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Physical and functional coupling of RNA-dependent RNA polymerase and Dicer in the biogenesis of endogenous siRNAs Suzanne R Lee & Kathleen Collins Many classes of small RNA (sRNA) involved in RNA silencing are generated by double-stranded RNA (dsRNA) processing. Although principles of sRNA biogenesis have emerged, newly identified classes of sRNAs have features that suggest additional biogenesis mechanisms. Tetrahymena thermophila expresses one such class, comprising sRNAs of 23 and 24 nucleotides (nt) with an absolute strand bias in accumulation. Here we demonstrate sRNA production by the T. thermophila Dicer Dcr2 and the RNAdependent RNA polymerase Rdr1, which purifies as a multisubunit RNA-dependent RNA polymerase complex (RDRC). Dcr2 and RDRC interact, stimulating Dcr2 activity. Moreover, Dcr2 specificity is influenced by RDRC beyond this physical interaction, as Dcr2 generates discrete 23- and 24-nt sRNAs only from dsRNA with a 5¢-triphosphate. These findings suggest that sRNA strand bias arises from Dcr2 processing polarity, conferred by physical and functional coupling of RDRC and Dicer enzymes.
RNA silencing pathways regulate gene expression, promote heterochromatin assembly and provide cellular defense against viruses and mobile elements1. Conserved enzymes are used in the biogenesis of 18- to 30-nt sRNA guides that assemble with Argonaute or Piwi proteins to form effector ribonucleoproteins (RNPs)2,3. Known sRNA biogenesis pathways depend on the endonuclease Dicer, which processes dsRNA precursors into sRNA duplexes. Precursors can be single-stranded RNA (ssRNA) transcripts with stem-loop structures, or dsRNA generated by transcript annealing or RNA-dependent RNA polymerase (RdRP) activity. Selection of one strand from each sRNA duplex for assembly into a silencing effector RNP involves Dicer coordination with an Argonaute or Piwi protein3–5. For some sRNAs, guide-strand selection is dictated by thermodynamic asymmetry in the sRNA duplex6,7. General principles of sRNA biogenesis have been developed that invoke processive Dicer cleavage and recognition of sRNA duplex ends by Dicerassociated dsRNA-binding proteins (dsRBPs)3. However, several sRNA classes have features that are inconsistent with known biogenesis mechanisms8–14, indicating that much remains to be learned about sRNA biogenesis. In many organisms, expansion of the protein families involved in RNA silencing has occurred15. Whereas Schizosaccharomyces pombe has one Argonaute, one Dicer and one RdRP that function in heterochromatin assembly, Arabidopsis thaliana has ten Argonautes, four Dicers and six RdRPs that mediate transcriptional and posttranscriptional silencing16. The genome of the ciliate T. thermophila encodes at least ten predicted members of the Piwi subclade of the
Argonaute/Piwi family, three Dicer-family proteins17,18 and a single predicted RdRP. Consistent with the high complexity of its RNA silencing machinery, T. thermophila expresses at least two classes of sRNA with distinct biogenesis pathways9. One class, comprising sRNAs of 27–30 nt, is expressed only during conjugation, the sexual phase of the T. thermophila life cycle. Accumulation of these sRNAs and their function in heterochromatin establishment depend on the Dicer Dcl1 and the Piwi protein Twi1 (refs. 19,20). The second sRNA class, comprising sRNAs of 23 and 24 nt, is constitutively expressed9. The sRNAs sequenced so far have an unusual origin: their genomic loci cluster in an unphased manner at B12 sites in the genome and show an absolute bias in strand polarity. This bias is not accounted for by the thermodynamic properties of predicted sRNA duplexes. Strand bias and lack of phasing are not unique to the T. thermophila 23- and 24-nt sRNAs: similar features have been reported for the Caenorhabditis elegans X-cluster sRNAs from an intergenic region on the X chromosome11, and for the germline-specific sRNAs associated with Piwi proteins in mammals and flies8,10. Biogenesis mechanisms underlying these unusual sRNA features remain to be understood. In T. thermophila, the 23- and 24-nt sRNAs accumulate from strands that are antisense to predicted genes, leading us to propose that they function to repress gene expression9. This silencing probably occurs at a post-transcriptional level, because heterochromatin-mediated silencing in T. thermophila has been detected only in conjugating cells. Success in developing experimental RNA interference for T. thermophila supports the idea that 23- and 24-nt sRNAs function in RNA degradation21.
