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Jun 9, 2013 - Here, we exploited DNA family shuffling, a molecular evolution technology, ..... with miR-122 and target RNA containing a perfect miR-122–binding site. ...... Ago2 sequence with a 27-nucleotide stuffer (annealed oligonucleotides #631/632 .... For quantification, we first normalized to either the precursor.
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Molecular dissection of human Argonaute proteins by DNA shuffling

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© 2013 Nature America, Inc. All rights reserved.

Nina Schürmann1, Leonardo G Trabuco2, Christian Bender3, Robert B Russell2 & Dirk Grimm1 A paramount task in RNA interference research is to decipher the complex biology of cellular effectors, exemplified in humans by four pleiotropic Argonaute proteins (Ago1–Ago4). Here, we exploited DNA family shuffling, a molecular evolution technology, to generate chimeric Ago protein libraries for dissection of intricate phenotypes independently of prior structural knowledge. Through shuffling of human Ago2 and Ago3, we discovered two N-terminal motifs that govern RNA cleavage in concert with the PIWI domain. Structural modeling predicts an impact on protein rigidity and/or RNA-PIWI alignment, suggesting new mechanistic explanations for Ago3’s slicing deficiency. Characterization of hybrids including Ago1 and Ago4 solidifies that slicing requires the juxtaposition and combined action of multiple disseminated modules. We also present a Gateway library of codonoptimized chimeras of human Ago1–Ago4 and molecular evolution analysis software as resources for future investigations into RNA interference sequence-structure-function relationships. Fifteen years after its discovery, RNA interference (RNAi) has become a complex research area whose intricacy is exemplified by the wealth of RNA and protein components confirmed experimentally or predicted from sequence homologies. Today, these comprise >25,000 mature micro RNAs (miRNAs) (miRBase v19, http://www.mirbase. org/), >40 Dicers1 and >60 Argonaute (Ago) proteins in numerous species2. This complexity puts growing demands on the RNAi community to devise and apply potent resources to unravel the role and molecular function of all these key players. A prominent example illustrating these needs is that of Ago1–Ago4, which form the functional core of the RNA-induced silencing complex (RISC) in mammals including humans2–5. A precise understanding of the molecular determinants characterizing and distinguishing these four proteins is vital from several perspectives: (i) basic biology, as Ago1–Ago4 exert pleiotropic activities in physiology or pathology, from roles in cell proliferation or differentiation to cancers or infections6–11; (ii) RNAi applications, as Ago1–Ago4 engagement by ectopic RNAi triggers can result in dysregulation of off-target genes or miRNAs, thus hampering data interpretation12; and (iii) clinical RNAi therapies, as Ago1–Ago4 saturation can accelerate tumorigenesis or cause organ damage and lethality in animals13–18, whereas ectopic Ago2 overexpression can boost and prolong RNAi in vitro and in vivo19,20. Accordingly, major efforts were made to dissect human Ago1–Ago4 on a molecular level by using traditional methodologies, such as mutagenesis21–23 or X-ray crystallography24–27. This led to the understanding that all four proteins share the signature domains N, PAZ, MID and PIWI, load and resolve small-RNA duplexes and then direct RISC to target mRNAs for inhibition, destabilization or cleavage and slicing4. The latter mechanism is inherent to only Ago2 and is linked to its

PIWI domain, which adopts an RNase H fold with a catalytic AspAsp-His (DDH) triad28. Besides, slicing is also involved in miRNA biogenesis and processing29–32. Still, numerous questions puzzle the community to date, as best exemplified by the conundrum of why human Ago3 is slicing deficient despite its conserved catalytic triad. This question and others have not been answered for four reasons: (i) the lack of comprehensive structural information except for human Ago2 and a few yeast or prokaryotic Ago proteins, which hampers rational mutagenesis; (ii) the restriction of standard techniques to manipulation or analysis of one molecule at a time; (iii) the largely undefined modes of action of the three human nonslicers, which complicates the interrogation of mutants of Ago1, Ago3 and Ago4; and (iv) the limited ­understanding of many Ago proteins in other species, which affects comparative analyses. To overcome these problems, some labs have made and studied hybrids between two proteins, for example, Arabidopsis Ago1Ago4 or Ago1-ZLL33,34 and human Ago1-Ago2 (refs. 35,36). Yet this approach remains low throughput and is limited by a need for detailed knowledge on at least one partner to rationalize domain swapping. The RNAi field would thus still substantially benefit from scalable, comprehensive and unbiased new strategies to systematically untangle protein sequence-structure-function relationships. To fill in this gap, we introduce DNA family shuffling (DFS), a molecular evolution technology whose hallmark is the controlled fragmentation of multiple related genes and subsequent reassembly based on partial sequence homologies37. In a few rapid reactions, it yields tens of thousands of chimeric cDNAs whose translation into hybrid proteins and subsequent screening in an arrayed or batch format permits isolation of desired phenotypes and identification

1Heidelberg

University Hospital, Cluster of Excellence CellNetworks, Department of Infectious Diseases, Virology, Heidelberg, Germany. 2Cluster of Excellence CellNetworks, Heidelberg University, Heidelberg, Germany. 3Translational Oncology at the University Medical Center of the Johannes Gutenberg University, Mainz, Germany. Correspondence should be addressed to D.G. ([email protected]). Received 14 February; accepted 13 May; published online 9 June 2013; doi:10.1038/nsmb.2607

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Creation and screening of a library of Ago2-Ago3 chimeras We next used DFS to create a pool of Ago2-Ago3 chimeras (Fig. 1b), using Ago3 in two-fold excess to increase our chances to identify regions outside of PIWI responsible for slicing activity. In tested clones, 116 of 136 (85%) showed a correct length and protein expression (data not shown), a result verifying stringent shuffling.

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RESULTS The Ago3 PIWI domain is slicing competent Initially, we focused on the paradox that human Ago3 lacks slicing capability despite the >90% identity of its PIWI domain to that of slicing-competent Ago2 (Supplementary Fig. 1a). We hypothesized that Ago2 possesses critical residues or domains beyond PIWI and postulated that their transfer into Ago3 may reconstitute slicing. First, however, we had to demonstrate that Ago3 PIWI is slicing competent. We thus created mutant N2P2M2P3 by fusing Ago3 PIWI (residues 575–860) to Ago2 N–PAZ–MID (residues 1–573). For all clones in this study, we used codon-optimized human Ago cDNAs expressing comparable protein levels19,40. As expected, Ago immunoprecipitates from cells cotransfected with a short hairpin RNA (shRNA) and Flagtagged wild-type Ago proteins and then incubated with an shRNA target showed Ago2 but not Ago3 cleavage activity. Notably, N2P2M2P3 mediated slicing similar to that of Ago2, and this verified the principal functionality of Ago3 PIWI (Fig. 1a and Supplementary Fig. 1b).

