform high throughput qPCR to directly measure the in vivo levels of different precursor transcripts to identify mutants that show defects in splicing. Because the intron landscape in S. pombe is more diverse than that of S. cerevisiae, and more closely resembles that of higher eukaryotes, it provides an opportunity to examine more complex mechanisms of splicing. Indeed, our lab has recently identified a subset of transcripts in S. pombe that undergo alternative splicing providing an opportunity to investigate how alternative splicing events are regulated mechanistically. After screening several precursor transcripts, we have identified candidates that are novel to the splicing pathway. Some of these proteins have previously annotated functions in other nuclear processes, such as chromatin remodeling and mRNA transport, while others have functions that are unknown. We have performed splicing sensitive microarrays on several of these candidates, and have shown that candidates identified in the screen exhibit either global splicing defects or transcript-specific splicing phenotypes. Some of the candidates also show an enhanced splicing phenotype for several transcripts. We have screened first, middle, and terminal introns within a single precursor transcript in order to identify factors that are specifically required for downstream splicing events. Further biochemical studies of these candidates will help elucidate how complex splicing pathways are regulated in higher eukaryotes.

346 Ensemble and Single Molecule Characterization of Brr2 Helicase Activity on the U4/U6 snRNAs

Sarah Ledoux, John Abelson, Haralambos Hadjivassiliou, Christine Guthrie University of California, San Francisco, USA Pre-mRNA splicing is a dynamic process that requires five snRNPs to bind and release from the spliceosome in an ordered manner. The U6 snRNA enters the splicing cycle as part of the U4/U6-U5 triple snRNP in which it is extensively base paired to the U4 snRNA. Catalytic activation of the spliceosome requires that this U4/U6 base pairing be disrupted to expose U6 snRNA residues that must base pair with the 5’ splice site of the pre-mRNA as well as the U2 snRNA. Unwinding of the U4/U6 helices requires Brr2, a member of the DExD/H family of RNA-dependent ATPases, which is stimulated by the C-terminus of Prp8 [1]. The Brr2 protein has a tandem repeat of helicase domains, although only the first helicase domain has the canonical residues in the ATP and RNA binding motifs. The lengthy base pairing interaction between U4 and U6 suggests Brr2 must act processively to unwind them. However, there is currently little mechanistic detail describing how Brr2 unwinds the U4/U6 snRNAs. We are performing a biochemical characterization of the Brr2 helicase activity using a minimal in vitro system. Single turnover ensemble helicase assays demonstrate that the C-terminal fragment of Prp8 increases both the apparent rate of U4/U6 unwinding and the overall fraction of U4/U6 snRNAs unwound. However, the Prp8 fragment does not increase the affinity of Brr2 for U4/U6. The rate of the Brr2 helicase reaction is highly dependent upon the monovalent cation concentration, decreasing exponentially as the concentration of salt increases. This is typical of helicase reactions since the duplex affinity increases with increasing monovalent cation while the protein’s affinity for the RNA decreases. Mutations within the putative “unwinding ratchet” of Brr2 result in a lower extent of total U4/U6 snRNAs unwound. We predict that these mutations decrease the processivity of Brr2 and result in more aborted attempts at unwinding. Using the conditions established in our ensemble assays, we are now performing single molecule Forster Resonance Energy Transfer (smFRET) assays to further interrogate the mechanism of Brr2 helicase activity and its stimulation by Prp8. 1. Maeder, C., A.K. Kutach, and C. Guthrie, ATP-dependent unwinding of U4/U6 snRNAs by the Brr2 helicase requires the C terminus of Prp8. Nat Struct Mol Biol, 2009. 16(1): p. 42-8. Poster Session 2: Splicing Mechanisms

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

347

Compositional analysis of the pre-spliceosome intermediates associating with Prp5 protein

348

The Role of Saccharomyces cerevisiae Prp5 in Splicing Fidelity Control

Sujin Lee, Scott Stevens University of Texas at Austin, Austin, TX, USA Pre-mRNA splicing is the process by which introns are removed from the primary transcript and exons are joined, giving rise to mature and translatable transcripts. The splicing process is achieved by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNAs (snRNAs), U1, U2, U4, U5, and U6 snRNAs, >150 proteins associating with these snRNAs (snRNP proteins), and non-snRNP protein factors. During the splicing cycle, the spliceosome undergoes multiple RNA-RNA and RNA-protein rearrangements facilitated by RNA helicases belonging to the DExD/H box protein family. In Saccharomyces cerevisiae, eight different RNA helicases of this family are required for the splicing process. Among these RNA helicases, Prp5p is known to be essential for the formation of prespliceosome (U1/U2/pre-mRNA), the first ATP-dependent step of the splicing cycle. Although it is known that Prp5p recruits U2 snRNP to the commitment complex (U1/pre-mRNA) by enabling the basepairing between U2 snRNA and the branchpoint region in pre-mRNA, the mechanism of how Prp5p performs its role is not fully understood yet. To map how Prp5p is involved in the prespliceosome formation, we propose a compositional analysis of the spliceosome intermediates at the time of Prp5p’s function. We screened conditionally defective prp5 mutants, some of which were shown to have first step splicing defects, and isolated the spliceosome intermediates associating with WT Prp5p and cold-sensitive prp5p, respectively. MuDPIT mass spectrometry analysis of the spliceosome intermediates revealed that prp5p associates with numerous protein components of U1 and U2 snRNPs as well as U4/U6·U5 components and the Prp19p-complex.

Wen-Wei Liang, Soo-Chen Cheng Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan The DEAD-box RNA helicase Prp5 is required for U2 activation and for prespliceosome formation. Recently, a role of Prp5 in splicing fidelity control has been demonstrated using Prp5 SAT mutants, which improve the splicing of branch site mutation, U257C, in vivo. However, the mechanism underlying Prp5 mediated-splicing fidelity control is poorly understood. To understand how Prp5 affects the splicing of U257 mutants, we analyzed the splicing reaction using the in vitro system. Splicing reactions were carried out with mutated actin pre-mRNA, followed by immunoprecipitation of the spliceosome. We found that the spliceosome was mainly arrested in the stage after U2 association, and, interestingly, Prp5 could associate with the spliceosome assembled on U257 mutant pre-mRNA but not on wild-type pre-mRNA. The uncharacterized Prp5-associated spliceosome that assembled on the branch site mutant pre-mRNA is assigned as Prp5associated (FA, Prp Five-associated) spliceosome. Investigation of FA spliceosome revealed that Prp5 and U2 required each other for stable association with the spliceosome, and U2 snRNA directly interacted with Prp5. Only a fraction of U2-associated spliceosome contained Prp5, and the level of FA spliceosome was inversely proportional to the splicing efficiency, suggesting that Prp5 stabilization on the spliceosome might impede tri-snRNPs recruitment. Moreover, the tri-snRNP-associated spliceosome appealed to contain no Prp5, and vice versa. Our results suggest that the U257 mutants spliceosome may be impeded for Prp5 release, leading to the block of tri-snRNPs recruitment.

Poster Session 2: Splicing Mechanisms

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

349 RNA-binding Protein GPKOW Interacts with Spliceosomal DExD/H-box Protein DHX16/ hPRP2 and is Required for Pre-mRNA splicing

Shengbing Zang1,2, Ting-Yu Lin1,3, Ren-Jang Lin1 Beckman Research Institute of City of Hope, Duarte, California, 2Fujian Medical University, Fuzhou, China, 3 National Taiwan University, Taipei, Taiwan Human GPKOW protein contains a glycine-rich G-patch domain and two KOW domains, and is a homologue of Arabidopsis MOS2 and yeast Spp2 protein. GPKOW is associated with human spliceosomes, but its involvement in splicing has not been elucidated. In the report, we showed with purified components that GPKOW interacts directly with the DHX16/hPRP2 protein and with RNA. Immuno-depletion of GPKOW from HeLa nuclear extracts abolished in vitro pre-mRNA splicing and adding back recombinant GPKOW restored splicing. GPKOW with mutations at a conserved G-patch motif lost DHX16 interaction, and GPKOW with mutations at a conserved KOW1 motif lost RNA interaction; both mutant proteins complemented depleted extract less efficiently. In addition, overexpression of GPKOW alleviated the inhibition of pre-mRNA splicing by DHX16 mutants in HEK293 cells. Our results indicated that GPKOW, and perhaps MOS2 as well, is a bona fide pre-mRNA splicing factor and a functional orthologue of yeast Spp2.

1

350

Identification of Critical Residues in the Pseudo-Helicase Domain of Brr2

Corina Maeder1, William Boswell1, Christine Guthrie2 1 Texas State University-San Marcos, San Marcos, TX, USA, 2University of California, San Francisco, California, USA In pre-mRNA splicing, the dynamic interactions and exchange of the splicing factors are facilitated by eight DExD/H-box ATPases. Brr2, one of these ATPases, functions to ensure that the spliceosome is catalytically active by facilitating the exchange of U4 for U2 in the U4/U6 duplex. During spliceosome disassembly, Brr2 then unwinds the U2/U6 duplex to allow for snRNP recycling. Because Brr2 functions at two steps during the splicing cycle and is an integral part of the U5 snRNP, it must be tightly regulated to ensure its accuracy. Previous work has identified the interactions with Prp8 and Snu114 as regulating factors of Brr2 (reviewed in (1)). However, the precise mechanism of this regulation is still unclear and specific interactions amongst Brr2, Prp8, and Snu114 are still elusive. Brr2 has a unique domain structure consisting of an uncharacterized N-terminal domain, followed by two helicase domains that are suggested to resemble the processive DNA helicase Hel308 (2,3). The first helicase domain has canonical DEIH motifs and had been implicated in the unwinding function of Brr2 (4). The second helicase domain, or pseudo-helicase domain, lacks key canonical DExD/H residues, and mutational analysis has suggested that this domain does not function to unwind the RNA duplexes (4). However, the pseudo-helicase domain is conserved, suggesting that it plays an important functional role. We sought to further understand the functional importance of Brr2’s pseudo-helicase domain. Using Saccharomyces cerevisiae, we created and screened an exhaustive library of brr2 mutations in the second pseudohelicase domain for both ts and cs phenotypes. From this screen, we have identified critical residues in a putative ATP binding pocket of the pseudo-helicase that results in both ts and cs phenotypes. These findings suggest a functional importance for the ATP binding region of this domain. 1. Hahn, D., and Beggs, J. D. (2010) Biochem Soc Trans 38, 1105-1109 2. Pena, V., Jovin, S. M., Fabrizio, P., Orlowski, J., Bujnicki, J. M., Luhrmann, R., and Wahl, M. C. (2009) Mol Cell 35, 454-466 3. Zhang, L., Xu, T., Maeder, C., Bud, L. O., Shanks, J., Nix, J., Guthrie, C., Pleiss, J. A., and Zhao, R. (2009) Nat Struct Mol Biol 16, 731-739 4. Xu, D., Nouraini, S., Field, D., Tang, S. J., and Friesen, J. D. (1996) Nature 381, 709-713 Poster Session 2: Splicing Mechanisms

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

351

Role of U2 snRNA stem IIb in Spliceosome Function

352

Identification of an Early ATP-Independent Role for Prp28 in Spliceosome Assembly

Alberto Moldon, Charles Query Albert Einstein College of Medicine, Bronx, (New York), USA Most eukaryotic genes contain introns that are removed post/co-transcriptionally to join the exons and form a mature mRNA. The machinery responsible for this process is the spliceosome, a RNP complex highly dynamic in composition and structure. After various rearrangements, the snRNPs U2, U5 and U6 form the core of the spliceosome. There has been extensive genetic and structural research to elucidate the interactions of the core. Previous studies reported structures representative of different points of the pathway, such as the formation of the branch-site loop when U2 recognizes the intron, toggling from U2 stem IIc to IIa during transitions between more stable steps, or the U2-U6 interactions in helices I, II and III to form the first catalytic reaction core. Elucidation of other secondary structures and interactions has been complicated because these interactions can be weak or transient. U2 snRNP is implicated in important roles along the assembly, catalysis and product release, but its conformational rearrangements are still not well understood. Here we show that stem IIb of U2 snRNA is important in multiple points of the splicing pathway. We combined U2 stem IIb mutants (shorter stem) with different ACT1-CUP1 reporters and we found that stem IIb mutants enhance defects of branch-flanking mutants, suggesting a role in assembly. This mutant also affects first and second step defective reporters, suggesting additional roles. Moreover, we found genetic interactions between U2 stem IIb and the ATPase/helicase Prp5. Combinations of stem IIb and Prp5 mutants were synthetically lethal. To further understand this phenotype, we screened for U2 mutants that, while having a short stem, suppress the synthetic lethality with Prp5 mutants. We obtained U2 alleles that resemble the length, shape and stability of the WT structure, consistent with the phylogenetic conservation of stem IIb structure but not sequence. The strongest suppressor, in addition to restoring viability, improves splicing of first- and second-step defective reporters compared to WT U2. We also show that stem IIb may be important for tertiary interactions with U2/U6 helix Ia. This study provides insights in the role of stem IIb of U2 snRNP in multiple events during the splicing pathway. Our hypothesis is that stem IIb interacts with the U2-U6 snRNA core and modulates splicing by tightening or relaxing the catalytic core, facilitating or impairing transitions between consecutive conformations.

Argenta Price, Christine Guthrie University of California, San Francisco, (CA), USA The spliceosome is a macromolecular machine that must identify and excise introns with single nucleotide precision. This is accomplished by a cascade of recognition and rearrangement events, catalyzed by members of the DExD/Hbox family of ATPases during spliceosome assembly, catalysis, and disassembly. According to the canonical model of spliceosome assembly, the U1 snRNP first binds the 5’ss, forming commitment complex 1, then BBP and Mud2 bind the branchpoint, forming commitment complex 2 (CC2). In the first ATP-dependent step of splicing, the U2 snRNP binds the branchpoint and displaces BBP and Mud2. Next, the U4/U6-U6 tri-snRNP associates. DEAD-box protein Prp28 then promotes the ATP-dependent switch from U1 to U6 snRNA bound to the 5’ splice site, releasing U1, and Brr2 promotes the ATP-dependent release of U4, allowing U6 to base-pair with U2. These activities form the U2/U6-U5 spliceosome, competent for activation. While previous work showed that Prp28 promotes the release of U1, it also showed that the cold-sensitive prp28-1 allele may prevent stable tri-snRNP association (Staley and Guthrie 1999). We further investigated the prp28-1 defect in order to determine how Prp28 affects tri-snRNP association. To identify the precise defect in the prp28-1 mutant, we monitored the kinetics of spliceosome assembly on biotinylated pre-mRNA, and unexpectedly found that U2 association, in addition to U5 and U6 association, is reduced in prp28-1 extracts. In addition, we monitored commitment complex abundance on native gels, and found that prp28-1 reduces the amount of CC2 that forms in reactions lacking ATP, well before Prp28 was previously thought to act. This defect in CC2 accumulation correlates with a decrease in formation of the U1-U2 pre-spliceosome upon addition of ATP, which could also explain the reduction in U2 and tri-snRNP associated with biotinylated pre-mRNA. We are currently testing how adding purified Prp28 protein, or Prp28 protein with mutations in ATP binding and hydrolysis motifs, affects CC2 formation in Prp28-depleted extracts. We propose that Prp28 plays two roles during spliceosome assembly: one ATPindependent role in which Prp28 helps recruit or stabilize BBP/Mud2 on the pre-mRNA, and a subsequent ATP-dependent role in which Prp28 facilitates the release of U1 following tri-snRNP association.

Poster Session 2: Splicing Mechanisms

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

353 A Conserved Serine of HnRNP L Mediates Depolarization-Regulated Alternative Splicing of Potassium Channels

Aleh Razanau1, Guodong Liu1, Yan Hai1, Jiankun Yu1, Muhammad Sohail1, Vincent Lobo1, Jiayou Chu2, Sam Kung1, Jiuyong Xie1 1 University of Manitoba , 2Chinese Academy of Medical Sciences and Peking Union Medical College In vertebrates, the Slo1 gene transcripts undergo extensive alternative splicing to generate diverse isoforms of BK channels that contribute to the membrane repolarization and after hyperpolarization of action potentials. One of the most intensively studied Slo1 exon is the stress-axis regulated exon (STREX). STREX inclusion is repressed by depolarization and activation of CaM Kinase IV through the CaM kinase responsive RNA element the 3’ splice site of STREX. However, proteins that mediate this regulation and how they are affected by CaMKIV are not known. Here we show that the heterogeneous nuclear ribonucleoprotein L (hnRNP L) is required for the depolarization-induced repression of STRXE inclusion. A highly conserved CaMKIV target serine (S513) of hnRNP L is critical for this regulation. Ser513 phosphorylation within the RNA recognition motif 4 (RRM4) enhanced hnRNP L interaction with CaRRE1 and inhibited binding of the large subunit of the U2 auxiliary factor U2AF65 to the upstream polypyrimidine tract. These data thus provide a physical link between CaMKIV and U2AF65, a critical component of early spliceosome assembly, for depolarization to control the variant subunit composition of potassium channels.

354

Structural basis for regulation of Brr2 incorporation into U5 snRNP by the Aar2 protein

Gert Weber1, Vanessa Cristão2, Flavia de L. Alves2, Karine Santos1, Nicole Holton1, Juri Rappsilber2, Jean Beggs2, Markus Wahl1 1 Free University of Berlin, Laboratory of Structural Biochemistry, Berlin, Germany, 2Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK Small nuclear ribonucleoprotein particles (snRNP) are major subunits of several eukaryotic RNA processing machineries, in particular of spliceosomes. The snRNPs comprise similar core structures, consisting of a set of seven Sm or LSm proteins bound in a ring-like fashion to a uridine-rich Sm site in the small nuclear RNAs (snRNAs), and a varying number of particle-specific proteins. Specialized molecular machineries guide the assembly of the Sm core RNPs in higher eukaryotes. However, presently little is known about when, where and how the specific proteins are incorporated. The particle-specific proteins of the U5 snRNP include Prp8, generally considered the master regulator of the spliceosome, and Brr2, an unconventional RNA helicase essential for RNP remodeling during spliceosome assembly and disassembly. U5 snRNP in yeast is assembled via a cytoplasmic precursor form that contains Prp8 but lacks Brr2. ‘Instead, pre-U5 snRNP includes the Aar2 protein, which is not found in mature U5 snRNP or in the spliceosome [1,2]. Previously, we have shown that Aar2 and Brr2 bind, respectively, to an RNase H-like and a Jab1/MPN-like domain in the C-terminal region of Prp8 [3]. Although these domains are connected by a long, flexible linker, Aar2 hinders binding of Brr2 to Prp8. However, the precise mechanism of this competition is not fully understood. We have now determined a crystal structure of Aar2 in complex with both C-terminal domains of Prp8. In the structure, Aar2 establishes a primary contact to the Prp8 RNase H domain via its globular body and uses a C-terminal extension to tie the Jab1/MPN to the RNase H-like domain. Perhaps surprisingly, the C-terminal tail of the Jab1/MPN domain, which represents a binding site for Brr2, remains available in the complex. These findings suggest that, to exclude Brr2, Aar2 either occupies another essential Brr2 interaction region in Prp8 or locks the Jab1/MPN and RNase H domains in an orientation that is incompatible with Brr2 binding. Our results further specify the function of Aar2 as a U5 snRNP assembly chaperone that safeguards against premature macromolecular interactions. References [1] Boon, KL, Grainger, RJ, Ehsani, P, Barrass, JD, Auchynnikava, T, Inglehearn, CF, and Beggs, JD. 2007. prp8 mutations that cause human retinitis pigmentosa lead to a U5 snRNP maturation defect in yeast. Nat Struct Mol Biol 14, 1077-1083. [2] Gottschalk, A, Kastner, B, Lührmann, R, and Fabrizio, P. 2001. The yeast U5 snRNP coisolated with the U1 snRNP has an unexpected protein composition and includes the splicing factor Aar2p. RNA 7, 1554-1565. [3] Weber, G., Cristao, V. F., Alves, F. L., Santos, K. F., Holton, N., Rappsilber, J., Beggs, J. D., Wahl, M. C. 2011. Mechanism for Aar2p function as a U5 snRNP assembly factor. Genes Dev. 25, 1601-1612. Poster Session 2: Splicing Mechanisms

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

355

Branched Spliceosome Assembly Pathway Revealed by Single Molecule Microscopy

Inna Shcherbakova1,3, Aaron Hoskins2, Larry Friedman3, Victor Serebrov1, Jeff Gelles3, Melissa Moore1,4 1 University of Massachusetts Medical School, Department of Biochemistry and Molecular Pharmacology, Worcester, MA, USA, 2University of Wisconsin-Madison, Department of Biochemistry, Madison, WI, USA, 3 Brandeis University, Department of Biochemistry, Waltham, MA, USA, 4Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA, USA Excision of introns from pre-mRNAs is carried out by the spliceosome, a multimegadalton machine composed of five small nuclear ribonucleoprotein particles (U1, U2, U4, U5 and U6 snRNPs), the protein-only Prp19 complex (NTC) and numerous transiently interacting splicing factors. For each round of splicing, an active spliceosome must be assembled so that precise and timely intron excision and exon ligation can occur. Ensemble experiments following the appearance and disappearance of stable complexes on a few well-studied splicing substrates have yielded a general understanding of the order of events involved in spliceosome assembly. According to the current model, U1 snRNP first recognizes the 5’ splice site followed by U2 snRNP binding the branch point region; subsequent joining of U5/U4¿U6 tri-snRNP is closely followed by the NTC. Using Colocalization of Single Molecules Spectroscopy (CoSMoS) we directly observe in whole cell extract the real-time dynamics of association of U1, U2 and the tri-snRNP labeled with fluorophores of different colors with surface-tethered pre-mRNA molecules. We find that binding of U1 and U2 snRNPs is not necessarily ordered. For one of the natural S. cerevisiae pre-mRNAs, RPS30A, U1->U2 and U2->U1 binding orders occurred with nearly equal frequencies, suggesting a branched pathway for U1/U2/pre-mRNA complex formation. The U1/U2/premRNA complexes formed on RPS30A by either assembly pathway are equally functional for subsequent tri-snRNP recruitment. Furthermore, a CoSMoS experiment on RPS30A pre-mRNA with fluorophore-labeled intron showed that spliceosomes assembled via the U1->U2 and U2->U1 pathways excised the intron with equal efficiencies. Taken together, our data suggest that the two assembly pathways are equally likely on RPS30A pre-mRNA, and that the resulting U1/ U2/pre-mRNA complexes are functionally identical. This branched assembly pathway could provide additional means for regulation of gene expression at the level of spliceosome assembly.

356 The U2AF35-related protein Urp contacts the 3’ splice site to promote U12-type intron splicing and the second step of U2-type intron splicing

xuexiu zheng zheng1, haihong shen2 Gwangju institute of Science and technology, 2gwangju institute of science and technology The U2AF35-related protein Urp has been implicated previously in splicing of the major class of U2-type introns. Here we show that Urp is also required for splicing of the minor class of U12-type introns. Urp is recruited in an ATP-dependent fashion to the U12-type intron 3’ splice site, where it promotes formation of spliceosomal complexes. Remarkably, Urp also contacts the 3’ splice site of a U2-type intron, but in this case is specifically required for the second step of splicing. Thus, through recognition of a common splicing element, Urp facilitates distinct steps of U2- and U12-type intron splicing.

1

Poster Session 2: Splicing Mechanisms

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

357

Role of a Unique RNA-RNA Long-distance Interaction in Spinal Muscular Atrophy

358

Spliceosome Product Release and Disassembly is Dependent Upon the 3’-end of U6 snRNA

Natalia Singh, Mariah Lawler, Daya Upreti, Ravindra Singh Iowa State University, Ames, Iowa, USA Targeted correction of aberrant pre-mRNA splicing with antisense oligonucleotides (ASOs) offers tremendous therapeutic potential. An ASO-based approach could also be used to uncover “splicing regulators” that cannot be identified by conventional methods. Employing an ASO-based approach, we have recently discovered a unique RNA-RNA longdistance interaction (LDI) that contributes to SMN2 exon 7 skipping (Singh et al., 2010). SMN2 exon 7 skipping is linked to Spinal Muscular Atrophy (SMA), a leading genetic cause of infant mortality. The LDI requires participation of an unpaired cytosine residue at the 10th intronic position (10C). Using overlapping deletions and site-specific mutations, we have now dissected the nature of this LDI. We have established that the inhibitory effect of 10C is dependent on a sequence composed of six residues and located more than 200 nucleotides away from 10C. Our results also support that 10 C engages in formation of a RNA structure within SMN2 intron 7. We show a strong correlation between 10C-mediated structural changes and modulation of SMN2 exon 7 splicing in vivo. Our findings underscore the utility of an immensely powerful approach to capture regulatory long-distance RNA-RNA interactions that drive the dynamic process of exon definition and pre-mRNA splicing. Reference: Singh NN, Hollinger K, Bhattacharya D and Singh RN (2010). An antisense microwalk reveals criticalrole of an intronic position linked to a unique long-distance interaction in pre-mRNA splicing. RNA, 16, 1167-1181.

Rebecca Toroney, Jonathan Staley The University of Chicago, Chicago, (IL) USA After the catalytic phase of pre-mRNA splicing, the spliceosome first releases the mRNA product and then releases the excised intron and disassembles to recycle components, which is essential for subsequent rounds of splicing. Although a number of protein factors that participate in product release and disassembly have been identified, such as the DExD/H-box proteins Prp22, Prp43, and Brr2, as well as Snu114, Ntr1 and Ntr2, the mechanism of disassembly, and in particular the role of snRNA components in this process, remains unclear. In order to investigate roles of snRNA segments at various stages of splicing, we have utilized RNase H to degrade regions of the snRNAs within assembled snRNP particles in order to assess the effects of snRNA truncations on splicing activity independent of effects on snRNP biogenesis. In particular, we observe that cleavage of nucleotides 90-112 from the 3’-end of U6 snRNA, which includes the Lsm binding site and nucleotides involved in U2/U6 helix II in the catalytic conformation, results in a defect in splicing efficiency and, most strikingly, an accumulation of excised intron. This observation is in agreement with a similar effect observed previously by in vitro reconstitution with 3’ truncated U6 snRNA.1 This intron accumulation is indicative of an apparent defect in Prp22-mediated mRNA product release, Prp43/Brr2-mediated intron release and spliceosome disassembly, or intron degradation, suggesting that nucleotides at the 3’-end of U6 snRNA, and, potentially, the associated Lsm proteins or U2/ U6 helix II, may be required for association or activity of various disassembly factors. We find that mRNA release from spliceosomes is unaffected by U6 90-112 cleavage, indicating that Prp22 activity is not dependent upon the Lsm proteins or the 3’-end of U6. By contrast, we find that excised intron release from spliceosomes is affected, which also argues against a trivial defect in intron degradation. Brr2 appears to be required for both U2/U6 unwinding during disassembly and U4/U6 unwinding during spliceosome assembly, but we find that Brr2 unwinding activity of U4/U6 is tolerant to U6 3’-end cleavage, suggesting that Brr2-mediated U2/U6 unwinding is not compromised by U6 3’-end cleavage. These results thus suggest a previously unknown role for the 3’-end of U6 snRNA, U2/U6 helix II, and/or the associated Lsm proteins in enabling the role of Prp43 in intron release and spliceosome disassembly. 1. Ryan, D.E., Stevens, S.W., and Abelson, J. (2002). RNA, 8, 1011-1033. Poster Session 2: Splicing Mechanisms

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

359 Backbone Oxygens in U6 snRNA Position a Catalytic Metal to Stabilize the Leaving Group During Exon Ligation

Nicole Tuttle, Sebastian Fica, Thaddeus Novak, Qing Dai, Jun Lu, Nan-Sheng Li, Jonathan Staley, Joseph Piccirilli University of Chicago, Chicago, IL, USA Because of the ubiquity and importance of pre-mRNA splicing, understanding the catalytic mechanism of splicing is critical for our understanding of eukaryotic gene expression. While extensive work has been done to define the components of the catalytically active spliceosome, the structural and mechanistic details of splicing catalysis are poorly understood. Previous work has shown that the human spliceosome employs a catalytic metal ion to stabilize the leaving group in each chemical step of splicing. Here we employ an approach based on metal ion rescue experiments to demonstrate that catalytic metal ions stabilize the leaving groups during both steps of splicing in yeast. Importantly, we have obtained functional evidence to identify specific atoms in the U6 small nuclear RNA that serve as ligands to these catalytic metal ions. These results represent the first assignment of a specific catalytic role to a particular component of the spliceosome.

360

Detecting Metal Ion Binding Pockets within the Group II Intron ai5γ in vivo

Michael Wildauer, Christina Waldsich Max F. Perutz Laboratories, Vienna, Austria Aside from proteins divalent metal ions are essential for RNA to adopt and maintain their tertiary structure. These cations can interact with RNA either as delocalized ions or they are bound to specific sites, thereby forming a discreet metal ion-pocket. Even though metal ion-binding pockets have been identified for several RNAs (including the Sc. ai5γ intron) in vitro, there is little information on whether Mg2+ binds to similar sites within RNA in vivo and on how proteins shape and alter such metal pockets. Given that efficient splicing of group II introns is closely linked to the ion homeostasis in yeast mitochondria, the Sc. ai5γ group II intron represents a good model system to address this important aspect of RNA structure formation. In addition, Mss116p promotes splicing of the Sc. ai5γ group II intron in vitro at near-physiological conditions, suggesting that it lowers the Mg2+ requirements for RNA folding. Therefore, we recently explored how this protein influences metal ion binding sites within RNA in the cell. In order to map intracellular metal ion binding pockets, a suitable methodology has to be established: in vivo Pb2+-induced cleavage. This technique allowed us to provide the first insights into differences in the metal ion binding sites within RNA in vitro and in vivo as well as to reveal the impact of the protein Mss116p on the formation of specific metal ion binding pockets within the ai5γ intron in vivo. In short, despite the presence of a protein cofactor, which in known to stabilize the intron RNA during folding, we observed that the metal ion binding sites in vivo correlate nicely with those observed in vitro, suggesting that Mss116p does not significantly influence the formation of metal binding pockets in vivo.

Poster Session 2: Splicing Mechanisms

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

361

Capturing DExD/H-box protein Prp28p in action

Fu-lung Yeh, Tien-Hsien Chang Genomics Research Center, Taipei, Taiwan Although DExD/H-box proteins are known to unwind RNA duplexes and/or modulate RNA structures in vitro, it seems plausible that, in vivo, some may function as RNPases to dissociate proteins from RNA or to remodel RNA-protein complexes. Precisely how the latter can be achieved remains unknown. We have been trying to approach this issue by using yeast Prp28p as a model system. Prp28p is an evolutionarily conserved DExD/H-box splicing factor that facilitates the U1/U6 switch at the 5’ splice site (5’ss) during spliceosomal assembly. We have previously shown that Prp28p can be made dispensable in the presence of specific mutations that alter U1C, Prp42p, Snu71p, Cbp80p, and Ynl187p. These data suggest a model that Prp28p counteracts the stabilizing effect by those proteins on U1 snRNP/5’ss interaction. To probe how Prp28p contacts its targets in a splicing-dependent manner, we have strategically placed a chemical cross-linker, ρ-benzoyl-phenylalanine (BPA), along the length of Prp28p in vivo using a nonsense-suppressor-mediated approach. Extracts prepared from these strains were then used for splicing at various ATP concentrations and for UV-activated crosslinking reactions. Prp28p appears to transiently interact with the spliceosome at low ATP concentration, which is known to accumulate A2-1 (or B; mammalian system) complex. Under such a condition, we observed that Prp28p cross-links with a small number of proteins and some of these cross-linked products are dependent on the presence of UV, ATP, RNA, and, importantly, functional 5’ss and branch site. Furthermore, the cross-linked products are resistant to RNase treatment. Using mass-spec and Western blotting analysis, we identified Prp8p as one that physically interacts with Prp28p via the K136 residue. This result is consistent with our genetic analyses showing that Prp28p and Prp8p functionally working in concert. Studies are ongoing to characterize the identity of other proteins, their timing of interaction with Prp28p and their dependency of various splicing components and conditions. We suggest this approach may very well be applicable for studying other DExD/H-box proteins functioning in a physiologically relevant environment, thereby shedding new lights on the longstanding question as to how DExD/H-box proteins act in vivo.

362 Genome-wide in vivo RNA binding sites of key spliceosomal protein Prp8 identified using HITS-CLIP and CRAC

Xueni Li, Wenzheng Zhang, Tao Xu, Jolene Ramsey, Jay Hesselberth, Rui Zhao University of Colorado Denver School of Medicine, Aurora, CO, US Pre-mRNA splicing is an essential step in the eukaryotic gene expression pathway. The splicing reaction is catalyzed by the spliceosome, a huge RNA protein complex that contains 5 snRNAs (U1, U2, U4, U5, U6) and numerous protein factors. Prp8 stands out among hundreds of splicing factors as a key regulator for spliceosome assembly/activation and potentially contributes functional groups to the splicing reaction. Prp8 is one of the largest and most conserved proteins in the nucleus. It is the only spliceosomal protein that extensively cross-links with all key RNA components of the splicing reaction. Numerous Prp8 mutants exacerbate or suppress other splicing mutants that are on the pre-mRNA substrates or on other splicing factors required for spliceosomal assembly/activation. We have performed CLIP (cross-linking and immunoprecipitation) and CRAC (cross-linking and analysis of cDNAs) experiments followed by Solexa deep sequencing to identify the genome-wide in vivo RNA-binding sites of Prp8. Our results revealed the first comprehensive in vivo Prp8 RNA footprints in the entire yeast genome, which contains all the previously known Prp8-RNA cross-linking sites identified by site-directed cross-linking and reveals many new sites. Analyses of mutations in the sequencing reads suggest direct cross-linking sites that may have important implications for the function of Prp8 in splicing.

Poster Session 2: Splicing Mechanisms

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

363

Self-Splicing of Group IIE and Group IIF Introns

Katherine Zhou, Vivien Nagy, Anna Pyle Yale University, New Haven, (Illinois), USA Among the largest known ribozymes in nature, the self-splicing group II introns are a valuable source of information on RNA structure and function. As mobile retroelements with high site-specificity, group II introns also constitute a potentially useful tool for gene therapy. Introns of the recently identified classes E and F are small and have a secondary structure very similar to that of the larger and more prevalent group IIB introns, making them likely candidates for biophysical studies. However, if they are to be used in structural studies or developed for gene therapy, group IIE and IIF introns must first satisfy the basic requirement of being functional introns with the ability to self-splice. To date, they have been identified bioinformatically, but never transcribed in-vitro or examined experimentally for biochemical activity. We find that the group IIE intron Cl.be.I2 from Clostridium beijerinckii exhibits robust splicing activity under a large variety of conditions, although it did not splice very rapidly under any of the conditions tested. Cl.be.I2 exhibited the most efficient splicing at high concentrations of KCl and spliced much more rapidly at 50 degC and 55 degC, respectively, than at 42 degC. The group IIF intron Pe.th.I2 from Pelotomaculum thermopropionicum was found to splice more efficiently than Cl.be.I2 and exhibited its highest reaction rate constant at high concentrations of KCl. Interestingly, Pe.th.I2 formed both lariat and linear intron in the presence of the monovalent salt NH4+, but primarily formed linear intron in the presence of K+. We are currently using mutant Cl.be.I2 and Pe.th.I2 constructs to test whether the putative EBS/IBS interactions are essential for the splicing activity of group IIE and IIF introns.

364 Reconstitution of the Sequential Pathway for 1-methyl Pseudouridine Formation in Archaeal tRNAs

Kunal Chatterjee1, Ian Blaby2, Patrick Thiaville2, Mrinmoyee Majumder1, Henri Grosjean3, Adam Yuan4, Valerie de Crecy-Lagard2, Ramesh Gupta1 1 Southern Illinois University, Carbondale, (Illinois), USA, 2University of Florida, Gainesville, (Florida),USA, 3 Universite Paris 11, IGM,CNRS,UMR 8621, Orsay, France, 4National University of Singapore, Singapore Most Bacteria and Eukarya contain m5U (riboT) at position 54 in the TΨC- loop of their tRNAs. However, majority of Archaea contain different modifications at this position, a common one being methylated pseudouridine (m1Ψ). Pus10 produces Ψ54, the first step in m1Ψ54 formation. Here, we have identified the enzyme responsible for the subsequent N1 methylation of the uracil ring of Ψ54. A potential candidate for the m1Ψ54 methyltransferase came from bioinformatic analyses of genes encoding putative AdoMet-dependent methyltransferases. One such enzyme belonging to COG1901, encompassing an α/β knot fold (also named SPOUT) superfamily of methyltransferases appeared as a valid candidate. Crystal structure of a COG1901 family member, Mj1640 from Methanocaldococcus jannaschii, showed structural similarities to another RNA m1Ψ methyltransferase Nep1, but had critical dissimilarities as well. We tested this candidate by deleting the COG1901 encoding gene, HVO_1989, from the Haloferax volcanii genome. Resulting strain showed only Ψ54, but not m1Ψ54 in its tRNAs. Transformation of this strain with a plasmid-borne copy of the COG1901 encoding gene from Halobacterium sp. NRC1, VNG_1980C, rescued m1Ψ activity. Furthermore, recombinant COG1901 and Pus10 proteins of M. jannaschii together produced m1Ψ54 both in full-size tRNA transcripts and TΨC-arm (17-mer) fragments. Archaeal COG1901 members are now named as TrmY.

Poster Session 2: Splicing Mechanisms & RNA Editing and Modification

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

365

Alu-transposons Determine Site-selective A-to-I Editing

Chammiran Daniel, Gilad Silberberg, Marie Ohman Stockholm University Stockholm Sweden Adenosine-to-inosine (A-to-I) RNA editing by adenosine deamination occurs in both coding and non-coding sequences within structures that are largely double stranded. In the mammalian brain, a number of transcripts have been found to be site selectively edited, recoding the read out of genes involved in neurotransmission. Although it is known that the ADAR enzymes that catalyse A-to-I editing require a double stranded structure in the vicinity of the edited site, other requirements for substrate recognition are largerly unknown. We have previously shown that the I/M site in the Gabra-3 transcript coding for the α3 subunit of the GABAA receptor requires a distant intronic cis-acting stem loop structure for editing to occur. This intronic stem also has the capability of inducing editing in RNA sequences normally not edited. In the present study we show that most other evolutionary conserved ADAR editing sites are accompained by distant conserved duplex structures. We show that these cis-acting stem loops also enhance editing at the nearby site-selectively edited site, similar to the I/M site in Gabra-3 transcript. Furthermore, in primates, long stem loop structures formed by non-coding inverted repeats of Alu transposable elements have been shown to be hyper-edited. We demonstrate that primate specific site-selective editing is facilitated by nearby hyper-edited elements, revealing a biological relevance of transposable elements. Our data suggests that nearby intronic stem loop structures helps recruiting the ADAR enzymes to the transcript and thereby increase the local concentration of the enzymes. Thus, we imply, that these duplex structures are used as a general mechanism of cis-acting elements to select and induce site selective A-to-I editing. Therefore, in future studies of A-to-I editing predictions, the presence of intronic stem loops nearby editing candidates should be taken account.

366 The Rapid tRNA Decay Pathway Acts Broadly on Modification-Deficient Yeast Strains and Competes with Elongation Factor 1A for Substrates

Joshua Dewe, Joseph Whipple, Irina Chernyakov, Laura Nemeth, Eric Phizicky University of Rochester, Rochester, NY, USA In the yeast Saccharomyces cerevisiae, tRNA stability is monitored by at least two quality control pathways: the nuclear surveillance pathway, which monitors pre-tRNAs; and the rapid tRNA decay (RTD) pathway, which monitors mature tRNAs. In the RTD pathway, the 5’-3’ exonucleases Rat1 and Xrn1 degrade certain hypomodified or destabilized tRNAs, and degradation is inhibited by met22 mutants, likely by an indirect mechanism. The RTD pathway is responsible for temperature sensitive phenotypes of trm8-Δ trm4-Δ mutants (lacking m7G and m5C) and tan1-Δ trm44-Δ mutants (lacking ac4C and Um), but the full scope of this pathway is unknown. RTD appears to act on charged tRNAs, suggesting a role for translation in the pathway, but it is largely unknown how the RTD pathway interacts with other translation and cellular components. To identify other proteins that are involved in the RTD pathway, we screened for suppressors of the temperature sensitivity of trm8-Δ trm4-Δ mutants. We find evidence in favor of a model in which EF-1A binding competes with RTD for substrate tRNAs. Thus, EF-1A acts as a high-copy suppressor of RTD in trm8-Δ trm4-Δ and tan1-Δ trm44-Δ mutants, based on suppression of their temperature sensitivity, and prevention of tRNA degradation. Conversely, reduced levels of EF-1A result in exacerbated temperature sensitivity of RTD-sensitive strains, which is suppressed by deletion of MET22, and which is accompanied by more complete degradation of RTD substrate tRNAs. In addition, we find spontaneous mutations in two pol III subunits, each of which suppresses RTD, based on examination of tRNA levels before temperature shift, and after temperature shift in the absence of transcription. To investigate the scope of modification mutants that elicit RTD, we examined other strains. We find that trm1-Δ trm4-Δ mutants (lacking m2,2G and m5C) are also subject to RTD, because they are temperature sensitive due to degradation of two tRNA species, and because the temperature sensitivity is suppressed by mutation of MET22. Surprisingly, we also find evidence that RTD occurs in trm8-Δ, tan1-Δ, and trm1-Δ single mutants, and targets the same tRNA species as in the corresponding double mutants, albeit at a reduced rate.

Poster Session 2: RNA Editing and Modification

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

367 The Editing Deaminase is Required for Anticodon Loop Methylation of Threonyl-tRNA in Trypanosoma brucei

Ian Fleming1, Kirk Gaston2, Zdenek Paris1, Kady Krivos2, Pat Limbach2, Mary Anne Rubio1, Juan Alfonzo1 1 Department of Microbiology and OSU Center for RNA Biology, The Ohio State University, Columbus, 43210 Ohio, USA, 2Department of Chemistry, Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati, Cincinnati, OH 45221, USA Transfer RNAs undergo numerous post-transcriptional modification and editing events important for structure and function. One such modification, 3-methylcytidine (m3C) is highly conserved at position 32 in the anticodon loop of eukaryotic tRNAThr. Interestingly, tRNAThr of Trypanosoma brucei also undergoes C to U editing at this position, a unique event not documented in other organisms. Database searches revealed the presence of two potential homologs (Tbm3A and Tbm3B), of the Saccharomyces cerevisiae m3C methyltransferase, a situation so far unique to the kinetoplastid lineage, which includes Trypanosoma and Leishmania. Using molecular and genetic approaches, we have established that C32 methylation occurs in the nucleus prior to tRNA export to the cytoplasm. Given our previous observation of both a nuclear and cytoplasmic localization of the TbADAT2/3 deaminase and its involvement in both A to I and C to U editing, we explored the possibility of a connection between editing and methylation. Using in vitro methylation assays, we show that recombinant Tbm3B can efficiently methylate C32 of a synthetic tRNA substrate but only in the presence of TbADAT2/3. While we have not identified a function for Tbm3A, down-regulation of the expression of either protein (Tbm3A or Tbm3B) leads to severe growth defects, a phenotype that is unique to the T. brucei system. These results highlight the tight interconnection between editing and modification pathways with great implications for the regulation of tRNA function.

368 Structural Features of Archaeal Pus10 Proteins That Specify Pseudouridine Formation at tRNA Position 54

Archi Joardar, Sujata Jana, Priyatansh Gurha, Elisabeth Fitzek, Mrinmoyee Majumder, Kunal Chatterjee, Matt Geisler, Ramesh Gupta Southern Illinois University, Carbondale, (IL), USA Universally conserved pseudouridine (Ψ) at position 55 in tRNAs is produced by homologous Ψ synthases TruB and Pus4 in Bacteria and Eukarya, respectively. However, Pus10, an unrelated Ψ synthase produces tRNA Ψ55 in Archaea. Previously, we have shown that archaeal Pus10, in vitro, in addition to Ψ55, can also produce tRNA Ψ54. This Ψ54 synthase activity of Methanocaldococcus jannaschii Pus10 (MjPus10) is very robust while that of Pyrococcus furiosus Pus10 (PfuPus10) is observed only under certain specific conditions. Here we demonstrate heterologous in vivo activities of these two archaeal Pus10 proteins by expressing them in different E. coli strains that contain unmodified U at position 54 or 55 or both. These two archaeal proteins can produce tRNA Ψ55, but only MjPus10 can produce tRNA Ψ54 in appropriate E. coli strains. Interestingly, MjPus10 but not PfuPus10, produces tRNA Ψ54, instead of m5U54 (rT54) in strains that have native TrmA (tRNA m5U54 methyltransferase). Homology modeling of these archaeal Pus10 proteins based on the catalytic domain of human Pus10 suggests that MjPus10 has a larger Forefinger loop than does PfuPus10. Forefinger and Thumb loops of the proteins are required for holding the substrate tRNA in place. Deletion of Forefinger loop and substitution mutations in Thumb loop of MjPus10 showed very little effect on Ψ55 activity, but resulted in loss of Ψ54 activity. Replacement of Forefinger and Thumb loops of PfuPus10 with those of MjPus10 resulted in some recovery of Ψ54 under certain specific conditions. Furthermore, alanine substitutions of certain amino acids of MjPus10 that are absolutely conserved in the catalytic core of all Ψ synthases, showed differential Ψ54 and Ψ55 activities. Some mutants lost or showed severely reduced Ψ54 activity, but most mutants retained nearly wild-type levels or showed only little reduction of Ψ55 activity. Overall, using both in vivo and in vitro assays, we have identified several previously uncharacterized residues and domains of archaeal Pus10 proteins that are involved in tRNA Ψ54 activity.

Poster Session 2: RNA Editing and Modification

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

369 Inverted Alu dsRNA Structures Alter Translation of Human mRNAs Independent of RNA Editing

Claire Capshew, Kristen Dusenbury, Heather Hundley Indiana University, Bloomington, IN, USA With over 1 million copies, Alu elements are the most abundant repetitive elements in the human genome. When transcribed, interaction between two Alu elements in inverted orientation give rise to long intramolecular RNA duplexes. These inverted Alu elements are present in a large number of cellular mRNAs, with enrichment in untranslated regions (UTRs). As 3’ UTR elements often direct post-transcriptional gene expression, we tested whether these dsRNA structures regulate gene expression in human cell lines. To dissect the function of these intramolecular duplexes on gene expression, we created a mammalian reporter assay where firefly luciferase is fused to the 3’ UTR of the human PSMB2 gene, which contains two Alu elements in inverted orientation. We compared it to reporters with 3’ UTRs of the same length but containing the reverse complement (RC) sequence of either the first or second Alu. The controls do not form dsRNA as the two Alu elements are in the same orientation. Using the PSMB2 reporters, we consistently observed a 2-3-fold decrease in firefly activity for mRNAs with the double-stranded 3’ UTR compared to the control mRNAs. As the reporter bearing the PSMB2 3’ UTR did not have significant differences in mRNA levels or localization compared to the control mRNAs, we propose that the inverted Alus regulate translation of human mRNAs. The intramolecular duplexes formed from inverted Alu elements are often edited by the adenosine deaminase that acts on RNA (ADAR) family. In fact, of the ~40,000 editing sites in the human transcriptome, the vast majority are present within inverted Alu elements. To test whether editing or ADAR binding to the 3’ UTRs are important for the effects on translation, we analyzed the reporters in mammalian cell lines that silence human ADAR1. Interestingly, we observed that the effects of the PSMB2 3’ UTR on translation are independent of editing. To determine both the biological significance and breadth of the translational control, we are currently monitoring both the PSMB2 reporters and endogenous transcripts that contain inverted Alus in a variety of cell lines and conditions. However, as inverted Alus are predicted to reside in over 5% of human protein-coding genes, we predict that these intramolecular dsRNA structures are important regulators of gene expression.

370

Multiple Functions of Thg1 Family Enzymes in Mitochondria of Dictyostelium discoideum

Yicheng Long, Maria Abad, Jane Jackman The Ohio State University The highly conserved tRNAHisguanylyltransferase (Thg1) enzyme uses an unusual 3’-5’ polymerase activity to play an essential role in maturation of cytoplasmic tRNAHis in eukaryotes. In this study we demonstrated that Thg1 family enzymes are involved in additional cellular functions that make use of their 3’-5’ polymerase activities. Most eukaryotes contain one unique Thg1 enzyme, but four Thg1 orthologs, DdiThg1 and three Thg1-like proteins (DdiTLP2-4), have been identified by BLAST in Dictyostelium discoideum. Here we show that theseThg1-like proteins are 3’-5’ polymerases that have multiple cellular functions, including maturation of both cytoplasmic and mitochondrial tRNAHis and potential 5’-tRNA editing activity in mitochondria. Both the cytoplasmic and mitochondrial tRNAHis in D. discoideum lack a genomically-encoded G-1 residue, which is known to be required for tRNAHis function in cells, and presumably require the post-transcriptional addition of this critical residue, as has been shown in yeast. We demonstrated that DdiThg1 and DdiTLP2 add the G-1residue to distinct tRNA substrates, cytoplasmic versus mitochondrial tRNAHis, respectively. Surprisingly both DdiThg1 and DdiTLP2 are highly specific for these individual tRNAHis substrates, both of which contain the GUG anticodon, and this substrate specificity for different tRNAHis has never been observed before among other Thg1 family members. Identification of the specificity elements on the two tRNAHis is underway. Thg1 family enzymes are also involved in a proposed 5’-tRNA editing process. Using in vitro activity assays, DdiTLP3 and DdiTLP4 have been shown to be capable of adding missing nucleotides to the 5’-end of tRNAs. The ability to catalyze tRNA 5’-end repair suggests that DdiTLP3 and/or DdiTLP4 may participate in an unusual 5’-tRNA editing reaction occurring in the mitochondria, in which 9 of 18 mitochondrial genome encoded tRNA genes contain predicted mismatched nucleotides at their 5’-ends. However, it remains unclear why multiple genes with tRNA 5’-end repair activity are encoded in D.discoideum. Results of a yeast two hybrid experiment indicated that DdiTLP3 and DdiTLP4 interact with each other in vivo in yeast, which infers that an editing complex might be formed in vivo. We are currently using genetic method in D. discoideum to investigate their activities in vivo. Poster Session 2: RNA Editing and Modification

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

371 Structural Features of Haloferax volcanii Cbf5 Protein Essential for In Vivo RNA-guided Pseudouridylation

Mrinmoyee Majumder, Ramesh Gupta Southern Illinois University, Carbondale, (IL), USA Pseudouridine (Ψ), the C-5 ribosyl isomer of uridine, is commonly found at several positions of stable RNAs of all organisms. In addition to single- or multi-site specific protein-only Ψ synthases, Eukarya and Archaea have specific ribonucleoprotein complexes that can also produce Ψ at many sites of different cellular RNAs. Each complex contains a distinct box H/ACA guide RNA and four core proteins. Cbf5 is the Ψ synthase in these complexes. In our previous study, we produced a Haloferax volcanii strain that had genomic deletion of the cbf5 homolog (HVO_2493). It showed absence of Ψ at 23S rRNA positions 1940, 1942 and 2605 (E. coli positions 1915, 1917 and 2572). A plasmid-borne copy of cbf5 could rescue the synthesis of these Ψs. We identified several potential residues and structures of functional importance in H. volcanii Cbf5 by comparing its sequence with other Cbf5 as well as by homology modeling based on the crystal structures of Pyrococcous furiosus Cbf5. We mutated these structures and determined their in vivo effects towards Ψ production at the three rRNA positions. Mutations of certain residues conserved in all Ψ synthases, including the catalytic Asp and some residues in the thumb loop resulted in absence of Ψ at all three positions, while mutations in other regions showed differences in their effects at the three positions.

372

3’-5’ Polymerization in tRNA Biogenesis

Fuad Mohammad, Maria Abad, Yicheng Long, Jane Jackman The Ohio State University, Columbus, (OH), USA In the process of translation, transfer RNAs function as a link between nucleic acid and amino acid sequences, allowing the ribosome to interpret the mRNA code into a functional polypeptide. Like most biomolecules, tRNAs have highly conserved secondary and tertiary structures that are critical for proper function. In all domains of life, the evolutionary pressure to maintain tRNA structure has led to diverse pathways in tRNA biogenesis that make use of 3’-5’ RNA polymerization catalyzed by the tRNAHis guanylyltransferase (Thg1) enzyme family. Here, we study two processes catalyzed by Thg1 enzymes. First, members of the Thg1 enzyme family, designated as Thg1-like proteins (TLPs), have been suggested to participate in a multistep tRNA editing pathway in several lower eukarya. Organisms such as Dictyostelium discoideum and Acanthamoeba castellanii encode in their mitochondria aberrant tRNAs with mismatches at the normally base-paired tRNA acceptor stem. Two TLPs in D. discoideum (DdiTLP3-4) are predicted participants in the tRNA editing pathway, and have robust in vitro 3’-5’ polymerization on all 9 truncated tRNA intermediates proposed to be edited in D. discoideum. The sequencing of one repaired tRNA product reaffirms previously observed preference for templated nucleotide addition. Although DdiTLP3 and DdiTLP4 share similar tRNA repair activities, the mechanisms for repairing truncated tRNAs differ. DdiTLP3 more sensitive to tRNA length, and can only incorporate nucleotides to the first 3 positions of the 5’ end of tRNAs. Alternatively, DdiTLP4 repairs larger 5’-end truncations than DdiTLP3, and is capable of incorporating excess nucleotides at the 5’ under sub-physiological ATP concentrations. The second process studied is the catalysis of G-1 addition to tRNAHis, a process though to occur in all eukarya and many archaea. D. discoideum uses two additional Thg1 proteins (DdiThg1 and DdiTLP2) for G-1 addition, and therefore, there is a division of labor between tRNA editing and tRNAHis maturation by Thg1 enzymes in the organism. In A. castellanii, however, there are only two encoded TLPs (AcaTLP1-2). Both AcaTLPs catalyze tRNA repair, but have very weak in vitro G-1 addition activity. Whether G-1 addition occurs or is a necessary activity in vivo is uncertain. In studying D. discoideum and A. castellanii Thg1 enzymes, the activities and mechanistic properties of 3’-5’ RNA polymerization provide insight to the flexibility of the Thg1 family. Poster Session 2: RNA Editing and Modification

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

373 Down-regulation of tRNA m1G Formation Affects Mitochondrial but not Cytoplasmic Protein Synthesis in Trypanosoma brucei

Zdenek Paris1, Eva Horakova2, Mary Anne Rubio1, Paul Sample1, Ian Fleming1, Julius Lukes2, Juan Alfonzo1 1 Department of Microbiology and OSU Center for RNA Biology, The Ohio State University, Columbus, Ohio, USA, 2Biology Centre, Institute of Parasitology, Czech Academy of Sciences and Faculty of Sciences, University of South Bohemia, Ceske Budejovice (Budweis), Czech Republic A general feature of transfer RNAs is a high number of modified nucleotides that are introduced enzymatically at the post-transcriptional level. In all organisms, 1-methylguanosine (m1G) is a highly conserved modification found at position 37 of several tRNAs. Due to a complete lack of tRNA genes in the mitochondrial genome of trypanosomatids, tRNAs are encoded in the nucleus and imported into the mitochondrion from the cytoplasm. Mitochondrial tRNA import is thus essential for organellar translation. Here we show by a combination of molecular, genetic and biochemical approaches, that m1G formation in tRNA plays a critical role in mitochondrial function. To this end, we have identified the putative TRM5 gene of T. brucei and show that TRM5 is responsible for m1G formation at position 37 in the anticodon loop of various tRNAs in both cytoplasm and mitochondria. Silencing of TRM5 resulted in a growth phenotype with pronounced decrease in mitochondrial translation, concomitant defects in respiration and increased production of reactive oxygen species (ROS). Surprisingly, cytosolic translation was unaffected. These observations are discussed in the context of the possible roles that tRNA import and differential intracellular localization of tRNA modification enzymes play in mitochondrial function.

374 A Comprehensive Analysis of Developmental Expression Patterns of RNA Modifying Enzymes in Zebrafish

Marion Pesch1, Jana Pfeiffer2, Erez Raz2,1, Sebastian Leidel1 Max Planck Institute for Molecular Biomedicine, Muenster, Germany, 2Institute for Cell Biology Westfaelische Wilhelms-Universitaet Muenster, Muenster, Germany The chemical modification of RNA nucleosides is a widespread phenomenon throughout evolution and occurs in all known taxa. Some of these modifications are known for many years and most enzymes required for their generation have been identified and characterized. Nevertheless, we know only little about the in vivo functions of most of these modifications. In lower eukaryotes, deletion of RNA modification pathways is often not essential. In contrast, deletion of RNA modifying enzymes like components of the ELP complex can lead to severe phenotypes in higher eukaryotes. Most research however, has been performed in single cell organisms and we lack insights into RNA modification pathways in vertebrates. Do RNA modification defects generally lead to more severe phenotypes in higher eukaryotes and are RNA modification enzymes differentially regulated in different tissues and during development? To gain first insights into the spatio-temporal regulation of RNA modification pathways, we undertake a comprehensive analysis of the expression patterns of RNA modification genes in zebrafish (Danio rerio) using in situ hybridization. First, we identified homologues of all known RNA modification genes in yeast. Subsequently, we generated a library of probes for the detection of these genes. Finally, we analyzed the expression patterns of this library throughout zebrafish development using embryos at 4h, 10h, 24h, 48h and 72h post fertilization. Here we present the expression patterns of 57 putative RNA modification genes in zebrafish. We found four striking staining patterns in the midbrain, spinal cord, eyes and pectoral fins. Our study is the first comprehensive analysis of the expression patterns of RNA modification enzymes in a vertebrate system and may provide further insights into their regulation.

1

Poster Session 2: RNA Editing and Modification

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

375 Novel RNA Editing Events Necessary for Endonuclease Processing of tRNAs in Trypanosoma brucei

Mary Anne Rubio1, Juan Alfonzo1, Chris Trotta2 The Ohio State University, 2PTC Pharmaceuticals In eukaryotes, precursor tRNAs contain introns whose cleavage is mediated by an evolutionarily conserved endonuclease complex that generates fully functional mature tRNAs. The Trypanosoma brucei genome encodes a single intron-containing tRNA (tRNATyr), responsible for decoding all tyrosine codons; therefore, intron removal is essential for protein synthesis and consequently cell viability. In this organism, little is known about the mechanism of intron processing, but owing to its early evolutionary divergence from other eukaryotes, T. brucei often reveals unexpected peculiarities. Here we show an unprecedented number of nucleotide differences within the intron-containing pre-tRNATyr and its genomic copy. Significantly, these differences occur at the RNA level and cannot be ascribed to canonical deamination-type editing. Intron editing is required for proper pre-tRNA processing, establishing its functional significance for production of the full complement of tRNAs needed for translation. Bioinformatic analyses revealed only one homolog of the four conserved canonical subunits of the multi-protein complex endonucleases required for tRNA splicing in other eukaryotes. The demonstration of a novel editing mechanism required for proper function of a highly divergent splicing endonuclease in kinetoplastids has great implications to our understanding of the evolution of tRNA processing across eukaryotes. 1

376

The Elongator Subcomplex Elp456 is a Hexameric RecA-like ATPase

Sebastian Glatt1, Juliette Létoquart2, Céline Faux2, Nicholas Taylor1, Bertrand Séraphin2, Christoph Müller1 1 EMBL, Heidelberg, Germany, 2IGBMC, Illkirch, France The Elongator complex was initially described as a RNA polymerase II associated factor, but has since been associated with a broad range of cellular activities. It has also attracted clinical attention due to its role in certain neurodegenerative diseases. We report the crystal structure of the Saccharomyces cerevisiae subcomplex of Elongator proteins 4, 5 and 6 (Elp456). The subunits share an almost identical RecA fold forming a hetero-hexameric ring-like structure that unexpectedly resembles hexameric RecA-like ATPases. This arrangement is supported by different complementary in vitro and in vivo approaches. We also observed that hexameric Elp456 subcomplex specifically binds to tRNAs in a manner regulated by ATP. Our results support a role of Elongator in tRNA modification and explain the importance of each of the Elp4, Elp5 and Elp6 subunits for complex integrity. Together with other data, the structure of the Elp456 subcomplex allows us to suggest a model for the architecture of the holoElongator complex.

Poster Session 2: RNA Editing and Modification

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

377

Substrate specificity of a multi-functional tRNA modification enzyme.

378

Genome-Wide Identification of RNA Editing Events in RNA-Seq Data

William Swinehart, Jane Jackman The Ohio State University, Columbus, OH, United States tRNA modification enzymes are often challenged with the complex task of identifying specific tRNAs from among a pool of similarly structured molecules. This task is accomplished in diverse ways, with some enzymes identifying specific residues, while others recognize a tertiary fold unique to the intended tRNA substrate. In S. cerevisiae, the tRNA methyltransferase Trm10, which catalyzes m1G9 methylation, recognizes a specific set of substrate tRNAs through an unknown mechanism. Interestingly, a human Trm10 ortholog, RG9MTD1, is implicated in a mitochondrial RNase P complex whose tRNA recognition pattern predictably contrasts with the apparently specific nature in which yeast Trm10 recognizes its substrates. The goal of this work is to identify the molecular basis for Trm10 recognition in yeast and provide a foundation for understanding the substrate selectivity of Trm10 in other organisms. Identifying sequence determinants is especially challenging since there is no obvious identity element shared among Trm10 substrate tRNAs. We have employed in vitro activity assays and primer extension to determine the complete set of in vitro and in vivo Trm10 substrates in yeast. Surprisingly, Trm10 does not efficiently distinguish between some substrate and non-substrate tRNAs in vitro, and when Trm10 is overexpressed in cells an increased number of tRNA species are observed to be methylated. Therefore, additional protein or RNA factors appear to participate in selective recognition of only some tRNAs in vivo in yeast, and the identity of these is currently under investigation. As one step toward elucidating Trm10 identity elements, we have shown that Trm10 does not methylate any tRNA which contains an extended variable loop. We are comparing in vitro activities of Trm10 with a series of tRNALeuCAA chimeras that contain domains from the known substrate tRNAGlyGCC. We have shown that, unlike the wild-type tRNALeuCAA, which is not a substrate for methylation, several of these chimeras are methylated by Trm10. Thus, we have identified an important tool for elucidating structural components of the tRNA responsible for Trm10 recognition. These data raise the possibility that the “leaky” Trm10-substrate recognition may be important for other Trm10-related activities in the cell.

Jae Hoon Bahn, Jae-Hyung Lee, Jason Ang, Xinshu Xiao University of California, Los Angeles, Los Angeles, CA, USA RNA editing enhances the diversity of gene products at the post-transcriptional level. Methods for genome-wide identification of RNA editing face two main challenges: separating true editing sites from false discoveries and accurate estimation of editing levels. We developed an approach to analyze transcriptome sequencing data (RNA-Seq) for global identification of RNA editing in cells for which whole-genome sequencing data are available. Using human RNA-Seq data, we showed that our approach is associated with a low false discovery rate of 5% and the estimated editing levels correlated well with those based on clonal sequencing. In human cancer cells, we identified around 10,000 DNA-RNA differences with the majority being putative A-to-I editing sites. Genes with predicted A-to-I editing were significantly enriched with those known to be involved in cancer, supporting the potential importance of cancer-specific RNA editing. In addition, different cancer transcriptomes demonstrated significant overlap in their editomes despite their difference in cell type, cancer type and genomic backgrounds. We also found evidence of other types of DNA-RNA differences, but with relatively low prevalence and validation rate. We will further discuss the technical aspects of applications of RNA-Seq to RNA editing studies in human and other species. Our approach enabled de novo identification of the RNA editome, which sets the stage for further mechanistic studies of this important step of post-transcriptional regulation.

Poster Session 2: RNA Editing and Modification

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

379

The Study of tRNA Modifications by Molecular Dynamics

Xiaoju Zhang, David Mathews University of Rochester, Rochester, (NY), USA Modified nucleosides are prevalent in tRNA. Experimental studies reveal that modifications play an important role in tuning tRNA activity. In this study, molecular dynamics (MD) simulations were used to investigate how modifications alter tRNA structure and dynamics. The X-ray crystal structures of tRNA-Asp, tRNA-Phe, and tRNA-iMet [1-3], both with and without modifications, were used as initial structures for 333 ns time scale MD trajectories with AMBER [4]. For each tRNA molecule, three independent trajectory calculations were performed. Force field parameters were built using the RESP procedure of Cieplak et al [5]. for 17 nonstandard tRNA residues. The global root mean square deviations (RMSDs) of atomic positions show that modifications only introduce significant rigidity to tRNA-Phe’s global structure. Interestingly, regional RMSDs of anticodon stem-loop suggest that modified tRNA has more rigid structure compared to the unmodified tRNA in this domain. The anticodon RMSDs of the modified tRNAs, however, are higher than those of corresponding unmodified tRNAs. These findings suggest that rigidity of the anticodon arm is essential for tRNA translocation in the ribosome complex, and, on the other hand, flexibility of anticodon might be critical for anticodon - codon recognition. We also measure the angle between the 3D L-shaped arms of tRNA; backbone atoms of acceptor stem and TψC stem loop are selected to indicate one vector, and backbone atoms of anticodon stem and D stem loop are selected to indicate the other vector. By measuring the angle between two vectors, we find that the initiator tRNA has a significantly narrower range of hinge motion compared to tRNA-Asp and tRNA-Phe, which are elongator tRNA. This suggests that elongator tRNAs, which require significant flexibility in this hinge to transition from the A to P site in the ribsome, have evolved to specifically to accommodate this need. 1. Westhof, E., Dumas, P., and Moras, D., Acta Crystallogr A 44(Pt2): 112(1988). 2. Shi, H., Moore, P.B., RNA 6: 1091(2000). 3. Basavappa, R. and Sigler, P.B., EMBO J. 10(10): 3105(1991). 4. Case D.A., Cheatham T.E. 3rd, Darden T., Gohlke H., Luo R., Merz K.M. Jr, Onufriev A., Simmerling C., Wang B., Woods R.J., J Comput Chem 26(16): 1668(2005). 5. Cieplak, P., Cornell, W.D., Bayly, C. and Kollman, P.A., J Comput. Chem. 16: 1357(1995).

380 Directed Translational Initiation by the Anticodon Mimicry Domain of a Viral Internal Ribosome Entry Site

Hilda Au, Eric Jan University of British Columbia, Vancouver, Canada The intergenic region internal ribosome entry site (IGR IRES) of the Dicistroviridae family adopts an overlapping triple pseudoknot structure to directly recruit the 80S ribosome in the absence of initiation factors. The pseudoknot I (PKI) domain of the IRES mimics a tRNA-like codon:anticodon interaction in the ribosomal P-site to direct translation initiation from a non-AUG codon in the A-site and directs translation in a specific reading frame. In this study, we examine the elements within the tRNA-mimicry domain that impact IRES translation and reading frame selection. We demonstrate that IRES-mediated translation can initiate at an alternate adjacent and overlapping start site, provided that base-pairing interactions within PKI remain intact. Consistent with this, IGR IRES translation can tolerate increases in the variable loop region that connects the anticodon- and codon-like elements within the PKI domain, as IRES activity remains relatively robust up to a 4-nucleotide insertion in this region. Finally, we show that elements from an authentic tRNA anticodon stem-loop can functionally supplant corresponding regions within PKI. These results provide insight into the role of specific tRNA-like PKI elements on IRES-mediated translation and reading frame selection and sheds light into how a viral RNA can manipulate the ribosome.

Poster Session 2: RNA Editing and Modification & Poster Session 2: Translational Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

381 The Structure of Intact E. coli RelBE Suggests a Structural Basis for Conditional Cooperativity

Andreas Bøggild1, Nicholas Sofos1, Ashley Easter2, Lori Passmore2, Ditlev Brodersen1 1 Aarhus University, Aarhus C, Denmark, 2MRC Laboratory of Molecular Biology, Cambridge, UK Type II toxin/antitoxin (TA) loci generally lead to expression of two proteins, one capable of down-regulating cellular metabolism (the toxin) and another that binds and inhibits the toxin under normal circumstances (the antitoxin). Activation of the toxin occurs during cellular stress through degradation of the labile antitoxin and subsequent release of the toxin. Furthermore, transcription from the TA loci is controlled through promoter binding by a DNA binding module on the antitoxin molecule. Such TA loci are surprisingly widespread in archaea and bacteria, in particular pathogenic bacteria, and are thus believed to constitute an important means of adaptation to changing environmental conditions for these organisms. Here, we present the intact crystal structure of the E. coli RelBE complex including the DNA-binding domain of RelB. The RelBE complex crystallized the space group P6122 and crystals diffracted to about 2.7Å. The structure was determined by multiple isomorphous replacement using anomalous scattering based on data from a Pt soak. The crystal structure shows a V-shaped RelB2E2 heterotetramer confirming that the two proteins are able to bind each other in a 1:1 ratio. The contents of the crystallographic asymmetric further suggested a complex higher order structure, where three RelB2E2 complexes intertwine to form a large spherical superstructure with the RelB DNA binding domains on the outside. Analytical ultracentrifugation and chemical cross-linking, however, indicate that the superstructure is not present in solution and more likely a result of crystallization. Studies of DNA binding by the RelBE complex have shown optimal DNA affinity at a 2:1 ratio (RelB:RelE), leading to a model for conditional cooperativity in which excess RelE is able to de-repress the promoter by diminishing overall DNA affinity1. The RelBE crystal structure presented here supports a model in which the symmetric promoter DNA with two palindromic binding sites is optimally bound by two adjacent RelB2E heterotrimeric units. Our data thus provide firm support for the model of conditional cooperativity and provides a structural and architectural basis for understanding the phenomenon at the molecular level. 1 Overgaard, M., Borch, J., Jorgensen, M. G., & Gerdes, K. (2008). Messenger RNA interferase RelE controls relBE transcription by conditional cooperativity. Molecular microbiology, 69(4), 841-57.

382 Thriving under Stress: HIV-1 mRNAs Exploit Nuclear Cap-Binding Protein to Sustain Translation during Viral Impairment of eIF4E Activity

Amit Sharma1,1, Alan Cochrane2, Kathleen Boris-Lawrie1 1 Ohio State University, 2University of Toronto Retroviruses are intracellular parasites that are dependent on the host cell’s gene expression machinery. The activity of the cell’s translation machinery fluctuates during cell cycle progression and is activity is attenuated during the mitosis. Ironically, HIV-1 gene products arrest cells at this point in the cycle. The potential for HIV-1 infection to disrupt cellular protein synthesis was investigated herein. Our results demonstrated that cellular protein synthesis is downregulated during progression of the viral infection, but synthesis of the viral proteins is sustained. The molecular basis of HIV-1 translation attenuation is impairment of eIF4E activity by reduced accumulation of P-eIF4E and P-4E-BP1. The viral gene necessary is the pathogenicity determinant, viral protein R (Vpr). To investigate why selected viral transcripts can sustain protein synthesis, we investigated components of HIV-1 RNPs by RNA-coprecipitation assays. The comparison was made between RNPs of the fully spliced HIV-1 transcripts or the viral unspliced and incompletely spliced transcripts, which require the CRM-1 nuclear export receptor. The fully spliced viral RNAs demonstrated efficient exchange of nuclear cap binding complex (CBC) for eIF4E in the cytoplasm. However, the unspliced and incompletely spliced viral transcripts retained CBC in the cytoplasm. These viral transcripts were loaded onto the polysomes and yielded virion structural proteins. The exploitation of CBC to overcome downregulation of eIF4E-dependent translation appears to represent viral adaptation of an cellular mechanism to generate functional mRNA templates during cell cycle progression. Our findings have uncovered that retention of CBC is a unique viral strategy to sustain viral protein synthesis during generalized downregulation of eIF4E activity.

Poster Session 2: Translational Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

383 Genome-wide Investigations of Translating mRNAs to Study the Cellular Functions of tRNA Nuclear-Cytoplasmic Dynamics in S. cerevisiae

Hui-Yi Chu, Anita Hopper The Ohio State University, Columbus, (OH), USA In eukaryotic cells tRNAs are transcribed in the nucleus followed by export to the cytoplasm for their essential role in protein synthesis. Surprisingly, we and another group showed that mature cytoplasmic tRNAs shuttle between nucleus and cytoplasm (Shaheen and Hopper, 2005; Takano et al., 2005). At least three members of β-importin family function for tRNA nuclear-cytoplasmic intracellular movement: (1) Los1 functions in both of the tRNA primary export and re-export processes; (2) Mtr10, directly or indirectly, is responsible for the constitutive retrograde import cytoplasmic tRNA to the nucleus; (3) Msn5 functions solely in re-export process (Murthi et al., 2010). Cytoplasmic tRNAs accumulate in the nucleus when cells are deprived of nutrients (Shaheen and Hopper, 2005; Hurto et al., 2007; Whitney et al., 2007). Our current efforts focus on the physiological role(s) of the tRNA nuclear retrograde pathway. One possibility is that nuclear accumulation of cytoplasmic tRNA serves to modulate translation of particular transcripts. To test this hypothesis, we compared expression profiles from non-translating mRNAs and polyribosome-bound translating mRNAs collected from msn5Δ and mtr10Δ mutants and wild-type cells, under fed or acute amino acid starved conditions. Our microarray data revealed that the sulfur assimilation pathway and arginine biosynthesis pathway are primary targets of the tRNA retrograde processes. We confirmed the microarray data by both Northern and western blot analyses; translation of all tested mRNAs in the sulfur assimilation pathway and arginine biosynthesis pathway is down-regulated when the tRNA nuclear import or re-export is disrupted. Interestingly, both affected pathways are upstream events for polyamines biogenesis and LCMS/MS analyses demonstrated that intracellular levels of polyamines were changed in tRNA retrograde mutants. Since polyamines are known to participate in numerous cellular functions, our study provides the first evidence that tRNA nuclear-cytoplasm dynamics is connected to cell physiology via control at the level of translation.

