npgrj_nProt_81 920..927

PROTOCOL

A protocol for PAIR: PNA-assisted identification of RNA binding proteins in living cells Fanyi Zeng1,2, Tiina Peritz1, Theresa J Kannanayakal1, Kalle Kilk3, Emelı´a Eirı´ksdo´ttir3, Ulo Langel3 & James Eberwine1 1Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA. 2Shanghai Institute of Medical Genetics, Shanghai

© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

Jiao Tong University, Shanghai 200040, China. 3Department of Neurochemistry, Stockholm University, SE-106 91 Stockholm, Sweden. Correspondence should be addressed to J.E. ([email protected]). Published online 3 August 2006; doi:10.1038/nprot.2006.81

All aspects of RNA metabolism are regulated by RNA-binding proteins (RBPs) that directly associate with the RNA. Some aspects of RNA biology such as RNA abundance can be readily assessed using standard hybridization technologies. However, identification of RBPs that specifically associate with selected RNAs has been more difficult, particularly when attempting to assess this in live cells. The peptide nucleic acid (PNA)-assisted identification of RBPs (PAIR) technology has recently been developed to overcome this issue. The PAIR technology uses a cell membrane–penetrating peptide (CPP) to efficiently deliver into the cell its linked PNA oligomer that complements the target mRNA sequence. The PNA will then anneal to its target mRNA in the living cell, and then covalently couple to the mRNA-RBP complexes subsequent to an ultraviolet (UV) cross-linking step. The resulting PNA-RNA-RBP complex can be isolated using sense oligonucleotide magnetic beads, and the RBPs can then be identified by mass spectrometry (MS). This procedure can usually be completed within 3 d. The use of the PAIR procedure promises to provide insight into the dynamics of RNA processing, transport, degradation and translation in live cells.

INTRODUCTION One of the primary goals of the field of genomics is the study of populations of RNA, often at the level of RNA abundances using microarray analysis. Although such information is important, the biology of RNA is significantly more complex than simple abundance measurements might imply. mRNA is made in the nucleus of the cell through the transcription process, during which the nascent RNA transcript is spliced to make a mature mRNA. The mRNA is then transported from the nucleus to the cytoplasm. In the cytoplasm the mRNA can be targeted to selective subcellular sites, and translated as well as degraded. Each of these steps in RNA metabolism is controlled through the activity of RBPs that directly bind to the RNA. These RBPs can (i) exert direct enzymatic activity on the RNA such as the adenosine deaminase protein’s ability to deaminate adenosine to create an inosine base1, (ii) translate the mRNA into protein such as eIF4 binding to RNA and associating with the ribosome2 or (iii) act as an intermediary facilitating the coupling of the RNA to the cytoskeleton so that the RNA can be moved from one cellular site to another3. It is clear from various studies that RBPs can bind to primary sequence in the RNA or to secondary or tertiary structures that form through intramolecular base pairing. These complex structures permit the possibility that multiple RBPs may bind to any individual RNA and that this binding may be regulated by various cellular modulators4–6. Given the fundamental role of RBPs in controlling mRNA function and metabolism, identification and quantitation of mRNA-RBP interactions should be a critical component of any genomics effort. One can use two general approaches to characterize mRNA-RBP interactions: (i) the identification of RBPs associated with a known RNA target or (ii) the complementary approach of identification of RNAs that are bound to a known RBP. The assays used include isolating known RNA and its associated RBP complex through affinity column purification using antisense RNA sequence as bait; 920 | VOL.1 NO.2 | 2006 | NATURE PROTOCOLS