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of the responsible DNA and protein regions. Previously, we and others have applied DFS to study and enhance various proteins or viral gene-therapy vectors37,38. Here, we used it to elucidate features of human Ago1, Ago3 and Ago4 that may explain their RNA slicing deficiency. We thus shuffled Ago2 (slicer) with Ago3 or Ago1, Ago3 and Ago4 and then screened the resulting libraries for hybrids exhibiting slicer versus nonslicer phenotypes. This was accompanied by the study of Ago1–Ago4 mutants that we rationally designed on that basis of our insights from the library clones. We discovered that (i) Ago3 PIWI is slicing competent when embedded in Ago2, (ii) two short N-terminal motifs support (in Ago2) or inhibit (in Ago3) ­slicing, (iii) the mere juxtaposition of these two Ago2 motifs with Ago3 PIWI restores Ago3 slicer activity, (iv) point-mutated Ago1 PIWI is slicing competent in the context of Ago1–Ago3 and (v) Ago4 is functionally distinct from Ago1–Ago3 and incompatible with ­slicing. We conclude that the microenvironment determining Ago phenotypes is highly complex and involves the coordinated action of multiple disseminated protein parts, explaining why subtle natural or experimental alterations can trigger the switch between slicer and nonslicer. Finally, we adapted our Ago1–Ago4 library to the Gateway rapid cloning system39 and implemented a public software for automated analyses of DFS products, to facilitate the broad application of our resources and to foster future attempts to unravel and harness RNAi in and across many species.

Relative knockdown efficiency

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Ago2 59 110 44 78 129 74 2 82 Ago3

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Motif II 134–166

Figure 1  Use of DFS to discover Ago motifs critical for slicing. (a) RISC cleavage assay with immunoprecipitates from HEK293T cells transfected with Flag-YFP or Flag-Ago plus specific (Ren3 shRNA, +) or control shRNA (−). Cleavage activity was calculated as a percentage of products (triangles) relative to total target RNA. Comparable protein inputs are verified in Supplementary Figure 1b. (b) Ago DFS protocol scheme (details in Online Methods). (c) Luciferase knockdown after Huh7 cotransfection with Renilla luciferase, Ago or YFP (control) and a Renilla luciferase (Ren3) shRNA or a control shRNA. P values were determined by Student’s t test. Error bars, s.d. (n = 8 biological replicates). Rlu, relative light units. (d) Functional performance of 116 Ago2-Ago3 chimeras in luciferase knockdown assays (in c). Clones with strong Ago2-like phenotypes (relative knockdown efficiency 0.15). To confirm the relevance of the two motifs for the Ago2 pheno- activation. Alanine mutation of motif I in Ago2 (Ago2IAAAAA) also type, we rationally designed additional Ago2-Ago3 mutants (Fig. 2a). ablated silencing and slicing. Reconstitution of miRNA-guided Indeed, changing motif I in Ago2 to five alanines (Ago2IAAAAA) or mRNA cleavage in Ago3 required simultaneous insertion of motifs I substituting motif II with the Ago3 counterpart (Ago2II3) largely abol- (at least Met47) and II, as evidenced by functional (Ago3I 2II2, ished shRNA-guided target cleavage (Fig. 2b,c). We observed the same Ago3IMetII2 and N2P3M3P3) versus inactive mutants (Ago3I2 and for an Ago2 derivative carrying the Ago3 N terminus (N3P2M2P2). Ago3II2). Ago2 with the Ago3 counterpart of motif II (Ago2II3) also The exact opposite chimera (N2P3M3P3) exhibited target cleavage sliced and mediated knockdown, contrary to the shRNA results. comparable to that of Ago2 (Fig. 2b,c), akin to N2P2M2P3 (Fig. 1a). Notably, we observed no enrichment of either mature miR-122 Notably, we could similarly evoke slicing capability by merely insert- guide strands in immunoprecipitated slicing-competent-Ago ing our two motifs instead of the entire Ago2 N domain into Ago3 complexes or star strands in complexes with slicing-incompetent (Ago3I2II2), whereas replacing each domain individually (Ago3I2 or Agos (Fig. 2g,h). The only exception was Ago2IAAAAA, which was characterized Ago3II2) had no effect. To further dissect the five residues in motif I, we performed an by an excess of star strands surpassing levels in Ago3 and all other alanine scan within the Ago2 context (Supplementary Figs. 3 and 4). mutants (Fig. 2h). Because Ago2IAAAAA also failed to accumulate Whereas four substitutions—F44A, F45A, E46A and D48A—were mature shRNA strands (Fig. 2c and Supplementary Fig. 3c), this phenotypically inconspicuous, the M47A mutation reduced Ago2 suggested that the mutation of Ago2 motif I to five alanines had knockdown and cleavage capability (Supplementary Fig. 3a–c and created pleiotropic effects on RISC activation and mRNA slicing Ago3

© 2013 Nature America, Inc. All rights reserved.

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data not shown). Conversely, introducing this methionine into the Ago3 variant containing the Ago2 motif II induced slicing activity (Supplementary Fig. 3b,c; comparison of Ago3 mutants II2, I2II2 and IMetII2), whereas inserting it into wild-type Ago3 had no effect (data not shown). From these and corroboratory data with three independent shRNAs (Supplementary Fig. 3d), we conclude that both Ago2 N-terminal motifs I and II are crucial and required for an Ago2like phenotype.

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Figure 4  Characterization of chimeric Ago1–Ago4 mutants. (a) Scheme depicting Ago1–Ago4 mutants (color-coded according to the four wild types on the left). The catalytic DEDH tetrad is indicated where reconstituted. (b) Knockdown efficiencies mediated by tethered Ago1–Ago4 mutants, analyzed by normalizing luciferase activities to the λN-fused YFP control. P values were determined by Student’s t test. Error bars, s.d. (n = 3 biological replicates). (c,d) Luciferase (top) and RISC cleavage assays (bottom) in all Ago1–Ago4 mutants with perfect (c; Ren1 shRNA) or imperfect (d; miR-122) small-RNA duplexes (experimental details as in Fig. 1).