Department of Molecular and Cell Biology, University of California – Berkeley, 142 Life Sciences Addition 3200, Berkeley, California 94720-3200, USA. Correspondence should be addressed to K.C. (
[email protected]). Received 23 March; accepted 22 May; published online 1 July 2007; doi:10.1038/nsmb1262
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Here we have examined the biogenesis pathway of the 23- and 24-nt sRNAs. We previously found that, of the T. thermophila Dicer genes, neither DCL1 nor DCR1 is required for accumulation of 23- and 24-nt sRNAs, but DCR2 is essential for viability9,17. We show here that the single RdRP gene in T. thermophila, RDR1, is also essential for viability. Using a biochemical approach, we demonstrate that Rdr1 and Dcr2 collaborate in generating 23- and 24-nt sRNAs. Endogenous expression of tagged Rdr1 or Dcr2 and affinity purification of their associated complexes reveals a physical interaction between Rdr1 and Dcr2 that stimulates Dcr2 activity on dsRNA substrates. Dcr2 cleavage specificity is also influenced by Rdr1 indirectly, because production of 23- and 24-nt sRNAs is enhanced by the presence of 5¢-triphosphates on dsRNA substrates. Physical and functional coupling of Dicer and RdRP provides a mechanism that could generate the strand asymmetry of T. thermophila sRNAs and sRNAs in other eukaryotes. RESULTS The T. thermophila RdRP gene RDR1 is essential Because the strand bias of sequenced 23- and 24-nt sRNAs is antisense to predicted transcripts, we hypothesized that biogenesis of the sRNAs might involve an RdRP. We identified and cloned the single T. thermophila gene predicted to encode an RdRP, which we named RDR1. Sequence alignment with eukaryotic RdRPs revealed extensive homology over the polymerase domain, including active site residues (Fig. 1a). Expression analysis showed that RDR1 is expressed throughout growth, starvation and conjugation (data not shown), consistent with a role in the biogenesis of constitutively expressed sRNAs. To test the function of Rdr1 at a genetic level, we attempted to generate RDR1 knockout strains by replacing RDR1 with an expression cassette conferring resistance to neomycin. In all of the putative knockout strains obtained, we found that the resistance gene had not fully replaced the RDR1 locus (data from two of these strains are shown in Fig. 1b), indicating that RDR1 is essential for viability.
Figure 1 T. thermophila RDR1 is essential. (a) Conserved active site residues of eukaryotic RdRPs. Identical and similar residues are shaded gray. Tt, T. thermophila; Sp, S. pombe; Nc, Neurospora crassa; Ce, C. elegans; At, Arabidopsis thaliana. (b,c) Construction of RDR1 knockdown strains (b) and RDR1 knockout strains carrying a BTU1-integrated transgene encoding Rdr1 with an N-terminal tag (c). A 3.2-kb region of RDR1 including sequence encoding the Rdr1 active site was replaced by the neo2 selectable marker cassette. DNA blot analysis of the RDR1 locus was performed by using genomic DNA from wild-type (WT), knockdown (KD) and knockout (KO) strains digested with XbaI and the indicated region (gray bar) as a probe. Neo-S and Taxol-S indicate phenotypic sensitivity to drug; Neo-R and Taxol-R indicate phenotypic resistance to drug.