gel-shift or supershift assays (to assess the RBP activity through antibodies recognizing the protein in the presence of RNA); RBP immunoprecipitation (IP), cross-linking IP7–10. The problem with these assays is that they only confirm the physical relationship between the RNAs and correlated RBPs in vitro, but fail to faithfully represent the RNA-RBP interaction under physiological conditions. In addition, detection of these interactions is quite difficult because of the chemical nature of the strongly charged RNA molecule. RNAs are ‘sticky’; they may bind nonspecifically to other charged molecules thereby making in vitro characterization cumbersome. Ideally, characterization of these interactions in living cells may provide more specificity, in that the endogenous RBP-RNA associations, which are highly specific, would be interrogated in a manner that does not disrupt these interactions. This has driven the development of the methodology termed the PAIR procedure11 that is detailed in this protocol (Fig. 1). The PAIR procedure permits the simultaneous analysis of multiple mRNAs and their associated RBPs in a quantitative manner that reflects the interactions occurring in living cells. The PAIR procedure was developed through a collaborative effort between scientists who specialize in molecular biology, neuroscience and chemistry. Such collaborations are important as genomics enters its next phase, the quantitative analysis of genomics processes in live cells. The chemistry portion of the protocol involves the synthesis of molecules with two distinct abilities, that of crossing cell membranes of live cells (CPP)12 and specific binding to selected endogenous RNAs (PNA)13,14. This combination creates an mRNA-specific probe that can enter living cells and hybridize to complementary sequences on a targeted RNA. Incorporation of p-benzoylphenylalanine (Bpa), a photoactivatable amino acid adduct, into PNA sequences makes possible the capture of adjacent RBPs by photoactivated cross-linking, and it places no additional restrictions on conventional solid-phase peptide and PNA synthesis

PROTOCOL CPP-Cys-SH +(Npys)Cys-Bpa-PNA

a

Bpa CPP– S –S–

PNA

CPP-Cys-S-S-Cys-Bpa-PNA

Plasma membrane

or

b

CPP-S-S-Bpa-PNA

CPP–SH

Figure 2 | Scheme for covalent conjugation of CPP to PNA oligomer.

Bpa PNA

Furthermore, the number of RBPs isolated by the procedure will be determined by how many RBPs are in proximity to the photoactivated amino acid on the PNA. Traditionally, it has been speculated that more RBPs tend to bind more often to the 3¢ and 5¢ untranslated regions (UTRs) of mRNAs than to the coding region, because the UTRs are thought to be the dominant regulatory sites in mRNA11. Therefore, it is generally advisable to direct the PNAs to these regulatory sites to enhance the ability to identify and characterize RBPs. Considering the short distance over which the photoactivatable amino acid is able to cross-link to a protein, it is probable that any PAIR PNA directed against a single sequence of a particular RNA might not identify all of the RBPs that are binding to that RNA at any given time. A complete characterization of all RBPs that bind to a particular RNA is possible, however. It would require the use of PAIR PNAs that bind to regions of the RNA spaced every 25 bases apart. These PAIR PNAs could be applied separately or as a mixture onto the cells to be interrogated. The only impediment to this study is the cost of PAIR-CPP-PNA synthesis. It is important to note that single PAIR PNA studies do permit an analysis of regulatory changes in RBP-RNA associations for any RBPs that bind in the area to which the PNA is annealed, and that the use of two to three PNAs11 permits an analysis of dynamic changes in RBP binding to different regions of the same type of RNA.

RBP


mRNA

c

UV

Bpa RBP

PNA

mRNA

d Bpa PNA

RBP mRNA

e Bpa PNA

Bpa

Bpa RBP1

PNA

RBP2

PNA

RBP3

Figure 1 | General scheme of PAIR technology. (a) PNA-Bpa is transferred intracellularly with the assistance of CPP (or by CPP); (b) the disulfide bond is reduced and CPP is separated from the PNA-Bpa; (c) PNA anneals to complementary mRNA sequence, and following UV irradiation, (d) the PNA-Bpa and adjacent RBPs will covalently cross-link; (e) after cell lysis the PNA-RBP complexes are isolated and identified by MS.

protocols. Conventional solid-phase peptide and PNA synthesis protocols are used to produce linear CPP and PNA oligomers15. The commercial purchase of required CPP and PNA oligomers can be recommended (e.g., peptides from Bachem and PNAs from Applied Biosystems). In general, PNA oligomers with a general sequence (Npys)CysBpa-PNA and the carrier peptide, CPP-Cys-SH, can be synthesized or purchased (Fig. 2). Modification of the cysteine residues by 3-nitro-2-pyridinesulphenyl (Npys) protection is recommended to achieve higher yields for the conjugation reaction. (Npys)Cys (Bachem) in a PNA oligomer is usually placed at the N terminus. In CPP the location of an extra cysteine is selected depending on the translocational properties of the CPP. In the case of TP10 (ref. 16), (Npys)Cys is either introduced at the N terminus or orthogonally to the lysine at position 7. The selectivity of PNA annealing to a particular endogenous RNA is dictated by the length and the sequence of the PNA.