and in turn yielded a stronger Ago3 phenotype. To further dissect this possibility and the role of motif I, we created two additional Ago2 mutants in which we replaced motif I with the corresponding residues from Ago3 (FFEMD→CFQVE, Ago2I3) or substituted both motifs I and II simultaneously (Ago2I 3II3) (Supplementary Fig. 3a). Of note, and unlike Ago2IAAAAA, Ago2I3 exhibited weak but detectable knockdown and slicing with shRNA and miRNA triggers (Supplementary Fig. 3e,f). In further contrast, Ago2I3 recapitulated potent RISC activation as evidenced by sense/antisense strand ratios that were identical to those for Ago2 (Supplementary Fig. 3g–i). The Ago2 mutant in which we had replaced both motifs simultaneously (Ago2I3II3) was deficient for RISC activation when loaded with the shRNA, but not the miRNA, thereby ­ mimicking Ago3 (Supplementary Fig. 3g–i and Supplementary Fig. 4). Further akin to Ago3, this mutant ­exhibited neither knockdown nor slicing, regardless of the RNAi trigger (Supplementary Fig. 3e,f). Overall, our data support a dual role of the Ago2 N domain in RISC activation and mRNA cleavage. We conclude that the concurrent juxtaposition of our two identified short Ago2 N motifs with either Ago2 or Ago3 PIWI is critical and sufficient for effective passenger-strand removal from perfect duplexes as a prerequisite for Ago2-like slicing and target knockdown. Only the Ago3I2II2 double mutant recapitulated an Ago2 phenotype, and, vice versa, concurrent replacement of motifs I and II in Ago2 was the sole strategy that fully mimicked 822

Ago3. Our data further imply that the different steps in RNAi are partly uncoupled in vivo and that our two motifs have synergistic but distinct roles. Particularly important during initial RISC activation may be motif II, as suggested by Ago2II3, which was defective for shRNA resolution, unlike Ago2I3, which was identical to Ago2 at this step. Additional evidence is that Ago2II3 became competent for knockdown and slicing when loaded with an imperfect miRNA whose activation is independent of passenger-strand slicing. Conversely, motif I seems indispensable for target slicing because Ago2I 3 was indistinguishable from Ago2 during shRNA loading and RISC activation but still deficient in luciferase knockdown and mRNA slicing. These two different but synergistic roles of Ago2 motifs I and II can explain why their conjunction in Ago3 was required to reconstitute a complete Ago2-like RNAi process, from potent RISC activation to mRNA slicing and target knockdown. Human Ago3 modeling predicts functions for motifs I and II To further investigate our two motifs, we performed homology modeling of human Ago3 (Fig. 3 and Supplementary Fig. 5a) on the basis of the two recently reported high-resolution structures of human Ago2–RNA complexes24,25. We also modeled a complete human Ago2 structure and noted a close correlation with the published crystal structures, confirming the quality of our modeling. Notably, we found our two motifs to reside adjacent to PIWI and in the lower part of the

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Sequences of 30 Ago1–Ago4 chimeras

Figure 5  DNA family shuffling of Ago1–Ago4. Ago1 (a) Alignment and annotation (generated Ago2 by Salanto) of 30 randomly chosen clones Ago3 Ago4 (each depicted as one horizontal bar) derived Mut from Ago1–Ago4 DFS and color-coded as indicated. Mut, mutations. (b) Homologies of N PAZ MID PIWI L1 L2 shuffled Ago1–Ago4 library clones to parental Ago proteins. For better visualization of the Ago1–Ago4 chimeras 1 2 3 4 5 6 7 9 10 11 12 14 15 16 17 18 19 20 23 26 27 28 29 30 3132 33 34 36 38 differences, residues identical in all Ago Ago1 1 2 3 4 proteins were excluded. (c) Diverse functionality Ago2 Ago3 of representative Ago1–Ago4 chimeras. Bars 4 Ago4 3.5 × 10 indicate enhanced (Ago2-like, –1) or decreased pIRES (Ago1-, Ago3- or Ago4-like, >0) knockdown 0% Homology 100% efficiencies in luciferase assays with perfect Catalytic tetrad 6 2.8 × 10 (Ren1 shRNA) or imperfect (miR-122) smallDEDR Ago1 DEDH Ago2 RNA triggers. The composition of the individual pENTR attL attL DEDH Ago3 clones and their catalytic tetrads are depicted DEGR Ago4 at left. (Full sequences of all ten clones #1 DEDR pDEST are shown in Supplementary Fig. 7c.) (d) A DEDH #3 Gateway-compatible library of codon-optimized #5 DEDH Delivery: Host: Fusion: - Fluorophore - Transfection - Mammalian DEDH #7 human Ago1–Ago4 cDNAs. The scheme depicts - λN peptide Infection... Insect DEDH #14 (top to bottom) the generation of a shuffled - HIS ... - Yeast DEDR #31 - Bacteria... Ago1–Ago4 library in the pIRES expressionDEDH #32 plasmid backbone and its transfer into the DEDR #33 pENTR vector, to become flanked with attL miR-122 #34 DEGH Ren1 shRNA #38 DEDH i ii iii iv v (attachment) sites for Gateway recombination. –2 –1 0 1 2 The subsequent choice of destination vector N L1PAZ L2 MID PIWI ? ! Relative knockdown allows for manifold applications, including efficiency fusion with fluorescent reporters for intracellular localization studies (i), dissection of domains governing Ago-RNA interactions (akin to the present proof-of-concept work) (ii), screens for, and isolation of, Ago-interacting cellular proteins (iii) or enrichment of Ago variants that specifically process certain small RNAs (iv). Moreover, the Gateway design permits the shuttling of Ago chimeras of interest between different plasmids, allowing, for example, fusion with affinity tags for purification and further biochemical characterization (v).

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N domain, and this probably affects positioning of the guide–target complex and unwinding of the small-RNA duplex (Fig. 3)45,46. Both motifs, moreover, encompass two conserved β-sheets in the L1 linker, which interact with an α-helix implicated in Ago-RNA interactions25. Additionally noteworthy is that eukaryotic Ago proteins may arrange the N domain such that it creates a nucleic acid–binding channel to permit complete guide-target pairing and mRNA positioning relative to the catalytic protein core47. Our modeling suggests that this channel is partly obstructed in Ago3, owing to a bulky loop formed by the eight additional residues in the equivalent of Ago2 motif II (Fig. 3b,c). Support comes from an Ago3 mutant (Ago3I2IIloop2) in which we merely replaced this loop instead of the entire motif II, which still exhibited catalytic activity (Supplementary Fig. 5b). Moreover, our Ago3 model suggests multiple interactions between residues in the PIWI and N domains (salt bridges, hydrogen bonds and hydrophobic contacts; Supplementary Fig. 5c,d and data not shown) that may increase protein rigidity or invoke other properties that contribute to Ago3’s slicing deficiency. Finally, it was recently proposed on the basis of a yeast Ago structure that rather than a triad, an amino acid tetrad including a previously unappreciated glutamate constitutes the active site of slicing Agos47. We thus mutated the corresponding glutamate (Glu637) in human Ago2 and found that it is indeed required for mRNA slicing with shRNAs and miRNAs, verifying the importance of an extended catalytic DEDH core in human slicer (Supplementary Fig. 5e). Notably, Glu637 probably requires a conformational protein change induced by RNA loading to enter the catalytic site47. We thus speculate that the unique structure of the Ago3 N domain prevents proper Glu637 repositioning and hence completion of the catalytic tetrad, and this in turn averts passenger or target RNA slicing.