Rdr1 assembles a multisubunit complex that recruits Dcr2 We adopted a biochemical approach to study Rdr1 and Dcr2 involvement in sRNA biogenesis. We created strains expressing an epitopetagged version of Rdr1. An epitope tag placed at the C terminus of Rdr1 generated a fusion protein that could not provide the essential function of Rdr1 (data not shown). By contrast, an N-terminally tagged version of Rdr1 expressed by transgene integration at the nonessential BTU1 locus (Fig. 1c) fully supported endogenous Rdr1 function, because RDR1 could be eliminated by gene knockout in the presence of the transgene (compare Fig. 1b and c). Affinity purification of Rdr1 tagged with tandem Protein A domains and a cleavage site for tobacco etch virus (TEV) protease (zz-Tev-Rdr1) reproducibly copurified a set of proteins specific to zz-Tev-Rdr1 that were not present in parallel mock affinity purifications from wild-type cell extract (Fig. 2). Mass spectrometry indicated that these associated proteins included two predicted nucleotidyltransferases of B65 kDa and a protein of B38 kDa with no known motifs (Supplementary Table 1 online). We could not dissociate these copurifying proteins from Rdr1 using high salt or other nondenaturing treatments. A putative catalytically dead Rdr1 bearing the amino acid substitution D1004A (Fig. 1a) copurified an identical set of polypeptides (Fig. 3a; see below). We conclude that the endogenous T. thermophila Rdr1 assembles into an RDRC. S. pombe Rdr1 also assembles as an RDRC22. Like T. thermophila RDRC, S. pombe RDRC has a putative nucleotidyltransferase subunit (Cid12); however, the putative helicase subunit (Hrr1) in S. pombe RDRC is not present in T. thermophila RDRC, and S. pombe RDRC lacks a subunit with homology to the T. thermophila protein of B38 kDa. No difference in the ratio of zz-Tev-Rdr1 to its associated proteins was detected in purifications from cell extracts with or without endogenous Rdr1 (data not shown), suggesting that Rdr1 is
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limiting for RDRC assembly. In addition, we did not detect any change in the composition of RDRC purified from cells in rapid growth, starvation or conjugation (data not shown). An additional protein of B230 kDa was detected in Rdr1 purifications performed with less stringent washes (Fig. 3a, lane 2). Sequence analysis of this protein by mass spectrometry showed that it was the essential Dicer protein Dcr2 (Supplementary Table 1). To confirm the Dcr2-RDRC interaction, we generated a strain expressing Dcr2 with a C-terminal tag from its endogenous locus. Tagged Dcr2 could fully substitute for endogenous Dcr2, because the DCR2 locus could be completely replaced (Fig. 3b). Purification of Dcr2 performed with low-stringency washes recovered Rdr1 and other RDRC subunits (Fig. 3a, lane 4, and Supplementary Table 1). The wash conditions that dissociated Dcr2 from tagged RDRC (Fig. 2) similarly dissociated RDRC from tagged Dcr2 (Fig. 3c). The Dcr2-RDRC association was not reduced by treatment with micrococcal nuclease or RNase A, indicating that the interaction is not mediated by RNA (data not shown).
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Figure 3 Dcr2 associates with RDRC. (a) Silver-stained denaturing gel of proteins obtained from purifications of tagged wild-type Rdr1, Rdr1-D1004A and Dcr2 performed with washes less stringent than those used in the purifications shown in Figure 2. Asterisk denotes a probable Rdr1 degradation product that was not consistently observed. (b) Construction of strains expressing Dcr2-TAP from the DCR2 locus. DNA blot analysis of the DCR2 locus was performed by using genomic DNA from wild-type (WT) and two knock-in (KI) strains digested with NsiI and AvaII and the indicated region (gray bar) as a probe. The faint signal from the wild-type DCR2 locus in knock-in strains derives from DCR2 in the transcriptionally silent micronucleus. (c) Tagged Dcr2 purified under conditions used for the Rdr1 purifications shown in Figure 2. In a and c, wild-type cell extract was used for the parallel mock purification.