Experimental design The PAIR protocol described here is designed to identify RBPs associated with the RNA of interest in living cells. To identify these RNA-RBP complexes under different pharmacological or physiological conditions, the cells can be pretreated with appropriate pharmacological agents or different culturing conditions before beginning the PAIR procedure on day 1. For example, cells can be incubated with dihydroxyphenyl glycol (DHPG) at a final concentration of 20 mM for 30 min before addition of PNA (day 1, Step 17) to identify different sets of RBPs that are important to the gene expression responding to DHPG treatment. The quality of the synthesized PNA is important to the PAIR procedure. MS analysis of the conjugate can be used as an optional step to further verify the identity of the desired product (an optional step after Step 8). Care must be taken not to reduce the disulfide bridge during preparation of samples or collection for MS. The coupling efficiency of the CPP and the PNA varies between sequences and depends on solubility and purity of the compounds; therefore, the 1:1 molar ratio should be modified and optimized in every case. A positive control for the specificity of the PAIR assay may be obtained by using overlapping but non-identical PNAs. These PNAs should yield comparable sets of RBP identities11. An appropriate negative control can be achieved by performing the PAIR procedure on living cells in the absence of PAIR-PNA (day 1, Step 10). NATURE PROTOCOLS | VOL.1 NO.2 | 2006 | 921

PROTOCOL In addition, other negative control experiments can be used to address signal-to-noise issues to optimize the procedure, such as processing an experimental sample without UV cross-linking (Step 20), or addition of streptavidin beads coupled to an antisense

oligonucleotide (rather than the sense oligonucleotide) to isolate PNA-RNA-RBP complexes from cell lysate (Steps 10–16 and Step 26) See Supplementary Table 1 online for a quick reference guide for steps 17–37 of the PAIR procedure.


MATERIALS REAGENTS . CPP with a Npys-derivatized Cys residue (Bachem) . PNAs with a free thiol group . Acetic acid buffer (0.01 M, pH 5.5) . Dimethylformamide (DMF) . Dimethylsulfoxide (DMSO) . 99.9% (vol/vol) Acetonitrile (AcN) . 0.1% (vol/vol) Trifluoroacetic acid (TFA) . Matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) matrix: a-cyano-4-hydroxycinnamic acid . Cell culture media and reagents: . Neurobasal medium with B27 supplement (NB/B27; Invitrogen), or any cell culture medium appropriate to the cell culture system of interest . Pharmacological or physiological agent for cell treatments (e.g., dihydroxyphenyl glycol (DHPG), brain-derived neurotrophic factor (BDNF), potassium treatments) . PNAs for the mRNA of interest (e.g., Applied Biosystems) . Biotinylated sense oligonucleotide complementary to the PNA sequence (to be coupled to streptavidin magnetic beads for RBP isolation) . Streptavidin magnetic beads (Pure Biotech) . RNase A (final concentration 1 mg ml–1) . Methanol . Chloroform . Nuclease-free water . 25 mM ammonium bicarbonate (NH4HCO3), pH 8.0 (freshly made) . 50 mM ammonium bicarbonate (NH4HCO3) . Acetic acid (1–2%) (vol/vol) . 25 mM HEPES . 0.75 mM Na2HPO4 . 0.1% (vol/vol) Triton X-100 . 20 mM glycerophosphate . 1.5 mM MgCl2 . 1 mM DTT . 1 mM benzamidine . 200 nM Na3VO4 . 1 mM PMSF . Leupeptin . Aprotinin . Pepstatin . NaN3 . Standard reagents for PAGE: . NuPAGE 10% Bis-Tris Gels and running buffer, SilverQuest Silver Staining Kit (Invitrogen) or Coomassie staining reagents that are compatible with MS. . Note: It is highly recommended that any staining kit used should be compatible with MS if protein gel slices are to be used for MS analysis. . Sequencing-grade trypsin

EQUIPMENT

. Mass spectrometer: e.g. MALDI-TOF and Q-TOF (Nanospray/Qstar-XL and Voyager-DE STR; Applied Biosystems)