384

Selection of Inhibitory Codon Combinations in Saccharomyces cerevisiae

Kimberly Dean, Elizabeth Grayhack University of Rochester Medical Center, Rochester, NY, USA There is a wealth of evidence that the choice of synonymous codons used to encode a polypeptide moderates translation efficiency, but neither the parameters that govern their efficacy nor the mechanism by which codons exert their effects are known. In a systematic study of 59 of the 61 codons in S. cerevisiae, we found that the arginine CGA codon is strongly inhibitory due to wobble decoding with its cognate tRNA. Furthermore, we demonstrated that two adjacent CGA codons are far more inhibitory than separated CGA codons, suggesting that it is codon combinations that modulate translation1. This idea is also supported by prior observations that codon pair bias is correlated with gene expression in E. coli2, and that recoding poliovirus with underused codon pairs results in reduced expression3. However, neither the identities nor properties that are necessary for inhibition have been elucidated. To identify inhibitory codon combinations other than CGA pairs in S. cerevisiae, we developed a screen for sequences that inhibit GFP expression when inserted into the coding sequence. Noise, measured by differences in expression from yeast bearing GFP reporters with a single sequence inserted, is minimized by integrating the reporter into the chromosome, and by using an RFP reporter driven from the same promoter to control for promoter activation. A chromosomally integrated reporter yields a robust GFP/RFP signal that is 270-fold above background. As expected, expression is decreased progressively by insertion of increasing numbers of CGA codons, and this inhibition is suppressed by the expression of the exact base pairing tRNAArgUCG. To determine if other inhibitory sequences could be found with this method, we inserted a semi-randomized threecodon (9 nucleotide) sequence at amino acid 6 of GFP and screened clones of this library for effects on expression. While most insertions have little effect on GFP expression, a fraction of the library exhibited reduced expression. From this fraction, we have obtained sequences that cause reduced GFP expression and remade these variants to confirm the dependence on the inserted sequence. 1. Letzring, D., Dean, K., and Grayhack, E. (2010). RNA 16: 2516-2528. 2. Gutman, G. and Hatfield, G.W. (1989). PNAS 86: 3699-3703. 3. Coleman, J.R., et al. (2008). Science 320:1784-1787. Poster Session 2: Translational Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

385 Investigating the Targeting and Regulation of Ded1, a DEAD-box ATPase, in Returning Repressed mRNAs to Translation

Angie Hilliker1, Roy Parker2 University of Richmond, Richmond, VA, US, 2University of Arizona, Tucson, AZ, US Translation regulation is important, especially during stress, in neurons, and in early development, where many mRNAs are stored in translationally repressed states. Repressed mRNAs can accumulate in cytoplasmic granules, such as processing (P) bodies and stress granules (SGs). These repressed mRNAs can be returned to translation, but this process is poorly understood. The RNA helicase, Ded1, is conserved from yeast to humans and has been shown to both promote and repress translation. In our previous work, we have developed a working model of Ded1 function that reconciles these two observations (Hilliker et al., 2011). Our data are consistent with Ded1 performing two sequential steps to return a repressed mRNA back into translation. First, Ded1 promotes the repression of mRNAs that can accumulate in SGs with eIF4F, but not the multi factor complex. Second, Ded1 uses its ATPase domain to allow mRNAs to leave SGs and return to translation. Our model suggests that Ded1 acts like an ATP-dependent switch move mRNAs from a SG-like storage state back into translation. To better understand how this switch is controlled, we wish to determine how Ded1 is regulated. Over-expression of Ded1 in vivo causes a severe growth defect and other phenotypes that suggest that Ded1 has accomplished its first role, but is unable to complete its second role, resulting in too many repressed mRNAs. By looking for over-expression suppressors of this growth defect, we can identify factors that either prevent Ded1’s first function or promote Ded1’s second, ATPdependent function. The preliminary results of this screen have identified factors that may modify Ded1 post-translationally. We have previously identified mutations in DED1 that are cold-sensitive; the phenotypes of these mutations suggests that Ded1 may target mRNAs in P-bodies to return them to translation. We are currently using these cold-sensitive mutants in genetic screens to identify in vivo targets for Ded1. If the cold-sensitive defect is due to an inability to destabilize its target, then mutations in the targets of Ded1 should suppress the cold-sensitive defect of these ded1 mutants. These types of genetic screens will allow us to better understand the role of Ded1 in promoting the translation of repressed mRNAs. Additionally, these screens will allow us to identify other factors involved in control of mRNA translation.

1

386

Post-transcriptional Regulation of Gene Expression by Khd1, Ccr4, and Pbp1

Kenji Irie, Yuichi Kimura, Xia Li, Tomoaki Mizuno University of Tsukuba, Tsukuba, JAPAN RNA-binding protein Khd1 and cytoplasmic deadenylase Ccr4 are involved in the post-transcriptional regulation of gene expression in yeast. We have previously found that Khd1 andCcr4 modulate a signal from Rho1 in the cell wall integrity pathway by regulating the expression of Rom2 and LRG1, encoding a guanine nucleotide exchange factor (GEF) and a GTPase-activating protein (GAP) for Rho1, respectively. The khd1Δccr4Δ double mutant, in which ROM2 expression is decreased and LRG1 expression is increased, shows severe cell lysis. Here we found that a mutation in a third gene PBP1, encoding a polyA-binding protein (Pab1)-binding protein, an yeast orthlog of human Ataxin-2, suppressed the growth defect of the khd1Δ ccr4Δ double mutant. While the pbp1Δ mutation did not affect the ROM2 expression, the pbp1Δ mutation suppressed an increased expression of Lrg1 in the khd1Δ ccr4Δ double mutant. We also found that Pbp1 associated with ribosomal proteins Rpl12A and Rpl12B as well as the known binding partners, Lsm12 and Mkt1. Although mutation in PBP4 or MKT1 did not suppress the growth defect caused by double deletions of KHD1 and CCR4, mutation in the gene encoding Rpl12A and Rpl12B did suppress the growth defect. Our results suggested that Pbp1 is involved in the Khd1-and Ccr4-mediated regulation of the cell wall integrity through the interaction with ribosomal protein.

Poster Session 2: Translational Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

387 Characterization of Translational Regulation during Hypoxia in Human Colon Cancer HCT116 Cells

Ming-Chih Lai, Shaw-Jenq Tsai, H. Sunny Sun National Cheng Kung University Medical College, Tainan, Taiwan Colon cancer, also called colorectal cancer, is one of the most common cancers in the world. In Taiwan, colon cancer has risen to the third leading cause of all cancer deaths. The incidence of colon cancer has been rising steadily in the last 20 years. Although the genetic process of carcinogenesis has been well documented, many patients with invasive/ metastatic colon cancers still die within five years due to treatment failure. Thus, new strategies and better therapeutics are still required for the therapy of human colon cancer. Hypoxia occurs in a wide variety of physiological and pathological conditions, including tumorigenesis. Because of rapid proliferation and aberrant tumor angiogenesis, tumors contain areas with various degrees of hypoxia. Tumor cells have to adapt to hypoxic stress by altering their gene and protein expression profiles. The presence of hypoxic cells in solid tumors is associated with a poor clinical prognosis. Indeed, previous studies have demonstrated that hypoxic tumor cells are more resistant to radiotherapy and chemotherapy. Thus, a better understanding of the regulation of gene expression during hypoxia might open perspectives for future tumor therapy. Hypoxia has been shown to inhibit general translation in various cell types. However, a subset of transcripts still remain efficiently translated under hypoxic conditions. These mRNAs may play crucial roles in cell adaptation to hypoxia. Using a genome-wide approach, we have identified a large number of mRNAs that are translationally activated during hypoxia (1% O2, 16 h) in human colon cancer HCT116 cells. In addition, we also found that hypoxia inhibits translation of general mRNAs mainly through modulating the activities of mTOR and PERK kinases. Our results might provide novel molecular targets for anti-cancer therapy by blocking tumor cell adaptation to hypoxia.

388 LARP1 Induces HeLa Cell Migration And Invasion By Activating Localised Protein Synthesis At The Cellular Leading Edge

Manuela Mura, Normala Abd Latip, Theodora Michael, Jacqueline Fok, Thomas Hopkins, Francesco Mauri, Roberto Dina, Edward Curry, Sarah Blagden Imperial College London UK LARP1 is a member of the LA-related protein (LARP) family, which shares with genuine LA protein the LA-motif (LAM) and the RNA recognition motif (RRM). All members of the LARP family play a role in mRNA translation or RNA homeostasis. LARP1 expression is dramatically increased in cervical, lung and ovarian cancer compared to normal tissue, and levels of LARP1 expression in cervical cancer positively correlates with disease progression. In a previous work we have shown that LARP1 RNAi decreases overall protein synthesis by 15%. We show here that after ectopic over-expression, LARP1 localises to pseudopodia in HeLa cells and invadopodia in SKOV3 cells, and cellular migration and invasion are respectively increased in these cell lines. Treatment with growth factors such as TGF-B and FGF increase the overall expression of LARP1 and cause its re-localisation to the leading edge of migrating cells where it co-localises with the translation regulators eIF4E and PABP. The interaction between LARP1, PABP and eIF4E, confirmed by co-IP, suggests these proteins together activate protein translation at the leading edge. RIP-CHIP analysis showed increased LARP1 binding for transcripts involved in migration and proliferation including many components MAPK and WNT signalling pathways.In light of this we postulate a role of LARP1 in cell migration and investigate its role in invasion and therefore in malignant transformation.

Poster Session 2: Translational Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

389 Efficient Ribosomal Frameshifting on a Non-canonical Sequence in a Herpes Simplex Virus Mutant is Promoted by Non-stop mRNA

Dongli Pan, Donald Coen Harvard Medical School, Boston, MA, USA Clinically relevant antiviral drug resistance requires mechanisms that retain viral pathogenicity. Acyclovir resistance in patients with herpes simplex virus disease is frequently due to frameshift mutations in the viral thymidine kinase (tk) gene, yet TK is important for pathogenicity. One such mutation is a one base deletion from a run of six cytosines (C-chord) in tk that generates a new reading frame that lacks stop codons (non-stop), yet the mutation does not abolish TK activity. To investigate how this mutant retains TK activity, we engineered viruses with epitope-tagged TK. Using a quantitative, sensitive immunoprecipitation-Western assay, we found that the mutant’s TK activity can be accounted for by low level expression of full-length TK. Deep sequencing of mutant mRNA failed to detect levels of altered tk transcripts that could account for the levels of full-length protein, indicating that -1 ribosomal frameshifting produces full-length TK. The mutant also expressed the polypeptide from the new reading frame generated by the deletion, but at levels only ten-fold higher than those of full length TK. Northern blot hybridization of tk mRNA, and studies using cycloheximide and MG132 indicated that the low levels of the out-of-frame polypeptide are largely due to inefficient protein synthesis with a smaller contribution from protein degradation. Thus, the efficiency of ribosomal frameshifting is relatively high, ~5%. Stop codons introduced into the new reading frame of the mutant greatly increased the level of the out-of-frame polypeptide, as expected for the product of a non-stop mRNA. However, unexpectedly, these stop codons greatly decreased the level of full-length TK, indicating that frameshifting is stimulated by a non-stop mechanism. Effects of stop codons engineered in the TK reading frame showed that frameshifting occurs in or near the C-chord. Remarkably, this region does not contain a canonical slippery sequence. Non-stop stimulation of frameshifting was also observed when the C-chord was replaced with the canonical slippery sequence from human immunodeficiency virus. Thus, non-stop is a new mechanism for stimulating -1 ribosomal frameshifting. We hypothesize that stalled ribosomes arising during translation of non-stop mRNA stimulates -1 slippage.

390

Physiological significance of tRNA thio-modification in protein translation

Vanessa Rezgui1, Kshitiz Tyagi2, Namit Ranjan1, Patrick Pedrioli2, Matthias Peter1 1 Institute of Biochemistry, ETH Zürich, Switzerland, 2SCILLS, University of Dundee, Scotland tRNA has a central role in biology as the adaptor between mRNA and the protein. Many modified nucleotides are found in tRNAs, and the modifications in or adjacent to the anticodon are thought to directly influence translation fidelity. In Saccharomyces cerevisiae and many other organisms the wobble Uridine (U34) of the anticodon of tRNALys (tKUUU), tRNAGln (tQUUG), and tRNAGlu (tEUUC) carries the modification 5-methoxy-carbonyl-methyl-2-thio (mcm5s2). Our laboratory and others identified the URM1-pathway responsible for the thiolation of U34, however the physiological significance of this modification is poorly understood. urm1Δ cells are sensitive to range of drugs and stress such as rapamycin, paromomycin, caffeine and diamide. Interestingly, overexpression of unmodified tQ and tK can differentially rescue urm1Δ cells from these sensitivities, indicating that lack of thiolation affects a distinct set of genes under these conditions. In order to identify the physiological substrates of thio-modification, we performed quantitative proteomics using SILAC for wild type and urm1Δ cells. Interestingly, we found that expression of several candidates enriched in tQ, tK and tE were down-regulated in urm1Δ cells, although their transcript levels as measured by Q-PCR are not affected. A specific defect in protein translation was also confirmed using specific reporter assays in vivo. To study the role of modified tRNAs during the decoding step of protein synthesis, we currently apply rapid quench-flow experiments to study binding of modified and unmodified tRNAs to the ribosome and the subsequent translocation step (in collaboration with M.V. Rodnina, MPI Göttingen). We hope that this study will help us to gain more insight into the role of modified tRNAs in protein translation in general, and in particular in response to different stress conditions.

Poster Session 2: Translational Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

391

Ribosome Profiling of Caulobacter crescentus Development

Jared Schrader1, Gene-Wei Li2, Jonathan Weissman2, Lucy Shapiro1 1 Stanford University, Stanford, CA, USA, 2University of California San Francisco, HHMI, San Francisco, CA USA Cellular differentiation is an essential process by which cells carrying identical genes develop into specialized cell types with distinct functions. An important goal in understanding cellular differentiationis to determine how the genetic information encoded in the genome is expressed properly in time and space to ensure the correct cell fate. The bacterium Caulobacter crescentus has proven to be an excellent model organism for studying cellular differentiation processes that occur as a function of the cell cycle. In Caulobacter each cell division is asymmetric, yielding a daughter with a different cell fate. This process requires rapid and specific changes in gene expression during the cell cycle that are controlled at many levels, including transcriptional regulation, transient DNA methylation, differential proteolysis and protein phospho-signaling. However, relatively little is known about the cell cycle control of mRNA translation. To understand the role of translation in Caulobacter cell cycle progression and cell differentiation, we are employing a recently developed method, ribosome profiling, to monitor genome-wide changes in translation throughout the cell cycle. Ribosomes are blocked during elongation by addition of chloramphenicol, and polysomes are treated with ribonuclease to partially digest the mRNAs yielding short fragments protected by monoribosomes. These ribosome-protected mRNA fragments are purified and prepared for high throughput sequencing to map the position of cellular ribosomes. Efforts to map the positions of ribosomes as a function of the cell cycle will be presented. This approach will provide a direct genome-wide insight into the control of mRNA translation in the Caulobacter cell cycle.

392 Biophysical Characterization Of The SLIP1-SLBP Complex Reveals New Insights Into The Role Of Oligomerization In Regulation Of Histone mRNAs

Nitin Bansal2, Minyou Zhang1,2, Aishwarya Bhaskar2, Patrick Itotia2, EunHee Lee3, Lyudmila Shlyakhtenko4, Joseph Luft2, TuKiet Lam5, Andrew Fritz6, Ronald Berezney6, Yuri Lyubchenko4, Walter Stafford3, Roopa Thapar2,1 1 Department of Structural Biology, SUNY at Buffalo, Buffalo, NY, USA, 2Hauptman Woodward Medical Research Institute, Buffalo, NY, USA, 3Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA, USA , 4Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA, 5WM Keck Foundation Biotechnology Resource Laboratory, Yale University, New Haven CT, USA , 6 Department of Biological Sciences, SUNY at Buffalo, Buffalo, NY, USA. In eukaryotes, the SLIP1-SLBP complex activates translation of replication-dependent histone mRNAs. As yet, no structural information is available for these proteins. The crystal structure of MIF4Gdb from Danio rerio that has 72% sequence identity to hSLIP1, has been solved as a protein of unknown function as part of the Structural Genomics Consortium. Each MIF4Gdb homodimer resembles the middle domain of eIF4G, consistent with MIF4Gdb being a molecular adaptor that could to help assemble components of the protein translation machinery. To gain insight into the mechanism of assembly of the SLBP-hSLIP1 complex at the 3’ UTR of histone mRNAs, we reconstituted the human SLBP-SLIP1 and MIF4Gdb-SLBP complexes using bacterially and baculovirus expressed proteins and characterized the biophysical properties of these complexes using a number of biophysical techniques. The stoichiometry of the complexes was examined using analytical ultracentrifugation and atomic force microscopy (AFM). Phosphorylated and unphosphorylated full-length SLBP proteins preferentially form a stoichiometric (2:2) high affinity heterotetramer with hSLIP1 that does not bind histone mRNA. In contrast, pre-binding of baculovirus expressed phosphorylated SLBP to histone mRNA can an “active” ternary complex with hSLIP1. Phosphorylation of the SLBP RNA binding domain is important for histone mRNA binding and also promotes dissociation of the heterotetramer to the SLIP1-SLBP heterodimer. Using alanine scanning mutagenesis and surface plasmon resonance we mapped the site of interaction on hSLIP1 with SLBP. A single hSLIP1 point mutant near the homodimer interface abolished interaction with SLBP in vitro and reduced histone mRNA abundance in vivo. Our studies using NMR spectroscopy and AFM reveal that uncomplexed SLBP is an intrinsically disordered phosphoprotein that fluctuates between folded, partly folded and unstructured states. The results suggest that oligomerization and phosphorylation of the SLBP-SLIP1 complex may regulate interaction with histone mRNA and histone gene expression. Poster Session 2: Translational Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

393

Novel Activities of Drosophila Pumilio Reveal New Mechanisms of mRNA Regulation

394

Characterizing the disorder in Tristetraprolin and its contribution to RNA binding specificity

Chase Weidmann, Aaron Goldstrohm University of Michigan PUF (Pumilio and FBF) proteins bind specific mRNAs to regulate protein expression. Repression of certain Drosophila mRNAs by the PUF protein Pumilio (Pum) is crucial for embryonic development, fertility, stem cell maintenance and memory formation. Pum is thought to recruit cofactors to elicit mRNA decay and inhibit translation of its targets. The RNA-binding domain (RBD) of Pum interacts with several cofactors and was previously thought to be responsible for mRNA regulation. In this research, we dissect Pum’s regulatory activities, the roles of cofactors and mechanisms that modulate Pum repression. We created a functional assay that specifically measures mRNA repression by Pum in Drosophila cells. Over-expression of Pum results in potent repression of protein expression and enhancement of mRNA degradation. Conversely, the RBD alone exhibits severely compromised repression. Because the RBD is not fully active, regions outside of the RBD must be required for activity. Indeed, we show that the N-terminus of Pum possesses robust repressive activity. Previously identified cofactors are not required for Pum activity, although the protein Nanos can enhance Pum repression. These facts indicate novel modes of repression. Three autonomous repression domains were identified in the protein’s N-terminus. The domain architecture and function of the Pum N-terminus is shared by human PUFs, PUM1 and PUM2. These repression domains are unique: they are not related to other known domains. In addition, we discovered two motifs that control Pum’s repressive activity via opposing inhibitory and activating functions. From these results and sequence analysis, we postulate that members of the PUF family have evolved novel mRNA regulatory mechanisms mediated by unique domains appended to the evolutionarily conserved PUF RNA binding domain. Previously, all PUFs were believed to function in the same manner, based on the conservation of the RNA binding domain. In contrast, our results show that for insect and vertebrate PUFs, the major regulatory functions are dictated by domains other than the RBD. Other PUF members may have evolved new functions through the addition of unique protein domains to the RBD. These sequences could confer unique functions that regulate translation, stability and localization of mRNAs. Laura Andersh, Francesca Massi UMass Medical School Worcester Tristetraprolin (TTP) and its homologues, TIS11b and TIS11d, are required for the post-transcriptional repression of over 100 different genes associated with chronic inflammation and cancer initiation and progression in mammals. The TTP family proteins are characterized by a tandem zinc finger (TZF) motif comprised of two CX8CX5CX3H zinccoordinating fingers separated by an 18 amino acid linker. TTP binds to essential cytokine and growth factor mRNAs at adenine-uridine rich elements (AREs) located in the 3’ untranslated regions (3’UTR), in order to promote their degradation. Data acquired by NMR spectroscopy indicate that the TZF domain of TTP is partially unfolded in its unbound state, but folds upon RNA binding. On the other hand, TIS11d is folded in both its RNA-free and bound states. For this reason, we used this pair of highly homologous proteins to investigate how the lack of a well-defined structure affects RNA-binding affinity and specificity. We constructed chimeric proteins of the TZF domains of TTP and TIS11d and characterized their structure and RNA-binding activity. We found that the N-terminal zinc finger and its interactions with the rest of the domain determine the structure and activity of the TZF domain.

Poster Session 2: Translational Regulation & RNA Turnover

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

395 High resolution analysis of the stable protein-associated RNA fraction in different nutrient starvation conditions in the yeast, Saccharomyces cerevisiae

Rodoniki Athanasiadou, David Gresham NYU, New York, (NY), USA Transcriptome composition changes accompany virtually any alteration in the environmental conditions experienced by cells. These changes lead to specific proteome changes, which in turn lead to specific physiological adaptations that allow the cell to survive in the new conditions and maintain homeostasis. In response to nutrient deprivation, eukaryotic cells enter a quiescent state, which is characterized by an arrest in cell division and distinct alterations in cell physiology and metabolism. The transcriptome of quiescent cells seems to partially depend on the exact environmental trigger for exiting the cell cycle. For example, the transcriptomes of cells starved for carbon, nitrogen or phosphate share many similarities reflecting the common physiology of the quiescent state, but there are also nutrient-specific starvation responses. When deprived of glucose, it has been demonstrated that quiescent cells hold a fraction of their transcriptome in an extractionresistant protease-labile form. This fraction of mRNAs is believed to be rapidly mobilized upon additional stress. We have characterized differences in the RNA composition of the extraction-resistant protease-labile fraction in quiescent cells starved for either carbon, nitrogen or phosphate. We performed high-throughput strand-specific RNA sequencing on RNA extracted in the presence or absence of proteases from quiescent cells. Our analysis provides new insight into the transcriptional programs associated with cell quiescence, including the role of differential UTR utilization and antisense transcripts, and broadens the definition of transcripts associated with protein complexes in quiescent cells with potential implications for our understanding of the function of P-bodies.

396

An eIF4E-Binding Protein Promotes mRNA Decapping and is Required for PUF Repression

Nathan Blewett, Aaron Goldstrohm University of Michigan, Ann Arbor, MI, U.S.A. PUF proteins are eukaryotic RNA-binding proteins that repress specific mRNAs to control important processes such as stem cell proliferation, development and memory formation. Multiple means of repression have been proposed, yet the co-repressors and mechanisms involved remain to be identified. In this study, we explored repression by two PUF proteins in S. cerevisiae. Puf5p and Puf4p repress the mRNA encoding the mating type endonuclease, HO. Both PUFs accelerate deadenylation of this mRNA, but Puf5p can also repress in the absence of deadenylation, indicating a second repression mechanism. We now report discovery of this deadenylation independent repression mechanism. We discovered that Puf5p specifically requires Eap1p to repress mRNAs. This functional requirement was specific, as Puf4p repression did not require Eap1p. Eap1p is an eIF4E-binding protein (4E-BP), belonging to a class of translational regulators proposed to inhibit initiation by binding eIF4E. Therefore, we explored the hypothesis that Eap1p inhibits translation of HO mRNA. Surprisingly, Eap1p had no effect on the translation state of HO or several other mRNAs. Instead, we discovered that Eap1p accelerates degradation of HO mRNA. Eap1p associates with HO mRNA. Deletion of Eap1 greatly stabilizes HO mRNA whereas over-expression of Eap1p enhances degradation. Eap1p interaction with eIF4E facilitates this activity. Deadenylation of HO mRNA is not affected by Eap1p. In contrast, our data demonstrate that Eap1p promotes decapping. When the EAP1 gene is deleted, decapping is dramatically reduced, resulting in the accumulation of capped mRNA with short poly(A) tails. Biochemical analysis reveals that Eap1p associates with Puf5p and decapping factors, providing physical connections linking HO mRNA repression and decapping. Based on these discoveries, we propose a new model: Puf5p binds to target mRNAs and recruits Eap1p and associated decapping factors to enhance decapping. The eIF4E binding activity of Eap1p facilitates decapping by targeting the decapping machinery to the 5’ cap and/or by altering the interaction of eIF4E with the 5’ cap structure. These findings have important implications for the functions of other 4E-BPs, which play roles in cancer, cell proliferation, development and neurological functions that were presumed to result from translational inhibition. Our results suggest that 4E-BPs may also inhibit gene expression by promoting specific mRNA degradation steps. Poster Session 2: RNA Turnover

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

397

Gene Expression Knock-Down by Forced Splicing-Dependent NMD

398

Psp1p Interacts with Rbp1p to Mediate the Recruitment of Rbp1p to P-bodies

Francesca Zammarchi, Gina Rocco, Sandra Vorlova, Clare LeFave, Luca Cartegni Memorial Sloan-Kettering Cancer Center, NY, (NY), USA Modified antisense oligonucleotides (ASOs) can be effectively used to re-direct alternative splicing patterns, to generate a desirable splicing isoform at the expense of another variant. This can lead to the expression of proteins with related structures, but changes in functions, varying from subtle modulation of activity to antagonistic or dominant negative properties. In some instances, alternative splicing patterns lead to the introduction of null-variants, for example when out-offrame changes or intron retention are associated to the incorporation of PrematureTermination Codons (PTCs) that trigger Nonsense-Mediated Decay (NMD). To exploit this aspect of splicing re-direction, we developed Forced Splicing-Dependent NMD (FSD-NMD), where ASOs are utilized to induce the frame-shifting skipping of a constitutive early exon in the Open Reading Frame (ORF) of a target gene. The ensuing PTC lead to the NMD-dependent effective ablation of the target. This approach is robust and leads to the specific, dose-dependent and rapid elimination >95% of endogenous RNA and protein targets. FSD-NMD can be used as a general alternative to RNAi or other knock-down approaches, as an additional method of knock-down to verify specificity of biological effects and as an ideal control of splicing re-direction experiments.

Lin-Chun Chang, Ying-Chieh Chu, Li-Ting Jang, Fang-Jen S Lee National Taiwan University, Taipei, Taiwan Rbp1p, a yeast RNA-binding protein, was shown to decrease the level of mitochondrial porin mRNA by enhancing its degradation. The recruitment of Rbp1p to P-bodies requires integrity of Rbp1-mRNP, through self-interaction and/ or unidentified signaling. We identified an Rbp1p-interacting protein, Psp1p by yeast two-hybrid screening. Psp1p was previously identified as a polymerase suppressor, and cause growth inhibition when overexpressed. However, the physiological function of Psp1p is largely unknown. In this study, we report that Psp1p helps the recruitment of Rbp1p to P-bodies. Psp1p directly interacts with Rbp1p through its C-terminal region and in an mRNA independent manner. GFP-tagged Psp1p co-localizes with mCherry-tagged Dhh1p and Dcp2p, indicating that Psp1p localizes to P-bodies. Psp1p is co-localized with Rbp1p when yeast is grown under glucose deprivation or treatment with KCl. Deletion of PSP1 delays the recruitment of Rbp1p to P-bodies. Furthermore, we demonstrate that expression of Psp1p, but not the Psp1p lacking Rbp1p interacting region, in psp1¿ mutants restored the recruitment of Rbp1p to P-bodies. Thus, we infer that Psp1p is involved in Rbp1p function by modulating the recruitment of Rbp1p to P-bodies.

Poster Session 2: RNA Turnover

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

399 Implication that SMG5-PNRC2 is a more functionally dominant complex than SMG5-SMG7 under normal conditions: Cellular substrates targeted for nonsense-mediated mRNA decay have their own preference for Upf1-binding players

Hana Cho1, Sisu Han1, Kyoung Mi Kim1, Seung Gu Park2, Sun Shim Choi2, Yoon Ki Kim1 1 Korea University, 2Kangwon National University In mammals, nonsense-mediated mRNA decay (NMD) functions in post-transcriptional gene regulation as well as mRNA surveillance. A key NMD factor Upf1 requires its binding players such as SMG5-7 or PNRC2, to trigger rapid mRNA degradation. Here we propose that Upf1, SMG5, and PNRC2 function all together at the same step of NMD and form a functionally predominant complex under normal conditions. Accordingly, microarray results reveal that SMG5dependent NMD substrates significantly overlap with PNRC2-dependent NMD substrates. Furthermore, our results reveal that, to some extent, endogenous NMD substrates have their own preferences for players.

400 The RNA Binding Protein Y14 Inhibits mRNA Decapping and Modulates Processing Body Formation

Tzu-Wei Chuang, Wei-Lun Chang, Kuo-Ming Lee, Woan-Yuh Tarn Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan The exon-junction complex (EJC), deposited on a newly spliced mRNA, plays an important role in subsequent mRNA metabolic events. Here, we show that an EJC core heterodimer, Y14/Magoh, specifically associates with mRNA degradation factors including the mRNA decapping complex and exoribonucleases, whereas another core factor, eIF4AIII/ MLN51, does not. Moreover, we found that overexpression of Y14 prolonged the half-life of a reporter mRNA. Our results demonstrated that Y14 interacted directly with the decapping factor Dcp2 and the 5’ cap structure of mRNAs via different but overlapping domains and that Y14 inhibited the mRNA decapping activity of Dcp2 in vitro. Therefore, Y14 may function independently of the EJC in preventing mRNA decapping and decay. Furthermore, we observed that depletion of Y14 disrupted the formation of processing bodies whereas overexpression of a phosphomimetic Y14 considerably increased the number of processing bodies. Coincidently, this phosphomimetic Y14 interacted strongly with the spliced mRNA and the mRNA degradation factors. In conclusion, this report provides unprecedented evidence for a role of Y14 in regulating mRNA degradation and processing body formation, and reinforces the influence of phosphorylation of Y14 on its activity in post-splicing mRNA metabolism.

Poster Session 2: RNA Turnover

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

401 Post-transcriptional Control of Stress Response mRNAs by a Zinc Finger Protein and AU-rich Elements

Dorothea Droll, Igor Minia, Aditi Singh, Abeer Fadda, Christine Clayton Zentrum für Molekulare Biologie, Heidelberg, Germany In Kinetoplastids, virtually all control of protein-coding gene expression is post-transcriptional, with mRNA degradation playing a central role. Unlike other eukaryotes, trypanosomes do not transcriptionally induce the expression of heat shock proteins upon elevated temperatures. It has previously been shown that heat shock protein transcripts are selectively stabilized under stress conditions, mediated by sequences in their 3’UTR, but the molecular mechanism had not been resolved. We show here that expression of the Trypanosoma brucei CCCH zinc finger protein ZC3H11 is induced upon heat shock and that it controls the levels of various stress-related mRNAs. ZC3H11 binds to mRNAs that encode homologues of the major heat shock protein and co-chaperone families. These transcripts share an AU-rich sequence element (UAUUAUUAU or similar) in their 3’UTRs. Steady-state levels of several targets, including the major cytoplasmic HSP70 mRNA, are decreased upon ZC3H11 depletion. Artificially tethering ZC3H11 to a reporter mRNA also increases reporter expression. In the bloodstream stage of the parasite (in the mammalian host, 37°C) ZC3H11 is essential and in the procyclic form (in the insect vector, 27°C) it is required for a heat shock response. Taken together our results indicate that the biological function of ZC3H11 is a selective stabilization of chaperone mRNAs upon stress. ZC3H11 interacts with a trypanosome homologue of yeast Mkt1, which in turn interacts with a trypanosome PBP1. In yeast, Mkt1 also interacts with Pbp1, which binds to the poly(A) binding protein Pab1. We propose that ZC3H11 stabilizes HSP70 and other stress-related mRNAs by recruiting MKT1 and PBP1 and thereby promoting retention of poly(A) binding protein on the mRNA.

402

The role of CNOT10 in the process of mRNA turnover

Valentin Faerber, Esteban Erben, Abeer Fadda, Christine Clayton Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Heidelberg, Germany mRNA degradation starts with deadenylation by the CAF1-NOT complex and is followed by either 5’->3’ or 3’->5’ digestion. In the protozoan parasite Trypanosoma brucei, gene expression is controlled mainly at the level of mRNA degradation. The trypanosome CAF1-NOT complex is built on the scaffold protein NOT1, to which the remaining subunits CAF1, NOT2, NOT5, DHH1 and a CNOT10-like protein are attached. CAF1 is the catalytic subunit and the functions of the other subunits are unclear. We investigated the role of trypanosome CNOT10. It is around 20 kDa smaller than its counterpart in humans and only has a sequence identity of around 22 %. We showed that CNOT10 is part of the complex and interacts directly with CAF1 and NOT1. Depletion of CNOT10 led to a proliferation defect, a decrease in NOT1 abundance and detachment of CAF1 from the complex. Using RNA sequencing to measure transcriptome-wide RNA degradation, we observed that in CNOT10- or CAF1- depleted cells mRNA degradation came to a halt. We speculate that the attachment of CAF1 to the NOT complex is required for its recruitment to mRNAs.

Poster Session 2: RNA Turnover

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

403 A novel form of nonsense-mediated mRNA decay revealed by studies on COL10A1 mutations

Yiwen Fang1,2, Jacqueline Tan1, Julian Mercer2, Shireen Lamandé1, John Bateman1 Murdoch Childrens Research Institute, Parkville, (Victoria), Australia, 2Deakin University, Burwood, (Victoria), Australia Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance system that functions in all eukaryotic cells to selectively target mRNA transcripts that carry premature termination codons (PTCs) for degradation. As a consequence, truncated dysfunctional proteins that would otherwise have catastrophic effects on cellular function will not be translated. We previously reported that COL10A1 NMD challenges the key rule of the generally accepted model of NMD, which is its dependence on a downstream multi-protein complex, the exon junction complex (EJC), deposited upstream of exonexon junctions during pre-mRNA splicing, to recognize PTCs. This therefore rules out NMD on transcripts carrying PTCs in the last exon. However, NMD-inducing PTC mutations from patients with metaphyseal chondrodysplasia, type Schmid, reside in the last exon of COL10A1. In addition, we also reported that this form of NMD occurs only in COL10A1 expressing chondrocytes from patients and not in lymphoblast or osteoblasts. To explore the mechanism of this intriguing form of NMD, we used RNAi to test the role of known NMD factors such as Upf1 in mouse chondrocytes transfected with a NMD-inducing PTC mutation. We also tested the need for pre-mRNA splicing and a downstream EJC by transfecting Col10a1 cDNA constructs with NMD-inducing PTC mutations into mouse chondrocytes and quantifying Col10a1 NMD. Lastly, we also characterized Col10a1 NMD in different cell types including fibroblasts, HeLa cells and chondrocytes. Our data revealed that Col10a1 NMD uniquely functions only in chondrocytes and it involves Upf1 but does not require splicing. This is an unusual phenomenon as splicing is required for NMD in most mammalian genes. To our surprise, Col10a1 NMD efficiency was also greater in cDNA constructs than in the genomic constructs. We propose that this novel form of Col10a1 “last-exon” NMD may be a failsafe pathway used by genes that may have similar gene structures as COL10A1 or have large 3’terminal exons. 1

404

Inhibition of Nonsense-mediated mRNA Decay by Retroviral RNA Elements

Zhiyun Ge, Stacey Baker, J. Robert Hogg National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA The nonsense-mediated mRNA decay pathway recognizes and degrades transcripts containing long 3’UTRs. Using an RNA-based affinity purification approach, we have observed that Upf1 accumulates in mRNPs in a 3’UTR lengthdependent manner. Preferential accumulation of Upf1 on mRNAs containing long 3’UTRs thereby increases the probability that Upf1 will interact with release factors during translation termination and initiate mRNA decay. We are investigating two classes of retroviral RNA elements capable of protecting mRNAs from Upf1-dependent decay. First, the RNA stability element (RSE) of Rous Sarcoma Virus stabilizes both retroviral and synthetic reporter mRNAs containing long 3’UTRs. This large RNA element resides immediately downstream of the viral gag termination codon, preventing it from being recognized as premature by the NMD machinery. Using biochemical and functional assays, we are exploring the mechanistic basis for the RSE’s protective activity. Second, we find that retroviral recoding elements that promote translational frameshifting or readthrough antagonize NMD at two distinct steps. Relatively frequent stop codon bypass can reduce steady-state accumulation of Upf1 in mRNPs, thereby disrupting its ability to monitor 3’UTR length. In addition, we find that less frequent readthrough events permit recovery of Upf1 binding to mRNPs but remain able to inhibit degradation of mRNAs containing long 3’UTRs. These findings point to a two-step model for Upf1-dependent recognition and decay of long 3’UTR-containing transcripts. In this model, Upf1 accumulation in mRNPs is a prerequisite for decay but is not sufficient to induce transcript destruction. Instead, the initiation of decay requires the completion of one or more additional rate-limiting steps susceptible to disruption by translational readthrough events. We are currently investigating the mechanism by which rare translational readthrough events prevent commitment to RNA decay despite efficient recruitment of Upf1.

Poster Session 2: RNA Turnover

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

405

EJC and NMD mutants in Cryptococcus neoformans

406

Analysis of archaeal RNase E-like protein, FAU-1 in Pyrococcus furiosus

Sara Gonzalez-Hilarion, Estelle Mogensen, Guilhem Janbon Insitut Pasteur, Paris, France The extent, the complexity, the regulation and the roles of splicing in fungi have been studied very little. Most of the studies published up today have been done on Saccharomyces cerevisiae which is an organism with very few introns (less than 5% of genes contain introns). In recent years, a large number of fungal genomes have been sequenced and the presence of predicted introns in genes has appeared less uncommon. For example, in Yarrowia lipolytica and in Schizosaccharomyces pombe up to 14.5% and 47% of genes contain introns, respectively. Cryptococcus neoformans is a capsular basidiomycete yeast responsible for opportunistic infections in patients presenting a cellular immunity deficiency (mainly AIDS patients). A striking feature of C. neoformans genome compared to those of other fungi is that genes are intron-rich. Indeed, more than 98% of them contain introns. Besides, alternative splicing has been reported to be very common in C. neoformans and intron retention represents its most common manifestation. Interestingly, alternative splicing has been shown to be involved in the post-transcriptional regulation of virulence factors gene expression, suggesting that the intron-dependent regulation of gene expression could play a role in C. neoformans biology and virulence. In order to get deeper insight into the RNA metabolism of this yeast, we carried out an analysis of the genomic data available for C. neoformans serotype D. We identified orthologous sequences for nearly all of the components of the RNA metabolism that are present in higher eukaryotes, including factors that are not found in S. cerevisiae. In particular, we identified homologous proteins for most of the components of the core Exon Junction Complex (EJC), and the nonsensemediated mRNA decay pathway (NMD) factors. Some of these components are highly conserved. For example, the eIF4A3 and Magoh proteins from C. neoformans share 94% and 82 % of similarity in their amino acid sequences with the human counterparts, respectively. We have constructed a collection of strains mutated in the different components of the EJC and the NMD pathway in C. neoformans. The mutant phenotypes and the consequences in the regulation of the expression of some virulence factors will be discussed.

Yoshiki Ikeda1,2, Shinnosuke Murakami1,2, Asako Sato1, Masaru Tomita1,2, Akio Kanai1,2 1 Inst. Adv. Biosci., Keio Univ., Tsuruoka, Yamagata, Japan, 2Syst. Biol. Prog. Grad. Sch. Media & Governance, Keio Univ., Tsuruoka, Yamagata, Japan Ribonuclease (RNase) is one of the key enzymes in RNA processing and degradation. In bacteria, RNase E is the major endoribonuclease and well studied especially in Escherichia coli (E. coli). For examples, RNase E is involved in processing of ribosomal RNA (rRNA), in maturation of transfer RNA (tRNA) and in degradation of messenger RNA (mRNA). While, RNases that involved in these steps are still not well understood in archaea. Previously, we identified an RNA-binding protein, called FAU-1, in the hyperthermophilic archaeon Pyrococcus furiosus. We showed that the N-terminal half of the FAU-1 protein had a degree of similarity (25%) with RNase E from E. coli, although we did not detect its nuclease activity (Kanai et al., Biochem. J., 2003). To analyze the role of the FAU-1, we constructed the recombinant FAU-1 protein with a His6 tagged sequence on plasmid DNA and FAU-1-His6 proteins were induced in E. coli. Then, we purified FAU-1-His6 proteins to near homogeneity by a His-affinity column chromatography, followed by a RESOURCE-Q ion exchange column chromatography. Using these fractions, we tested whether the FAU-1-His6 protein was able to digest known targets of E. coli RNase E or not. As a result, the fractions possessing the peak of the FAU-1 protein showed endoribonuclease activity againist pre-5S ribosomal RNA (rRNA) sequence and manganese ion enhanced its activity in vitro. It is proposed that 5’ end of the archaeal 5S rRNA is processed by tRNase Z (Hölzle et al., RNA, 2008), however a processing enzyme for its 3’ end remains unknown. Our result may suggest that the FAU-1 protein is a possible processing enzyme for the pre-5S rRNA. Now, we construct a series of plasmids carrying mutant fau-1 gene encoding internal deleted FAU-1 protein to confirm the RNase activity. These results will be discussed in the conference.

Poster Session 2: RNA Turnover

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

407

Copper tolerance of Saccharomyces cerevisiae Nonsense-Mediated mRNA decay mutants

408

Questioning the relevance of the interaction between Pab1 and eRF3

Xuya Wang, Bessie Kebaara Baylor University The nonsense-mediated mRNA (NMD) pathway, present in most eukaryotic cells is a specialized pathway that leads to the recognition and rapid degradation of mRNAs with premature termination codons and some natural mRNAs. The regulation of natural mRNAs by NMD has been observed in organisms ranging from yeast to humans. Global expression profiling of the effect of NMD on mRNA levels in Saccharomyces cerevisiae, Drosophila melanogaster and humans show that approximately 10% of the transcriptome is affected when NMD is inactivated. The regulation of natural mRNAs by NMD has been most extensively studied in S. cerevisiae and has been shown to have physiological consequences. First, nmd mutants have altered chromosome structure and grow at reduced rates on some non-fermentable carbon sources. Second, nmd mutants are sensitive to Calcofluor White, a cell wall disruptor. Third, CPA1 mRNA regulation by NMD is regulated by arginine, where addition of arginine causes ribosomal stalling at an upstream Open Reading Frame (uORF) termination codon, leading to the destabilization and NMD-mediated degradation of CPA1 mRNA. Fourth, thiamine starvation has been shown to induce alternative transcripts involved in thiamine metabolism and some of these transcripts are degraded in an NMD dependent manner. We have also shown that nmd mutants tolerate higher copper concentrations relative to wild-type yeast cells. The tolerance to high copper levels by nmd mutants is dependent on the presence of CTR2. CTR2 encodes a copper transporter of the vacuolar membrane that controls the flux of copper into the vacuole. Additional genes involved in copper metabolism in S. cerevisiae may also be regulated by NMD. We examined the mechanism targeting the CTR2 mRNA for NMD mediated degradation.

Marie Cerciat, Sylvain Roque, Isabelle Gaugue, Liliana Mora, Emmeline Huvelle, Miklos de Zamaroczy, Valerie Heurgue-Hamard, Stephanie Kervestin UPR9073 du CNRS, Institut de Biologie Physico-Chimique, Paris, France The relationship between translation and mRNA turnover is complex and still under investigation. After translation, the mechanism that initiates mRNA decay by activating deadenylation, and thus inhibiting translation, is still unknown. Pab1, the poly(A) binding protein, is at the crossroads between translation and mRNA turnover. It activates translation initiation by promoting the closed loop structure via eIF4G interaction. Pab1p is also thought to play a role in translation termination through its association with the translation termination factor eRF3. Finally, Pab1p is required for mRNA stabilization even though its terminal domain (406-477 aa) activates deadenylation. The interaction between Pab1p and eRF3 suggests the existence of a link between translation termination and mRNA decay, while protecting normal mRNA from the activation of the NMD pathway. To gain insight into the exact functions of Pab1-eRF3 interaction in S. cerevisiae, we generated yeast strains expressing eRF3 variants that do not interact with Pab1, either by deletion of the interaction domain (1-253 aa) or containing a triple mutation blocking Pab1 interaction. Analysis of the phenotype of these strains reveals that the interaction between Pab1p and eRF3 is not required for protecting an mRNA from deadenylation. These data also suggest that NMD activation is not solely based on the absence of interaction between Pab1 and eRF3. In parallel, we unravel a role of the eRF3 NM (1-253) domain in the efficiency of translation termination. We are now investigating if this function is linked to Pab1 interaction. Finally, to decipher if the interaction between Pab1 and eRF3 is relevant, we generated haploid strains containing Pab1 and eRF3 factors deleted for their mutual interaction domain. These strains are viable and we are now studying their phenotype.

Poster Session 2: RNA Turnover

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

409

Functional Analysis of Mtr4 as a Component of TRAMP Complex

410

The Human La-related Protein 4 mRNA Contains a Functional AU-rich Element

Yan Li, James Anderson Marquette University, Milwaukee, WI, USA Degradation of RNA can modulate gene expression or help cells eliminate old and abnormally formed RNAs. In Saccharomyces cerevisiae, nuclear RNA degradation is initiated by Trf4/5p-Air1/2p-Mtr4ppolyadenylation complex (TRAMP). TRAMP is composed of a poly (A)polymerase Tr4/5p, an RNA binding protein Air1/2p and a member of DExH RNA helicase superfamily, Mtr4p. Mtr4 plays an important role in TRAMP function that has been thus far linked to itsRNA-dependent ATPase and RNA-displacement activities. Previous studies in our lab indicate that Mtr4 is involved in degradation of hypomodified tRNAiMetby the nuclear exosome, as a component of TRAMP complex. Three-dimensional structure analysis of Mtr4 reveals a novel arch domain, which is important for 5.8s rRNA processing and exosome activation invivo, however, the arch domain role in TRAMP stability and activity is unknown. To begin studying function of Mtr4 arch domain, a library of Mtr4 arch domain mutants was generated by error-prone PCR mutagenesis and a dominant-negative screen in yeast. More than 90% of the mutants showed obvious reversal of hypomodified tRNAiMet degradation. 7of these Mtr4 single mutants were expressed in a well-characterized mtr4-20 strain, which is ATPase and helicase defective and scored for complementation of mtr4-20. Three of them failed to complement mtr4-20, suggesting that these mutations have reduced Mtr4 function invivo. Mutant S672N showing the strongest phenotype was cloned into a His-tagged expression vector to obtain purified recombinant Mtr4-S672N protein. ATPase assays showed that Mtr4-S672N exhibits wild-type ATPase activity in vitro. Because the ATPase activity of Mtr4 is dependent on RNA and a vast molar excess of tRNA is used in the ATPase assay, we hypothesized that the Arch-domain promotes RNA binding of Mtr4 to stimulate its ATPase activity. Mtr4-S672N has been expressed by autoinduction to obtain large amounts of purified protein for use in a coupled enzyme assay to measure ATP hydrolysis. Different RNA substrates will be used in this assay to test if RNA of different sequence or structure differentially stimulates Mtr4. Achromosomal Mtr4 knockout strain in a hypomodified tRNAiMet background was created to use plasmid shuffling for expression of Mtr4 mutants in a genetic background for tracking tRNAiMet degradation. Mtr4-S672N shows modest tRNAiMetdegradation defects. More experiments are being done to evaluate growth phenotypes of the Mtr4-S672N mutant in cells. In future studies, Mtr4 mutant activity for 5.8s rRNA processing, 5’ETS and CUTs degradation will be tested to more specifically assign a function to this part of the Arch domain of Mtr4. I will also test whether Mtr4 mutants interact with other TRAMP subunits normally by reconstituting TRAMP from recombinant proteins, and by doing coimmunoprecipitations from yeast cell extracts expressing epitope tagged TRAMP components. Sandy Mattijssen, Richard Maraia NIH / NICHD, Bethesda (MD), USA La-related protein 4 (LARP4) is a newly described member of the family of La-domain containing proteins. The La domain consists of an N-terminal La motif (LAM) and an RNA recognition motif (RRM). Unlike the genuine La-protein, which binds 3’ oligo(U) to stabilize its small nuclear RNA processing intermediates, LARP4 binds poly-A preferentially, apparently involved in mRNA stability. In addition, it interacts with the MLLE domain of polyA binding protein (PABP) via a variant PAM2 motif (PAMw) in its N-terminus [1]. LARP4 is found associated with a large number of diverse mRNAs and is important for general translation. Human LARP4 mRNA levels are very low consistent with preliminary evidence from ours and another lab that suggest tight regulation and that over expression may be toxic under some conditions and associated with cancer progression. Thus understanding the mechanisms by which LARP4 is acting and how its expression is regulated is important but unclear. By using the online ARED organism search engine, we found a possible AU-rich element (ARE) in the 3’-UTR of the LARP4 mRNA. AREs are mammalian cis-acting sequences of 50-150 nt that reside in the 3’-UTR of 5-8 % of human mRNAs. These mRNAs mostly encode proteins that need to be tightly regulated, e.g. transcription factors, cytokines and cell-cycle genes. It has been shown that in colorectal cancer (CRC), ARE-containing genes are enriched in expression. Interestingly, LARP4 is also found to be upregulated in CRC. To determine if the predicted LARP4 ARE is functional, we used a Tet-off system to monitor the stability of specific mRNAs. Inserting the putative LARP4 ARE in the 3’-UTR of a B-globin mRNA reporter decreased the half-life from over 6 hours to 100 minutes. Point mutation of the ARE rescued the stability of the reporter, demonstrating that it is functional. We are currently investigating under which conditions this mRNA is expressed. In conclusion, this is the first evidence that the mRNA stability of a La-related protein family member is regulated by an ARE. [1] Yang, R., Gaidamakov, S., Xie, J, Lee, J., Martino, L., Kozlov, G., Crawford, A., Russo, A., Conte, M., Gehring, K. and Maraia, R., “La-Related Protein 4 Binds Poly(A), Interacts with the Poly(A)-Binding Protein MLLE Domain via a Variant PAM2w Motif, and Can Promote mRNA Stability,” Mol. Cell Biol. (2011)

Poster Session 2: RNA Turnover

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

411 Dissecting the Mammalian Nonsense-Mediated mRNA Decay Pathway by a Combined Tethering / Knockdown Approach

Pamela Nicholson, Oliver Mühlemann University of Bern, Dept. of Chemistry and Biochemistry, Bern, Switzerland Nonsense-mediated mRNA decay (NMD) is an important quality control mechanism that degrades aberrant mRNAs with premature termination codons (PTCs), but also contributes to post-transcriptional gene regulation by targeting 2 – 10% of the cell’s physiological mRNAs. According to current models (Amrani et al., NRM 2006; Nicholson et al., CMLS 2010), NMD occurs on mRNAs with the stop codon in an unfavorable environment for efficient translation termination. Although the phenomenon of NMD and its impact on gene expression and genetic diseases is well documented, the understanding of the underlying molecular mechanisms is still incomplete. The list of factors required for mammalian NMD comprises more than a dozen proteins, which assemble on target mRNAs into a dynamic complex that finally recruits RNA nucleases. The interactions among most of the NMD factors have been thoroughly characterized and for some complexes, even crystal structures are available. However, the temporal order of the protein-protein interactions on the target mRNA are not well understood. We combine MS2-mediated tethering of the NMD factors UPF1, UPF2, UPF3b, SMG5, SMG6, SMG7 and PNRC2 with RNAi-mediated knockdowns of other NMD factors. The simplistic rationale for this approach is that NMD of the reporter transcript triggered by direct tethering of a NMD factor should bypass the requirement for factors acting upstream in the pathway, but still be dependent on those factors with a function downstream of the tethered protein. In addition to the wild-type proteins, we also tether mutant versions of UPF1, UPF2, UPF3b, SMG5, SMG6 and SMG7. The testing of all possible combinations is ongoing, but we have already obtained some interesting results. The SMG6 PIN domain and its endonuclease activity are necessary but not sufficient to induce RNA decay in the tethering assay, and knockdown of UPF1 or SMG1 prevent tethered full-length SMG6 from degrading the reporter transcript, suggesting that the presence of phosphorylated UPF1 is required for SMG6 to cleave the mRNA. On the other hand, tethered wild-type UPF1 can induce decay independently of SMG6, indicating the existence of an alternative decay pathway. However, tethering of a UPF1 mutation that cannot bind SMG5/SMG7 anymore requires the presence of SMG6, which supports the model of two redundant degradation pathways contributing to mammlian NMD, one involving the endonuclease SMG6 and the other requiring SMG5/SMG7.

412

Biochemical Localization of mRNA Repression and Degradation Factors

Susanne Brettschneider, Tracy Nissan Umeå University, Umeå, Sweden Messenger RNA that sediment deep within sucrose gradients are often assumed to be associated with many ribosomes and therefore highly translated. However, we have observed mRNA that rapidly sediments, but is not translated. Since non-translating mRNA are enriched in RNA granules, one possibility is that the mRNA migrate rapidly in gradients due to their association with the granules, such as cytoplasmic P-Bodies. They contain translational repressors, nontranslating mRNA and decay intermediates as well as many components of the mRNA degradation machinery. As they are often found in P-Bodies, we have examined many proteins involved in translation repression and mRNA decay in yeast. Many of these proteins are found in the same fractions as the mRNA. However, the sedimentation of these proteins is independent of visible RNA aggregation in granules and does not require interaction with RNA. Instead, the sedimentation of the translational repression and mRNA decay factors was due to membrane association, raising the possibility that membranes may act as a site for recruitment of mRNA for repression or degradation.

Poster Session 2: RNA Turnover

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

413

Degradation of rRNA in Bacteria

Anton Paier, Tanel Tenson, Ülo Maiväli Institute of Technology, University of Tartu, Estonia Ribosomes are ribozymes that synthesize all cellular proteins. As their function is a key factor for cell growth and survival, elucidating their turnover and stability is of utmost importance for understanding cell physiology. Ribosomes are widely believed to be stable in bacteria, but this notion has until recently never been tested in bacteria growing in nutrient rich media. We developed an experimental system to directly measure ribosomal stability in Escherichia coli. We showed that ribosomes are stable when cells are grown at a constant rate in the exponential phase but more than half of the ribosomes made during exponential growth are degraded during slowing of culture growth preceding the entry into stationary phase. Ribosomes are stable for many hours in the stationary phase. Furthermore we are studying ribosomal stability in presence of mutations in intersubunit bridges. We found that at constant growth rate, differently from what occurs in WT ribosomes, the 23S rRNA containing is degraded. This suggests that intersubunit bridges play an important role in ribosome stability.

414 Structural and Functional Analysis of the Rrp6-Rrp47 Interaction in RNA Degradation Processes

Benjamin Schuch1, Monika Feigenbutz2, Claire Basquin1, Phil Mitchell2, Elena Conti1 1 Max Planck Institute of Biochemistry, Martinsried, Germany, 2University of Sheffield, Sheffield, UK The eukaryotic exosome is a multiprotein complex involved in the processing, turnover and surveillance of many RNAs in the cell, both in the cytoplasm and the nucleus. The nine core subunits that form the barrel-like structure of the eukaryotic exosome are catalytically inactive. Two associated RNases, Rrp44 and Rrp6, endow the exosome with hydrolytic nuclease activities. Rrp6 is restricted to the nuclear compartment in yeast and interacts with the protein Rrp47 (also known as Lrp1 and as C1D in higher eukaryotes). Rrp6 has a multidomain architecture. The N-terminal domain binds Rrp47, the central domain has 3’ exoribonuclease activity and the C-terminal domain binds to the exosome. The structures of the central domain of yeast and human Rrp6 have been reported, showing it has an RNase D core flanked by an HRDC domain. The catalytic activity of Rrp6 is promoted by Rrp47, but the mechanisms are currently unclear. On one hand, Rrp47 is thought to facilitate binding of RNA substrates and to target them to the nuclease active site. On the other hand, Rrp47 is thought to be a binding platform to recruit proteins that facilitate RNA processing. To obtain insights into the contribution of Rrp47 to Rrp6 activity and function, we have solved the X-ray crystal structure of a complex containing the interacting domains of Rrp6 and Rrp47 at 2.6 Å resolution. The structure shows that the two proteins are intertwined and together form a globular domain. Using the structural information, we are currently investigating the Rrp6-Rrp47 interaction both in vitro (using biochemical and biophysical approaches) and in vivo (using molecular genetics techniques) in yeast.

Poster Session 2: RNA Turnover

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

415

Regulation of CAF1-dependant deadenylation under stress conditions

416

D-foci - Sites for RNA Decay in Human Mitochondria

Sahil Sharma, Georg Stoecklin German Cancer Research Center, Heidelberg, Germany Gene expression is regulated at multiple levels, wherein cells integrate extra- and intracellular signals in order to properly decode the information in the genome. The control of mRNA degradation is a major mechanism by which gene expression can be rapidly turned on and off. The removal of poly-A tails is a key event that initiates degradation of most mRNAs. There is substantial evidence that under stress conditions, many mRNAs are stabilised, yet the mechanism remains elusive. In this study, we investigate the regulation of the major cytoplasmic deadenylase in metazoans, CAF1. Using two different assays, 1) a tethering approach whereby human (h)CAF1a is forced to bind a reporter mRNA within cells, and 2) an in vitro deadenylation assay, for which hCAF1a is purified from cells that were subjected to stress, we observed that hCAF1a is regulated. Arsenite-induced oxidative stress accelerated deadenylation via hCAF1a, whereas anisomycin-induced ribotoxic stress slows deadenylation. Using Mass spectrometry, we identified more stable interactions within the components of CCR4-CAF-NOT complex after arsenite treatment. We also discovered phosphorylation on hCAF1a at serine 201 under anisomycin stress. Mutating the residue to alanine or aspartic acid abolished interactions with BTG2, an activator of hCAF1a. This demonstrates the importance of serine 201. In the tethering assay, mutations at serine 201 caused hCAF1a to be less efficient when BTG2 was over-expressed. Co-immunoprecipitation experiments confirmed that post-translational modifications regulate hCAF1a by modulating the interaction with BTG2. In summary, we describe two different mechanisms by which CAF1-dependant deadenylation is regulated under different stress conditions.

Lukasz Borowski1, Andrzej Dziembowski1,2, Monika Hejnowicz2, Piotr Stepien1,2, Roman Szczesny1,2 1 Institute of Genetics and Biotechnology, Warsaw University, Warsaw, Poland , 2Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland RNA decay is usually mediated by protein complexes and can occur in specific foci like P-bodies in the cytoplasm of eukaryotes. In human mitochondria nothing is known about the spatial organization of the RNA decay machinery and the ribonuclease responsible for RNA degradation has been unidentified. We demonstrate that silencing of human polynucleotide phosphorylase (PNPase) causes accumulation of RNA decay intermediates and increases the half-life of mitochondrial mRNA. Combination of fluorescence lifetime imaging microscopy with Förster resonance energy transfer as well as bimolecular fluorescence complementation experiments proves that PNPase and hSuv3p helicase (Suv3, hSuv3, SUPV3L1) form a complex in vivo. This complex, referred to as the degradosome, is formed only in specific foci which co-localize with mitochondrial RNA and nucleoids. Notably, interaction between PNPase and hSuv3p is essential for efficient mitochondrial RNA degradation. This provides indirect evidence that mitochondrial RNA decay takes place in foci present in the mitochondrial matrix, which we shall call D-foci as they contain the degradosome.

Poster Session 2: RNA Turnover

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

417

Proteasome-independent Ubiquitin Signaling in HuR-mediated mRNA Stability Control

Hua-Lin Zhou, Hua Lou Case Western Reserve University, Cleveland, Ohio, USA

RNA-binding proteins (RBPs) recognize specific cis-regulatory RNA elements to form ribonucleoprotein (RNP) complexes, which control every aspect of the life journey of an mRNA molecule, from its production, transport, function in translation, to turnover. The assembly and disassembly of mRNP are dynamic processes that occur during the entire life of an RNA molecule. However, how these processes are regulated inpost-transcriptional control of gene expression is not understood. HuR plays a vital role in mammalian stress response, affecting proliferation and survival of cells that are exposed to stress conditions. It has been demonstrated that various stress conditions induce a massive change of association of HuR with a large number of its target RNAs, which usually contain AU-rich elements (AREs), and consequently affect mRNA stability and/or translation of the target mRNAs. Thus, HuR provides an excellent model system to study the regulatory mechanisms of the dynamic assembly/ disassembly of mRNPs in post-transcriptional control. Recently, we discovered a novel connection between HuR and an AAA ATPase p97/VCP as well as the ubiquitin receptor UBXD8. Importantly, siRNA knockdown of UBXD8 or p97/VCP in HeLa cells increased mRNA half-life of p21, a previously well established HuR target, leading to accumulation of higher levels of p21 mRNA and protein. We showed that this effect depends on the HuR protein and the ARE in the 3’-UTR of p21. An RNA-IP experiment indicated that knockdown of UBXD8 or p97 increased association of HuR with the endogenous p21 mRNA, while over-expression of UBXD8 or p97 decreased this association. Next, we investigated how the interaction between HuR and p97-UBXD8 modulates association of HuR with p21mRNA to control mRNA stability and made several important findings. We found that HuR can be ubiquitinated in a proteasome-independent fashion and the ubiquitin chain forms through lysine 29 (K29) on ubiquitin. Extensive domain and point mutagenesis analyses of the HuR protein indicated that K313 and K326 on the RRM3 of HuR are responsible for the observed HuR ubiquitination. We then designed in vitro mRNP disassembly assay to test the hypothesis that the p97–UBXD8 complex regulate mRNA stability through releasing ubiquitinated HuR from p21 mRNA. We found that ubiquitinated HuR, but not unubiquitinated HuR, was specifically released from the mRNP formed on p21 RNA by p97–UBXD8 complex. Importantly, the release is dependent on ATP, the ATPase activity of p97, as well as the K29-linked polyubiquitin chain attached to lysines 313 and 326 of the HuR protein. Disruption of the two lysines on HuR blocked releasing of HuR from p21 RNA in vitro and increased p21 mRNA stability in cells. These studies represent the first example of mRNA stability regulation by proteasome-independent ubiquitin signaling. They reveal a new paradigm in RNA biology:ubiquitintination of RBPs in modulating their activity.

418 Microsecond Timescale Molecular Dynamics SImulation of Ligand-Induced Strand Migration in an S-adenosyl methionine Riboswitch

fareed aboul-ela1, Wei Huang1,4, Vamsi Boyapati1, Joohyun Kim2, Shantenu Jha3 1 Louisiana State University, Baton Rouge, LA, USA (former), 2Louisiana State University, Baton Rouge, LA, USA , 3Rutgers University, Piscataway, NJ, USA, 4Case Western Reserve University, Cleveland, OH (current) Folding dynamics is crucial for RNA function. Riboswitches are a classic example. A typical riboswitch senses the cellular concentration of a small molecule. By refolding itself into a new structure, the riboswitch converts that information into changes in rates for synthesis of related metabolites. Understanding how the small molecule physically changes RNA structure can help us to target riboswitches, which occur mainly in bacteria, for drug design, or to engineer new riboswitches. This understanding has been blocked because 1) we cannot view intermediate stages experimentally and 2) simulations cannot reach the timescale for the structural conversion. Recent advances in RNA structure modelling enable us to model intermediate states. A new computer specialized for long timescale molecular dynamics (MD) simulations, called Anton, helps us to extend the simulation timescale. We modelled intermediate riboswitch structures for the S-Adenosyl Methionine (SAM)-I riboswitch, focusing on a reduced segment of the structure-switching region, in order to reduce the time required for a transition. Experimentally, we showed that model RNAs mimicking the intermediate conformations bind the ligand in a manner similar to that of the SAMbound aptamer, allowing us to use X-ray coordinates to model the core of the starting RNA structure. In an unprecedented three μs trajectory, we observed a strand migration event. Altogether, a series of long MD trajectories in the presence and absence of SAM indicate that the presence of the ligand tends to extend the length of the P1 helix, characterisitic of the transcription OFF state, at the expense of the antiterminator helix, characteristic of the transcription ON state. References: 1. Wei Huang, Fareed Aboul-ela, Shantenu Jha, and Joohyun Kim, A Role of Small Molecule in Strand Switching from RNA Modeling and Molecular Dynamics Simulation. In preparation 2. Lindorff-Larsen, K, Piana, S, Dror, RO, Shaw, DE. 2011. How Fast-Folding Proteins Fold. Science, 334: 517-520. 3. Vamsi Krishna Boyapati, Wei Huang, Jessica Spedale, and Fareed Aboul-ela, Basis for Ligand Discrimination between ON and OFF State Riboswitch Conformations: the Case of the SAM-I Riboswitch. RNA, 2012, In Press. 4. Wei Huang, Joohyun Kim, Shantenu Jha, Fareed Aboul-ela, Conformational Heterogeneity of the SAM-I Riboswitch Transcriptional ON State: A Chaperone-like Role for S-adenosylmethionine. Journal of Molecular Biology, 2012, In Press Poster Session 2: RNA Turnover & RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

419

Highlighting Features of the B-Z Transition in DNA

Heidi Alvey, Hashim Al-Hashimi University of Michigan, Ann Arbor, (Michigan), USA Z-DNA’s role in nature is becoming more defined since its discovery four decades ago. The mechanism of B-Z transition, however, remains unclear. Conversion of B-DNA into Z-DNA requires anti-to-syn transition of purine bases. Recently, we showed that purine bases in canonical duplex DNA transiently sample the syn conformation to form Hoogsteen base-pairs. Here, we report studies directed at exploring the potential role of Hoogsteen base-pairs as intermediates along the B-to-Z DNA transition pathway. We combine NMR relaxation dispersion for characterizing lowly populated (less than 5%), short-lived (lifetimes less than 1.5 ms) transient Hoogsteen states in GC rich DNA sequences with single atom substitutions, that either favor or destabilize Hoogsteen base-pairs, and CD experiments for kinetic and thermodynamic measurements of the B-to-Z transition. Our results suggest a link between the tendency to spontaneously form Hoogsteen base-pairs and to undergo the B-to-Z transition.

420 SAXS and ITC Analyses Reveal Important Role of the K-turn in Folding of Glycine Riboswitches

Nathan Baird, Adrian Ferré-D’Amaré National Heart, Lung and Blood Institute, Bethesda, MD USA The glycine riboswitch is widely distributed in bacteria. Most examples are comprised of two homologous glycine binding aptamer domains repeated in tandem. Although this riboswitch has been the subject of considerable biochemical, biophysical and crystallographic characterization, the modest affinity for glycine of tandem aptamer domains under physiological conditions has remained a puzzle. Recently, two groups independently reported the discovery of a conserved kink turn (K-turn) element that forms between the linker joining the tandem aptamer domains, and a hitherto undetected segment 5’ to the first aptamer domain1,2. Here, we investigate the effect of this K-turn in modulating global structural responses to glycine binding by the F. nucleatum and B. subtilis tandem glycine riboswitches. Using small-angle X-ray scattering (SAXS) we probe size and shape changes of these riboswitches upon ligand binding, in the presence of near-physiological magnesium cation concentrations. In the absence of glycine, inclusion of the K-turn results in a moderate compaction of the riboswitches relative to constructs lacking the K-turn. The addition of glycine folds the F. nucleatum RNA to maximal compaction, regardless of whether the K-turn is included. In contrast, the K-turn is required for complete compaction of the B. subtilis RNA. We recently reported the first structures of bacterial K-turn binding proteins, B. subtilis YbxF and YlxQ3. To evaluate a potential role for YbxF in glycine riboswitch function, we carried out isothermal titration calorimetry (ITC) experiments yielding the thermodynamic parameters of glycine binding in the absence and presence of this cognate K-turn-binding protein. Binding YbxF to the K-turn-containing B. subtilis glycine riboswitch provides a modest (~2-fold) increase in binding affinity to glycine. This is the first demonstration of a cognate RNP interaction assisting in ligand binding by a riboswitch. A similar improvement in binding affinity is observed when YbxF is bound to the F. nucleatum riboswitch. Thus, the formation of the K-turn in glycine riboswitches, and its stabilization by K-turn binding proteins, leads to higher affinity glycine binding under physiological conditions. 1. Kladwang W, Chou FC, Das R. J Am Chem Soc. 134:1404-7, 2012. 2. Sherman EM, Esquiaqui J, Elsayed G, Ye JD. RNA. 18:496-507, 2012. 3. Baird NJ, Zhang J, Hamma T, Ferré-D’Amaré AR. RNA. 18:759-70, 2012. This research was supported by the Intramural Research Program of the NIH, NHLBI. Poster Session 2: RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

421

Structural Similarities at the Active Sites of the VS and Hairpin Ribozymes

422

Correcting Pervasive Errors in RNA Crystallography

Eric Bonneau1, Genevieve Desjardins1, Nicolas Girard1, Jerome Boisbouvier2, Pascale Legault1 1 Département de Biochimie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, Québec, H3C 3J7, Canada, 2Comissariat à l’énergie atomique (CEA), Centre National de la Recherche Scientifique (CNRS) and Institut de la Biologie Structurale Jean-Pierre Ebel, Université Joseph-Fourier, Grenoble, France. The VS ribozyme is a naturally occurring nucleolytic ribozyme for which there is no complete high-resolution structure determined yet. The VS ribozyme adopts an H-shaped secondary structure with sixstem-loops (SL): SLI contains the cleavage site and is considered as the substrate domain, whereas SLII-SLVI forms the catalytic domain. Although the VS ribozyme has a unique secondary structure, it was postulated that its active site is formed by the interaction of two internal loops in a manner similar to the hairpin ribozyme loops A and B. In the VS ribozyme context, the A730 loop of SLVI must interact with the SLI internal loop to form the active site. The SLI cleavage site internal loop contains the important G638 residue proposed to act as the general base in the cleavage mechanism, whereas the A730 loop contains the important A756 residue proposed to act as the general acid. In order to get insights into the VS ribozyme active site, we determined the high-resolution NMR structure of an RNA fragment containing the A730 loop. The structure showed that the A730 loop adopts an S-turn motif stabilized by a cis-WC-WC G-A base pair in the presence of Mg2+-ions. In this structure, residue A756 protrudes into a broadened minor groove with its Watson-Crick face exposed for catalysis. Interestingly, an S-turn motif is found within the hairpin ribozyme active site and also helps protrude an adenine residue proposed to act as the general acid in the cleavage mechanism. Furthermore, additional structural similarities are found between the cleavage site internal loops of the VS and hairpin ribozymes, indicating that these two ribozymes share important structural features at the active site. Given these structural similarities, we modelled the pre-catalytic active site of the Neurospora VS ribozyme based on the crystal structure of the hairpin ribozyme.

Fang-Chieh Chou, Parin Sripakdeevong, Rhiju Das Stanford University, USA RNA crystallographic models contain pervasive local errors due to ambiguities in manually fitting RNA backbones into experimental density maps. To resolve these ambiguities, we have developed a new Rosetta structure prediction tool (ERRASER: Enumerative Real-space Refinement ASsisted by Electron density under Rosetta) and coupled it to MolProbity validation and PHENIX diffraction-based refinement. On 22 crystallographic datasets for ribozymes, riboswitches, ribosomal domains, and other RNAs, ERRASER/PHENIX corrects the majority of identifiable sugar pucker errors, steric clashes, suspicious backbone rotamers, and incorrect bond lengths/angles, while, on average, improving R(free) correlation to set-aside diffraction data by 0.008. As further confirmation of improved accuracy, the refinement enhances agreement between models with low resolution diffraction data and subsequently released high resolution models. Finally, we demonstrate successful application of ERRASER on coordinates for an entire 30S ribosomal subunit. By rapidly and systematically disambiguating RNA model fitting, ERRASER enables RNA crystallography with significantly fewer errors.

Poster Session 2: RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

423

Structure and folding of a rare kink turn with an A•A pair at the 2b•2n position

424

SHAPE Analysis of VSV RNA Transcipts

Peter Daldrop1, Kersten Schroeder2, Scott McPhee1, David M. J. J. Lilley1 1 University of Dundee, Dundee, UK, 2LECOM, Bradenton (Florida), USA Kink-turns, present in many functional RNA species, alter the axial trajectory of duplex RNA by the introduction of a tight kink. The typical k-turn comprises a bulge followed by consecutive G•A and A•G pairs. The minor groove edges of the adenine bases are directed into the minor groove of the opposing helix, in a classic A-minor interaction. The kinked conformation may be stabilized by 1) the presence of metal ions, 2) the binding of proteins such as L7Ae, and 3) tertiary interactions in the RNA. Natural k-turn sequences exhibit significant departure from the consensus, even in the A•G pairs despite their critical role in the structure. Kt-23 is found in the small ribosomal subunit of ribosomes from all domains of life. We analysed > 6,000 Kt-23 sequences, finding that the frequency of occurrence at the 2n position (i.e. the G of the second G•A pair in the consensus k-turn, present on the non-bulged strand) is U>C>G>A. Less than 1% of sequences have an A at the 2n position. This raises the question of how the k-turn is stabilized with a putative A•A pair at the 2b•2n position, yet no structural data were available for such a k-turn. Kt-23 of Thelohania solenopsae is an example of such a k-turn. This sequence is very weakly induced to fold into the kinked conformation as a simple duplex RNA by addition of divalent metal ions, but the same species could be fully folding by binding Archeoglobus fulgidus L7Ae protein. Folding could also be induced by tertiary interactions; a hybrid SAM-I riboswitch containing the T. solenopsae k-turn bound retained the ability to bind its SAM ligand. We have solved the crystal structure of the SAM-I riboswitch carrying the T. solenopsae Kt-23 sequence, finding that the k-turn adopts the normal k-turn fold with retention of critical hydrogen bonds. However, the unusual A2n•A2b pair lacks the hydrogen bonds between the 2b sugar edge and the 2n Hoogsteen edge that are characteristic of conventional k-turns. Thus T. solenopsae Kt-23 is a genuine k-turn, providing insight into how the k-turn motif can adapt to deviations form the canonical sequence. K. T. Schroeder and D. M. J. Lilley Ion-induced folding of a kink turn that departs from the conventional sequence Nucleic Acids Res. 37, 7281-7289 (2009). K. T. Schroeder, S. A. McPhee, J. Ouellet and D. M. J. Lilley A structural database for k-turn motifs in RNA RNA 16, 14631468 (2010). K. T. Schroeder, P. Daldrop and D. M. J. Lilley. RNA tertiary interactions in a riboswitch stabilize the structure of a kink turn. Structure 19, 1233-1240 (2011).

Adam Davidson, Rebecca Alexander Wake Forest University, Winston-Salem, (NC), USA Vesicular Stomatitis Virus (VSV) is a non-segmented negative strand virus of the Rhabdoviridae family. Genomic VSV is reverse transcribed by virally encoded RNA-dependent RNA polymerase to generate the mRNA responsible for protein synthesis in the host cell. As a weak human pathogen, it is a model organism for the more infectious viral agents influenza A and rabies. Additionally, VSV is a player in oncolytic viriotherapy. Through inhibition of host cell machinery during viral transcription, VSV is capable of tumor suppression through cellular hypoxia, inflammatory cytokine release and direct cell lysis. Efforts are underway in our lab to understand the structure of the VSV genome using SHAPE (Selective 2’-Hydroxyl acylation Analyzed by Primer Extension) analysis of both genomic and antigenomic RNA transcripts. For both the sense and antisense structures, current analysis is centered on the nucleocapsid gene encoding the nucleoprotein (N). Following proof-of-principle structure analysis on transcripts, we will move to structure probing of authentic viral RNA isolated from viral particles and to direct in virio structure probing.

Poster Session 2: RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

425 Fragment-Based Drug Discovery: X-ray Structures of the TPP Riboswitch in Complex with Lead Compounds

Katherine Deigan1,2, Alison Smith3, Chris Abell2, Adrian Ferré-D’Amaré1 National Heart, Lung and Blood Institute, NIH, Bethesda, MD, USA, 2Department of Chemistry, University of Cambridge, Cambridge, UK, 3Department of Plant Sciences, University of Cambridge, Cambridge, UK Riboswitches are structured regions of mRNA that control gene expression as a function of the intracellular concentration of a variety of small molecules. Riboswitches control essential genes in many pathogenic bacteria and are attractive targets for the development of novel antibiotics. The TPP-binding riboswitch controls transcription, translation, or splicing in response to binding thiamine pyrophosphate (TPP) and was the first riboswitch to be discovered in eukaryotes, including fungi, algae and higher plants. The riboswitch folds into a three-helix junction. Two of the helices are bridged by the bound TPP, with one helix responsible for recognizing the aminopyrimidine of TPP and another binding to the pyrophosphate as a chelate with two divalent cations. Previously, fragment-based screening methods identified multiple small molecule “fragments” that displace thiamine from the E. coli thiM TPP-binding riboswitch in equilibrium dialysis experiments; these fragments have KD’s in the micromolar range1. We have determined crystal structures of the E. coli thiM riboswitch in complex with a number of these lead compounds, suggesting that crystals can be grown and structures determined for the riboswitch in complex with small molecules with weak affinity. These results provide new direction for elaboration of the fragments as well as insights into the minimal requirement for folding of the TPP riboswitch. 1. Cressina, E., et al. Chem. Sci. 2011, 2, 157-165.