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Rdr1 makes dsRNA that Dcr2 cuts into 23- and 24-nt duplexes Using purified RDRC and Dcr2 complexes, we tested the functional interdependence of RdRP and Dicer activities. First, we characterized the RNA synthesis activity of T. thermophila RDRC in various assay conditions, which in general produced similar results. For ease of comparison, the reactions reported here were performed with buffer conditions similar to those used in another study23. Reactions contained a limiting concentration of ssRNA template (Supplementary Fig. 1a online) and radiolabeled cytidine triphosphate (CTP) to visualize products. We tested several ssRNA templates that were distinct in length and sequence, including a set derived from a putative mRNA antisense to endogenous 23- and 24-nt sRNAs (Fig. 4a; line thickness indicates the position of shorter templates within longer ones). Preliminary experiments with ssRNA and dsRNA markers established that nonstandard denaturing gel RDRC/Dcr2 RDRC S1 – conditions were required to eliminate product RNA structure during electrophoresis. In this gel system (Methods), RNA mobility was largely unaffected by the presence or absence of one or more phosphate groups on the 5¢ end (data not shown). As expected, RDRC reaction products were dependent on the presence of Rdr1 and ssRNA template (Fig. 4b, lanes 1–3, 5 and 6) and were sensitive
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Figure 4 Dcr2 generates 23- and 24-nt sRNA duplexes from dsRNA products of Rdr1. (a) RNA templates used in RdRP assays. Lines of the same width represent sequence shared between templates. (b–e) Products of RdRP assays using purified RDRC, with or without RNA and copurifying Dcr2, resolved on denaturing acrylamide gels. Lower panels show longer exposures of small products. Products were untreated in b, or were digested with increasing amounts of S1 nuclease (five-fold steps) in c or increasing amounts of RNase I (ten-fold steps) in d and e. M, RNA length markers. (f) Model of dsRNA synthesis and production of 23- and 24-nt sRNAs by RDRC and Dcr2. Thick line represents the strand synthesized by RDRC.
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to treatment with RNase V1 (data not shown), which preferentially degrades dsRNA. RDRC products from any given template showed some heterogeneity in length but were not as short as endogenous T. thermophila sRNAs. The main RDRC products were dependent on Rdr1 catalytic activity, because they were not observed in reactions with Rdr1D1004A, the variant predicted to be catalytically dead (Fig. 4b, lane 4). Dcr2 does not greatly influence the catalytic activity of Rdr1 in vitro, because the main RDRC products were similar in the presence or absence of copurified Dcr2 (Fig. 4b, compare lanes 2 and 3). Curiously, a small but reproducible amount of radiolabeled product was generated in RDRC assays containing Rdr1-D1004A in a manner that required input ssRNA. Because this minor product was degraded by nucleases that act on single-stranded nucleic acid, it probably derives from the activity of a nucleotidyltransferase subunit of RDRC rather than Rdr1. Across reactions with different templates (Fig. 4a), the main RDRC products were longer than the template itself (Fig. 4b–e). To determine whether the whole length of the product was double stranded, we degraded ssRNA segments with S1 nuclease or RNase I in 300 mM NaCl. Treatment with either enzyme shifted the product profile to lengths less than or equal to the input template (Fig. 4c–e). The length of the nuclease-resistant product increased