. Standard tissue culture equipment and environment, including hood . UV box for cross-linking (15-W Sylvania Germicidal light bulb: l ¼ 254 nm) . Cell rotator . Magnetic separator stands (Promega) . Electrophoresis equipment . Benchtop thermomixer REAGENT SETUP To obtain a heterodimeric disulfide bridge, a derivatized cysteine residue of one component, either the PNA or peptide, is recommended. Npys-derivatized Cys is specifically reactive towards free thiols. Npys-labeled cysteine is commercially available and with special cautions can be assembled into a peptide chain like a commonly protected amino acid. Cells This procedure can be used for any cell culture system of interest. For example, rat cortical neurons are plated on 10-cm tissue culture plates at a density of 4 105 neurons ml–1 in NB/B27. Note that depending on the intracellular abundances of the RNAs to be assayed, a range of 2.4 106 to 1.5 107 rat cortical cells can be used for this procedure, with the lower abundance RNAs requiring more cells. HEPES-buffered saline (HBS) 25 mM HEPES, 0.75 mM Na2HPO4, 70 mM NaCl. pH ¼ 7.4, and store at 20 1C. TX-100 lysis buffer 25 mM HEPES, pH 7.4, 0.1% Triton X-100, 300 mM NaCl, 20 mM glycerophosphate, 1.5 mM MgCl2, 1 mM DTT, 2 mM EDTA, pH 8.0. Add fresh protease inhibitors for a final concentration of 1 mM benzamidine, 200 nM Na3VO4, 1 mM PMSF, 2 mg ml–1 leupeptin, 2 mg ml–1 aprotinin, 1.4 mg ml–1 pepstatin. Salt-free lysis buffer Same as TX-100 lysis buffer, except that there is no NaCl in the solution. Streptavidin magnetic bead storage buffer 0.1% BSA (wt/vol), 0.2% NaN3 (wt/vol) in PBS, pH 7.4. m CRITICAL Store at 20 1C. Eluants for reverse-phase high-performance liquid chromatography (RPHPLC) Eluant A: 99.9% AcN + 0.1% TFA; eluant B: 99.9% H2O + 0.1% TFA. Washing solution for MS (Step 45) 50 mM ammonium bicarbonate (NH4HCO3) in 50% AcN. EQUIPMENT SETUP C18 columns for RP-HPLC For example, Discovery, 25 cm 10 mm, 5 mm (Supelco; Sigma-Aldrich). Gradient for RP-HPLC Isocratic 20% eluant A for 5 min, followed by gradiental increase of eluant A to 100% in 40 min. 20% AcN in the beginning prevents unreacted PNA from interacting with the stationary phase in the column, and it is washed out together with solvents (DMSO and DMF). Conjugated PNA precedes the peptide peak. Detection wavelengths 218 nm is the absorbance maximum for peptide bonds, and 260 nm indicates PNA nucleobases. (For single-wavelength detector use 260 nm.)

PROCEDURE Synthesis of the CPP-PNA compound 1| Weigh 1 molar equivalent (0.5–2 mg) of peptide and 1 molar equivalent of PNA in separate microcentrifuge tubes. 2| Dissolve PNA in 200 ml deoxygenated DMSO. 3| Dissolve peptide in 100 ml of 0.01 M acetic acid buffer, pH 5.5. 4| Add 200 ml of DMF to both of the solutions, and mix the two solutions by vortexing thoroughly. 5| Stir the mixture overnight, or for at least 4 h, at room temperature (RT, 20–25 1C). Protect from light. 6| Separate reaction products by RP-HPLC using a C18 column. 922 | VOL.1 NO.2 | 2006 | NATURE PROTOCOLS

PROTOCOL 7| Collect fractions absorbing at both wavelengths. 8| Freeze-dry fraction(s), and store protected from light at 20 1C. Note: as an optional step one can verify the correct mass with MALDI-TOF MS. ! CAUTION The disulfide bridge partially breaks down in the mass spectrometer, resulting in three peaks: the conjugate and the two starting compounds.