We propose that conformational differences in Ago2 and Ago3 N domains fundamentally affect protein structure, flexibility and activity. Particularly unexpected was that Ago3 possesses an inhibitory motif that blocks slicing, contrary to our initial hypothesis that Ago3 merely lacked activating functions present in Ago2. Also notably, neither our models nor the published structures predict major shifts of motif I or Met47 in Ago3 or their direct role in Ago2. This highlights the potential of DFS to identify functionally relevant features that are not apparent in static sequences or even in three-dimensional structures. Directed molecular evolution of all four human Ago proteins To gain further insights into the elements distinguishing slicers from nonslicers, we expanded DFS to human Ago1 and Ago4. To validate the feasibility of shuffling all four Agos, we initially generated Ago1–Ago4 domain-swapping mutants (Fig. 4a,b). Moreover, we engineered Ago1 and Ago4 to contain the catalytic PIWI DEDH tetrad. Following our previous workflow, we then tested all mutants for knockdown, cleavage and RNA binding activity with perfect or imperfect duplexes (Fig. 4c,d and Supplementary Fig. 6a–c). Notably, we observed marked differences between the Ago chimeras, depending on the small-RNA trigger. Whereas N2P3M3P3 and N2P2M2P3 were the only mutants besides Ago2 that sliced efficiently with the shRNA, the results profoundly differed after miRNA loading (Fig. 4c,d). There, we detected slicing with all mutants containing Ago1 or Ago2 N domains fused with PIWI from Ago1DEDH, Ago2 or Ago3. Accordingly, even Ago1DEDH, differing from wild-type Ago1 by one residue (R805H), was slicing competent with the miRNA yet not with the shRNA. The Ago1 N domain performed best when juxtaposed with cognate PAZ and MID (for example, N1P1M1P2 outperformed N1P2M2P2), and this again exemplifies the importance of

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articles proper intermolecular-domain interactions. Unlike Ago1, Ago4DEDH and all chimeras containing Ago4DEDH PIWI or Ago4 N-terminal domains (N4P2M2P2, N4P3M3P3, N4P4M4P2 and N4P4M4P3) were slicing deficient, regardless of the RNA trigger (additional data in Supplementary Fig. 6d,e). Similar differences were apparent when we analyzed the smallRNA binding capacity of the Ago1–Ago4 chimeras (Supplementary Fig. 6a–c). Mutants with reconstituted Ago1 PIWI showed signals for both shRNA strands, a result hinting at impaired RISC activation and thus extending our data with the slicing-incompetent Ago2-Ago3 chimeras. That these mutants became slicing proficient when loaded with the miRNA underscores that duplex activation and mRNA cleavage are separate processes dictated by different structural or functional Ago features. In this respect, Ago1DEDH PIWI is different from Ago2 or Ago3 PIWI because the latter could resolve shRNA duplexes by cleavage (when juxtaposed with our two Ago2 motifs). Concurrently, these findings support that it is PIWI in combination with regulatory N-terminal (and other) determinants rather than PIWI alone that defines slicing proficiency of the whole protein. It is also noteworthy that chimeras with Ago4 N-terminal domains were defective in RISC activation (even with the miRNA; for example, N4P2M2P2 in Supplementary Fig. 1b,c) and sometimes also in RNA loading (for example, N 4P3M3P3 in Supplementary Fig. 1a). This highlights the pleiotropic role of the Ago N domain and emphasizes the unique position of human Ago4 within the Ago protein family. A versatile Gateway library to dissect Ago1–Ago4 functionality The variety of mutant phenotypes prompted us to apply DFS to create a library of Ago1–Ago4 chimeras. This second library comprised >35,000 cDNA expression plasmids, of which >85% (41 of 48 tested clones) showed restriction patterns of full-length sequences (data not shown). Complete sequencing of 30 randomly chosen clones and alignment to the four parental cDNAs confirmed efficient shuffling (8.33 ± 2.41 crossovers per clone and no domain-specific enrichment of individual parents) and a high library diversity (Fig. 5a,b). To validate library functionality, we tested ten randomly chosen chimeras in knockdown assays with an shRNA or miRNA. We observed a trigger-dependent spectrum of RNAi activation (Ago2 phenotype) or inhibition (Ago1, Ago3 or Ago4 phenotype) (Fig. 5c), as with our Ago2-Ago3 library. Clones 3, 5 and 38, all of which contain the Ago2 or Ago3 PIWI DEDH tetrad (Supplementary Fig. 7), underscore the different abilities of the Ago1 and Ago2 N domains to confer target knockdown depending on the RNA trigger. That clone 32 was inactive despite the Ago2 N domain’s juxtaposition with Ago3 PIWI hints at additional critical residues or domains with which the N or PIWI domains must interact that were perturbed in this chimera by replacement with Ago1 or Ago4 sequences. Alternatively, it is conceivable that these Ago1 or Ago4 features inhibit slicing. This would be in line with the inhibitory role of Ago3 motif II and could explain the inactivity of Ago1DEDH (when loaded with shRNAs). Either way, this clone illustrates the prospect of our Ago1–Ago4 library to identify and dissect complex structure-function relationships involving determinants from all four human Ago proteins, in a manner that exceeds conventional methodologies. Last, to facilitate the future custom application of this library, we rendered it Gateway compatible (Fig. 5d). Therefore, we transferred the complete cDNA pool into the pENTR vector, to yield a library of 2.8 × 106 clones. After shuttling 48 chimeric cDNAs into two different pDEST vectors, we found that all resulting plasmids contained full and diverse Ago1–Ago4 sequences. Together with effects on 824