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426

Predicting RNA-RNA Interactions with Consideration for Competing Self Structure

Laura DiChiacchio, David Mathews University of Rochester, School of Medicine and Dentistry, Rochester, NY, US RNA duplex formation is an important step in catalytic and regulatory processes in the cell, including pathways of mRNA degradation, post-transcriptional editing, and small nuclear RNA assembly. RNA-RNA interactions are often complex, including intramolecular secondary structures that compete with intermolecular structure formation. Computational methods for predicting secondary structure of single-stranded RNA have become both sophisticated and accurate, and are widely used to evaluate probable RNA structures. Prediction of RNA-RNA interactions, however, has remained a computational challenge, and new approaches to model the competition between intramolecular and intermolecular structure formation are needed. Here, we describe a novel algorithm for predicting RNA-RNA interactions that utilizes a heuristic for considering the competition between single stranded-self structure and bimolecular structure. This extension to standard free energy minimization uses a single-stranded partition function calculation to determine the probability that each nucleotide is involved in self-structure. A pseudo-energy penalty is added to each nucleotide forming an intermolecular base pair, where the energy is scaled by the nucleotide’s probability of forming self-structure. To test the algorithm, we built a database of seventeen sets of interacting RNA sequences, as determined by sequence comparison or experiment. We find that this algorithm provides a statistically significant increase in sensitivity from 44% to 73% as compared to prior available algorithms. We conclude that this method for considering the competition between intramolecular and intermolecular structure formation provides a benefit in the modeling of RNA-RNA interactions.

Poster Session 2: RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

427

Abstract Withdrawn

428

Previously Overlooked CoA Aptamers Revealed by High-Throughput Analysis

Kyle Hill, Mark Ditzler, Collin Luebbert, Donald Burke University of Missouri, Columbia, MO, USA RNA aptamers that stabilize the pantotheine arm of coenzyme A are excellent candidates for engineering ribozymes that catalyze acyl transfer. CoA is structurally composed of an adenosine group and a pantotheine arm, which together act as an acylgroup carrier for metabolic reactions in all organisms. Enzymes such as HistoneAcetyltransferase (HAT) make extensive interactions with the pantotheine arm of CoA to reduce conformational entropy during acyltransfer catalysis. The identification and characterization of a class of RNA aptamers that preferentially bind the pantotheine arm in place of a protein enzyme would contribute to our understanding of RNA molecule interactions. Through Systematic Evolution of Ligands by Exponential Enrichment (SELEX), RNA aptamers have been identified previously to interact with CoA immobilized through either the adenine N6 (Burke 1998) or via the sulfur on the pantotheine arm (Saran2003). The dominant species of aptamer revealed through positive selection steps had strong affinity for adenosine or AMP moieties however subtractive selection with AMP washes removed most RNA with this affinity. While some of these species may interact with the pantotheine arm, no enrichment of specific sequences or structural motifs were evident from low-throughput sequence (LTS) data. Here we revisit all of the CoA aptamer populations using high-throughput sequencing (HTS). HTS provides an opportunity to identify population dynamics and rare aptamers that would otherwise be obscured, including unambiguous enrichment of sequence and structural motifs along multiple selection trajectories. As with previous LTS analysis, the HTS data for population 70Arnd8 is dominated by a single structural motif (the ‘CAUG’ motif), which has been extensively characterized biochemically. However, the sequence cluster that is most enriched in this set does not conform to the ‘CAUG’ motif; thus, aptamers of interest can be easily overlooked by LTS methods.The most highly expressed sequence in each of six selection populations were assayed by terbium (Tb3+)-mediated cleavage in the presence of AMP and CoA. Several of these aptamers contain regions that are protected by CoA but not by AMP, potentially indicating interactions with the pantotheine arm. These results highlight the power of comparative HTS analysis, especially when combined with biochemical validation. Initial analysis of these potential pantotheine aptamers will be presented. Poster Session 2: RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

429 SHAPEclash: A Biochemical and Computational Approach for RNA Tertiary Structure Analysis and Refinement

Philip Homan1, Feng Ding2, Nikolay V. Dokholyan2, Kevin Weeks1 1 Department of Chemistry, University of North Carolina, Chapel Hill, USA, 2Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, USA The diverse functional roles of RNAs are enabled to a significant degree by the complex secondary and tertiary structures they form. Accurate, high-resolution models of these structures are therefore central to a comprehensive understanding of RNA function. Current high-resolution approaches for determining RNA tertiary structures, X-ray crystallography and NMR spectroscopy, are restricted to readily crystallizable and small RNAs, respectively. For most other RNAs, functionally important tertiary structures must be probed using biochemical approaches, and the resulting data are often insufficient to constrain nucleotide-resolution models. Here we introduce a combined biochemical and computational approach for creating high-quality models for RNA tertiary structure. SHAPEclash identifies nucleotides in structurally crowded regions of an RNA structure by the ability of a bulky adduct at the 2’-hydroxyl position to disrupt the overall RNA structure. These data are then incorporated as experimental constraints in discrete molecular dynamics (DMD) simulations to obtain experimentally informed, three-dimensional models. We applied this approach to a test set of RNAs with lengths up to 200 nts and were able to obtain high-resolution tertiary structure models with high statistically significant agreement with crystal structures. The test set included several “hard” RNAs for which chemical probing approaches were otherwise insufficient to direct accurate three-dimensional fold refinement. We envision that SHAPEclash will greatly expand the range of RNAs for which we can accurately refine tertiary structure models useful for guiding new hypotheses about biological functions carried out by RNA.

430 Using a Non-Redundant Dataset of RNA Crystal Structures to understand RNA Structural Flexibility for Rational Design of RNA Molecules and RNP Complexes

Swati Jain1, Laura Murray2, Jane Richardson1, Bruce Donald1 Duke University, 2Yale University RNA molecules undergo significant conformational changes upon binding to proteins and other small molecules. However, the nature of these conformational changes is not clearly understood and characterized. We have assembled a quality-conscious, hand-annotated dataset of RNA crystal structures (all with a resolution of 3 Å or higher), called RNA11, to study and characterize RNA structure and its intrinsic features, both in terms of backbone and base dihedral angles. Our analysis on the dataset shows significant differences in the range of values observed for the χ angle for C3’-endo versus the C2’-endo pucker, as well as for purines versus pyrimidines. These values are now used in RNA structure validation in MolProbity, and for RNA structure refinement in PHENIX. Our analysis of the backbone dihedral angles has led to the addition of some distinct backbone conformers to the initial list of all-angle RNA backbone conformers, published by the RNA Ontology Consortium. Additionally, the design software developed by Bruce Donald’s lab at Duke University has been extended to design RNA molecules and RNP interfaces. The design process now accommodates RNA base flexibility, by searching over a range of χ angle values observed in RNA11, specific to the ribose pucker and the base identity. This will help in incorporating RNA structural flexibility during the rational design of RNA molecules, RNP complexes, or small molecules targeting RNA and RNPs, and is a step forward to achieve better and more realistic results.

1

Poster Session 2: RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

431 A Novel RNA Structural Motif with a Conserved Backbone Structure but non-Conserved Base Sequence

Gary Kapral, Swati Jain, Jane Richardson, David Richardson Duke University RNA structural motifs have always played an important role in RNA function, from stabilizing ribozymes to providing binding sites for proteins and ligands. Bases are the most dominant identifier for motifs, but with the discovery that the RNA backbone is rotameric, the inclusion of backbone conformations in motif definitions became a possibility. Analysis of strings of RNA backbone conformations using the modular nomenclature described in Richardson, 2008(1), yielded many conserved backbone motifs. Though many “suitestrings” (deriving from the suite unit of backbone division from Murray, 2003(2)) corresponded to other previously described motifs, we discovered that there are suitestrings that correspond to novel structural motifs, having highly conserved backbone structure but a non-conserved base sequence. The pentaloop described here is a novel structure motif, characterized by the suitestring 1b4b6p2a; besides the common suitestring, the pentaloop has a conserved H-bond between n 2’OH to n+4 2’OH, thus giving the motif it’s name: the OHOH pentaloop. Out of 19 instances observed in our dataset, there are 9 different base sequences, the most common sequence accounting for only a third of the total. The sequences are also internally diverse, showing no significant preferences for conserved bases at any given position in the pentaloop. The functions of this motif are similarly diverse. It can act as a junction between two helices, or be a standard stem-loop; it even appears as a 5-nucleotide bulge in the glmS ribozyme, interrupting an A-form helix and returning to standard A-form helix with no other significant structural changes. We believe this represents the first discovery of a backbone-defined RNA motif with a diverse sequence and diverse function, which opens up the possibility of other conserved RNA structures with diverse base sequence. Combined with techniques that target the RNA backbone, such as SHAPE, we look forward to finding other examples of RNA backbone motifs in the future and learning how to harness them to improve our understanding of RNA structure and function. (1) JS Richardson, B Schneider, LW Murray, GJ Kapral, RM Immormino, JJ Headd, DC Richardson, D Ham, E Hershkovits, LD Williams, KS Keating, AM Pyle, D Micallef, J Westbrook and HM Berman (2008) “RNA Backbone: Consensus All-angle Conformers and Modular String Nomenclature (an RNA Ontology Consortium contribution)” RNA 14 :465-481. (2) LJ Murray, WB Arendall III, DC Richardson, and JS Richardson (2003) “RNA Backbone Is Rotameric” PNAS-USA 100, 13904-13909.

432 Model Building in RNA Structural Biology: The RCrane Approach for Crystallographic Applications Kevin Keating1, Anna Marie Pyle1,2 Yale University, New Haven, CT, USA, 2Howard Hughes Medical Institute

1

X-ray crystallography is currently the most common technique for determining the atomic-level details of RNA structures. However, these structural studies present a large number of challenges. One notable complication arises from the low resolutions typical of RNA crystallography, which results in electron density maps that are imprecise and difficult to interpret. While 1-2 Å resolutions are common in protein crystallography, over 60% of the crystal structures in the Nucleic Acid Database were solved at 2.5-3.5 Å. Although phosphates can be reliably located at these low resolutions, sugar locations and puckers are exceedingly difficult to determine. Furthermore, bases are frequently difficult to orient precisely. These problems are exacerbated by the lack of computational tools for RNA modeling. Many of the tools commonly used in protein crystallography have no equivalents for RNA structure. Structure determination is further complicated by the flexibility of the RNA backbone, as each nucleotide possesses six variable torsion angles that must be accurately modeled. Because of these complexities, RNA crystallographic model building is a difficult and time-consuming task, and these complications can lead to inaccuracies in published structures. To address this, we have developed RCrane , [1,2] a method for accurately building the RNA backbone into maps of up to 4 Å resolution. We have implemented this method as a plugin for Coot , a popular program for crystallographic model building. This plugin assists the crystallographer in locating phosphates and bases within electron density. After this initial trace of the molecule, an accurate backbone structure can be built without further user intervention. To accomplish this, backbone conformers [3] are first predicted using the RNA pseudotorsions [4] and base-phosphate perpendicular distances. Detailed backbone coordinates are then calculated to conform both to the predicted conformers and to the previously located phosphates and bases. These conformer predictions are highly accurate: one of the first three predictions is correct 98% of the time, and the first prediction is correct 84% of the time. Additionally, these predictions are largely insensitive to the expected imprecision of the phosphate and base coordinates. We have also begun to fully automate the model building process by incorporating the RCrane methodology with the helix finding techniques available in the Phenix crystallographic software suite. This process will allow an initial model to be built from an experimentally phased density map without crystallographer intervention. Additionally, this technique holds promise for atomic-level interpretations of cryoelectron microscopy maps. As the resolution received from cryo-EM experiments continues to improve, it may soon be possible to locate individual phosphates and bases using cryo-EM techniques. The RCrane method could then be used to accurately determine the full backbone structures of these molecules. [1] Keating and Pyle. PNAS (2010) 107 : 8177-8182. [2] Keating and Pyle. Acta Cryst D, manuscript submitted . [3] Richardson, et al. RNA (2008) 12 : 533-541. [4] Wadley, Keating, Duarte, and Pyle. JMB (2007) 372 : 942-957.

Poster Session 2: RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

433 The Role of Salt Concentration and Magnesium Binding in HIV-1 Subtype-A and Subtype-B Kissing Loop Monomer Structures

Taejin Kim, Bruce Shapiro National Cancer Institute, Frederick, Maryland, USA The subtype-B monomers of the human immunodeficiency virus type-1 (HIV-1) have experimentally been shown to dimerize at high salt concentration or in the presence of magnesium, while the dimerization of the subtype-A monomers requires magnesium binding at the G273 or G274 phosphate groups regardless of salt concentration. We used explicit solvent molecular dynamics (MD) simulations to investigate the conformational changes in subtype-A and -B monomers in different salt concentrations, and we found that our MD simulation results are consistent with those of experiments. At low salt concentration, hairpin loop structures of both subtypes were deformed and bases in the hairpin loop were turned inside. At high salt concentrations, the subtype-B monomer maintained the hairpin loop shape and most bases in the hairpin loop pointed out, while the subtype-A monomer showed a severe deformation. We also found that the flanking bases in the subtype-B stabilize the hairpin loop with non-Watson-Crick hydrogen bond interactions or water bridges mediated by Na+ ions. However, the flanking base G273 in the subtype-A caused a significant deformation at low and high salt concentration. A bound magnesium at the G273 or G274 phosphate groups controlled the behavior of the G273 base and prevented the subtype-A monomer from deformation. We applied restraints to both subtypes to examine the role of high salt concentration or magnesium binding. While restraints were applied, both subtypes at 0M salt concentration maintained their shapes. However, when restraints were removed, they deformed significantly and the shape of structure returned to those at 0M salt concentration. Therefore, we suggest that the dimerization of both subtypes may be induced by the proper conformation of the monomers with the appropriate ionic concentrations or magnesium binding.

434

Abstract Withdrawn

Poster Session 2: RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

435

The Hairpin Ribozyme, A Multiconformer Ribozyme?

436

Restoring SNP Disrupted RNA Structural Ensembles with LNAs

Matthew Marek, Berhanegebriel Assefa, Nils Walter University of Michigan, Ann Arbor, Michigan, USA In the complex world of RNA folding the hairpin ribozyme has emerged as a model system exemplifying multiple stable and functional structures. The hairpin ribozyme is composed of two helix-loop-helix structures (A and B) which must dock utilizing inter-domain tertiary interactions. These docking/undocking events have shown heterogeneous but stable docking rates, resulting in differential cleavage rates. Recent work has begun to elucidate the origin of this heterogeneous behavior. Utilizing a newly developed native purification technique, we have been able to eliminate this heterogeneity from in vitro purified samples. While causality has not yet been fully elucidated, damage from ultraviolet irradiation in the course of standard purifications has been strongly implicated. Additionally, the molecular basis of this phenomenon is being probed utilizing selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) to localize structural variations.

Joshua Martin2, Justin Ritz2, Lauren Neulander1, Chetna Gopinath3, Alain Laederach2 1 Developmental Genetics and Bioinformatics, Wadsworth Center, Albany, NY, USA, 2University of North Carolina, Chapel Hill, NC, USA, 3Biomedical Sciences, University of Albany, Albany, NY, 12208 The ensemble of structures adopted by the 5’ Untranslated Region (UTR) of the mRNA of the human Ferritin Light Chain (FTL) gene can be altered by single nucleotide polymorphisms (SNPs). The hyperferritinemia cataract syndrome associated SNP, U22G, alters the ensemble of structures as confirmed by high-accuracy single nucleotide resolution chemical mapping. Using this same technique we are able to observe how the ensemble of structures for WT and U22G are altered by Locked Nucleic Acid (LNA) binding. We are able to restore the structure of a critical regulatory element in the UTR (an Iron Responsive Element) by specifically targeting alternative conformations of the RNA. LNA titrations reveal the relative thermodynamic parameters for binding to WT and U22G and provide further evidence for the presence of alternative secondary structures in eukaryotic mRNAs.

Poster Session 2: RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

437 Single-Molecule Studies of HIV-1 Dimerization Initiation Sequence Kissing Interaction and its Resolution to a Stable Extended Duplex

Hansini Mundigala1, Jonathan Michaux1, Eric Ennifar2, Andrew Feig1, David Rueda1 Department of Chemistry, Wayne State University, Detroit, MI, USA, 2Architecture et Réactivité de l’ARN, Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes F-67084 Strasbourg, France. The Dimerization Initiation Sequence (DIS) is a conserved hairpin motif on the 5’ UTR of the HIV-1 genome. It plays an important role in genome dimerization through formation of a “kissing complex” intermediate between two homologous DIS sequences. This bimolecular kissing complex ultimately leads to the formation of an extended RNA duplex. Understanding the kinetics of this interaction is key to exploiting DIS as a possible drug target against HIV. We wish to report a novel study that makes an important contribution to understanding the dimerization mechanism of HIV-1 RNA in vitro. Our work has employed single-molecule fluorescence resonance energy transfer to monitor the dimerization of a minimal HIV-1 RNA sequence containing DIS. Most significantly, we observed a previously uncharacterized folding intermediate that plays a critical role in dimerization mechanism. Our data clearly show that the dimerization mechanism involves three distinct steps in dynamic equilibrium and Mg2+ ions regulate these dynamics. Two of the steps correspond to previously proposed structures. Mutations in the highly conserved purines flanking the DIS loop destabilize the formation of this intermediate, indicating that these purines may play an important role in the HIV-1 RNA dimerization in vivo. 1

438 Solution NMR and X-ray Crystallographic Examination of RNA Plasticity Using and Aptamer for Ribosomal Protein S8

Edward Nikonowicz, James Donarski, Jiachen Wang, Yousif Shamoo Rice University The bacterial ribosomalprotein S8 protein is an ~132 amino-acid protein that binds the 16S rRNA in the central domain and plays a critical role in the early stages of 30S ribosomal subunit assembly. The primary rRNA binding site for protein S8 is located on helix-21 of 16S rRNA and is sufficient for specific and high-affinity binding.The binding site is composed of a core of several phylogenetically conserved nucleotides that are distributed in an asymmetric internal loop. A pair of stacked base triples is formed by six of the conserved nucleotides and a nearly universally conserved adenine forms the only base-specific contact. We have used protein S8 to explore the plasticity of RNA and to examine possible alternative modes of S8-RNA binding. An RNA selection experiment (SELEX) was performed using the B. subtilis S8 protein which is well-ordered in the free and RNA-bound forms. An aptamer, with secondary structure motif that differs from the native binding site on helix-21, was chosen from the selected apatmer pool for further analysis. In solution, the RNA aptamer forms a helix with G-A and U-U base pairs and an A-A mismatch and no evidence for minor conformations of the RNA could be detected in the NMR spectral data. Similarly, the structure of the 16SrRNA binding site is well-ordered in the free state, but does require magnesium to form a single stable conformation. However, unlike the RNA aptamer, the structure adopted by the native binding site in the free state is very similar to the structure of the RNA in the complex, two base triples and an unpaired adenine. Interestingly, the binding of the RNA aptamer to the S8 protein leadsto a dramatic change in the RNA conformation. The non-canonical G-U and G-Ainter actions dissolve to create a G-(G-C) base triple with a Watson-Crick G-Cbase pair and a base quartet composed of uridine and adenine bases. Although several interactions present in the native S8-RNA complex are present in the complex between S8 and the RNA aptamer, including a base-specific contact involving an unpaired adenine, hydrogen bonds to the ribose 2’-hydroxyl groups, and ionic interactions between the phosphate backbone and amino acid sidechains, the topology of the aptamer RNA in the complex differs from that of helix-21 in complex with S8.

Poster Session 2: RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

439 Transcriptional pausing coordinates folding of the aptamer domain and the expression platform of a riboswitch.

George Perdrizet II1, Irina Artsimovitch2, Ran Furman2, Tobin Sosnick1, Tao Pan1 University of Chicago, Chicago, (IL), USA, 2Ohio State University, Columbus, (OH), USA RNA folding and conformational rearrangement is a fundamental mechanism in controlling gene expression. Bacterial riboswitches are cis-elements that sense the cellular pool of metabolites to regulate transcriptional termination or translational initiation. Riboswitch performs regulatory control with an upstream aptamer domain capable of binding its cognate metabolite and a downstream expression platform that contains a transcriptional terminator or the ribosome binding site of the gene. Despite a large body of work on the structure and folding of the aptamer domains from many riboswitches, how riboswitch promotes folding and conformational rearrangement when both aptamer and expression platform are present is still poorly understood. Riboswitch function depends on its folding into complex RNA structures, which occurs during transcription. It is thus critical to elucidate the role of transcription in riboswitch folding. RNA polymerase pausing is a fundamental property of transcription that can influence RNA folding. Here we show that pausing plays an important role in the folding and conformational rearrangement of the Escherichia coli btuB riboswitch during transcription by the E. coli RNA polymerase. This riboswitch consists of an approximately 200 nucleotide, coenzyme B12 binding aptamer domain and an approximately 40 nucleotide expression platform that controls ribosome access for translational initiation. We found that transcriptional pauses at strategic locations facilitate folding and structural rearrangement of the full-length riboswitch, but have minimal effect on the folding of the isolated aptamer domain. Pausing at these regulatory sites blocks the formation of alternate structures and plays a chaperoning role that couples folding of the large aptamer domain and the small expression platform. The mechanism and structural basis of the observed inter domain coordination effect is under investigation. Transcriptional pausing may be a general mechanism for coordination of ligand binding and RNA conformational rearrangement which allows riboswitches to respond to environmental cues. Therefore this research is being expanded to include other riboswitches of various sizes and structural complexity. 1

440 Mutations in The UTRs of SERPINA1 Transcripts Are Involved in The Disease Associated Mechanisms

Gabriela Phillips, Chetna Gopinath, Mat Halvorsen, Justin Ritz, Joshua Martin, Alain Laederach University of North Carolina, Chapel Hill, USA Single Nucleotide Polymorphisms (SNPs) are found throughout the human genome. SNPs implicated in diseaseassociated mechanisms that map to coding regions of the genome generally alter the function of the protein. However, disease-associated SNPs that map to non-coding regions can affect the translation regulation and/or stability of the mRNA. Novel high-throughput mappings of RNA binding protein sites found that most UnTranslated Regions (UTR) of mRNA are often targeted by proteins involved in the regulation of translation, stability of mRNA, and mRNA localization. Not only that many RNA binding proteins recognize specific primary sequences of the mRNA, it is also now well established that the secondary structure plays a critical role in the accessibility of a binding site. Thus, SNPs that significantly alter the structure of UTRs could affect post-transcriptional regulation. Alpha1-antitrypsin deficiency (A1AD) is an autosomal recessive genetic disorder caused by the defective production of alpha 1-antitrypsin (A1AT), a protein encoded by SERPINA1. Severe deficiency of A1AT causes panacinar emphysema or Chronic Obstructive Pulmonary Disease (COPD) as well as various liver diseases. It has been shown that certain mutations (Glu342Lys) in the coding regions of SERPINA1 led to severe cases of COPD. A recent genome wide-association study identified a SNP in the 5’ UTR of SERPINA1 associated with increased risk of developing COPD. SHAPE structure mapping analysis reveals that this SNP alters RNA structure acting forming a RiboSNitch – a SNP that induces a large conformational change of RNA resulting in an altered function of RNA. Thus, SNP induced RNA structure change likely plays an important role in COPD predisposition.

Poster Session 2: RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

441 Site-specific Crosslinking Using Cis-diamminedichloroplatinum (II) to Probe the Tertiary Structure of Complex RNAs

Kory Plakos1, Erich Chapman2, Elaine Chase3, Barbara Golden3, Victoria DeRose1 1 University of Oregon, Eugene, (OR), USA, 2University of Colorado Denver, Denver, (CO), USA, 3Purdue University, West Lafayette, (IN), USA

Chemical crosslinking is an important tool for identifying RNA tertiary interactions, which are essential to understanding RNA structure and function (Juzumeine et al., Methods 2001). Cis-diamminedichloroplatinum (II), or cisplatin, is an anticancer drug known to preferentially bind N7 of purine nucleotides in both DNA and RNA (Kozelka et al., Coordin Chem Rev 1999), a property which our lab is utilizing toward developing new crosslinking methods for determining complex RNA structure. Typically crosslinks form between adjacent purine nucleotides, most commonly guanine residues, with a rare population of interstrand crosslinks observed. Recently, we have shown that incorporation of a phosphorothioate substitution can drive cisplatin toward forming site-specific interstrand crosslinks between the substituted sulfur and local Pt(II) binding partners in certain contexts. This technique was first developed by our lab in the hammerhead ribozyme (HHRz) (Chapman and DeRose, JACS 2012). In the HHRz, crosslinking efficiency was shown to be dependent on proper tertiary structure formation, and crosslinking between the scissile phosphorothioate and nucleobases G8 (major) and G10.1 (minor) demonstrated the specificity of this method as a ‘baited’ crosslinking tool with a predicted ~8 angstrom reach. Currently, we are further exploring our technique in the hepatitis delta virus ribozyme (HDV). A recent crystal structure (PDB 3NKB, Chen et al., Biochemistry 2010) revealed a magnesium ion located in the active site. This structure lacked electron density 5’ to the scissile phosphate, but modeling in the nucleotides immediately surrounding the cleavage site from the hammerhead ribozyme (PDB 2OEU Martick and Scott, Cell 2006) suggests that a nonbridging oxygen on the scissile phosphate is important for coordinating the magnesium ion, which also appears to contact a guanine N7 on the enzyme strand. This model presents an excellent opportunity test to our site-specific cisplatin crosslinking technique to explore active site metal coordination in the HDV ribozyme. We have made a phosphorothioate substitution to a non-bridging oxygen at the cleavage phosphate on a short substrate strand and successfully crosslinked it using cisplatin to the HDV ribozyme enzyme strand. Thus far we have observed phosphorothioate-specific cisplatin crosslinking between the two strands and established that it is in competition with hexamminecobalt (III) chloride, a hydrated magnesium ion mimic known to compete with magnesium in the active site of the HDV ribozyme. This evidence suggests that cisplatin is crosslinking in the expected binding pocket. We are undertaking mapping studies to determine to which nucleotide on the enzyme strand cisplatin is crosslinking. Future targets that may benefit from this novel crosslinking technique include other complex RNAs and RNA-protein complexes.

442 Structural Changes of a Group II Intron ID3 Stem Loop Associated with Binding of its Target Exon 1 Sequence

Milena Popović1,2, Nancy Greenbaum2 1 Florida State University, 2Hunter College of CUNY RNA-RNA interactions involved in recognition associated with ribozyme catalysis are essential for ribozyme function. Research described here focuses on RNA-RNA interactions at the 5’ splice site of a self-splicing yeast mitochondrial group II intron aI5γ. At the functional core of group II introns is the pairing of the EBS1-IBS1 sequences (exon binding sequence one and intron binding sequence one, respectively), which is essential for preserving fidelity of the splice site. The EBS1 guide sequence is a part of an 11-nucleotide loop at the terminus of the ID3 stem loop, which is a subdomain of Domain one (D1), the largest of the group II intron domains. The goal was to investigate the structural features of the ID3 stem loop and the ID3-IBS1 complex. We investigated the effects of the ID3 stem loop structure on the EBS1-IBS1 pairing. A question addressed was whether the large 11-nucleotide loop forms a stable structure. We tested whether the EBS1-IBS1pairing forms in solution and what structural changes the ID3 loop undergoes upon formation of the EBS1IBS1 pairing. We wanted to determine the effects of ID3 structure on the availability of bases of EBS1 for base pairing and thus for the 5’ splice site selection. Solution NMR structure of the ID3 stem loop shows a structured stem and a fairly structured base of the loop, as well as an unstructured or dynamic loop, involving residues of the EBS1 sequence. NMR spectroscopic study of the ID3-IBS1 complex in solution indicates that the unstructured region of the ID3 loop becomes structured upon interaction with the IBS1 sequence, in an apparent induced-fit mechanism, by which both the guide sequence and the target become structured upon interaction. An important observation here is that the double stranded EBS1-IBS1 region ends at the 5’ splice site, placing it at the single/double stranded junction, which may play an important role in recognition and/or accessibility of the 5’ splice site. The placement of the EBS1 sequence in the specific structural context of the ID3 loop may be an important feature, which aids the recognition of the 5’ splice site. We show here that by virtue of being placed within the loop of a certain size, the two potential base pairs downstream of the 5’ splice site, which could form in a free duplex, do not form in the context of the loop. These findings are important because we show that positioning of a guide sequence within a loop determines the availability of bases for pairing and controls the extent of base pairing and thus the position of the 5’ splice site. Poster Session 2: RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

443 Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling

Xiaojun Zhang, Peter Qin Univeristy of Southern California, Los Angeles, CA The condensation of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shaped protein/RNA complex, and its function requires an RNA component called packaging RNA (pRNA). Current structural information on pRNA is limited, which hinders studies of motor function. Here, we used site-directed spin labeling to map the conformation of a pRNA three-way junction that bridges binding sites for the motor ATPase and the procapsid. The studies were carried out on a pRNA dimer, which is the simplest ring-shaped pRNA complex and serves as a functional intermediate during motor assembly. Using a nucleotide-independent labeling scheme, stable nitroxide radicals were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 inter-nitroxide distances spanning the three-way junction were measured using Double Electron-Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select 3,662 viable three-way junction models from a pool of 65 billion. The results reveal a similar conformation among the viable models, with two of the helices (Ht and Hl) adopting an acute bend. This is in contrast to a recently reported pRNA tetramer crystal structure, in which Ht and Hl stack onto each other linearly. The studies establish a new method for mapping global structures of complex RNA molecules, and provide information on pRNA conformation that aids investigations of phi29 packaging motor and developments of pRNA-based nanomedicine and nanomaterial.

444

Two Protein Cofactors Co-opt To Facilitate bI5 Intron Folding In Yeast Mitochondria

Nora Sachsenmaier, Christina Waldsich University of Vienna MFPL, Vienna, Austria RNA is important for most of the processes in living cells. To be able to carry out its function RNA has to adopt a specific three dimensional shape. Therefore it is very important to investigate the structure of RNA to get a better insight into the mechanism of RNA-dependent processes. By studying folding of many RNAs of diverse function it will be possible to derive the principles underlying RNA structure formation in general. While RNA folding has been studied in vitro in great detail, little is known about how RNAs acquire their structure in vivo. Here we want to contribute to the understanding of RNA folding in the complex cellular environment. Catalytic RNAs, like group I introns, are a very well suited model system to study RNA folding in the living cell, because one can measure formation of its native state as a function of catalysis. Another advantage in choosing a group I intron as a working model is that there is already large amount of data available about their structures and folding pathways in vitro. I am focusing on determining the intracellular structure of the yeast mitochondrial bI5 group I intron, which depends on two nuclear-encoded proteins, Mss116p and Cbp2, for efficient splicing in vivo. In contrast, in vitro Cbp2 is required but also sufficient to promote intron folding at near-physiological conditions. While the bI5-Cbp2 complex has been well characterized in vitro, the function of Mss116p in bI5 folding remains enigmatic. By monitoring Mss116p- and Cbp2-induced conformational changes within the bI5 group I intron in vivo, I will provide the first mechanistic insights into how these two proteins co-opt to facilitate bI5 folding and in turn splicing in yeast mitochondria. In brief, the aim of my thesis is to explore the principles underlying the interplay of RNA folding and RNP assembly. Since the methods to study RNA structure and folding in vivo are limited, it is essential to develop novel techniques. Ultimately, this challenging project will advance us in exploiting the medical and biotechnological potential of catalytic RNA.

Poster Session 2: RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

445 Dynamic Nature of pri-miRNA Revealed by Molecular Dynamics Simulations and Biochemical Methods

Debashish Sahu, Kaycee Quarles, Scott Showalter Pennsylvania State University Drawing parallels from the dynamic nature of protein structures in determining their functions, RNA molecules are clearly conformationally dynamic. This dynamic nature can be captured by various biochemical and enzymatic approaches as well as through computational methods, if suitable initial structural information is available. Multiple classes of non-coding RNAs (e.g. RNAs that do not code for proteins) are also encoded by genes and often transcribed to serve structural, catalytic and regulatory functions. Once transcribed, they are further processed by nuclear machineries as a part of canonical processing pathway in creating small RNA molecules called primary micro RNA (pri-miRNA). Biochemical studies conducted in our laboratory point to dynamic regions on the pri-miRNA as having a direct role in establishing Drosha cut site location. Here we perform molecular dynamic simulations on segments spanning the stem region of pri-miRNA structures predicted form MC-Pipeline as the atomistic structures are currently unavailable. The pri-miRNA structures analyzed here are pri-mi16-1, pri-mi107 and pri-mi30a. Each is efficiently processed by the Drosha/DGCR8 microprocessor complex that produces pre-miRNA, excised ~11 base-pairs from the single strand-double strand junction and the cut site is present near a region of enhanced dynamics predicted by MC-Sym calculations. We hypothesize that these “hot spot” regions are necessary structural features recognized by microprocessor-complexes to initiate processing. We obtain vital information on slower time scale dynamics by the characterization of pri-miRNA structures predicted by MC-Sym. On a much more detailed atomistic scale, our molecular dynamics simulations indicate that these “hot spot” regions undergo a large scale motions in the ns-ps time scale. In this study we obtain a broad structural perspective in the role of dynamic regions on the predicted structures of miRNA in proper cut site recognition by Drosha/DGCR8 microprocessor complex for efficient miRNA processing.

446

Two Retinoblastoma Associated SNVs in RB1 form a RiboSNitch

Wes Sanders, Matt Halvorsen, Justin Ritz, Joshua Martin, Alain Laederach UNC Chapel Hill, Chapel Hill, NC, USA Retinoblastoma (RB1) is a negative regulator of the cell cycle and also involved in tumor suppression. Recent computational analysis of known, disease-associated Single Nucleotide Variants (SNVs) in the human genome suggest that two disease-associated SNVs found in the 5’ UTR of the RB1 gene alter the mRNA transcript structure. These two mutations, G17C and G18U in RB1’s 5’ UTR, are also predicted to affect the structure of a putative Internal Ribosome Entry Site (IRES). Using Selective 2’-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) we have analyzed both the wild type (WT) RB1 and the RB1 mutants G17C and G18U. The data reveal significant changes in SHAPE reactivity in both mutants compared to the WT along with structural changes to the IRES site, consistent with the computational prediction. The RB1 5’ UTR is thus a RiboSNitch, and our data suggest that SNV induced conformational change in mRNA is likely a drive of oncogenesis.

Poster Session 2: RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

447

Structural and Biochemical Studies of the dG Riboswitch

Olga Pikovskaya2, Anna Polonskaia2, Dinsnaw Patel2, Alexander Serganov1,2 1 New York University School of Medicine, New York, USA, 2Memorial Sloan-Kettering Cancer Center, New York, USA Riboswitches are evolutionarily conserved mRNA regions capable of highly specific interactions with various cellular metabolites. Purine riboswitches play an essential role in genetic regulation of purine metabolism in bacteria. This family includes the 2’-deoxyguanosine (dG) riboswitch, which is involved in feedback control of deoxyguanosine biosynthesis. To understand the principles that define dG selectivity, we determined crystal structures of the natural Mesoplasma florum riboswitch bound to cognate dG as well as to noncognate guanosine, deoxyguanosine monophosphate, and guanosine monophosphate. Comparison with related purine riboswitch structures reveals that the dG riboswitch achieves its specificity through modification of key interactions involving the nucleobase and rearrangement of the ligand-binding pocket to accommodate the additional sugar moiety. In addition, we observe new conformational changes beyond the junctional binding pocket extending as far as peripheral loop-loop interactions. It appears that re-engineering riboswitch scaffolds will require consideration of selectivity features dispersed throughout the riboswitch tertiary fold, and structureguided drug design efforts targeted to junctional RNA scaffolds need to be addressed within such an expanded framework. The study was supported by the US National Institutes of Health grant GM66354 to DJP and NYU start-up funds to AS.

448 Non-Nearest Neighbor Dependence of Stability for Group III RNA Single Nucleotide Bulge Loops

Martin Serra, Christina Hasson, Brandon Panaro, Dan Phillips, Michael McCann Allegheny College, Meadville, (PA), USA Thirty-five RNA duplexes containing single nucleotide bulge loops were optically melted in 1M NaCl, and the thermodynamic parameters ΔHo, ΔSo, ΔGo37, and TM for each sequence were determined. The bulge loops were of the group III variety, where for example, the bulged nucleotide is either a 5’AG/3’U or 5’CU/3’G, leading to ambiguity to the exact position and identity of the bulge. The complete set of all possible group III bulge loops were examined. The data was used to develop a model to predict the free energy of an RNA duplex containing a group III single nucleotide bulge loop. The destabilization of the duplex by the group III bulge could be modeled as though the bulge nucleotide leads to the formation of the Watson-Crick base pair rather than the wobble base pair. By assuming the formation of the Watson-Crick pair, the position and identity of the bulge is no longer ambiguous and can therefore be treated as a group I bulge loop (Blose et al., (2007) Biochemistry 46, 15123). Therefore, the destabilization of an RNA duplex caused by the insertion of a group III bulge is primarily dependent upon non-nearest neighbor interactions and was shown to be dependent upon the stability of less stable stem of the duplex.

Poster Session 2: RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

449

Single Molecule Dynamics of Functional Spliceosomes

450

Abstract Withdrawn

Amanda Solem1, Yu-Chih Tsai2, Jonas Korlach2, David Rueda1 1 Wayne State University, Detroit, MI, USA, 2Pacific Biosciences, Menlo Park, CA, USA The spliceosome is a highly dynamic assembly of five small nuclear RNAs (snRNAs) and a large number of proteins that catalyzes splicing. U2 and U6 are two spliceosomal snRNAs strictly required for both steps of splicing. U2/U6 can adopt multiple conformations, and U6 likely must fold specifically to create metal binding sites for catalysis. Multiple groups have proposed that conformational changes in U2/U6 have a role in regulating splicing activity or rearrangement of the active site between the two steps of splicing. However, the conformational dynamics of U6 in the context of the holo-spliceosome remain unclear. We have used a single molecule fluorescence assay based on Zero Mode Waveguide (ZMW) technology to monitor U6 dynamics in yeast splicing extract. ZMWs enable observation of single spliceosomes by blocking the background fluorescence from the cell extract. We observe several different classes of spliceosomes exhibiting distinct conformational dynamics. Two of the classes dynamically sample FRET states. We are using mutant pre-mRNAs that block the second step of splicing to distinguish between different classes of molecules. Our preliminary data show that we can monitor conformational dynamics of U6 in holo-spliceosomes that may yield valuable mechanistic details.

Poster Session 2: RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

451 Influence Of Ligand Binding On The Loop-Loop Interaction Of The Adenine Riboswitch Aptamer

Patrick St-Pierre1, Juan Penedo2, Daniel Lafontaine1 1 Groupe ARN/RNA Group, Département de biologie, Faculté des sciences, Université de Sherbrooke, Québec, Canada, 2School of Physics and Astronomy & Biomedical Sciences Research Complex, University of St. Andrews, St. Andrews, Fife, UK Riboswitches are regulatory elements usually found in the 5’ UTR of messenger RNAs. Upon the binding of a small metabolite to their aptamer region, riboswitches regulate the expression of the genes located downstream on the messenger RNA. Although a lot of information is available on the bound and unbound structures of many aptamers, there is still little known about riboswitch folding pathways. To get a better understanding about riboswitch conformational changes, single molecule Förster Resonance Energy Transfer (sm-FRET) was used to monitor the folding in real time of a riboswitch aptamer. The adenine-binding aptamer of the add riboswitch was used as a model for these experiments because of its relative simplicity and the amount of data available on the structure of this aptamer. In these assays, we monitored the folding of the loop-loop interaction of the add aptamer at different concentrations of magnesium and ligand. In absence of ligand, the aptamer adopts two conformations corresponding to the unfolded and folded states (1), consistent with the aptamer being highly flexible in absence of ligand. However, in presence of either adenine, 2-6 diaminopurine or 2-aminopurine, we found that the aptamer preferentially adopts the folded conformation, as expected based on our previous study (1). By analyzing single-molecule dynamics, we found that ligand binding mostly produces an increase of the dwell time of the folded state, consistent with the aptamer spending more time in the folded state when bound to the ligand. We also found that each ligand does not affect the dwell time to the same level. In contrast, we observed that the dwell time of the unfolded state is not significantly affected by the presence of any ligand showing that the RNA conformational change is affected by the nature of the bound ligand. Taken together, these results suggest that ligand binding on the aptamer leads to the “conformational capture” of the loop-loop folded state, a situation similar to what we recently observed for the SAM-I riboswitch (2). Importantly, our data do not rule out the possibility of a ligand induced fit mechanism involving the core region of the add riboswitch. (1) Lemay et al, Chem Biol, 2006 13:8. (2) Heppell et al, Nature Chem Biol, 2011, 7:384.

452

Antibodies as RNA crystallization chaperones

Nikolai Suslov, Joseph Piccirilli University of Chicago, Chicago, IL, USA The preparation of well-ordered crystals is usually the rate-limiting step in the determination of the three-dimensional structure of RNA by X-ray crystallography. This has proven more challenging for RNA than for proteins. For example, there are currently 72813 protein structures deposited in the PDB compared to a meager 888 RNA and 1271 RNP structures. It was hypothesized that introduction of a chemically differentiated surface, such as those provided by RNA-binding proteins, may help position molecules in a regular register, and hence facilitate the growth of well-ordered crystals. The first successful strategy involved replacing a nonessential region of an RNA with the ten-nucleotide sequence recognized by the U1A protein and crystallizing the RNA in complex with the U1A protein 23. We sought to expand the repertoire of crystallization chaperones by developing a phage-displayed Fab platform for generation of RNA specific Fabs. These antibodies are being used to crystallize several novel RNA targets.

Poster Session 2: RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

453 Characterization of Structural Changes Associated with HIV-1 Genomic RNA Dimerization Using Fluorescence, NMR, and SANS

Andrea Szakal1, John Marino1, Susan Krueger2 Institute for Bioscience and Biotechnology Research of the National Institute of Standards and Technology and the University of Maryland, Rockville, MD, USA, 2National Institute of Standards and Technology, Center for Neutron Research, Gaithersburg, MD, USA During virus assembly, all retroviruses, including HIV-1, specifically encapsidate two copies of full-length viral genomic RNA in the form of a non-covalently linked RNA dimer. A number of studies suggest that dimerization is mediated through a highly conserved 35-nucleotide RNA stem-loop, the dimerization initiation site (DIS), in the 5’-untranslated region (UTR) of the genomic RNA via an intermolecular kissing interaction between two DIS loops. The structures formed by the 5’-UTR RNA and the in vivo mechanism by which genome dimerization occurs are still not well understood. We will present results from measurements using fluorescence, nuclear magnetic resonance (NMR), and small-angle neutron scattering (SANS) which are being used to analyze the structure of the 5’-UTR of the genomic HIV-1 RNA and guide the building of structural models. We are using these techniques to probe the 5’-UTR as both a monomer and a dimer, and also in the absence and presence of the HIV-1 nucleocapsid protein, NCp7, which has been implicated as an RNA chaperone in the genome dimerization process. Knowledge of these structures could potentially guide the development of novel therapeutics that target HIV-1 genomic RNA before packaging and/or upon introduction to the cell after infection, and may lead to new treatments for other retroviruses as well.

1

454

Single Molecule Studies of Prp24-Dependent Folding Dynamics of the U2/U6 Complex

Chandani Warnasooriya1, Zhuojun Guo1, Samuel Butcher2, David Brow3, David Rueda1 1 Chemistry, Wayne State University, Detroit, MI, USA, 2Biochemistry, University of Wisconsin-Madison, Madison, WI, USA, 3Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, USA Splicing is catalyzed by the spliceosome, which consists of five small nuclear ribonucleoprotein particles (snRNPs: U1, U2, U4, U5 and U6) and numerous proteins. Several of these proteins facilitate structural rearrangements in the spliceosome. Proper assembly of spliceosomal components is critical for function, and thus, defects in assembly can be lethal. We have previously used single-molecule Fluorescence Resonance Energy Transfer (smFRET) to show that a protein free U2/U6 complex can adopt at least three distinct conformations in dynamic equilibrium. We are now testing how spliceosomal proteins affect these dynamics. Prp24 is an essential factor in U6 snRNP and it has been proposed to help in the formation and unwinding of the U4/U6 complex. Prp24 consists of four RNA recognition motifs (RRMs 1-4). Our single molecule data show that Prp24 binds U2/U6 with high affinity and stabilizes a low FRET conformation of U6. Removal of RRM1 reduces the binding affinity, in agreement with previous results. Interestingly, our results show that Prp24 binding induces U2 unwinding from the U2/U6 complex, suggesting that a possible role for Prp24 may be to disassemble the U2/U6 complex, prior to U6 recycling.

Poster Session 2: RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

455

Orientational Information from FRET in the Structural Analysis of RNA

456

Towards Structural Insights Into Human Telomerase

Timothy Wilson, Stephanie Kath-Schorr, Jonathan Ouellet, Asif Iqbal, David Lilley University of Dundee, Dundee, United Kingdom Fluorescence is a highly sensitive spectroscopic method that can be applied to single molecules or living cells, and fluorescence resonance energy transfer (FRET) provides distance information on the macromolecular scale. FRET has been extensively employed in the analysis of the structure and folding of RNA species. FRET arises from dipolar coupling between the transition dipoles of donor and acceptor fluorophores attached to the molecule of interest, and is a function of both distance and orientation. We have shown that the cyanine fluorophores Cy3 and Cy5 have a strong propensity to stack onto the ends of DNA and RNA helices and have determined their structures using NMR. We have shown that FRET efficiency between Cy3 and Cy5 fluorophores terminally attached to ds DNA or RNA is dependent on the distance between them, and is modulated by their relative orientation in a predictable way. This is true irrespective of the length of the tether and the presence or absence of sulfonyl groups. Ignoring fluorophore orientation may lead to significant errors in distance measurements. However, we show that exploitation of these effects provides a new and reliable source of orientational information from FRET measurements using the cyanine fluorophores. Furthermore, the treatment of orientation effects allows us to calculate distance information with greater accuracy. A. Iqbal, S. Arslan, B. Okumus, T. J. Wilson, G. Giraud, D. G. Norman, T. Ha and D. M. J. Lilley. Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally-attached to double-stranded nucleic acids. Proc. Natl. Acad. Sci. USA 105, 11176-11181 (2008). J. Ouellet, S. Schorr, A. Iqbal, T. J. Wilson and D. M. J. Lilley. Orientation of cyanine fluorophores terminally attached to DNA via long, flexible tethers. Biophys. J. 101, 1148-1154 (2011). L. Urnavicius, S. A. McPhee, D. M. J. Lilley and D. G. Norman. The structure of sulfoindocarbocyanine 3 terminally attached to dsDNA via a long, flexible tether. Biophysical J. 102, 561-569 (2012).

Georgeta Zemora1, Samuel Coulbourn Flores2, Christina Waldsich1 1 University of Vienna MFPL, Vienna, Austria, 2Department of Cell and Molecular Biology, Uppsala, Sweden Telomerase is a large ribonucleo-protein (RNP) complex that adds telomeric DNA repeats to the end of eukaryotic chromosomes. Telomerases from all species are composed of the reverse transcriptase catalytic subunit (TERT), an essential RNA component (TR) and several accessory proteins. TR has at least two functions: it provides a scaffold for binding of associated proteins and carries the template sequence that is copied into telomeric DNA by TERT. Telomerase is significantly up-regulated in cancer cells and mutations in human TR, TERT and associated proteins have been linked to the genetic diseases, like Dyskeratosis Congenita. Despite its bio-medical importance, the structure of human telomerase complex remains unknown. Since understanding an RNAs fold is instrumental in understanding its function, we are interested in exploring the structure and folding landscape of human telomerase RNA in vitro and in vivo and to characterize the protein-induced conformational changes within hTR. The cellular compartment differs significantly from the artificial in vitro refolding conditions. Therefore, it is our goal to explore the telomerase RNA structure in human cell culture by employing an DMS chemical probing technique. Mutations in the telomerase complex disrupt either nucleic acid binding or catalysis, and are the cause of numerous human diseases. Parts of the complex from Tribolium castaneum and Tetrahymena thermophila however have been solved crystallographically. We use these fragments to predict the structure of experimentally undetermined fragments of the human TERT and Telomerase RNA binding domain (TRBD) by homology modeling and validate the results with functional assays.

Poster Session 2: RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

457

mRNA Localization Mechanisms are Conserved in Trypanosomes

Lysangela Alves, Arthur Oliveira, Samuel Goldenberg, Bruno Dallagiovanna Inst. Carlos Chagas, ICC-FIOCRUZ

Asymmetric mRNA localization represents a sophisticated tool to regulate and optimize protein synthesis and sustaining cell polarity. Molecular mechanisms involved in the regulated localization of transcripts have been described in higher eukaryotes and in fungi but not in protozoa. Trypanosomes are ancient eukaryotes that have branched early in eukaryote evolution. We hypothesized that the general mechanisms of mRNA specific localization should be active in trypanosomes. Hence, we have performed FISH assays using probes against well characterized proteins in terms of localization within the parasite, namely, beta-tubulin, paraflagellar rod protein 2 (PFR2) and cruzipain. The results showed discrete localization of their mRNAs in the cytoplasm, being directly associated with protein expression. Alpha tubulin showed a perinuclear and slightly granular pattern while PFR2 localization was mostly concentrated at the anterior pole of the cell, next to and along the flagellum where the encoded protein is localized. The cruzipain mRNA was distributed in granules in a location resembling the reservosomes where this proteinase is localized. In the insect non-infective epimastigotes under nutritional stress, the distribution pattern of β-tubulin changed to a granular localization, suggesting the mobilization of this mRNA to storage or degradation granules. This mobilization of transcripts to RNA granules was even more evident in the case of PFR2 mRNA, On the other hand, cruzipain transcripts maintained their localization in the posterior region of the cytoplasm reinforcing the idea that they are localized in the interior of this vacuolar organelle. mRNA localization was also investigated in the infective metacyclic trypomastigotes. In metacyclics, the overall rate of transcription decreases drastically and because of this the signal of the probes is weaker as compared to the epimastigote forms. Notwithstanding, it was possible to observe that the subcellular localization of tubulin mRNA in the perinuclear region was maintained. This was not the case for the PFR2 transcripts which appeared to lose the anterior localization and became widely distributed along the parasite body. The cruzipain mRNA was not detected in metacyclics in accordance with the absence of the reservosome vacuoles in the infective forms. We next asked if these mechanisms of mRNA localization in the cytoplasm could also be observed in other trypanosomes. The β-tubulin distribution pattern in T. brucei was very similar to that observed in T. cruzi, though the perinuclear localization was not as evident as in T. cruzi epimastigotes. TbPFR2 mRNA was also observed clearly concentrated in the proximity of the flagellum, which in T. brucei extends from the posterior towards the anterior end attached to the cell body. Altogether, these results indicate a mechanism of mRNA localization in this parasite. Transfection assays with reporter genes showed that, as in higher eukaryotes, the 3’UTRs are responsible for guiding mRNAs to their final location as observed for the beta-tubulin and PFR2 3’ UTRs. Our results strongly suggest the presence of a core and basic mechanism of mRNA localization in protozoa. This kind of mRNA controlled transport is ancient in eukaryotic evolution and highlights the importance of trypanosomes as model organisms. Financial Support Fundacao Araucaria, CNPq, FIOCRUZ

458 Association, Recruitment and Function of TREX Components Aly, THO and UAP56 are Interdependent

Binkai Chi, Qingliang Wang, Guifen Wu, Min Shi, Lantian Wang, Hong Cheng Institute of Biochemistry and Cell Biology, Shanghai, China The highly conserved mRNA export complex TREX mainly contains the multi-subunit THO complex (THO) and proteins UAP56, Aly and Tex1. It is unclear whether TREX components are recruited sequentially or as a preformed complex. Here, we show that Tex1 is co-immunodepleted, co-knocked down and co-fractionated with THO, indicating that Tex1 is a component of the THO sub-complex. Using the combination of immunodepletion and immunoprecipitations, we found that depletion of any one of Aly, THO and UAP56 from the HeLa nuclear extract disrupts the interaction between the other two, indicating that assembly of TREX requires Aly, THO and UAP56. Interestingly, Aly and THO are required for efficient recruitment of each other and UAP56 to the spliced mRNA. Consistent with these biochemical results, Aly, THO and UAP56 are all required for cytoplasmic accumulation of polyA+ RNA and spliced mRNA. Together, our data indicate that association, recruitment and function of Aly, THO and UAP56 are interdependent and suggest that TREX is recruited to mRNA as a preformed complex.

Poster Session 2: RNA Transport and Localization

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

459 p180 Promotes the Ribosome-Independent Localization of a Subset of mRNA to the Endoplasmic Reticulum

Xianying (Amy) Cui, Alexander Palazzo University of Toronto, Toronto, Canada In metazoans, the majority of mRNAs coding for secreted and membrane bound proteins are translated on the surface of the endoplasmic reticulum (ER). Although the targeting of these transcripts to the surface of the ER can be mediated by the translation of a signal sequence, and their maintenance is mediated by interactions between the ribosome and the translocon, it is becoming increasingly clear that additional ER-localization pathways exist. To investigate this, we used a specialized cellular membrane extraction and FISH staining protocol to directly visualize the mRNA localization in mammalian cells. We found that >50% of the mRNAs were maintained at the ER in a translation independent manner. We then investigated the localization of individual transcripts and found that, CALR and ALPP mRNAs, but not t-ftz and INSL3 mRNAs, can be first targeted and later maintained at the ER independent of ribosomes. Using a mass-spectrometry analysis of proteins that associate with ER-bound polysomes, we identified putative mRNA receptors that may mediate this alternative mechanism including p180, an abundant, positively charged membrane-bound protein. We demonstrate that p180 is required for the efficient ER-anchoring of certain transcripts, such as ALPP and CALR, and that its overexpression can enhance the association of generic mRNAs with the ER. In addition, we showed that the Lysine rich region within p180 is capable of binding to RNAs in vitro. In summary, we provide mechanistic details for an alternative pathway to target and maintain mRNA at the ER. It is likely that this alternative pathway not only enhances the fidelity of protein sorting, but also localizes mRNAs to various subdomains of the ER, and thus contributes to cellular organization.

460 Antibodies Specific for Active Spliceosomes Reveal the Global Extent and Subnuclear Location of co- and post-Transcriptional Splicing

Cyrille Girard1, Cindy Will1, Jianhe Peng2, Evgeny Makarov3, Berthold Kastner1, Ira Lemm1, Henning Urlaub1, Klaus Hartmuth1, Reinhard Lührmann1 1 Max Planck Institute for Biophysical Chemistry, Goettingen, Germany, 2CRUK Clinical Centre, Leeds, United Kingdom, 3Division of Biosciences School of Health Science and Social Care, Uxbridge, United Kingdom Most pre-mRNA introns are thought to be removed by the spliceosome while the pre-mRNA is still being transcribed. However, there is little quantitative information as to how much splicing occurs co-transcriptionally and how much posttranscriptionally, and it remains unclear where splicing exactly occurs in the nucleus. To determine the global extent of co- and post-transcriptional splicing, as well as their respective subnuclear locations, we generated antibodies that specifically recognize phosphoepitopes in the U2 snRNP-associated SF3b155 protein (anti-P-155), which are found only in catalytically-activated/active spliceosomes. Fractionation of Hela cell nuclei into chromatin and nucleoplasmic fractions followed by western blotting with anti-P-155 antibodies indicated that minimally 85% of all splicing events occur co-transcriptionally, whereas maximally 15% occur post-transcriptionally. Immunofluorescence (IF) microscopy studies demonstrated that active spliceosomes localize mainly to regions of decompacted chromatin and are preferentially found at the periphery of or (to a lesser extent) within nuclear speckles. By blocking both transcription (with DRB) and splicing at a stage after SF3b155 phosphorylation (via RNAi knockdown of CDC5L), post-transcriptional spliceosomes containing polyadenylated pre- mRNA were shown by IF to accumulate in speckles. Addition of CDC5L to CDC5Ldepleted cells reversed the accumulation of both active spliceosomes and poly(A)+ RNA in speckles, whereas knockdown of the export factor Aly prevented the release of spliced mRNA from speckles. Our data suggest a new role for nuclear speckles, namely as sites involved in posttranscriptional splicing, and that splicing completion triggers the release of newly spliced mRNA from speckles and its export to the cytoplasm.

Poster Session 2: RNA Transport and Localization

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

461

Regulation of tRNA nucleus-cytoplasm distribution by nutrient availability

462

MicroRNA-Mediated Translational Repression of a Localized mRNA in Xenopus Oocytes

Rebecca Hurto, Anita Hopper The Ohio State University, Columbus (OH), USA Transport of tRNAs has traditionally been thought to be unidirectional, from the nucleus to the cytoplam. However, it is now known that cytoplasmic tRNAs constitutively move in a retrograde fashion back into the nucleus and are reexported to the cytoplasm in S. cerevisiae and mammalian cells (Review: Phizicky and Hopper, 2010). The intracellular distributions of tRNAs change in response to acute loss of various nutrients. Previous studies determined that acute loss of all 20 amino acids (aa) results in tRNA nuclear accumulation. The yeast strains studied contained mutations in one or more aa biosynthetic pathways, causing them to be auxotrophic for some aa and prototrophic for others. To determine how the loss of each type of aa affect tRNA nucleus-cytoplasm (n/c) distribution in cells auxotrophic for 3 aa, fluorescence in situ hybridization was used to detect tRNA n/c distribution before and during time courses of starvation for various combinations of aa. After 30 min of acute starvation for 17 aa that the cells are prototrophic for, cells exhibited a tRNA n/c ratio that is lower than cells starved for all 20 aa. Therefore, the tRNA distribution that occurs during acute loss of all 20 aa is the result of acute loss of both types of aa. After 120 min of acute starvation for the same 17 aa, the tRNA n/c distribution returns to a distribution similar to the cells grown in nutrient replete conditions. We call this “restoration” of tRNA subcellular distribution. In contrast, starvation for either all 20 aa or a single aa that the cells are auxotrophic for caused cells to exhibit elevated tRNA n/c ratios for more than 3 hrs indicating that the inability to acquire aa results in prolonged tRNA nuclear accumulation. We propose that the restoration of the tRNA n/c distributions for cells undergoing prototrophic starvation to pre-starvation ratios is due to new amino acid biosynthesis. This is supported by data showing that lack of the major starvation-induced transcription factor for aa biosynthetic genes (Gcn4) causes a delay in the restoration of tRNA subcellular distribution.

Catherine Pratt, Kimberly Mowry Brown University, Providence, (RI), USA RNA localization is a widely conserved mechanism for generating polarized protein expression, and in order to prevent spatially inappropriate protein expression, localized mRNAs are maintained in a translationally silent state until they reach their destination. In oocytes of the frog, Xenopus laevis, localization of specific mRNAs along the animalvegetal axis is critical for proper embryonic patterning. A major focus of our studies is Vg1 mRNA, which is localized to the vegetal cortex during stages III-IV of oogenesis. Importantly, translation of Vg1 mRNA does not begin until stages V and VI, after localization is complete. A translational control element (TCE) within the Vg1 3’ UTR has been shown to repress translation of reporter RNAs, but the mechanistic basis has remained unclear. To investigate whether translation of Vg1 RNA is repressed by a miRNA-dependent mechanism, we first looked for the presence of the effector Argonaute proteins in Vg1 transport particles. We found that Argonaute proteins interact with the Vg1 mRNP and are enriched at the vegetal cortex early in oogenesis. Within the Vg1 TCE, we identified conserved binding sites for let-7 family members that are expressed during oogenesis, suggesting a potential role in translational repression of Vg1 mRNA. Indeed, both removal of the putative target sites and blocking let-7 miRNA function resulted in relief of repression. Notably, we find that Vg1 mRNA is no longer associated with miRISC after stage IV of oogenesis. Taken together, these data suggest a mechanism whereby let-7 miRISCs repress translation before and during localization. After localization is complete, release of the mRNA from miRISC thus triggers spatially-restricted translation.

Poster Session 2: RNA Transport and Localization

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

463

The Dynamics of Splicing Factor Interactions with Active Genes in Living Cells

464

SRp20 Plays An Essential Role in the Regulation of HIV-1 RNA Processing

Noa Neufeld1, Yehuda Brody1, Itamar Kanter1, Shai Carmi1, Eva-Maria Böhnlein2, Karla Neugebauer2, Yaron ShavTal1 1 Bar-Ilan University, Ramat Gan, Israel, 2Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Splicing factors are co-transcriptionally recruited to the nascent transcript as it emerges from the active polymerase. Using tandem gene arrays that contain genes with introns and exons we could show that spliceosome assembly occurs at the active site of transcription. To obtain information about the recruitment kinetics of splicing factors to active genes in vivo we used live-cell microscopy in which both the splicing factors and the produced mRNAs could be fluorescently visualized. Typically, studies that have followed splicing factor diffusion dynamics in the nucleus of living cells have utilized fluorescently-tagged splicing factors that were expressed from viral promoter-driven constructs (e.g. CMV or SV2). Overexpression of splicing factors might shift the endogenous balance of splicing factors and could influence the outcome of splicing decisions. We therefore used recombineered BACs containing GFP-splicing factor genes, i.e. these splicing factors were expressed from their endogenous promoters, leading to physiological levels of expression of the fluorescently tagged splicing factors. Each of these BACs was separately integrated into U2OS Tet-On cells already harboring the gene array. Using a combination of techniques for measuring intra-cellular kinetics, namely, fluorescence recovery after photobleaching (FRAP), fluorescence loss after photobleaching (FLIP), and fluorescence correlation spectroscopy (FCS), we measured the kinetics of a variety of splicing factors in the nucleoplasm, nuclear speckles and when engaged with the actively transcribing gene. Using computer simulations we provide measurements of the engagement times of splicing factors with active genes, and show that splicing factors exhibit different dynamics depending on their nuclear location.

Maria Calimano, Annie Mao, Raymond Wong, Alan Cochrane University of Toronto, Toronto, Ontario, Canada Control of RNA processing plays a central role in the replication of HIV-1. From a single transcript over 40 mRNAs are generated by suboptimal splicing to produce three classes of viral RNAs; unspliced, singly spliced and multiply spliced RNAs. Changes in the balance of HIV-1 RNA splicing will result in changes to viral protein synthesis and failure to assembly new virions. To identify host factor that play major roles in regulating this process, we have examined the effects of overexpression and depletion of various SR proteins on HIV-1 protein expression and viral RNA processing. Overexpression assays determined that increased levels of SRp20 and two isoforms of Tra2beta (beta1 and beta3) induced mark reductions in unspliced viral RNA accumulation as well as corresponding decreases in HIV-1 structural proteins. Overexpression of each factor also induced changes in splice site usage that would also alter expression of key viral regulatory proteins Tat, Rev and Nef. Parallel analysis of the effects of depletion of each of these factors on viral RNA revealed significant roles in HIV-1 RNA processing. In particular, depletion of SRp20 resulted in increased accumulation of HIV-1 multiply spliced RNAs and reduced levels of unspliced and singly spliced viral RNAs. Examination of viral RNA splice sites revealed alterations in usage inverse to that seem upon SRp20 overexpression. Together these findings reveal SRp20 to be a key regulator of HIV-1 RNA processing and replication, too much or too little of the factor resulting in changes in viral RNA splicing incompatible with new virion assembly. This central role of SRp20 in regulating HIV-1 replication was further validated upon demonstrating that it is one of the SR proteins that are modified upon treatment of cells with digoxin, an inhibitor of HIV-1 replication. Changes in HIV-1 RNA processing upon digoxin treatment are comparable to those observed upon SRp20 overexpression. Current efforts are directed at developing a more detailed understanding of how SRp20 achieves its effects and how to manipulate its function to suppress HIV-1 replication.

Poster Session 2: RNA Transport and Localization & Viral RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

465 Gammaherpesvirus 68 Exemplifies a Novel Biogenesis Pathway to Produce Biologically Functional miRNAs

Kevin Diebel, Linda van Dyk University of Colorado School of Medicine, Aurora, CO, USA Canonical biogenesis of microRNAs (miRNAs) begins with RNA polymerase II transcription of a primary-microRNA (pri-miRNA) that undergoes sequential cleavage by the endonucleases Drosha and Dicer, resulting in a ~22 nucleotide long, maturely processed miRNA. The production of biologically functional miRNAs from gammaherpesvirus 68 (γHV68), however, represents an alternative transcription and processing pathway. Production of γHV68 pri-miRNA transcripts begins via transcription by RNA polymerase III (pol III) initiated from novel RNA pol III type 2-like promoters. These γHV68 pol III transcripts contain a 5’ tRNA-like domain immediately upstream to precursor-miRNA (pre-miRNA) stem-loops. Here we demonstrate that the production of the γHV68 pri-miRNA transcripts utilizes multiple RNA pol III termination signals and that the maturation of the γHV68 pri-miRNA transcripts is dependent upon the enzymatic activity of the tRNA processing enzyme RNaseZL coupled with the presence of the protein La. Additionally, we characterize the novel RNA pol III type 2-like promoter sequences, composed of overlapping and apparently redundant A boxes, and demonstrate that they are found in genomes across all three domains of cellular life. Further, we show that these novel pol III type 2-like promoters are required in their entirety to initiate transcription of pri-miRNA transcripts. In total, transcription and processing of the γHV68 miRNAs demonstrate an alternate means of miRNA generation that share particular features reminiscent of tRNA biogenesis.

466

IFIT1 is an antiviral protein that recognises 5’-triphosphate RNA

Andreas Pichlmair1,2, Matthias Habjan1, Caroline Lassnig3,4, Cathleen Holze1, Carol-Ann Eberle2, Keiryn L Bennett2, Jacques Colinge2, Thomas Rülicke4,5, Friedemann Weber6, Mathias Müller3,4, Giulio Superti-Furga2 1 Max-Planck Institute of Biochemistry, Martinsried near Munich, Germany, 2CeMM- Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria, 3Institute of Animal Breeding and Genetics, VetMedUni Vienna, Austria, 4Biomodels Austria, VetMedUni Vienna, Austria, 5Institute of Laboratory Animal Science, VetMedUni Vienna, Austria, 6University of Freiburg, Germany Antiviral innate immunity relies on pattern recognition receptors (PRRs) that recognise microbial structures and orchestrate cellular responses that culminate in expression of interferon stimulated proteins with antiviral activity. One such critical microbial structure is viral RNA that carries a triphosphate group on its 5’terminus (PPP-RNA). In an affinity proteomics strategy using PPP-RNA as bait and subsequent protein-protein interaction analysis we identified a protein complex consisting of members of interferon induced proteins with tetratricopeptide repeats (hIFITs), which are among most strongly interferon induced proteins. In this complex hIFIT1 was the only protein directly and specifically binding to PPP-RNA. Structure modelling and mutational analysis suggest that binding of hIFIT1 required a highly charged batch in a groove formed by tetratricopeptide repeats. Human cells treated with siRNAs against individual human IFIT family members as well as primary cells isolated from mice lacking the mIfit1 gene were impaired in containing growth of PPPRNA generating viruses. We propose that IFIT proteins assemble a barrier for viral replication by efficiently engaging PPP-RNA. Thus, similar to PRRs that sense specific microbial structures, interferon response genes may have evolved analogous specificity to clear viral infections.

Poster Session 2: Viral RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

467

Effect of single nucleotide substitutions in Pepino mosaic virus genome on its virulence

468

A Non-Canonical Ribosomal Frameshift Signal in a Plant Viral RNA

Beata Hasiow-Jaroszewska, Julia Byczyk, Henryk Pospieszny, Natasza Borodynko Institute of Plant Protection-National Research Institute, Poznan, Poland Pepino mosaic virus is a dangerous pathogen infecting tomato worldwide. Its characteristic future is high level of genetic variability and ability to creating new variants which induced symptoms from mild mosaic to severe necrosis. Site directed mutagenesis was used to create a collection of PepMV clones, each carrying a different mutation chosen basing on sequence comparison between isolates representing different genotypes. Infectious clone of mild P22 and necrotic P22 isolates were used. Solanum lycopersicum, Datura inoxia and Nicotiana benthamiana plants were inoculated with in vitro obtained transcripts. Competition experiments between each mutants and the ancestral nonmutated clone were performed. The effect of incorporated mutation in RNA PepMV on its virulence was explored. Successful infection was produced by all the mutants and no lethal mutations were observed. Most of the mutations were neutral so far however mutations introduced in triple gene block 3 and coat protein genes influence on induced symptoms significantly.

Alice Hui1, Vijayapalani Paramasivan1, Norma Wills2, Betty Chung3, Andrew Firth3, John Atkins2, W. Allen Miller1 1 Iowa State University, Ames, IA, USA, 2University of Utah, Salt Lake City, UT, USA, 3University of Cambridge, Cambridge CB2 1QP, United Kingdom The RNA genome of potyviruses is translated as one large polyprotein from which about 10 mature proteins are cleaved proteolytically. The viral genome region encoding the P3 protein contains a small overlapping open reading frame (ORF), called pipo, within the central region. Immunoblot results show that pipo is expressed as a fusion with the N terminus of P3 (P3N-PIPO), indicating that pipo is translated by ribosomal frameshifting. However, the sequence around the suspected frameshift site does not resemble the conventional -1 frameshift signal. Instead, a highly conserved G1-2A6-7 motif at the 5’ end of the pipo ORF is suspected to cause the -1 or +2 change in reading frame required for translation of the PIPO protein. To identify the sequence required for ribosomal frameshifting, we used a dual luciferase system to study translation in wheat germ extract and in plant protoplasts. We found that the sequence in the 5’ end of the pipo ORF facilitates a net -1/+2 change in reading frame in both assays. In protoplasts, the frameshift efficiency appears higher than in cell-free extracts. Point mutations in the G1-2A6-7 motif reduced frameshifting to background levels. Constructs containing only this motif potentiate frameshifting nearly as well as constructs containing a much larger viral context. This is interesting because the predicted frameshift site lacks both the canonical shifty heptanucleotide and conserved RNA secondary structure known to be required for -1 ribosomal frameshifting in RNAs of other viruses.

Poster Session 2: Viral RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

469 Structural Studies on the Panicum mosaic virus-like Cap-independent Translation Element: an Uncapped RNA That Tightly Binds the Cap-binding Protein eIF4E

Jelena Kraft, Mariko Peterson, W. Allen Miller Iowa State University, Ames, Iowa, USA Plant viral RNAs often contain diverse cap-independent translation elements (CITEs) in the 3’ untranslated region (UTR) that facilitate translation initiation at the 5’ end of the RNA. Based on sequence and structure, the 3’ CITEs are divided into several classes. All characterized CITEs bind translation initiation factor eIF4F, which in plants is composed of the cap-binding protein eIF4E and the scaffolding protein eIF4G. The mechanisms by which these CITEs recruit initiation machinery are poorly understood. Here we describe the dynamic structural properties of the Panicum mosaic virus-like elements (PTEs) and report preliminary crystallization conditions for Panicum mosaic virus RNA. The PTEs form a tight 3-way helical junction with a pseudoknot between a C-rich bulge at the branch point and a G-rich bulge in the main helix (Wang et al. 2009 JBC 284, 14189). This pseudoknot is recognized and bound by eIF4E without major reorganization of the RNA structure (Wang et al.,2011 Structure 19, 868). Progressive truncations of the Panicum mosaic virus RNA revealed that the 86 nucleotide PTE RNA construct spanning bases 4117-4197 of the viral genome was the minimal functional CITE. This was used in crystallization trials. The polydispersity index of PTE4117-4197 sequence measured by dynamic light scattering was 41.4%, indicating the presence of oligomeric states. These alternative oligomeric forms were visible on native PAGE but were significantly relieved by annealing the RNA in the presence of 5mM magnesium ions. This RNA is currently used for crystal screening using commercial screens. The PTE is unique in its ability to bind eIF4E in the absence of a m7G cap structure. Understanding of its structure will provide insight into mechanisms of translation factor, and ultimately ribosome, recruitment.

470

Discovery of RNA Secondary Structure in Influenza Virus

Walter Moss, Salvatore Priore, Lumbini Dela-Moss, Tian Jiang, Douglas Turner University of Rochester Influenza virus represents a serious threat to human health and economy. Seasonal flu causes an estimated three to five million severe infections world-wide with up to 500,000 deaths. Type A influenza also has the possibility of causing wide-spread pandemic outbreaks; the most severe example being the 1918 Spanish flu that killed between 20 and 100 million people. Despite using RNA throughout its replication, most therapeutic strategies focus on protein. A better understanding of influenza RNA structure could help in the design of therapeutics that target viral RNA. We have undertaken a complete survey of potential secondary structure in the three influenza virus genera: type A, B, and C. This work has uncovered secondary structures that are evolutionarily conserved, affect the evolution of protein coding potential, and have implications for function (e.g. control of mRNA splicing). This work has also uncovered global trends in host-species specific mRNA stability.

Poster Session 2: Viral RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

471 In vivo Functional Selection and SELEX Reveal Flexible Length of the H2 Stem Loop in a Satellite RNA

Allison Murawski1, Tareq Azad1, Johnathan Nieves1, Holleh Tajalli1, Nina Jean-Jacques1, Biology 419 Students1, Megan Young2, Anne Simon2, David Kushner1 1 Department of Biology, Dickinson College, Carlisle, PA, USA, 2Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA Satellite C (satC) is a 356 nt subviral RNA that associates with Turnip Crinkle Virus (TCV) and serves as a model to analyze specific cis-acting sequences and structures that play a role in viral RNA replication. For example, the satC 5’ end is thought to control (+)-strand synthesis from (-)-strand replication intermediates, as well as the monomer to dimer ratio of progeny. However, specific sequences or structures in the 5’ end important for the fitness of satC have yet to be defined. Stem loops H2 (nt 48-123) and H6 (nt 125-145) may be important for satC function (movement of viral RNAs through plants and enhancement of symptoms caused by TCV). Thus, satCs containing randomization of H2 nts without or with truncation to one-half or one-fourth its wt length were co-inoculated with TCV onto three-week old turnip plants and total RNA was extracted from new plant leaves three weeks post-inoculation in order to clone and sequence satC. Five rounds of these in vivo SELEXes resulted in the recovery of different H2 sequences of various lengths. For example, replacing wt H2 (76 nt) with either 76, 38, or 19 random nt resulted in recovery of satC with 3839 nt H2. Moreover, in planta competition of satCs with 19 nt vs 38 nt for H2 resulted in recovery of satC with 38-nt H2. satC molecules lacking H2 (ΔH2) or the adjacent stem loop H6 (ΔH6) each were co-inoculated with TCV onto turnip plants to ascertain the necessity of either stem loop in satC function. Self-evolution occurred over three rounds. The majority of these satC molecules remained ΔH2 or ΔH6 in early rounds, although most ΔH2 satCs had second-site mutations within the 20 nt directly 5’ or 3’ of H2, potentially promoting the formation of a different stem loop (Mfold). An insertion in H2 composed of H2-flanking wt nt 124-40, 27-47 also was observed in some satC molecules. Although the H2 region may not be required for satC movement through plants, together these data suggest that H2 is flexible and may favor a nonspecific sequence in order to act as a spacer and promote the pathogenesis of the satellite, as stem loop H2 with at least 38 nt appears to be most beneficial for in planta fitness. These data, along with results of the ongoing ΔH6 studies, will be presented.