9| Before proceeding to the PAIR procedure, dissolve the lyophilized CPP-PNA conjugates in HBS at a stock concentration of 5 mM. They can be stored at 20 1C for Z12 months. Coupling of biotinylated sense oligonucleotide to streptavidin magnetic beads (PAIR day 0) 10| To begin the PAIR procedure, aliquot 10 mg of streptavidin magnetic beads and magnetically separate them from the storage solution. Place the tube containing the streptavidin beads on the magnetic stand for 10 min at RT. Carefully remove the buffer and discard. 11| Wash the beads twice with PBS (pH 7.4), each time resuspending the beads in 1 ml of PBS, and placing the tube on the magnetic stand for 10 min before removing PBS. 12| Resuspend the beads to a final volume of 1 ml in PBS containing 50 mg biotinylated sense oligonucleotide complementary to the PNA sequence. Rotate the solution at RT for 1 h at 8–10 r.p.m. 13| After magnetic separation as just described, wash the beads with 1 ml PBS five times to remove any unbound biotinylated oligonucleotide. For each wash, rotate the tube at RT for 10 min at B8–10 r.p.m., and then put on magnetic stand for 10 min before removing the washing solution. 14| Resuspend the beads in 1 ml PBS containing 0.2% (wt/vol) BSA to block nonspecific binding to the beads. Rotate the tubes at RT for 1 h at B8–10 r.p.m. 15| Wash the bead-streptavidin-biotin-oligo complex three times with 1 ml PBS at RT, 10 min per wash. 16| Resuspend the bead complex in 1 ml TX-100 lysis buffer (a final concentration of 10 mg ml–1) if it is to be used immediately; alternatively, store at 4 1C at 10 mg ml–1 in storage buffer. Binding of PNA to RBPs under pharmacological or physiological conditions in living cells (PAIR day 1) 17| Incubate the cells of interest in the appropriate cell culture medium with and without pharmacological or physiological manipulations. For example, use 4.8 106 healthy rat cortical neurons between 7 and 22 d old that are plated on 10-cm tissue culture plates in fresh NB/B27 medium. 18| For each sample, incubate 2 106 to 1 107 cells with 4 ml of pre-warmed fresh NB/B27 medium containing 50 nM CPP-PNA conjugate for 90 min at 37 1C. 19| Replace medium with 4 ml HBS to rinse the cells once. Aspirate out the medium and replace with 4 ml HBS. 20| Bridge the UV box facing down on top of two pipette tip boxes and subject the mixture to UV cross-linking at a distance of 2.5 in. (6 cm) for 2.5 min. Note: the height of standard pipette tip boxes is roughly the appropriate distance for this procedure. 21| Immediately replace HBS with 4 ml ice-cold TX-100 lysis buffer (with freshly added protease inhibitors), harvest the cells using cell scrapers and transfer the cell lysate into a 15-ml conical tube. Take a 30-ml aliquot of this lysate, label as TCL (total cell lysate) and store at 80 1C for later gel analysis. m CRITICAL STEP Make every effort to minimize the time spent on this step. Take caution to reduce keratin contamination by following strict tissue culture guidelines, and minimize exposure to human skin and hair. While scraping the cells and transferring the cell lysate, minimize bubbles. ’ PAUSE POINT It is possible to store the cell lysate at 80 1C and proceed to Step 22 the next day or at a later time. 22| Add 1 ml of RNase A (1 mg ml–1) per milliliter of lysate to the remainder of the lysate to digest RNA from the PNA-protein covalent complexes. Rotate the tubes at 37 1C for at least 20 min at B8–10 r.p.m. 23| Freeze the cell lysate on dry ice or store at 80 1C. m CRITICAL STEP It is important that cells are frozen at 80 1C before the next step to help solubilize the PNA-protein complex. To continue with the procedure the same day, let the cell lysate freeze on dry ice or remain at 80 1C for at least 1 h. ’ PAUSE POINT At this point the cell lysate can be stored frozen at –80 1C for r6 months before proceeding to the next step. NATURE PROTOCOLS | VOL.1 NO.2 | 2006 | 923

PROTOCOL Isolation of RBPs from cell lysate (PAIR day 2) 24| Thaw the cell lysate in a water bath at RT (20–25 1C). 25| Add to each tube of lysate 100 mg (10 ml of 10 mg ml–1 stock) of streptavidin magnetic beads that were previously coupled to sense oligos (day 0). Rotate the tubes at 37 1C for at least 30 min at B8–10 r.p.m. and then at RT for 30 min. The beads with sense oligo should now be annealed to the PNAs cross-linked to RBPs.