t­ arget knockdown in a range of over one order of magnitude (data not shown), this validates the integrity, functionality and usefulness of our Ago1–Ago4 Gateway library. DISCUSSION We have established and validated a fresh concept to investigate sequence-structure-function relationships of RNAi factors, using human Ago1–Ago4 for proofs of principle. With its feasibility to annotate residues or domains in a simple, rapid, scalable and unbiased manner, DFS expands the current arsenal of molecular, biochemical and modeling tools in the RNAi field. Here, we exemplified its potential with our new findings on the significance of two Ago2 and Ago3 N-terminal motifs for RNA loading, duplex resolution and target cleavage and with our insights into the distinct roles of Ago1 and Ago4 domains. Our results complement and extend prior studies on human Ago biology including a recent paper that used conservation-driven alanine scanning and in vitro assays to identify single Ago2 N-terminal residues affecting RISC assembly and activity46. Notably, the two conceptually different screening strategies converge on Met47. This suggests that this particular residue is critical downstream of duplex loading (at a step that can be mimicked in vitro), in line with our notion that Ago2M47A was defective in mRNA slicing but not in RNA loading or RISC activation. It is generally notable that the Ago2 N domain in vitro seems to be required mostly for duplex unwinding46, whereas our results suggest a more complex in vivo scenario in which the N domain is also crucially involved in target slicing, as implied by our RISC assembly-competent yet cleavage-deficient mutants. These additional insights reflect the power of our DFS approach, which was devised to concurrently screen for loss and gain of phenotype and to allow study of all four Ago proteins and all domains simultaneously. The potential of our methodology is further illustrated by our discovery of two complete motifs that both need to be juxtaposed with an active PIWI, a result that helps to explain why human Ago3 is slicing deficient. Although our findings support the idea that non-PIWI residues are important for slicing, the nature and extent of the regulatory mechanisms that we unraveled were unexpected. Previously we and others predicted that Ago3’s functional differences stemmed simply from its missing critical residues or subdomains from Ago2. Instead, our results suggest that slicer functionality is actually defined by two opposing functions: the presence of activating regions (Ago2 motif I) coupled with the absence of inhibitory regions (Ago3 motif II). From our Ago2 and Ago3 models, we postulate that the two motifs govern multiple Ago properties that permit correct RNA duplex positioning toward the catalytic tetrad and thereby regulate slicing. Although motif II seems critical during early perfect duplex unwinding and motif I during later target cleavage, only their combination renders PIWI fully slicing competent. More generally, we conclude that PIWI itself not only determines whether an Ago acts as a slicer but also embeds in a slicing-proficient microarchitecture that enables intricate intramolecular interactions and thus fine-tunes Ago activity in a versatile and modular manner. This concept is backed up further by our data with the second library, which imply that many more regulatory features and interactions await discovery. The analysis of our hybrids and libraries in other labs and with physiologically relevant readouts could thus help to further unravel and consolidate the biology of all four human Agos35,42,43,48. To complete the annotation of their sequencestructure-function relationships, it will also be highly rewarding to compare results and clone compositions with the human Ago1, Ago3 and Ago4 crystal structures once they are available.

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articles Our conclusion that Ago functionality is defined by complex interactions of modular (sub)domains has implications for Ago evolution. Notable in this regard is that slicing-competent Ago3 PIWI is rendered inactive by minimal motifs or that a single residue determines whether Ago1 can slice mRNAs (when loaded with imperfect duplexes) in vivo (our work) and in vitro48. Whether this reflects nature’s means to subdue excessive slicing, whether the loss or repression of Ago1–Ago3 slicing is a byproduct of other emerging features and whether regulatory mechanisms such as allostery fine-tune Ago activities49,50 remain unanswered questions. Also notable is that no chimera containing Ago4 domains was slicing competent, a result implying that Ago4 is evolutionarily most distinct and least compatible with the intermolecular foldings and interactions needed to activate PIWI. We also envision innovative means to exploit Ago modularity to advance biological or medical RNAi applications akin to our viralvector work38. These include screens of our Gateway Ago library in a high-throughput and/or high-content format to produce chimeras exerting functions not found in nature, such as preferred loading of therapeutic small RNAs (for example, miR-451 derivatives30–32). Their judicious overexpression could maximize RNAi potency, specificity and safety while overcoming notorious adverse events such as cytotoxicity15. Notably, DFS can readily be extended to other factors or species, provided that the cDNAs are sufficiently similar (>50% total), such as plant Ago1, Ago5 and Ago10 or Caenorhabditis elegans Alg1 and Alg2. Equally promising will be to produce and study chimeras across species borders, for example, between human and fly Ago1 (58.9% DNA identity and >40 stretches of at least eight identical contiguous nucleotides). Besides, codon optimization will further increase homologies and likelihoods of recombination to even enable crosskingdom shuffling. Notably, because Salanto represents an easily accessible open community resource, it could guide others to adapt DFS for any custom needs and to annotate their lead candidates. In summary, directed molecular evolution technology has the potential to untangle many of the still-enigmatic RNAi processes in normal or diseased cells, and we thus believe that its broader implementation and use will considerably advance basic and applied RNAi research. Methods Methods and any associated references are available in the online version of the paper. Note: Supplementary information is available in the online version of the paper. Acknowledgments The authors greatly appreciate funding of their group and work by the Heidelberg University Cluster of Excellence CellNetworks (grant number EXC 81) as well as by the Chica and Heinz Schaller Foundation. L.G.T. was supported by an European Molecular Biology Organization fellowship (EMBO ALTF 676-2010). We moreover thank H.-G. Kräusslich, R. Bartenschlager, G. Stöcklin and all lab members for helpful discussions and suggestions. In addition, we are indebted to S. Ruedel (University of Regensburg, Regensburg, Germany) for kindly providing her protocol for slicer assays as well as to W. Filipowicz (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland) for his much appreciated gifts of Ago2-knockdown cells and of various plasmids for the tethering assays. Finally, we are grateful to E. Wiedtke for technical support as well as to S. Grosse for her extensive critical testing of our Salanto software and for providing sundry useful ideas for improvements. AUTHOR CONTRIBUTIONS N.S. and D.G. designed the experiments in this work and wrote the manuscript. N.S. conducted all experiments. L.G.T. and R.B.R. contributed the Ago2 and Ago3 Rosetta models and assisted in their interpretation. C.B. performed the

bioinformatical analyses of the DFS reactions and products, using the Salanto software tool that he, together with N.S., developed for this work. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. 1. Mukherjee, K., Campos, H. & Kolaczkowski, B. Evolution of animal and plant dicers: early parallel duplications and recurrent adaptation of antiviral RNA binding in plants. Mol. Biol. Evol. 30, 627–641 (2013). 2. Höck, J. & Meister, G. The Argonaute protein family. Genome Biol. 9, 210 (2008). 3. Hutvagner, G. & Simard, M.J. Argonaute proteins: key players in RNA silencing. Nat. Rev. Mol. Cell Biol. 9, 22–32 (2008). 4. Su, H., Trombly, M.I., Chen, J. & Wang, X. Essential and overlapping functions for mammalian Argonautes in microRNA silencing. Genes Dev. 23, 304–317 (2009). 5. Ender, C. & Meister, G. Argonaute proteins at a glance. J. 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Cheloufi, S., Dos Santos, C.O., Chong, M.M. & Hannon, G.J. A dicer-independent miRNA biogenesis pathway that requires Ago catalysis. Nature 465, 584–589 (2010). 31. Cifuentes, D. et al. A novel miRNA processing pathway independent of Dicer requires Argonaute2 catalytic activity. Science 328, 1694–1698 (2010).