472 Involvement Of PSF In The Recognition Of An RNA Promoter Derived From The HDV RNA Genome

Martin Pelchat University of Ottawa, Ottawa, (Ontario), Canada The hepatitis delta virus(HDV) consists of a small (~1,700 nucleotides, nt) single-stranded, circularRNA genome, and uses the host DNA-dependent RNA polymerase II (RNAP II) for its transcription and replication. Using coimmmunoprecipitations and UVcross-linking assays, we have determined that PSF directly binds to the regions on the HDV RNA genome recognized by RNAP II. RNA affinity chromatography indicated that both PSF and RNAP II simultaneously bind an HDV-derived RNA promoter in vitro, and that the binding of PSF to both HDV RNA and the C-terminal domain (CTD) of RNAP II is required for efficient RNA recognition by RNAP II. PSF knockdown experiments and mutagenesis of PSF further indicated that HDV RNA accumulation requires the N-terminal portion of PSF to interact with RNAP II and the C-terminal portion to interact with HDV RNA. Taken together, our results suggest that PSF acts as a transcription factor on HDV-derived RNA promoters by interacting with both the RNAP II CTD and RNA simultaneously during the pre-initiation complex formation.

Poster Session 2: Viral RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

473 Comparison of SIVmac239 and HIV-1NL4-3 Genomic RNA Structures: Stabilization and Reformation of Structure Throughout Sequence Evolution

Elizabeth Pollom1, Kristen Dang1, Elizabeth Potter1, Robert Gorelick2, Christina Burch1, Kevin Weeks1, Ronald Swanstrom1 1 University of North Carolina, Chapel Hill, (NC), USA, 2AIDS and Cancer Virus Program, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, (MD), USA Regions of RNA secondary structure play essential roles in the replication cycle of HIV-1. The SHAPE-determined RNA secondary structure of the HIV-1NL4-3 genome has shown many elements of structure, but only a fraction of these have been previously studied. One tool to assess the importance of these structures is to determine the extent to which they are conserved over evolutionary time. To this end, we used SHAPE technology to develop a secondary structure model for the genomic RNA of a second primate lentivirus, simian immunodeficiency virus (SIVmac239), which shares 50% sequence identity at the nucleotide level with HIV-1NL4-3. In both genomes approximately 60% of the nucleotides are paired within the coding region (8,738 nucleotides). However, only about half of these paired nucleotides are paired in both sequences, and only 61 base pairs form with the same partner in the coding region of both sequences. Thus on average the RNA secondary structure is evolving at a much faster rate than the sequence. Some structures are conserved between HIV-1NL4-3 and SIVmac239, including the 5’ untranslated region (5’ UTR), the Rev responsive element (RRE), a pseudoknot to sequester the 5’ polyadenylation sequence, the polypurine tracts (PPT and cPPT) that begin plus-strand synthesis, and the stem-loop structure that includes the first splice acceptor site. Structure at the Gag-Pro-Pol frameshift site is maintained but in a significantly altered form. As with all lentiviruses, the HIV-1NL4-3 and SIVmac239 genomes areadenosine-rich and cytidine-poor. Approximately two-thirds of the cytidines, uridines, and guanosines are base-paired while only one-third of adenosines are base-paired, leading to concentration of adenosines in single-stranded regions (55% of the unpaired nucleotides). Thus the base composition of the structured regions is very different from either the unpaired regions or the genome as a whole. Structures with adenosine content equal to or greater than the number of guanosines had higher SHAPE reactivity and were not conserved between the two genomes. By contrast, those structures in which guanosines were more abundant than adenosines had lower SHAPE reactivity and structure was maintained, although still undergoing significant evolution. We conclude that much of the secondary structure reflects pairing in a state which allows the RNA to form and reform interactions throughout evolution of the sequence. However, regions of the structure that perform necessary functions within the viral replication cycle seem to have a high guanosine content, which stabilizes these structures and allows them to remain intact even through the course of sequence evolution.

474 Novel microRNAs and Alternative mRNA Isoforms Arising During Human Cytomegalovirus Infection

Thomas Stark1,2, Brett Roberts1,2, Justin Arnold1,2, Deborah Spector1, Gene Yeo1,2 1 Department of Cellular and Molecular Medicine, 2Stem Cell Program and Institute for Genomic Medicine, University of California at San Diego, La Jolla, CA Human cytomegalovirus (HCMV) is the main viral cause of birth defects, often leading to neurological disorders, but the underlying molecular mechanisms are poorly characterized. To advance our understanding of the complex virus-host transcriptional landscape present during infection, we have completed a suite of comprehensive transcriptome-focused studies using HCMV-infected human fibroblasts and neural stem cells. Through deep sequencing analysis of small RNAs, we have refined viral miRNA annotations, identified novel HCMV miRNAs, and observed significantly upregulated host miRNAs. We also sequenced Argonaute-associated RNAs (Ago CLIP-seq) to obtain direct evidence for incorporation of all HCMV miRNAs into the endogenous host silencing machinery. High-throughput sequencing of polyadenylated mRNAs (RNA-seq), in combination with splicing-sensitive microarray analysis, has enabled us to identify novel spliced HCMV transcripts and hundreds of human genes that are alternatively spliced upon infection. Our findings will enable the elucidation of improved therapeutic targets in the near future.

Poster Session 2: Viral RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

475 Physical Interactions Between eIF4G and the 3’ Cap-independent Translation Element of Barley yellow dwarf virus (BYDV) RNA

Krzysztof Treder1, Jelena Kraft1, Zhaohui Wang1,2, W. Allen Miller1 Iowa State University, Ames, Iowa. USA, 2Present address: University of Texas Southwestern Medical Center, Dallas, Texas, USA Many viral genomes are composed of positive sense ssRNA, acting as both genome and mRNA. These RNAs are often uncapped and not polyadenylated, yet they translate efficiently. For a large group of positive sense plant viral genomes, translational activity depends on specific elements present in their 3’ UTRs, called 3’ cap-independent translation elements (CITEs). All known 3’ CITEs bind the eukaryotic translation initiation factor complex eIF4F and deliver it to 5’ end of the mRNA, usually by specific base pairing to a short stretch of nucleotides in the 5’ UTR. We showed previously that the 3’ CITE of Barley yellow dwarf virus (BTE) binds the eIF4G subunit of eIF4F. To understand how the BTE acts, we tested the ability of a series of N-terminal truncation mutants of eIF4G to restore translation in wheat germ extract (wge) that had been depleted of eIF4F by m7G-sepharose chromatography. Construct p86, which lacks the PABP and eIF4E binding sites was sufficient for cap-independent translation mediated by the BTE, while construct p70, with a slightly larger N-terminal deletion, had no activity. Thus, a region between amino acids 766-863 in wheat eIF4G (immediately upstream of the MIF4G domain) is required to bind the BTE and facilitate translation. Confirming this, a protein, p18 consisting only of amino acids 766-863 was found to bind the BTE, but also 18S rRNA, indicating it is a nonspecific RNA binding region, when out of the eIF4G context. To determine the sites on the BTE that are bound by eIF4G, we performed footprinting by examining protection of the BTE by functional truncations of eIF4G (p100, p86) from modification by SHAPE reagents or nucleases. A distinct tract of nucleotides, including a 17 nt highly conserved sequence and flanking bases in the helical junction were protected by functional deletion mutants of eIF4G, but not by the nonfunctional proteins such as p70. In contrast, loop 3 of the BTE remained highly solvent accessible in all cases, as we predicted, because it must base pair to the 5’ UTR while the BTE simultaneously binds eIF4G, according to our model. Addition of eIF4E enhanced protection in the presence of eIF4G, consistent with our observation that eIF4E stimulates eIF4G by 20-30% in BTE-mediated translation. These data provide strong support for our model that the BTE simultaneously binds eIF4F and base pairs to the 5’ UTR via loop 3, in order for eIF4F to recruit the ribosome to the 5’ UTR.

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476

Kinetic Analysis of the Double-stranded RNA Formation in Qβ RNA Replication.

Kimihito Usui1, Norikazu Ichihashi1,2, Yasuaki Kazuta1, Tomoaki Matsuura1,4, Tetsuya Yomo1,3 1 JST, ERATO, Yomo Dynamical Micro-scale Reaction Environment Project, Suita, Osaka, Japan, 2Department of Bioinformatic Engineering, Graduate School of Information Science and Techanology, Osaka University, Suita, Osaka, Japan, 3Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan, 4 Graduate school of Engineering, Osaka University, Suita, Osaka, Japan Qβ replicase is an RNA-dependent RNA polymerase responsible for replication of the RNA genome of bacteriophage Qβ. The active form of the Qβ replicase was heterotrimer enzyme composed of β-subunit, which is encoded in Qβ phage genome, elongation factor Tu (EF-Tu) and elongation factor Ts (EF-Ts). In the presence of excess Qβ replicase, a template RNA can be synthesized autocatalytically. Because of this unique autocatalytic synthesis activity of template RNA, Qβ replicase has been used in several applications. However, the autocatalytic replication of RNA is inhibited mainly the double-stranded RNA formation of template RNA. Therefore, it is very important to elucidate the mechanisms of duplex formation during RNA replication. However, at present, there is no kinetic model to account for the duplex formation process by Qβ replicase. In this study, we propose a kinetic model that is able to account for duplex formation process and determined rate and equilibrium constants. The key to our proposed model is the consideration of the concentration effect of ribosome on duplex formation and the two independent routes for the duplex formation. [Materials and Methods] Plasmid pUC-U1 was constructed by insertion of the cDNA fragment of Qβ phage genome into pUC-MDV(-)β(+). Template RNA were prepared by in vitro transcription from linearized plasmid DNA, using T7 RNA polymerase. RNA replication was carried out at 37 °C by adding template RNA and purified Qβ replicase to the amino acids-free cell free translation system. The amounts of synthesized RNA were measured by incorporation of [32P]-UTP during synthesis and measurement of the band intensity on agarose gel electrophoresis in TBE buffer at 4 °C. [Results and Discussions] The content of the double-stranded RNA in the total amounts of synthesized RNA markedly decreased when a part of Qβ phage genome was inserted into MDV(-)β(+) and was not affected by the concentrations of template RNA. In the presence of ribosome, the affinity of Qβ replicase to the template RNA and the rate of total and single-stranded RNA synthesis increased. All kinetic parameters of the double-stranded RNA synthesis markedly decreased when U1 RNA was used as a template. This study provides new insight into the biology of Qβ phage and also provides useful information for the design of the amplifiable RNA by Qβ replicase. Poster Session 2: Viral RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

477 The Rous Sarcoma Virus RNA Stability Element Acts As an Insulator to Prevent Recognition of Unspliced Retroviral RNA by Host Cell Decay Machinery

Johanna Withers1, B. Lin Quek1, Nicholas Ingolia2, Karen Beemon1 Johns Hopkins University, Baltimore, (MD), USA, 2Carnegie Institution for Science, Baltimore, (MD), USA For simple retroviruses, such as the Rous sarcoma virus, RNA elements interact with cellular host proteins to regulate viral RNA splicing, export, stability and packaging into virions. One such element, known as the Rous sarcoma virus stability element (RSE), is required for maintaining stability of the full-length unspliced RNA. This viral RNA serves as the genome packaged in progeny virions and also the mRNA for the Gag and Pol proteins. When the RSE is deleted from the viral RNA, the unspliced RNA becomes unstable and is degraded in a translation and Upf1-dependent manner. This strongly suggests that the RSE acts as an insulator to the nonsense-mediated mRNA decay (NMD) pathway, thereby preventing at least one of the required functional steps that target an mRNA for degradation. Observations from our lab suggest that the RSE inhibits recognition of the viral gag termination codon by NMD. We propose that a direct interaction between the RSE RNA and a host cell factor facilitates RSE insulator activity. However, the presence or absence of this insulator activity does not dramatically alter the distribution of elongating or terminating ribosomes, as assayed by ribosome footprint profiling experiments. Furthermore, RSE-like activity appears not to be unique to the Rous sarcoma virus. Preliminary results suggest the potential for RSE-like elements in related retroviruses.

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478 Requirement of Ded1p, a Conserved DExD/H-Box Translation Factor, in Yeast L-A Virus Replication

Chung-Shu Yeh, Tien-Hsien Chang Genomics Research Center, Academia Sinica, Taipei, Taiwan In comparison to their host genomes, virus genomes are considerably smaller. As a result, viruses must recruit host factors to complete their life cycles. Identification and characterization of these host factors are thus critical for revealing the complex virus-host interactions. L-A is a commonly found dsRNA virus residing in the cyctoplasm of the budding yeast. It possesses a 4.6-kb genome that encodes only two proteins: Gag, the coat protein, and Gag-Pol, the RNA-dependent RNA polymerase. We have previously shown that Ded1p, a conserved DExD/H-box protein, is associated with the L-A virus particle through the interaction with Gag in vivo and accelerates the viral RNA replication in vitro. This and the fact that Ddx3p, the Ded1p’s human orthologue, also plays a role in human hepatitis C virus (HCV) replication thus raise a possibility that Ded1p may play a fundamental role in the life cycles of several key RNA viruses. To understand how Ded1p mechanistically promotes L-A replication, we have employed cryo-EM to deduce the stochiometric ratio of Ded1p and L-A virus particle. Furthermore, systematically truncated ded1 alleles are used to screen for Ded1p mutants that may abolish L-A virus replication without compromising Ded1p’s essential cellular functions. We are also testing a hypothesis that Ded1p may play a role in facilitating the extrusion of the newly synthesized RNA from the L-A particle, on the basis that DExD/H-box proteins are thought to be able to remodel RNA-protein complexes. Because Ded1p is evolutionally conserved, the mechanism as to how Ded1p facilitates the viral replication in yeast is expected to provide a conceptual platform for the role of Ddx3p in HCV and other related virus’ replication.

Poster Session 2: Viral RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

479

Self-cleavage Mechanism of the Hairpin Ribozyme

Berhanegebriel Assefa, Matt marek, Nils Walter University Of Michigan, Ann Arbor, (MI), US Ribozymesare ideal model systems for the vast number of non-protein coding RNAs found in all domains of life. They have an easily detectable biological function –catalysis. They also are of high biological and biotechnological relevance in their own right for their roles in the processing and regulation of genetic information. The hairpin ribozyme is a selfcleaving and ligating RNA enzyme found in the minus strand of the satellite RNA from the Tobacco Ringspot Virus and similar viruses. It is a member of the family of small nucleolytic ribozymes including the hammerhead, HDV (Hepatitis delta virus), VS (Varkud satellite) and glmS ribozymes. The hairpin ribozyme is active at a length of ~50 nucleotides and consists of the two independently folding helix-loop-helix domains A and B that need to dock to induce catalytic activity. It has been shown that there are several tertiary undocking subpopulations that each can undergo cleavage. A native purification technique developed by our lab circumvents the exposure of RNA to UV light and denaturation. Generally, site-specific backbone cleavage is taking place when the 2’-oxygen attacks the phosphodiester bond to form a 2’,3’-cyclic phosphate ring as the 5’-oxygen leaves. MD simulation data suggest that A38 may serve as general acid. I hypothesize that A38H+ involves in the cleavage mechanism by serving as a general acid.

480 Defective RNAs associated with Tomato black ring virus isolates collected from zucchini plants

Beata Hasiow-Jaroszewska, Natalia Rymelska, Henryk Pospieszny, Natasza Borodynko Institute of Plant Protection-National Research Institute, Poznan, Poland Two new isolates of Tomato black ring virus (TBRV), representative of Nepovirus genus, have been collected from zucchini plants in Poland recently. The virus caused plant yellowing and leaves malformation. Both isolates were propagated in Chenopodium quinoa and after serial of passages RNA was isolated from purified viral preparations. The analysis of viral RNA obtained from plants after first and 10th round of passages reveled that original isolates did not possess any additional RNA particular besides two RNAs corresponding to genomic TBRV RNA. After serial of passages additional small RNAs appeared. They were subjected to RT-PCR reaction, cloned and sequenced. Obtained sequenced were compared with other sequence of defective RNAs described previously for TBRV. Sequence analysis showed that small RNAs derived from two zucchini isolates were identical and shared 95% identity with isolates obtained from tomato and liliac. Like others defective RNAs of TBRV they originated from RNA1. In comparison, in another isolate collected in 2007 from zucchini plants even after 30 passages none small RNAs appeared. It suggests that there is a mechanism leading to defective RNAs during viral replication which is independent from the host.

Poster Session 3: Small RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

481 Regulation of Drosha by Microprocessor-independent Viral miRNAs in Cells Latently Infected by Herpesvirus saimiri

Demian Cazalla, Joan Steitz Yale University, New Haven, (CT), USA MicroRNAs (miRNAs) are short non-coding RNAs that constitute an important layer of regulation of gene expression. Most miRNAs are generated through sequential cleavage by two RNAse III-type enzymes: Drosha, which cleaves primary miRNA transcripts in the nucleus to release a precursor miRNA (pre-miRNA) and Dicer, which cleaves the pre-miRNA in the cytoplasm. Examples of miRNAs that bypass the use of either of these two RNAse III-type enzymes have been described, although an explanation for the existence of such alternative pathways is missing. One example of alternative miRNA biogenesis pathways is provided by Herpesvirus saimiri (HVS). HVS is a member of the oncogenic γ-Herpesvirus family that infects T cells in New World primates causing aggressive leukemias, lymphomas, and lymphosarcomas. During latency in marmoset T cells, HVS produces seven Sm-class small nuclear RNAs called HSURs, for Herpesvirus saimiri U RNAs. We have recently identified six novel HVS-encoded miRNAs (1). These viral miRNAs derive from three stem-loop structures located downstream of HSURs. HVS miRNAs do not require the Microprocessor complex that acts to generate most host miRNAs, but instead rely on both Sm-class processing signals and machinery to create precursor miRNAs during the nuclear stage of miRNA processing. We have identified target sites for HVS miRNAs in the mRNA encoding marmoset Drosha. Overexpression of HVS miRNAs showed that these miRNAs can downregulate the levels of Drosha. Transient knockdown of these miRNAs in virally infected T cells results in upregulation of Drosha levels, indicating that these miRNAs do indeed regulate Drosha abundance during latent infection. By expressing Drosha-independent miRNAs that target Drosha mRNA, HVS may globally affect the processing of miRNAs and therefore downregulate the levels of most miRNAs in latently infected cells. Global downregulation of miRNA levels has been observed in different types of cancers and correlates with high cell proliferation (2). These results provide a possible mechanism that could contribute to the oncogenic potential of HVS, as well as the first explanation for the existence of alternative miRNA biogenesis pathways in mammalian cells. 1. D. Cazalla, M. Xie, J. A. Steitz, A Primate Herpesvirus Uses the Integrator Complex to Generate Viral MicroRNAs. Molecular cell 43, 982 (Sep 16, 2011).2. M. S. Kumar, J. Lu, K. L. Mercer, T. R. Golub, T. Jacks, Impaired microRNA processing enhances cellular transformation and tumorigenesis. Nat Genet 39, 673 (May, 2007).

482

Design and Synthesis of Small Molecules for RNA Internal Loop

Takeo Fukuzumi, Asako Murata, Yasue Harada, Kazuhiko Nakatani ISIR Osaka University, Osaka, Japan MicroRNAs (miRNA) are known to have multiple roles in critical cellular functions. Thus, there is a great potential for miRNA-binding small molecules as both therapeutic agents and cellular probes. Recently, miRNA targeted drug screening has become paid attention. A high throughput screening (HTS) method for RNA targeted screening has made possible to find out lead compounds more efficiently. Despite all advances, however, little work has been reported on HTS study for targeted miRNA. To address this, we have developed a fluorescent indicator displacement (FID) assay to identify RNA-ligand interactions. Herein we report FID assay to identify the pre-miRNA binding small molecules from chemical library. These small molecule ligands for the screening were designed for targeted pre-miRNA internal loops. A twisted backbone is possible to provide with substantial affinity and specificity for pre-miRNA internal loop structure. Also, it can avoid a non-specific intercalation for the RNA double helix region. The twisted structure was constructed by Suzuki coupling reaction. The small molecule library was chemically synthesized by us. This chemical library was used for our FID assay procedure to find molecules that bind to specific pre-miRNA. In this presentation, the obtained several candidates and binding affinities will be described./p>

Poster Session 3: Small RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

483

Phosphorylation Regulates MiRNA Biogenesis

484

A Role for AGO4 in the Male Mouse Germ Line

Kristina Herbert, Genaro Pimienta, Suzanne DeGregorio, Joan Steitz Yale University, New Haven, CT, USA Since their discovery, microRNAs (miRNAs) have been shown to be critical in regulating gene expression for a variety of cellular processes including developmental timing, cell growth, apoptosis, and differentiation. Therefore, it is important to determine how miRNA biogenesis is regulated. The Microprocessor complex is made up minimally of Drosha, an RNaseIII-like enzyme, and DGCR8, a dsRBD containing protein. The Microprocessor is responsible for the first step in miRNA biogenesis, cleavage of the pri-miRNA to release a stem-loop structure known as the pre-miRNA, which contains the embedded mature miRNA. Using a novel peptide fractionation protocol and phosphopeptide enrichment strategies coupled to high-resolution mass-spectrometry and Maxquant software data analysis, we have systematically mapped 23 sites of phosphorylation in DGCR8 expressed in either insect or HEK cells. Expression of phospho-mimic DGCR8 or inhibition of phosphatases increased the levels of expression of DGCR8 and Drosha, as well as pri-miRNA processing. This indicates that signaling cascades may play an important role in regulating the Microprocessor complex.

Stephanie Hilz, Andrew Modzelewski, Rebecca Holmes, Andrew Grimson, Paula Cohen Cornell University AGO proteins interact with small non-coding regulatory RNAs such as siRNAs (small interfering RNAs) and microRNAs (miRNAs). Together, these AGO-small RNA complexes function to regulate gene expression in eukaryotes; regulation can be transcriptional and post-transcriptional, and a variety of different mechanisms have been described. Almost all animal genomes encode multiple AGO proteins, and both human and mouse genomes encode four: AGO14. While unique roles for different AGO proteins have been identified in model systems such as C. elegans and D. melanogaster, little is known about functional specialization of mammalian AGO proteins. To explore the role of AGO4 in mice, in which this protein is expressed at the highest levels in the male germ line, we have generated an Ago4 knockout mouse line. Ago4 knockout mice are viable but have a variety of defects within the male germ line. Ago4 knockout male mice have a reduced sperm count and smaller testis, likely as a result of increased apoptosis in meiotic germ cells. Further investigation revealed that these fertility phenotypes in Ago4 knockout mice result from a failure to correctly silence the sex chromosomes during meiosis in males, a process referred to as meiotic sex chromosome inactivation (MSCI), a defining feature of meiosis in male mammals. As AGO proteins function together with small RNAs, we isolated and sequenced small RNAs from the germ lines of Ago4 knockout and wild-type males. We found a global reduction of microRNAs in the absence of AGO4, and down regulation of specific microRNA families, including those with previously established roles in the male germ line. To understand how the altered Ago4 knockout small RNA populations might change patterns of gene expression, we performed a series of transcriptome sequencing experiments. We isolated and sequenced mRNAs from multiple pre-meiotic and meiotic germ cell populations from both Ago4 knockout and wild-type mice. Analysis of mRNA expression levels confirmed signatures of defective sex chromosome silencing. Surprisingly, our analysis of the pre-meiotic sequencing data indicated that loss of AGO4 drives premature entry into meiosis, a hypothesis validated histologically. Together, our results indicate that AGO4 has specialized roles in the male germ line, contributing to sex chromosome silencing in meiosis, and regulating meiotic entry. Poster Session 3: Small RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

485 Characterization of Stage-Specific Small RNAs during Development of Triops cancriformis (Tadpole Shrimp)

Yuka Hirose1,2, Kahori Takane1,2, Emiko Noro1, Kiriko Hiraoka1, Masaru Tomita1,2, Akio Kanai1,2 Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan, 2Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa, Japan Small RNA is one of key factors in gene regulation, and has been implicated in various cellular processes. Recently, it has been reported that microRNA (miRNA), which is the almost minimum size of regulatory RNA (approximately 20-22 nucleotides (nt) long), is deeply involved in cell differentiation and development. While, the biological importance of other sizes of small RNAs on the cell differentiation and development still remain unclear. Thus, we focused on small RNAs ranging in length from 25 to 45 nt. We selected Triops cancriformis (tadpole shrimp) as our target organism, since morphology of T. cancriformis larva dramatically changes during development. In order to identify stage-specific small RNAs in T. cancriformis development, small RNA libraries in each six developmental stages (egg, 1st to 4th instar larvae, and adult) were constructed. By deep sequencing, we first obtained approximately 46 million reads ranging from 12 to 45 nt in length and extracted reads of 25-45 nt small RNAs. Then, we conducted expression pattern analysis and detected 57 small RNA candidates that express in any one of the six developmental stages with more than one thousand reads number. After the nucleotide sequence comparison of these candidates with that of known non-coding RNAs in other species, we found that eight transfer RNA (tRNA) fragments, one ribosomal RNA fragment and one piwi-interacting RNA are stage-specifically expressed in T. cancriformis development. Furthermore, four of eight tRNA fragments were derived from the 5’-end region of tRNA and three of eight tRNA fragments were derived from the 3’-end region of tRNA. Since it is suggested that tRNA fragments may involved in cell proliferation in several recent papers, these stage-specific tRNA fragments with high reads numbers may have a some important function(s) in T. cancriformis development.

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486

Real-time Dynamics of RISC-mRNA Interaction Observed via Single-Molecule FRET

Seung-Ryoung Jung1,3, Eunji Kim2, Ji-Joon Song2, Sungchul Hohng1,4 1 Department of Physics and Astronomy, Seoul National University, Seoul, South Korea, 2Department of Biological Sciences, KAIST, Daejeon, South Korea, 3National Center of Creative Research Initiatives, Seoul National University, Seoul, South Korea, 4Department of Biophysics and Chemical Biology, Seoul National University, Seoul, South Korea RNA molecules are not simple information carriers, but play critical regulatory roles in eukaryotic gene expression via the mechanism called RNA interference. Two main types of small eukaryotic regulatory RNA molecules—microRNA and small interfering RNA (siRNA)—are generated through distinct biogenesis pathways, but to perform their functions, they are incorporated into the same effector complexes, called RNA-induced silencing complexes (RISCs). Argonaute is the key component of the RISC, which is responsible for target transcript recognition and their destructions. The target recognition and processing mechanisms of Argonaute have been investigated using conventional biochemical and biophysical techniques such as gel electrophoresis and x-ray crystallography. Although these techniques have significantly advanced our understanding of the operational mechanism of Argonaute, they provide just static pictures of an otherwise dynamic process. To overcome the limit of conventional experimental tools, we developed single-molecule FRET assay to visualize in real time the interaction of RISC with its various targets. Consistently with the conventional view that the nucleation starts from the 5’-end of the guide strand, mismatches in the first 6 bases of the 5’-end of the guide strand exhibited huge deceleration in the binding rate. The nucleation step was greatly accelerated in the presence of Mg2+, but such an effect was not observed when the phosphate at the 5’-end of the guide strand or the valine residue at the C-terminal of the Argonaute was removed, suggesting that anchoring of the guide strand to the MID domain is critical for the nucleation step. RISC-target stability was not affected by mismatches introduced to the 3’-end of the guide strand, but was in other regions, suggesting that base paring between guide and target strands was not complete, but limited to the 16th base. After finding the target, the 3’-end of the guide strand was repeatedly released from the PAZ domain, and trapped again. Mismatches introduced in the 12 - 17 bases from the 3’-end of the guide strand had significant effects on the repetitive release dynamics of the 3’-end, supporting that these bases are involved in the dynamic unwinding process. Poster Session 3: Small RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

487 The Profile of snoRNA-derived MicroRNAs that Regulate Expression of Variant Surface Proteins in Giardia lamblia

Wei Li, Ashesh Saraiya, Ching Wang Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158-2280 In the current investigation, we analyzed all the known small nucleolar RNAs (snoRNAs) in the deeply branching protozoan parasite Giardia lamblia for potential microRNAs (miRNAs) that might be derived from them. Two putative miRNAs have since been identified by Northern blot, primer extension, 3’-RACE and co-immunoprecipitation with Giardia Argonaute (GlAgo), and designated miR6 and miR10. Giardia Dicer (GlDcr) is capable of processing the snoRNAs into the corresponding miRNAs in vitro. Potential miR6 and miR10 binding sites in Giardia genome were predicted bioinformatically. A miR6 binding site was found at the 3’-untranslated regions (UTR) of 44 variant surface protein (vsp) genes, whereas a miR10 binding site was identified at the 3’-end of 159 vsp open-reading frames. Thirtythree of these vsp genes turned out to contain binding sites for both miR6 and miR10. A reporter mRNA tagged with the 3’ end of vsp1267, which contains the target sites for both miRNAs, was translationally repressed by both miRNAs in Giardia. Episomal expression of an N-terminal c-myc tagged VSP1267 was found significantly repressed by introducing either miR6 or miR10 into the cells and the repressive effects were additive. When the 2’-O-methyl antisense oligos (ASOs) of either miR6 or miR10 was introduced, however, there was an enhancement of tagged VSP1267 expression suggesting an inhibition of the repressive effects of endogenous miR6 or miR10 by the ASOs. Of the total 220 vsp genes in Giardia, we have now found 178 of them carrying putative binding sites for all the miRNAs that have been currently identified, suggesting that miRNAs are likely the regulators of VSP expression in Giardia.

488

The mir-35-41 Family of MicroRNAs Regulates RNAi Sensitivity in C. elegans

Katlin Massirer, Saida Perez, Vanessa Mondol, Amy Pasquinelli UCSD, San Diego, CA, USA MicroRNAs (miRNAs) play important roles in many biological pathways, although very few miRNA genes have been shown to be essential for viability. In C. elegans , loss of the mir-35-41 cluster of miRNAs in the mir-35-41(gk262) deletion mutant results in temperature sensitive embryonic lethality. These seven miRNAs share the same seed sequence (nucleotides 2-7) and are predicted to regulate common genes. While investigating potential targets of the miR-35-41 miRNAs, we discovered that the mir-35-41(gk262) strain is hypersensitive to RNA interference (RNAi). In contrast to miRNAs that typically pair imperfectly to target mRNAs, resulting in reduced stability and translation, the RNAi mechanism utilizes small interfering RNAs (siRNAs) that usually pair perfectly to their targets and induce mRNA cleavage or transcriptional silencing. The siRNA guides can originate from exogenous (exo-RNAi) or natural endogenous (endoRNAi) sources of double-stranded RNA (dsRNA). In C. elegans, inactivation of genes that function in the endo-RNAi pathway can result in enhanced silencing of genes targeted by siRNAs from exogenous sources, indicating cross-regulation between the pathways. Our observation that loss of miR-35-41 increases RNAi sensitivity reveals a new connection between these small RNA pathways. Here we show that the hypersensitivity of mir-35-41 is dependent on the canonical RNAi pathway and is similar to the enhanced RNAi exhibited by lin-35/Rb mutants. In worms lacking miR-35-41, there is reduced expression of lin-35/Rb, the C. elegans homolog of the tumor suppressor Retinoblastoma gene. Genome wide microarray analyses show that targets of endo-siRNAs are up-regulated in mir-35-41 mutants, a phenotype also displayed by lin-35/Rb mutants. Furthermore, overexpression of lin-35/Rb specifically rescues the RNAi hypersensitivity of mir35-41 mutants. Although the mir-35-41 miRNAs appear to be exclusively expressed in germline and embryos, their effect on RNAi sensitivity is transmitted to multiple tissues and stages of development. Additionally, we demonstrate that maternal contribution of mir-35-41 or lin-35/Rb is sufficient to reduce RNAi effectiveness in progeny worms. Our results reveal that miRNAs can broadly regulate other small RNA pathways and, thus, have far reaching effects on gene expression beyond directly targeting specific mRNAs. This work was supported by CAPES-Brazil, the Emerald Foundation and NIH (GM071654). Poster Session 3: Small RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

489 Global miRNA profiling and Ago2 RIP-Chip identifies consistently deregulated miRNAs in neuronal adaptive and death responses induced by MPP+

Elena Miñones-Moyano1, Birgit Kagerbauer1, Michaela Beitzinger2, Gunter Meister2, Xavier Estivill1, Eulalia Marti1 1 Centre for Genomic Regulation, Barcelona, Spain, 2Regensburg Univeristy, Regensburg, Germany MicroRNAs (miRNAs) are small non-coding RNAs that modulate gene expression, and they are particularly abundant in the nervous system, where they play key roles in neuronal differentiation and maintenance. Transcriptome modulation is crucial for the development of stress responses, enabling the activation of specific gene expression networks, which will trigger adaptive responses or death pathways depending on the strength of the stimuli. Typically, exposure to a mild stress will trigger an adaptive response that will result in increased survival rates to a subsequent exposure to higher levels of the same type of stress. In this study we have aimed to assess the role of miRNAs in the development of such stress responses induced by MPP+ intoxication in postmitotic neurons. We profiled global miRNA expression as well as miRNA deregulation within Ago2 complexes (Ago2 RIP-Chip) upon either mild or acute intoxication and in basal conditions. Significant and consistent miRNA deregulation was detected by both techniques and the deregulation of several miRNAs from miR-29 and miR-30 families was validated using rRT-PCR. Furthermore iTRAQ proteomic profiling of the same physiological conditions showed significant deregulation of proteins which are predicted targets for the deregulated miRNAs, and revealed an inverse correlation between them. Our results show that changes in the expression of several miRNAs are wider and stronger in neuronal death responses compared to adaptive responses, suggesting that the extent of miRNA deregulation could be involved in progressively switching gene expression from neuronal adaptive responses to cell death pathways.

490

Substrate Specificity In Small RNA Silencing Pathways

Milijana Mirkovic-Hoesle, Klaus Foerstemann Gene Center, Munich, Germany RNA silencing (RNAi) is a conserved mechanism for posttranscriptional gene regulation executed by double-stranded RNAs (dsRNAs) suppressing specific transcripts in a sequence-dependent manner. Two classes of these guide molecules are known: microRNAs (miRNAs) and small interfering RNAs (siRNAs). The latter include exogenous RNAs (exosiRNAs) produced during viral infection or transgenic RNAi and endogenous small RNAs (endo-siRNAs) derived from transposable elements. Exo-siRNA biogenesis depends on Dicer 2 (Dcr 2) and a double-stranded RNA binding protein (dsRBP) called R2D2 while endo-siRNAs are produced by Dcr2 and a specific splice variant of Loquacious, Loqs-PD. The puzzling conclusion is that the cells must be able to distinguish the precursor despite the fact that their structure is identical. How is this possible? I will use Drosophila melanogaster as a model organism to combine genetic studies with next-generation sequencing approaches to respond to this question.

Poster Session 3: Small RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

491

Abstract Withdrawn

492

Characterization of Small RNAs by Using a Ribosome-enriched Fraction in Escherichia coli

Shinnosuke Murakami1,2, Yoshiki Ikeda1, Emiko Noro1, Masaru Tomita1,2, Kenji Nakahigashi1, Akio Kanai1,2 1 Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan, 2Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa, Japan Small RNAs (sRNAs) are important part of post-transcriptional regulators of gene expression in prokaryotes. Recently, we reported a deep sequencing analysis of a sRNA fraction in E. coli and suggested that there are still many potential transcriptional units for candidates of novel sRNAs in E. coli genome (Shinhara, A. et al., BMC Genomics, 2011). While it was quite difficult to find out biological function(s) of these sRNA candidates, we predicted that at least 104 sRNA candidates possessed potential open reading frames and may encode small peptides. Therefore, in this study, we further conducted deep sequencing analysis of RNAs prepared from ribosome (polysome)-enriched fractions (an average of 24.6 M reads, n=2) of E. coli and compared relative numbers of RNAs with those from total RNA fractions (an average of 19.6 M reads, n=3). As expected, a number of RNAs from CDS regions, tRNA genes and rRNA genes are detected in the ribosome-enriched fractions. Besides, at least 10 possible sRNA candidates from intergenic regions are also detected in the fraction. The result suggested that these candidates are expected to translate to small peptides or could have function related to translation. Comparing to the ribosome-enriched fraction, total RNA fraction possessed a huge number of RNA reads from intergenic regions of tRNA or rRNA operons. It is noted that at least 7 novel sRNA candidates as well as some previously known sRNAs (csrB/C, sibB/C/D/E, glmY/Z and so on) are enriched in the total RNA fraction, suggesting that these sRNA candidates are expected to have some biological role(s). Now, we are confirming the expression of these candidates during E. coli growth by northern blotting and are constructing overexpression plasmids of those sRNA candidates for biological analysis.

Poster Session 3: Small RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

493

Regulatory small RNAs generated from miRNA loops

494

Position of Hfq Distal Face Binding Site on RNA Controls the Rate of RNA Annealing

Katsutomo Okamura1,2, Erik Ladewig1, Eric Lai1 1 Memorial Sloan-Kettering Cancer Center New York, USA, 2Temasek Lifesciences Laboratory, Singapore microRNAs (miRNAs) are an extensive family of short regulatory RNAs that are collectively essential for development and physiology in higher eukaryotes. Most miRNAs are generated from stepwise processing of primary miRNA (primiRNA) transcripts. The pri-miRNA is initially cleaved in the nucleus by Drosha into a ~60-70 nt pre-miRNA hairpin, and then cleaved in the cytoplasm by Dicer to yield a miRNA/miRNA* duplex. The miRNA strand is preferentially incorporated into an Argonaute protein and guides it to target transcripts. In addition to the canonical pathway, several alternative pathways exploit other ribonucleases to bypass one of the core miRNA processing steps. The steady-state levels of miRNA strands are often much greater than their partner miRNA* strands, which has led to the perception that miRNA* strands lack function. However, we observed that many miRNA* strands have detectable activity and contribute to functional miRNA regulatory networks. Here, we examined another species widely assumed to be a byproduct of miRNA biogenesis, the terminal loop of the pre-miRNA that is released upon Dicer cleavage. Although loop reads are usually quite rare, select miRNA loops are present at non-trivial levels and even occur in small RNA libraries prepared from Drosophila Argonaute1 complexes. We verified the association of several miRNA loops with Argonaute1 using Northern analysis, and further observed that expression of their cognate pri-miRNAs repressed luciferase reporters bearing complementary loop target sequences. We recapitulated the loading of these miRNA loops using an in vitro processing assay, and the analysis of different miRNAs and mutagenized hairpins suggests that loop loading acts selectively on specific miRNA genes. Moreover, miRNA loop loading appears to be evolutionary conserved, since we could detect several miRNA loops in mammalian Argonaute complexes. These observations reveal an unexpected mechanism by which a subset of miRNA hairpins generates regulatory RNAs from their loop regions. This finding again broadens the range of miRNA target regulation, and raises mechanistic questions about how these presumably single-stranded species are loaded into Argonaute proteins.

Subrata Panja, Sarah Woodson Johns Hopkins University, Baltimore, MD, USA Regulation of bacterial gene networks by small non-coding RNAs (sRNAs) requires basepairing between the sRNA and its mRNA target. The RNA chaperone protein Hfq facilitates this base pairing and is necessary for gene regulation by sRNAs ingram negative bacteria. In vitro, rapid binding and release of two RNA strands from the Hfq ternary complex accelerates helixinitiation 30-10,000 times above the Hfq-independent rate. Although mRNAs regulated by sRNAs frequently contain A-rich Hfq binding sites, it is not clear how the location of Hfq binding affects sRNA base pairing and the efficiency of gene control. Using fluorescent “toy” RNAs engineered to contain a strong Hfq binding site at different distances from the complementary target region, we show that RNA annealing is fastest when Hfq binds adjacent to the target sequence. When this distance reaches 20-40 nucleotides, the advantage of tethering Hfq to the RNA substrate is lost and the annealing rate returns to the basal level. We further show that Hfq-dependent annealing requires an A-rich sequence that binds the distal face of Hfq, while a strong U-rich proximal binding sequence inhibits annealing to the target sequence. Finally, we show that adding A18 ectopically to a minimal rpoS mRNA dramatically increases annealing of DsrA sRNA in the presence of Hfq. Our results show that tethering Hfq to target RNAs through its distal face results in efficient strand annealing independently of sequence context, and that the annealing rate is inversely proportional to the distance between Hfq and the sRNA binding site.

Poster Session 3: Small RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

495 Complexity of murine cardiomyocyte miRNA biogenesis, sequence variant expression and function

Jennifer Clancy1, David Humphreys2, Carly Hynes1, Hardip Patel1, Thomas Preiss1 1 ANU, Canberra, ACT, Australia, 2VCCRI, Sydney, NSW, Australia microRNAs (miRNAs) are critical to heart development and disease. Emerging research indicates that regulated precursor processing can give rise to an unexpected diversity of miRNA variants. We subjected small RNA from murine HL-1 cardiomyocyte cells to next generation sequencing to investigate the relevance of such diversity to cardiac biology. ~40 million tags were mapped to known miRNA hairpin sequences as deposited in miRBase version 16, calling 403 generic miRNAs as appreciably expressed. Hairpin arm bias broadly agreed with miRBase annotation, although 44 miR* were unexpectedly abundant (>20% of tags); conversely, 33 -5p/-3p annotated hairpins were asymmetrically expressed. Overall, variability was infrequent at the 5’ start but common at the 3’ end of miRNAs (5.2% and 52.3% of tags, respectively). Nevertheless, 105 miRNAs showed marked 5’ isomiR expression (>20% of tags). Among these was miR-133a, a miRNA with important cardiac functions, and we demonstrated differential mRNA targeting by two of its prevalent 5’ isomiRs. Analyses of miRNA termini and base-pairing patterns around Drosha and Dicer cleavage regions confirmed the known bias towards uridine at the 5’ most position of miRNAs, as well as supporting the thermodynamic asymmetry rule for miRNA strand selection and a role for local structural distortions in fine tuning miRNA processing. We further recorded appreciable expression of 5 novel miR*, 38 extreme variants and 8 antisense miRNAs. Analysis of genome-mapped tags revealed 147 novel candidate miRNAs. In summary, we revealed pronounced sequence diversity among cardiomyocyte miRNAs, knowledge of which will underpin future research into the mechanisms involved in miRNA biogenesis and, importantly, cardiac function, disease and therapy. Humphreys et al. PLoS One. 2012;7(2):e30933

496 Transition of MicroRNA Function from Repression to Activation Depending on the Extent of Base Pairing with the Target Site

Ashesh Saraiya, Ching Wang University of California-SF, San Francisco, CA, USA MicroRNAs (miRNAs) are major post-transcriptional regulators of gene expression that bind to their target sites in gene transcripts and repress translation or induce mRNA cleavage. Here we show in the ancient protozoan Giardia lamblia a snoRNA-derived 26-nucleotide(nt) miRNA, miR3, that represses the translation of a histone H2A mRNA carrying an imperfectly matched target site. However, when the target site was made fully complimentary to miR3, it enhanced the translation of the transcript by 2-fold, whereas the 2’-O-methyl miR3 anti-sense oligo caused a ~25% repression. Both of these effects are dependent on the non-slicing Argonaute protein in this organism. A stepwise mutational analysis of the 26 nt fully-complementary target site for miR3 was conducted. The activating effect of miR3 was significantly reduced, when a single nt at the 5’-end of the target site was altered. The residual activating effect of miR3 (~20%) persisted up to a loss of base-pairing with 10 of the 3’ nts in miR3. The repressive effect of miR3 became detectable when 12 of the 3’ nts in miR3 have lost their base-pairings to the target. The maximum level of repression was reached when only the 8 nt seed sequence at the 5’-end of miR3 remained complementary to the target. The 8-nt seed sequence in miR3 was synthesized and found capable of exerting a similar extent of Argonaute-dependent translational repression. Thus, a full complementation between miR3 and the target site results in activation, whereas binding of only the 8 nt seed sequence of miR3 to the target site leads to repression. This is the first report showing a correlation between base pairings and drastic functional changes in a miRNA. The potential mechanisms behind this intriguing phenomenon are postulated.

Poster Session 3: Small RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

497 Comparative Profiling of Prostate Epithelial and Stromal Cell MicroRNA Transcriptome by Deep Sequencing

Girish Shukla1, Guru Jagadeeswaran2, Zheng Yun3, Kavleen Sikand4, Ramanjulu Sunker2 1 Cleveland State University, Cleveland, OH USA, 2. Department of Biochemistry and Molecular Biology, Oklahoma State University, Still Water, Oklahoma, USA, 3Institute of Developmental Biology and Molecular Medicine and School of Life Sciences, Fudan University, Shanghai, 200433, China, 4Center for Stem cell and regenerative medicine, Postgraduate Institute of medical Research, Chandigarh, India Differential expression of miRNAs has been implicated in Prostate cancer (PCa). Prostate epithelial and stromal cells interactions appear to play an important role in prostate cancer progression. For experimental purposes, commercially available Prostate Epithelial (PrEC) and stromal (PrSC) cells as controls are being used widely in numerous studies focused on prostate carcinogenesis. However, miRNA complement of these cells has not been investigated yet. We utilized these commercially available human PrEC and stromal PrSC to generate size fractionated small RNA libraries and established the expression of miRNAs by deep sequencing. Over 50 million reads for PrEC were analyzed in which 860,468 were unique sequences. Similarly, nearly 76 million reads for PrSC were analyzed and we found over 1 million unique reads. A large number of miRNAs were found to be expressed in both PrEC and PrSC primary cell lines. Let-7 family was found to be highly expresses in both cell lines. We have also identified PrEC and PrSC specific novel miRNAs. Further validation and characterization of expression data and expression of novel miRNAs is in progress.

498

Mapping Targets for sRNAs in Pathogenic E.coli

Jai Tree, Sander Granneman, David Gally, David Tollervey University of Edinburgh EnterohaemorrhagicE. coli (EHEC) strains express multiple virulence factors including a type III secretion system (T3SS), which is essential for colonization of the host, and Shiga toxins that are responsible for tissue damage during human infection. Analyses of heterogeneity in the expression of virulence factors indicated the presence of post-transcriptional regulation and implicated the RNA chaperone Hfq in this process. To better understand the post-transcriptional control mechanism, Hfq binding sites were identified across the transcriptome using UV-crosslinking (CRAC) of RNA-protein complexes in actively growing, pathogenic E.coli. Hfq commonly mediates translational control by facilitating base pairing between mRNAs and small RNAs (sRNAs). We located Hfq binding binding sites within mRNAs for the T3SS and identified 8 novel small RNAs encoded within horizontally acquired DNA regions. Novel sRNAs are encoded within the T3SS loci and in the prophage that encodes Shiga toxin 2; the roles of these sRNAs in gene regulation are being functionally characterized. In silico predictions of the mRNA targets of an sRNA are prone to generating high numbers of false positives. Only a small region of complementarity is required for Hfq-dependent base pairing and the interactions can occur at diverse sites on both the mRNA and sRNAs. UV-crosslinking and analysis of hybrid cDNA sequences (CLASH) has recently been developed to identify RNA:RNA interactions using a modification of the CRAC protocol. Preliminary data indicate that interacting sRNA:mRNA pairs can be experimentally identified by CLASH with Hfq, potentially allowing global mapping of sRNA targets in vivo.

Poster Session 3: Small RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

499

The Period protein homolog LIN-42 negatively regulates microRNA biogenesis in C. elegans

500

Comprehensive Analysis of Small RNAs in Oryzias latipes and Takifugu rubripes

Priscilla Van Wynsberghe1,2, Emily Finnegan2, Amy Pasquinelli2 1 Colgate University, 2University of California - San Diego MicroRNAs (miRNAs) are essential small RNAs that post-transcriptionally regulate development in C. elegans and other species. They are encoded in the genome and transcribed into primary (pri-) miRNAs that are capped and polyadenylated. Processing of a pri-miRNA by the RNase III enzyme Drosha produces the ~70 nt precursor (pre-) miRNA, which is further processed by a second RNase III enzyme, Dicer, in the cytoplasm to form the mature miRNA. In order to produce the appropriate amount of a particular miRNA in the correct location at the correct time, proper regulation of miRNA biogenesis is crucial. Here we present our work that identifies the Period protein homolog LIN42 as a new regulator of miRNA biogenesis in C. elegans. We mapped a spontaneous suppressor of the normally lethal let-7(n2853) allele to the C terminal region of lin-42. Mutations in this allele (ap201) or a second lin-42 allele (n1089) caused increased mature let-7 miRNA levels at all time points throughout the 3rd and 4th larval stages (when mature let-7 miRNA is normally expressed). These results indicate that lin-42 normally represses the accumulation of let-7 miRNA. This inhibition of mature miRNA expression by LIN-42 is not specific to let-7, as mature levels of lin-4, mir35 and mir-58 are also increased in lin-42 mutant worms. Thus we propose that LIN-42 is a global regulator of miRNA biogenesis. Here we present our results characterizing the mechanism by which LIN-42 regulates miRNA biogenesis and the extent to which LIN-42 regulates mature miRNA levels as seen through RNA sequencing analysis in lin-42 mutant worms. Since LIN-42 is the homolog of Drosophila and mammalian Period proteins, our results raise the possibility that these proteins may share a conserved function in regulating miRNA biogenesis to ultimately control rhythmic processes.

Chaninya Wongwarangkana1, Ryota Terai1, Kosuke Negoro1, Masaki Akiba1, Kazuhiro E. Fujimori2, Shigeharu Kinoshita1, Atsushi Shimizu4, Kosuke Sakai3, Sabine K. Kojima4, Susumu Mitsuyama3, Ikuya Kikuzato5,6, Morimi Teruya5,7, Maiko Nezuo5,8, Shuichi Yano5,8, Yukino Miwa5,8, Yumi Imada5,8, Yuki Sato5,6, Tsukahara Masatoshi5,8, Jun Kudoh3,9, Takashi Hirano2,6, Nobuyoshi Shimizu9, Shugo Watabe1, Shuichi Asakawa1 1 Grad. Sch. Agri. Life Sci., Univ. Tokyo., 2AIST. Res. Inst. Cell Eng., 3Lab. Gene Med., Keio Univ. Sch. Med., 4 Dept. Mol. Biol., Keio Univ. Sch. Med., 5Okinawa Cutting-Edge Genome PJ(OCGP), 6Okinawa Sci. Tech. Prom. Ctr (OSTC), 7Okinawa Ind. Tech. Ctr (OITC), 8Tropical Tech. Ctr (TTC), 9Adv. Res. Ctr. GSP, Keio Univ. Objective: Recent development of high-throughput sequencing technology has uncovered a landscape of small RNAs in eukaryote. Various aspects of small RNAs, such as origins, structures, associated effector proteins, and biological roles have led to the general recognition of three main categories: short interfering RNAs (siRNAs),microRNAs (miRNAs), and piwi-interacting RNAs (piRNAs). Many studies hint that human and mouse cells contain a large number of the small RNAs, with potential to regulate expression of almost all the human and mouse genes. While, only small number of fish small RNAs has been reported comparing with other animal categories. Our current study is designed to explore torafugu (Takifugu rubripes) and medaka (Oryzias latipes) small RNAs, which their entire genomes have already been sequenced. In this study, in order to understand how specific small RNAs regulate gene expression in the various organs of fishes and get insight into their biological functions, we used a next-generation sequencer (SOLiD3) and fully characterized torafugu and medaka small RNA population. Results: In torafugu, we categorized miRNAs based on their seed motifs. miRNAs with a same seed motif 5’-end 2-8 nucleotides are grouped into the same family. According to this classification, we were able to categorize miRNAs into more than 300 seed families. miRNAs from the same family tend to have similar expression patterns. miRNAs were mainly expressed in tissues other than ovary and testis. However, some miRNA families were highly abundant in gonads. In addition to those miRNAs, a large number of other small RNA species were also found in this study. In ovary and testis, small RNAs with larger size were much more abundantly expressed. These results would be consistent with previously reports of other organisms, in which miRNAs were mainly expressed in tissues other than ovary and testis, whereas piRNAs were superiorly expressed in the ovary and testis. In addition to torafugu, we also performed comprehensive analysis medaka small RNAs. We found about 400 known miRNAs. Some of the miRNAs showed strong tissue specific expression. In ovary and testis, population of 25-31 nucleotides small RNAs were expressed much more from repetitive sequences when compared with other tissues. The result was in agreement with previous studies, in which piRNAs were derived from repetitive elements in the germ line of various species such as Drosophila and mouse. When compared size distribution pattern of torafugu with medaka, we found that the sizes of small RNAs which mainly expressed in gonads were different between torafugu and medaka. We will also discuss the difference between torafugu and medaka in other aspects in this meeting.

Poster Session 3: Small RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

501 The General Role of Drosha Processing in the Regulation of microRNA Expression and Lessons Beyond microRNA Biogenesis

Yong Feng, Xiaoxiao Zhang, Yan Zeng University of Minnesota, Minneapolis, MN, USA microRNAs (miRNAs) are first transcribed as long, primary transcripts, which then undergo a series of processing steps to generate the single-stranded, mature miRNAs. Here, we show that Drosha cleaves hundreds of human primary miRNA (pri-miRNA) transcript substrates with different efficiencies in vitro. The differential Drosha susceptibility of the pri-miRNAs significantly correlates with the expression of the corresponding, mature miRNAs in vivo. Conserved miRNAs are more efficiently expressed in vivo, and their pri-miRNAs are also better Drosha substrates in vitro. Combining secondary structure prediction and statistical analyses, we identify features in human primary miRNA transcripts that predispose miRNAs to efficient Drosha processing in vitro as well as to better expression in vivo. We propose that the selectivity of Drosha action contributes greatly to the specificity and efficiency of miRNA biogenesis. Moreover, this study serves as an example of relative substrate specificity of a biochemical reaction having a functional consequence at a global scale in vivo. Additional evidence is presented to support such a relative specificity hypothesis in diverse, complex biochemical systems.

502

The Butterfly Effect of Translationally-acting Riboswitches

Laurène Bastet, Antony Lussier, Audrey Dubé, Daniel Lafontaine Département de biologie, Faculté des sciences, Groupe ARN/RNA Group, Université de Sherbrooke, Sherbrooke, Québec, Canada From signal detection to gene expression regulation, bacteria make use of complex networks to quickly adjust to environmental changes. Among the major players of the adaptive response are riboswitches, which are cis-acting regulatory elements located in the 5’UTR of mRNAs. The binding of a metabolite to a riboswitch leads to mRNA conformational changes that control the downstream gene expression at a transcriptional or translational level. Because they regulate at the mRNA level, transcriptionally-acting riboswitches can control gene expression of very large operons. In contrast, riboswitches acting at the level of translation should control translation initiation of the gene located immediately downstream to the riboswitch. However, numerous examples have shown that translation inhibition often leads to mRNA degradation, thus suggesting, that translationally-acting riboswitches may also modulate gene expression by controlling the level of mRNA. To shed light on the fate of mRNAs regulated by translationally-acting riboswitches, we focus here on the Escherichia coli btuB and thiM riboswitches,which recognize coenzyme B12 and thiamine pyrophosphate, respectively. Using lacZ transcriptional fusions and Northern blot experiments, we have observed that the addition of ligand to the growth medium causes rapid mRNA degradation. Using key mutant constructs, we established that the inhibition of translation initiation is a prerequisite to the mRNA level decrease. Moreover, we also determined using in vivo and in vitro assays that the RNA degradosome recognizes a similar region found in both btuB and thiM ORF. Which corresponds to a cleavage site that is located nearby the AUG start codon. Interestingly, our results also show that a stem-loop structure positioned upstream of the cleavage site plays a crucial role in the degradation activity and could constitute an internal entry site for the RNA degradosome. Taken together, our study provides the first experimental evidence that translationally-acting riboswitches can also regulate mRNA stability by controlling translation initiation. Indeed, like a butterfly effect, ligand binding to the riboswitch induces RBS sequestration into a stem loop structure,which thus leads to ribosomes clearing and mRNA degradation. Surprisingly, while most riboswitches operating at the translational level are proposed to be reversible, here we show that the tight coupling between translation modulation and mRNA degradation makes the repression irreversible. Poster Session 3: Small RNAs & RNA Catalysis and Riboswitches

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

503

A small kinase ribozyme with unusual dependence on pH and Cu2+ for dual-site activity

504

Mechanism for Gene Control by a Natural Allosteric Group I Ribozyme

Elisa Biondi, Raghav Poudyal, Andrew Sawyer, Adam Maxwell, Donald Burke University of Missouri Phosphoryl transfer onto backbone hydroxyls is a recognized catalytic activity of nucleic acids, but relatively little is known about how ribozymes position donor and acceptor substrates and effect phosphoryl transfer between them. We demonstrate that kinase ribozyme K28 possesses a complex active site that promotes (thio)phosphorylation of two residues that are widely separated in primary sequence. After allowing the ribozyme to radiolabeled itself by phosphoryl transfer from [γ-32P]GTP, DNAzyme-mediated cleavage yielded two radiolabeled cleavage fragments, indicating separate phosphorylation sites within each fragment. Enzymatic digestion and mutational analysis identified specific nucleotides important for activity and established that the active structure comprises a constrained pseudoknot with unusual connectivity. Nuclease sensitivities for several nucleotides near the pseudoknot core were altered in the presence of GTPγS, indicating donor-induced folding. To reveal the chemical strategies used by ribozyme K28 to enhance reaction rates, the metal ion and pH requirements were determined for a fully-active, 58nt form of the ribozyme. Importantly, K28 is strongly dependent upon Cu2+, which it binds with sub-micromolar affinity. The possibility that Cu2+ dependence is solely due to interactions with the GTPγS thiophosphoryl group was ruled out by the observation that phosphoryl transfer from GTP also required Cu2+, consistent with a specific role for copper in catalysis of phosphoryl transfer. Replacing Mg2+ with [Co(NH3)6]3+ significantly reduced net activity, indicating contributions from inner-sphere coordination with Mg2+. However, the role of Mg2+ is structural rather than catalytic, as the observed first-order rate constant in CoHex/Cu2+ is indistinguishable from the rate constant in Mg2+/Cu2+. Unlike previously studied kinase ribozymes, the reaction rate for the truncated form of K28 increased with pH in log-linear fashion (n=1), indicating participation of a species that deprotonates with an apparent pKa of 8.0±0.1. We propose that this ribozyme may utilize Cu2+ in its catalytic mechanism to promote phosphoryl transfer through formation of Lewis acid catalytic sites, deprotonation of a catalytic copper-bound water or modulation of nucleobase pKa through direct Cu2+-nucleobase interactions.

Andy G. Y. Chen, Narasimhan Sudarsan, Ronald Breaker Yale University, New Haven, CT, USA An allosteric ribozyme consisting of a metabolite-sensing riboswitch and a group I self-splicing ribozyme was recently found in the pathogenic bacterium Clostridium difficile (1). The riboswitch senses the bacterial second messenger c-diGMP, thereby controlling 5’-splice site choice by the downstream group I ribozyme. The proximity of this allosteric ribozyme to the open reading frame (ORF) for CD3246, a putative virulence gene in C. difficile, suggests that coenzymemediated regulation of splicing controls gene expression. In the presence of c-di-GMP, the allosteric ribozyme in the CD3246 precursor mRNA generates a spliced transcript that retains the riboswitch aptamer and the coding sequence. In the absence of c-di-GMP, the ribozyme mediates an alternative GTP attack that results in a truncated transcript (alternative GTP-attack product). Using lacZ translational fusion constructs in the surrogate organism Escherichia coli, we investigated the difference in gene expression between the spliced product and the alternative GTP-attack product. We provide evidence (2) that CD3246 gene expression is activated if the aptamer binds c-di-GMP. In this case, allosteric ribozyme splicing creates a ribosome binding site (RBS) at the junction of the splice site, thereby driving translation from a conserved UUG start codon. In addition, in-line probing analysis shows that the riboswitch aptamer, even after splicing, can undergo structural changes in response to c-di-GMP. Using phosphodiesterase-deletion mutants of E. coli, further genetic analysis suggests that the riboswitch may control CD3246 expression by revealing or occluding the newlyformed RBS. Overall, this tandem riboswitch-ribozyme architecture extends the timespan of regulation by allowing the riboswitch to control translation after splicing, and adds a backup mechanism for suppression of translation in the event of misregulated splicing. 1. Lee ER, Baker JL, Weinberg Z, Sudarsan N, Breaker RR(2010). An Allosteric Self-Splicing Ribozyme Triggered by a Bacterial Second Messenger. Science329: 845-848. 2. Chen AG, Sudarsan N, Breaker RR (2011). Mechanism of gene control by a natural allosteric group I ribozyme. RNA 17:1967-1972. Support provided by the Yale Center for RNA Science and Medicine Poster Session 3: RNA Catalysis and Riboswitches

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

505 Characterizing the Contribution of a Reverse G-U Wobble Pair to Metal Ion Catalysis in the HDV Ribozyme

Ji Chen1, Abir Ganguly2, Zulaika Miswan1, Sharon Hammes-Schiffer2, Philip Bevilacqua2, Barbara Golden1 1 Department of Biochemistry, Purdue University, West Lafayette, Indiana, USA, 2Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania, USA The heptatitis delta virus (HDV) ribozyme catalyzes a self-cleavage reaction using both nucleobase- and metal ionmediated strategies. Previous mechanistic and crystallographic studies have suggested that a partially hydrated Mg2+ ion serves as a Lewis acid to activate the 2’-OH nucleophile, while a protonated cytosine (C75) acts as a general acid to stabilize the leaving group. A reverse G25U20 wobble observed in a recent crystal structure of the HDV ribozyme was found to interact with a Mg2+ ion through water molecules [1]. Nonlinear Poisson-Boltzmann calculations suggest that this site is significantly negatively charged and thus is able to accommodate positively charged metal ions [2]. To characterize the role of the reverse G25U20 wobble in metal ion catalysis, we replaced this base pair with an isosteric neutral A25C20 wobble (HDV AC mutant) and studied the reaction of this variant. Cleavage assays showed that the HDV AC mutant cleaves to at least 80% of completion in a monophasic fashion, but with a rate constant ~100-fold less than that of the wild-type ribozyme. Continuing studies will explore how this reverse wobble base pair contributes to metal ion catalysis in the HDV ribozyme cleavage reaction. References: [1] Chen, J. H., Yajima, R., Chadalavada, D. M., Chase, E., Bevilacqua, P.C. & Golden, B. L. (2010). A 1.9 Å crystal structure of the HDV ribozymepre-cleavage suggests both Lewis acid and general acid mechanisms contribute to phosphodiester bond cleavage. Biochemistry 49, 6508-6518. [2] Veeraraghavan, N., Ganguly, A., Chen, J. H., Bevilacqua, P. C.,Hammes-Schiffer, S. & Golden, B. L. (2011). Metal binding motif in theactive site of the HDV ribozyme binds divalent and monovalent ions. Biochemistry 50, 2672-2682.

506

Efficiency and Fidelity of Splicing at the 3’-Splice Site of RmInt1 Group II Intron

Isabel Chillon1,2, Olga Fedorova1,2, Francisco Martinez-Abarca3, Anna Pyle1,2, Nicolas Toro3 1 Yale University, New Haven (CT), USA, 2Howard Hughes Medical Institute, 3Estacion Experimental del Zaidin - Consejo Superior de Investigaciones Cientificas, Granada, Spain Group II introns are catalytic retroelements which promote their self-splicing from precursor RNAs. They are organized in six domains that undergo a stable tertiary structure stabilized by long-range interactions. The efficiency of splicing and the accuracy of splice site selection are determined by specific base pairing interactions between group II introns and their exons. In particular, the 3’ splice site (3’ SS) is comprised of two interactions immediately upstream of the splice site, the Watson-Crick γ-γ’ interaction and a non-canonical interaction between the first and penultimate positions of the intron, and one interaction just downstream of the splice site, the EBS3-IBS3 (Exon and Intron Binding Site 3) interaction. In this work, we have investigated the role of each of these 3’ SS interactions using the RmInt1 group II intron from the bacterium Sinorhizobium melitoti. We found that disrupting these interactions usually reduced the splicing efficiency, but remarkably we could identify i) mutations that compensated the defects and ii) mutations that increased the rate of lariat formation. Our results suggest that group II introns have an intrinsic structural plasticity, which enables them to improve their catalytic capabilities. This plasticity decisively contributes to make group II introns efficient regulators of gene expression and it has likely played a crucial role in determining the function of these retroelements in evolution.

Poster Session 3: RNA Catalysis and Riboswitches

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

507

The kinetics of the interaction between the btuB riboswitch and coenzymeB12

508

Sequence-Specific Multicolor Imaging of Nucleic Acid Nanostructures

Pallavi Choudhary, Roland K. O. Sigel Institute of Inorganic Chemistry,University of Zurich,Switzerland Riboswitches are small conserved sequences in the 5’-untranslated region of bacterial mRNA that bind certain metabolites with high affinity and specificity. The 202 nucleotide long btuB riboswitch of E.coli undergoes a structural rearrangement upon interaction with coenzyme B12 and some of its derivatives [1-4]. Although being one of the earliest reported riboswitches, little is known about the mechanism of interaction between the btuB riboswitch and its ligand. To get into the details of the interaction between the btuB riboswitch and coenzyme B12, we applied the surface plasmon resonance (SPR) spectroscopy. SPR is a powerful method to study the real time kinetics of binding events and to elucidate the single steps of this docking interaction. With the help of SPR, we were able to measure the association and dissociation constants of the btuB-coenzyme B12 system. Moreover, the comparison of the kinetic constants has also revealed that the slow rate of dissociation makes coenzyme B12 bind stronger to the btuB riboswitch compared to a related ligand, Vitamin B12. Acknowledgement: Financial support by the Swiss National Science Foundation, the European Research Council (ERC) and the University of Zurich is gratefully acknowledged. References: [1] X. Nou, R. J. Kadner, Proc. Natl. Acad. Sci. USA 2000, 97, 7190 [2] A. Nahvi, N. Sudarsan, M. S. Ebert, X. Zou, K. L. Brown, R. R. Breaker, Chem. Biol. 2002, 9, 1043 [3] S. Gallo, M. Oberhuber, R. K. O. Sigel, B. Kraeutler, ChemBioChem. 2008, 9, 1408 [4] S. Gallo, S. Mundwiler, R. Alberto, R. K. O. Sigel, Chem. Commun. 2011, 47, 403-405.

Alexander Johnson-Buck1, Jeanette Nangreave2, Hao Yan2, Nils Walter1 1 University of Michigan, Ann Arbor, MI, USA, 2Arizona State University, Phoenix, AZ, USA Super-resolution fluorescence microscopy (SFM), while typically lacking the spatial resolution of AFM and cryoEM, boasts tunable chemical specificity and mild sample perturbation. Here, we demonstrate the power of an SFM technique called DNA-PAINT to acquire detailed images of structurally heterogeneous nucleic acid objects. We show that DNA-PAINT can reveal the internal structure of 60-by-100 nm origami tiles, obtaining images of the presence and accessibility of specific oligonucleotide sequences on the tile at a resolution of ~10 nm. Imaging before and after cleavage of oligonucleotide substrate patterns on the origami by a deoxyribozyme demonstrates the ability to visualize chemical changes in a single object over time. There is also evidence of non-uniform accessibility of sequences in densely populated arrays. In summary, we demonstrate the feasibility of obtaining sequence-specific multicolor images of nucleic acid objects at the nanoscale with DNA-PAINT.

Poster Session 3: RNA Catalysis and Riboswitches

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

509

Substrate Interaction And RNase P RNA Mediated Cleavage

Leif Kirsebom, Shiying Wu, Guanzhong Mao, Abhishek Srivastava Uppsala University, Uppsala, Sweden The tRNA genes are transcribed as precursors and ribonuclease P is responsible for the trimming of the 5’ end of tRNA in the cell. Bacterial RNase P consists of one protein and one RNA (RPR). The RPR can process tRNA precursors correctly in the absence of the protein. The T-stem/loop (TSL) interacts with a region in RPR (TBS, TSL binding site) forming the TSL/TBS-interaction. We have provided a model in which a productive TSL/TBS-interaction influences the positioning of chemical groups and/or Mg(II)-ions at the cleavage site (1, 2). We use model hairpin loop substrates corresponding to the acceptor, T-stem and T-loop of a precursor tRNA to study the importance of the structure of the substrate and RPR with respect to this model. We are particularly interested in to investigate and define the link between the TSL/TBS-interaction and events at the cleavage site. In our studies we use primarly Escherichia coli RPR and we study the reaction in the absence of protein. We will provide data supporting a functional link between a productive TSL/TBS-interaction and events at the cleavage site. Collectively our findings emphasize the interplay between separate regions upon formation of a productive RPR-substrate complex that leads to efficient and accurate cleavage. Our data provides support for an induced fit mechanism in bacterial RPR-mediated cleavage at the correct site. 1. Brannvall, M., Kikovska, E., Wu, S. and Kirsebom, L.A. (2007). Evidence for induced fit in RNase P RNA mediated cleavage. J. Mol. Biol. 372, 1149-1164. 2. Wu, S., Chen, Y., Lindell, M., Mao, G. and Kirsebom, L.A. (2011). Functional coupling between a distal interaction and the cleavage site in bacterial RNase P RNA mediated cleavage. J. Mol. Biol. 411, 384-396.

510 Helix-length Compensation Studies for Cleavage of Alternate Substrates by the Neurospora VS Ribozyme

Julie Lacroix-Labonté, Nicolas Girard, Sébastien Lemieux, Pascale Legault Université de Montréal, Montréal, Québec, Canada Ribozymes are useful tools for cleavage of specific target RNAs, but they are generally designed to cleave singlestranded RNAs. However, most functional RNAs adopt complex secondary and tertiary structures, and it would be interesting to develop ribozymes that target folded RNAs. The Neurospora VS ribozyme represents a good candidate for recognition of folded RNAs because it recognizes and cleaves a hairpin substrate. Substrate recognition by the VSribozyme involves a loop-loop interaction between the stem-loop I substrate and stem-loop V from the catalytic domain and is important for the cleavage activity of the ribozyme. In this project, we tested the possibility for the VS ribozyme to recognize substrates of different lengths by investigating helix-length compensation between stem-loops I and V. To accelerate the characterization of the different substrate/ribozyme mutants (kcat/KM),we validated a method for simultaneous kinetic characterization of multiple substrates. Several active substrate/ribozyme pairs were identified, indicating the presence of limited substrate promiscuity for stem Ib mutants and helix-length compensation between stems Ib and V. Three-dimensional models of the I/V interaction were generated that are compatible with the kinetic data and provide global structural information on the I/V kissing-loop interaction. This study further illustrates the adaptability of the VS ribozyme architecture for substrate cleavage and represents a first step in the design of mutant VS ribozymes for cleavage of alternate substrates.

Poster Session 3: RNA Catalysis and Riboswitches

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

511 SELEX Experiment Suggests that the Coenzyme-Dependent glmS Ribozyme Evolved from a Self-Cleaving Ribozyme Requiring Only Magnesium for Catalysis

Matthew Lau, Adrian Ferré-D’Amaré National Heart, Lung and Blood Institute, NIH, Bethesda, Maryland, USA Five structurally distinct classes of catalytic RNAs that self-cleave through an internal transesterification reaction have been discovered in nature: the glmS, hairpin, hammerhead, HDV, and VS ribozymes. Among these, the glmS ribozymeriboswitch, which is widespread in Gram-positive bacteria, is unique in requiring a small-molecule coenzyme, glucosamine6-phosphate (GlcN6P) for catalysis. While this requirement is consistent with the role of the glmS ribozyme-riboswitch in controlling expression of the protein enzyme GlcN6P synthetase, it contrasts with the ability of the other four classes of self-cleaving ribozymes to catalyze the same reaction without any cofactors (except Mg2+ in the case of the HDV ribozyme). Moreover, SELEX experiments have shown that coenzyme-independent self-cleaving ribozymes are widespread in RNA sequence space (1). Did the glmS ribozyme evolve from an ancestral GlcN6P-independent self-cleaving ribozyme of similar overall structure? We addressed this question by subjecting a pool of mutagenized glmS ribozyme RNAs to in vitro selection for specific self-cleavage in the absence of any small molecule cofactors. Analysis of sequences after ten rounds of SELEX indicates that just three point mutations suffice to confer GlcN6P-independent cleavage activity. We find that GlcN6P, various organic buffers, or cobalt (III) hexammine do not support self-cleavage of this triple mutant, which appears to have a specific requirement for Mg2+ for optimal catalytic activity. Crystallographic analysis demonstrates that the triple-mutant RNA adopts the same overall fold as the wild-type glmS ribozyme, and that the mutations abrogate the GlcN6P binding pocket. Kinetic analysis of RNAs containing the three point mutations in various combinations shows that conversion of any of the three nucleotides from the mutant ribozyme to wild-type results in the mutant ribozyme losing its ligand independent self-cleavage activity, while conversion of one of the three nucleotides to wild-type results in recovery of substantial GlcN6P-dependent activity. This study illustrates how RNAs of distinctly different biochemical functionality (GlcN6P-dependent and independent self-cleavage, in this case) populate adjacent portions of sequence and structure space. This property might provide the basis for the facile evolution of RNAs with novel activities. This research was supported by the Intramural Research Program of the NIH, NHLBI. 1. Ferré-D’Amaré, A. R., and Scott, W. G. (2010) Small Self-cleaving Ribozymes, CSH Perspect Biol 2.

512

Structural Diversity in Riboswitches that Respond to Pre-Queuosine1

Jonathan Liang, Phillip McCown, Ronald Breaker Yale University, New Haven, CT, USA Pre-queuosine1 (preQ1) is a hypermodified nucleobase that is incorporated into certain tRNAs at the wobble position and converted to queuosine, which plays a key role in translational accuracy. Representatives of two riboswitch classes, called preQ1-I and preQ1-II, both bind specifically to preQ1 using RNA aptamers with unrelated structures and regulate genes that function in the biosynthesis or transport of preQ1. We have investigated the diversity of the preQ1-I and preQ1II riboswitches and identified naturally-occurring variants of both classes across substantial taxonomic ranges. These variants differ in sequence and secondary structure from previously-known members of those classes. They show little loss of affinity for preQ1, but some exhibit increased ligand specificity, rejecting ligands similar in chemical structure to preQ1. The discovery of these variants suggests that some natural riboswitches are more diverse in sequence and structure than previously known.

Poster Session 3: RNA Catalysis and Riboswitches

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

513 Thermodynamic characterization of Mg2+ function in the binding of tRNA to the T box riboswitch antiterminator

Jia Liu, Jennifer Hines Ohio University, Athens, (Ohio), USA The T box riboswitch is a gene-expression controlling mechanism found in many gram-positive bacteria. In the T box mechanism, transcription is controlled by whether the terminator or antiterminator secondary structure forms in the 5’ untranslated region of the mRNA. The antiterminator is stabilized by tRNA binding and facilitates gene expression. Isothermal titration calorimetry (ITC) measures the heat change during binding interactions and determines thermodynamic properties. ITC was used to investigate how Mg2+ affected the tRNA-antiterminator binding by titrating antiterminator model RNA (AM1A) into tRNA at different Mg2+ concentrations. No binding interactions were observed in the absence of Mg2+. With Mg2+, the binding reaction was endothermic (positive ΔH) with positive entropy change. As the Mg2+ concentration was increased from 5 mM to 20 mM, the binding affinity of tRNA and AM1A remained unchanged while both ΔH and ΔS increased positively. These results indicate that increased Mg2+ facilitates a binding interaction that might cause conformational changes in the tRNA and/or antiterminator RNA, which lead to the entropy and enthalpy increase. Also, the ITC results at higher Mg2+ levels (above 15mM) were dependent on titration rates, indicating that these higher levels of Mg2+ might induce a slow conformational change. These results are consistent with previous non-ITC binding studies, which indicated that higher Mg2+ concentrations were required to form all four base pairs between the tRNA and antiterminator. The effect of spermidine was also investigated and found to bind AM1A with good affinity. In addition, ITC results for tRNA binding to AM1A in the presence of 5mM Mg2+ and 5mM spermidine were comparable to those for binding in the presence of 15 mM Mg2+, suggesting that spermidine might serve similar stabilization functions as Mg2+.