26| Centrifuge the tubes at 2,500 r.p.m. for 30 s to 1 min to collect the magnetic bead complex. Save a 30-ml aliquot (labeled as FT for flowthrough) of the supernatant for future gel analysis and store at 80 1C. Carefully remove the supernatant until 500 ml of lysate remains. Transfer the PNA-coupled beads in 500 ml lysate to a 1.5-ml microcentrifuge tube. 27| Separate the beads from the lysate by placing the microcentrifuge tube on the magnetic stand for 10 min and then carefully aspirating the supernatant. 28| Wash the beads with 500–1,000 ml TX-100 lysis buffer containing freshly added protease inhibitors to remove any uncoupled lysate proteins. Resuspend the beads in 1 ml of TX-100 lysis buffer. Rotate the tube at RT (8–10 r.p.m.) for 20 min, and place on magnetic stand for 10 min before carefully removing the buffer. Repeat wash once. 29| At this point one can either continue with Steps 30–40 (chloroform-methanol precipitation followed by MS) or resuspend beads in 30 ml TX-100 lysis buffer and gel-isolate protein bands for MS (Steps 41–49). Protein elution, chloroform-methanol precipitation17 and trypsin digestion for MS (PAIR days 2 and 3) 30| Resuspend the beads in 100 ml of salt-free TX-100 lysis buffer (with freshly added protease inhibitors) pre-warmed to 50 1C to elute the PNA-RBP complex. Incubate the beads at 50 1C in a benchtop thermomixer (B450 r.p.m.) for 20–30 min. 31| Spin the beads for 1 min at RT at maximum speed in a benchtop centrifuge. Place the tubes on the magnetic stand and transfer the eluate to a new microcentrifuge tube. The eluate now contains the RBPs bound to the specific PNA. ’ PAUSE POINT It is possible to store these samples at –80 1C and continue with chloroform-methanol precipitation the next day. 32| Add 400 ml methanol to the tube containing 100 ml eluate from Step 31. Vortex for 10 s, and then centrifuge at 10,000 r.p.m. for 10 s. 33| Add 100 ml chloroform to the same tube with the methanol-eluate mixture. Vortex for 10 s and then centrifuge at 10,000 r.p.m. for 10 s. 34| Add 300 ml nuclease-free water to the same tube with the chloroform-methanol-eluate mixture. Vortex for 10 s and centrifuge at 10,000 r.p.m. for 3 min at 4 1C. m CRITICAL STEP If phase separation is not observed after this step, add 100 ml chloroform and repeat Steps 34 and 35. 35| The interphase now contains the proteins. Aspirate and discard the upper phase and leave r20 ml of the upper phase in the tube. Take care not to disturb the interphase. 36| Add 300 ml methanol to the remaining interphase and lower phase. Vortex for 10 s and centrifuge at 13,000 r.p.m. for 10 min at 4 1C. 37| Discard the supernatant and air dry the pellet for 10 min or until liquid droplets have evaporated but the pellet is still moist. Resuspend the pellet in 50 ml of freshly prepared 25 mM NH4HCO3. Take 20 ml of the isolated RBPs (label E for eluate) for gel analysis. m CRITICAL STEP NH4HCO3 should be made fresh each time. Alternatively, aliquots of the NH4HCO3 can be stored at 80 1C and freshly thawed each time. ’ PAUSE POINT Store the samples at 80 1C while performing electrophoresis analysis of TCL, FT and E, before MS identification of RBPs. 38| Perform standard PAGE on the TCL, FT protein and E protein (bound protein) using NuPAGE 10% Bis-Tris Gels (Invitrogen), and then stain using SilverQuest Silver Staining Kit (Invitrogen) for protein verification and confirmation (see ANTICIPATED RESULTS section). 39| Perform in-solution trypsin digestion for MS by first adding sequencing-grade trypsin to chloroform-methanol precipitated eluate (from Step 37) at a final concentration of 12.5 ng ml–1, and incubate the mixture overnight at 37 1C. Dry the samples in a vacuum centrifuge, and dissolve the digested peptides in 10 ml of 5% AcN and 0.1% TFA. 924 | VOL.1 NO.2 | 2006 | NATURE PROTOCOLS