with template length, independent of reaction time or template concentration, suggesting that dsRNA synthesis by Rdr1 in the context of RDRC is fairly processive (see Discussion). We conclude that a typical reaction product of T. thermophila RDRC in vitro is composed of a segment of dsRNA equal, or nearly equal, in length to template and a segment of ssRNA added as a 3¢ tail (Fig. 4f, middle). Although dsRNA synthesis by RDRC did not require the presence of Dcr2, only reactions containing Dcr2 generated RNA products of 23 and 24 nt (Fig. 4b–e, bottom). These products matched the size of constitutively expressed T. thermophila sRNAs in vivo9. As expected for Dicer products, the 23- and 24-nt RNAs produced in vitro were largely resistant to S1 nuclease and RNase I (Fig. 4c–e, bottom). In addition, the sRNA products migrated on nondenaturing gels with the mobility of an RNA duplex and not of ssRNA (Supplementary Fig. 1). Treatment with S1 nuclease increased the mobility of the sRNAs slightly on denaturing gels (Fig. 4c and Supplementary Fig. 1a), consistent with loss of the characteristic 3¢ overhang ends
Figure 5 RDRC stimulates Dcr2 activity. Dicing reactions were performed on an 81-bp RNA duplex bearing 3¢ 2-nt overhangs using purified Dcr2 with or without separately purified RDRC (Dcr2+RDRC) or with copurified RDRC (Dcr2/RDRC). Vertical bars on the right denote products of dicing. RNA markers with the indicated lengths were resolved in parallel. Quantification of dicing is presented as the fold difference in sRNA product intensity relative to that obtained with Dcr2 alone. (a) SYBR Gold stain of unlabeled products. (b) Products generated with internally labeled RNA duplex. ‘Preinc’ indicates coincubation of the proteins for 15 min at 30 1C before adding them to the reaction. The amounts of RDRC in the Dcr2+RDRC (lanes 2–3) and Dcr2/RDRC (lane 6) reactions were B1.5- and 3-fold less, respectively, than that used in lane 3 of a. M, RNA length marker.
resulting from Dicer cleavage. We conclude that T. thermophila Dcr2 processes RDRC products to generate 23- and 24-nt sRNA duplexes (Fig. 4f). RDRC influences Dcr2 activity In the above assays, Dcr2 activity was dependent on RDRC for dsRNA synthesis. To determine whether Dcr2 cleavage requires ongoing dsRNA synthesis or can occur after synthesis, we assayed the activity of purified Dcr2 on annealed dsRNA substrates. To prevent dsRNA synthesis by any RDRC molecules that might remain associated with Dcr2 after stringent washes, reactions were performed without nucleotides or with only ATP. On a preannealed dsRNA substrate, Dcr2 generated 23- and 24-nt products that could be visualized by direct SYBR Gold staining of denaturing gels (Fig. 5a) or by using internally radiolabeled dsRNA (Fig. 5b). Different lengths of input dsRNA yielded similar 23- and 24-nt products but also distinct, longer products, which had sizes consistent with Dcr2 processing from dsRNA substrate ends24. Identical results were obtained for reactions performed in the presence (Fig. 5) and absence (data not shown) of ATP. Purified dsRNA synthesized in RDRC reactions without Dcr2 could also be processed by purified Dcr2 to yield sRNA products (Supplementary Fig. 2 online). Substrates with 2-nt 3¢ overhangs (Fig. 5) or blunt ends (data not shown) generated comparable amounts and profiles of Dcr2 products. Although Dcr2 without copurified RDRC retained dsRNA-cleavage activity, Dcr2 with copurified RDRC consistently yielded more diced
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Figure 7 Model of T. thermophila 23- and 24-nt sRNA biogenesis (see text).