514 Development of efficient methods to prepare natively folded RNA using photocleavable biotin-modified nucleotides

Yiling Luo, Nadukkudy Eldho, Herman Sintim, Kwaku Dayie University of Maryland, College Park, MD, US RNA, as one of the essential macromolecules in life, plays an active role in gene expression, gene regulation, or catalysis. The ability to adopt complex 3D structures mediated by dynamics is vital for the proper functioning of RNAs. A problem encountered when preparing RNAs for structural or enzymatic studies is the difficulty in obtaining homogeneous population of natively folded RNAs. The traditional method for preparing RNA involves phenol chloroform exaction of associated proteins, ethanol precipitation, heating in urea, denaturing polyacrylamide gel electrophoresis (PAGE) separation, gel elution retrieval, and extensive refolding. However, the preparation of natively folded RNA without going through these processes of denaturing and refolding is important to obtain maximal RNA biological function. Here we present a simple strategy using ‘click’ chemistry to couple biotin to a “caged” photocleavable (PC) guanosine monophosphate (GMP). This biotin-PC-GMP is readily accepted by T7 RNA polymerase to transcribe “natively folded” RNA. This PC-Biotin-GMP has been used to transcribe RNAs ranging in size from 27 to 493 nucleotides. Furthermore we show, using an in gel fluorescence assay, that the natively prepared 160 kDa minimal group II intron ribozyme has enhanced catalytic activity over the same RNA purified via denaturing conditions and refolded. This facile approach for native RNA preparation should benefit preparation of RNAs for biophysical and therapeutic applications.

Poster Session 3: RNA Catalysis and Riboswitches

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

515

The Diversity of Architectures in Lysine and TPP Riboswitches

516

An In Vitro Assay for the Function of a Guanine Riboswitch

Phillip McCown, Adam Roth, Ronald Breaker Yale University, New Haven, Connecticut, USA Riboswitches are RNA structures that typically bind to small molecules and regulate gene expression. Riboswitches are placed into distinct classes based on their different secondary and tertiary structures and based on their ligands. We have been exploring the diversity of riboswitch structures and functions by seeking natural variants of known riboswitch classes. In a previous study, we found glmS riboswitch variants that carry unique sequences and structural features compared to those discovered previously, yet these newfound RNAs retain efficient ligand binding and self-cleavage activities (McCown et al. 2011). More recently, we have identified additional variants belonging to the lysine and thiamin pyrophosphate (TPP) riboswitch classes that appear to have significant structural changes. Some of these newly discovered RNAs exhibit altered ligand specificity and are likely to form new RNA-ligand interactions. These findings indicate that some riboswitch classes are structurally and functionally malleable and that additional variants might exist with greater diversity of structures and functions. McCown PJ, Roth A, Breaker RR. 2011. An expanded collection and refined consensus model of glmS ribozymes. RNA 17:728-736.

Colin Nevins, Daniel Morse United States Naval Academy, Annapolis, MD, USA Riboswitches are RNA elements found in non-coding regions of messenger RNAs that regulate gene expression through a ligand-triggered conformational change (for review, see ref. 1). In, B. subtilis and other gram positive bacteria, purine metabolism is regulated by riboswitches that bind to guanine (2). The best studied of the guanine riboswitches regulates the transcription of the B. subtilis xpt-pbuX operon. Guanine binding to this riboswitch inhibits transcription by stabilizing an alternative conformation that allows formation of a premature transcriptional terminator. In order to study guanine-triggered structure switching, we have developed a fluorescence quenching assay for the function of the guanine riboswitch. Our system consists of the guanine-binding domain (aptamer domain) of the riboswitch (xpt RNA), and two DNA oligonucleotides that are related to the 5’ and 3’ halves of the terminator stem (5’T and 3’T). xpt RNA is labeled at its 3’ end with a fluorophore and 5’T is labeled at its 5’ end with a fluorescence quencher. When 5’T is annealed to a complementary sequence at the 3’ end of xpt RNA, fluorescence is quenched. Guanine binding triggers a strand-exchange reaction that mimics the formation of the terminator in the full-length riboswitch. 5’T dissociates from xpt RNA and anneals to 3’T. The strand-exchange reaction causes an increase in fluorescence intensity as the quencher moves away from the fluorophore. Using this assay, we can reproducibly detect as little as 5 nM guanine. As expected, our system does not detect hypoxanthine, adenine, or guanosine. Results of experiments with ligands previously tested for antibiotic activity (3, 4) will also be presented. We are currently examining the function of a variant aptamer domain with a point mutation (C74U) known to change the ligand specificity from guanine to adenine (5, 6). We are also testing the effect of magnesium ions on the rate, specificity, and sensitivity of the assay. These results and potential applications of the assay will be discussed. 1. Smith, A.M., Fuchs, R.T., Grundy, F.J., Henkin, T.M., RNA Biol. 7, 104 (2010). 2. Mandal, M., Boese, B., Barrick, J.E., Winkler, W.C., Breaker, R.R., Cell 113, 577 (2003). 3. Kim, J.N., Blount, K.F., Puskarz, I., Lim, J. Link, K.H., Breaker, R.R., ACS Chemical Biology 4, 915 (2009). 4. Mulhbacher, J., Brouillette, E., Allard, M., Fortier, L-C., Malouin, F., and Lafontaine, D.A., PLOS Pathogens 6, e1000865 (2010). 5. Mandal, M. and Breaker, R. R., Nat. Struct. Mol. Biol., 11, 29 (2004). 6. Serganov, A., et al., Chemistry & Biology 11, 1729 (2004). Poster Session 3: RNA Catalysis and Riboswitches

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

517

In Vivo Evolution of Trans-Splicing Group I Intron Ribozymes

Karen Olson, Gregory Dolan, Zhaleh Amini, Ulrich Müller University of California San Diego, La Jolla, CA, U.S.A. Group I intron ribozymes are cis-splicing ribozymes that can be converted to trans-splicing ribozymes. These transsplicing ribozymes replace the 3’-fragments of substrate RNAs both in vitro and in vivo. Here we utilize this system to study the evolutionary transition from cis- to trans-acting ribozymes, and to improve them for possible therapeutic applications. Three central questions are addressed. First, can these ribozymes evolve higher trans-splicing efficiency, and if yes, which mutations can achieve that? To identify efficient ribozymes, the ribozyme from Tetrahymena was evolved over 20 rounds with our in vivo evolution method. One resulting ribozyme with strongly increased in vivo efficiency was analysed in detail. This ribozyme contains four mutations that are necessary and sufficient for high in vivo activity, but it does not show increased in vitro activity. Instead, the ribozyme appears to recruit a cellular protein, thereby showing the evolution from a cis-splicing ribozyme to a trans-splicing RNP. This allows an interesting comparison to natural cisand trans-acting ribozymes. Second, we asked whether and how different evolutionary parameters affect this evolution. Four lines of evolution were carried out in parallel that differed in the application of mutagenesis and recombination. The activities of the evolving ribozyme populations revealed, how and when do these parameters allow and benefit evolution. Third, we tested whether the much smaller group I intron ribozyme from Azoarcus could be converted to an efficient trans-splicing ribozyme. We found that this ribozyme can indeed catalyze trans-splicing in vivo but that it has different evolutionary characteristics. Our results provide insight into our understanding of RNA evolution in vivo and have implications on the possible development of therapeutic ribozymes.

518 Characterization of the Trans Watson-Crick GU Base Pair Located in the Catalytic Core of The Antigenomic HDV Rbozyme.

Dominique Lévesque, Cedric Reymond, Jean-Pierre Perreault Université de Sherbrooke, Sherbrooke (Quebec) Canada The HDV ribozyme’s folding pathway is, by far, the most complex folding pathway elucidated to date for a small ribozyme. This pathway includes at least 6 different steps that must occur before the chemical cleavage can take place. That said, it is likely that other steps remain to be discovered. One of the most critical of these steps is the formation of the trans Watson-Crick GU base pair within loop III. The U23 and G28 nucleotides that form this base pair are perfectly conserved in all natural variants of the HDV ribozyme, and therefore are considered as being part of the signature of HDV-like ribozymes. A set of experiments, including direct mutagenesis, the site-specific substitution of chemical groups, kinetic studies, chemical probing, magnesium-induced cleavage and MC-Sym structure prediction, were performed with the express goal of characterizing this trans Watson-Crick GU base pair in an antigenomic HDV ribozyme. Both U23 and G28 can be substituted for by nucleotides that likely preserve some part of interaction between the residues in addition to the binding of a Mg2+ ion. The formation of this base pair is shown to be a post-cleavage event, a result that sheds some light on an earlier controversy. We believe that the formation of this base pair acts as a driving force on the chemical cleavage by favouring a more stable ground state of the product-ribozyme complex. To our knowledge, this represents the first demonstration of an essential role for a post-cleavage event in a ribozyme catalyzed reaction.

Poster Session 3: RNA Catalysis and Riboswitches

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

519 Targeted Modifications of Glucosamine-6-phosphate to Affect the Binding and Activation of the glmS Ribozyme

Jeffrey Posakony, Adrian Ferré-D’Amaré National Institutes of Health (NHLBI), Bethesda, (MD), USA The glmS ribozyme is a catalytic RNA found in the 5’-untranslated region of the mRNA encoding the glucosamine6-phosphate synthase gene in numerous Gram-positive and a few Gram-negative bacteria. This ribozyme functions as a cis-acting regulatory element, controlling the translation of GlmS, but it differs from other, similar RNA-based regulatory elements (riboswitches) through its catalytic activity. In the presence of sufficient glucosamine-6-phosphate (GlcN6P), the RNA self-cleaves yielding a 2’-3’-cyclic phosphate and 5’-OH. The latter is then degraded by a ribonuclease resulting in abrogation of gene translation.1 GlcN6P synthase (GlmS) is an attractive target for novel antibiotics because it is a component of the pathway used to synthesize the bacterial peptidoglycan cell wall.2 The activity of GlmS might be affected by disrupting the normal activity of the glmS ribozyme, which could lead to GlmS misregulation. In order to understand the mechanism of the glmS ribozyme and its potential as a drug target, numerous analogues of GlcN6P have been prepared and evaluated through self-cleavage assays and biochemical characterization.3-5 Results from these experiments along with X-ray crystallographic investigations have revealed that the ribozyme has high specificity for the D-gluco stereoisomers, the 2-amino group is critical for its activity and the 6-phosphate significantly improves the binding of the sugar. We will describe the preparation and subsequent biochemical characterization of our most recent D-gluco amino sugars and sugar mimetics that have modifications to the 2, 3, or 6-positions. These modifications were designed to provide altered hydrogen bond contacts with the ribozyme and altered amino group pKa as well as to take advantage of pockets of open space observed in the X-ray crystal structures of the glmS ribozyme. This research was supported in part by the Intramural Research Program of the NIH, NHLBI. (1) Ferré-D’amaré, A. R. Quart. Rev. Biophys. 2010, 43, 423–447. (2) Gautam, A.; Vyas, R.; Tewari, R. Critical Reviews in Biotechnology 2011, 31, 295–336. (3) Lim, J.; Grove, B. C.; Roth, A.; Breaker, R. R. Angew. Chem. Int. Ed. 2006, 45, 6689–6693. (4) Blount, K.; Puskarz, I.; Penchovsky, R.; Breaker, R. RNA biology 2006, 3, 77–81. (5) Mccarthy, T.; Plog, M.; Floy, S.; Jansen, J.; Soukup, J.; Soukup, G. Chemistry & Biology 2006, 13, 683–683.

520 Differential Conformational Selection and Induced Fit of Structurally Similar Single Transcriptional and Translational Riboswitches

Arlie Rinaldi, Krishna Suddala, Jun Feng, Anthony Mustoe, Charles Brooks III, Nils Walter University of Michigan, Ann Arbor, (MI), USA The family of transcriptional and translational preQ1 riboswitches contains some of the smallest known metabolitesensing aptamers in nature, making them ideal models for studying these gene regulatory RNA elements. Crystal and NMR structures of the transcriptional Bacillus subtilis (Bsu) and translational Thermoanaerobacter tengcongensis (Tte) preQ1 riboswitch aptamers show them to adopt structurally similar pseudoknots, but their modes of genetic regulation and ligand-free states have been proposed to be distinct. Here, we use single molecule fluorescence resonance energy transfer microscopy to study the conformational dynamics of these two riboswitches. In this work, we show that both riboswitches adopt a conformationally-similar prefolded state to achieve the fully folded ligand-bound state. We observe that Tte utilizes this prefolded state to sense increasing ligand concentrations. Contrary to previous studies, we show that both riboswitches exist in this prefolded state in the absence of ligand in near-physiological buffer. Together with atomistic simulations, we show that the distinction between the two riboswitches lies in their folding pathways via conformational selection (Bsu) or induced fit (Tte). Finally, we show that mutation of a single nucleotide distal from the ligand binding pocket alters the properties of the riboswitches. Ultimately, we plan to use PAINT single molecule probing techniques to investigate the binding and dissociation of 30S subunits to the translational riboswitch (Tte) in the context of the full mRNA.

Poster Session 3: RNA Catalysis and Riboswitches

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

521 Programming the highly structured HDV ribozyme into complex allostery with RNA oligonucleotides.

Samuel Rouleau, Jean-Pierre Perreault Université de Sherbrooke, Sherbrooke, Quebec, Canada The advent of syntheticbiology, a novel field that seeks to mimic biological behaviour through the use of synthetic molecules, will accelerate the meaningful health applications of RNA knowledge. RNA is a highly malleable molecule, making it an attractive tool with which to drive a programmable function. Moreover, it is possible to combine different functions in a single RNA molecule. Therefore, it is feasible to couple the ligand sensing properties of aptamers with the catalytic activity of ribozymes. These ribozyme/aptamer fusions (aptazymes) can be used to construct RNA devices that, when inserted in the untranslated regions (UTR) of mRNA, can regulate gene expression in a ligand-dependant manner. Our goal is to modulate the HDV ribozyme with aptamers that recognize small RNA oligonucleotides. First, we fused the ribozyme with an 18 nucleotides hairpin (HP18) structure which totally abolishes the ribozyme self-cleaving activity. The binding of an effector (E), a 14 nucleotides RNA oligomer, to the HP18 rescues the self-cleavage in a concentration-dependant manner. We inserted this aptazyme in the 5’ UTR of a luciferase reporter gene and showed that we can modulate its expression in HEK 293 cells with the effector. This aptazyme, by being active only when the effector is present, corresponds to a YES Boolean Logic Gate. We decided to design other aptamers that could create new aptazymes corresponding to other logic gates. Aptazymes respecting the NOT, OR, NOR, AND and NAND gates were designed. Each one could correctly modulate the ribozyme self-cleavage in vitro and moderately regulate gene expression in cellulo. This system could eventually be used to tightly modulate whole sets of genes in a coordinated way and thus present a great potential tool for synthetic biology.

522

Mg2+-Dependent Folding of the btuB Riboswitch by Single Molecule FRET Studies

Michelle F. Schaffer , Roland K.O. Sigel Institute of Inorganic Chemistry, University of Zürich, Switzerland Riboswitches are complex folded RNA domains in the 5’-untranslated region of bacterial mRNA that serve as specific receptors for metabolites. Conformational changes of the 202nt long btuB riboswitch of E.coli induced by coenzyme B12 (AdoCbl) and some of its derivatives lead to an altered gene expression of the downstream btuB gene [1]. The interaction of the riboswitch with its ligand strongly depends on the initial tertiary structure of the RNA and is moreover affected by Mg2+ ions. Despite ongoing studies on this system [2, 3, 4], the binding mode on the atomic level and the folding pathway still remain unknown. Therefore, we investigate the folding pathway of this system in the presence of Mg2+ to characterise the initial conditions needed for coenzyme B12 binding to the riboswitch. In our study, we focus on single molecule Förster Resonance Energy Transfer (smFRET) measurements. FRET observed upon excitation of Cy3/Cy5 labelled RNA provides distance information about inter-dye distances, which are related to distinct conformations [5]. For successful FRET studies it is crucial to find an optimal position for labelling of a large RNA. Hence, we created three different constructs with modified internal loops and extended 3’- and 5’-ends to attach a pair of fluorophore-labelled DNA-oligonucleotides (Cy3 and Cy5). FRET allows us then to monitor the interdomain movements of the btuB riboswitch and to study the effects of Mg2+ ions on its folding. Financial support by the Swiss National Science Foundation, the European Research council (ERC starting grant) and the University of Zurich is gratefully acknowledged. [1] X. Nou, R.J. Kadner, Proc. Natl. Acad. Sci. USA 97, 7190 (2000) [2] S. Gallo, M. Oberhuber, R.K.O. Sigel, B. Kraeutler, ChemBioChem 9, 1408 (2008) [3] G.A. Perdrizet 2nd, I. Artsimovitch, et al. Proc. Natl. Acad. Sci. U S A 109(9) 3323-8 (2012) [4] A. Nahvi, J.E. Barrick, R.R. Breaker, Nucleic Acids Res., 32, 143-150 (2004) [5] R. Zhao and D. Rueda. Methods 49, 112-117 (2009) Poster Session 3: RNA Catalysis and Riboswitches

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

523 A Newly Characterized Version of the Hepatitis Delta Virus Ribozyme Binds its Substrate Less Efficiently than Previous Versions

Kamali Sripathi1, Pavel Banáš2, Wendy Tay1, Jiſí­Šponer3, Michal Otyepka2, Nils Walter1 1 University of Michigan, Ann Arbor, MI, 48103, 2Faculty of Science, Palacky University, Olomouc, Czech Republic, 3Institue of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic The hepatitis delta virus (HDV) is the only known human pathogen to contain a catalytic RNA motif (ribozyme) in its genome. Until recently, the ribozyme was thought to undergo significant conformational changes upon cleavage. The latest precursor crystal structure of the HDV ribozyme refutes this notion1. This new structure also seems to indicate that the highly controversial role of the catalytic C75 is that of a general acid during catalysis. We are investigating the solution behavior of the newly crystallized version of the HDV ribozyme using fluorescence resonance energy transfer (FRET). We are concurrently also probing various conformations of the active site using molecular dynamics (MD) simulations on the most recent crystal structure. Solution studies indicate that this new version of the HDV ribozyme does undergo small but detectable conformational changes upon cleavage. Additionally, our solution studies seem to suggest that this version of the ribozyme shows a lower affinity for its substrate than have previous ribozymes. Our MD simulations indicate that catalytic fitness2 is greatly disrupted by deprotonation of C75 and alternate active site conditions. Simulations also show that the magnesium ion resolved near the scissile phosphate results in favorable catalytic geometry and a more stable L3 loop. Although simulation results indicate that the new crystal structure represents a viable conformation of the ribozyme with C75 poised to act as a general acid, our results from solution study imply that this crystal structure instead may represent a less active version of the ribozyme. 1. Chen, J. H., Yajima, R., Chadalavada, D. M., Chase, E., Bevilacqua, P. C. & Golden, B. L. A 1.9 A crystal structure of the HDV ribozyme precleavagesuggests both Lewis acid and general acid mechanisms contribute tophosphodiester cleavage. Biochemistry 49,6508-18. 2. Soukup, G. A. & Breaker, R. R. (1999). Relationship between internucleotide linkagegeometry and the stability of RNA. RNA 5,1308-25.

524 Exploring the Significance of a Water-Involving Hydrogen Bonding Network Within the Hairpin Ribozyme

Wendy Tay, Nils Walter University of Michigan, Ann Arbor, MI, USA In 2006, from studies involving molecular dynamics simulations and X-ray crystal structures, a chain of five to seven long-residing, ordered water molecules was discovered within the otherwise solvent-protected core of the hairpin ribozyme(1,2). This water chain allows for the formation of an extended hydrogen bonding network within the ribozyme. Such a network could potentially be involved in mediating long-range communication important for ribozyme catalysis between the RNA residues within the ribozyme core. However, until now, the functional relevance of this water network has still not yet been explored. Molecular dynamics (MD) simulations combined with single molecule experiments are being used to gain insight into how the water chain and hydrogen bonding network may be important for catalytic activity. Specifically, MD is used to computationally scan for subtle mutations in core residues, such as A38, A10 and U42, that disrupt the hydrogen bonding network. Suitable mutations, as predicted by MD, are then incorporated into constructs to be characterized experimentally using a single molecule kinetic fingerprinting protocol(3). This protocol allows for determination of each of the rate constants for the known steps of the hairpin ribozyme kinetic mechanism and thus can pinpoint which kinetic steps are affected by disruption of the hydrogen bonding network. Using a combination of computational and experimental techniques, we are able to investigate further the catalytic significance of the hydrogen bonding network within the hairpin ribozyme. (1) Rhodes, M. M.; Reblova, K.; Sponer, J.; Walter, N. G. Proc Natl Acad Sci U S A 2006, 103, 13380-5. (2) Salter, J.; Krucinska, J.; Alam, S.; Grum-Tokars, V.; Wedekind, J. E. Biochemistry 2006, 45, 686-700. (3) Liu, S.; Bokinsky, G.; Walter, N. G.; Zhuang, X. Proc Natl Acad Sci U S A 2007, 104, 12634-12639.

Poster Session 3: RNA Catalysis and Riboswitches

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

525 Interaction between the Scissile Phosphate and a Putative Catalytic Metal Ion in the HDV Ribozyme

Pallavi Thaplyal1, Barbara Golden2, Philip Bevilacqua1 1 The Pennsylvania State University, University Park, (PA), USA, 2Purdue University, West Lafayette, (IN), USA Early studies on the hepatitis delta virus (HDV) ribozyme suggested that the non-bridging proR oxygen at the scissile phosphate may play a role in the active ribozyme-substrate complex, but were unable to establish a thio effect or a metal ion rescue (1). In contrast, a recent 1.9 Å crystal structure suggests that the proR oxygen acts as an inner sphere ligand for a potentially catalytic Mg2+ ion (2). Our present study probes the function of the non-bridging oxygen atoms, including their possible interaction with this putative catalytic metal ion. We utilized phosphorothioate substitutions at the scissile phosphate and subsequent metal ion rescue experiments. Our experiments were conducted on a fast-reacting version of the HDV ribozyme, under conditions wherein chemistry is rate limiting and pH does not denature the ribozyme. The Rp and Sp diastereomers from the phosphorothioate substitution were separated using reverse phase HPLC. The Rp substrate exhibited a biphasic kinetic profile, with the fast- and slow-reacting phases displaying thio effects of ~5 and ~800, respectively, suggesting that the proR non-bridging oxygen affects the cleavage rate. The Sp substrate, in contrast, did not exhibit a thio effect and was indistinguishable from the oxo substrate in its kinetic properties. In an effort to establish whether there is an interaction between the proR oxygen and a metal ion, metal ion rescue studies were carried out in presence of the Rp substrate and various thiophilic metal ions. Rescues of ~10- and ~30-fold were observed for the fast- and slow-reacting phases, respectively. These studies support a functionally important interaction between the proR oxygen on the scissile phosphate and the metal ion observed in active site of the crystal structure of the HDV ribozyme. References 1) H. Fauzi, J. Kawakami, F. Nishikawa, S. Nishikawa; Nucleic Acids Res. , 1997 , 25 , 3124. Analysis of the cleavage reaction of a trans-acting human hepatitis delta virus ribozyme. 2) J. H. Chen, R. Yajima, D. M. Chadalavada, E. Chase, P. C. Bevilacqua, B. L. Golden; Biochemistry , 2010, 49, 6508. A 1.9 Å crystal structure of the HDV ribozyme precleavage suggests both Lewis acid and general acid mechanisms contribute to phosphodiester cleavage.

526 The Effects of Expression Platform Stability on Riboswitch Mediated Gene Expression Control

Nakesha Smith, Shanelle Graham, Carla Theimer University at Albany, SUNY Bacteria use RNA riboswitches to control the expression of key metabolic genes in response to chemical factors in their environment. Riboswitches are naturally occurring regulatory RNA structures typically found in the 5’ untranslated regions of certain bacterial (and some eukaryotic) messenger RNAs. Although the mechanism has not been fully characterized, naturally occurring riboswitches have been shown to control metabolic gene expression in a number of medically relevant bacteria. The preQ1 riboswitch regulates gene expression in response to preQ1, the biosynthetic precursor of queosine, an essential hypermodified guanine nucleotide in the tRNAs of a number of amino acids. This switch represents a good model system for biophysical characterization due to its relatively small size and the availability of high resolution structural information for the ligand-bound aptamer domain. NMR and X-ray crystallographic studies revealed an H-type RNA pseudoknot that sequesters a portion of the 3’ A-rich tail that would otherwise form a transcriptional anti-terminator stem. In this study, we characterize the interplay between the conformational states of the RNA structural elements associated with representative members of the transcriptional control preQ1 riboswitch family (type 1). Thermodynamic measurements, native gel electrophoresis, and NMR solution structure methods are used to characterize the individual structural elements, combined in different permutations, to assess their individual properties and the interplay of the different structural elements in the expression platform. The properties of the various riboswitch expression platforms in vitro have been correlated with gene expression in an in vivo bacterial reporter system in the presence and absence of ligand.

Poster Session 3: RNA Catalysis and Riboswitches

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

527

Metal-Dependent Folding and Catalysis of a Native Hammerhead Ribozyme

528

The Hairpin Ribozyme: Metal Dependence to Tight Intermolecular Docking

Luke Ward, Dan Morris, Matthew Hendricks, Victoria DeRose University of Oregon, Eugene, Oregon, USA The factors that determine RNA structure formation, stability, and dynamics are inextricablylinked to RNA function. The Hammerhead ribozyme (HHRz) has long served as a model for studying metal-dependent folding and catalysis in RNA. The HHRz consists of 3 helices meeting at a common junction of conserved nucleotides that form the active site of the ribozyme. Current models of metal-dependent HHRz function involve a requirement for divalent metals to globally fold the ribozyme at low metal concentrations, followed by a second metal-dependent process which activates the HHRz for catalysis. The exact role of metal ions in activating HHRz catalysis is still a subject of investigation. We used 2-aminopurine substitutions near the active site of the ribozyme to determine if this second metal-dependent process involves a conformational rearrangement in the core of the ribozyme. We find evidence for a conformational change beyond global folding in the core of the ribozyme that not only correlates with metal activated catalysis but is also sensitive to the identity of the metal ions used for folding. Though phosphorothioate substitutions indicate that a ground-state coordination of a catalytic metal to the scissile phosphate is required for efficient catalysis (Ward and DeRose, RNA, 2012), our folding studies show that this coordination event is not absolutely required for folding of the HHRz core. To investigate possible roles for metal ions in general acid-base catalysis, we tested the pH dependence of the HHRz rate using a variety of metal ions. We find the pH dependent rate profile of the ribozyme is shifted by transition metal ions, whereas other group II metals show similar profiles to Mg2+. Combined these data suggest there is a non-specific requirement for divalent metal ions to fold the core of the ribozyme, while the specific characteristics of metal ions affect the architecture of the active site and dictate the effects of metals on the catalytic mechanism.

Neil White, Minako Sumita, Charles Hoogstraten Michigan State University, East Lansing, MI, USA The hairpin ribozyme is a well studied self-cleaving catalytic RNA. It contains two domains, loop A and loop B. In nature cleavage occurs on a specific site in loop A when loop A and loop B are docked. The loop A – loop B interaction represents a pure RNA tertiary structure interface, with dramatic structural rearrangements between the undocked and docked states. For docking to occur metal ions such as magnesium or cobalt (III) hexamine (Co(NH3)63+) must be present. To study docking kinetics we used a trans –docking system, meaning loop A and loop B are on separate molecules. We performed the study with submillimolar [Co(NH3)3+]. The appropriate metal ion concentration was previously determined by a novel difference circular dichroism (CD) assay developed by our lab. We are now examining the bimolecular interaction using surface plasmon resonance (SPR). We find unusually slow association and dissociation rates ((1.86 ±0.23)x103 M-1s-1 and (7.4 ±1.1)x10-4 s-1, respectively) with submicromolar affinity of docking. The results are consistent with the proposed “double conformational capture” mechanism for docking, in which both components undergo fluctuations to a structure resembling their docked form. We are now turning our attention to mutant forms of loop B, including substitutions and the conserved A38 position that are hypothesized to dock but not cleave. Confirming and extending understanding of the behavior of variants at this site will guide further structural and dynamic studies using NMR, with the ultimate goal of further testing the proposed docking mechanism.

Poster Session 3: RNA Catalysis and Riboswitches

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

529

Effect of Spermidine on The T Box Riboswitch Antiterminator Model RNA

Chunxi Zeng, Shu Zhou, Jennifer Hines Department of Chemistry and Biochemistry, Ohio University, Athens, (Ohio), USA The T box riboswitch is found in the mRNA 5’ untranslated region (UTR) of many bacterial genes and regulates transcription by binding with cognate tRNA. Formation of the T box antiterminator structure is a critical step to allow transcription read-through. Spermidine is a polyamine that is able to interact with RNA and induce RNA structural changes. To investigate the effect of spermidine on the secondary structure of the antiterminator model RNA (AM1A), fluorophore and quencher labeled AM1A was examined using a fluorescence-monitored thermal denaturation assay. In addition, the detailed effect of spermidine on each nucleotide of the AM1A was mapped by in-line probing using 5’ radiolabeled AM1A. The results from both assays indicated that spermidine had two distinct effects on the AM1A: at low concentrations, spermidine enhanced the stability of the AM1A; at high concentrations, spermidine dramatically destabilized the AM1A. These results suggested that spermidine may bind and stabilize a specific region of the AM1A at low concentrations, while at high concentrations, spermidine likely binds the antiterminator non-specifically leading to denaturation. Further study is in progress to explore how spermidine affects the AM1A in the presence of cognate tRNA, to unveil the possible role of spermidine in antitermination of the T box riboswitch.

530 Base-pairing, Base-stacking, and Steric Occlusion Mediate Recognition of a Nonaminoacylated tRNA by a T-box Riboswitch Jinwei Zhang, Adrian Ferré-D’Amaré National Heart, Lung, and Blood Institute, NIH, Bethesda,MD, USA.

Faithful translation requires stable intracellular pools of aminoacylated tRNAs(aa-tRNAs). In Gram-positive bacteria, evolutionarily conserved T-box riboswitches control aa-tRNA levels. These riboswitches are 200-300 nucleotideslong and usually precede genes for aminoacyl-tRNA synthetases (aaRSs). T-boxRNAs are comprised of the conserved elements Stem I and Antiterminator Stem connected by a variable linker. Stem I contains a loop that base pairs with the tRNA anticodon to confer tRNA specificity, while the Antiterminator Stem presents a bulge that base pairs with the tRNA 3’-XCCA terminus. T-box riboswitches act by coupling the direct binding of non-aminoacylated tRNAs with formation of a transcription antiterminator allowing expression of downstream aaRSs, thereby completing a negative feedback loop.1 Although several thousand T-boxes have been described, little is known about this tRNA-mRNA interaction beyond base-pair complementarity. The mechanisms by which T-boxes recognize the overall structure of tRNA and importantly, discriminate aminoacylated vs non-aminoacylated tRNAs, or how non-aminoacylated tRNA stabilizes the antiterminator, are unknown. Here, we report direct, specific binding of full-length Bacillus subtilis tRNAGly to its cognate GlyQS T-box Stem I, with KD~180 nM. Mutational studies reveal essential roles of the Stem I terminal loop for tRNA binding, explaining an earlier report that certain Stem I terminal loop substitutions abolish tRNA-mediated antitermination in vivo.2 Intracellular aa-tRNAs are almost always bound by the abundant translation factor EF-Tu; thus, in vivo the T-boxes only need to discriminate non-aminoacylated tRNAs from aa-tRNAs complexed with EF-Tu. To test if T-boxes are capable of discriminating aminoacylated from non-aminoacylated tRNAs independent of EF-Tu, we developed a flexible, scalable method to prepare homogeneous aminoacylated tRNA, using flexizyme3 for aminoacylation, and a N-pentenoyl group4 as a protecting group and a hydrophobic purification handle. Glycyl-tRNAGly fails to induce antitermination in vitro, indicating that T-boxes can distinguish aminoacylated from non-aminoacylated tRNAs independent of EF-Tu. We further find that tRNA variants carrying terminal 3’- or 2’-deoxyadenosine efficiently induce antitermination, whereas tRNA variants carrying a 3’-phosphate, 2’,3’-cyclic phosphate, or puromycin are unable to induce antitermination.These results suggest that T-boxes reject aa-tRNAs by steric occlusion, rather than by actively recognizing the 3’-OH on non-aminoacylated tRNA.

1. Grundy, F.J. et al., 1993, Cell, 74(3), 475–482. 2. Winkler W., 2002, OSU Ph.D thesis. 3. Murakami, H. et al., 1996, Nature Methods, 3(5), 357–359. 4. Lodder, M. et al., 2005, Methods 36(3), 245–251. This research was supported in part by the Intramural Research Program of the NIH, NHLBI.

Poster Session 3: RNA Catalysis and Riboswitches

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

531

Development of a New Fluorescent Toolbox for Imaging RNA in Live Cells

532

Preparation of Large RNAs by Splintless RNA Ligation

Tucker Carrocci, Jacquelyn Turri, Aaron Hoskins University of Wisconsin - Madison In vivo fluorescent labeling has proven useful in gaining a molecular understanding of many biological systems. Extension of this technique to RNA systems has been plagued by the lack of fluorescent reporter motifs. The Singer group has previously developed a method using fluorescent proteins to study messenger RNA (mRNA) localization in living yeast cells. This method utilizes RNA binding sites of the MS2 bacteriophage coat protein cloned into an untranslated region of the target RNA molecule and the coexpression of green fluorescent protein (GFP) fused MS2-binding protein. Despite the successful application of this method to a number of biological questions, it is not without drawbacks such as background fluorescence from unbound MS2-GFP, the large size of the reporter system, and weak fluorescence of GFP compared to organic fluorophores that emit light in the visible and near-IR spectrums. Alternative methods would prove useful for studying systems not amenable to visualization by MS-GFP. In one promising approach, the background signal can be reduced by splitting the fluorescent signal between two RNA-binding proteins. The PP7 bacteriophage coat protein and the corresponding stem loop were selected to use in conjugation with the MS2-system. We are in the process of creating tools in which RNA can be visualized by small molecule or protein fluorescence and either by direct excitation or fluorescence resonance energy transfer (FRET). This new RNA visualization toolbox significantly extends the capabilities of fluorescence microscopy for imaging RNA in live cells.

Kevin Desai, Aaron Hoskins, Ronald Raines University of Wisconsin-Madison, Madison, WI, USA Current methods for preparation of large, modified RNAs often rely on enzymatic ligation of one or more fragments by either T4 DNA ligase, T4 RNA ligase 1 or T4 RNA ligase 2. These methods often require annealing of a DNA splint prior to ligation in order to create a substrate for the ligase, remove interfering secondary structures, and avoid unwanted side reactions. However, synthesis of the splint and optimization of annealing conditions can be both time-consuming and costly. To circumvent these issues, we have developed a novel, splintless RNA ligation protocol. We have recently shown that the RNA ligase RtcB catalyzes the GTP-dependent ligation of RNA with 3’-phosphate and 5’-hydroxyl termini. The ease of synthesizing RNA strands possessing these termini has motivated us to exploit RtcB for practical applications in RNA biochemistry and molecular biology. We present methods for the facile cloning of RNA using RtcB from a thermophilic archaeon. This ligase is highly adept at ligating single-stranded RNAs of various lengths and base compositions in the absence of a splint. Moreover, the ability of this thermostable enzyme to function at elevated temperatures will melt problematic RNA secondary structures that can hinder ligation. We believe that RNA ligation methods using this thermophilic RtcB will be a superior alternative to current methods that use T4 RNA ligase.

Poster Session 3: Chemical and Synthetic Biology of RNA

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

533

Kinetic Analysis of Aptazyme-regulated Gene Expression in a Cell-free Translation System

534

Defining RNA-Platinum Adducts within the Eukaryotic Ribosome

Shungo Kobori Osaka University, Osaka, Japan Aptazymes are widely utilized as RNA-based switches of gene expression responsive to several types of compound. One of the most important properties of the switching ability is the Signal/Noise (S/N) ratio, i.e., the ratio of gene expression in the presence to that in the absence of ligand. The present study was performed to gain a quantitative understanding of how the aptazyme S/N ratio is determined by factors involved in gene expression, such as transcription, RNA self-cleavage, RNA degradation, protein translation, and their ligand dependencies. We performed switching of gene expression using two on-switch aptazymes with different properties in a cell-free translation system, and constructed a kinetic model that quantitatively describes the dynamics of RNA and protein species involved in switching. Both theoretical and experimental analyses consistently demonstrated that factors determining both the absolute value and the dynamics of the S/N ratio are highly dependent on the route of translation in the absence of ligand, either ligand-independent cleavage or leaky translation from the non-cleaved RNA. The model obtained here is useful to assess the factors that restrict the S/N ratio of aptazymes and to improve their efficiency.

Maire Osborn, Jonathan White, Victoria DeRose University of Oregon, Eugene, OR, USA Cis-diamminedichloroplatinum(II), or cisplatin, is a universally prescribed anticancer agent that forms exchange-inert complexes with a variety of biomolecular targets, particularly nucleic acids. Irreparable intrastrand crosslink formation on DNA is the primary effector in the induction of programmed cell death in several eukaryotic models. Platination has also been shown to disrupt essential RNA-dependent processes such as splicing and translation. The potential of cisplatin-derived RNA damage to contribute to the overall cytotoxicity of the drug has not been directly investigated in detail and is a current focus of our lab. In our investigation of RNA-Pt adduct formation, we quantified Pt accumulation on different RNA species isolated from cisplatin-treated S. cerevisiae. Interestingly, we approximate a 4-20 fold excess of Pt on total cellular RNA when compared to total DNA (Hostetter et al, ACS Chem. Biol., 2012). At present, we are defining populations of specific Pt-RNA adducts within cytoplasmic and mitochondrial rRNA, while also attempting to distinguish binding sites where accumulation is functionally tolerated versus those where platination disrupts endogenous activities, such as translation. To date, we have demonstrated that cisplatin targets purine residues within the universally conserved sarcin-ricin loop (SRL) in S. cerevisiae 25S rRNA. This loop scaffolds several critical protein-RNA interactions within the ribosome, including that of the translation elongation factor eEF2 (EF-G in E. coli). Recent experiments highlighting the major Pt binding sites within the peptidyl transferase center and adjacent solvent accessible helices will also be discussed. Taken together, our results indicate sequence- and structure-specific Pt(II) drug binding to ribosomal RNA and the potential for ribotoxic response in chemotherapies. In pursuit of a high-throughput method of Pt target identification, we have synthesized novel azide- and alkynefunctionalized Pt(II) reagents capable of nucleotide binding, which have potential to be used for extraction of Pt targets via a 1,3 Huisgen azide-alkyne cycloaddition (‘Click’) reaction. Such techniques may also be extended to broad identification of drug targets within whole cell RNA populations. Poster Session 3: Chemical and Synthetic Biology of RNA

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

535 RNA

Use of Fluorescence Spectroscopy for High-Throughput Quantification of pKa Shifting in

536

HOW, an RNA Binding Protein, Regulates Alternative Splicing in D. melanogaster

Jennifer Wilcox, Philip Bevilacqua The Pennsylvania State University, University Park, PA, USA In order for RNA to be efficient in catalysis, the pKas of certain nucleobases, which are typically near 4 and 9, must be shifted toward neutrality. Understanding the driving forces for pKa shifting would be extremely beneficial to deciphering the mechanisms of RNA catalysis. In this study, the pH dependence of the emission of the fluorescent nucleotide analog 2-aminopurine (2AP) has been used to determine the pKa of protonated adenine in A+*C base pairs in model oligonucleotides. In order for the 2AP to report on ionization, a 2AP-T base pair was placed near the ionizing A+*C base pair. The pKa was measured by standard fluorescence spectroscopy, high-throughput (HT) fluorescence spectroscopy via a plate reader, and 31P NMR spectroscopy. The three values are in good agreement, differing by only 0.3 pH units after addition of the 2AP. The HT method should allow for use of a 96-well plate and analysis of up to 8 different RNA sequences in approximately 1 hour. The pKa of an A+*C base pair between G-C and A-T base pairs showed a shifted pKa of 7.9, which is a drastic shift from that of adenine at 3.5. This supports the argument that nucleobases have the potential to participate in general acid-base catalysis at physiological pH. The fluorescence method will continue to be utilized to rapidly determine pKa’s of different nucleic acids under varying conditions to find the ideal environment for formation of a protonated nucleobase.

Nehemiah Alvarez1,2, Malcolm Cook2, Marco Blanchette2 1 Department of Pathology and Laboratory Medicine, Division of Cancer and Developmental Biology, University of Kansas Medical Center, Kansas City, KS 66160, 2Stowers Institute for Medical Research, Kansas City, MO 64110 Regulation of gene expression through alternative splicing of pre-mRNAs is important for the neuronal development of a large number of organisms. In Drosophila melanogaster the RNA binding protein held out wings (HOW), the homologue of mammalian Quaking (QKI), is required for proper glial cell migration. Here we have interrogated the role of HOW as an alternative splicing regulator. We designed a data analysis scheme that uses RNA-seq data to identify changes in alternative splicing. Applying our scheme to RNA-seq from a how RNAi, in S2 cells, revealed that HOW affects the alternative splicing of hundreds of genes. We have observed that HOW can act as both an enhancer and repressor of alternative splicing events. To gain further insight into HOW’s role in alternative splicing we turned to CLIP-seq. Our CLIP-seq revealed that HOW directly associates with genes affected in a how RNAi. We have observed positional effects of HOW binding that correlates with its repressing and enhancing activity in alternative splicing. Our initial observations indicate that when HOW binds upstream of an alternative splice site it enhances it usage, while binding downstream causes repression.

Poster Session 3: Chemical and Synthetic Biology of RNA & Splicing Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

537

Pseudouridines induced during filamentous growth in yeast

538

Splicing stimulates biogenesis of plant microRNAs

Anindita Basak, Charles Query Albert Einstein College of Medicine Filamentous growth in many fungal species in response to environmental stress, especially nutrient starvation, is characterized by changes in yeast cell morphology–most notably the extension of filaments into the growth media to forage for nutrients. Both haploid and diploid yeast cells can undergo filamentous growth, termed as haploid invasive growth and pseudohyphal growth respectively. Historically observed in the virulent form of the pathogenic fungus Candida albicans, filamentous growth can also be observed in Saccharomyces cerevisiae. Four major signaling pathways regulate filamentation – (1) rat sarcoma/protein kinase A (RAS/PKA) pathway (2) sucrosenon-fermentable (SNF) pathway (3) target of rapamycin (TOR) pathway and (4) mitogen-activated protein kinase(MAPK) pathway. Pseudouridines (Ψ) are C5-glycoside isomers of uridines that are thought to enhance local RNAstacking. Besides the canonical Ψ’s that occur in a vast majority of ribosomal RNAs and some in spliceosomal RNAs (snRNAs), Ψs have recently been shown to be induced in yeast upon stress– e.g. heat shock or nutrient starvation in liquid culture. In Saccharomyces cerevisiae, we have found a novel Ψ residue on U6 snRNA, which is induced under conditions akin to filamentous growth. We have identified Pus1, which pseudouridylates 8 positions in tRNAs and also position U44 in U2 snRNA, as the Ψ-synthase for this novel modificationsite. We are investigating the effect(s) of this induced Ψ on splicing. We postulate that such pseudouridine(s) may contribute to alternative growth programs by fine-tuning splicing. We also show that the transcription factor Whi2, which binds to the STRE (stress response elements) in the promoters of stress-response genes, might be at the top of a signal transduction cascade that works through Pus1 as the effector enzyme. We are currently investigating the intriguing mechanism of such a pathway. References Gimeno C. J., Ljungdahl P. O., Styles C. A., Fink G.R., Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS. Cell, 1992, 68: 1077–1090. Kaida D,Yashiroda H,Toh-e A, Kikuchi Y. Yeast Whi2 and Psr1-phosphatase form a complex and regulate STRE-mediated gene expression. Genes Cells, 2002 Jun;7(6):543-52. Guowei Wu, Mu Xiao, Chunxing Yang, Yi-Tao Yu. U2 snRNA is inducibly pseudouridylated at novelsites by Pus7p and snR81 RNP. The EMBOJournal (2011) 30, 79 – 89. Dawid Bielewicz1, Lukasz Sobkowiak1, Katarzyna Raczynska1, Daniel Kierzkowski1, Maria Kalyna2, Andrea Barta2, Franck Vazquez3, Artur Jarmolowski1, Zofia Szweykowska-Kulinska1 1 Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland, 2Max F. Perutz Labratories, Vienna, Austria, 3Botanical Institute of the University of Basel, Basel, Switzerland Plant microRNA genes are usually long, and very often contain introns. In the majority of cases a miRNA/miRNA* hairpin is found in the first exon of pri-miRNAs, but there are also miRNA genes where a miRNA is encoded in the second or third exon. Biogenesis of miRNAs is a multistep and complex process. miRNA maturation steps include: constitutive and alternative splicing of pri-miRNAs, alternative polyadenylation of miRNA primary transcripts, miRNA-containing hairpins excision, miRNA/miRNA* duplex formation, and miRNAs incorporation into the RISC complex. Our studies on A. thaliana and barley miRNA genes have revealed that miRNA genes organization is similar in both plant species, and probably reflects general miRNA genes organization in higher plants. Our results show that alternative miRNA precursor isoforms are specific to the particular organ and/or developmental stage of A. thaliana. To answer the question whether splicing plays an essential role in the efficiency of mature miRNA production, we analyzed processing of five different intron-containing A. thaliana primiRNAs in several Arabidopsis SR protein mutants. The data show that in some of the SR mutants tested the level of mature miRNAs originated from intron-containing genes is significantly decreased. Next, we asked the question whether the observed changes in the level of mature miRNAs were caused by direct or rather indirect effects. To test it, we introduced three variants of the Arabidopsis MIR163 gene: a native form containing one intron, a gene containing mutated 5` and 3`ss, and an intronless variant, into the A. thaliana mir163-2 mutant (SALK_034556), in which T-DNA insertion had disrupted the endogenous MIR163 gene. Introduction of the wild type form of MIR163 showed the same level of pri-miRNA 163 and its mature form, as it was observed in wild type plants. In the case of the intronless MIR163 construct we observed the accumulation of pri-miRNA 163, while the level of mature miRNA 163 was decreased about three times. In the case of transgenic plants containing the MIR163 gene with mutated both splice sites, the level of mature miRNA 163 was again lower when compared to wild type plants. Altogether, our results show that splicing stimulates biogenesis of plant miRNAs derived from intron-containing genes. In addition, we showed that two factors involved in miRNA biogenesis in plants, SERRATE (SE) and AtCBC (AtCBP20/ AtCBP80) are key players in the crosstalk between miRNA biogenesis and splicing. Poster Session 3: Splicing Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

539

A Subset of Introns is Abundant in poly-A mRNA

540

Combinatorial Control of Alternative Splicing by SR Protein Regulatory Networks

Paul Boutz, Jesse Zamudio, Xuebing Wu, Phillip Sharp Massachusetts Institute of Technolgy, Cambridge, MA, USA Many studies support a model in which introns are spliced out of pre-mRNA co-transcriptionally. We performed strandspecific RNA-seq on poly-A selected mRNA from mouse ES cells, and observed a number of introns that have higher read coverage than other introns within the same genes. In many cases, the same introns show this pattern in multiple RNA-seq data sets from human and mouse, cell lines and tissues. We quantified the relative coverage of all introns from our mouse ES cell data, as well as from four human cell lines in the ENCODE data sets. Introns with high read coverage that flank constitutive exons are characterized by weak 5’ and 3’ splice sites and shorter intron length. Interestingly, the 3’ splice site of the intron upstream of a high coverage intron, and the 5’ splice site of the downstream intron, also tend to be weak. This suggests a cross-exon effect on the splicing of high coverage introns. In contrast, introns in the lowest coverage bin, regardless of splice site strength, are typically flanked by introns with strong splice sites across the common exon. Introns in the highest 20% of the read coverage distribution are significantly more likely to have a high ratio of read coverage compared to the next downstream intron, consistent with these introns being retained in the transcript after the downstream intron has been spliced out. A subset of alternatively spliced exons are flanked by high read coverage introns, and most exhibit high read coverage in the upstream or downstream intron, but not both, which may indicate the order of intron removal. We treated ES cells with flavopiridol, which inhibits new transcriptional elongation, to track the fate of introns in the absence of continuing transcription. We observed that the high coverage introns were significantly more stable compared to the flanking introns on either side, but that eventually they also decreased in abundance, consistent with their being spliced out with slower kinetics than the other introns in the same transcripts. To determine whether the high coverage intron-containing transcripts are subject to nonsense-mediated decay (NMD), we treated mouse ES cells with cyclohexamide. The abundance of transcripts containing the high-coverage introns was not significantly enhanced, indicating that they are not normally subject to NMD. High-coverage introns are significantly overrepresented in gene ontology categories related to cell proliferation as well as RNA-binding proteins. These data suggest that a subset of introns is removed post-transcriptionally and that this may occur most commonly in genes required for proliferation, perhaps indicating a core function for splicing regulation in the decision of a cell to divide. Todd Bradley1,2, Malcolm Cook1, Marco Blanchette1,2 1 Stowers Institute for Medical Research, Kansas City, MO USA, 2Dept. of Pathology and Laboratory Medicine,University of Kansas School of Medicine,Kansas City,KS USA The SR proteins have been identified as an essential class of proteins involved in the regulation of splice site selection in higher eukaryotes, and have been implicated as key regulators during other stages of mRNA metabolism. Here we define the specific RNA-binding maps of the entire family of SR proteins in the trascriptome of Drosophila S2 cells using single-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP). We find that SR proteins bind a distinct, but functionally diverse, class of RNAs that includes mRNAs, both constitutive and alternatively spliced. Closer analysis of the bound transcripts revealed that while individual SR proteins can bind unique transcripts, multiple SR protein family members bind a majority of the target RNAs. Global analysis of SR-dependent splicing by RNAseq reveals that SR proteins are required for the regulation of many types of alternative splicing events, and can act as positive or negative regulators of splice site choice depending on their binding location on the target RNA. In addition, a vast majority of regulated targets require multiple SR protein members for regulation. This comprehensive analysis indicates that SR proteins bind and control the alternative splicing of a distinct set of pre-MRNAs through combinatorial regulation between multiple members of the SR protein family.

Poster Session 3: Splicing Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

541 Genome-wide Analysis of Splicing Regulation in Drosophila melanogaster by RNAi Depletion of 58 RNA Binding Proteins

Angela Brooks1,2, Gemma May3, Li Yang3, Michael Duff3, Jane Landolin4, Kenneth Wan4, Jeremy Sandler4, Susan Celniker4, Brenton Graveley3, Steven Brenner1, Fly Transcriptome Group 4 1 University of California, Berkeley, CA, USA, 2Currently at: Broad Institute of MIT and Harvard, Cambridge, MA, USA, 3University of Connecticut Health Center, Farmington, CT, USA, 4Lawrence Berkeley National Laboratory, Berkeley, CA, USA Pre-mRNA splicing is generally regulated by RNA-binding proteins that recognize sequence elements in the RNA and either activate or repress splicing of adjacent exons. To gain a better understanding of splicing regulation, it is important to identify the full set of splicing regulatory proteins, exons regulated by each protein, and determine if the protein is acting to enhance or repress splicing. Towards this goal, 58 RNA-binding proteins were individually depleted in a D. melanogaster cell line using RNAi and affected splicing events were identified using RNA-Seq. The RNA binding proteins examined include 7 SR proteins, 13 hnRNPs, 22 additional proteins that are known to affect splicing, 3 components of the exon junction complex, 12 proteins previously unknown to regulate splicing, and one essential protein in the nonsense-mediated mRNA decay pathway. We found a regulatory network where depletion of one protein significantly affects splicing and gene expression of many other regulators. Through the integration of modENCODE CAGE data, we have identified splicing regulators that significantly affect alternative first exons, which involves differential promoter usage. A majority of splicing events are significantly affected by only one or a few proteins, indicating that most splicing events are not regulated by the combinatorial action of a large set of proteins. However, we observe that for a subset of splicing events, SR and hnRNP proteins tend to act coordinately together, while act antagonistically with other RNA binding proteins. In total, this work has identified exons that are regulated by individual RNA-binding proteins in fly at an unprecedented level of resolution and is aiding in our understanding of splicing regulation.

542

Functional Analysis of the Conserved AU Di-nucleotides at the 5’-end of the U1 snRNA

Jui-Hui Chen, Tien-Hsien Chang Genomics Research Center, Academia Sinica, Taipei, Taiwan Binding of the U1 snRNP to pre-mRNA 5’ splice site (5’ss) plays a critical role in splicing by committing the pre-mRNA substrate to the splicing pathway. The interaction between U1 snRNP and the 5’ss is mediated in part by Watson-and-Crick basepairing of the U1 snRNA’s 5’-end (from the 3rd to the 9th positions) to the relatively conserved 5’ss (GUAUGU, in the budding yeast). Intriguingly, despite no apparent participation of the first two AU residues of the U1 snRNA in interacting with the 5’ss, these two positions are highly conserved from fungi to metazoans. To investigate the role of these two residues in splicing, we systematically mutate them, at the U1 snRNA gene (SNR19) level, to all possible combinations. Quantitative analysis reveals that the majority of these mutants exhibit no statistically difference in terms of fitness from that of the wild-type cells, suggesting that the AU dinucleotides may not be crucial. However, the AU-to-UU (AU>UU) mutant is greatly compromised in fitness and exhibits a cold-sensitive phenotype. Primer extension analysis shows that transcription initiation for U1 snRNA in all mutants is often altered in a seemingly predictable pattern, with the AU>UU mutant giving rise to the most dramatic defect. Biochemical analysis of the U1(AU>UU) snRNP and its interaction with the pre-mRNA 5’ss is now underway. Genetic suppression and synthetic-lethal screens are also being pursued in the hope to uncover novel factors that may regulate U1 snRNP’s functions either at the transcription or the splicing level.

Poster Session 3: Splicing Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

543

SC35 and SF2/ASF Regulate Stress-Responsive Alternative Splicing of MDM2

544

Modifiers of SMN Splicing in Spinal Muscular Atrophy

Daniel Comiskey, Ravi Singh, Aixa Tapia-Santos, Dawn Chandler Department of Pedatrics, College of Medicine, The Ohio State University, Columbus, OH, United States The MDM2 oncogene encodes a protein that negatively regulates p53 by targeting it for proteasome-mediated degradation. Through the induction of DNA damage and in cancer, MDM2 is alternatively spliced into a variety of isoforms. The MDM2-ALT1 isoform, comprised of exons 3 and 12, is observed in over 85% of rhabdomyosarcomas and generated in cells in response to genotoxic stress. MDM2-ALT1 lacks a p53 binding domain and abrogates fulllength MDM2 from binding p53 by sequestering it. This leads to the stabilization of p53, causing cell cycle arrest and/ or apoptosis. Paradoxically, the mouse homolog Mdm2-Alt1 has been shown to accelerate tumorigenesis in a mouse model, indicating a potential role in cancer. In order to understand the mechanisms by which MDM2 is alternatively spliced, we developed an in vitro splicing system using MDM2 minigenes and normal and cisplatinum-treated HeLa nuclear extracts. The MDM2 3-11-12 minigene, comprising regions conserved between mouse and human MDM2, predominantly excludes exon 11 under cisplatinum treatment both in vivo and in vitro. Using ESEfinder 3.0, we identified putative binding sites for splicing regulators SC35 and SF2/ASF in exon 11 of the MDM2 minigene. We then performed a series of mutations in the predicted binding sites for SC35 and SF2/ASF and examined the alternative splicing of these mutant constructs both in vivo and in vitro. Our in vitro data demonstrates that disrupting the SC35 binding sites in exon 11 leads to greater exclusion of exon 11 under normal conditions and disrupting the SF2/ASF binding site promotes the inclusion of exon 11, even under damaged conditions. This is consistent with the canonical role of SC35 as a positive regulator of splicing, but suggests a negative regulatory role for SF2/ASF. However, in MCF7 and HeLa cells transfected with MDM2 3-11-12 minigenes, both SC35 and SF2/ASF site mutants promote the inclusion of exon 11 under normal, UV and cisplatinum treatment. We confirmed the affinity of these splicing regulatory proteins for their predicted target sequences through RNA oligonucleotide pull downs using the wild-type and mutant sequences of each binding site. We also performed immunodepletion of SF2/ASF in normal and damaged nuclear extracts to assay the effect of SF2/ASF on the alternative splicing of our wild-type MDM2 construct.

Catherine Dominguez, Thomas Bebee The Ohio State University Spinal Muscular Atrophy (SMA) is a neurodegenerative disease that is one of the greatest sources of genetic mortality in infants. SMA is caused by low levels of Survival Motor Neuron (SMN) protein, encoded by two genes, SMN1 and SMN2. SMA is the result of an incomplete rescue by SMN2 expression in individuals lacking SMN1. A key difference in these two genes is a single nucleotide substitution in exon 7. This leads to increased skipping in SMN2 transcripts during pre-mRNA splicing. Therefore, while SMN1 produces almost exclusively full-length protein, SMN2 produces few full-length and many truncated proteins, which are degraded. This work will investigate modifiers of SMN splicing. Our first aim is to use antisense oligonucleotides (ASOs) to interfere with SMN splicing to develop a novel mouse model of SMA that has an intermediate disease phenotype. Respiratory dysfunction and hypoxia are causes of acceleration in SMA disease progression and the only treatment of the disease is through the use of ventilation support. Therefore, our second aim is to assess the effects of hypoxia as a modifier of SMN splicing. We have developed a mild SMA mouse model with the mouse Smn gene mutated to resemble human SMN2. This mild SMA mouse will be used to produce an SMA mouse model with an intermediate SMA phenotype through increasing Smn exon 7 skipping through ASOs, which target an important splicing enhancer. To determine hypoxic alteration of splicing, we compared cellular splicing levels under hypoxic and normoxic conditions. In addition, we designed SMN minigenes for mutation analysis to determine the importance of splicing sites in hypoxia-induced skipping. In 293T cells and SMA patient fibroblasts, ASO treatment caused an increase in skipping levels at 24, 48, and 96hrs, with protein knock down in fibroblasts. In hypoxic cells hnRNPA1 and hnRNPA2/B1 accumulated after 24hrs and mutation of splicing silencer sites for these factors was conducted. Individually, mutants were not sufficient to eliminate hypoxic skipping induction, but when all chosen sites were mutated in conjunction, hypoxic induction was eliminated. We conclude that the ASO is able to decrease protein levels and will next move into our mouse model. In our hypoxiatreated cells, we observed important factors for skipping induction as well as important sites.

Poster Session 3: Splicing Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

545 Dissecting Minimal Domains Necessary for Alternative Splicing by Muscleblind-like Proteins

Christopher Edge, Clare Gooding, Chris Smith University of Cambridge Muscleblind-like proteins are RNA binding proteins containing 4 zinc fingers arranged in two tandem arrays, and are well established regulators of alternative spicing. Known to play a crucial role in the transition from embryonic to adult splicing patterns, deregulation of which leads to crippling pathologies such as Myotonic Dystrophy, muscleblind-like proteins bind YGCY motifs, and are now accepted as activators, as well as repressors of alternative splicing, depending on binding context. Using cell culture techniques and MS2 tethering, we examined the minimal protein domains necessary to activate, and repress exon inclusion, and using targeted mutations tested the importance of RNA binding in this tethered context. We found an expressed protein of ~100 amino acids consisting of the first tandem zinc finger array and a small portion of linker sequence is capable of affecting splicing to the same degree as the full length 382 amino acid protein, and is still dependent on its RNA binding capacity. Furthermore this sequence is sufficient for repression and activation of alternative splicing. Using RNA binding and proteomic analysis, we then show this region of the protein is sufficient to bind RNA at high affinity, and to interact with possible protein partners PTB, SF3B3, and DAZAP1.

546

First splicing step in Saccharomyces cerevisiae requires Prp45

Ondrej Gahura, Zdenek Cit, Anna Valentova, Frantisek Puta, Petr Folk Department of Cell Biology, Faculty of Science, Charles University in Prague, Prague, Czech Republic Prp45/SNW1/SKIP is a conserved part of the NTC/CDC5 spliceosomal sub-complex that is required for splicing. It associates with sequence specific transcription factors as well as the components of elongating RNAPolII complex in higher eukaryotes. Previously, we reported that the C-terminus of Prp45 is required for the proper interaction of essential second step helicase Prp22 with the spliceosome and that Prp45 affects the splicing efficiency of substrates with noncanonical splicing signals (Gahura O. et al., 2009). Here, we document the effects of Prp45 on the first step of splicing. The truncation of Prp45 - prp45(1-169) - resulted in pre-mRNA accumulation of multiple intron containing genes but only some of the corresponding mRNAs were significantly decreased. We employed Mer1-dependent reporter substrates, SpR and ExR, which yielded translated product when either spliced or not spliced, respectively. Results of SpR/ExR assayed in cells expressing truncated Prp45 support a defect before the first transesterification. In addition, we examined Mer1-dependent pre-mRNA and mRNA levels of endogenous meiotic genes, including MER2. prp45(1-169) cells accumulated significantly higher levels of pre-mRNA than wild-type cells only when Mer1 was present. Chromatin immunoprecipitation revealed that Prp45 recruitment to intron-containing genes with long second exons is impaired in prp45(1-169) cells and that the maximum of the Prp45 signal is shifted towards the 3’ end of the transcript. Our data support the involvement of Prp45 in the first step of splicing. We assume that Prp45 is required for both splicing steps, perhaps as an adaptor that contributes to the regulation of the helicases involved. This work was supported by Czech Ministry of Education, Youth and Sports grant MSM0021620858 and the Grant Agency of the Charles University grant 441711.

Poster Session 3: Splicing Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

547 De Novo Prediction of PTB Binding and Splicing Targets Reveals Unexpected Features of Its RNA Recognition and Function

Areum Han1, Peter Stoilov2, Yu Zhou3, Xiang-Dong Fu3, Douglas Black1 1 UCLA, Los Angeles, CA, USA, 2WVU, Morgantown, WV, USA, 3UCSD, La Jolla, CA, USA The splicing regulator Polypyrimidine Tract Binding Protein (PTB) has four RNA binding domains that each interact with a short pyrimidine element, usually containing a CU or a UC dinucleotide. Slight differences RNA recognition by each domain and their connection by sometimes flexible interdomain linkers allow the protein to bind a wide variety of pyrimidine-rich sequences. This variation makes it difficult to predict PTB binding to particular elements, to rank sequences for probable affinity, and thus to identify PTB target exons. We have developed computational models that predict the binding and splicing targets of PTB. Using a set of known PTB-bound RNA sequences from cross linking immunoprecipitation (CLIP-seq) data, we constructed a Hidden Markov Model (HMM) to score probable PTB binding sites. Scores from this model are highly correlated (ρ=-0.9) with experimentally determined dissociation constants. Notably, we find that the protein is not strictly pyrimidine specific, as interspersed G residues are well tolerated within PTB binding sites, whereas A residues reduce binding score. This model thus uncovers many previously unrecognized PTB binding sites, and using it we can now accurately score PTB binding sites across the transcriptome in the absence of CLIP data. To examine the placement and role of these PTB binding sites in controlling alternative exons, we trained a multinomial logistic model on sets of PTB regulated and unregulated exons. Applying this model across the mouse transcriptome, we ranked exons for probable PTB regulation. High scoring exons included known PTB targets that were not in the training set, as well as many new exons confirmed as PTB repressed by RT-PCR and RNA-seq after PTB depletion. We find that PTB dependent exons are diverse in structure and do not all fit previous models for the placement of PTB binding sites. Our study provides new tools for predicting PTB dependent regulation across the transcriptome. This approach can be applied to other multi-RRM domain proteins, including nPTB, to assess binding site degeneracy and multifactorial splicing regulation.

548 Identification and Characterization of RBM38 Regulated Alternative Splicing Events in Hematopoietic Cell Development

Laurie Heinicke1, Behnam Nabet2, Russ Carstens1 1 University of Pennsylvania, Philadelphia, Pennsylvania, USA, 2Northwestern University, Evanston, IL, USA Alternative splicing of pre-mRNA transcripts can produce protein isoforms with cell-type specific functions that are essential for development and homeostasis. We previously performed a cell-based high throughput, genome-wide cDNA expression screen that identified numerous novel mammalian splicing factors. One of several mammalian RNA binding proteins that was shown for the first time to be a robust splicing regulator was RNA binding motif protein 38 (RBM38; also known as RNPC1). In humans, RBM38 has only been shown to bind and stabilize the 3’UTR of cell cycle regulators, such as p21 and p63. In C. elegans, RBM38 ortholog, SUP-12, has been shown to function in muscle-specific alternative splicing. Together with the known splicing function of the worm RBM38 ortholog, SUP-12, our laboratory has clearly established for the first time that this protein is a definitive mammalian splicing regulator and yet the endogenous targets and cellular functions of these regulators now require elucidation. We are attempting to identify targets and functions of RBM38 using erythropoietic cell lineages, where RBM38 RNA expression levels are high and there is evidence from CHIP-seq data that RBM38 is a target of erythroid transcription factor, GATA1. Using Affymetrix Human Exon Junction Array data, we identified numerous RBM38 regulated splicing events and have further validated these events in response to RBM38 knockdown by RT-PCR. In addition to identifying alternatively spliced pre-mRNA transcripts, we are elucidating the mechanism of RBM38 regulated alternative splicing events by RNA-tethering assays and TAP-TAG purification of RBM38 protein complexes. Using a FGFR2 minigene containing a known RBM38 enhanced exon, we have replaced the endogenous downstream intronic sequence with an RNA hairpin Box B sequence. We have shown that co-transfection of the above minigene and an expression vector containing RBM38 tethered λN peptide recapitulates native splicing. In addition, we are generating truncated forms of RBM38 to examine the functional role of each region in splicing using the above tethering assay. Together, these studies will lay the groundwork for future investigations to more directly study the functions of RBM38 in vivo, to assess the developmental consequences of RBM38 ablation at specific steps during hematopoietic cell development, and to provide a better mechanistic model of RBM38 mediated splicing events. Poster Session 3: Splicing Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

549 MDM2 Stress Responsive Splicing: An Intricate Interplay of Positive and Negative Elements and Splicing Regulatory Factors

Aishwarya Jacob, Ravi Singh, Dawn Chandler The Ohio State University, Columbus, (Ohio), USA Alternative splicing networks are important in all major physiological processes within the cell and perturbations in splicing pathways have been implicated in a number of diseases including cancer. The oncogene MDM2 is part of a stress-response splicing network and generates shorter isoforms by differential inclusion of its internal exons under stress. Interestingly, a number of these MDM2 splice isoforms, which lack the p53-binding domain, have been identified constitutively in various cancer types. However, the mechanisms regulating this alternative splicing event remain elusive. MDM2-ALT1 is a splice isoform that is generated in cells upon UV, Cisplatin treatment and is also observed in >85% RMS tumors. Its expression has been shown to accelerate lymphomagenesis in Eμmyc mice indicating a role in oncogenesis. Further, we have observed that MDM2-ALT1 affects the p53 pathway by modulating the functions of full-length MDM2. It is therefore, crucial to understand the regulation of MDM2-ALT1 splicing and its induction upon genotoxic stress treatment is a fortuitous event that enables us to model this splicing event in cancer with stress. We hypothesize that under stress and in cancer, differentially modified splicing regulatory trans factors coordinate with cis elements on the MDM2 mRNA to induce MDM2-ALT1. To identify the cis elements and trans factors that dictate the damage-induced alternative splicing of MDM2, we used an MDM2 minigene that is stress-responsive in an in vitro, cell-free splicing system. We constructed chimeric minigenes by swapping the introns and exons of MDM2 and a nonstress responsive p53 minigene and assayed them for damage-induced alternative splicing. Our results show that intron 11 contains elements necessary for mediating efficient full-length splicing of the MDM2 minigene. Using RNA affinity chromatography, we identified trans factors FUBP1 and PTBP1 and their stress-specific modified forms binding intron 11 differentially under stress. Our immunointerference in vitro splicing assays indicate positive roles for these factors in MDM2 splicing. Interestingly, exon 11 of the MDM2 minigene is sufficient to regulate its own exclusion under stress even in a heterologous p53 context suggesting that exon 11 of MDM2 contains additional elements important for regulating MDM2-ALT1 splicing. Our study aims to establish the factors dictating MDM2 alternative splicing and will help develop novel therapies tailored to modulate the MDM2-p53 network through splicing modification.

550 Contribution of Chromatin Marks to Alternative Splicing Regulation by Rbfox2 in Mouse Embryonic Stem Cells

Mohini Jangi, Paul Boutz, Phillip Sharp MIT Recent advances in the characterization of RNA-protein interactions in vivo have greatly increased our understanding of the mechanisms of alternative splicing regulation. Analyses of CLIP-seq datasets have addressed on a global scale the extent to which the presence of an RNA motif and its cognate RNA binding protein can determine the outcome of an alternative splicing event. In the context of tissue- and cell type-specific splicing patterns across development, an area that remains to be explored is the contribution of the chromatin environment to making and maintaining these splicing decisions. A growing body of evidence suggests there may be extensive crosstalk between chromatin and splicing machinery. Candidate gene analyses have shown specific, causal relationships between the location of individual histone marks and splicing outcome, mediated through a chromatin-binding adaptor that recruits a splicing factor to its site of action. We sought to investigate the contribution of chromatin signals to the regulation of alternative splicing by the splicing regulator Rbfox2. We performed shRNA-mediated knockdown of Rbfox2 in mouse embryonic stem cells followed by deep sequencing of mRNA, revealing hundreds of Rbfox2-mediated splicing events. To find chromatin marks that may regulate Rbfox2 splicing activity, we have cross-correlated these Rbfox2-dependent splicing events with published ChIP-seq datasets mapping histone modifications across numerous human and mouse cell lines as well as RBFOX2 CLIP-seq data from human embryonic stem cells. Ongoing experiments are focused on determining whether modulation of candidate chromatin marks affects Rbfox2-dependent splicing activity. Such causal links between chromatin dynamics and splice site selection will further have clear implications in determining the mechanism of cell type-specific splicing regulation during development.

Poster Session 3: Splicing Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

551

Regulated Use of Alternative Splice Sites During Stress in S.cerevisiae

552

Understanding how PTB and NPTB Direct Different Splicing Outcomes

Tadashi Kawashima, Stephen Douglass, Guillaume Chanfreau UCLA, Los Angeles, (Ca), USA Using RNA-Seq analysis of wild-type and NMD deficient yeast strains, we show that about one half of intron containing genes in S. cerevisiae show alternative splice site use, most of which resulting in transcripts degraded by NMD. Both cryptic 5’ and 3’ splice sites were identified in NMD mutants. Cryptic 5’ splice sites deviate predominantly at the 4th and 6th position from the consensus, suggesting a weaker binding of the U1 snRNP. Indeed, when the U1associated factors, Nam8p and Mud1p are deleted, decreased cryptic 5’-splice site usage was observed in both RPL22B and RPS14B. Cryptic 3’-splice site usage shows a more complex requirement for 2nd-step splicing factors, suggesting diverse rules for 3’-splice site selection for different transcripts. Finally, we found that the use of some of these alternative splice sites increase under conditions of stress, such as heat shock, amino acid starvation, and rapamycin treatment. Heat shock clearly shows the largest increase in cryptic splice site usage for some intron-containing genes, especially RPL22B. These results show that regulated alternative splicing is frequent in the lower eukaryote S. cerevisiae, but that most of the alternatively spliced isoforms are degraded by NMD, allowing for an additional level of gene expression control during growth in stress conditions.

Niroshika Keppetipola, Douglas Black University of California, Los Angeles, CA, USA The Polypyrimidine tract binding protein controls the splicing of many exons that are induced during neuronal development. PTB is expressed in neuronal progenitor cells (NPCs) but is down regulated during neuronal differentiation and replaced with its homolog neuronal PTB (nPTB, brain PTB, PTBP2). This transition reprograms a set of exons during neuronal development. For a group of neuronally expressed exons, PTB acts as a repressor but nPTB does not, allowing their splicing in differentiating neurons where nPTB is expressed but PTB is absent. Other exons are affected by both proteins. One well characterized exon that is differentially affected by PTB and nPTB is the N1 exon of the c-src pre-mRNA. N1 is excluded in non-neuronal cells but included in neurons. There is not yet a clear understanding of how these two proteins can direct different splicing outcomes. The two proteins share 74% primary structure similarity, have similar RNA binding domains and near identical RNA interacting residues. Both PTB and nPTB bind with high affinity to the CU elements upstream and downstream from the N1 exon. However, the two proteins differ in their ability to maintain binding under certain conditions. Previous studies reveal that a complex is formed downstream of the N1 exon in the presence of nPTB but not PTB containing several other regulatory proteins such as KSRP, hnRNP H, and F. Thus, the different regulatory effects of the two proteins are thought to stem from differences in their primary structure, their protein-protein and protein-RNA interactions. We are carrying out biochemical characterization of PTB and nPTB. We have made chimeric PTB/nPTB constructs and identified region(s) of PTB that confer splicing repression activity on nPTB in vivo. The chimeras have been recombinantly expressed and are currently being analyzed in the in vitro splicing system. Protein- protein interactions of PTB and nPTB are being analyzed in detail to identify differences in their interactions. The chimeras that have similar repression activity to PTB will also be analyzed for protein- protein interactions that differ from those of nPTB. These will be assessed as potential regulatory factors that contribute to splicing repression. In other experiments, we are examining the higher order protein/RNA complexes of PTB and nPTB for differences in structure that might lead to different splicing outcomes.

Poster Session 3: Splicing Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

553

Mammalian Neuronal Development and Maturation Require the Splicing Regulator PTBP2

Qin Li1, Chia-Ho Lin2, Peter Stoilov3, Lily Shiue4, Manuel Ares Jr.4, Douglas Black1,2 1 Dept. MIMG/University of California Los Angeles, Los Angeles, CA, USA, 2Howard Hughes Medical Institute/ University of California Los Angeles, Los Angeles, CA, USA, 3Dept of Biochemistry, School of Medicine, West Virginia University, Morgantown, WV, USA, 4Dept of MCDB, University of California Santa Cruz, Santa Cruz, CA, USA Alternative splicing plays essential roles in the development and maintenance of mammalian nervous system, and is partly mediated by tissue-specific expression of splicing regulators. In the developing brain, neural progenitor cells express PTB but switch to PTBP2 upon differentiation to neurons. The switch between these two closely related splicing regulators leads to widespread alternative splicing reprogramming. Compared to PTB, PTBP2 has been shown to be a weaker repressor of a number of neuron-specific alternative exons. However, the functional significance of replacing PTB with PTBP2 in neurons has not been characterized in vivo. We generated a conditional knockout mouse of PTBP2 where PTBP2 can be selectively inactivated in all or select regions of the nervous system. Loss of PTBP2 expression early in the embryonic central nervous system leads to lethality in neonates without causing gross developmental patterning defects. By splice-junction microarrays, high throughput RNA-sequencing and quantitative RT-PCR, we identified many alternatively spliced exons misregulated in PTBP2 knockout brains, many with important functions in neurite growth, synapse formation and synaptic transmission. These findings support our hypothesis that PTBP2 regulates many alternative splicing events and a neuron-specific splicing program is essential for neuronal differentiation. Interestingly, many of these splicing events show premature inclusion of exons that are normally spliced at postnatal stages, suggesting PTBP2 may function as a temporary splicing repressor to prevent splicing of a subset of exons specific to mature neurons. Conditional inactivation of PTBP2 in the projecting neurons of the cortex and hippocampus led to slow postnatal growth and lethality around weaning. Lethality is likely a result of widespread neuronal deaths in the targeted brain regions. Neurons lacking PTBP2 also fail to survive beyond 3 weeks in dissociated culture and display defects in neuronal polarity and axon specification. Therefore PTBP2 expression and regulation of its target splicing events are critical to both embryonic and postnatal brain development.