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articles 41. Joern, J.M., Meinhold, P. & Arnold, F.H. Analysis of shuffled gene libraries. J. Mol. Biol. 316, 643–656 (2002). 42. Gu, S. et al. Thermodynamic stability of small hairpin RNAs highly influences the loading process of different mammalian Argonautes. Proc. Natl. Acad. Sci. USA 108, 9208–9213 (2011). 43. Petri, S. et al. Increased siRNA duplex stability correlates with reduced off-target and elevated on-target effects. RNA 17, 737–749 (2011). 44. Winter, J. & Diederichs, S. Argonaute proteins regulate microRNA stability: increased microRNA abundance by Argonaute proteins is due to microRNA stabilization. RNA Biol. 8, 1149–1157 (2011). 45. Wang, Y. et al. Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes. Nature 461, 754–761 (2009). 46. Kwak, P.B. & Tomari, Y. The N domain of Argonaute drives duplex unwinding during RISC assembly. Nat. Struct. Mol. Biol. 19, 145–151 (2012). 47. Nakanishi, K., Weinberg, D.E., Bartel, D.P. & Patel, D.J. Structure of yeast Argonaute with guide RNA. Nature 486, 368–374 (2012). 48. Janas, M.M. et al. Alternative RISC assembly: binding and repression of microRNA-mRNA duplexes by human Ago proteins. RNA 18, 2041–2055 (2012). 49. Djuranovic, S. et al. Allosteric regulation of Argonaute proteins by miRNAs. Nat. Struct. Mol. Biol. 17, 144–150 (2010). 50. Hur, J.K., Zinchenko, M.K., Djuranovic, S. & Green, R. Regulation of Argonaute Slicer activity by guide RNA 3′ end interactions with the N-terminal lobe. J. Biol. Chem. 288, 7829–7840 (2013).

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32. Yang, J.S. et al. Conserved vertebrate mir-451 provides a platform for Dicerindependent, Ago2-mediated microRNA biogenesis. Proc. Natl. Acad. Sci. USA 107, 15163–15168 (2010). 33. Mallory, A.C. et al. Redundant and specific roles of the ARGONAUTE proteins AGO1 and ZLL in development and small RNA-directed gene silencing. PLoS Genet. 5, e1000646 (2009). 34. Zha, X., Xia, Q. & Yuan, Y.A. Structural insights into small RNA sorting and mRNA target binding by Arabidopsis Argonaute Mid domains. FEBS Lett. 586, 3200–3207 (2012). 35. Wang, B. et al. Distinct passenger strand and mRNA cleavage activities of human Argonaute proteins. Nat. Struct. Mol. Biol. 16, 1259–1266 (2009). 36. Yuan, Y.R. et al. Crystal structure of A. aeolicus argonaute, a site-specific DNAguided endoribonuclease, provides insights into RISC-mediated mRNA cleavage. Mol. Cell 19, 405–419 (2005). 37. Crameri, A., Raillard, S.A., Bermudez, E. & Stemmer, W.P. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391, 288–291 (1998). 38. Grimm, D. et al. In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses. J. Virol. 82, 5887–5911 (2008). 39. Hartley, J.L., Temple, G.F. & Brasch, M.A. DNA cloning using in vitro site-specific recombination. Genome Res. 10, 1788–1795 (2000). 40. Valdmanis, P.N. et al. Expression determinants of mammalian argonaute proteins in mediating gene silencing. Nucleic Acids Res. 40, 3704–3713 (2012).