PROTOCOL 40| The precipitated peptide samples are now ready for MS (Q-TOF, LC purification and LC/MS/MS analysis) to identify the RNA binding proteins. Protein extraction from preparation gels and trypsin digestion for MS (PAIR days 2 and 3) 41| If you choose to gel-isolate protein bands for MS, resuspend the streptavidin magnetic beads that are coupled to the PNA-RBP complexes from Step 29 of the PAIR protocol in 30 ml of salt-free TX-100 lysis buffer (with freshly added protease inhibitors). 42| Perform standard PAGE using all 30 ml of the eluate.


43| Perform silver staining using the SilverQuest kit or other MS–compatible staining procedures (e.g., Coomassie staining and destaining using standard protocols). 44| Carefully excise the protein bands enriched for the bound proteins. One can store the protein slices in 1–2% (vol/vol) acetic acid at 20 1C until trypsin digestion is performed for MS. 45| Before trypsin digestion for MS, wash the gel slices with 100 ml of HPLC grade water, vortex for 15 min, discard solution, and then wash with 100 ml of 50 mM NH4HCO3 in 50% AcN, vortex 30 min, discard solution. 46| Shrink the gel slices by dehydration in 100 ml 99.9% AcN for 5 min followed by solvent removal in a vacuum centrifuge. Repeat once. 47| Rehydrate the gel slices in 15 ml of 25 mM NH4HCO3 containing 12.5 ng ml–1 sequencing-grade trypsin, and incubate the mixture overnight (12–16 h) at 37 1C. 48| Extract the peptides by adding 50 ml of HPLC-grade water/acetonitrile/TFA (10:10:1; vol/vol/vol) and then subjecting the samples to sonication for 10 min and brief centrifugation to collect supernatant. Perform two more extractions with 50 ml of HPLC-grade water/acetonitrile/TFA (10:10:1; vol/vol/vol), and combine the supernatants. 49| Dry samples using speed vacuum and dissolve the peptides in 10 ml of 5% AcN and 0.1% TFA. The extracted and digested peptide samples are now ready for MS (Q-TOF, LC purification and LC/MS/MS analysis) to identify the RNA-binding proteins.

TIMING The average time to complete the PAIR procedure is 3 d. This does not include synthesizing the CPP-PNA conjugates and MS analysis. It is not necessary to repeat coupling of the biotinylated sense oligonucleotides to streptavidin magnetic beads (day 0, 4 h) every time. Synthesis of the CPP-PNA compound: Steps 1–9: 5 h, or 2 h plus overnight (see Step 5). Coupling of biotinylated sense oligonucleotide to streptavidin magnetic beads (PAIR day 0): Steps 10–16: 3.5–4 h. Binding of PNAs to RBPs under physiological or pharmacological conditions in living cells (PAIR day 1): Steps 17–23: 3 h plus overnight (plus drug treatment) or 4 h total (if cells are frozen for 1 h instead of overnight; see Step 23) (plus drug treatment). Isolation of RBPs from total protein lysate (PAIR day 2): Steps 24–29: 2 h. Protein elution, chloroform-methanol precipitation17 and trypsin digestion for MS (PAIR days 2 and 3) Steps 30–40: 4 h (day 2) plus overnight (plus gel electrophoresis for protein confirmation) plus 5–6 h (day 3). Protein extraction from preparation gels and trypsin digestion for MS (PAIR days 2 and 3): Steps 41–49: 4 h (day 2) plus overnight plus 5–6 h (day 3). The length of this procedure can be adjusted according to the pause points in the protocol and the steps indicated in this paragraph to allow more flexibility. ? TROUBLESHOOTING Troubleshooting advice can be found in Table 1. NATURE PROTOCOLS | VOL.1 NO.2 | 2006 | 925

PROTOCOL TABLE 1 | Troubleshooting table. PROBLEM POSSIBLE REASON No CPP-PNA The low solubility of PNA causes it to remain in pellet conjugate obtained and not be available for the conjugation reaction. Low quality of the CPP and PNA.