554 PTB and nPTB regulated splicing events during neural progenitor maintenance and motor neuron development.

Anthony Linares, Douglas Black UCLA, Los Angeles, CA, USA RNA binding proteins (RBPs) regulate alternative splicing in a tissue-specific manner. Our laboratory studies several RBPs that control large sets of exons during neuronal development and in mature neurons. Two of these, polypyrimidine tract-binding protein (PTB) and neuronal PTB (nPTB), control splicing events that assist neuronal differentiation and maturation. During in vitro motor neuron development PTB-nPTB expression profiles suggest that two transitions in splicing occur. PTB expression declines and disappears during neurogenesis, while nPTB expression peaks during differentiation and neurite outgrowth. As motor neurons mature, nPTB expression declines.These results parallel previous observations in cultures of neural progenitor cells and neurons from embryonic brain. Using RNA-sequencing, we have profiled alternative splicing events in populations of mouse and human embryonic stem cells, neural progenitor cells, and mature motor neurons. Hundreds of significant splicing events during each developmental transition have been observed, including the differential splicing of several transcription factors and chromatin remodelers. In parallel,we are identifying all the PTB and nPTB binding sites across the expressed RNA during the differentiation process using the crosslinkingimmunoprecipitation (CLIP-seq) method. Comparing our data base of the observed changes in splicing with sites of PTB binding, we will identify PTB and nPTB-regulated splicing events. We are particularly interested in the alternative splicing of factors that may regulate neuronal development. We hypothesize that PTB promotes a progenitor state, while nPTB assists neuronal maturation.When PTB regulation is withdrawn, new splicing patterns promote isoform switches in factors that define a neuron-specific phenotype.Through these studies, we hope to understand the post-transcriptional regulatory events that program neuronal self-renewal, differentiation, and maturation, and particularly help define motor neurons from other neuronal lineages. 

Poster Session 3: Splicing Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

555 Analysis of Site-Specific Phosphorylation Events and Their Influence on Splicing in Schizosaccharomyces pombe

Michael Marvin, Jesse Lipp, Kevan Shokat, Christine Guthrie UCSF, San Francisco, (CA), USA The essential process of pre-mRNA splicing is carried out by the spliceosome, which is composed of 5 snRNPs as well as many non-snRNP proteins. In addition, splicing can be influenced by post-translational modification of spliceosomal proteins. Phosphorylation by several kinases is known to be one essential post-translational modification that is required at different steps in the splicing cycle yet an unbiased analysis of splicing-related kinase substrates is currently lacking. One of the known substrates of splicing kinases includes the evolutionarily conserved serine/arginine (SR) RNA binding proteins, which play vital roles in both constitutive and alternative splicing. SR proteins have been shown to require phosphorylation of their SR repeats in order to bind regulatory elements such as exonic splicing enhancer (ESE) sequences that have been shown to compensate for variations in intron splice sites by enabling bound SR proteins to make contacts with core spliceosome components, many of which also have RS-rich regions that can also be phosphorylated. The fission yeast S.pombe serves as an excellent system in which to study how phosphorylation affects splicing. Unlike S. cerevisiae, S. pombe contains the essential Prp4 kinase as well as two bona fide SR proteins. In addition, the SRPK1 homolog Dsk1 as well as Clk/Sty LAMMER kinase Kic1 are also present and have been shown to influence splicing. In addition, while less then 5% of budding yeast genes have introns almost half of fission yeast genes posses at least one intron as well as ESEs. Further, there have also been reports of alternative splicing by intron retention as well as exon skipping in S. pombe. In order to analyze the substrates of the splicing related kinases in S. pombe we are taking a chemical-genetic approach using analog-sensitive kinases. Gatekeeper mutations, which we currently have for Dsk1 with Kic1 and Prp4 kinases in progress, are added to S. pombe extract in the presence of bulky modified ATP analogs in order to map specific phosphorylation sites using mass spectrometry. Thus, by combining site-specific mutations at phosphorylation sites as well as kinase deletions or temperature sensitive alleles, we will investigate how phosphorylation affects both in vitro spliceosome assembly and splicing genome-wide using splicing microarrays. Our results from yeast will also inform parallel studies in humans by providing an evolutionary view of how phosphorylation influences splicing.

556 Investigating the Function of Tissue-dependent Alternative Splicing in the Mammalian Circadian Clock

Nicholas McGlincy, Inge van Bussel, Johanna Chesham, Jernej Ule, Michael Hastings MRC Laboratory of Molecular Biology, Cambridge, U.K. The vast majority of mammalian multi-exon genes undergo tissue-dependent alternative splicing; in humans this fraction is estimated to be >80%. Despite this, for many cellular systems, the functional ramifications of alternative splicing are unknown. Of particular interest is the mammalian circadian clock. The circadian clock orchestrates daily cycles of gene expression that underpin circadian rhythms in metabolism, physiology and behaviour. Circadian rhythms are necessary for the proper temporal organisation of metabolism, and tune physiology and behaviour to anticipate regular changes in the environment. Indeed, rhythm dysfunction is associated with major psychiatric and metabolic disorders (e.g. unipolar depression and obesity). At the cellular level, the mammalian circadian clock comprises approximately 20 genes. A core transcriptional negative feedback loop is augmented by a number of regulatory proteins that help to determine the clock’s period, and additional transcriptional feedback loops that provide the clock with robustness. Using the Exonmine database and public exon-array data, we identified potential alternative exons in the murine core clock genes. For seven of these exons we observed tissue-dependent alternative splicing in mouse tissues by RT-PCR. This raises two possibilities. First, that the clock is constructed differently in different cells types, and secondly, that alternative splicing is a mechanism by which the clock achieves tissue specificity in its output. Many of the identified exons are highly conserved in mammals, and we are examining whether the orthologous human exons also show tissuedependent inclusion. In order to examine the function of these exons we have identified phosphorodiamidate morpholino oligonucleotides (PMOs) that inhibit inclusion of Clock exon 18 & Csnk1d exon 9 in NIH3T3 cells. We will use these PMOs to examine the importance of exon inclusion for the proper functioning of the circadian clock in cells. This will shed light on the role of tissue-dependent alternative splicing in the core clock genes.

Poster Session 3: Splicing Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

557

Evolution of alternative splicing regulation

Joel McManus1, Joseph Coolon2, Michael Duff3, Jodi Mains3, Patricia Wittkopp2, Brenton Graveley3 1 Carnegie Mellon University, Pittsburgh, PA, 2University of Michigan, Ann Arbor, MI, 3University of Connecticut Health Center, Farmington, CT Alternative splicing is regulated via networks composed of cis-acting regulatory sequences and trans-acting factors. Variation of splicing cis-regulatory sequences is a well-established source of phenotypic diversity. In contrast, much less is known regarding how variability in trans-acting splicing factors (e.g. RNA binding proteins that recognize ciselements) relates to phenotypic diversity within and between species. Furthermore, the relative contributions of changes in cis- and trans-acting splicing components towards species-specific splicing are unknown. Using mRNA-seq, we investigated differences in exon skipping and intron retention in D. simulans, D. sechellia, and three strains of D. melanogaster. While a modest number of splicing differences were observed between D. melanogaster strains (~4-10%), roughly twice as many splicing events were divergent between species (~9-21% of all events). We further investigated the contributions of cis-and trans-acting changes in splicing regulatory networks by comparing allelespecific splicing in F1 interspecific hybrids. In F1 nuclei, each allele is subjected to the same set of trans-acting factors. Thus differences in allele-specific splicing reflect changes in cis-regulatory element activity. We find that changes in cisregulatory elements contribute more to species-specific intron retention and alternative splice site usage, while changes in trans-acting factors contribute more to species-specific exon skipping differences. Intriguingly, the modENCODE consortium’s analysis of the Drosophila transcriptome throughout development suggests that exon-skipping is more dynamically regulated than intron retention and alternative splice site usage. Taken together, these results suggest that exon skipping may have more trans-acting regulators than other types of alternative splicing, and this in turn may affect the evolution of splicing regulation. We are currently expanding on this work to investigate the evolution of sex-specific alternative splicing in Drosophila head tissue.

558 Using a Drosophila genetic model to study crosstalk between chromatin and alternative splicing

Michael Meers, A. Gregory Matera The University of North Carolina, Chapel Hill, NC Alternative splicing of pre-mRNA transcripts in complex eukaryotes is a crucial processing function that combinatorially enriches the proteomic diversity yielded directly from encoded genes. Recent work suggests that chromatin dynamics may play an important role in defining exons and modulating alternative splicing, though little is known about the potential mechanism of action. While it has been shown that trimethylation at lysine 36 of the H3 histone subunit (H3K36me3) is enriched at constitutive exons, and that an H3K36me3-binding “adaptor” may play a role in modulating splicing via RNA binding protein recruitment, other studies have suggested that a causal link between H3K36me3 and splicing is spurious. Here we propose to utilize data and resources from the ModENCODE consortium to evaluate the correlation betweenH3K36me3 and exon inclusion across developmental time in Drosophila melanogaster. Time-course correlation information will guide candidate gene approaches, in which well-characterized exons whose splicing varies across developmental time will be evaluated for their inclusion sensitivity to H3K36me3 depletion. We will study the genome-wide contribution of K36 trimethylation knockout to alternative splicing experimentally through a whole-organism histone mutagenesis system being developed in the Matera lab.

Poster Session 3: Splicing Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

559

Silent Effects of Splicing: Conservation of Splicing Signals in Coding Exons

William Mueller, Klemens Hertel UC Irvine Genetic mutations can alter multiple aspects of RNA processing. In the case of pre-mRNA splicing, mutations found in splice sites change splice patterns by modulating the affinity of the splicing machinery to exons. Mutations flanking splice sites can still influence splicing patterns by interfering with the appropriate binding of splicing regulatory proteins. The high level of conservation in exons makes it difficult to differentiate between what is conserved due to protein coding and codon bias and what is conserved due to the requirement for efficient splicing. To determine the influence of silent mutations on exon inclusion we generated a library of systematic mutations at wobble positions across the coding exon 7 of SMN. All possible permutations of wobble position mutations within a 6-nucleotide window walking across the exon were created, transfected into HeLa cells, and the resulting spliced mRNAs were sequenced. Many mutants altered the level of exon inclusion, especially in the context of a hexamer. To correlate the mutational analysis with conservation strain, we determined PhyloP scores at each position across the exon. The results demonstrate that wobble positions with high conservations scores associate with observed prominent exon inclusion changes. These observations were particularly striking within the context of hexamers, the sequence span typically required for splicing regulatory binding. Our data demonstrate that the high conservation observed within coding exons is in part due to the requirement for faithful splicing defined through hexamer sequences that most likely represent binding sites for splicing regulatory proteins.

560 Evolution and Functional Analysis of the Antisense Overlap Between mRNAs Encoding Two Mammalian Nuclear Receptors, TRα2 and Rev-erbα

Stephen Munroe1, Christopher Morales1, Cynthia Aguilar1, Paul Waters2, Jennifer Graves3 Marquette University, Milwaukee, WI, USA, 2The Australian National University, Canberra, Australia, 3La Trobe University, Melbourne, Australia Eutherian mammals encode a non-hormone-binding variant, TRα2, from the gene THRA, which also encodes the α-thyroid hormone receptor, TRα1. TRα2 is remarkable in sharing an antisense overlap with the final exon of Rev-erbα, another nuclear receptor protein that plays critical roles in embryonic development, cellular differentiation, energy metabolism and regulation of circadian rhythms. The overlap between Rev-erbα and TRα2 is extremely well conserved among eutherian species. However, TRα2 is not expressed in marsupial mammals despite an otherwise high degree of conservation (BC Rindfleisch et al., 2010, BMC Mol. Biol.). To better understand the evolution and functional role of the antisense overlap with Rev-erbα, the homologous region of the platypus genome was sequenced. Since the platypus is a prototherian mammal (a monotreme) representing a third distinct line of extant mammalian descent, the comparison of the platypus sequence with those of eutherians (placental mammals) and marsupials reveals divergent features within regions invariant in the other mammals. Multiple alignments support the conclusion that the antisense overlap between TRα2 and Rev-erbα is unique to the eutherian lineage. Differences between platypus and marsupial species highlight the conservation of sequences that appear as intermediate stages in the evolution of the alternatively spliced exon of TRα2, including sequences homologous to core splicing elements at both the 3’ and 5’ splice sites. In addition, intronic and exonic splicing enhancers for TRα2 have evolved in the context of 3’UTR elements required for the processing and regulation of Rev-erbα mRNA. Of particular interest is a G-rich region of about 30 nts within the coding sequence of TRα2 mRNA and antisense to the 3’UTR of Rev-erbα. Closely spaced deletions and substitutions within this region have dramatically different effects on TRα2 splicing depending on their precise structure and position. Interestingly, this sequence is poorly conserved in marsupials but resembles a G-rich region downstream of the Rev-erbα coding sequence in platypus. In summary, comparisons of disparate mammalian sequences in marsupials and monotremes reveal cis-acting elements important for the alternative processing and stability of the physiologically significant nuclear receptor proteins encoded by two highly conserved and closely linked genes. 1

Poster Session 3: Splicing Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

561

The Tumor Suppressor p53 Controls Alternative Splicing In Mammary Epithelial Cells

Ryan Percifield, Daniel Myrphy, Peter Stoilov West Virginia University, West Virginia, USA Tumor formation has been associated with a dramatic shift in splicing patterns. Here, we show that the previously described tumor specific splicing patterns are unlikely to be related to carcinogenesis, as they consist mostly of epithelial specific transcript variants. This bias is caused by the epithelial cell lineage composition of most solid tumors, which is a significant departure from the mix of cell types forming the normal tissue. To understand how alternative splicing is regulated in carcinogenesis we propose an approach where expression levels of tumor suppressors and oncogenes are manipulated to produce tumorigenic cells while preserving their epithelial phenotype. We show that p53 controls the expression of several splicing factors previously associated with tumor formation. Consequently, p53 loss has a broad effect on alternative splicing patterns and produces mRNA variants at ratios that resemble those in tumors.

562 Identifying and Characterizing the Mechanisms and Consequences of Nervous System Alternative Splicing

Adam Norris, John Calarco Harvard University, Cambridge, (MA), USA Alternative splicing is an important and pervasive means of increasing the transcriptomic and proteomic diversity of an organism, and is often differentially regulated across tissue types. However, despite the importance of spatiotemporal alternative splicing (AS) regulation, little is known about the factors or mechanisms regulating this process in vivo, and it remains an important goal to understand the physiological impact of splice variants in development. The nervous system is a particularly interesting model due to its high level of AS and its composition of many diverse cells with customized gene regulation and specialized function. I am performing unbiased forward-genetic screens in C. elegans using two-color splicing reporters to identify and characterize novel regulators of AS in the nervous system. I expect to identify previously uncharacterized RNA binding proteins and spliceosomal components plus novel classes of genes not previously implicated in AS. Novel regulators of AS will be further analyzed using genome-wide approaches to determine the AS regulatory networks they control. In an initial screen of 7,000 haploid genomes via EMSmutagenesis, I have uncovered eight mutants, which are now being mapped and sequenced. In additional work, I have been interested in understanding the behavioral and functional consequences of developmentally-regulated AS events. I will present the results of a detailed investigation of a developmentally regulated splicing event in transcripts encoding UNC-62/Meis, a homeodomain-containing transcription factor involved in proper coordination, axon pathfinding, egg laying and lifespan regulation in C. elegans. Using isoform-specific rescuing constructs and phenotypic analyses I have shown that the two UNC-62 isoforms contribute differentially and in non-redundant ways to lifespan regulation and coordination. I have shown that the DNA sequence binding specificity is identical between the two isoforms, and I am now preparing to determine whether the two isoforms bind at distinct sites in vivo via Chromatin Immunoprecipitation experiments. Together these experiments will help us understand the mechanisms and consequences of cell-type specific and developmentally regulated AS.

Poster Session 3: Splicing Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

563 Loss of a Positive Regulator Within a Deep Intronic Sequence of SBP2 Contributes to a Genetic Disorder

Eric Ottesen, Joonbae Seo, Senthilkumar Sivanesan, Natalia Singh, Ravindra Singh Iowa State University, Ames, Iowa, USA Selenium is a trace element that is essential to human health. Its biological role is realized primarily through cotranslational incorporation of selenocysteine (Sec; the 21st amino acid) into ~25 selenoproteins. SECIS binding protein 2 (SBP2) is an RNA binding protein that serves as the key factor for selenoprotein synthesis. The coding sequence of SBP2 gene is comprised of 17 exons. Several exons of SBP2 undergo alternative splicing. We have recently reported that transcripts lacking a part of exon 3 (exon 3a) represent the most abundant spliced variant of SBP2. We have also shown that skipping of SBP2 exon 3a serves as a regulatory step for limiting the synthesis of full-length SBP2. Recently, a patient suffering from a multi-system disorder has been shown to carry a number of mutations within SBP2 intron 2. However, the impact of these mutations on splicing of any of the SBP2 exons has not been assessed. Similarly, cis-elements and trans-acting factors associated with these mutations remain unknown. Here we describe the role of one of such mutation that is characterized by the loss of two adenosine residues within a 24-nucleotide long tract of adenosines(abbreviated as “24A-Tract”). We show that sequences immediately down stream of 24A-Tract play a critical role in inclusion of exon 3 of SBP2. Consistently, an 18-nucleotide antisense oligonucleotide that sequestered the last 5 residues of 24A-Tract and the downstream sequences led to skipping of SBP2 exon 3a. Our results suggest that the length of 24A-Tract is critical for inclusion of exon 3 as well as synthesis of full-length SBP2, which is the master regulator of selenoprotein synthesis. Our findings underscore the impact of a deep intronic cis-element on splice-site selection and bring novel insight into splicing regulation of exons flanked by large intronic sequences.

564

Identification of New Splicing Inhibitors

Andrea Pawellek1, Stuart McElroy2, Timur Samatov3, Reinhard Luehrmann3, Angus Lamond1 1 Wellcome Trust Centre for Gene Regulation & Expression, College of Life Sciences, Dundee, DD1 5EH, Scotland, UK, 2Drug Discovery Unit, College of Life Sciences, Dundee, DD1 5EH, Scotland, UK, 3Max Planck Institute for biophysical Chemistry, Department of Cellular Biochemistry, D-37077 Goettingen, Germany Eukaryotic pre-mRNA splicing enables a large proteome to be encoded by a relatively small genome. Defects in premRNA splicing are an important cause of disease. Approximately 15% of single base pair mutations that cause human genetic diseases are thought to be linked to pre-mRNA splicing defects and there are also increasing indications that aberrant pre-mRNA splicing events might play an important role in human cancer. Therefore splicing is increasingly regarded as an attractive target for drug discovery. However the identification of specific and selective splicing inhibitors/ modifiers would not only be useful for potential therapeutic applications but would also be extremely valuable for research purposes, e.g. as tools for dissecting the splicing mechanism. We recently screened 71504 small chemical compounds by using an high throughput in vitro splicing assay and identified 13 new compounds that modify splicing in vitro. Interestingly 10 out of these 13 compounds also inhibit premRNA splicing in HeLa cells. We are currently investigating the effect of these compounds on splicing, cell growth and localization of nuclear proteins in greater detail.

Poster Session 3: Splicing Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

565

A model in vitro system for co-transcriptional splicing

566

Identification of epigenetic regulators of alternative pre-mRNA splicing

YONG YU, Rita Das, Eric Folco, Robin Reed Harvard Medical School, Boston, MA, USA A hallmark of metazoan RNA polymerase II transcripts is the presence of numerous small exons surrounded by large introns. Abundant evidence indicates that splicing to excise introns occurs co-transcriptionally, prior to release of the nascent transcript from RNAP II. Here, we established an efficient model system for co-transcriptional splicing in vitro. In this system, CMV-DNA constructs immobilized on beads generate RNAP II transcripts containing two exons and an intron. Consistent with previous work, our data indicate that elongating nascent transcripts are tethered to RNAP II on the immobilized DNA template. We show that nascent transcripts that reach full length, but are still attached to RNAP II, are efficiently spliced. When the nascent transcript is cleaved within the intron using RNase H, both the 5’ and 3’ cleavage fragments are detected in the bound fraction, where they undergo splicing. Together, our work establishes a system for co-transcriptional splicing in vitro, in which the spliceosome containing the 5’ and 3’ exons are tethered to RNAP II for splicing.

Maayan Salton, Ty Voss, Tom Misteli NCI/NIH, Bethesda, Maryland, USA Splicing of precursor mRNA (pre-mRNA) is an important regulatory step in gene expression. Alternative splicing (AS) allows the production of multiple protein isoforms from one pre-mRNA molecule, thereby contributing to proteomic diversity. Novel high-throughput sequencing technology has recently revealed that more than 90% of human genes undergo AS and AS is crucial in differentiation and tissue-specific gene expression. Despite the importance of AS, how mRNA splicing machinery identifies the precise location of generally short exons in the context of the much larger introns and how AS decisions are made, particularly in a cell type- and tissue-specific fashion, remains largely unclear. Recent evidence points to the possibility that epigenetic marks and higher order chromatin structure play a key role in AS regulation. In order to identify novel chromatin regulators of AS, we have used a cell-based in-vivo assay for high-throughput screening. The assay consists of a two-color fluorescent reporter based on alternative splicing of the microtubule-associated protein TAU, whose mis-splicing cause frontotemporal dementia. We have adapted the TAU reporter assay to a 384-well format and have screened a siRNA library of ~400 chromatin-related proteins using high-throughput imaging. We confirmed our results using a validation assay with a dual read-out of imaging and qPCR. Identified factors associate with the TAU reporter as demonstrated by chromatin immunoprecipitation. We are currently mapping the genome wide effect of each of our candidates on global splicing outcome. Interestingly, all identified candidates shift splicing of the TAU reporter in a way that would correct the splice anomaly in frontotemporal dementia. Thus our candidates are not only shedding light on the emerging role of epigenetic regulators in the process of AS, but also have the potential to define new therapeutic directions for frontotemporal dementia.

Poster Session 3: Splicing Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

567

The role of Rbfox proteins during skeletal muscle differentiation

Ravi Singh1, Auinash Kalsotra1, Chris Bland1,Tomaž Curk4, Jernej Ule5, Liguo Wang2, Wei Li2, Thomas A. Cooper1,2,3 Departments of 1Pathology and Immunology, 2Molecular and Cellular Biology, and 3Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA. 4Faculty of Computer and Information Science, University of Ljubljana, Trzaska cesta 25, SI-1000 Ljubljana, Slovenia. 5Medical Research Council (MRC) Laboratory of Molecular Biology, Cambridge, UK. In metazoans, splicing is an essential aspect of gene expression and alternative splicing is critical for generating protein diversity from a limited set of genes. Regulation of alternative splicing is important for generating isoforms of genes that have tissue specific functions to fulfill critical physiological needs. Alternative splicing events occur in different temporal clusters during skeletal muscle differentiation in culture suggesting coordinated control. However, factors controlling these splicing events are not known. Computation prediction of sequences flanking alternative exons identified enriched and conserved nucleotide motifs of known RNA binding proteins i.e. Rbfox, CELF, MBNL, andSTAR family of proteins (Nucleic Acids Res 38, 7651). Rbfox (RNA binding protein, Fox-1 homolog of C. elegans) family of proteins bind to consensus motif UGCAUG and are expressed highly in brain, skeletal muscle, and heart. These motifs are enriched and conserved near exons that undergo splicing changes during skeletal muscle differentiation in culture in avian and mammalian species. Rbfox3 is neuron specific, however, Rbfox1 and Rbfox2 are expressed in various tissues including skeletal muscle. In this study, we will map the alternative splicing network of Rbfox1 and Rbfox2 during skeletal muscle differentiation in culture. To this end, we have performed iCLIP (individual nucleotide resolution cross-linking and immunoprecipitation) with antibodies specific to Rbfox1 and Rbfox2. Additionally, we have also performed RNA-Seq using RNA from cells with individual and double knockdown of Rbfox1 and/or Rbfox2 to distinguish Rbfox1 and Rbfox2 dependent changes during skeletal muscle differentiation. Overlapping iCLIP data with RNA-Seq data will help us identify specific transcriptome changes including Rbfox regulated networks that will be important for skeletal muscle differentiation. This study will shed light on importance of transcriptome changes mediated by the Rbfox family of proteins in skeletal muscle differentiation. The knowledge gleaned from this study will uncover previously unknown function of Rbfox proteins in mediating transcriptome transitions during skeletal muscle differentiation.

568 Pyrvinium Pamoate Regulates Alternative Splicing Of The Serotonin Receptor 2C pre-mRNA By Changing RNA Structure

Manli Shen1, Peter Stoilov2, Stefan Stamm1 1 University of Kentucky, Lexington, KY, USA, 2University of West Virginia, Morgantown, WV We are analyzing the regulation of the serotonin receptor 2C pre-mRNA, as it is disturbed in patients with PraderWilli syndrome and other psychiatric diseases. The serotonin receptor pre-mRNA undergoes alternative splicing at a distal 5’ splice site. Only usage of this distal 5’ splice site makes a functional protein. The pre-mRNA forms a stable secondary structure around the two competing 5’ splice sites that spans about 200 nts. This structure undergoes A->I editing, demonstrating that it is forming in vivo. RNA editing and the action of processed snoRNAs from the HBII-52 cluster promote usage of the distal site. As HBII-52 is missing in patients with Prader-Willi syndrome, we developed a two-color reporter system and screened a chemical library of 6,000 compounds. We identified pyrvinium pamoate as a substance that promotes distal splice site usage at 6 µM concentration in two hours. The action of pyrvinium pamoate is independent of protein synthesis and works in heterologous sequence contexts. CD (circular dichroism) spectrum analysis shows that pyrvinium pamoate binds directly to serotonin pre-mRNA in vitro and changes the tertiary structure of the RNA. Chemical probing (SHAPE) RNA structure analyses and RNAse T1 assays showed that the distal splice site is mostly double stranded and flanked by two stem-loop structures. Addition of pyrvinium pamoate changes the conformation of the pre-mRNA. The downstream loop is altered towards a double stranded conformation and the upstream loop structure is opened up. In addition, the distal splice site is slightly opened up, which likely allows U1 binding and exon recognition. Genome-wide array analysis identified several other alternative exons that are activated by pyrvinium pamoate and show strong secondary RNA structures. The data show that an RNA intercalating drug promotes alternative splicing by changing the secondary structure, which is similar to the action of a riboswitch in response to a small ligand. The identification of other pre-mRNAs containing secondary structures that respond similar to pyrvinium pamoate suggests a more widespread existence of such riboswitch-like structures in humans, indicating that they might have natural ligands. Poster Session 3: Splicing Regulation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

569 Increased Accumulation of Glucose-6-Phosphate Dehydrogenase mRNA Due to Enhanced Binding of the Splicing Factor SRSF3 In Response to Nutrients

Amanda Suchanek, Callee Walsh, Travis Cyphert, Lisa Salati West Virginia University, Morgantown, WV, USA Alternative splicing as a major source of protein diversity, yet the mechanisms regulating the process by exogenous factors remains largely unexplored. We have identified RNA splicing as an intracellular target for nutrient regulation of gene expression. Our laboratory has demonstrated that starvation of animals and treatment of hepatocytes in culture with polyunsaturated fatty acids reduces the efficiency of splicing of the glucose-6-phosphate dehydrogenase (G6PD) transcript. This change in splicing efficiency results in reduced expression of the enzyme due to a type of alternative splicing called intron retention. In contrast, feeding a high carbohydrate, low fat diet to rodents or treatment of hepatocytes with insulin induces efficient splicing and increases the cellular content of G6PD. We have identified a splicing regulatory element within exon 12 of the G6PD transcript that is required for splicing related changes in G6PD expression in hepatocytes in culture. Using this paradigm, splicing regulatory factors were identified that respond to nutritional and hormonal status. In this report, we present several lines of evidence that SRSF3 is required for nutrient regulated splicing. First, using in vitro binding assays, SRSF3 binds specifically to the exon 12 regulatory element. Furthermore, SRSF3 binding is increased in nuclear extracts prepared from the livers of fed mice as opposed to starved mice. Second, knockdown of SRSF3 reduces G6PD expression and the accumulation of spliced G6PD mRNA, while knockdown of other SR proteins did not change G6PD expression. Third, both phosphorylation and amount of SRSF3 are increased in primary hepatocytes in culture following treatment with insulin and these effects are diminished upon addition of arachidonic acid; treatments which increase and decrease G6PD expression, respectively. Finally, SRSF3 binding to exon 12 was enhanced 7-fold over background in RNA immunoprecipitation experiments in the livers of refed mice, while almost no binding (25 ribosome biogenesis factors, combined with Northern blotting and pulse-chase experiments, we have characterised ITS1 processing in human cells. We demonstrate that two processing sites in human ITS1, 2a and 2, are analogous to the yeast A2 and A3 cleavages, respectively. The majority of human pre-RNA is processed via a pathway that differs from that described in yeast: an endonucleolytic cleavage at site 2 is followed by exonucleolytic processing to site 2a, ~25nt from the 3’ end of 18S. This exonucleolytic processing is mediated by the RRP6-exosome, an enzyme that does not participate in yeast ITS1 removal. RRP6 is essential for 18S rRNA accumulation implying this is a major pathway. However, a small fraction of the human prerRNA is processed by an alternative pathway, similar to that described in yeast, involving serial endonucleolytic cleavages at sites 2 and 2a. The initial ITS1 cleavage at site 2, like yeast A3, is dependent on factors linked to the biogenesis of the large subunit, such as BOP1, NOL12 and RBM28 (Erb1, Rrp17 and Nop4, respectively in yeast). However, while site A2 is the main ITS1 cleavage site in yeast, the A3-like site 2 cleavage is the primary processing site in humans. Interestingly, we found that site 2 cleavage is not dependent on the yeast A3 endonuclease, RNase MRP. The genetic disease Cartilage-hair hypoplasia, which is caused by defects in the RNase MRP complex is classified as a ribosomopathy, but our data suggest this is not the case. We conclude that the early stages of human ITS1 processing utilise both different processing mechanisms and nucleases than those described in yeast.

602 The RNA kinase CLP1 is required for efficient tRNA splicing and regulates p53 activation in response to oxidative stress

Stefan Weitzer, Toshikatsu Hanada, Barbara Mair, Josef Penninger, Javier Martinez IMBA (Institute of Molecular Biotechnology of the Austrian Academy of Sciences), Vienna, Austria Both in mammals and archaeabacteria CLP1 proteins have been identified as kinases that phosphorylate 5’-hydroxyl ends of RNA molecules. Human CLP1 is a component of the mRNA 3’-end cleavage and polyadenylation machinery and associates in mammals with the tRNA splicing endonuclease (TSEN) complex. TSEN proteins remove an intronic region present within the anticodon loop of numerous pre-tRNAs generating 5’ and 3’ tRNA exon halves. Within the TSEN complex, CLP1 has been shown to phosphorylate the 5’-hydroxyl group of 3’ tRNA exons in vitro, potentially contributing to tRNA splicing in mammals. Here we characterize tRNA metabolism in kinase-dead Clp1 (Clp1K/K) mutant mice that contain a point mutation within the ATP binding motif of the genomic Clp1 locus. We show that extracts prepared from Clp1K/K embryonic fibroblasts display reduced TSEN cleavage activity, revealing an unexpected role for CLP1 during tRNA exon generation. Affinity purified kinase-dead CLP1 containing TSEN complexes were deficient in pre-tRNA cleavage, most likely due to reduced levels of TSEN2, TSEN34 and TSEN54 subunits. Thus CLP1 is an integral component of the TSEN complex and ATP binding and/or hydrolysis is crucial for complex assembly. Interestingly, loss of CLP1 activity causes increased cell death upon H2O2 challenge and leads to accumulation of an entirely novel set of small tRNA fragments, comprising 5’ leader and 5’ exon sequences derived from aberrant processing of tyrosine pretRNA. Overexpression of such tRNA fragments results in enhanced p53 activation in response to H2O2 challenge. We thus hypothesize that inactivation of CLP1’s kinase activity sensitizes cells to oxidative stress-induced p53 activation and p53-dependent cell death.

Poster Session 3: tRNA, snRNA, snoRNA, rRNA

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

603 A Genome-wide Analysis to Identify Novel Genes Involved in tRNA Metabolism and Subcellular Trafficking

Jingyan Wu, Anita Hopper The Ohio State University, Columbus, (OH), USA tRNAs are major components of the cell’s protein synthesis machinery. In addition to this essential role, tRNAs are involved in protein degradation, apoptosis, cellular response to stress, and tumorigenesis. It is known that nascent tRNAs are transcribed in the nucleus. After the removal of the 5’ and 3’ ends and addition of some modifications, tRNAs are exported to the cytoplasm where they fulfill their functions. In both yeast and vertebrate cells, the subcellular movement of tRNAs involves the initial export of tRNAs from the nucleus to the cytoplasm, retrograde nuclear import of cytoplasmic tRNAs, and re-export of the imported tRNAs back to the cytoplasm. Although tRNAs have been studied for decades, some major players in tRNA metabolism and subcellular movement remain unknown. For example, there is an unknown nuclear export pathway for intron-containing tRNAs in yeast. Moreover, the molecular mechanisms for regulating tRNA processing, subcellular trafficking, stability, and turnover remain unclear. The overall aim of my research is to identify and characterize all the missing gene products involved in tRNA biology. Using yeast as a model organism and the yeast deletion and temperature-sensitive collections, I am conducting a genome-wide assessment of how every gene affects tRNAs. To rapidly analyze tRNAs in each strain in the collections, I developed a procedure that allows for RNA extraction of ~80-90 strains within 3 hours. I also developed a highly sensitive nonradioactive Northern analysis method to detect the levels of tRNA processing intermediates, mature tRNA, tRNA halves and introns. To date, I’ve analyzed ~2000 out of 4848 genes in the deletion collection; 3 candidates that affect tRNA biology have been identified. Additional candidates will be identified by analyzing the remaining members of the collections. This study will uncover important factors that function in tRNA metabolism and intracellular trafficking, which will contribute to a better understanding of the complexity of tRNA biology.

604

DUX4 Induces Global Dysregulation of RNA Processing in Skeletal Muscle

Stephen Tapscott, Zizhen Yao, Robert Bradley Fred Hutchinson Cancer Research Center, Seattle, WA, USA Facioscapulohumeral dystrophy (FSHD) is a common form of inherited muscular dystrophy whose underlying genetic cause and molecular pathology are imperfectly understood. Recent results suggest that aberrant expression of DUX4, a retrotransposed gene encoding a double homeobox transcription factor, may give rise to FSHD in the context of a permissive genetic background. Normally restricted to the germ line, DUX4 is aberrantly expressed in a subset of skeletal muscle cells of FSHD patients, but not healthy individuals. In this study, we investigated the consequences of aberrant DUX4 expression in primary and immortalized human skeletal muscle cells with deep RNA-seq. Strikingly, 30% of the genes consistently and significantly up-regulated following DUX4 induction are involved in RNA metabolism, including many canonical splicing regulators such as SR and hnRNP genes. We observed correspondingly broad and biased changes in RNA processing, such as a global shift towards usage of intron-proximal 3’ splice sites in the context of competing sites. Taken together, our results suggest that altered RNA processing driven by DUX4 expression is a likely contributor to the molecular pathology of FSHD.

Poster Session 3: tRNA, snRNA, snoRNA, rRNA & RNAs in Diseases

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

605 Human microRNA Expression Profile in Amyotrophic Lateral Sclerosis: Role of microRNAs in the Regulation of Neurofilament Levels

Danae Campos-Melo, Kathryn Volkening, Michael Strong University of Western Ontario, London, ON, Canada The motor neuron degeneration that is the core feature of Amyotrophic Lateral Sclerosis (ALS) is associated with the formation of neurofilament aggregates and a selective suppression of low molecular weight neurofilament (NFL) mRNA. The preferential localization of NFL mRNA to degradative granules (P-bodies) in ALS affected spinal motor neurons suggests that the suppression of NFL mRNA levels is related to an increase in RNA degradation. Our preliminary data showed that RNA species of some type contribute to NFL mRNA stability. MicroRNAs (miRNAs) are small endogenous non-coding RNAs that participate in mRNA degradation through base pairing interactions in the mRNA 3’ untranslated region (UTR). The mechanism of action of miRNAs and its critical role in neurodegeneration, make them prime candidates as mediators of NFL mRNA stability in ALS. We performed a human miRNA expression profile in ventral lumbar spinal cord (SC) of sALS and controls using TaqMan array. After the analysis of 664 miRNAs we found that a large group of miRNAs is dysregulated in SC in sALS. Interestingly, the majority of them (246) have decreased expression while a small group (10) is up-regulated in sALS. Ingenuity Pathway Analysis (IPA) showed that dysregulated miRNAs in sALS are linked with nervous system function, cell cycle and cell death. Two computational algorithms were used to develop a panel of dysregulated miRNAs in sALS that have recognition elements within hNFL mRNA 3’UTRs of different length. We investigated the functional relevance of these miRNAs using reporter gene assays and rqRT-PCR. HEK293T cells were co-transfected with one of three plasmids containing different hNFL mRNA 3’UTRs linked to the fLuciferase gene and miRNAs predicted to interact with the 3’UTR. Our data suggest a potential role of miRNAs 146a*, 524-5p and 582-3p in the selective decrease of NFL mRNA observed in ALS that could contribute to the etiology of neurofilamentous aggregates and the pathology of ALS.

606

Hypoxia is a Modifier of SMN2 Splicing and Disease Severity in a Severe SMA Mouse Model

Thomas Bebee, Catherine Dominguez, Somayeh Samadzadeh-Tarighat, Dawn Chandler 1 The Center for Childhood Cancer at the Research Institute at Nationwide Childrens Hospital, Columbus, Ohio, USA. 2 The Department of Pediatrics, The Ohio State University, Columbus, Ohio, USA Spinal muscularatrophy (SMA) is a progressive neurodegenerative disease associated with the loss of spinal motor neurons and atrophy of proximal muscles. In severe SMA patients muscle atrophy leads to respiratory deficiency and mortality. SMA is due to low SMN levels due to loss of the SMN1 gene and only partial compensation by SMN2. SMN2 splicing efficiency is reduced by a C>T mutation in exon 7 that results in only low levels of functional SMN protein. Therefore, SMA disease severity can be modulated by changes in the efficiency of splicing thereby altering the levels of SMN protein. Global analysis of the severe SMNΔ7 SMA mouse model revealed that alterations in splicing patterns and increased levels of stress induced transcripts, such as the hypoxia inducible transcript hif3alpha, occur at late stages of disease progression. As severe SMA patients also develop respiratory deficiency during disease progression, we sought to evaluate whether hypoxia contributes to SMA disease by altering SMN2 exon 7 splicing and if increased oxygenation could modulate disease severity in a severe SMA mouse model. To evaluate the impact of hypoxia on SMN2 exon 7 splicing we treated cells in culture and observed increased SMN2 exon 7 skipping that reduced SMN protein levels. Conversely, treatment of SMNΔ7 severe SMA mice with hyperoxia increased inclusion of SMN2 exon 7 in skeletal muscles and resulted in improved motor function. To evaluate the regulatory elements or factors involved in hypoxia induced skipping of exon 7 we utilized transfection splicing assays of SMN minigenes. Mutational analysis revealed hypoxia induced skipping is dependent upon poor exon definition by the C>T mutation and suboptimal 5’ss. Furthermore, hypoxia treatment in cell culture led to increased hnRNP A1 levels, a negatively acting exon 7 splicing factor. Mutation of hnRNP A1 binding sites prevented hypoxia induced skipping of SMN exon 7 in minigene assays. These results implicate hypoxic stress as a modulator of SMN2 exon 7 splicing that can be prevented to alter disease progression by increased oxygenation in severe SMA mice. Additionally, poor exon definition and hnRNP A1 binding sites sensitize SMN2 exon 7 to hypoxia induced skipping in minigene assays, which implicate hnRNP A1 in hypoxia induced skipping.

Poster Session 3: RNAs in Diseases

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

607 Splice Isoform Switching: A New Mechanism Controlling EMT and Breast Cancer Progression

Rhonda Brown1, Lauren Reinke1, Yilin Xu1, Marin Damerow1, Denise Perez2, Lewis Chodosh2, Jing Yang3, Chonghui Cheng1 1 Department of Medicine, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, 2Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, 3Department of Pharmacology and Pediatrics, School of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093 Epithelial-mesenchymal transition (EMT) is a tightly regulated process that is critical for embryogenesis. When abnormally activated, EMT promotes cancer metastasis and recurrence. Here we show that splice isoform switching constitutes a novel mechanism that controls EMT. We identified a shift in CD44 expression from variant isoforms (CD44v) to the standard isoform (CD44s) during EMT. This isoform switch to CD44s, which is regulated at the level of alternative splicing, is essential for cells to undergo EMT and is required for the formation of breast tumors that display EMT characteristics in mice. Analysis of patient breast tumors showed that CD44s expression is upregulated in high-grade breast tumors and correlates with the level of the mesenchymal marker N-cadherin in these tumors. We are currently investigating mechanisms by which CD44 alternative splicing is regulated during EMT. We have determined sequence motifs that recruit splicing factors to promote or inhibit CD44 alternative splicing. Experiments are underway to determine the regulation of these splicing factors in EMT and breast cancer progression.

608 Rho Guanine Nucleotide Exchange Factor is a NFL mRNA Destabilizing Factor that Forms Cytoplasmic Inclusions in Amyotrophic Lateral Sclerosis

Cristian Droppelmann, Brian Keller, Danae Campos-Melo, Kathryn Volkening, Michael Strong University of Western Ontario, London, ON, Canada Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive disorder characterized by degeneration of motor neurons. Although the cause of the disease remains elusive, protein aggregate formation, including neurofilamentous aggregates, in motor neurons is a neuropathological hallmark. Recent evidence supports the hypothesis that alterations in RNA metabolism in motor neurons can lead to the development of these aggregates. In mice, p190RhoGEF, a guanine nucleotide exchange factor, is involved in neurofilament protein aggregation in a RNA-triggered transgenic model of motor neuron disease. Here, we observed that Rho Guanine Nucleotide Exchange Factor (RGNEF), the human homologue of p190RhoGEF, binds NFL mRNA and affects its stability via 3’UTR destabilization. We observed that the overexpression of RGNEF in a stable cell line significantly decreased the level of NFL protein. Furthermore, we observed RGNEF cytoplasmic inclusions in ALS spinal motor neurons that co-localized with ubiquitin, p62/Sequestosome-1 and TDP-43. Our results provide further evidence that RNA metabolism pathways are integral to ALS pathology. This is also the first described link between ALS and a RNA binding protein with aggregate formation that is also a central cell signalling pathway molecule.

Poster Session 3: RNAs in Diseases

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

609 Induced Pluripotent Stem Cells from Diamond Blackfan Anemia Patients Show Defects in Ribosome Biogenesis

Jingping Ge, Loic Garcon, Marisa Apicella, Jason Mills, Paul Gadue, Deborah French, Mitch Weiss, Monica Bessler , Philip Mason Children’s Hospital of Philadelphia, Philadelphia, PA, USA Diamond Blackfan Anemia (DBA) is a severe red cell aplasia, often associated with other developmental abnormalities such as short stature, cleft pallet, craniofacial and thumb anomalies. In all known cases DBA is caused by haploinsufficiency of one of several ribosomal proteins, including RPS19 and RPL5. The mechanism by which defects in a housekeeping function such as ribosome biogenesis lead to the specific abnormalities seen in DBA is not known. As a cellular model for DBA, we generated induced pluripotent stem cells (iPSCs) from DBA patients with RPS19 or RPL5 mutations. In polysome profile study, cells with an RPS19 mutation showed a dramatic reduction in the ratio of 40S/60S ribosome subunits, indicating the excess of large 60S ribosome subunits, while cells with an RPL5 mutation showed a similar increase of 40S/60S ratio indicating an excess of 40S subunits. In both cases, the imbalanced ratio of 40S and 60S led to a decrease of mature 80S subunits. At the level of rRNA processing, cells with an RPS19 mutation were defective in 18S rRNA production, with a sharp decrease in the 18SE pre-RNA molecules. Cells with an RPL5 mutation were surprisingly defective in both 18S rRNA and 5.8S rRNA production, leading to the accumulation of 18SE and 12S preRNA molecules, and significantly decreased levels of 21S pre-RNA. These findings suggest that RPL5 is required for both 18S and 5.8S rRNA production in iPSCs, while RPS19 is required for 18S rRNA. No p53 activation was detected in those iPSCs, though the p53 pathway is induced by ribosomal stress in somatic cells. We conclude that iPSCs from DBA patients effectively model the defects in ribosome biogenesis that underlie the development of DBA.

610 Noncoding Consequences of Disease Associated Mutations Set Against a Backdrop of Multiple Transcriptomic SNVs

Matthew Halvorsen, Joshua Martin, Gabriela Phillips, Justin Ritz, Wes Sanders, Alain Laederach University of North Carolina at Chapel Hill The advent of faster, cheaper and increasingly more reliable sequencing technologies has led to a sharp increase in the number of publicly available human genomic sequences and variants. The sheer amount and variety of human sample sequencing data presently available represents an opportunity to utilize a very large sample size in order to make population-wide statistical inferences regarding regions of sequence and structural conservation in the known transcriptome. Collections of SNVs from 1094 individuals in the 1000 genomes project were analyzed using an optimized variant of the SNPfold algorithm. The resultant data revealed transcriptome-wide sequence and structural variance in specific “wildtype” human RNAs. Areas observed to be high in sequence conservation, as well as predicted to have low variance in average base accessibility, are theorized to be regions of functional significance at the post-transcriptional level. Significant overlap is seen between these regions and experimentally derived RNA-binding protein and miRNA binding sites. Sets of disease-associated mutations cataloged in the Online Mendelian Inheritance of Man (OMIM) and the Human Gene Mutation Database were compared with the described transcriptomic variant data in order to further narrow down functional regions of significance on a per-disorder basis. The conservation of the sites in question were analyzed in the presence of detected variants in Cystic Fibrosis patients, as well as somatic mutations detected in various cancer patient samples, and various regions where normally conserved sequence and structure are disrupted are reported.

Poster Session 3: RNAs in Diseases

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

611

Analysis of Novel NFL targeting MicroRNAs in Amyotrophic lateral Sclerosis (ALS)

Muhammad Ishtiaq, Danae Campos Melo, Kathryn Volkening, Michael Strong University of Western Ontario, University of Western Ontario, University of Western Ontario, University of Western Ontario ALS is a progressive neurodegenerative disease in which microRNA (miRNA) expression patterns are altered when compared with control tissues. NFL mRNA levels are selectively decreased in ALS, and in previous studies we have shown that in ALS affected spinal cords 304 miRNA are differentially expressed. Of these, 48 (30 known, 18 novel) miRNAs were predicted to target NFL mRNA, of which 12 were down-regulated and the remaining were up-regulated in ALS patients compared to controls. miRNA target prediction was performed manually for novel miRNA. miRNAs with 7 or more matching seed sequence bases with NFL 3’UTR were considered potential candidates. Of these novel miRNA, real time PCR analyses showed that all but 2 miRNA (sblock659, block5539544) showed increased expression in ALS patients. Additionally, our laboratory has determined that there are 3 predicted isoforms of NFL differing in the length of their 3’UTRs: NFL short (286 bp), NFL medium (1380 bp) and NFL long (1838 bp). NFL long is expressed in the human spinal cord and therefore we have concentrated on this isoform in our analyses. Sblock3998 was predicted to have multiple interaction sites in this isoform of NFL. A subset of our candidate miRNAs was also predicted to target multiple ALS related mRNA, indicating a complex role in RNA metabolism. Sblock294 was predicted to target 5 ALS related mRNAs including NFL, RGNEF, TDP-43, FUS/TLS and SOD1. Block7043946 was predicted to target mRNA for NFL, FUS/TLS, TDP-43 and RGNEF. Both these miRNAs showed increased expression in small RNA derived from ALS affected spinal cord tissue, suggesting that there may be a more wide-reaching effect on regulation of ALS associated protein mRNA than previous thought.

612 A BIM deletion polymorphism contributes to resistance against targeted cancer therapy by promoting splicing of non-apoptotic BIM variants

Wen Chun Juan1, King Pan Ng1, Tun Kiat Ko1, Axel Hillmer2, Charles Chuah3, Yijun Ruan2, Xavier Roca4, Sin Tiong Ong1,5 1 Duke-NUS Graduate Medical School, Singapore, Singapore, 2Genome Institute of Singapore, Singapore, Singapore, 3Department of Haematology, Singapore General Hospital, Singapore, Singapore, 4Division of Molecular Genetics & Cell Biology, School of Biological Sciences, Nanyang Technological University, Singapore, Singapore, 5Department of Medicine, Duke University Medical Center, Durham, NC BIM is a pro-apoptotic member of the BCL-2 family of proteins that trigger apoptosis by interacting and sequestering the prosurvival members of the family (BCL-2, BCL-xL, MCL1), or by directly binding the distal apoptotic effectors, BAX and BAK. Critically, BIM expression is essential for sensitivity towards tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML) and EGFR-mutated non-small cell lung cancer (EGFR NSCLC). Alternative splicing of BIM transcripts produce variants that either contain exon 3 (E3) or exon 4 (E4) in a mutually exclusive manner due to the presence of a stop codon and a polyadenylation signal within E3. Because the pro-apoptotic BH3 domain that is critical for protein interaction is encoded exclusively by E4, only E4-containing BIM isoforms are pro-apoptotic. Using paired-end DNA sequencing, we recently discovered a novel 2.9kb deletion polymorphism in intron 2 of the BIM gene that was associated with TKI resistance in CML patients. Due to the close proximity of the deletion to E3, we postulated that the intronic deletion would result in preferential splicing of E3. Indeed, deletion of the 2.9kb intronic region in a minigene favored splicing to E3 over E4 by several folds. To study the function of this deletion, we have identified CML and EGFR NSCLC cell lines that harbor the deletion. Compared to non-deletion containing cells, the cells with the deletion exhibited preferential expression of BIM transcripts containing E3 over E4 and decreased expression of E4-containing transcripts and proteins after TKI exposure. Consequently, these cells also demonstrated impaired apoptotic signaling. We also aimed at identifying cis-regulatory elements within the deleted fragment that suppress splicing to E3. Using serial deletion analysis, we have mapped the cis-regulatory elements to a 150-nucleotide region within the deletion that is sufficient for E3 splicing repression. Ongoing analysis is expected to further narrow down the region sufficient for splicing repression, as well as to reveal the splicing factors that regulate this alternative splicing. Collectively, these results provide a novel mechanism by which an inherited polymorphism mediates TKI resistance in targeted cancer therapy. Importantly, our results also emphasize the increasing importance of aberrant pre-mRNA splicing in human genetic diseases. Poster Session 3: RNAs in Diseases

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

613 Expanded CUG Repeat RNA reactivates the embryonic gene program in Myotonic dystrophy

Auinash Kalsotra, Ravi Singh, Chad Creighton, Thomas Cooper Baylor College of Medicine, Houston, Texas, USA Myotonic dystrophy type 1 (DM1) is a dominantly inherited disease that affects multiple organ systems. The second leading cause of death in DM1 patients is cardiac sudden death due to conduction defects, arrhythmias and dilated cardiomyopathy. The causative mutation is a CTG expansion in the 3’ untranslated region of DMPK gene resulting in aberrant expression of expanded CUG repeat RNA that accumulates into nuclear foci and causes misregulated alternative splicing. From global expression analyses of micro- and messenger RNAs in a heart-specific and an inducible mouse model of DM1 we report that the expanded CUG repeat RNA reactivates the embryonic gene program in adult DM1 hearts which is distinct from the general hypertrophic stress response. Using q-PCR TaqMan arrays, we identified 54 miRNAs that were differentially expressed in DM1 mouse hearts one week following induction of CUG repeat RNA. Interestingly, 83% (45/54) exhibited a developmental shift in expression towards the embryonic pattern. Because over 90% (41/45) were down regulated within 72 hr after induction of repeat RNA and only 2 of 22 examined decreased in two unrelated mouse models of heart disease, we conclude reduced expression of the majority of miRNAs is specific to DM1 rather than a general response to cardiac injury. MBNL1 and CELF1 are two RNA binding proteins disrupted in DM1 and known to have physiological consequences via disrupted alternative splicing. Using heart tissue from loss of MBNL1 or gain of CELF1 DM1 mouse models, we found that these proteins are not driving the miRNA misregulation. Ten out of ten primary miRNA transcripts examined were down-regulated in CUG-repeat expressing hearts in parallel with their mature miRNAs, indicating that loss of expression is not due to a processing defect. Instead, we discovered that adult-to-embryonic shift in expression of select micro- and messenger RNAs in DM1 heart is due to inactivation of a MEF2 transcriptional program. MEF2 are evolutionarily conserved proteins that drive cardiac muscle gene expression during development through binding to specific cis-regulatory elements. Both in hearts of DM1 patients and our mouse model there is a significant loss of MEF2A and MEF2C proteins. This leads to reduced MEF2 interactions with its response elements on target genes including microRNAs resulting in their decreased steady state levels.

614

S6K1 alternative splicing modulates its oncogenic activity and regulates mTORC1

Vered Ben Hur1, Polina Denichenco1, Avraham Maimon1, Adrian Krainer2, Ben Davidson3, Rotem Karni1 1 Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel, 2Cold Spring Harbor Laboratory, NY, USA, 3Oslo University Hospital, Norwegian Radium Hospital, Oslo, Norway Ribosomal S6 Kinase 1 (S6K1) is a major mTOR downstream signaling molecule which has been implicated in the regulation of cell size and translation efficiency. Here we report that short isoforms of S6K1 are over-produced in breast cancer cell lines and tumors. Overexpression of mouse or human S6K1 short isoforms induces transformation of human breast epithelial cells. Surprisingly, the long S6K1 variant (isoform-1) induced opposite effects: its overexpression inhibited Ras-induced transformation and tumor formation, while its knockdown induced transformation suggesting that isoform-1 has a tumor suppressor activity. We further found that S6K1 short isoforms bind to mTOR and activate the mTORC1-4E-BP1 axis downstream of Akt, elevating 4E-BP1 phosphorylation and cap-dependent translation. Both a phosphorylation-defective 4E-BP1 mutant and the mTORC1 inhibitor rapamycin blocked the oncogenic effects of S6K1 short isoforms, indicating that these are mediated by mTORC1 and 4E-BP1. Thus, alternative splicing of S6K1 acts as a molecular switch in breast cancer cells; down-regulating a tumor suppressive isoform and elevating oncogenic isoforms that activatemTORC1.

Poster Session 3: RNAs in Diseases

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

615 Identification Of New Factors Associated With Translationally Repressed RNAs In Plasmodium Berghei

Natalia Kozlova1, Edwin Lasonder2, António Mendes3, Céline Carret1, Ana Guerreiro1, Gunnar Mair1 1 Molecular Parasitology Unit, Institute of Molecular Medicine, Lisbon, Portugal, 2Centre for Molecular and Biomolecular Informatics, NCMLS, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, 3Malaria Unit, Institute of Molecular Medicine, Lisbon, Portugal Translational repression (TR) is an evolutionary conserved mechanism of post-transcriptional regulation of gene expression. In the rodent malaria Plasmodium berghei a number of mRNAs synthesized in the female gametocytes are translationally quiescent and activated for protein synthesis only after fertilization in the mosquito midgut. This mechanism allows synchronized synthesis of proteins required for rapid post-fertilization development and protects parasite from attack of mammalian immune response, which can be initiated by premature translation of Plasmodium surface antigens. Previous studies identified protein complex in vivo associated with translationally repressed RNAs. Integrity of the complex was shown to be essential for stability of quiescent RNAs, sexual development and transmission of the parasite. However, no factors were found whose absence would activate premature translation. Here we provide a first attempt to identify proteins targeting RNAs for TR. We took advantage of in vitro assembly of RNAprotein complexes on the regions known to target RNAs for TR and tagged with three MS2 binding sites, which allow purification of the complexes by MS2-MBP affinity chromatography. Mass spectrometry analysis of the purified complexes identified several proteins enriched or only associated with translationally repressed versus translation-competent mRNAs. Importantly, 15 out of 16 proteins known to associate with repressed RNAs in vivo were found in the purified complexes, corroborating specificity of the assay. A Plasmodium ortholog of PTB (polypyrimidine tract binding protein), previously characterized as a splicing factor and translational repressor, and a protein of so far unknown function (PBANKA_133210) were abundantly associated with TR-targeting regions. To study the role of these proteins in TR gene disruption clones were generated. Integrity of repressed RNAs was not affected in the mutant clones in contrast to the typical KO phenotype of previously described components of TR machinery. This together with the fact that the proteins of interest were not found in previously described repression complex suggests existence of a new regulatory complex performing a distinct function in TR. The functional studies of the mutants are currently in progress. Our preliminary data demonstrated that transfer of the sporozoites from oocysts to the salivary glands is partially affected in the mutant clone bearing gene disruption of PBANKA_133210. This suggests the role of the protein in de-repression of maternally synthesized transcripts on late stages of parasite development.

616 Identification of altered MicroRNA expression in response to salmonella infection during early stage in intestinal epithelial cells

Xingyin Liu, Jun Sun University of Rochester, NY, USA Salmonellainfection is not only a public health concern also known to increase the risk of inflammatory bowel diseases and cancer. MicroRNAs (miRNAs) are noncoding small RNAs that function as an endogenous regulator of gene expression. Their dysregulation has been implicated in roles in eukaryotic host responses to viruses and extracellular bacterial pathogens. Therefore, it is important to understand how Salmonella works in targeting eukaryotic miRNA expression in intestinal epithelial infection. In this study, the 3D-Gene miRNA microarray platform (Toray, Kamakura, Japan) was used to investigate the miRNA responses of the human intestinal epithelial cells to different Salmonella strains during early stage of infection. Different from that of late stage infection of reported by Schulte LN (2011), expression pattern in all of miRNAs have no significant change during the early stage of infection. 5 miRNAs (hsa-miR-638, hsa-miR-494, hsa-miR-149, hsa-miR-1979 and hsa-miR-27b ) showed mild up-regulated change compared to control group. Three miRNAs (hsa-miR-130a, hsa-miR-1308 and hsa-miR-BART21) showed mild down-regulated. Quantitative reverse transcription-polymerase chain reaction also demonstrated that hsa-miR-494, hsa-miR-149, and hsa-miR-27b showed mild over-expressed in two infection groups compared to control group. Interestingly, we found the expression level of these mild degregulated miRNA in SL1134 infection group are lower than that of SB1117 infection group, the result suggested that Salmonella effector, AvrA maybe inhibit miRNA response through unknown pathway. Analysis of their putative targets suggested that these mild differentially expressed miRNAs molecules may have a potential role in regulating early inflammation signaling in response to salmonella invasion.

Poster Session 3: RNAs in Diseases

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

617 A Drosophila Model of Spinal Muscular Atrophy Uncouples the snRNP Biogenesis Functions of Survival Motor Neuron from Locomotion and Viability Defects

Kavita Praveen, Ying Wen, A. Gregory Matera University of North Carolina, Chapel Hill, NC, USA Spinal muscular atrophy (SMA) is caused by loss-of-function mutations in survival motor neuron 1 (SMN1). SMN protein has well characterized roles in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. SMN has also been implicated in tissue specific functions, however, it remains unclear which of these is important in the etiology of SMA. Here, we present a new Drosophila model system to study SMA patient-derived loss-of-function mutations in the background of an Smn null allele. Smn null mutants display a modest reduction in the levels of a subset of snRNAs and considerable defects in larval locomotion. Despite these reductions in snRNP levels, we found no appreciable defects in the splicing of mRNAs containing minor class (U12-type) introns in Smn null mutants. Incontrast, minor-intron splicing defects were readily apparent in U6atac (positive control) mutants. Transgenic expression of FLAG-tagged wild-type SMN rescues larval locomotion and organismal viability but, surprisingly, fails to rescue snRNA levels. Expression of an SmnT205I construct (which mimics SMNT274I in humans) also rescues the larval motility and viability defects, but the majority ofthese animals die as pupae with an snRNA profile nearly identical to that ofthe wild-type transgenics. We conclude that the observed decreases in snRNA levels in Smn null animals are not major contributors to the organismal phenotype. These findings have major implications for SMA etiology because they show that SMN’s role in snRNP biogenesis can be uncoupled from the lethality and locomotor defects in the organism.

618

Role of double-stranded RNA-dependent protein kinase (PKR) in metabolic disease

Takahisa Nakamura, Brenna Baccaro, Gökhan Hotamisligil Department of Genetics and Complex Diseases, Harvard University, School of Public Health, Boston, (MA), USA In the past two decades, significant advances have uncovered chronic inflammation as a critical mechanism underlying metabolic diseases such as obesity, insulin resistance, and type 2 diabetes. We recently showed that double-stranded RNA-dependent protein kinase (PKR), an established pathogen sensing protein, is involved in the critical aspects of metabolic diseases1. To further investigate the impact of PKR on metabolism and the underlying mechanisms, we generated genetically obese mice (ob/ob) with PKR deficiency. In this setting, body weight was not affected by the absence of PKR, yet PKRdeficiency still resulted in significantly improved insulin sensitivity along with reduced JNK activation. Hence, PKR is an obligatory mediator or obesity- and ER stress-induced JNK activity. Intriguingly, we also observed a significant reduction of obesity-induced eIF2α phosphorylation in PKR-deficient ob/ob mice, suggesting that PKR is a primary factor to regulate protein translation in metabolic stress. More recently, through mass spectrometry analyses, followed by biochemical and molecular validation experiments, we determined that PKR forms complexes with components of the RNA-induced silencing complex (RISC), which plays a central role in generating microRNAs. These complexes preferentially assemble when PKR is activated by inflammation, excess nutrients, and in the obese but not lean liver. The interactions between PKR and components of RISC require PKR’s RNA-binding domains, which are essential for PKR activation in these conditions. Our data demonstrate that PKR plays a central role in coordinating multiple networks to integrate metabolism with stress signals through formation of a metabolic inflammatory complex, a metaflammasome, which contains RNA-binding proteins, and RNA species in metabolic diseases. 1. Nakamura T., et al. Cell 140 (3): 338-348 (2010)

Poster Session 3: RNAs in Diseases

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

619 Aberrant mRNA processing, a major disease mechanism that precedes symptoms in a mouse model of Amyotrophic Lateral Sclerosis (ALS)

Ramesh Narayanan, Marie Mangelsdorf, Robyn Wallace Queensland Brain Institute, Brisbane, Queensland, Australia Splicing is a mechanism by which eukaryotes remove introns to produce a mature transcript. Alternative splicing(AS) is a process by which a variety of mature transcripts (isoforms) are made from the same pre-mRNA transcript resulting in a variety of isoforms of the proteins they encode. Aberrant AS has been reported in various neurodegenerative disorders including amyotrophic lateral sclerosis (ALS) and sporadic Alzheimer disease [1]. TDP-43 is an RNA binding protein known to regulate pre-mRNAsplicing, RNA transport and stability. In the central nervous system of rodents TDP-43 binds to various mRNA targets [2,3]. Mutations in TDP-43 have been reported to cause ALS in humans and inclusions containing TDP-43 have been found as a pathological feature of various neurological diseases [4]. We have utilised a transgenic mouse that overexpresses human TARDBP, the gene encoding TDP-43 that contains an ALS causing mutation (A315T). These mice develop motor neuron disease with symptoms similar to humans [5]. RNA from wild type and transgenic mouse brain and spinal cord tissue was hybridised to exon microarrays to elucidate the global effect of mutated TDP-43 on AS in the CNS of transgenic mice. Microarray results were verified in selected genes by PCR and qPCR. Pre-mRNA splicing of various transcripts, previously identified as RNA binding partners of TDP-43, were affected in the presence of the A315T mutation. Exon array analysis on brain and spinal cord tissue from TDP-43 transgenic mice showed that global aberrant splicing leads to changes in the ratio of isoforms of several genes. Our results suggest aberrant pre-mRNA splicing as one of the major disease mechanisms that precedes symptoms of ALS in TDP-43 mice. We hypothesize that ALS is an RNA metabolism disorder and that targeting RNA processing pathways for drug development and therapy might prove valuable for treating patients with ALS. 1. Anthony, K. and J.M. Gallo, Brain research, 2010. 1338: p. 67-77 2. Polymenidou,M., et al., Nature neuroscience, 2011. 14(4):p. 459-68. 3. Sephton,C.F., et al., J Biol Chem, 2010. 286(2):p. 1204-15. 4. Neumann,M., et al., Arch of Neurol., 2007. 64(10):p. 1388-94. 5. Wegorzewska,I., et al., PNAS, 2009. 106(44): p.18809-14.

620 A Novel Interplay Between the NMD Mechanism and ER Stress Response Under Normal Conditions and in Human Diseases

Yifat Oren1, Tamar Geiger2, Miriam Manor1, Matthias Mann3, Batsheva Kerem1 1 Hebrew University, Jerusalem, Israel, 2Tel Aviv University, Tel Aviv, Israel, 3Max-Planck-Institute for Biochemistry, Martinsried, Germany

Nonsense mediated mRNA decay (NMD) is a surveillance mechanism which detects and selectively degrades transcripts carrying premature termination codons (PTCs), therefore preventing accumulation of truncated proteins that might be nonfunctional or deleterious. Variability in NMD efficiency is found among different cell lines, tissues and among individuals, indicating that the efficiency of NMD is an inherent character of cells. The NMD is not only a quality control mechanism but also has a crucial post-transcriptional regulatory role that affects the expression of broad classes of physiologic transcripts that encode functional proteins. Different cellular and environmental conditions can perturb the NMD mechanism (hypoxia, UV irradiation and amino acid starvation). NMD substrates that encode membrane or secreted proteins are translated and processed in the endoplasmic reticulum (ER). Under conditions that perturb the NMD mechanism these truncated proteins cannot be folded correctly and accumulate in the ER, activating the Unfolded Protein Response (UPR). The UPR senses the misfolded protein stress and alters the transcriptional and translational cellular programs to enable the cells to cope with the stressful condition and resolve the protein-folding defect. We hypothesize that: 1. Inefficient NMD leads to accumulation of truncated proteins in the ER and activates the UPR. 2. The UPR further inhibits the NMD mechanism (which is translational dependent). Hence, the interplay between these two homeostasis mechanisms is expected to regulate the level of many important physiologic transcripts and transcripts carrying stop mutations leading to genetic diseases. Results: NMD down-regulation by siRNA directed against hUPF1 induced the UPR, as indicated by increased levels of UPR markers (spliced XBP, peIF2α). Importantly, UPR activation by DTT inhibited the NMD as revealed by increased levels of physiological NMD substrates. We further show that NMD inhibition together with UPR activation enhanced both the ER stress and NMD inhibition compared to each treatment alone, indicating a novel interplay between these two homeostatic mechanisms. In recent years an effort is being made to develop treatment for PTCs causing human genetic diseases which promote translational read-through of the PTC to generate full-length functional proteins. We have previously shown that the NMD efficiency regulates the response to read-through. Therefore, the novel interplay between NMD and UPR mechanisms might affect the response of patients to read-through treatments. As a model system we have studied two sisters homozygous for a PTC in the CFTR gene, who differ in their CF disease severity, response to the read-through treatment and CFTR transcript level. Using SILAC we compared the entire proteome between cells derived from these sister patients. Out of 6000 analyzed proteins, 440 showed a significant different level between the cell lines. At least 44 were ER proteins functioning in the UPR. Strikingly, a ~2 fold higher level of each of these proteins was found in the responding sister, indicating a higher UPR activation. Conclusion: these results support our hypothesis and highlight the importance of UPR and NMD positive feedback loop under normal conditions and in human diseases.

Poster Session 3: RNAs in Diseases

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

621 Novel Human Variation in MicroRNAs associated with Disease, Biomarkers, and Drug Metabolism.

Renata Rawlings, Sarah Tishkoff University of Pennsylvania MicroRNAs (miRNA) are evolutionarily conserved regulators of gene expression, which have a significant role in human development and disease. Under and over-expression of specific miRNA have been associated with a wide variety of diseases and disorders including but not limited to leukemia, myopathy, melanoma, lupus, Alzheimer’s, neurodegenerative diseases and most forms of cancer. Additionally, there are several clinical trials pursuing the utility of miRNAs as biomarkers, gene therapy agents, and effectors of drug metabolism. As miRNA progress toward therapeutics, population specific variation in miRNA sequences could answer questions concerning viability of drug targeting, and variability of drug metabolism among different populations. Here we analysis a worldwide panel of participants representing 15 distinct populations from around the world, with a focus on African populations, to assess novel human variation in miRNAs of interest.

622

Serum non-coding RNAs as Biomarkers for Osteoarthritis Progression After ACL Injury

Maozhou Yang1, Liang Zhang1, Mark Hurtig2, Lawrence White3, Paul Marks3, Gary Gibson1 1 Henry Ford Hospital, Detroit, MI, USA, 2University of Guelph, Guelph, ON, Canada, 3University of Toronto, Toronto, ON, Canada Osteoarthritis (OA) is a progressive joint disorder associated with pain and disability, compromising everyday life for approximately 20 million Americans. The diagnosis of OA is based on clinical and radiographic changes, which occur generally late during disease progression and have poor sensitivity for monitoring disease progression. Currently there are no simply measured biomarkers that provide an early diagnosis of OA or disease progression. This study explored the potential of serum non-coding RNAs as biomarkers for cartilage damage and OA progression associated with anterior cruciate ligament (ACL) injury. Serum was obtained from 80 patients one year after surgery for ACL injury and 60 normal donors without overt skeletal injury. Serum RNA was isolated and small RNAs analyzed by target-specific TaqMan arrays and quantitative RT-PCR. Cartilage damage was assessed by semi-quantitative MRI with detailed WORMS scoring. Initial miRNA array analysis did not show any significant change in the miRNA profile. However, an increase of a serum snoRNA, U48, in patients post ACL injury compared to that of normal donors was observed. Independent quantitative RT-PCR analysis of snoRNAs in serum from all patients and normal donors showed a strong association between the serum level of U48 and an additional snoRNA, U38, with cartilage damage in ACL injury patients and clear distinction of ACL injury patients and normal donors. This study suggests the analysis of serum snoRNAs is able to indicate the presence cartilage damage after ACL injury and thus a potential biomarker of post-ACL injury osteoarthritis.

Poster Session 3: RNAs in Diseases

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

623

Identification of Cellular dsRNA Required for PKR Activation during Metabolic Stress

624

DRAGins: Drug Binding Aptamers For Growing Intracellular Numbers

Osama Youssef2, Takahisa Nakamura1, Gökhan Hotamisligil1, Brenda Bass2 1 Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA, 2Department of Biochemistry, University of Utah, Salt Lake City, Utah PKR (Protein Kinase RNA activated) is a member of the stress-response kinase family. It inhibits protein synthesis by phosphorylating the eukaryotic initiation factor, eIF2α. PKR contains a regulatory dsRNA-binding domain at its N-terminus and a catalytic kinase domain at its C-terminus. PKR catalytic activity is stimulated under conditions of metabolic stress in mice (1). Under these conditions, PKR catalytic activity depends not only on the kinase domain but also on the dsRNA-binding domain. However, the cellular dsRNA responsible for the activation of PKR during metabolic stress in mouse is unknown. We are investigating this question using Mouse Embryonic Fibroblast (MEF) cells expressing wildtype PKR (PKRwt), PKR with a point mutation in the kinase domain (PKR-KDmt) or PKR with a point mutation in each dsRNAbinding motif (PKR-RDmt). Cells were incubated in the presence or absence of palmitic acid (PA), to mimic a high-fat or regular diet, respectively, followed by immunoprecipitation of PKR. PKR immunopurified RNAs were subjected to high-throughput sequencing. RNA immunopurified with PKR-KDmt and PKR-RMmt after PA treatment were used as controls for non-specific binding. We found that over 500 exons and 2000 introns were upregulated by 2 or more fold in PKRwt samples after PA treatment compared to the untreated sample (Qvalue less than or equal 0.001). Interestingly, 11% of the enriched exons encode non-coding RNAs. Ongoing research is aimed at analyzing biological replicates and validation of the most promising candidates. 1) Double-stranded RNA-dependent Protein Kinase Links Pathogen Sensing with Stress and Metabolic Homeostasis. Nakamura T, Furuhashi M, Li P, Cao H, Tuncman G, Sonenberg N, Gorgun CZ, Hotamisligil GS. Cell. 2010 Feb 5; 140 (3): 338-348.

Muslum Ilgu1, Supipi Auwardt1, Robert Feldges2, Khalid Boushaba1, Howard Levin1, Marit Nilsen-Hamilton1 1 Iowa State University,Ames,Iowa,USA, 2Florida Gulf Coast University,Fort Myers,Florida,USA A challenge in cancer treatment is to maintain high enough intracellular drug concentration to kill the cancer cells. This is because cancer cells upregulate the expression of multi-drug transporters. This phenomenon leads to the need for higher extracellular concentrations of drugs to compensate for the drug pumps for effective cell killing. Modern drug synthesis research has addressed this issue by many means, mainly based on “push mechanisms”. However, the extent to which the extracellular drug concentration and the cell permeability to the drug can be increased is limited. Therefore, we tested a different approach based on a “pull mechanism” by expressing molecules that can bind drugs inside the cells and increase their intracellular concentrations. These expressed molecules are single-stranded RNAs called aptamers selected for tight and specific binding to their drug targets. By mathematical modeling we predicted that the presence of mobile aptamers inside the cells will increase the intracellular concentration of the target drug compared to in the absence of aptamers (Levine, H. A.; Boushaba, K.; Nilsen-Hamilton, M. Math Biosci , 2009, 220, 131). In this context, the intracellularly expressed aptamers are referred to as DRAGins(Drug Binding Aptamers for Growing intracellular numbers). To test this hypothesis experimentally, we used bacteria that are sensitive to killing by aminoglycosides (the drugs) and aminoglycoside aptamers to increase cellular sensitivity to the drugs. E. coli BL21 strain was transformed with aptamer-expressing plasmids and incubated with different concentrations of aminoglycosides to determine the effect of aptamer expression on efficacy of cell killing. In this study, we were able to lower the IC50 values by 2 to 5 fold with several aminoglycoside-binding aptamers for several aminoglycoside antibiotics. The results of these studies suggest that DRAGins might be a useful means of increasing the efficacy of drugs.

Poster Session 3: RNAs in Diseases & Therapeutic RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

625 Robust Suppression of HIV Replication by Intracellularly-expressed RNA Aptamers is Independent of Ribozyme Processing

Margaret Lange1, Angela Whatley1, Tarun Sharma2, Marc Johnson1, Donald Burke1 1 University of Missouri, 2Indian Institute of Technology, Roorkee, India RNA aptamers that bind HIV-1 RT inhibit viral replication, making them attractive candidates for novel therapies and for revealing new insights into viral pathogenesis. However, it is not yet well understood how aptamers integrate into cellular RNA pathways or how the aptamer-expression context governs accumulation and antiviral bioactivity. Previous work described a CMV promoter-driven cassette (RNA Pol II) in which two minimal-core hammerhead ribozymes (mcHHRz) were introduced to “release” the aptamer from the transcript and to facilitate folding. However, using this construct, we observed only weak viral suppression in single-cycle infectivity assays with pseudotyped HIV. Ribozyme cleavage products are generally thought to degrade rapidly, potentially decreasing aptamer availability in this design. This work evaluates the roles of flanking ribozymes. The mcHHRz were substituted with either inactivated or highly-active, extended hammerhead ribozymes (eHHRz) that include stabilizing tertiary interactions. Inhibition of pseudotyped virus was much stronger for each of the eHHRz constructs than for the mcHHRz, and it correlated with RNA accumulation in viral, total, cytoplasmic, and nuclear RNA fractions (from RT-qPCR). Surprisingly, viral suppression and RNA accumulation were independent of cleavage in vitro. These results were confirmed by FISH. Clonal, aptamer-expressing, stable cell lines were constructed to monitor replication of infectious virus in serial passage assays. The eHHRz and mcHHRz constructs all strongly suppressed infectious virus at low MOI, but the eHHRz constructs inhibited more strongly at higher MOI. Our results suggest: (1) that ribozyme-mediated cleavage is unnecessary for inhibition of HIV by anti-RT aptamers, (2) that tertiary stabilization of flanking RNA structures greatly increases RNA accumulation, and (3) that increased accumulation is responsible for the observed improvements in antiviral bioactivity, and (4) that the antiviral bioactivity by intracellularly expressed RNA aptamers is greatest at low viral loads. We speculate that the bioactive form of the aptamer is likely one of the uncleaved or partially-cleaved products, and that tertiary stabilization increases transcript stability by reducing exonuclease degradation.