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ONLINE METHODS

Plasmids. Plasmids expressing human wild-type Ago2, Ago3 or YFP (negative control) were from Addgene (Ago2, 10822; Ago3, 10823; YFP, 10825). Plasmids encoding Ago2DEAH and codon-optimized human Ago1–Ago4 cDNAs were described previously19,40. All new Ago mutants are based on these cDNAs and were cloned into the parental pIRES backbone by NotI/EcoRI. Domain swapping or point mutants were generated by overlap-extension PCR (OE-PCR). Briefly, 5′, 3′ and middle fragments were produced by regular PCR and then used as OEPCR templates. (Supplementary Tables 1 and 2 list primers and templates.) The final reaction (50 µl) contained 0.5 µl each of 5′, 3′ and (if needed) middle PCR fragments, 1 µl HotStar HiFidelity DNA polymerase (Qiagen), 10 µl 5× HotStar HiFidelity PCR buffer and 1 µM each of primers #459/517 (Supplementary Table 2). Cycling conditions were 5 min at 95 °C and 40 cycles of 15 s at 94 °C, 1 min at 53 °C and 3 min at 72 °C, with a final 10-min extension step at 72 °C. Plasmid psi2-miR-122perf was generated by cloning one perfect miR-122–binding site (annealed oligonucleotides #102/103, Supplementary Table 2) into psiCheck2 (Promega) through XhoI/NotI. Plasmids encoding Renilla luciferase shRNAs or human α-1-antitrypsin shRNAs (control) were as described19. The Ren4 shRNA– expressing construct was made by cloning annealed oligonucleotides #189/190 (Supplementary Table 2) into the same plasmid backbone (through BbsI) as for all other shRNAs13. For tethering assays, five boxB elements were PCR-­amplified from pCIneoRL-5BoxB51 by primers #872/873 (Supplementary Table 2) and cloned into the Renilla luciferase 3′ UTR in psiCheck2 (XhoI/NotI) to yield psiCheck2-5boxB. To create λN-peptide-tagged Ago mutants, we first inserted annealed oligonucleotides #614/615 (Supplementary Table 2) into EcoRI/NotIdigested pλN-HA-Ago2 (ref. 51), inverting the NotI/EcoRI sites in this plasmid (pCINeo-λN-HA-N/E) and permitting subsequent Ago-mutant transfer from the pIRES backbone through NotI/EcoRI. Shuffled Ago1–Ago4 cDNAs were cloned into a pIRES-Ago2 derivative (pIRES-Oligo) in which we had replaced the Ago2 sequence with a 27-nucleotide stuffer (annealed oligonucleotides #631/632 (Supplementary Table 2) cloned into EcoRI/NotI). DNA family shuffling of Ago cDNAs. DFS was performed as a two-step reaction including controlled fragmentation of the parental cDNAs, a primerless PCR to reassemble full-length sequences (Fig. 1b) and a second PCR for their amplification. Codon-optimized Ago2 and Ago3 cDNAs were mixed in a 1:2 (Ago2/Ago3) ratio because we expected an excess of slicing-incompetent Ago3 to improve the likelihood of identifying the minimally required residues or domains for slicer activity in our subsequent functional assays. This mixture was next digested with DNaseI in a 50-µl reaction containing 2.5 µg Ago cDNAs, 0.25 U DNase I and 5 µl DNaseI reaction buffer (both from Invitrogen). The reaction was incubated for 2 min at 24 °C and then heat inactivated (65 °C) for 10 min. Fragments of 0.1–1.0 kb were gel-purified and reassembled in a 50-µl PCR containing 400 ng digested DNA, 10 µl Phusion GC buffer (Finnzymes), 1 µl dNTP mix (10 mM each, Fermentas), 3% DMSO and 1 U Phusion Hot Start II polymerase. PCR conditions were 30 s at 98 °C and 40 cycles of 10 s at 98 °C, 30 s at 50 °C and 45 s at 72 °C, with a final 10-min extension step at 72 °C. To amplify full-length cDNAs, we spiked 2 µl into a second PCR containing 5× HotStar HiFidelity PCR buffer, 0.1 U/µl HotStar HiFidelity DNA Polymerase and 1 µM each of primers #459/460 (Supplementary Table 2). Reassembled chimeric Ago cDNA sequences were cloned into pIRES through NotI/EcoRI. Fragmentation of Ago1–Ago4 cDNAs was performed in a 130-µl reaction containing a total of 6.8 µg DNA (1.7 µg of each Ago cDNA) in a Covaris S2 (5% duty cycles, intensity 3, 200 cycles per burst, 90 s). Proper fragmentation was confirmed by a 2100 Bioanalyzer (Agilent Technologies, DNA 1000 Chip). Fragments of 0.1–1.0 kb were purified and used in a reassembly PCR as above. For the second PCR (conditions as above), primers #602/603 (Supplementary Table 2) were used. The reassembled chimeric Ago1–Ago4 cDNAs were cloned into pIRES-Oligo through NotI/EcoRI. Our final library contained >3.5 × 104 plasmid clones. Generation of a Gateway-compatible Ago1–Ago4 library. To render our Ago1– Ago4 library Gateway compatible, we exploited an existing pENTR-Ago2 vector (generated by PCR amplification (primers #602/603, Supplementary Table 2) of the codon-optimized Ago2 cDNA, including Flag/HA tag and NotI/EcoRI restriction sites, and subsequent BP reaction–based insertion into pDONR207 (Invitrogen)). We replaced the Ago2 sequence in this plasmid with annealed oligonucleotides #616/617 (Supplementary Table 2, cloned into EcoRI/NotI)

doi:10.1038/nsmb.2607

to yield pENTRN/E-Oligo. The complete shuffled Ago1–Ago4 library was then transferred from pIRES into pENTRN/E-Oligo through NotI/EcoRI (to yield 2.8 × 106 clones). Cell culture and transfections. HEK293T cells were grown in DMEM with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. Huh7 medium was complemented with nonessential amino acids. HEKAgo2kd52 cells were grown in Huh7 medium containing 2 µg/ml puromycin, 10 µg/ml blasticidin and 1 µg/µl doxycycline. For RISC cleavage assays, HEK293T cells (­preferred for large-scale formats because of their high transfectability) were seeded at 5 × 106 cells per 140-mm plate and transfected 2 d later with polyethyleneimine (PEI). A total of 44 µg DNA containing shRNA- and Ago-expressing plasmids (1:4 ratio) was diluted in 790 µl H2O and combined with 790 µl 300 mM NaCl before PEI solution (352 µl PEI, 438 µl H2O and 790 µl 300 mM NaCl) was added dropwise and mixed by vortexing. After 10 min at room temperature, the entire transfection mix was added dropwise to the cells. For luciferase assays and western blot analyses, cells were seeded in 96-well plates and transfected with 100–200 ng plasmid DNA with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Luciferase reporter assays. All Ago proteins were tested in at least three independent experiments in triplicates. Error bars in the figures indicate s.d. HEK293T, Huh7 and HEKAgo2kd cells were transfected with 10 ng psiCheck2, 50 ng shRNA plasmid and 140 ng Ago or YFP plasmid (per well of a 96-well plate). For tethering experiments, transfection mixes contained 10 ng psiCheck2-5boxB plasmid and 100 ng vector encoding λN-peptide fusion proteins. Luciferase assays were performed 2 d post-transfection with the Dual-Luciferase Reporter Assay kit (Promega). Renilla luciferase activity was normalized to Firefly luciferase activity, and shRNA or miRNA effects were compared to those of an unrelated control small RNA. To better visualize influences of Ago overexpression on knockdown efficiency, we normalized luciferase values to YFP controls containing the specific small RNA. Usually, YFP, Ago2 and Ago3 values were subsequently set to 0, –1 and 1, respectively, and sample values were adjusted accordingly. For shuffled Ago2-Ago3 chimeras, all experimental data were eventually combined to generate a final phenotypic ranking of the clones (Fig. 1d). HEKAgo2kd data were counted as double compared to Huh7 data, as enhanced knockdown efficacies were clearer in HEKAgo2kd cells. Clones with Ago3-like phenotypes were ranked according to their performance in Huh7 cells. Western blotting and immunopurification of Flag-tagged Ago complexes. HEK293T cells seeded in 96-well format were transfected with 200 ng Ago plasmid and lysed 2 d later. Proteins were separated on an 8% polyacrylamide gel, transferred to a nitrocellulose membrane (Whatman) and incubated with antiFlag primary antibody (1:1,000, cat. no. F1804, Sigma). For detection, a peroxidase-conjugated anti-mouse secondary antibody (1:10,000, cat. no. 115-035-068, Jackson ImmunoResearch) and an enhanced chemiluminescence system (Perkin Elmer) were used. For immunopurification (IP) of Flag-Ago complexes, transfected HEK293T cells (one 140-mm plate) were washed twice with cold PBS 2 d after transfection and resuspended in 1 ml PBS. Cell suspension (20 µl) was taken for western blot analysis and mixed with 200 µl 2× protein sample buffer. For small-RNA northern blotting, 80 µl cell suspension was centrifuged and resuspended in 1 ml Qiazol (Qiagen). Remaining cells were pelleted and incubated in 350 µl lysis buffer (150 mM KCl, 25 mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.5% NP-40, 1 mM NaF, 0.5 mM DTT and 2 mM Pefabloc SC (Roche)) for 5 min on ice. Cytoplasmic extracts were cleared by centrifugation at 17,000g for 30 min at 4 °C and subsequently mixed with PBS to a total volume of 1,400 µl. Per IP, 20 µl Flag M2 agarose beads (A2220, Sigma) were incubated with the entire cytoplasmic extract for 2 h at 4 °C on a rotating wheel. Next, the beads were washed three times with IP buffer containing 300 mM NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM MgCl2 and 0.05% NP-40. Finally, the beads were washed (PBS) and aliquoted for western blotting (10% of the beads), RISC cleavage assay (45%) or small-RNA northern blotting (45%). For western blotting or RNA extraction, the beads were resuspended in 80 µl 2× protein sample buffer or 1 ml Qiazol, respectively. To determine ­successful Ago IP, 10 µl protein sample (1% of immunoprecipitate or 0.1% of cell suspension) was separated by 8% SDS-PAGE and analyzed as above. All western blots that were performed as loading controls can be found in Supplementary Figure 2.