SOLUTION Add a few microliters of TFA or guanidinium chloride solution to the PNA pellet. Warming for r5 min at 55 1C may also help.

Perform an additional HPLC purification step for the com-


pounds before conjugation. This should be done in a low-light environment so that the Npys group does not become detached because of continuous exposure to light or oxidative agents.

Keratin and other contaminants

The correct product peak in HPLC is invisible as a result of overlap with other peaks.

Change HPLC gradient. For best advice consult a specialist.

Keratin is a probable contaminant from human skin and hair. A baseline amount of BSA, trypsin and trypsin precursors, could also be present as part of the proteins identified, because they are introduced to the reaction during the PAIR procedure.

Minimize cell exposure to these contaminants by wearing gloves and long sleeves and hair protector.

Minimize the time spent on each step of the procedure, especially during day 1 (Steps 10–14).

Make sure to open cell culture dishes only in a ventilated cell culture hood.

Also try to minimize contamination during MS analysis. Low yield or no proteins identified

Selection of PNA and PNA design: Not all PNAs will work equally well.

See INTRODUCTION for more details on PNA design. PNA

Cells are exposed to non-physiological conditions during manipulation:

It is critical to minimize the amount of time live cells

Leaving the cell culture outside the incubator for

Minimize the length of time cells are outside of the incubator during day 1 (Steps 10–14).

an extended periods of time may alter the culture pH, which, in turn, will change the charge on the PNAs and alter the PNA annealing characteristics.

Similarly, leaving cells at RT for extended periods of

length and charge identity, as well as the targeted RNA regions play important roles in the success of the procedure and may require optimization. In addition, the use of multiple PNAs to the same RNA can help identify a more complete set of RBPs that interact with a particular RNA and can potentially improve the reliability of the assay. (To cover the whole length of the RNA, design PNAs to bind every 25 base pairs apart.) are exposed to non-physiological conditions such as higher pH or lower temperature during the procedure (e.g., during addition of PNA to the culture, scraping of cells).

Optimize experimental conditions (see below).

time may influence the effectiveness of the CPP-PNA entry into the cells, as well as the efficiency of PNA binding with RNA. Experimental conditions may not be optimal for the particular PNA of interest.

Possible parameters to be optimized include cell number, PNA concentration, incubation time for PNA to enter the cells and bind to RNA, length of time and temperature for streptavidin magnetic beads to bind to the PNA-RNA-RBP complexes. For example, increase (or decrease) number of cells to be used (day 1, Step 10); increase (or decrease) PNA final concentration and/ or incubation time during day 1, Step 11; or adjust temperature for day 2, Step 25 (see detailed notes in the PROCEDURE).

ANTICIPATED RESULTS PAIR is an efficient assay used to identify RBPs associated with the RNA (PNA) of interest in live cells. The results using this procedure will be specific to the RNA targeted, as well as to the type and number of cells used to identify the relevant RBPs. For example, when we used PAIR to identify RBPs interacting with ankylosis mRNA11, we typically observed 410 distinct protein bands on a silver-stained gel, and these normally produced 10–20 statistically significant protein identifications after MS analysis with sequencing by Nanospray/Qstar-XL; for other mRNAs, differing numbers of RBPs may be identified. Note that the final output in terms of the sensitivity of the PAIR procedure to identify RBPs that are bound to a particular RNA is dependent on the abundance of the RNA, the ability of the PNA to anneal to the RNA and the sensitivity of the MS technology that is used to identify the RBP protein sequences. Overall, although the number of proteins may vary with different PNAs, physiological replicates should generally be consistent for the same PNA in the same type of cells. 926 | VOL.1 NO.2 | 2006 | NATURE PROTOCOLS

PROTOCOL Note: Supplementary information is available via the HTML version of this article. ACKNOWLEDGMENTS We thank Jennifer Zielinski for helping to pioneer this technology. We thank Margie Maronski for help with culturing the neurons, and Dr. Chao Xing Yuan and Elena Blagoi for help with mass spectrometry. This work was funded by National Institutes of Health grants AG9900 and MH58561 to J.E.; and the Swedish Science Foundation Grants Med and NT, and European Community Grant QLRT-2001-01989 to U.L. COMPETING INTERESTS STATEMENT The authors declare competing financial interests.


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