626 Therapeutic Application of Antisense Compounds to Target Pathogenic RTKs for Cancer Therapy

Lee Spraggon, Sandra Vorlova, Gina Rocca, Luca Cartegni Memorial Sloan Kettering Cancer Center, New York, New York, USA Activation of the receptor tyrosine kinase (RTK) signaling pathways represents a key aspect of tumorigenesis in a broad range of human cancers. The targeting of pathogenic RTKs in cancer has become an attractive therapeutic approach for cancer therapy, with the generation of molecular inhibitors including small molecular inhibitors and antibodies. However, as is common in cancer therapy, resistance to the therapies emerges over time. We recently identified a number of novel secreted soluble decoy RTKs isoforms (sdRTKs) generated by a U1-snRNPdependent alternative intronic polyadenylation mechanism. We also reported the development of an antisense-based method to effectively activate the expression of these sd RTKs. Therapeutically, the generation of endogenous dominant negative variants of RTKs which can specifically effect the signaling of pathogenic RTKs provides a novel cancer therapy approach. We here present data using this approach to target the epidermal growth factor receptor (EGFR). Aberrant activity or expression of EGFR has been identified as an important factor inthe biology of many human epithelial malignancies, including head and neck squamous-cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). Employing EGFRspecific antisense compounds designed to activate intronic polyA sites, this approach generates truncated EGFR isoforms, which contain the extracellular ligand-binding domain but lack the intracellular signaling domain. We used these compounds to inhibit the EGFR pathway in clinically relevant cancer models, providing the basis for a novel approach to target and inhibit oncogenic EGFR signaling in a variety of cancers.

Poster Session 3: Therapeutic RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

627 Nanoparticle Delivery of MicroRNA Inhibition to Treat OncomiR-Addicted Lymphoma Tumors

Christopher Cheng, Imran Babar, Mark Saltzman, Frank Slack Yale University, New Haven, CT, USA The purpose of this study was to develop a therapeutic nanoparticle-based system that delivers anti-miRs to antagonize oncogenic microRNAs (miRNAs), or oncomiRs. Ubiquitously expressed in different cells and tissues, miRNAs have critical roles in various diseases, including cancer. Previously, we have shown that lymphomas can become addicted to specific oncomiRs such that cancer is induced by overexpression of the miRNA, and withdrawal of the miRNA leads to abrogation or even reversal of the malignant phenotype. Current strategies for inhibiting the function of miRNAs are limited by toxicity and target specificity concerns. Encapsulating anti-miRs into polymer nanoparticles (NP) represents a novel delivery approach toward targeting oncomiRs in tumors. Furthermore, these NP can be enhanced by coating their surface with PEG and the cell-penetrating peptides to improve delivery and tumor cell uptake. In order to evaluate the ability of these NP to inhibit miRNA in addicted lymphoma tumors, we developed a miRNA-induction model in which miR-155 overexpression leads to the development of disseminated lymphoma comprising neoplastic pre-B cells. These aggressive lymphocytes form solid tumors that are acutely sensitive to miR-155 inhibition. NP with a mean diameter between 100-200 nm were densely loaded with anti-miR-155 peptide nucleic acid (~500 pmoles/mg), and coated with approximately 2000 penetratin molecules per NP. Uptake of penetratin-enhanced NP by hard-to-transfect pre-B cells and tumor tissue was verified by confocal microscopy and flow cytometry. IVIS was used to monitor passive targeting of the PEGylated NP to hyper-vascularized pre-B cell tumors. Attenuation of tumor growth resulted from both systemic and local treatment with anti-miR-155-loaded NP. Relative to scramble control, systemic treatment reduced growth by 50% over 5 days, while local treatment delayed growth by 80% after one week. qPCR and a luciferase reporter system were used to verify the specificity and degree of miR-155 inhibition. Immunoblot of the pro-apoptotic miR-155 target, SHIP1, and TUNEL staining revealed that the anti-tumor mechanism was in part, due to augmentation of apoptosis. Currently, we are investigating the role of miR-155 in tumorigenesis and addiction using high throughput RNA expression analysis (RNA-seq) of this inducible tumor model. This work introduces a robust and versatile nanotherapeutic platform that exploits miRNAs as potent tools for the treatment of cancer.

628 The Potential of Nanoparticles Composed of RNA-Bolaamphiphile Complexes as a Therapeutic siRNA Delivery Vehicle

Taejin Kim, Kirill Afonin, Eliahu Heldman, Mathias Viard, Selene Sparks, Robert Blumenthal, Bruce Shapiro National Cancer Institute, Frederick, MD, USA Specific siRNA that are designed to silence oncogenic pathways can be used for cancer therapy. However, naked siRNAs have short half-lives in the blood stream and have difficulties in crossing biological membranes due to their negative charges. These biological barriers can be overcome by using siRNA-bolaamphiphile complexes. Bolaamphiphiles have two positively charged hydrophilic head groups connected by a hydrophobic chain and they have a relatively low toxicity and long persistence in the blood stream. In this study, we investigated the interactions between bolaamphiphiles and siRNA and correlated these interactions with gene silencing efficacy. Two different types of bolaamphiphiles have been studied, GLH19 and GLH20, each having different head group structures. Our explicit solvent molecular dynamics (MD) simulations showed that these bolaamphiphiles associate with RNA due to (1) electrostatic interactions, (2) hydrogen bond interactions and (3) hydrophobic interactions. MD simulation results also showed that the binding energy between RNA and GLH20 was stronger than that of GLH19 at 0M salt concentration. However, at 0.15M NaCl, GLH19 showed stronger binding energy than GLH20. Studies using agarose gel electrophoresis indicated weaker interactions of GLH20 with siRNA as compared to the interaction between siRNA and GLH19. In the presence of Triton X100, siRNA was released from GLH20 complexes, while the siRNA-GLH19 complex remained intact. In silencing experiments, the siRNA-GLH20 complex was more effective than siRNA-GLH19 complex. These MD simulations and experimental results suggest that siRNA is released from the siRNA-GLH20 complex within the cells more easily than from the siRNAGLH19 complex, enabling more effective gene silencing when the siRNA-GLH20 complex is used for the transfection. Therefore, we propose that siRNA-GLH20 complexes are strong candidates as vehicles for therapeutic purposes.

Poster Session 3: Therapeutic RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

629 Using Unlocked Nucleic Acid (UNA) Substitutions to Alter Strand Selection By the RNA Induced Silencing Complex

Nicholas Snead1, Julie Escamilla-Powers2, Sabrina Shore2, Natasha Paul2, John Rossi1, Anton McCaffrey2 Beckman Research Institute of the City of Hope, Duarte, CA, USA., 2Trilink Biotechnologies, San Diego, CA, USA Entry of the desired (antisense) strand into the RNA Induced Silencing Complex (RISC) is a critical determinant of small interfering RNA (siRNA) potency and specificity. For most synthetic siRNA duplexes, both strands enter RISC to some degree. Undesired RISC sense strand entry can cause off-target silencing of genes with sense strand homology. Offtarget effects can result from strand cleavage or microRNA-like interactions between the seed region of the incorporated strand and seed matches in off-target mRNAs. Here, we utilized Unlocked Nucleic Acid (UNA) substitutions to alter strand selection to favor incorporation of the desired antisense strand. UNAs are non-nucleotide analogs in which the C2’-C3’ bond of the ribose ring is absent. Previous studies have shown that strategic UNA substitutions at the 5’ end of the passenger strand and at position seven of the guide strand can reduce off-target effects by >90% (NAR (2011) 39, 1823). Previously, we conducted a detailed examination of the IC50 for the antisense and sense strands of a series of siRNAs targeting hnRNPH (NAR (2010) 39, 1510). We found that shifting an siRNA by a single nucleotide dramatically altered the relative potency of the antisense and sense strand as measured by luciferase target reporters. Here, we tested whether UNA substitution could “make a bad siRNA good.” As a test case, we chose hnRNPH siRNA H5, for which strand selection is highly biased toward undesired sense strand incorporation (IC50 of 71.6 pM for the sense strand and 9.75 pM for the antisense strand). 5’ UNA substitution of the sense strand greatly reduced sense strand silencing while increasing on-target silencing by the antisense strand; in effect, converting a non-functional siRNA to a functional one. Thus, UNA substitution can be used drive incorporation of the antisense strand into RISC for improved on-target silencing and presumably reduced off-target effects by the sense strand. When targeting highly conserved viral regions or single nucleotide polymorphisms, the potential sequence space for siRNA selection is highly constrained. In these cases, UNA modification may be a useful tool to expand the selection of potential siRNA sequences. In summary, UNAs are a non-nucleotide modification that is useful for manipulating RISC strand selection and reducing the potential for off-target effects. 1

630

Gene Expression From Pseudouridine and 5-methylcytidine Modified Messenger RNAs

Jiehua Zhou1, Julie Escamilla-Powers2, Anton P. McCaffrey2, John Rossi1 1 Beckman Research Institute of the City of Hope, Duarte, CA USA, 2Trilink Biotechnologies, San Diego, CA, USA Recently, there has been significant interest in the use of messenger RNA (mRNA) based expression systems for gene therapy applications as well as for the generation and manipulation of stem cells. Several groups have shown that mRNAs are attractive vehicles for therapeutic gene expression in mammals (Kormann et al. Nat. Biotechnol (2011) 29, 154; Kariko et al. Molecular Therapy (2012) Epub ahead of print). Additionally, Warren et al. demonstrated highly efficient induced pluripotent stem cell (iPSCs) generation by transfection of mRNAs encoding reprogramming factors (Warren et al. Cell Stem Cell (2010) 7, 618). Because they are RNAs, mRNAs do not have any risk of insertional mutagenesis and subsequent oncogenesis. For this reason, the authors suggested that iPSCs generated in this manner should be safer than iPSCs derived by plasmid transfection or viral transduction A key insight for the development of mRNA expression system was the recognition that mRNAs induce innate immune responses in transfected cells. Kariko et al. showed that substitution of uridine and cytidine residues with pseudouridine and 5-methlycytidine dramatically reduced innate immune recognition of mRNA (Kariko et al. Molecular Therapy (2008) 16, 1833). They also showed that pseudouridine modified RNA was translated more efficiently and had increased nuclease resistance. These studies highlight the importance of development of stable, non immunogenic mRNA to support these applications. In this current study, we synthesized capped pseudouridine and 5-methlycytidine modified eGFP mRNAs at milligram scales. Fluorescence activated cell sorting (FACS) demonstrated >95% transfection in HEK-293 cells. Expression in human CEM T-cells and CD34+ hematopoietic stem cells was also examined. Transfected mRNAs showed a surprising long duration of expression (up to 8 days post-transfection). Gene therapy applications of mRNA will require scalable purification methods that are able to produce mRNAs at gram scales. Recently, it was shown that purification of mRNA by high-performance liquid chromatography (HPLC) dramatically reduced innate immune responses relative to unpurified mRNA (Kariko et al. Nucleic Acids Research 39, e142). Here we compare mRNA purified by classical silica membrane chromatography to HPLC purified materials. Poster Session 3: Therapeutic RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

631 Therapeutic Application of RNA based Compounds to Target Pathogenic RTKs for Cancer Therapy

Lee Spraggon1, Sandra Vorlova1, Gina Rocco1, Luca Cartegni1,2 Molecular Pharmacology and Chemistry, Memorial Sloan Kettering Cancer Center, New York, (NY), USA, 2 Experimental Therapeutics Center, Memorial Sloan Kettering Cancer Center, New York, (NY), USA Activation of the receptor tyrosine kinase (RTK) signaling pathways represents a key aspect of tumorigenesis in a broad range of human cancers. The targeting of pathogenic RTKs in cancer has become an attractive therapeutic approach for cancer therapy, with the generation of molecular inhibitors including small molecular inhibitors and antibodies. However, as is common in cancer therapy, resistance to the therapies emerges over time. We recently identified a number of novel secreted soluble decoy RTKs isoforms (sdRTKs) generated by a U1-snRNPdependent alternative intronic polyadenylation mechanism. We also reported the development of an antisense-based method to effectively activate the expression of these sdRTKs. Therapeutically, the generation of endogenous dominant negative variants of RTKs which can specifically effect the signaling of pathogenic RTKs provides a novel cancer therapy approach. We here present data using this approach to target the epidermal growth factor receptor (EGFR). Aberrant activity or expression of EGFR has been identified as an important factor in the biology of many human epithelial malignancies, including head and neck squamous-cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). Employing EGFR specific antisense compounds designed to activate intronic polyA sites, this approach generates truncated EGFR isoforms, which contain the extracellular ligand-binding domain but lack the intracellular signaling domain. We used these compounds to inhibit the EGFR pathway in clinically relevant cancer models, providing the basis for a novel approach to target and inhibit oncogenic EGFR signaling in a variety of cancers.

1

632

Effective RNAi Therapeutics to Treat Chronic Hepatitis B Virus Infection

Christine Wooddell1, Matthias John2, Markus Hossbach2,3, Jochen Deckert2,3, Philippe Hadwiger2,3, Holly Hamilton1, Qili Chu1, Darren Wakefield1, Daniel Sheik1, Jason Klein1, Kerstin Jahn-Hofmann2, Alan McLachlan4, Hans-Peter Vornlocher2,3, David Rozema1, David Lewis1 1 Arrowhead Research Corporation, Arrowhead Madison, Madison, WI, USA, 2Roche Kulmbach, Kulmbach, Germany, 3Axolabs, Kulmbach, Germany, 4Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA Hepatitis B virus infection is a global health threat that results in 1,000,000 deaths annually from hepatocellular carcinoma, cirrhosis or liver failure. None of the current therapeutics reduce viral antigen expression. However, RNA interference (RNAi) has the potential to knock down expression of viral RNA, the pregenomic RNA and the replicative intermediates that arise from it, and also the viral proteins that result in disease and hinder the immune system’s ability to eradicate the virus. We designed 145 small interfering RNAs (siRNAs) that target conserved sequences in HBV and screened them for efficacy in cultured cells. The most potent of these were formulated in our siRNA delivery vehicle named Dynamic PolyConjugates (DPCs) and injected intravenously into mouse models of HBV infection. Single DPC injections in a replication-competent, transiently transgenic HBV mouse model resulted in greatly reduced levels of serum HBsAg, serum HBV DNA, and HBV RNA and DNA in liver. Serum HBeAg and HBV RNA and DNA in liver were also dramatically reduced in a transgenic mouse model of chronic HBV infection. Four biweekly injections of anti-HBV siRNA DPCs in mice carrying a hepatocyte-specific reporter gene fused to HBV sequences resulted in a 3-4 log reduction in gene expression over 2 months without changes in clinical chemistries. Safety and tolerability studies using rats and nonhuman primates revealed that DPCs have a wide safety margin. Formulation of potent anti-HBV siRNAs in DPCs allowed highly effective reduction of viral protein production and viral load in mouse models of HBV infection. DPCs were well tolerated in all species tested, which will enable study of anti-HBV siRNA DPCs in patients with chronic HBV infection.

Poster Session 3: Therapeutic RNAs

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

701

Molecular cloning and gene expresión of Fibrillarin in Phaseolus vulgaris

Jesus Cortes, Josefina Huerta, A. Fco. Cabral, E. E. De Leon Universidad Autonoma de Zacatecas, Zacatecas, Zacatecas, Mexico. (Fb ) is a major nucleolar protein that plays an essential role in ribosomal biogenesis , including pre-rRNA processing and methylation at position 2’-O-ribose rRNA , in association with snoRNAs of box C/D U3, U8, U13 forms, the nucleolar Ribonucleoproteins (snoRNPs). Fibrillarin is an evolutionarily conserved protein in eukaryotes, genes encoding cDNAs Fb have been cloned and sequenced in different organisms. The first plant genes Fb were cloned from Arabidopsis thaliana. The amino acid sequence of these cDNAs shows several conserved domains, including mainly the central domain binding RNAs. This protein is part of a large family with biochemical characteristics with a high degree of homology and a molecular weight ranging from 33 to 38 kDa , all seem to have key role in ribosomal biogenesis . Was isolated , amplified and sequenced the cDNA encoding fibrillarin in Phaseolus vulgaris that has not yet been characterized. Fb expression was compared with the cDNA from Arabidopsis thaliana AtFbr 1 of 1220 bp which encodes a protein of 310 aa. Expression of fibrillarin was monitored in embryos of Phaseolus vulgaris in early stages of germination by Western blot. Subsequently carried out the extraction of total RNA, mRNA was amplified by RT -PCR fibrillarin using oligonucleotides specific for the Arabidopsis thaliana AtFbr 1 :. The PCR product was purified and inserted into the cloning vector pGEM -T, electroporation, bacterial transformation in E. coli, plasmid DNA extraction and sequencing is being carried out .

702 Single-RNA FISH reveals that nonsense-mediated mRNA decay in human cells occurs in the cytoplasm near the nucleus

Tatjana Trček Pulisic1,2, Hanae Sato2,3, Robert H. Singer2, Lynne E. Maquat3 1 HHMI, The Skirball Institute of Biomolecular Medicine, NYU, NYC (NY), USA, 2Albert Einstein College of Medicine, Bronx (NY), USA, 3Center for RNA Biology, University of Rochester, Rochester, NY, 14642, USA Nonsense-mediated mRNA decay (NMD) is a quality-control mechanism responsible for “surveying” mRNAs during translation and degrading those that harbor a premature termination codon (PTC). Currently the intracellular spatial location of NMD and the kinetics of its decay step in mammalian cells are under debate. To address these issues, we used single-RNA FISH and measured the NMD of PTC-containing β-globin mRNA in intact single cells after the induction of β-globin transcription. This approach preserves spatial and temporal information of the NMD process, both of which would be lost in an ensemble study. We determined that decay of the majority of PTC-containing mRNA occurs soon after its export into the cytoplasm with a half-life of less than one minute; the remainder is degraded with a half-life of greater than 12 hours, similar to the half-life of normal PTC-free β-globin mRNA, indicating that it had evaded NMD. The rapid, PTC-dependent decay of β-globin mRNA agrees well with the time it would take translationally active ribosomes to reach the PTC. Finally, we show that NMD takes place within 430 nm of the nuclear envelope, suggesting that it could involve the nuclear pore complex. Importantly, NMD does not occur within the nucleoplasm, thus countering the long-debated idea of nuclear decay. We provide a spatial and temporal model for the biphasic decay of NMD targets. This work was supported by a Fellowship from the Japan Society for the Promotion of Science (H.S.), the NIH R01 GM84364 (R.H.S.) and NIH R01GM59614 (L.E.M).

Unassigned Posters: Ribonucleoproteins Structure, Functions and Biosinthesis & RNA Turnover

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

703

A Direct Role for Quaking in Muscle-Specific Alternative Splicing

W. Samuel Fagg, Megan Hall, Roland Nagel, Melissa Cline, Lily Shuie, Manuel Ares UC-Santa Cruz, Santa Cruz, CA, USA Quaking (Qk) is a member of the highly conserved Signal Transduction and Activator of RNAmetabolism (STAR) family that integrates cues from cellular signals with post-transcriptional gene regulation. Qk has been demonstrated to control RNA stabilization and translation by binding to ACUAA-containing sequences in the3’UTRs of target mRNAs. In oligodendrocytes, Qk appears to indirectly influence splicing by controlling expression of hnRNPA1 (1, 2). Although ACUAA sequences also occur near regulated mammalian exons, a general role for Qk directly influencing pre-mRNA splicing has not been demonstrated. In a study of tissue-specific alternative splicing in mouse, we recognized an enrichment of ACUAA containing motifs downstream of exons activated in muscle (3). To follow up on this finding, we used mouse C2C12 myoblasts that can differentiate into muscle fiber-like myotubes in vitro and examined alternative splicing using microarrays. This study (unpublished) and another (4) found that, as in adult muscle, many exons activated during myoblast to myotube differentiation have downstream ACUAA motifs. Once such event, exon 9 of Capzb (mm9 chr4:138,844,776-138,844,888), an actin filament capping protein, was selected for further study. Here we show that Qk controls the inclusion of Capzb, exon 9, as well as several hundred other myoblast exons, through ACUAA motifs near the adjacent exons. Several lines of evidence lead to this conclusion: First, mutation of the ACUAA motifs downstream of Capzb exon 9 in the context of a beta-globin based reporter construct results in skipping of this exon. Second, RNA-affinity chromatography coupled to Mud-PIT mass spectrometry using sequences downstream of the Capzb exon 9 results in the enrichment of Qk protein from C2C12 nuclear extract, whereas affinity chromatography-Mud-PIT using a similar sequence lacking ACUAA motifs does not. Third, RNAi-mediated Qk knockdown in myoblasts followed by genome-wide splicing sensitive microarray analysis reveals hundreds of exons whose inclusion is increased (having ACUAA motifs enriched upstream) upon Qk depletion. Although splicing is greatly affected by Qk knockdown in myoblasts, very few if any mRNA levels change, suggesting that the primary effect of Qk loss in myoblasts is on splicing rather than mRNA stability. Fourth, by transfection studies using tagged-Qk, we show that Qk activates Capzb exon 9 inclusion, in a fashion dependent on the downstream ACUAA motifs. Furthermore, Ptb represses Capzb inclusion, in opposition to Qk. During differentiation of myoblasts into myotubes, Ptb and nPtb levels are down regulated (5), as Qk levels increase (this study). We propose a model whereby Capzb exon 9 and other exons are activated during muscle differentiation combinatorially through both the relief of Ptb repression and an augmentation of Qk activation.

704 Saccharomyces cerevisiae General Splicing Factor Required for the Stable U2 snRNP Binding to pre-RNAs

Patricia Coltri, Carla Oliveira University of Sao Paulo, Sao Paulo, SP, Brazil Pre-RNA splicing is an essential process for the control of gene expression. Specific conserved sequences in premature transcripts are important to recruit the spliceosome machinery. The Saccharomyces cerevisiae catalytic spliceosome is composed of about 60 proteins and 5 snRNAs (U1, U2, U4/U6 and U5). Among these proteins, there are core components and regulatory factors, which might stabilize or facilitate splicing of specific substrates. Assembly of a catalytic complex depends on the dynamic of interactions between these proteins and RNAs. Cwc24p is an essential S. cerevisiae protein, originally identified as a component of the NTC complex, and later shown to affect splicing in vivo. In this work, we show that Cwc24p also affects splicing in vitro. We show that Cwc24p is important for the U2 snRNP binding to pre-RNAs, co-migrates with spliceosomes, and that it interacts with Brr2p. Additionally, we show that Cwc24p is important for the stable binding of Prp19p to the spliceosome. We propose a model in which Cwc24p is required for U2 association with pre-RNAs, and therefore, especially important for splicing of RNAs containing non-consensus branchpoint sequences.

Unassigned Posters: RNA-Protein Interactions & Splicing Mechanisms

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

705

Use of 19F-NMR to measure RNA structural dynamics

706

Direct Interaction between RanBP2/Nup358 and ALREX-Promoting SSCR RNA Elements

Caijie Zhao, Matthew Devany, Nancy Greenbaum CUNY-Hunter College, New York, (NY), USA During biological activity, functional RNA molecules may adopt alternative secondary structures and/or may undergo structural interconversion; thus, there may be more than one conformation present at a given time or under certain conditions. This structural heterogeneity and interconversion between alternative structures may be critical for function as it can affect both the kinetics and the thermodynamics of the processes. Therefore, it is important to probe the various conformations and their interconversion in order to provide insight into the mechanism by which RNA molecules mediate biochemical activity. This research focuses on characterization of the secondary structure fold and dynamics of RNA molecules that can adopt two alternative folds. Toward this goal, a novel 19F NMR technique, utilizing the fact that the fluoro nucleus in a 19F-labeled pyrimidine residue has different chemical shifts whether it is located within a singleor double-stranded RNA environment, is developed and tested on a model RNA stem loop capable of interconverting between two conformations. Data obtained from NOESY experiments acquired at different mixing times suggest that the interconversion between the two observed folds in the model molecule is in the range of 400 milliseconds. We have extended this approach to a biologically important RNA complex, the U2-U6 small nuclear (sn)RNA complex from the human spliceosome; the timescale of interconversion between two alternative folds is approximately 500 milliseconds. This dynamic information will help us understand the process of spliceosome assembly and the relationship between structure and function.

Hui Zhang1, Serge Gueroussov1, Kohila Mahadevan2, Can Cenik3, Frederick Roth3, Alexander Palazzo2, Alexander Palazzo2 1 University of Toronto, Toronto, ON Canada, 2University of Toronto, Toronto, ON Canada, 3Harvard Medical school, Boston, USA In higher eukaryotes, most mRNAs that encode secreted or membrane-bound proteins contain elements that promote an alternative mRNA nuclear export (ALREX) pathway. These elements are found at the 5’end of the open reading frame in the signal sequence coding region (SSCR), and are present in approximately 15% of all human protein-coding genes. Previously we found that these elements have a second function, they potentiate the efficient translation of the mRNA. Here we report that these RNA elements interact directly, and likely co-evolved with, the zinc finger repeats of RanBP2/Nup358, which is present on the cytoplasmic side of the nuclear pore. It was previously reported that these zinc fingers bind to the small GTPase Ran, and we now demonstrate that this interaction disrupts RNA-binding. Finally we demonstrate that RanBP2/Nup358 is required for the efficient global synthesis of proteins targeted to the ER. Thus upon the completion of export, mRNAs containing ALREX-elements likely interact with RanBP2/Nup358, and this step is required for the eventual translation of these mRNAs in the cytoplasm. ALREX-elements thus act as nucleotide platforms to coordinate various steps of post-transcriptional regulation for the majority of mRNAs that encode secreted proteins.

Unassigned Posters: RNA structure and folding & RNA Transport and Localization

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

707 Characterization of guanine riboswitches for the development of selective analog inhibitors

Anne-Marie Lamontagne, Jerome Mulhbacher, Daniel Lafontaine Universite de Sherbrooke, Sherbrooke (Quebec), Canada Riboswitches are mostly found in untranslated regions of bacterial mRNA and are involved in gene expression regulation. They are composed of two domains: an aptamer and an expression platform. The aptamer is involved in ligand recognition and the expression platform controls gene expression by adopting two mutually exclusive conformations depending on ligand binding. Riboswitches represent a novel solution to multiple drug resistance (MDR) since they can be considered as antimicrobial targets when agonistic ligands are employed to knockdown the expression of the associated gene(s).  Guanine riboswitches are involved in the regulation of transport and biosynthesis of purine metabolites, which are critical for the nucleotides cellular pool. Upon guanine binding, these riboswitches stabilize a 5’-untranslated mRNA structure that causes transcription attenuation of the downstream open reading frame. In principle, any agonistic compound targeting a guanine riboswitch could cause gene repression even when the cell is starved for guanine. Antibiotics binding to riboswitches provide novel antimicrobial compounds that can be rationally designed from riboswitch crystal structures. We have found that a pyrimidine compound (PC1) binding guanine riboswitches showed bactericidal activity against a subgroup of bacterial species including well-known nosocomial pathogens (1). This selective bacterial killing was only achieved when guaA, a gene coding for a GMP synthetase, is under the control of the riboswitch. Among the bacterial strains tested, several clinical strains exhibiting multiple drug resistance were inhibited suggesting that PC1 targets a different metabolic pathway. Finally, the structure-activity relationship of various guanine riboswitches found in infectious organisms was characterized as a way to determine what might constitute good antibiotic targets. Mulbacher et al, PLoS Pathogens, 2010.

708 Kramers Rate Theory Describes the Viscosity Dependent Folding Kinetics of the TetraloopReceptor Motif

Nicholas Dupuis, David Nesbitt JILA, University of Colorado, Boulder, CO, USA It is widely known that there exists a strong relationship between the solvent, including co-solutes (e.g. cations), and RNA that cooperatively defines the kinetics of tertiary structure formation. Viscosity, a solvent property that is coupled to dynamics, may be critical in RNA folding due to the propensity for populating kinetically trapped species on a rough free energy landscape. Viscous effects are especially relevant to understanding RNA folding in the cytosol, where viscosities can me 2-3 times larger than aqueous solvents. Here, Kramers’ rate theory is used to describe the viscosity dependence of the folding and unfolding rate constants of tetraloop-receptor docking at the single molecule level. Specifically, kfold and kunfold are measured at various glycerol percentages to manipulate the solvent viscosity. Both rate constants decrease with increasing viscosity, indicating that molecular motions along the reaction coordinate are overdamped processes. Temperature dependent measurements show that the thermodynamics of docking are not perturbed (ΔH°= -23(2) kcal/ mol and ΔS°= -76(6) cal/molK) even in up to 50% glycerol. Additionally, by accounting for the temperature dependence of the solvent viscosity, the thermodynamic parameters of the transition state are also found to be independent of glycerol content. These results indicate that the full free energy surface, including the transition state, is unperturbed by glycerol, and kinetics are slowed by reducing the rate at which the tetraloop and receptor diffusively sample the free energy surface on approach to the transition state. 

Unassigned Posters: RNA Catalysis and Riboswitches and RNA structure and folding

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

709 Conformational Selection of Initiation Factor 3 Signals Proper Substrate Selection During Translation Initiation

Margaret Elvekrog, Ruben Gonzalez, Jr Columbia University Prokaryotic translation initiation factor 3 (IF3) is an essential protein that binds to the small, 30S, ribosomal subunit and regulates the fidelity of initiator N-formylmethionyl-transfer RNA (fMet-tRNA(fMet)) and messenger RNA (mRNA) start codon selection during translation initiation [1, 2]. IF3 largely accomplishes this through two negative regulatory activities: destabilization of tRNA binding at the peptidyl-tRNA binding (P) site of the 30S initiation complex (30S IC) and inhibition of large, 50S, ribosomal subunit docking to the 30S IC to form an intact 70S initiation complex (70S IC) [2-4]. The molecular mechanism through which specific recognition of a properly base-paired fMet-tRNA(fMet) anticodon-start codon interaction within a completely assembled 30S IC results in relaxation of IF3’s subunit anti-association activity, and possibly its tRNA destabilization activity, have been elusive, however, precluding a precise understanding of the mechanisms that control the fidelity of translation initiation. Using a dual fluorescently labeled IF3 and single-molecule fluorescence resonance energy transfer (smFRET), here we show that 30S IC-bound IF3 exists in a dynamic equilibrium between at least three conformations during translation initiation. We find that specific recognition of an fMet-tRNA(fMet) anticodon-start codon interaction within a fully assembled 30S IC dramatically alters this conformational equilibrium by significantly destabilizing certain 30S IC-bound IF3 conformations. Taken together with the available biochemical data, we conclude that exclusion of these 30S IC-bound IF3 conformations is the structural corollary to relaxation of the subunit anti-association activity, and perhaps the tRNA destabilization activity, of IF3 and provides a structural rationale for IF3’s role in establishing the fidelity of translation initiation. References 1. Hartz, D., et al. (1989) Genes Dev, 3, 189912 2. Risuleo, G., et al. (1976) Eur J Biochem, 67, 6033 3. Subramanian, A.R. and Davis B.D. (1970) Nature, 228, 1273-5 4. Gottleib M., et al. (1975) Proc Natl Acad Sci USA, 72, 4238-42

710

Regulation of Ribosomal 70S Initiation Complex Stability during Translation Initiation

Daniel MacDougall, Ruben Gonzalez Department of Chemistry, Columbia University, New York, (NY), USA During prokaryotic translation initiation, a ribosomal 70S initiation complex (70S IC) is assembled at the start codon of a messenger RNA (mRNA) template under the direction of three essential initiation factors (IFs): IF1, IF2, and IF3. A key event in the 70S IC assembly pathway is docking of the large, 50S, ribosomal subunit to a small, 30S, ribosomal subunit carrying the IFs, mRNA, and initiator N-formylmethionyl-transfer RNA (fMet-tRNAfMet), termed the 30S initiation complex (30S IC). 30S IC-bound IF2 plays a critical role in promoting 50S subunit docking by establishing direct interactions with elements of the 50S subunit’s GTPase-associated center (GAC). The precise molecular mechanisms through which IF2-GAC interactions guide 50S subunit docking and 70S IC formation, however, remain unclear. In order to characterize IF2-GAC interactions during 50S subunit docking in real time, we have developed a single-molecule fluorescence resonance energy transfer (smFRET) signal between fluorescently labeled variants of IF2 and ribosomal protein L11, an important component of the GAC. Our results reveal that, following 50S subunit docking but prior to IF2’s dissociation from the 70S IC, the IF2-bound 70S IC can reversibly sample at least two distinct conformational states that are characterized by different distances between IF2 and L11. Comparison of smFRET experiments conducted in the absence and presence of IF3 suggests a functional role for these conformational changes. Inclusion of IF3 within the 30S IC renders 50S subunit docking highly reversible, consistent with IF3’s known anti-subunit association activity. Furthermore, two distinct types of reversible 50S subunit docking events were observed in the presence of IF3: short-lived docking events that result in formation of an unstable 70S IC and longer-lived docking events that result in formation of a relatively stable 70S IC. The degree of 70S IC stability was correlated with a shift in the relative occupancy of the two conformational states probed by the IF2-L11 smFRET signal, suggesting that 70S IC conformational rearrangements that modulate IF2’s interactions with the GAC may play a role in regulating 70S IC stability. Our data is consistent with a mechanistic model in which an underlying conformational equilibrium of the IF3-bound 30S IC regulates the stability of 50S subunit docking events during translation initiation.

Unassigned Posters: Ribosomes and Translation

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

711

Cloning, Expression, and Purification of the S. cerevisiae Sub2 ATPase

Yuliang Sun and Aaron A. Hoskins U. Wisconsin-Madison, Madison, (WI), USA Sub2 is a member of the DEAD-box family of RNA-dependent ATPases and has important roles in both pre-mRNA splicing and mRNA export. While many biochemical, genetic, and structural experiments have been carried out on the yeast Sub2 protein or its human homolog UAP56, the exact function of Sub2 in splicing and its mechanism of action remain unclear. In order to elucidate Sub2 biochemistry, we have cloned the gene encoding Sub2 from S. cerevisiae. The Sub2 gene was subsequently subcloned into a pGEX plasmid for heterologous expression in E. coli as a GST fusion. We have successfully expressed and purified a soluble GST-Sub2 fusion from E. coli. In addition, we have shown that that the GST tag can be removed by the TEV protease. The purified Sub2 ATPase will prove useful for biochemical and biophysical studies of Sub2 action during pre-mRNA splicing and RNA metabolism.

712 The Development of Methods for the Site-Specific Fluorescent Labeling of Spliceosomal Proteins for use in Single-Molecule Studies

Matthew Kahlscheuer, Ramya Krishnan, Mario Blanco, Nils Walter University of Michigan, Ann Arbor, (MI), USA Spliceosomes are multi-megadalton ribonucleoprotein (RNP) complexes responsible for catalyzing the removal of noncoding introns from eukaryotic precursor messenger RNA (pre-mRNA) transcripts and ligating the flanking coding exon sequences to produce a mature messenger RNA (mRNA). Spliceosomal assembly and catalysis require a highly dynamic coordination of protein and RNA with the purpose of producing mature mRNA that can be properly transcribed by the ribosome. Due to the complexity of the process and a lack of suitable tools, there is still much unknown about the assembly of the spliceosome. Single molecule fluorescence microscopy tools have recently been developed for studies of spliceosome assembly and dynamics of the pre-mRNA substrate throughout splicing. In an effort to advance the study of splicing and delineate the exact timing and dynamics required to achieve splicing, we are currently investigating methods for the site-specific labeling of the protein components of the spliceosome. A more specific approach we are utilizing is the labeling of single cysteine-containing proteins such as Cwc25 and single cysteine mutant proteins such as Prp28. A more general approach we are developing, which would allow for the labeling of a wide range of proteins, is protein trans-splicing (PTS) using a split intein. Protein splicing is a naturally occurring process in which a protein editor, called an intein, excises itself out of a host protein in which it is embedded creating a new peptide bond between its two flanking regions, the exteins. PTS uses an artificially or naturally split intein to create a new peptide bond between flanking exteins similar to protein splicing1. Upon incubation of the two components, protein splicing will take place, resulting in the ligation of the two extein regions. By fluorescently labeling one of the extein components, we hope to use PTS to site-specifically label several spliceosomal proteins with the ultimate goal of correlating the association and dissociation of spliceosomal components with pre-mRNA dynamics in order to gain a better understanding of the mechanism of pre-mRNA splicing. 1. Vila-Porollo, M., Muir, T.W. Cell 143, 191-200 (2010)

Unassigned Posters: Splicing Mechanisms

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

NOTES

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

AUTHOR INDEX (Note: Numbers refer to abstract numbers, not page numbers) A Aas, Per A......................................... 251 Abad, Maria.............................. 370, 372 Abd Latip, Normala.......................... 388 Abdelmohsen, Kotb............................ 21 Abell, Chris....................................... 425 Abelson, John...................... 68, 344, 346 Abeysirigunawardena, Sanjaya C....... 29 Aboul-ela, Fareed.............................. 418 Adamiak, Ryszard W........................ 229 Adams, Mark..................................... 217 Adamska, Dorota.............................. 599 Afonin, Kirill A................................. 172 Afonin, Kirill..................................... 628 Afroz, Tariq....................................... 120 Agris, Paul F..................................... 233 Aguilar, Cynthia................................ 560 Ahlquist, Paul.................................... 237 Akef, Abdalla............................ 148, 161 Akiba, Masaki................................... 500 Akiyama, Hideo................................ 194 Albring, Michael....................... 216, 320 Alemán, Elvin................................... 191 Alexander, Rebecca W.............. 424, 580 Alfonzo, Juan D........ 367, 373, 375, 598 Al-Hashimi, Hashim M....... 39, 123, 419 Allain, Frederic................................. 120 Allen, Mary A.................................... 183 Almada, Albert E.............................. 253 Almutairi, Mashal M......................... 285 Alramini, Hussein............................. 246 Alshiekh, Alak................................... 239 Altuntop, Mediha E........................... 306 Alvarez, Nehemiah........................... 536 Alves, Flavia de L............................. 354 Alves, Lysangela R........................... 457 Alvey, Heidi S................................... 419 Amacher, Sharon L........................... 164 Amini, Zhaleh N............................... 517 Amrani, Nadia..................................... 62 Andersh, Laura M............................. 394 Anderson, David................................... 7 Anderson, Eric G................................ 72 Anderson, James T............................ 409 Anderson, John R................................ 26 Anderson, Vernon................................. 7 Anderssen, Endre.............................. 251 Andreeva, Irena................................... 30 Andrews, Kristie L.............................. 70 Androsavich, John R................. 129, 254 Andrzej, Zielezinski.......................... 238 Ang, Jason......................................... 378 Anthony, Jon..................................... 265

Apicella, Marisa................................ 609 Ares, Jr., Manuel....... 105, 137, 553, 571 Ares, Manuel............................. 342, 703 Arnold, Justin D................................ 571 Arnold, Justin............................ 225, 474 Arribere, Joshua A............................. 171 Artavanis-Tsakonas, Spyros.............. 146 Artsimovitch, Irina............................ 439 Artur, Jarmolowski............................ 238 Arumemi, Fortuna............................. 320 Aruscavage, Philip J.......................... 242 Asahara, Haruichi............................. 286 Asai, Kiyoshi...................................... 47 Asakawa, Shuichi.............................. 500 Ashar, Ami J...................................... 185 Ashley, James.................................... 147 Assefa, Berhanegebriel............. 435, 479 Assmann, Sarah M............................ 187 Ataman, Bulent................................. 147 Athanasiadou, Rodoniki.................... 395 Atkins, John.............................. 301, 468 Au, Hilda H....................................... 380 Auwardt, Supipi L............................. 624 Azad, Tareq....................................... 471

B Babar, Imran...................................... 627 Babiano, Reyes................................. 335 Baccaro, Brenna................................ 618 Bachu, Ravichandra.......................... 336 Bacikova, Veronika........................... 121 Bacusmo, Jo Marie............................ 287 Badertscher, Lukas............................ 288 Bahn, Jae Hoon......................... 177, 378 Bai, Yong........................................... 136 Baird, Nathan J.................................. 420 Baker, Brett J..................................... 214 Baker, Stacey L................................. 404 Balakrishnan, Rohan......................... 289 Balcer, Zuzanna................................ 178 Baltzinger, Mireille........................... 152 Bammert, Lukas................................ 290 Banáš, Pavel.............................. 159, 523 Banerjee, Bidisha.............................. 203 Banfield, Jillian F.............................. 214 Bansal, Nitin..................................... 392 Barbosa, Isabelle................................. 10 Barkan, Alice..................................... 218 Barrass, David..................................... 71 Barraud, Pierre.................................. 139 Barria, Romina.................................. 147 Barta, Andrea.................................... 538 Bartel, David P.................................... 13

Basak, Anindita................................. 537 Basquin, Claire.................................. 414 Bass, Brenda L................ 1, 78, 242, 623 Bastet, Laurène......................... 155, 502 Bateman, John F................................ 403 Baumann, Peter................................... 23 Baumbusch, Lars O........................... 260 Baumgärtner, Marc............................. 60 Bawankar, Praveen.............................. 58 Beaudoin, Jean-Denis....................... 274 Bebee, Thomas W..................... 544, 606 Bec, Guillaume................................. 152 Becker, Kevin G.................................. 21 Beckmann, Benedikt M........................ 5 Beeharry, Yasnee............................... 204 Beemon, Karen L...................... 163, 477 Beggs, Jean D.............................. 71, 354 Beh, Leslie........................................ 309 Behm, Mikaela.................................... 81 Behrman, Edward J........................... 197 Beitzinger, Michaela......................... 489 Belardinelli, Riccardo......................... 30 Belasco, Joel G.................................... 53 Bellaousov, Stanislav........................ 176 Bellur, Deepti L................................. 337 Belmont, Brian J................................. 93 Ben Hur, Vered.................................. 614 Benjamin, Julie-Anna M........... 135, 264 Bennett, C Frank............................... 107 Bennett, Keiryn L.............................. 466 Berezhna, Svitlana Y......................... 223 Berezin, Igor..................................... 328 Berezney, Ronald...................... 323, 392 Berg, Michael G................................ 169 Bergkessel, Megan............................ 142 Berglund, Andrew............................. 106 Berndt, Heike...................................... 82 Bernick, David L....................... 166, 577 Bertrand, Edouard............................. 138 Bessler, Monica......................... 330, 609 Bevilacqua, Philip C........ 115, 125, 187, 505, 525, 535 Bhaskar, Aishwarya.......................... 392 Bhaskaran, Hari......................... 300, 578 Bhat, Balkrishen................................ 254 Bickel, Peter J................................... 310 Bieberstein*, Nicole.......................... 141 Bielewicz, Dawid.............................. 538 Biesinger, Jacob................................ 167 Billaud, Marc.................................... 224 Bindewald, Eckart............................. 172 Biondi, Elisa...................................... 503 Birch, Christina M.............................. 88 Birmingham, Amanda....................... 176 Author Index – 1

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

Blaby, Ian K...................................... 364 Black, Douglas L........ 69, 137, 547, 552, 553, 554, 572, 575 Blagden, Sarah P............................... 388 Blanchette, Marco............. 325, 536, 540 Blanco, Mario R.................. 68, 344, 712 Blech-Hermoni, Yotam..................... 205 Bleckley, Samuel................................. 51 Blewett, Nathan H..................... 396, 592 Blower, Michael................................ 255 Blumenthal, Robert........................... 628 Bodner, Micah J................................ 106 Boeke, Jef D........................................ 42 Boesen, Thomas.................................. 59 Bøggild, Andreas............................... 381 Bogna, Szarzynska............................ 238 Boguta, Magdalena........................... 600 Böhnlein, Eva-Maria......................... 463 Boisbouvier, Jerome.......................... 421 Boisset, Sandrine............................... 132 Boland, Andreas.................................. 58 Bolisetty, Mohan T............................ 163 Bompiani, Kristin.............................. 102 Boniecki, Michal T..................... 52, 593 Bonneau, Eric.................................... 421 Boots, Jennifer L............................... 273 Borah, Sumit....................................... 27 Boris-Lawrie, Kathleen............. 206, 382 Borodynko, Natasza.................. 467, 480 Borowski, Lukasz S.......................... 416 Børresen-Dale, Anne-Lise................. 260 Bose, Debojit....................................... 89 Boswell, William............................... 350 Bottoms, Christopher A....................... 89 Boushaba, Khalid.............................. 624 Boutz, Paul L............................. 539, 550 Bouvette, Jonathan............................ 212 Bowers, Heath................................... 215 Boyapati, Vamsi................................ 418 Bradley, Robert K............................. 604 Bradley, Todd C................................ 540 Bravo, Jeronimo................................ 304 Bray, Walter...................................... 342 Breaker, Ronald R............. 504, 512, 515 Brenner, Steven E.... 146, 310, 311, 312, 313, 541 Brenner, Sydney................................ 571 Brenowitz, Michael........................... 336 Bresson, Stefan M............................... 65 Brettschneider, Susanne.................... 412 Brodersen, Ditlev E..................... 59, 381 Brody, Yehuda................................... 463 Brooks 3rd, Lionel............................ 207 Brooks III, Charles L.......... 39, 123, 520 Brooks, Angela N.............. 312, 313, 541 Brooks, Angela.................................. 146 Brow, David A............. 70, 331, 338, 454 Brown, Ben....................................... 146 Author Index – 2

Brown, Jeremy.................................. 279 Brown, Michael................................. 195 Brown, Rhonda................................. 607 Brumbaugh, Justin............................ 100 Bubulya, Athanasios......................... 314 Bubulya, Paula A............................... 314 Bucheli, Miriam E............................. 103 Budnik, Vivian.................................. 147 Bühler, Marc..................................... 139 Bujnicki, Janusz M...................... 52, 178 Burch, Christina L............................. 473 Burke, Donald H......... 89, 428, 503, 625 Burke, Jordan E................................. 338 Burley, Glenn A................................. 339 Burlingame, Al.................................... 73 Burnouf, Dominique......................... 152 Busch, Anke...................................... 167 Buschmann, Juliane............................ 84 Butcher, Samuel E........ 37, 70, 331, 338, 454 Butler, J. Scott..................................... 99 Byczyk, Julia..................................... 467 Byrd, R. Andrew............................... 119

C C. Wahl, Markus............................... 108 Cabral, A. Fco................................... 701 Caceres, Javier F................................. 25 Calarco, John..................................... 562 Calimano, Maria............................... 464 Campbell, Frank.................................... 7 Campos Melo, Danae........................ 611 Campos-Melo, Danae............... 605, 608 Cao, Lijuan.......................................... 43 Capewell, Paul.................................. 334 Capshew, Claire R............................. 369 Carmack, Charles S........................... 173 Carmi, Shai....................................... 463 Carmo-Fonseca, Maria........................ 76 Caron, Marie-Pier............................. 155 Carret, Céline K................................ 615 Carrier, Marie-Claude............... 135, 264 Carrillo Oesterreich, Fernando.......... 141 Carrocci, Tucker J............................. 531 Carstens, Russ........................... 548, 573 Cartegni, Luca................... 397, 626, 631 Carvalho, Célia................................... 76 Cascio, Duilio................................... 116 Cass, Danielle................................... 208 Castello, Alfredo................................... 5 Cazalla, Demian................................ 481 Cech, Thomas................................... 271 Celniker, Susan E...................... 146, 541 Cenik, Can............................. 9, 161, 706 Cerciat, Marie................................... 408 Cernak, Paul...................................... 156 Chakraborty, Anirban........................ 589 Chakraborty, Saikat..................... 40, 256

Chalkley, Robert J............................... 73 Chamberlain, Chris........................... 105 Chan, Elton....................................... 136 Chan, Mandy..................................... 301 Chan, Patricia P................................. 579 Chandler, Dawn S............. 543, 549, 606 Chanfreau, Guillaume F.................... 551 Chanfreau, Guillaume....................... 231 Chang, Keng-Ming........................... 580 Chang, Lin-Chun............................... 398 Chang, Roger K................................ 128 Chang, Sean Shang-Lin.................... 184 Chang, Tien-Hsien.... 184, 361, 478, 542 Chang, Wei-Lun................................ 400 Chang, Young-Tae............................. 147 Chapman, Erich................................. 441 Chartier, Nicolas............................... 224 Chase, Elaine..................................... 441 Chatterjee, Kunal...................... 364, 368 Chau, Nelson..................................... 254 Chen, Andy G Y................................ 504 Chen, Changbin................................. 140 Chen, Chunlai..................................... 33 Chen, Chunxia................................... 176 Chen, Doris....................................... 273 Chen, Ji..................................... 115, 505 Chen, Jui-Hui.................................... 542 Chen, Rui.......................................... 104 Chen, Stella....................................... 235 Chen, Weijun..................................... 185 Chen, Xinbin..................................... 236 Chen, Ying.......................................... 58 Chen, Yu............................................ 160 Chen, Yuan-Chuan............................ 114 Chen, Zugen...................................... 177 Cheng, Chonghui...................... 143, 607 Cheng, Christopher J......................... 627 Cheng, Hong..................................... 458 Cheng, Soo-Chen........................ 75, 348 Cherbas, Peter................................... 146 Chernyakov, Irina.............................. 366 Chesham, Johanna E......................... 556 Chi, Binkai........................................ 458 Chilakalapudi, Durga Rao................. 340 Chillon, Isabel................................... 506 Chinen, Madoka................................ 250 Chiou, Ni-ting..................................... 66 Chirn, Gung-wei............................... 130 Cho, Eun Jeong................................. 193 Cho, Hana......................................... 399 Chodosh, Lewis................................ 607 Choi, Sun Shim................................. 399 Chong, Shaorong.............................. 286 Chorghade, Sandip G........................ 340 Chou, Fang-Chieh............................. 422 Choudhary, Pallavi............................ 507 Chow, Christine S..................... 209, 291 Christian, Henning.............................. 74

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

Chu, Hui-Yi....................................... 383 Chu, Jiayou....................................... 353 Chu, Qili............................................ 632 Chu, Ying-Chieh............................... 398 Chua, Hon Nian................................ 161 Chuah, Charles.................................. 612 Chuang, Tzu-Wei.............................. 400 Chuang, Tzu-Ying............................. 103 Chung, Betty WY.............................. 468 Cit, Zdenek........................................ 546 Clancy, Jennifer L............................. 495 Clark, Tyson A........................... 190, 195 Clayton, Christine..................... 401, 402 Clerici, Marcello............................... 138 Cleveland, Don W............................. 107 Cline, Melissa................................... 703 Clingman, Carina C............................ 12 Cloutier, Sara C................................. 319 Cochrane, Alan.......................... 382, 464 Coen, Donald M................................ 389 Cofré, Axel........................................ 326 Cohen, Paula E.................................. 484 Cole, Brian S............................. 145, 210 Colinge, Jacques............................... 466 Coller, Jeffery M............................... 217 Collins, Kathleen............................... 116 Coltri, Patricia P................................ 704 Combs, Joshua.................................. 227 Comiskey, Daniel F........................... 543 Conboy, John G................................. 164 Conboy, John..................................... 225 Conrad, Nicholas K............................. 65 Conti, Elena................................. 60, 414 Cook, Atlanta G................................ 211 Cook, Malcolm E.............................. 325 Cook, Malcolm......................... 536, 540 Cook, William B............................... 218 Coolon, Joseph D.............................. 557 Coon, Josh......................................... 100 Coonrod, Leslie A............................. 106 Cooper, Thomas A..................... 567, 613 Cooperman, Barry S............................ 33 Corbett, Anita H................................ 324 Corcoran, David................................ 202 Cordero, Pablo.................................. 176 Corey, David R.......................... 127, 240 Cornett, Ashley................................. 275 Cornish, Peter...................................... 36 Correa, Alejandro.............................. 326 Cortes, Jesus...................................... 701 Coulbourn Flores, Samuel................ 456 Crawford, Amanda K........................ 592 Crecy-Lagard, Valerie de.................. 364 Creighton, Chad................................ 613 Cristão, Vanessa F............................. 354 Csúcs, Gábor..................................... 288 Cui, Xianying (Amy)........................ 459 Cunningham, Philip.......................... 295

Curk, Tomaž...................................... 567 Curran, Elizabeth C............................. 70 Curry, Edward W.............................. 388 Curtis, Daniel M................................ 303 Cusack, Stephen........................ 138, 593 Cvetesic, Nevena............................... 300 Cyphert, Travis J............................... 569 Cyphert, Travis.................................. 341

D Dahlberg, Albert E.............................. 34 Dai, Qing................................... 190, 359 Daldrop, Peter................................... 423 Dallagiovanna, Bruno............... 326, 457 Damerow, Marin............................... 607 Damha, Masad............................ 97, 227 Damianov, Andrey............................ 575 Dang, Kristen K................................ 473 Daniel, Chammiran........................... 365 Darnell, Robert B.......................... 6, 144 Das Sharma, Sohani.................. 203, 293 Das, Mom.................................... 87, 292 Das, Rhiju................................. 176, 422 Das, Rita............................................ 565 Dator, Romel P.................................. 186 Daugeron, Marie-Claire.................... 304 Davidson, Adam S............................ 424 Davidson, Ben................................... 614 Davis-Neulander, Lauren.................. 176 Dawid, Bielewicz.............................. 238 Dawson, Wayne.................................. 47 Dayie, Kwaku T................................ 514 de la Cruz, Jesus................................ 335 de la Grange, Pierre........................... 257 De Leon, E. E.................................... 701 de Silva, Chamaree........................... 191 de Zamaroczy, Miklos....................... 408 De, Supriyo......................................... 21 Dean, Kimberly M............................ 384 Debreuck, Nadège..............................111 Deckert, Jochen................................. 632 Dedic, Emil......................................... 59 DeGregorio, Suzanne J..................... 483 Deigan, Katherine E.......................... 425 Del Campo, Mark................................ 14 Dela-Moss, Lumbini I....................... 470 Demirci, Hasan................................... 34 Deng, Zhihui..................................... 314 Denichenco, Polina........................... 614 Dennis, Patrick P....................... 166, 577 DeRose, Victoria J............ 441, 527, 534 Derti, Adnan...................................... 161 Deryusheva, Svetlana........................ 581 Desai, Kevin K.................................. 532 Deshpande, Atul................................ 228 Desjardins, Alexandre....................... 212 Desjardins, Genevieve...................... 421 Deutscher, Murray P......................... 276

Devany, Matthew.............................. 705 Devaraj, Aishwarya........................... 297 Deveau, Laura M................................ 12 Devkota, Ashwini K.......................... 193 Dewe, Joshua.................................... 366 Dhungel, Nripesh................................ 96 Di Giulio, Massimo........................... 214 DiChiacchio, Laura........................... 426 Dickinson, Rachel L.......................... 339 Dickson, David A.............................. 220 Diebel, Kevin W............................... 465 Dietrich, Rosemary C........................ 110 Dina, Roberto.................................... 388 Ding, Feng........................................ 429 Ding, Ye............................................ 173 Ding, Yiliang..................................... 187 Ditzler, Mark A................................... 89 Ditzler, Mark..................................... 428 Dixon, Neil.......................................... 92 Dohm, Juliane C.................................. 82 Dokholyan, Nikolay V...................... 429 Dolan, Gregory F.............................. 517 Dolata, Jakub..................................... 238 Dominguez, Catherine E........... 544, 606 Donald, Bruce R................................ 430 Donarski, James................................ 438 Donohue, John Paul.... 28, 107, 296, 571 Doudna, Jennifer A...... 16, 243, 245, 259 Douglass, Stephen............................. 551 Dowell, Robin................................... 183 Doyle, Francis................................... 269 Drabløs, Finn.................................... 251 Dreyfuss, Gideon.............................. 169 Drinnenberg, Anna............................ 140 Droll, Dorothea................................. 401 Droppelmann, Cristian A.................. 608 Dubé, Audrey............................ 155, 502 Duc, Anne-Cecile E.......................... 213 Duelli, Dominik M.................... 109, 258 Duff, Michael O................ 146, 541, 557 Dulic, Morana................................... 300 Dumas, Philippe................................ 152 Dumesic, Phillip A.................... 140, 244 Duncan, Caia..................................... 176 Duncan, John N................................... 92 Dunn, Josh.......................................... 48 Dupuis, Nicholas............................... 708 Dusenbury, Kristen L........................ 369 Dutta, Tanmay................................... 276 Dybkov, Olexandr............................... 44 Dziembowski, Andrzej...................... 416

E E. Fujimori, Kazuhiro....................... 500 Easter, Ashley D................................ 381 Eberle, Carol-Ann............................. 466 Edge, Christopher............................. 545 Effenberger, Kerstin.......................... 342 Author Index – 3

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

Eichelbaum, Katrin............................... 5 Eichhorn, Catherine D...................... 123 Eichhorn, Katie D............................... 39 Eizentstat, Linda................................. 42 Ekdahl, Ylva........................................ 81 Eldho, Nadukkudy V......................... 514 Elmroth, Sofi KC.............................. 239 Elvekrog, Margaret M....................... 709 Emmerth, Stephan............................. 139 Ennifar, Eric.............................. 152, 437 Eperon, Ian C.................................... 339 Erben, Esteban.................................. 402 Ermolenko, Dmitri N.................. 28, 294 Esakova, Olga................................... 328 Escamilla-Powers, Julie R........ 629, 630 Espinosa, Joaquin.............................. 183 Estivill, Xavier.................................. 489 Etienne, Chris.................................... 228 Eyras, Eduardo............................ 25, 257

F F. Santos, Karine............................... 108 Fabrizio, Patrizia........................... 44, 74 Fadda, Abeer............................. 401, 402 Faerber, Valentin............................... 402 Fagg, W. Samuel............................... 703 Fahlman, Richard P........................... 582 Fak, John J........................................ 144 Falaleeva, Marina.............................. 257 Fan, Jianmeng................................... 330 Faner, Martha A................................. 134 Fang, Xianyang................................. 119 Fang, Yiwen...................................... 403 Farabaugh, Phil................................. 592 Farahani, Hossein S........................... 81 Farhat, May D................................... 295 Farris, Hamilton E............................. 109 Fast, Naomi M.................................. 165 Faux, Céline...................................... 376 Fedorova, Olga.................................. 506 Fedosova, Natalya U........................... 59 Feig, Andrew L................................. 134 Feig, Andrew..................................... 437 Feigenbutz, Monika.......................... 414 Feigon, Juli........................................ 116 Feldges, Robert................................. 624 Feng, Jun............................. 39, 123, 520 Feng, Yong........................................ 501 Fenn, Katelyn.................................... 334 Ferré-D’Amaré, Adrian R........ 150, 420, 425, 511, 519, 530 Fica, Sebastian M.............................. 359 Fichtenbaum, Jeremy.......................... 41 Ficner, Ralf.................................... 74, 84 Fields, Stanley..................................... 97 Fierke, Carol A.............. 4, 160, 188, 584 Filipowicz, Witold............................. 262 Filonava, Liudmila.............................. 30 Author Index – 4

Finnegan, Emily F............................. 499 Firth, Andrew............................ 301, 468 Fischer, Bernd....................................... 5 Fitzek, Elisabeth................................ 368 Fleming, Ian MC............................... 367 Fleming, Ian...................................... 373 Florens, Laurence.............................. 249 Flores, Samuel..................................... 57 Flynn, Ryan A................................... 253 Foerstemann, Klaus........................... 490 Fok, Jacqueline................................. 388 Folco, Eric G..................................... 565 Folk, Petr........................................... 546 Forsyth, Ellen.................................... 122 Foster, Leonard................................. 301 Foster, Mark...................................... 232 Fourmann, Jean-Baptiste..................... 74 Francis, Sandrea M........................... 304 Frazer, Michelle................................ 112 Fredrick, Kurt...................... 35, 289, 297 French, Courtney E................... 312, 313 French, Deborah................................ 609 Friedman, Larry J........................ 72, 355 Friedman, Larry.................................. 62 Frilander, Mikko J....................... 95, 570 Fritz, Andrew............................ 323, 392 Fritz, Sarah........................................ 206 Fu, Xiang-Dong................................ 547 Fu, Yinghan....................................... 174 Fujii, Kotaro...................................... 101 Fujishima, Kosuke............ 214, 268, 583 Fukuzumi, Takeo....................... 189, 482 Fulton, Bruce....................................... 90 Fung, Angela WS.............................. 582 Furman, Ran...................................... 439

G Gadue, Paul....................................... 609 Gagnon, Keith T........................ 127, 240 Gahura, Ondrej.................................. 546 Gaidatzis, Dimos............................... 139 Gall, Joseph G................................... 581 Gallagher, Thomas L......................... 164 Gallivan, Justin P.............................. 154 Gally, David L................................... 498 Gamalinda, Michael.......................... 335 Gancarz, Brandi................................ 237 Ganguly, Abir............................ 115, 505 Gao, Zhaofeng................................... 215 Garcia, Ivelitza.................................. 216 Garcia-Perez, Jose L........................... 25 Garcon, Loic..................................... 609 Garfinkel, David J............................... 42 Garrey, Stephen................................... 97 Gas, María-Eugenia.......................... 304 Gaston, Kirk W......................... 367, 598 Gaucher, Eric A................................. 286 Gaugue, Isabelle................................ 408

Gautam, Amit...................................... 71 Ge, Jingping.............................. 330, 609 Ge, Zhiyun........................................ 404 Gee, Sherry....................................... 225 Geerlings, Torsten............................... 92 Geiger, Tamar.................................... 620 Geisler, Matt...................................... 368 Geisler, Sarah J................................. 217 Geissmann, Tom................................ 132 Gelles, Jeff............................ 62, 72, 355 Genga, Ryan M................................... 12 Ghanem, Dana................................... 225 Ghanem, Eman.................................... 49 Gibson, Gary..................................... 622 Gilbert, Wendy V.............................. 171 Gilfillan, Gregor................................ 251 Gilman, Benjamin............................... 14 Gingeras, Thomas............................... 17 Gingeras, Tom................................... 146 Girard, Cyrille................................... 460 Girard, Nicolas.......................... 421, 510 Givan, Scott A..................................... 89 Glatt, Sebastian................................. 376 Gleghorn, Michael L........................... 61 Golden, Barbara L..... 115, 441, 505, 525 Goldenberg, Samuel.................. 326, 457 Goldfless, Stephen J............................ 93 Goldman, Yale E................................. 33 Goldstrohm, Aaron............ 100, 393, 396 Gong, Chenguang............................... 61 Gong, Hao................................. 114, 136 Gonzalez-Hilarion, Sara.............111, 405 Gonzalez, Jr, Ruben L....................... 709 Gonzalez, Ruben L............................ 710 Gooding, Clare.................................. 545 Gopalan, Venkat........................ 197, 329 Gopinath, Chetna...................... 436, 440 Gorelick, Robert J............................. 473 Gorospe, Myriam................................ 21 Goss, Dixie J............................. 203, 293 Gostan, Thierry................................. 138 Gouw, Joost....................................... 301 Goyal, Akanksha................................. 30 Grabinski, Sarah E............................ 144 Graham, Lauren T............................. 137 Graham, Shanelle T........................... 526 Grainger, Richard J............................. 71 Granneman, Sander........................... 498 Graveley, Brenton R......... 146, 195, 541, 557 Graves, Jennifer A............................. 560 Gray, Joe........................................... 225 Grayhack, Elizabeth J....................... 384 Green, Rachel.................................... 298 Greenbaum, Nancy L........ 336, 442, 705 Greenberger, Ben.............................. 103 Gregory, Brian..................................... 19 Gregory, Steven T............................... 34

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

Greimann, Jaclyn C........................... 142 Gresham, David........................ 175, 395 Grimson, Andrew W......................... 198 Grimson, Andrew.............................. 484 Grisdale, Cameron J.......................... 165 Grosjean, Henri................................. 364 Gruic-Sovulj, Ita............................... 300 Grzechnik, Pawel...................... 591, 599 Gu, Bai-Wei...................................... 330 Guarna, Marta................................... 301 Guedich, Sondes............................... 152 Guenther, Ulf-Peter....................... 7, 217 Gueroussov, Serge............................. 706 Guerreiro, Ana M.............................. 615 Guo, Eric............................................. 64 Guo, Yanwen....................................... 63 Guo, Zhuojun.................................... 454 Gupta, Ramesh.......... 364, 368, 371, 586 Gurha, Priyatansh.............................. 368 Guthrie, Christine....... 68, 142, 343, 344, 346, 350, 352, 555 Guy, Michael P.................................... 86

H Ha, Taekjip.......................................... 29 Habjan, Matthias............................... 466 Hackermüller, Jörg............................ 260 Hadad, Christopher M................. 87, 292 Hadjivassiliou, Haralambos A.......... 343, 346 Hadwiger, Philippe............................ 632 Hagihara, Masaki.............................. 189 Hai, Yan............................................. 353 Haley, Michael M.............................. 106 Hall, Kathleen B................................ 162 Hall, Megan P................................... 703 Hallais, Marie.................................... 138 Halvorsen, Matthew......... 176, 440, 446, 610 Hamashima, Kiyofumi...................... 583 Hamilton, Holly L............................. 632 Hammani, Kamel.............................. 218 Hammes-Schiffer, Sharon......... 115, 505 Hammond, Ming C............................. 83 Han, Areum....................................... 547 Han, Jongyoon.................................... 88 Han, Sisu........................................... 399 Hanada, Toshikatsu........................... 602 Hansen, Matthew.............................. 103 Haque, Nazmul................................. 325 Harada, Yasue............................ 189, 482 Harms, Scot A................................... 325 Harnisch, Christiane............................ 82 Harris, Michael..................................... 7 Hart, P. John...................................... 227 Hartmuth, Klaus................................ 460 Hartwich, Agnieszka......................... 267 Hasiow-Jaroszewska, Beata...... 467, 480

Hasson, Christina L........................... 448 Hastings, Michelle L......... 109, 258, 556 Hauer, Michael.................................. 259 Havens, Mallory A............................ 258 Hay, Samantha A................................. 12 He, Fang............................................ 112 Hegre, Siv A...................................... 251 Heiner, Monika................................... 43 Heinicke, Laurie................................ 548 Hejnowicz, Monika S........................ 416 Heldman, Eliahu............................... 628 Hendricks, Matthew.......................... 527 Hennelly, Scott P................................. 22 Hennigan, Jennifer A......................... 315 Henriksson, Niklas.............................. 57 Hentze, Matthias W............................... 5 Heras, Sara R...................................... 25 Herbert, Kristina M........................... 483 Hertel, Klemens J.............................. 559 Hertz, Marla I.................................... 305 Hesselberth, Jay.......................... 97, 362 Heurgue-Hamard, Valerie................. 408 Hexner, Elizabeth O.......................... 234 Heyd, Florian.................................... 316 Higa-Nakamine, Sayomi................... 589 Hill, Kyle........................................... 428 Hilliker, Angie................................... 385 Hillmer, Axel..................................... 612 Hilton, Cameron L............................ 106 Hilz, Stephanie.................................. 484 Himmelbauer, Heinz........................... 82 Hines, Jennifer V....................... 513, 529 Hinrich, Anthony J............................ 109 Hintersteiner, Martin......................... 262 Hirano, Takashi................................. 500 Hiraoka, Kiriko......................... 268, 485 Hirose, Yuka.............................. 268, 485 Hobor, Fruzsina......................... 121, 219 Höchsmann, Matthias....................... 577 Hoffmann, Søren V............................. 59 Hogg, J. Robert................................. 404 Hohng, Sungchul...................... 151, 486 Holl, Eda........................................... 102 Holloway, Stephen............................ 227 Holmes, Andrew D............................ 166 Holmes, Rebecca J............................ 484 Holton, Nicole................................... 354 Holze, Cathleen................................. 466 Homan, Philip J................................. 429 Hong, Hannah................................... 105 Hoogstraten, Charles G............. 220, 528 Hoon, Shawn..................................... 571 Hoopengardner, Barry......................... 79 Hopkins, Thomas G.......................... 388 Hopper, Anita K......... 96, 383, 461, 585, 590 Hopper, Anita.................................... 603 Hoque, Mainul.................................. 168

Horakova, Eva................................... 373 Horn, Friedemann............................. 260 Horos, Rastislav.................................... 5 Horvath, Peter................................... 288 Hoskins, Aaron A....... 72, 331, 355, 531, 532, 711 Hoskins, Roger.................................. 146 Hossbach, Markus............................. 632 Hotamisligil, Gökhan S..................... 623 Hotamisligil, Gökhan........................ 618 Hotz, Hans-Rudolf............................ 139 Hou, Han Wei...................................... 88 Hou, Ya-Ming................................... 597 Howard, Michael J........................ 4, 584 Hoy, Julie A......................................... 90 Hrit, Joel............................................ 100 Hrossova, Dominika......................... 219 Hsieh, Ping-kun.................................. 53 Huang, Haiyan.................................. 310 Huang, Hsiao-Yun............................. 585 Huang, Qing........................................ 42 Huang, Wei........................................ 418 Huarte, Maite...................................... 21 Hudson, Stephen W........................... 278 Huelga, Stephanie C.......... 107, 235, 571 Huerta, Josefina................................. 701 Hui, Alice Y...................................... 468 Hui, Jingyi........................................... 43 Humphreys, David T............... 5, 85, 495 Hundley, Heather A........................... 369 Hunicke-Smith, Scott.......................... 49 Hurtig, Mark..................................... 622 Hurto, Rebecca L.............................. 461 Hutt, Kasey R.................................... 107 Hüttelmaier, Stefan............................. 82 Huvelle, Emmeline........................... 408 Hynes, Carly J................................... 495

I Ibarra, Edgar..................................... 103 Ibba, Michael...................................... 32 Iben, James R.................................... 592 Ichihashi, Norikazu........................... 476 Ichikawa, Makiko............................. 194 Ideue, Takashi................................... 250 Ikeda, Yoshiki........................... 406, 492 Ilagan, Janine...................................... 73 ILGU, Muslum............................ 91, 624 Im, Seongwang................................. 189 Imada, Yumi...................................... 500 Ingolia, Nicholas T............................ 477 Ingram, Ebone................................... 103 Iqbal, Asif.......................................... 455 Irie, Kenji.......................................... 386 Ishtiaq, Muhammad.......................... 611 Itoh, Takashi...................................... 588 Itotia, Patrick..................................... 392 Ivanova, Natalia................................ 296 Author Index – 5

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

Iyer, Vishwanath................................. 49 Izaurralde, Elisa.................................. 58

J J. Lilley, David M. J.......................... 423 Jackman, Jane E....... 370, 372, 377, 594, 596 Jacob, Aishwarya G.......................... 549 Jacobson, Allan S................................ 62 Jaeger, Luc........................................ 172 Jafarifar, Faegheh.............................. 110 Jagadeeswaran, Guru........................ 497 Jahn-Hofmann, Kerstin..................... 632 Jain, Saumya......................................... 8 Jain, Shashank................................... 102 Jain, Swati................................. 430, 431 Jakovljevic, Jelena............................ 335 Jambhekar, Ashwini.......................... 255 Jameson, Nora E................................. 94 Jan, Eric..................................... 301, 380 Jana, Sujata............................... 368, 586 Janbon, Guilhem............................... 405 Jang, Li-Ting..................................... 398 Jangi, Mohini.................................... 550 Jankowsky, Eckhard........ 7, 15, 215, 217 Jarmolowski, Artur............................ 538 Jayaseelan, Sabarinath...................... 269 Jean-Jacques, Nina B........................ 471 Jensen, Jan K....................................... 59 Jeschke, Grace R............................... 234 Jeske, Mandy....................................... 84 Jha, Shantenu.................................... 418 Ji, Xinjun........................................... 277 Ji, Zhe................................................ 168 Jia, Jieshuang.....................................111 Jiang, Lihua....................................... 104 Jiang, Tian......................................... 470 Jiang, Xiaohong................................ 114 Jinek, Martin..................................... 259 Joardar, Archi............................ 368, 586 Jodelka, Francine M.......................... 109 Jogl, Gerwald...................................... 34 Johansen, Steinar D........................... 118 John, Matthias................................... 632 Johnson, John E................................ 333 Johnson, Marc C............................... 625 Johnson, Richard............................... 227 Johnson-Buck, Alexander................. 508 Joiner, Cassandra.............................. 134 Jokhi, Vahbiz..................................... 147 Jones, Christopher P............................ 41 Jones, Thomas E............................... 149 Jonstrup, Anette T............................... 59 Juan, Wen Chun................................ 612 Jung, Seung-Ryoung......................... 486 Jurica, Melissa S................................. 73 Jurica, Melissa................................... 342 Jurkin, Jennifer.................................. 587 Author Index – 6

K K. Kojima, Sabine............................. 500 Kaboord, Barbara.............................. 228 Kagerbauer, Birgit............................. 489 Kahlscheuer, Matthew L............. 68, 344 Kai, Zoya S....................................... 128 Kaida, Daisuke.................................. 169 Kalantari, Roya......................... 127, 240 Kahlscheuer, Matthew...................... 712 Kalsotra, Auinash.............................. 613 Kalyna, Maria................................... 538 Kamba, Pakoyo F.............................. 220 Kanai, Akio...... 214, 268, 406, 485, 492, 583, 588 Kang, Hyun-Seo................................ 576 Kannan, Krishna................................. 38 Kanter, Itamar................................... 463 Kapral, Gary J................................... 431 Karbstein, Katrin................... 31, 45, 299 Karkusiewicz, Iwona......................... 600 Karni, Rotem..................................... 614 Kasack, Katharina............................. 260 Kastner, Berthold........................ 74, 460 Kaszynska, Aleksandra..................... 267 Kath-Schorr, Stephanie............. 158, 455 Katolik, Adam................................... 227 Kaufman, Thom................................ 146 Kaur, Moninderpal............................ 209 Kavaliauskas, Darius........................... 33 Kawai, Gota........................................ 47 Kawashima, Tadashi R...................... 551 Kaymak, Ebru................................... 221 Kazan, Hilal...................................... 235 Kazuta, Yasuaki................................. 476 Keating, Christine D......................... 125 Keating, Kevin S............................... 432 Kebaara, Bessie W............................ 407 Keegan, Liam P................................... 80 Keller, Brian A.................................. 608 Kenmochi, Naoya............................. 589 Keppetipola, Niroshika A.................. 552 Kerem, Batsheva............................... 620 Kern, Thomas.................................... 576 Kervestin, Stephanie......................... 408 Khanova, Elena................................. 328 Khromykh, Alexander A..................... 26 Kielkopf, Clara L................................ 61 Kierzkowski, Daniel......................... 538 Kikuzato, Ikuya................................. 500 Kim, Eunji......................................... 486 Kim, Hajin........................................... 29 Kim, Joohyun.................................... 418 Kim, Kyoung Mi............................... 399 Kim, Sang Hoon............................... 318 Kim, Taejin................................ 433, 628 Kim, Yoon Ki.................................... 399 Kim, Yoon-Jin................................... 318 Kimura, Yuichi.................................. 386