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RNA extraction and small-RNA northern blotting. RNAs from cell lysates or Ago IPs were isolated with Qiazol (Qiagen) according to the manufacturer’s protocol. Beforehand, IP samples were supplemented with 20 µg naive HEK293T RNA to monitor equal RNA extraction and PAGE loading with a probe against U6 snRNA. Immunoprecipitated RNA was resuspended in 15 µl RNA sample buffer, and 5 µl denatured isolated RNA (or 10 µg RNA from cell lysates) was separated by 12% denaturing PAGE and then transferred to a Hybond-N+ membrane (Amersham, GE Healthcare). For probe generation, 50 pmol DNA oligonucleotides (Supplementary Table 4) was 5′ labeled with 25 µCi [γ-32P]ATP at 37 °C for 30 min with T4 polynucleotide kinase (NEB) and purified (Nucleotide removal kit, Qiagen). The probe was hybridized to the membrane in PerfectHyb Plus hybridization buffer overnight at 37 °C. Both strands of the small RNA used were detected by sequential probing. Radioactive signals were detected by 24–48 h exposure to a phosphor screen and scanning with a Personal Molecular Imager (Bio-Rad). For quantification, we first normalized to either the precursor small-RNA band in the Ago3 sample that was detected by both probes (graphs in Fig. 2e,h) or to oligonucleotides that we had loaded on the same gel as references and that were likewise complementary to our probes (graphs in Supplementary Figs. 3i and 6c). Subsequently, all values were normalized to Ago2 to allow for a better comparison between strand ratios from shRNA and miRNA samples. Modeling. Homology models for complete human Ago2 and Ago3 were built with a human Ago2 crystal structure as template (either PDB 4Ei1 (ref. 25) or 4F3T (ref. 24)). Sequences were aligned by ClustalW54 and MultiSeq55, and the alignment was manually optimized on the basis of structure information. The models were constructed with the threading protocol in Rosetta 3.2 (ref. 56),

with fragment libraries generated by the Robetta web server57. Each model was ­subjected to several rounds of optimization by Rosetta’s relax protocol. All structure analyses and visualizations were performed with VMD58. Statistical analysis. Pairwise comparisons of measurements were done by Student’s t test. We tested enrichment of Ago2- or Ago3-derived sequences in Ago2-like chimeras for each amino acid position by using Fisher’s exact test. P values were corrected for multiple testing with the method of Benjamini and Hochberg59. Sequence analysis of Ago chimeras. Sequences of chimeras and parental Agos were aligned by ClustalX2 and analyzed by Salanto (shuffling alignment analysis tool), a program that we designed specifically for the analysis of chimeric DNA or protein sequences generated by DFS. A parsimonious heuristic approach was implemented to compute the composition of single chimeras in terms of their reference sequences and to calculate homologies to each individual parent. Further modules were written to provide summary statistics on overall clone composition, cross-over frequencies or position-wise composition of all chimeras. This freely available Java program and a manual containing a complete description of the software can be downloaded from https://bitbucket.org/benderc/salanto/wiki/Home/.

51. Pillai, R.S., Artus, C.G. & Filipowicz, W. Tethering of human Ago proteins to mRNA mimics the miRNA-mediated repression of protein synthesis. RNA 10, 1518–1525 (2004). 52. Schmitter, D. et al. Effects of Dicer and Argonaute down-regulation on mRNA levels in human HEK293 cells. Nucleic Acids Res. 34, 4801–4815 (2006). 53. Stoehr, J. & Meister, G. In vitro RISC cleavage assay. Methods Mol. Biol. 725, 77–90 (2011). 54. Thompson, J.D., Higgins, D.G. & Gibson, T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 (1994). 55. Roberts, E., Eargle, J., Wright, D. & Luthey-Schulten, Z. MultiSeq: unifying sequence and structure data for evolutionary analysis. BMC Bioinformatics 7, 382 (2006). 56. Leaver-Fay, A. et al. ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. Methods Enzymol. 487, 545–574 (2011). 57. Kim, D.E., Chivian, D. & Baker, D. Protein structure prediction and analysis using the Robetta server. Nucleic Acids Res. 32, W526–W531 (2004). 58. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 , 27–28 (1996). 59. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. Roy. Statist. Soc. Ser. B 57, 289–300 (1995).

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In vitro RISC cleavage assay. Template DNAs containing perfect binding sites for selected small RNAs were PCR amplified with primers (Supplementary Table 3) to introduce a T7 promoter sequence. One microgram of purified PCR product was incubated in a 40-µl reaction containing 1.25 U/µl T7 RNA polymerase (NEB), 40 mM Tris-HCl, 6 mM MgCl2, 10 mM DTT, 2 mM spermidine, 500 µM each ATP, GTP and CTP, 12 µM UTP, 5 µl [α-32P]UTP (3,000 Ci/mmol) and 1 U/µl murine RNase inhibitor (NEB) at 37 °C for 3 h. Subsequently, radioactively labeled products were run on an 8% acrylamide gel at 300 V for 2 h, purified and used in RISC cleavage assays as described53. Samples from RISC cleavage assays were loaded onto an 8% acrylamide gel together with a 32P-end-labeled RNA marker and run for 2 h at 300 V. Radioactive signals were detected on a Personal Molecular Imager and quantified with Quantity One software (both from Bio-Rad).

nature structural & molecular biology

doi:10.1038/nsmb.2607