Kinoshita, Shigeharu......................... 500 Kirchhausen, Tomas............................ 76 Kirsebom, Leif A............................... 509 Kitabatake, Makoto........................... 101 Kleckler, Megan M............................. 91 Kleiman, Lawrence............................. 41 Klein, Jason J.................................... 632 Knejzlik, Zdenek............................... 332 Knight, Rob....................................... 176 Knudsen, Charlotte R.......................... 33 Ko, Tun Kiat..................................... 612 Kobori, Shungo................................. 533 Kohlbacher, Oliver............................ 222 Koo, Bon-Kyung............................... 116 Koon, Alex........................................ 147 Koreny, Ludek................................... 598 Korlach, Jonas................... 190, 195, 449 Korostelev, Andrei...................... 28, 296 Kotolik, Adam..................................... 97 Koutmos, Markos.......................... 4, 584 Kowal, Justyna.................................. 600 Kozak, Karol..................................... 288 Kozlova, Natalia V............................ 615 Kraft, Jelena...................... 293, 469, 475 Krainer, Adrian.................................. 614 Krämer, Angela................................. 576 Kramer, Emily B............................... 590 Kramer, Katharina....................... 44, 222 Krans, Amy....................................... 112 Krasilnikov, Andrey S....................... 328 Krijgsveld, Jeroen................................. 5 Krishnan, Nithya............................... 323 Krishnan, Ramya................. 68, 344, 712 Krishnan, Vishalakshi....................... 241 Krishnan, Yamuna....................... 40, 256 Krivos, Kady L.................................. 367 Krokan, Hans E................................. 251 Krueger, Susan.................................. 453 Kruk, Jennifer A.................................. 11 Krzyszton, Michal............................. 261 Kubicek, Karel.......................... 121, 219 Kucukural, Alper................................... 9 Kudoh, Jun........................................ 500 Kuersten, Scott.............................. 48, 49 Kufel, Joanna.................... 261, 591, 599 Kulathinal, Rob................................. 103 Kuligovski, Crisciele......................... 326 Kumar, Amit...................................... 134 Kumar, Sandeep.......................... 87, 292 Kundu, Sucharita............................... 242 Kung, Sam........................................ 353 Kushner, David B.............................. 471 Kushner, Sidney R.............................. 54 Kutay, Ulrike............................. 288, 290 Kutscha, Paul D................................ 269 Kuttan, Ashani..................................... 78 Kwok, Chun Kit................................ 187

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

L Lach, Grzegorz.................................... 52 Lackey, Patrick E................................ 98 Lacroix-Labonté, Julie...................... 510 Ladd, Andrea N................................. 205 Ladewig, Erik.................................... 493 Laederach, Alain...... 124, 176, 344, 436, 440, 446, 610 Lafontaine, Daniel..... 155, 451, 502, 707 Lagergren, Jens................................... 81 Lagier-Tourenne, Clotilde................. 107 Lai, Eric C......................................... 493 Lai, Lien B................................ 197, 329 Lai, Ming-Chih................................. 387 Lal, Ashish.......................................... 20 Lam, Hugo........................................ 104 Lam, TuKiet T........................... 323, 392 Lamandé, Shireen R.......................... 403 Lambowitz, Alan M...................... 14, 49 Lamichhane, Rajan........................... 223 Lamichhane, Tek N........................... 592 Lamm, Monica H.......................... 90, 91 Lamond, Angus................................. 564 Lamontagne, Anne-Marie................. 707 Lancaster, Laura.......................... 28, 307 Landolin, Jane M.............................. 541 Landry, Dori M................................. 305 Landweber, Laura............................. 309 Lang, Andrew...................................... 37 Lange, Margaret J............................. 625 Langer, Creitekya.............................. 320 Lareau, Liana F................................. 311 Larralde-Ridaura, Rosa....................... 94 Larson, Amy M................................. 345 Lasonder, Edwin............................... 615 Lassnig, Caroline.............................. 466 Lau, Matthew WL............................. 511 Lau, Nelson....................................... 130 Lawler, Mariah.................................. 357 Lays, Claire....................................... 132 Le Borgne, Mailys............................ 224 Le Grice, Stuart F................................ 42 Le Hir, Herve...................................... 10 Ledoux, Sarah................................... 346 Lee, EunHee...................................... 392 Lee, Fang-Jen S................................. 398 Lee, Ho Young.................................. 243 Lee, Jae-Hyung......................... 177, 378 Lee, Jaewoo....................................... 102 Lee, Ju Youn........................................ 56 Lee, Kuang-Yung.............................. 105 Lee, Kuo-Ming.................................. 400 Lee, Lawrence................................... 195 Lee, Sujin.......................................... 347 LeFave, Clare.................................... 397 Legault, Pascale................ 212, 421, 510 Legiewicz, Michal............................... 42 LeGrice, Stuart F............................... 119

Lei, Wei............................................. 567 Leidel, Sebastian............................... 374 Lejeune, Fabrice.................................111 Lemieux, Sébastien........................... 510 Lemm, Ira.......................................... 460 Lena, Sokol....................................... 262 Lener, Daniela................................... 138 Lenis, Diana A................................... 272 Lentz, Jennifer J................................ 109 Leong, Kam....................................... 102 Leontis, Neocles B.............. 50, 176, 180 Leppek, Kathrin.................................. 83 Leszyk, John......................................... 9 Létoquart, Juliette............................. 376 Leulliot, Nicolas................................ 113 Lévesque, Dominique....................... 518 Levin, Howard A............................... 624 Lewis, David L.................................. 632 Lewis, Helen..................................... 339 Li, Gang............................................ 177 Li, Gene-Wei..................................... 391 Li, Hua.............................................. 249 Li, Jingyi Jessica............................... 310 Li, Li................................................. 593 Li, Moyi............................................ 105 Li, Nan-Sheng........................... 158, 359 Li, Q. Quinn...................................... 284 Li, Qian............................................. 147 Li, Qin............................................... 553 Li, Wei............................................... 487 Li, Wencheng.................................... 168 Li, Xia............................................... 386 Li, Xueni........................................... 362 Li, Yan............................................... 409 Li, Yihang.......................................... 147 Li, Yue............................................... 306 Li, Zhi............................................... 593 Li, Zhongwei....................................... 55 Liang, Jonathan J.............................. 512 Liang, Tiffany Y................ 225, 235, 571 Liang, Wen-Wei................................ 348 Libri, Domenico................................ 219 Licatalosi, Donny D.......................... 144 Liebhaber, Stephen A........................ 277 Liger, Dominique.............................. 113 Lilley, David M......................... 158, 455 Lim, Wan Hsin.............................. 4, 584 Limbach, Patrick A............ 186, 367, 598 Lin, Chia-Ho............................. 553, 575 Lin, Dana............................................. 94 Lin, Ren-Jang.................................... 349 Lin, Ting-Yu...................................... 349 Linares, Anthony J............................ 554 Lindell, Magnus.................................. 57 Linneman, Jan................................... 335 Linsley, Peter..................................... 254 Lioliou, Efthimia............................... 132 Lipchock, James................................ 574

Li-Pook-Than, Jennifer..................... 104 Lipovich, Leonard............................. 270 Lipp, Jesse......................................... 555 Liu, Chaochun................................... 173 Liu, Fei.......................................... 15, 24 Liu, Fenyong............................. 114, 136 Liu, Guodong.................................... 353 Liu, Jessica F....................................... 93 Liu, Jia............................................... 513 Liu, Jui-wen........................................ 51 Liu, Qi................................................. 35 Liu, Qiang......................................... 169 Liu, Qinghua..................................... 242 Liu, Wei............................................... 33 Liu, Xin............................................. 188 Liu, Xingyin...................................... 616 Lobo, Vincent.................................... 353 Logan, Charlotte............................... 130 Long, Dang....................................... 173 Long, Yicheng........................... 370, 372 Lotti, Francesco................................. 282 Lou, Hua........................................... 417 Louis, Elan........................................ 112 Lovci, Michael.................................. 225 Lowe, Phillip....................................... 92 Lowe, Todd M................... 166, 577, 579 Lu, Jun................................ 63, 158, 359 Lu, Laura........................................... 131 Lu, Sangwei.............................. 114, 136 Lu, Zhipeng....................................... 226 Luciano, Dan....................................... 53 Luebbert, Collin................................ 428 Luft, Joseph....................................... 392 Lührmann, Reinhard............ 44, 74, 108, 117, 460, 564 Lui, Lauren M................................... 166 Łukasik, Anna................................... 178 Lukeš, Julius............................. 373, 598 Lünse, Christina E............................. 263 Luo, Guangxiang............................... 305 Luo, Wenting..................................... 168 Luo, Yiling........................................ 514 Luptak, Andrej............................ 94, 302 Lussier, Antony......................... 155, 502 Lusvarghi, Sabrina.............................. 42 Luthey-Schulten, Zan........................ 593 Lutz, Carol S..................................... 275 Luuk, Tiit.......................................... 593 Lyle, Robert....................................... 251 Lynch, Kristen W........ 66, 145, 210, 574 Lyons, Shawn...................................... 98 Lyubchenko, Yuri L........................... 392

M Ma, Enbo........................................... 245 Ma, Wai Kit....................................... 319 Ma, Yinghong...................................... 63 MacDougall, Daniel D...................... 710 Author Index – 7

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

Machado-Pinilla, Rosario................. 113 Macias, Sara........................................ 25 Madhani, Hiten D.............................. 244 Madhani, Hiten................................. 140 Madl, Tobias...................................... 576 Maeder, Corina.................................. 350 Mahadevan, Kohila................... 148, 706 Mahoney, John M.............................. 207 Maimon, Avraham............................. 614 Mains, Jodi E.................................... 557 Mair, Barbara.................................... 602 Mair, Gunnar R................................. 615 Maiväli, Ülo...................................... 413 Majumder, Mrinmoyee..... 364, 368, 371 Mak, Ho Yi........................................ 249 Makarov, Evgeny M.......................... 460 Makino, Debora L............................... 60 Malhotra, Arun.................................. 276 Mallam, Anna L.................................. 14 Mallick, Bibekanand......................... 173 Manchanda, Mini.............................. 105 Mangelsdorf, Marie........................... 619 Manivannan, Sathiya N..................... 329 Mankin, Alexander S.................. 38, 285 Mann, Matthias................................. 620 Manor, Miriam.................................. 620 Mao, Annie........................................ 464 Mao, Guanzhong............................... 509 Maquat, Lynne E......................... 61, 702 Maracci, Cristina................................. 30 Maraia, Richard J...................... 410, 592 Marchfelder, Anita............................ 591 Marcia, Marco..................................... 67 Marek, Matt....................................... 479 Marek, Matthew S............................. 435 Marino, John P.................................. 453 Markmiller, Sebastian....................... 107 Marks, Paul....................................... 622 Maroney, Patricia.............................. 281 Marr, Henry....................................... 225 Marti, Eulalia.................................... 489 Martin, Joshua S...... 124, 344, 436, 440, 446, 610 Martindale, Jennifer L......................... 21 Martinez, Javier................. 587, 595, 602 Martinez-Abarca, Francisco.............. 506 Martinis, Susan A.............................. 593 Marvin, Michael C............................ 555 Marzluff, William F............ 98, 323, 392 Masatoshi, Tsukahara........................ 500 Mason, Philip J......................... 330, 609 Masquida, Benoît.............................. 118 Massé, Eric........................ 135, 155, 264 Massi, Francesca......................... 12, 394 Massirer, Katlin B............................. 488 Massirer, Katlin................................. 235 Masuda, Stuart J.................................. 94 Masuda, Takeshi........................ 583, 588 Author Index – 8

Mateju, Daniel................................... 332 Matera, A. Gregory........... 226, 558, 617 Mathews, David H....... 46, 99, 174, 176, 379, 426 Matsuda, Emiko.................................. 42 Matsuura, Tomoaki........................... 476 Matthews, Keith R............................ 334 Mattijssen, Sandy.............. 410, 592, 601 Matts, Jessica.................................... 130 Matuszek, Zaneta.............................. 599 Matylla-Kulinska, Katarzyna............ 273 Mauri, Francesco............................... 388 Maxwell, Adam WR......................... 503 May, Gemma............................. 146, 541 Mayer, Günter................................... 263 Mayerle, Magan.................................. 29 Mazur, Curt....................................... 107 McCaffrey, Anton P.................. 629, 630 McCaffrey, Kate E............................ 109 McCann, Michael D.......................... 448 McClory, Sean P............................... 297 McCown, Phillip J.................... 512, 515 McDonald, Megan E......................... 298 McElroy, Stuart................................. 564 McGlincy, Nicholas J........................ 556 McGurk, Leeanne............................... 80 McKay, David................................... 271 McLachlan, Alan............................... 632 McManus, Joel.................................. 557 McNally, Mark T............................... 278 McNeil, Bonnie A............................. 157 McPhee, Scott A................................ 423 McReynolds, Larry........................... 201 Mechtler, Karl................................... 587 Meera, Pratap.................................... 137 Meers, Michael................................. 558 Mehtab, Shabana......................... 40, 256 Meier, Scott....................................... 228 Meier, U. Thomas............................. 113 Meisler, Miriam H............................. 137 Meisner-Kober, Nicole...................... 262 Meister, Gunter................................. 489 Mele, Aldo......................................... 144 Mendes, António............................... 615 Mentis, George.................................. 282 Mercer, Julian F................................ 403 Meyer, Mélanie................................. 118 Meyer, Michelle M........................... 265 Mias, George..................................... 104 Michael, Theodora............................ 388 Michal, Szczesniak........................... 238 Michaux, Jonathan............................ 437 Micklefield, Jason............................... 92 Midura, Devin H............................... 303 Mikolajczak, Katarzyna.................... 178 Milanowska, Kaja....................... 52, 178 Milcarek, Christine........................... 320 Milkereit, Philipp.............................. 335

Millar, David P.................................. 223 Miller, Christopher S......................... 214 Miller, Jason E.................................... 11 Miller, W. Allen................. 468, 469, 475 Miller, W.Allen................................. 293 Mills, Jason A.................................... 609 Milón, Pohl......................................... 30 Mina, Mazdak..................................... 90 Minderman, Hans.............................. 323 Minia, Igor........................................ 401 Miñones-Moyano, Elena................... 489 Mirkovic-Hoesle, Milijana................ 490 Mishler, Dennis M............................ 154 Misteli, Tom...................................... 566 Miswan, Zulaika............................... 505 Mitchell, Michelle............................. 119 Mitchell, Phil.................................... 414 Mitchell, Sarah F................................... 8 Mitsuyama, Susumu.......................... 500 Miwa, Yukino.................................... 500 Mizuno, Tomoaki.............................. 386 Mjelle, Robin.................................... 251 Mlynsky, Vojtech.............................. 159 Moda, Rachel.................................... 103 Modzelewski, Andrew J.................... 484 Mogensen, Estelle............................. 405 Mohammad, Fuad............................. 372 Mohan, Apoorva............................... 105 Mohandas, Narla............................... 164 Mohanty, Bijoy K................................ 54 Mohi El Din, Hatem.......................... 279 Mohr, Sabine....................................... 49 Molden, Rosalynn C......................... 125 Moldon, Alberto................................ 351 Mondol, Vanessa....................... 266, 488 Monecke, Thomas............................... 84 Montemayor, Eric J........................... 227 Moon, Stephanie L.............................. 26 Moore, Melissa J...... 9, 62, 72, 147, 161, 185, 192, 355 Mora, Liliana..................................... 408 Morales, Christopher A1724H.......... 560 Moresco, James......................... 140, 244 Morita, Misato................................... 250 Moritz, Bodo....................................... 84 Morris, Dan....................................... 527 Morris, Quaid.................................... 235 Morrissey, David............................... 262 Morse, Daniel P................................. 516 Moss, Walter N......................... 179, 470 Mouzakis, Kathryn.............................. 37 Mowry, Kimberly L.......................... 462 Mozaffari Jovin, Sina................ 108, 117 Mu, Shirong........................................ 43 Mueller, Berndt................................. 280 Mueller, William F............................ 559 Mühlemann, Oliver........................... 411 Mulhbacher, Jérôme.......................... 707

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

Müller, Christoph W......................... 376 Müller, Mathias................................. 466 Müller, Ulrich F................................ 517 Mundigala, Hansini R....................... 437 Munroe, Stephen H........................... 560 Mura, Manuela.................................. 388 Murakami, Shinnosuke............. 406, 492 Murata, Asako........................... 189, 482 Murawski, Allison M........................ 471 Murigneux, Valentine.......................... 10 Murphy, Frank V................................. 34 Murray, Laura................................... 430 Musier-Forsyth, Karin.......... 41, 87, 191, 232, 287, 292 Mustoe, Anthony M.................. 123, 520 Myrphy, Daniel................................. 561

N Nabet, Behnam.................................. 548 Nagel, Roland................................... 703 Nagy, Vivien...................................... 363 Nair, Satish........................................ 593 Nakahigashi, Kenji............................ 492 Nakamori, Masayuki......................... 106 Nakamura, Shingo............................... 47 Nakamura, Takahisa.................. 618, 623 Nakanishi, Kotaro............................... 13 Nakao, Mistuyoshi............................ 250 Nakatani, Kazuhiko................... 189, 482 Nangreave, Jeanette.......................... 508 Narasimhan, Jay................................ 208 Narayanan, Ramesh.......................... 619 Natarajan, Prashanthi................ 140, 244 Neff, Ashley T..................................... 56 Negoro, Kosuke................................ 500 Nelson, Hosea................................... 196 Nelson, Stanley F.............................. 177 Nemeth, Laura................................... 366 Nesbitt, David J................................. 708 Neufeld, Noa..................................... 463 Neugebauer, Karla M................ 141, 463 Neulander, Lauren............................. 436 Nevins, Colin E................................. 516 Nezuo, Maiko.................................... 500 Ng, King Pan..................................... 612 Ni, Ting............................................. 202 Nicholson, Pamela.................... 280, 411 Nielsen, Henrik................................. 118 Niemela, Elina H................................. 95 Nieves, Johnathan L.......................... 471 Niewoehner, Ole............................... 259 Nikonowicz, Edward P..................... 438 Niles, Jacquin C............................ 88, 93 Nilsen, Timothy W............................ 281 Nilsen-Hamilton, Marit......... 90, 91, 624 Nilsson, Per......................................... 57 Nimjee, Shahid.................................. 102 Nishimura, Kanako........................... 250

Nissan, Tracy.................................... 412 Nissbeck, Mikael................................. 57 Noland, Cameron.............................. 245 Noller, Harry F............ 28, 294, 296, 307 Noro, Emiko...................... 268, 485, 492 Norris, Adam..................................... 562 Nousch, Marco.................................... 85 Novak, Megan N......................... 31, 299 Novak, Thaddeus.............................. 359 Novikova, Irina V............................... 22 Novoa, Eva M................................... 170 Novotny, Ivan.................................... 332 Nunnari, John.................................... 147

O O’ Loughlin, Kieran.......................... 323 Obar, Robert A.................................. 146 O’Brian, Janelle E............................. 137 O’Connell, Mary A.............................. 80 Oh, Seok Yoon.................................. 112 Ohler, Uwe........................................ 202 Ohman, Marie............................. 81, 365 Ohmayer, Uli..................................... 335 Ohno, Mutsuhito............................... 101 Okamura, Katsutomo........................ 493 Olejniczak, Mikolaj........................... 267 Oliveira, Arthur V............................. 457 Oliveira, Carla C............................... 704 Oliver, Brian...................................... 146 Olson, Erik D...................................... 41 Olson, Karen E.................................. 517 Olson, Sara................................ 146, 195 Omichinski, James G........................ 212 Oney, Sabah...................................... 102 Ong, Sin Tiong.................................. 612 Opperman, Kay................................. 228 Oren, Yifat S..................................... 620 Osborn, Maire F................................ 534 Otis, Thomas S.................................. 137 Ottesen, Eric W................................. 563 Otyepka, Michal........................ 159, 523 Ouellet, Jonathan............................... 455 Oviedo, Mariana............................... 133

P Pachulska-Wieczorek, Katarzyna..... 229 Padgett, Richard A.................... 110, 321 Paier, Anton....................................... 413 Palazzo, Alexander F....... 148, 161, 459, 706 Palencia, Andrés................................ 593 Pan, Dongli....................................... 389 Pan, Tao..................... 149, 170, 190, 439 Panaro, Brandon................................ 448 Panchapakesan, Shyam S.................. 133 Panja, Subrata................................... 494 Paramasivan, Vijayapalani................ 468 Paris, Zdenek............................. 367, 373

Park, Kyung SO................................ 320 Park, Seung Gu................................. 399 Parker, Brian J..................................... 85 Parker, Roy.................................... 8, 385 Paro, Simona....................................... 80 Parra, Marilyn K............................... 164 Parra, Marilyn................................... 225 Pasquinelli, Amy E... 128, 266, 488, 499 Passmore, Lori A............................... 381 Pasulka, Josef.................................... 121 Patel, Anooj....................................... 116 Patel, Dinshaw J.................................. 13 Patel, Dinsnaw J................................ 447 Patel, Hardip R............................ 85, 495 Patel, Krishna.................................... 594 Patrick, Eric M.................................. 191 Patwardhan, Anand R.................. 40, 256 Paul, Natasha..................................... 629 Paulson, Henry L............................... 112 Pavon-Eternod, Mariana................... 170 Pawellek, Andrea.............................. 564 Pedrioli, Patrick................................. 390 Pei, Shermin...................................... 265 Pelchat, Martin.......................... 204, 472 Pellizzoni, Livio................................ 282 Pena, Vladimir............................ 44, 117 Penedo, Juan C.................................. 451 Peng, Guangdun................................ 177 Peng, Jianhe...................................... 460 Peng, Weiqun.................................... 202 PenÌ¿a-Diaz, Javier........................... 251 Penninger, Josef................................ 602 Percifield, Ryan................................. 561 Perdrizet II, George A....................... 439 Perederina, Anna............................... 328 Perez, Denise..................................... 607 Perez, Saida G................................... 488 Pergoli, Roberto................................ 219 Perona, John.............................. 300, 578 Perreault, Jean-Pierre........ 274, 518, 521 Perrett, Andrew J............................... 339 Perriman, Rhonda............................. 342 Pesch, Marion................................... 374 Peter, Matthias.................................. 390 Peterson, Mariko............................... 469 Petrov, Anton I............................ 50, 180 Pfeiffer, Jana..................................... 374 Philbin, Padriac................................. 323 Philips, Anna....................................... 52 Phillips, Dan...................................... 448 Phillips, Gabriela....................... 440, 610 Phizicky, Eric M.......................... 86, 366 Piccirilli, Joseph A............ 158, 359, 452 Pichlmair, Andreas............................ 466 Pierce, Niles A................................... 196 Pikovskaya, Olga.............................. 447 Pimienta, Genaro............................... 483 Pinto, Anna Maria............................. 169 Author Index – 9

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

Pitchiaya, Sethu................................. 129 Pitchiaya, Sethuramasundaram......... 241 Pitoc, George..................................... 102 Plakos, Kory...................................... 441 Plass, Mireya....................................... 25 Podell, Elaine.................................... 271 Podyma, Brandon M........................... 86 Polioudakis, Damon............................ 49 Pollom, Elizabeth.............................. 473 Polonskaia, Anna............................... 447 Polymenidou, Magdalini................... 107 Pontvianne, Frédéric......................... 138 Popović, Milena................................ 442 Popow, Johannes....................... 587, 595 Porrua-Fuerte, Odil........................... 219 Posakony, Jeffrey.............................. 519 Pospieszny, Henryk................... 467, 480 Pospisek, Martin............................... 283 Post, Christina................................... 130 Potabathula, Divya............................ 314 Potter, Elizabeth L............................. 473 Poudyal, Raghav R............................ 503 Prasanth, Kannanganattu V................. 20 Prasanth, Supriya G............................ 20 Pratt, Catherine A.............................. 462 Praveen, Kavita................................. 617 Preiss, Thomas........................ 5, 85, 495 Price, Argenta.................................... 352 Priore, Salvatore F.................... 179, 470 Pruijn, Ger JM............................. 95, 601 Pugh, B. Franklin................................ 11 Purzycka, Katarzyna J................. 42, 229 Puta, Frantisek................................... 546 Putman, Andrea................................. 215 Putnam, Andrea................................... 15 Pyle, Anna M.............. 67, 363, 432, 506

Q Qin, Peiwu.......................................... 36 Qin, Peter.......................................... 443 Qin, Yidan........................................... 49 Qiu, Caihong....................................... 63 Quan, Chao....................................... 328 Quarles, Kaycee A............. 122, 248, 445 Quek, B. Lin...................................... 477 Query, Charles C............... 185, 351, 537

R Raczynska, Katarzyna D................... 538 Radek, Agnes...................................... 48 Raines, Ronald T............................... 532 Rammelt, Christiane........................... 82 Ramos, Silvia B................................ 230 Ramsey, Jolene.................................. 362 Ranjan, Namit................................... 390 Ranji, Arnaz...................................... 206 Rao, Bhalchandra S........................... 596 Rappsilber, Juri................................. 354 Author Index – 10

Rasche, Nicolas................................... 44 Rattray, Alexander............................. 280 Rawlings, Renata A........................... 621 Raz, Erez........................................... 374 Razanau, Aleh................................... 353 Reed, Robin....................................... 565 Reenan, Robert A................................ 79 Reese, Joseph C.................................. 11 Refaei, Maryanne.............................. 232 Regehr, Monika................................. 316 Reich, Ashley A................................. 258 Reiche, Kristin.................................. 260 Reinke, Lauren.................................. 607 Reitter, Sonja....................................... 83 Rempinski, Donald........................... 323 Ren, Qian.......................................... 301 Rennie, William A............................. 173 Renoux, Abigail................................ 112 Reymond, Cedric.............................. 518 Reynolds, Noah M.............................. 32 Rezgui, Vanessa................................ 390 Rhind, Nick....................................... 185 Ribas de Pouplana, Lluís................... 170 Richards, Jamie................................... 53 Richardson, David C......................... 431 Richardson, Jane S.................... 430, 431 Richie, Ashley C......................... 70, 331 Rieder, Leila E.................................... 79 Rigo, Frank....................................... 109 Riley, Kasandra................................... 64 Rinaldi, Arlie J.......................... 123, 520 Rino, José............................................ 76 Ritz, Justin....... 124, 176, 436, 440, 446, 610 Robert, Marie-Cécile......................... 138 Roberts, Brett.................................... 474 Robertson-Anderson, Rae M............ 223 Robinson, Christopher J...................... 92 Roca, Xavier..................................... 612 Rocca, Gina....................................... 626 Rocca-Serra, Philippe....................... 176 Rocco, Gina............................... 397, 631 Rodgers, Margaret L......................... 331 Rodnina, Marina V.............................. 30 Roest Crollius, Hugues....................... 10 Roll, James E.................................... 180 Romby, Pascale................................. 132 Romilly, Cedric................................. 132 Roque, Sylvain.................................. 408 Rosenberg, Oren S............................ 343 Rossi, John J............................. 629, 630 Roszak, Anna.................................... 267 Roth, Adam....................................... 515 Roth, Frederick P...................... 161, 706 Rother, Kristian........................... 52, 178 Rouleau, Samuel G........................... 521 Rouskin, Silvi...................................... 48 Rowley, Jordan.................................... 19

Roy, Christian K................................ 192 Roy, Kevin........................................ 231 Rozema, David B.............................. 632 Ruan, Yijun....................................... 612 Rubio, Mary Anne T......... 367, 373, 375 Rudolph, Thomas................................ 84 Rueda, David..... 191, 295, 437, 449, 454 Rülicke, Thomas............................... 466 Ruminski, Dana J.............................. 302 Russell, J. Eric................................... 234 Russnes, Hege................................... 260 Ryder, Sean P...................................... 12 Ryder, Sean....................................... 221 Rye, Inga H....................................... 260 Rymelska, Natalia............................. 480 Rymond, Brian.................................. 257

S Saadatmand, Jenan.............................. 41 Sachsenberg, Timo............................ 222 Sachsenmaier, Nora.......................... 444 Sætrom, Pål....................................... 251 Sahu, Debashish........................ 122, 445 Saieva, Luciano................................. 282 Sakaguchi, Reiko.............................. 597 Sakai, Kosuke................................... 500 Sakai, Tokie....................................... 101 Sakakibara, Yogo.............................. 291 Sakata, Tomoko................................. 101 Saks, Margaret E............................... 303 Salati, Lisa M.................................... 569 Salati, Lisa......................................... 341 Salton, Maayan................................. 566 Saltzman, Mark................................. 627 Samadzadeh-Tarighat, Somayeh....... 606 Samatov, Timur................................. 564 Sample, Paul J........................... 373, 598 Samuel, Nebiyou............................... 103 Sanbonmatsu, Karissa Y..................... 22 Sanders, Wes............................. 446, 610 Sandler, Jeremy E............................. 541 Sanford, Brianne............................... 232 Santos, Karine F........................ 117, 354 Saraiya, Ashesh A...................... 487, 496 Sashital, Dipali G........................ 16, 338 Sato, Asako....................... 214, 406, 588 Sato, Hanae....................................... 702 Sato, Yuki.......................................... 500 Sattler, Michael................................. 576 Sauliere, Jerome.................................. 10 Sawyer, Andrew W..................... 89, 503 Schaffer, Michelle F.......................... 522 Schagat, Trista................................... 100 Schiller, Benjamin............................. 140 Schlatterer, Joerg C........................... 336 Schleiffer, Alexander......................... 595 Schmidt, Karyn................................... 99 Schmidt, Magnus S........................... 263

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

Schmitzova, Jana................................. 44 Schmitzová, Jana................................. 74 Schott, Johanna................................... 83 Schrader, Jared M............................... 33 Schrader, Jared.................................. 391 Schroeder, Kersten T......................... 423 Schroeder, Renée.............................. 273 Schroeder, Susan J.............................. 51 Schuch, Benjamin............................. 414 Schuermann, Jonathan...................... 227 Schultz, J. S....................................... 228 Scott, Fraser J.................................... 263 Sedaghat, Yalda................................. 107 Sen, Dipankar.................................... 156 Sen, Taner Z........................................ 91 Seo, Joonbae..................................... 563 Séraphin, Bertrand.................... 304, 376 Serebrov, Victor.......................... 62, 355 Serganov, Alexander......................... 447 Serra, Martin J................................... 448 Seweryn, Paulina................................. 59 Shaffer, Scott......................................... 9 Shamoo, Yousif................................. 438 Shandilya, Shivender.......................... 12 Shankarling, Ganesh........... 66, 145, 210 Shapiro, Bruce A............... 172, 433, 628 Shapiro, Lucy.................................... 391 Sharma, Amit.................................... 382 Sharma, Sahil.................................... 415 Sharma, Shalini........................... 69, 572 Sharma, Tarun K............................... 625 Sharp, Phillip A......... 131, 253, 539, 550 Shav-Tal, Yaron................................. 463 Shcherbakova, Inna........................... 355 She, Meipei........................................... 8 Sheeter, Dennis................................... 49 Sheik, Daniel..................................... 632 Shen, Haihong................................... 356 Shen, Manli............................... 257, 568 Shen, Zhen.......................................... 20 Shephard, Lindsay............................. 133 Sherman, Eileen M............................ 153 Shi, Mary............................................ 27 Shi, Min............................................ 458 Shi, Yongsheng................................. 167 Shimizu, Atsushi............................... 500 Shimizu, Nobuyoshi.......................... 500 Shiue, Lily......... 105, 107, 137, 553, 571 Shlyakhtenko, Lyudmila S................ 392 Shokat, Kevan M.............................. 555 Shore, Sabrina................................... 629 Showalter, Scott A............. 122, 248, 445 Shu, Mei-Di........................................ 27 Shuie, Lily......................................... 703 Shukla, Girish C................................ 497 Shulha, Hennady P............................ 185 Sibbritt, Tennille.................................. 85 Sidote, David J.................................... 14

Siefert, Soenke.................................. 119 Sigel, Roland K.O..................... 507, 522 Sikand, Kavleen................................ 497 Silberberg, Gilad............................... 365 Simcox, Amanda............................... 329 Simon, Anne E.................................. 471 Simon, Dawn M................................ 157 Simoneau-Roy, Maxime................... 155 Singer, Robert H............................... 702 Singh, Aditi....................................... 401 Singh, Guramrit..................................... 9 Singh, Jarnail..................................... 321 Singh, Larry N.................................. 169 Singh, Mahavir.................................. 116 Singh, Natalia N........................ 357, 563 Singh, Ravi K............ 543, 549, 567, 613 Singh, Ravindra N..................... 357, 563 Singh, Upinder.................................. 246 Sintim, Herman O............................. 514 Sivanesan, Senthilkumar................... 563 Skorupski, Marcin............................. 178 Skowronek, Ewa............................... 591 Skrisovska, Lenka............................. 120 Slack, Frank...................................... 627 Slevin, Michael................................... 98 Sloan, Katherine E............................ 601 Smith, Alison G................................. 425 Smith, Chris WJ................................ 545 Smith, Lee Ann................................... 79 Smith, Nakesha L...................... 269, 526 Smith, Whitney................................... 49 Snead, Nicholas M............................ 629 Snyder, Michael................................ 104 Sobkowiak, Lukasz........................... 538 Sofos, Nicholas E.............................. 381 Sohail, Muhammad........................... 353 Solem, Amanda................................. 449 Song, Ji-Joon..................................... 486 Sorenson, Matthew R........................ 193 Sosnick, Tobin R............................... 439 Spalitta, Matthew J............................ 109 Sparks, Selene................................... 628 Späth, Bettina.................................... 591 Spears, Jessica L............................... 233 Spector, Deborah............................... 474 Speese, Sean D.................................. 147 Spollen, William................................. 89 Sponer, Jiri........................................ 159 Šponer, Jiſí........................................ 523 Spraggon, Lee........................... 626, 631 Squires, Jeffrey E................................ 85 Srikantan, Subramanya....................... 21 Sripakdeevong, Parin........................ 422 Sripathi, Kamali................................ 523 Srivastava, Abhishek S...................... 509 Stadanlink, Jason E........................... 213 Stafford, Walter F.............................. 392 Staley, Jonathan P............. 337, 358, 359

Stamm, Stefan........................... 257, 568 Stanek, David.................................... 332 Stark, Caren J.................................... 233 Stark, Thomas........................... 235, 474 Stauffer, Eva...................................... 322 Stebler, Michael................................ 288 Stefaniak, Agnieszka......................... 229 Stefl, Richard............................. 121, 219 Steinmetz, Lars M................................. 5 Steitz, Joan A......... 27, 64, 272, 481, 483 Stepien, Piotr P.................................. 416 Stevens, Scott W....................... 193, 315 Stevens, Scott............................ 227, 347 Stillwagon, Samantha J..................... 205 Stimamiglio, Marco A....................... 326 Stoecklin, Georg......................... 83, 415 Stöhr, Nadine...................................... 82 Stoilov, Peter..... 137, 547, 553, 561, 568 Stoltz, Brian...................................... 196 Stombaugh, Jesse.............................. 176 Stone, Jonathan W............................... 51 St-Pierre, Patrick............................... 451 Strein, Claudia....................................... 5 Strong, Michael J...................... 605, 608 Strong, Michael................................. 611 Strulson, Christopher A..................... 125 Strunk, Bethany S............................... 31 Strunk, Bethany................................. 299 Stunnenberg, Rieka........................... 139 Suchanek, Amanda L........................ 569 Suckling, Colin................................. 263 Sudarsan, Narasimhan....................... 504 Suddala, Krishna C................... 123, 520 Sudo, Hiroko..................................... 194 Sugahara, Junichi...................... 214, 583 Sullenger, Bruce................................ 102 Sumita, Minako................................. 528 Sun, H. Sunny................................... 387 Sun, Jun............................................. 616 Sun, Yuliang...................................... 711 Sunker, Ramanjulu............................ 497 Superti-Furga, Giulio........................ 466 Surface, Justin................................... 257 Suslov, Nikolai B.............................. 452 Suter, Catherine M.............................. 85 Suzuki, Takeo.................................... 589 Suzuki, Tsutomu................................ 589 Sveda, Martin.................................... 332 Swami, Sajani..................................... 48 Swamy, Sajani..................................... 49 Swanson, Maurice............................. 105 Swanstrom, Ronald........................... 473 Swett, Rebecca.................................. 134 Swinehart, William E........................ 377 Sylwia, Alaba.................................... 238 Szakal, Andrea L............................... 453 Szczepaniak, Sylwia A...................... 599 Szczesny, Roman J............................ 416 Author Index – 11

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

Szweykowska-Kulinska, Zofia......... 538 Szymczyna, Blair R.......................... 333

T Tajalli, Holleh F................................ 471 Takane, Kahori.......................... 268, 485 Takesue, Kanako............................... 214 Takizawa, Satoko.............................. 194 Tambe, Akshay.................................. 217 Tan, Jacqueline T.............................. 403 Tang, Hua.......................................... 104 Tang, Wen........................................... 23 Tani, Tokio........................................ 250 Tapia-Santos, Aixa S......................... 543 Tapscott, Stephen J............................ 604 Tarn, Woan-Yuh................................ 400 Tarnawsky, Stefan............................. 161 Tat, Trinh T....................................... 281 Taurog, Rebecca E............................ 333 Tay, Wendy................................ 523, 524 Taylor, Alexander B.......................... 227 Taylor, J. Paul.................................... 112 Taylor, John P.................................... 571 Taylor, Nicholas MI.......................... 376 Tedesci, Frank A................................ 217 Tenenbaum, Scott A.......................... 269 Tenson, Tanel.................................... 413 Terai, Ryota....................................... 500 Teruya, Morimi................................. 500 Thapar, Roopa........................... 323, 392 Thaplyal, Pallavi....................... 115, 525 Theimer, Carla A................. 24, 269, 526 Thiaville, Patrick C........................... 364 Thompson, James.............................. 140 Thompson, Sunnie R......................... 305 Thornton, Charles A.......................... 106 Tian, Bin...................................... 56, 168 Tisdale, Sarah.................................... 282 Tishkoff, Sarah A.............................. 621 Todd, Peter K.................................... 112 Tokunaga, Kazuaki........................... 250 Tollervey, David.................. 18, 498, 601 Tomita, Masaru........ 214, 268, 406, 485, 492, 583, 588 Toro, Nicolas..................................... 506 Toroney, Rebecca.............................. 358 Trakhanov, Sergei............................. 307 Tran, Elizabeth J............................... 319 Tran, Elizabeth.................................. 217 Tran, Vy............................................. 246 Trang, Phong..................................... 114 Travers, Kevin................................... 195 Trček Pulisic, Tatjana........................ 702 Treba, Christine N............................... 70 Treder, Krzysztof.............................. 475 Tree, Jai J.......................................... 498 Tripathi, Vidisha.................................. 20 Trotta, Chris R................................... 375 Author Index – 12

Tsai, Shaw-Jenq................................ 387 Tsai, Yu-Chih............................ 190, 449 Tsao, Lulu......................................... 103 Tseng, Chi-Kang................................. 75 Tuck, Alex C....................................... 18 Turner, Douglas H..................... 179, 470 Turner, Stephen W............................ 190 Turowski, Tomasz W........................ 600 Turri, Jacquelyn S............................. 531 Turunen, Janne J............................... 570 Tuschl, Thomas................................. 126 Tuttle, Nicole.................................... 359 Tyagi, Kshitiz.................................... 390 Tycowski, Kazimierz T....................... 27

U Uechi, Tamayo.................................. 589 Ueda, Yoji......................................... 194 Uhlenbeck, Olke C.................. 2, 33, 303 Uhm, Heesoo..................................... 151 Ule, Jernej......................................... 556 Umemoto, Shiori............................... 189 Underwood, Jason G......................... 195 Unrau, Peter J.................................... 133 Upreti, Daya...................................... 357 Urlaub, Henning............ 44, 74, 222, 460 Usui, Kimihito.................................. 476

V Vaidyanathan, Ramesh........................ 48 Valentova, Anna................................ 546 Vallandingham, Jim........................... 249 van Bussel, Inge................................ 556 van der Schans, Edwin JC................. 223 van Dyk, Linda F.............................. 465 Van Etten, Jamie L............................ 100 Van Wynsberghe, Priscilla M............ 499 van Zalen, Sebastiaan........................ 234 Vanacova, Stepanka.......................... 219 Vandenesch, François........................ 132 Vander Meulen, Kirk........................... 37 Vargas-Rodriguez, Oscar.................... 87 Varia, Sapna...................................... 314 Vazquez, Franck................................ 538 Vazquez-Laslop, Nora......................... 38 Verheggen, Céline............................. 138 Verma, Bhupendra............................. 570 Vertino, Paula M............................... 324 Viard, Mathias................................... 628 Vieregg, Jeffrey R............................. 196 Vilfan, Igor D.................................... 190 Vincent, Florence.............................. 132 Virkler, Katherine F............................ 89 Virtanen, Anders................................. 57 Vishnu, Melanie................................ 277 Volkening, Kathryn........... 605, 608, 611 von Pelchrzim, Frederike.................. 273 Vopalensky, Vaclav........................... 283

Vorlova, Sandra................. 397, 626, 631 Vornlocher, Hans-Peter..................... 632 Voss, Ty............................................. 566 Vu, Anthony Q.................. 107, 235, 571 Vu, Gia-Phong........................... 114, 136 Vu, Michael M.................................... 94

W Wachter, Andreas.............................. 322 Wahl, Markus C........................ 117, 354 Wahle, Elmar................................. 82, 84 Wakefield, Darren H......................... 632 Waldsich, Christina........... 360, 444, 456 Wallace, Andrew J............................ 197 Wallace, Robyn................................. 619 Walrad, Pegine B.............................. 334 Walsh, Callee M................................ 569 Walter, Nils G............. 68, 123, 129, 159, 241, 254, 344, 435, 479, 508, 520, 523, 524, 712 Wan, Ji............................................... 277 Wan, Kenneth H................................ 541 Wan, Lili........................................... 169 Wancewicz, Edward.......................... 107 Wandersleben, Traudy....................... 108 Wang, Ching C.......................... 487, 496 Wang, Jiachen................................... 438 wang, Jie........................................... 284 Wang, Jinbu....................................... 119 Wang, Lantian................................... 458 Wang, Liguo...................................... 567 Wang, Qing....................................... 301 Wang, Qingliang............................... 458 Wang, Tianjiao.................................... 90 Wang, Xuya....................................... 407 Wang, Yi............................................ 119 Wang, Yuhong................................... 306 Wang, Yun-Xing................................ 119 Wang, Zhaohui.................................. 475 Wang, Zhen......................................... 10 Wang, Zhonghua............................... 116 Ward, Luke........................................ 527 Warnasooriya, Chandani................... 454 Watabe, Shugo.................................. 500 Waters, Paul D.................................. 560 Watkins, Nick J................................. 601 Weber, Friedemann........................... 466 Weber, Gert............................... 117, 354 Weeks, Kevin M................ 176, 429, 473 Wegener, Jeffrey................................ 190 Wei, Gang................................. 312, 313 Wei, Wenjuan...................................... 43 Weichenrieder, Oliver......................... 58 Weidmann, Chase..................... 100, 393 Weiler, Jan......................................... 262 Weisberg, Chloe................................ 149 Weiss, Adam..................................... 273 Weiss, Mitch..................................... 609

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

Weissman, Jonathan................ 3, 48, 391 Weitzer, Stefan.......................... 587, 602 Wen, Ying.......................................... 617 Weng, Zhiping............................... 9, 185 Westhof, Eric..................................... 118 Whatley, Angela S....................... 89, 625 Whipple, Joseph................................ 366 White, Jonathan D............................. 534 White, Lawrence............................... 622 White, Neil A............................ 220, 528 Whitfield, Michael L......................... 207 Wiedenheft, Blake............................... 16 Wierzbicki, Andrzej T......................... 19 Wiest, David L.................................. 213 Wigington, Callie P........................... 324 Wilbert, Melissa................................ 235 Wilcox, Jennifer L............................. 535 Wild, Thomas.................................... 288 Wildauer, Michael............................. 360 Wilhelmi, Ilka................................... 316 Will, Cindy........................................ 460 Williams, Sandra G........................... 162 Williamson, James R................... 77, 333 Willis, Anne E................................... 305 Wills, Norma..................................... 468 Wilson, Timothy J..................... 158, 455 Wilusz, Carol J.............................. 26, 56 Wilusz, Jeffrey.............................. 26, 56 Wilusz, Jeremy E.............................. 131 Winberg, David E................................ 13 Winnas, Randall................................ 119 Wissink, Erin M................................ 198 Withers, Johanna B........................... 477 Wittkopp, Patricia J........................... 557 Wittmann, Valentin........................... 263 Wohlbold, Lara................................... 58 Wohlschlegel, James......................... 572 Wojciech, Karlowski......................... 238 Wolenc, Adam................................... 173 Wolkowicz, Urszula M..................... 211 Wong, Raymond............................... 464 Wong, Winnie.................................... 216 Wongpalee, Somsakul P.............. 69, 572 Wongwarangkana, Chaninya............ 500 Wood, Emily J................................... 270 Wooddell, Christine I........................ 632 Woodson, Sarah A....................... 29, 494 Woolcock, Katrina J.......................... 139 Woolford, John L.............................. 335 Wostenberg, Christopher........... 122, 248 Wower, Iwona K............................... 199 Wower, Jacek.................................... 199 Wu, Guifen........................................ 458 Wu, Han............................................ 202 Wu, Jingyan...................................... 603 Wu, Ming-Cheng................................ 92 Wu, Ruobin....................................... 136 Wu, Shiying...................................... 509

Wu, Xuebing..................................... 539

Z

X

Zakrzewska-Placzek, Monika........... 261 Zammarchi, Francesca...................... 397 Zamore, Phillip D.............................. 192 Zamudio, Jesse R...................... 253, 539 Zang, Shengbing............................... 349 Zemora, Georgeta............................. 456 Zeng, Chunxi..................................... 529 Zeng, Yan.......................................... 501 Zhan, Ming.......................................... 21 Zhang, Chaolin.............................. 6, 144 Zhang, Chen-Yu................................ 114 zhang, Hanbang................................. 246 Zhang, Hui........................ 148, 161, 706 Zhang, Jinwei.................................... 530 Zhang, Liang..................................... 622 Zhang, Liye......................................... 11 Zhang, Minyou.................................. 392 Zhang, Wenzheng.............................. 362 Zhang, Xiaoju................................... 379 Zhang, Xiaojun................................. 443 Zhang, Xiaoxiao................................ 501 Zhang, Yanhong................................ 236 Zhang, Ying....................................... 249 Zhang, Yong...................................... 213 Zhang, Yun........................................ 576 Zhang, Zhenxi................................... 169 Zhao, Caijie....................................... 705 Zhao, Rui........................................... 362 Zhelkovsky, Alexander..................... 201 Zheng, Dinghai................................. 168 Zheng, Qi............................................ 19 Zheng, Xuexiu Zheng....................... 356 Zhou, Hong....................................... 572 Zhou, Hua-Lin................................... 417 Zhou, Jie...................................... 28, 307 Zhou, Jiehua...................................... 630 Zhou, Katherine I.............................. 363 Zhou, Shu.......................................... 529 Zhou, Ying........................................ 286 Zhou, Yu............................................ 547 Zhu, Jianyu........................................ 296 Zhu, Jun............................................ 202 Zimmerly, Steven.............................. 157 Zimmermann, Bob............................ 273 Zirbel, Craig L..................... 50, 176, 180 Zirkel, Anne........................................ 82 Zisoulis, Dimitrios G........................ 128 Zofia, Szweykowska-Kulinska......... 238 Zong, Xinying..................................... 20 Zou, Xiaobing..................................... 36 Zuo, Xiaobing................................... 119 Zwieb, Christian................................ 199 Zywicki, Marek................................. 273

Xi, Linghe......................................... 271 Xiao, Ming........................................ 306 Xiao, Xinshu............................. 177, 378 Xie, Jiuyong...................................... 353 Xie, Mingyi....................................... 272 Xie, Xiaohui...................................... 167 Xing, Yi............................................. 277 Xu, Tao.............................................. 362 Xu, Yilin.................................... 143, 607 Xu, Zhenjiang............................... 46, 99

Y Yadavalli, Srujana S............................ 32 Yan, Hao............................................ 508 Yan, Jing............................................ 185 Yan, Wensheng.................................. 236 Yandek, Lindsay.................................... 7 Yang, Ao............................................ 212 Yang, Edward.................................... 136 Yang, Huan........................................ 249 Yang, Jing.......................................... 607 Yang, Li............................................. 541 Yang, Maozhou................................. 622 Yang, Quansheng.............................. 237 Yang, Wenjing................................... 202 Yang, Xiaoling.................................... 21 Yang, Yueqin..................................... 573 Yano, Masato.................................... 144 Yano, Shuichi.................................... 500 Yao, Chengguo.................................. 167 Yao, Zizhen....................................... 604 Yarosh, Christopher A....................... 574 Yates III, John........................... 140, 244 Ye, Jingdong...................................... 153 Ye, Xuecheng.................................... 242 Yeh, Chung-Shu........................ 184, 478 Yeh, Fu-lung...................................... 361 Yennamalli, Ragothaman M............... 91 Yeo, Gene W..... 107, 225, 235, 474, 571 Yi, Chengqi....................................... 190 Ying, Yi............................................. 575 Yomo, Tetsuya................................... 476 Yoon, Je-Hyun.................................... 21 You, Bei............................................ 168 Young, Crystal L........................... 31, 45 Young, Lisa....................................... 262 Young, Megan Y............................... 471 Younis, Ihab...................................... 169 Yourik, Paul...................................... 594 Youssef, Osama A............................. 623 Yu, Dongmei....................................... 36 Yu, Jiankun........................................ 353 Yu, Yong............................................ 565 Yuan, Adam Y................................... 364 Yun, Zheng........................................ 497

Author Index – 13

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

NOTES

Author Index – 14

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

KEYWORD INDEX (Note: Numbers refer to abstract numbers, not page numbers) 1-methyl Pseudouridine.................... 364 2’-O-methylation............................... 586 2’-phosphotransferase......................... 96 3’ end processing................................. 60 3’ processing...... 23, 138, 168, 169, 202, 262, 272, 274, 277, 281, 283, 284, 591, 599 3’ splice site recognition..................... 43 3’-5’ polymerization.......................... 372 3D RNA modeling............................ 122 3’-tRNase.......................................... 276 5’ capping............................................ 23 5’ end processing............................... 599 5-methylcytosine................................. 85 5’UTR............................................... 171 8-oxo-G............................................... 55

A absolute quantitation......................... 186 ADAR................................................. 79 adipogenesis...................................... 318 Allele specific expression................. 104 allosteric ribozyme............................ 533 AlREX Element................................ 706 ALS........................................... 611, 619 alternative splicing...... 43, 157, 543, 547 aminoacyl-tRNA synthetase.............. 287 Amyotrophic Lateral Sclerosis........ 605, 608 antibiotics.......................................... 707 Antisense Oligonucleotides.............. 109 Antisense.... 42, 114, 166, 239, 253, 270, 397, 544, 556, 560, 579, 626 antitermination.................................. 529 antiterminator.................................... 513 Aptamer........ 88, 89, 90, 91, 93, 94, 199, 428, 438, 512, 515, 521, 533, 625 aptamers in therapeutics.................... 624 Arabidopsis PTB............................... 322 Arabidopsis....................................... 284 archaea.............................................. 166 archaeal sRNA.................................. 577 Argonaute.......................................... 127 AU-rich elements and AU-BPs......... 340 autophagy............................................ 80 autoregulation................................... 128

B base-flipping........................................ 78 Bioinformatics................................... 432 Bioinformatics: covariation.................................... 256

motif searches...... 6, 27, 50, 163, 180, 225 phylogenetic analysis............ 168, 265 secondary structure prediction....... 46, 51, 124, 172, 174, 176, 179, 424, 426, 436, 440, 470, 610 sequence analysis........... 89, 174, 177, 185, 226, 235, 251, 378, 497, 559 tertiarty structure prediction........ 180, 204, 431 Biosensing......................................... 199 btuB riboswitch................................. 507 btuB................................................... 522

C Cajal body................................. 332, 581 Calcium/calmodulin dependent protein kinase IV............................ 353 Cancer....................................... 388, 612 cap-binding complex......................... 599 Capping............................................. 630 Carcinogenesis.................................. 561 Catalysis............................................ 159 Catalytic Mechanism........................ 158 Ccr4 Pbp1......................................... 386 Ccr4-Not complex......................... 11, 58 cell cycle........................................... 251 cell-free translation system............... 533 Chemical biology..... 176, 189, 193, 228, 339, 503, 519, 531, 532, 535, 544 Chemical synthesis.................... 482, 519 Chimeric Proteins.............................. 552 chromosome segregation.................. 250 circulating miRNA............................ 194 clustered motif sites.............................. 6 Co- and post-transcriptional splicing localization....................... 460 Co-factor effects on RNA helicases.... 45 colon cancer...................................... 387 complementation assay....................... 69 Conformational changes.... 90, 151, 291, 307, 418, 439, 445, 574, 705, 710 conservation.......................................... 6 cooperativity..................................... 153 Co-transcriptional folding................. 439 co-transcriptional splicing................. 565 CRISPR....................................... 16, 259 crosslinking immunoprecipitation.... 173 CTR2................................................. 407

D database............................................. 178

DDX1................................................ 223 DEAD-box protein............................ 319 deadenylation...................................... 82 Ded1p................................................ 215 Deep sequencing............................... 168 degradation.......................................... 55 Development.... 105, 205, 238, 268, 310, 484, 553, 589, 613 developmental gene expression........ 485 Diagnostics.................. 49, 194, 196, 199 Diamond Blackfan Anemia,.............. 609 Dicer.................................................. 243 DICER1 mutation............................. 491 Differentiation................................... 234 Disease....... 80, 105, 106, 107, 109, 110, 112, 183, 254, 260, 388, 389, 467, 480, 491, 498, 568, 571, 604, 607, 608, 609, 610, 612, 614, 619, 622 DNA replication................................ 251 Double-straded RNA......................... 476 Drosophila......................................... 329 Drug discovery.................................. 193 drug................................................... 568 dsRNA binding protein..................... 243 dsRNA............................................... 369 dynamics............................................. 90

E early embryo developemnt................ 230 Editing.... 78, 79, 81, 104, 287, 292, 300, 365, 369, 370, 378 EF-Tu.................................................. 33 eIF4E downregulation....................... 382 eIF4E................................................. 215 eIF4G................................................ 215 elegans....................................... 128, 249 elongation.......................................... 320 EMT.................................................. 143 endonuclease..................................... 591 endo-siRNAs..................................... 490 enrichment analysis........................... 173 Enzymes: deaminase............................... 80, 367 helicase............ 14, 74, 117, 215, 216, 217, 237, 315, 346, 361, 385, 587 ligase..................... 201, 532, 588, 595 nuclease... 57, 60, 178, 261, 375, 400, 406, 584 polymerase............ 321, 372, 476, 596 RNA modification.......... 86, 113, 285, 365, 368, 371, 377, 592 transferase............... 85, 211, 364, 582 tRNA splicing endonuclease........... 96 Keyword– 1

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

tRNA synthetase...... 32, 87, 232, 287, 292, 300, 580, 593 Epigenetic control...... 19, 141, 250, 309, 566 epigenetics......................................... 251 EPR spectroscopy............................. 443 ESRP1............................................... 573 Evolution............ 47, 150, 311, 467, 471, 517, 557 exon junction complex............ 9, 10, 314 exon-intron organization................... 141 exo-siRNAs....................................... 490 Exosome...................................... 59, 279 Expression profiling/microarray analysis............................ 49, 193, 196

F FAU-1............................................... 406 fidelity............................................... 593 fluorescence polarization.................. 188 Folding: dynamics.................. 40, 91, 394, 419, 434, 437, 449, 454, 522, 705 kinetics.................. 152, 419, 578, 708 mechanism.................... 123, 153, 360 methodology......................... 187, 578 thermodynamics.... 47, 179, 448, 578, 708 FR3D................................................. 180 Frameshifting................ 36, 37, 389, 468 FRET................................. 151, 344, 486 FSD-NMD........................................ 397 FSHD................................................ 604 function............................................... 90

G gene expression regulation................ 202 gene expression......................... 168, 251 Gene regulation.......... 5, 8, 25, 103, 114, 134, 136, 139, 141, 142, 198, 234, 236, 240, 257, 264, 270, 271, 309, 310, 316, 318, 319, 320, 324, 326, 334, 383, 391, 439, 447, 457, 487, 504, 521, 526, 530, 556 gene therapy...................................... 625 genetic code...................................... 583 genome defense................................. 140 Genomic tRNA Gene Content.......... 170 Genomics........... 56, 146, 167, 175, 184, 251, 273, 289, 325, 542, 621 G-quadruplex.................................... 274 group II introns................................... 67 growth-rate........................................ 175 GTPase.............................................. 304

H half-a-tetratricopeptide...................... 218 Keyword Index – 2

Hepatitis B virus............................... 632 Herpesvirus saimiri........................... 272 Hfq.................................... 267, 494, 498 High-throughput Screening....... 188, 189 High-throughput sequencing.......... 9, 10, 11, 48, 49, 81, 99, 104, 130, 169, 171, 173, 177, 183, 185, 187, 195, 202, 207, 210, 217, 268, 271, 312, 313, 362, 402, 428, 485, 498, 539, 554, 623 HIV-1 DIS Dimerization and Mg Binding.......................................... 433 HIV-1 DIS......................................... 437 HIV-1........................................ 233, 464 HIV-2 leader RNA............................ 229 hnRNP....... 143, 145, 322, 341, 353, 570 hypoxia.............................................. 387

I i6A:isopentenyl adenosine................ 592 iCLIP-seq.......................................... 167 in vitro assay..................................... 516 in vitro evolution............................... 511 in vitro................... 88, 94, 156, 273, 286 In vivo RNA structural mapping....... 187 in vivo............................................... 471 infection............................................ 616 Influenza.................................... 179, 470 inhibitors........................................... 188 Initiation of translation...................... 217 interaction......................................... 458 interdependency................................ 458 intersubunit rotation............................ 36 IRES.................................................. 301 ITC............................................ 420, 513

J JAR3D.............................................. 180

K kinetic model..................................... 533 Kinetic-ITC....................................... 152 Kinetics/enzymology......... 15, 115, 159, 160, 188, 215, 267, 276, 506, 507, 510, 528, 584, 594 K-loop............................................... 586 K-turn................................................ 586

L L1 stalk................................................ 36 L-A virus........................................... 478 La...................................................... 279 LARP4.............................................. 410 Ligand analogs.................................. 707 LINE-1 retrotransposon...................... 25

Localization........ 93, 125, 246, 314, 457, 459, 460, 461, 508, 573, 603 long-distance interaction................... 357 long-non coding RNAs..................... 260 Loqs.................................................. 490

M m3C................................................... 367 m7G-cap............................................ 138 macrolide........................................... 285 malachite green................................... 90 mass spectrometry............................. 186 MDM2...................................... 543, 549 Membrane depolarization................. 353 Metal ion interactions........ 52, 160, 239, 360, 433, 513, 525, 527 methylation....................................... 373 microarray......................................... 383 microRNA binding sites.................... 173 microRNA processing and regulation....................................... 262 microRNA.......... 64, 129, 194, 266, 268, 269, 445, 474, 481, 487, 488, 489, 496, 500, 605, 611, 613, 616, 621, 627 microRNA: activation...................................... 496 biogenesis................ 25, 40, 122, 128, 238, 243, 248, 256, 258, 266, 272, 465, 483, 491, 493, 495, 499, 538 isomiRs......................................... 495 other...................... 245, 275, 324, 496 Polysome Binding........................ 254 RNA degradation............ 63, 254, 611 target identification...... 128, 173, 201, 486, 495 translation arrest........................... 462 microRNA-27..................................... 64 Microscopy........ 76, 129, 463, 508, 531, 702 minor spliceosome............................ 110 mir-35................................................ 488 miRNA processing............................ 256 miRNA................................................ 81 mitochondrial RNA decay................. 416 mitochondrial tRNA.......................... 598 mitochondrion................................... 373 model system.................................... 565 Modeling........................... 172, 422, 433 modification....... 85, 190, 233, 291, 483, 537, 602 Molecular Dynamics......................... 379 molecular modeling............................ 91 motif search......................................... 94 Motif................................... 50, 198, 547

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

mRNA degradation: poly(A) and decapping...... 57, 58, 84, 98, 99, 100, 230, 396, 400, 402, 408, 415 mechanisms........... 26, 53, 57, 61, 98, 131, 135, 155, 264, 386, 414, 620 ncRNA-dependent........................ 502 nonsense-mediated decay.......62, 111, 171, 311, 312, 313, 397, 403, 404, 405, 407, 411, 702 other.......... 95, 97, 175, 410, 412, 416 mRNA export.................................... 458 mRNA expression vectors................. 630 mRNA Processing.... 165, 192, 195, 274, 314, 320, 321, 398, 405, 474, 619 mRNA regulation.............................. 385 mRNA splicing regulation................ 117 mRNA Splicing: exon definition..... 163, 357, 539, 565, 606 factors.............. 43, 97, 208, 210, 250, 331, 337, 349, 350, 536, 540, 541, 545, 546, 548, 549, 553, 561, 562, 563, 567, 569, 574, 575, 703 functional consequences............... 316 mechanism................... 66, 68, 69, 70, 71, 74, 75, 76, 108, 337, 339, 342, 343, 349, 350, 351, 358, 359, 546, 549, 555, 562, 564 regulation................. 6, 20, 43, 66, 71, 142, 143, 144, 146, 164, 165, 258, 341, 353, 357, 464, 536, 540, 541, 545, 547, 548, 550, 552, 553, 554, 557, 558, 561, 564, 566, 567, 568, 569, 570, 573, 575, 576, 604, 606, 607, 612, 614, 703 signals.................. 140, 340, 550, 559, 560, 563 spliceosome assembly....... 69, 70, 73, 75, 336, 338, 342, 343, 344, 351, 352, 355, 361, 460, 463, 565, 576 mRNP identification.............................. 8 MS2 tethering system....................... 411 mTOR S6K1 signal transduction...... 614 muscle............................................... 703 Mutation............................................ 109 mutual modulation............................ 215

N NA chaperones.................................. 229 Nab3.................................................. 599 nanoparticle....................................... 627 Nanotechnology................................ 508 NCp8 binding.................................... 229 Nematoda.......................................... 583 neurodegenration NFL...................... 611

neuronal development....................... 554 Neuronal PTB................................... 552 neuronal stress responses.................. 489 NMD................................................. 702 non-coding pathogenic RNA............. 613 non-coding RNA transcription termination..................................... 219 non-coding RNA.................... 16, 18, 19, 20, 21, 24, 27, 39, 131, 132, 133, 134, 135, 136, 166, 174, 189, 192, 253, 259, 260, 261, 270, 319, 440, 443, 465, 485, 492, 497, 538, 577, 586 Non-Redundant Dataset.................... 430 Novel RNA discovery...... 147, 470, 492, 583 NPCs................................................. 702 Nrd1.................................................. 599 nuclear exosome.................................. 95 Nuclear pore complex............... 139, 148 nuclear transporter............................ 383 Nucleotidase........................................ 84 Nucleotide metabolism....................... 84 nucleotide repeat disorder................. 112 nucleus.............................................. 373

processing......................................... 406 Prostate Epithelial and stromal cells................................................ 497 Protein Motif: DEAD/H box..... 15, 45, 72, 184, 216, 352, 478 ds RNA binding...... 61, 211, 231, 242 other.............................. 218, 233, 368 RBD/RRM................ 12, 44, 162, 248 Zn finger................. 44, 208, 394, 401 protein synthesis................................ 286 Prp24................................................. 454 Prp43................................................. 358 Prp45................................................. 546 prp8 in vivo RNA binding sites........ 362 Psedouridine synthase....................... 368 Pseudoknots and suboptimal structure........................................... 47 Pseudouridine synthase..................... 371 pseudouridine.................................... 330 pseudouridylation.............................. 537 PTB........................................... 547, 572 PUF................................................... 393 Pyruvate decarboxylase.................... 156

O

Q

oncomiR............................................ 627 osteoarthritis...................................... 622 oxidation............................................. 55

Quality control.................................. 101

P P-bodies............................................ 398 P-body....................... 244, 326, 395, 416 pentatricopeptide repeat.................... 220 Phi29 Packaging RNA...................... 443 Phosphorylation........ 234, 323, 555, 576 phylogenetic analysis........................ 214 piRNA....................................... 130, 500 platinum............................................ 239 poly(A)-specific ribonuclease............. 82 polyadenylation........................... 54, 281 polyadenylation: cytoplasmic..... 82, 120 polyadenylation: nuclear.... 65, 275, 284, 599 polyamines........................................ 383 Polypyrimidine tract binding protein (PTB)................................. 552 polysomes................................. 304, 383 Post-Transfer editing......................... 287 Potassium channels........................... 353 PPARγ.............................................. 318 preference............................................ 78 pre-mRNA dynamics........................ 344 Pre-transfer editing............................ 287 Primary miRNA................................ 256 pri-miRNA expression database....... 238 pri-miRNA splicing........................... 538

R R2D2................................................. 490 RanBP2............................................. 706 recruitment........................................ 458 resistance gene.................................. 285 retrotransposition................................ 67 Retrotransposons............................... 302 RF3 GDPNP mRNA......................... 307 ribonucleases..................................... 178 Ribonuclease P.................................. 188 Ribonucleoprotein complex: 3’ processing........... 59, 167, 279, 282 binding................ 12, 16, 42, 207, 325 other............ 5, 22, 224, 404, 615, 701 splicing...................................... 9, 10, 73, 108, 110, 162, 329, 340, 356, 359, 362, 542, 572, 617, 704 structure........ 116, 186, 259, 398, 586 transcription.......................... 133, 472 translation...... 48, 206, 213, 295, 296, 334, 385 transport................................ 147, 462 Ribosnitches...................................... 436 Ribosome Biogenesis................ 288, 609 ribosome............................................ 294 Ribosome: active sites.................................... 306 antibiotics................... 34, 38, 52, 209 Keyword– 3

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

assembly............ 29, 31, 45, 290, 299, 335, 601 elongationn................ 33, 35, 38, 289, 297, 306, 384, 468 initiation.................. 30, 293, 709, 710 mechanism.. 33, 35, 36, 286, 294, 709 proteins........... 29, 186, 286, 335, 476 rRNA.... 101, 285, 297, 330, 406, 413, 534, 601, 609 structure.................. 34, 296, 307, 431 Riboswitch....... 123, 124, 150, 151, 152, 153, 154, 155, 263, 418, 420, 423, 425, 434, 447, 451, 482, 504, 511, 512, 513, 513, 515, 516, 520, 522, 526, 529, 707 ribozyme engineering........................ 510 Ribozymes: Group I.......................................... 517 Group II................ 157, 363, 442, 506 Hairpin.. 158, 159, 435, 479, 524, 528 Hammerhead................. 125, 441, 527 Hepatitus Delta.... 115, 441, 505, 518, 521, 523, 525 Other............ 118, 150, 156, 263, 302, 328, 421, 452, 479, 503, 510, 511, 533, 535 RNase P......... 7, 160, 197, 328, 329, 509 RISC.................................. 127, 244, 629 RNA 3D motifs................................... 50 RNA binding protein......................... 224 RNA catalysis...................................... 67 RNA damage....................................... 55 RNA degradation: polyadenylation..... 99 RNA Design...................................... 430 RNA Granule.................................... 326 RNA helicase.................................... 215 RNA interference............... 13, 127, 139, 140, 240, 241, 243, 249, 480, 488, 629, 632 RNA map.......................................... 547 RNA metabolism............................... 178 RNA Nanobiology............................ 172 RNA nuclear export.......................... 147 RNA recognition................................. 44 RNA secondary structure...................... 6 RNA Stability: AU-rich elements.... 230, 262, 401, 410, 417 RNA stability: non-coding RNA-mediated................................. 26 RNA Stability: regulation...... 53, 56, 65, 83, 95, 323, 393, 396, 407, 411, 413, 415, 477, 600, 605, 608 RNA structure................... 365, 430, 518 RNA structure/function....................... 42 RNA structure-function..................... 593 RNA-binding motif............................. 43 RNA-binding protein - miRNA interplay......................................... 324 Keyword Index – 4

RNA-ligand interaction..................... 418 RNA-ligand interactions..................... 52 RNA-metal ion interactions.............. 359 RNAP II promoter............................. 204 RNA-phage display........................... 209 RNA-protein interaction................... 323 RNA-protein interactions.................. 392 RNA-protein interactions: assembly....... 214, 223, 332, 356, 417 binding................. 7, 18, 59, 107, 144, 145, 203, 213, 218, 220, 225, 226, 228, 229, 231, 235, 242, 267, 269, 315, 466, 547, 571, 581 other..... 8, 11, 22, 277, 280, 377, 388, 395, 623 structure....................... 14, 51, 60, 79, 83, 120, 121, 122, 204, 219, 222, 227, 229, 232, 333, 371 RNase E............................................ 406 RNAse III.......................................... 231 RNase MRP....................................... 328 RNase P............................................. 114 RNase Z............................................ 276 RNase.......................................... 55, 406 RNA-Seq................................... 378, 547 RNP biosynthesis.............................. 354 RNPs in Development....................... 334 Rnt1................................................... 599 rRNA degradation............................. 413 rRNA modification............................ 371

S S/N ratio............................................ 533 Saccharomyces cerevisiae................. 215 Salmonella......................................... 136 SAXS................................................ 420 SC35.................................................. 543 screening........................................... 482 Second messenger............................. 504 Secondary structure........................... 336 Selection................ 88, 94, 156, 273, 471 SF2/ASF............................................ 543 SHAPE analysis................................ 256 SHAPE.............................................. 424 Signal recognition particle................ 279 simtron.............................................. 258 single cells......................................... 702 Single Molecule Fluorescence Microscopy...................................... 62 single molecule FRET......................... 29 Single particle tracking..................... 129 single-molecule FRET...................... 434 Single-RNA FISH............................. 702 siRNA delivery vehicles................... 628 siRNA................ 241, 245, 246, 436, 628 Site-directed spin labeling................. 443 SLBP................................................. 207 small angle X-ray scattering............. 119

small molecule...................................111 Small RNA Binding Proteins............ 484 Small RNA sequencing..................... 579 Small RNA........................................ 109 Small RNAs...................................... 136 smFRET............................................ 306 SmpB................................................ 296 snoRNA biogenesis........................... 599 snoRNA...... 82, 113, 257, 330, 487, 577, 581, 589, 622 snoRNP............................................. 113 snRNA....... 336, 338, 358, 449, 537, 617 snRNP assembly surveillance........... 332 snRNP............... 110, 282, 332, 346, 704 spermidine......................................... 529 Spinal Muscular Atrophy.......... 544, 606 splice site selection........................... 356 Splice Sites........................................ 506 spliceosome disassembly.................... 74 Spliceosome...................................... 336 Spliceosome: footprinting................. 185 splicing and disease........................... 337 splicing catalysis................................. 44 Splicing code..................................... 547 Splicing Efficiency............................ 506 Splicing Factor 1............................... 576 splicing........................ 67, 109, 168, 464 SR proteins............................ 9, 278, 555 sRNA................................................. 494 SRp20................................................ 464 sR-tMet+F38..................................... 586 ssRNA dynamics................................. 39 stability element.................................. 27 Staphylococcus aureus...................... 132 stem cells........................................... 554 Stem-loop recognition element........... 83 Stepwise photobleaching.................. 129 Steroid Receptor RNA Activator...... 271 Streptomycin Dependence.................. 34 Streptomycin Resistance..................... 34 Stress response.................................. 149 Structure Analysis: biophysical methods................ 24, 39, 84, 293, 333, 392, 420, 438, 443, 453, 523, 524 EM 432, 572 FRET................ 36, 68, 437, 454, 455 NMR..................................... 421, 442 single-molecule.............. 72, 190, 191, 223, 295, 331, 344, 355, 451, 455, 473, 486, 520 X-ray.........13, 58, 116, 117, 118, 119, 227, 304, 354, 376, 381, 414, 422, 423, 425, 432, 452 Structure........................................ 40, 90 Sugar-phosphate Self-cleavage......... 159 synergistic......................................... 153 synthetic riboswitch mechanism (?).. 154

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

T T box................................................. 529 telomerase RNA.................................. 23 telomerase................................... 24, 116 Tertiary Interactions.......................... 506 tertiary structure.................................. 79 Therapeutics......106, 109, 111, 114, 534, 624, 627, 628, 630, 632, 707 thermodynamic................................. 513 Thermus thermophilus...................... 286 Thg1-like protein (TLP).................... 370 Three Dimensional Topology of Large RNA................................ 119 tmRNA.............................................. 296 TRAMP............................................. 279 Transcript Leader.............................. 171 Transcription antitermination............ 530 Transcription termination......... 103, 121, 219, 516 transcriptional regulation.......... 133, 253 Transcriptional Riboregulation......... 273 transcriptome..................................... 202 translation factors.............................. 289 Translation of secreted and membrane proteins......................... 459 Translation regulation......................... 93 Translation: IRES............. 301, 302, 305, 380, 387 Mechanism.. 112, 203, 206, 294, 303, 305, 469, 475 Regulation by ncRNAs.. 21, 132, 265, 502 Regulation................ 30, 37, 100, 148, 149, 170, 236, 322, 369, 380, 382, 384, 386, 387, 389, 390, 391, 393, 459, 615 Ribosome shunting........................... 305

translational fidelity............................ 87 Translational repression.................... 615 translocation...................................... 295 Transport: factors.............. 138, 304, 392 Transport: mechanisms..................... 119 trans-translation................................. 296 TREX................................................ 458 Trm Y................................................ 364 TRM5................................................ 373 tRNA biosynthetic pathway.............. 598 tRNA Decay...................................... 600 tRNA editing+F1377......................... 367 tRNA import..................................... 373 tRNA ligase......................................... 96 tRNA mimicry................................... 380 tRNA modification.................... 368, 373 tRNA properties................................ 303 tRNA re-export.................................. 585 tRNA repair....................................... 596 tRNA......................... 367, 373, 461, 529 tRNA: charging..... 32, 54, 87, 149, 580, 583, 593 modification........... 86, 170, 364, 366, 367, 376, 379, 390, 579, 592, 597, 598, 600 processing................ 54, 96, 188, 197, 214, 276, 370, 372, 375, 509, 585, 587, 588, 591, 595, 596, 602, 603 recognition.............. 41, 530, 582, 597 tRNAHis guanylyltransferase (Thg1).................................... 370, 594 tRNA-like element.............................. 41 Trypanosoma brucei.......................... 220 Trypanosoma............................. 367, 373 Trypanosome..................................... 457

Tyw................................................... 598

U U1 snRNP................................... 69, 169 U12 introns......................................... 95 U12-dependent snRNAs................... 110 U2-U6 snRNA................................... 336 ubiquitin............................................ 343 Unfolded Protein Response............... 620

V variable arm...................................... 583 Variant Surface Protein..................... 487 Viral RNA Structure............................ 51 Virulence factors............................... 136 virus replication................................ 464 Virus/Viroid: other...................................... 466, 468 replication..... 237, 471, 472, 478, 625 retrovirus.41, 278, 453, 464, 473, 477 gene regulation....... 64, 333, 382, 481

W Wybutosine....................................... 598

X X-ray crystal structure......................... 61 X-ray structures................................... 67

Y YB-1.................................................... 43 yeast.......................................... 376, 594

Keyword– 5

RNA 2012 • Ann Arbor, Michigan, USA • May 29–June 2, 2012

NOTES

Keyword Index – 6 View publication stats