product. The stimulation was modest when quantified as a comparison of Dcr2 purified with or without associated RDRC (Fig. 5b, lanes 5 and 6, and Supplementary Fig. 3 online), but an enhancement greater than two-fold was reproducibly observed if highly purified Dcr2 was combined with highly purified RDRC (Fig. 5a, lane 3; Fig. 5b, lanes 2 and 3; and Supplementary Fig. 3). The reactions containing Dcr2 with copurified RDRC probably under-represent the level of RDRC-induced stimulation because the Dcr2 preparations used contained a mixed population of Dcr2 alone and Dcr2-RDRC (Fig. 3a, lane 4). Control reactions combining Dcr2 with heatinactivated RDRC or a mock purification from wild-type cell extract showed no stimulation of Dcr2 activity (Supplementary Fig. 3). The 5¢-triphosphate groups on the in vitro–transcribed dsRNA substrates used above mimic the expected dsRNA end structure at Rdr1 sites of initiation. To investigate whether the phosphorylation status of dsRNA 5¢ ends has an impact on Dcr2 activity, we generated internally labeled duplexes of identical sequence with 5¢-triphosphate, 5¢-monophosphate and 5¢-hydroxyl groups. We found that only dsRNAs with 5¢-triphosphate ends yielded discrete products of 23 and 24 nt (Fig. 6a, lanes 1–3). Substrates modified to have 5¢-monophosphate or 5¢-hydroxyl groups yielded heterogeneously sized sRNA products, including some longer than 24 nt (Fig. 6a, lanes 4–9). These size differences were observed in assays using both highly purified Dcr2 (Fig. 6a, lanes 2, 5, 8) and Dcr2 with copurified RDRC (Fig. 6a, lanes 3, 6 and 9). Product treatment with alkaline phosphatase to remove the variable number of 5¢-phosphate groups before resolution on denaturing gels did not alter the relative differences in product mobility (data not shown). To confirm the altered size of Dcr2 products from substrates lacking a 5¢-triphosphate, we tested Dcr2 activity on substrates end-labeled with a 5¢-monophosphate such that the only detectable products would be generated from 5¢-monophosphate ends. From this substrate, Dcr2 generated heterogeneous products longer than 24 nt (Fig. 6b). The relaxed cleavage specificity of Dcr2 on substrates with 5¢-monophosphate and 5¢-hydroxyl ends may be linked to its lack of an obvious DUF283, PAZ or dsRNA-binding motif, each of which in other Dicer enzymes is proposed to contact dsRNA substrate or contribute to cleavage site specificity, or both9,25–27. The T. thermo-
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phila Dcr2 dual-mode dicing specificity expands the potential range of sRNA products that can be generated by an individual Dicer and suggests that efficient production of 23- and 24-nt sRNAs could depend on Dcr2 processing of RDRC products from ends bearing the Rdr1 initiation site. DISCUSSION Coupling of RdRP and Dicer for sRNA biogenesis We have shown that T. thermophila RDR1, like DCR2, is essential for viability, consistent with the involvement of Rdr1 and Dcr2 in the same sRNA biogenesis pathway. Affinity purifications of Rdr1 and Dcr2 revealed their physical association and a dependence of Dcr2 on RDRC at a biochemical level: cleavage activity and specificity are enhanced by RDRC through physical interaction and RDRC product structure, respectively. Stimulation of Dcr2 cleavage activity by RDRC could occur through protein interaction or through RDRCmediated recruitment of dsRNA substrates. If Dcr2 has low dsRNA-binding affinity and cleavage specificity, its physical and functional coupling to RDRC should be essential for biological function. Indeed, this coupling could have evolved to limit Dcr2 activity on dsRNAs that are not RDRC products. The modest level of Dcr2 stimulation by RDRC in vitro may be limited by missing downstream factors. Our results suggest a model for the biogenesis of 23- and 24-nt sRNAs in T. thermophila (Fig. 7). The pathway begins with RDRC recognition of ssRNA transcripts expressed from specific sites in the genome. RNA synthesis by Rdr1 converts the ssRNA into dsRNA, and RDRC association with Dcr2 commits the dsRNA to sRNA duplex production. If sRNAs derived from the Rdr1 product strand are selectively stabilized, the efficiency and specificity of silencing should be enhanced because effector RNPs will carry only sRNAs that are antisense to the initiating transcripts. The heterogeneity of Rdr1 dsRNA product lengths in vitro suggests that Rdr1 may initiate at several sites near the 3¢ end of an ssRNA template, as has been proposed for other eukaryotic RdRPs12,14,28, which might account for the lack of phasing of endogenous T. thermophila 23- and 24-nt sRNAs. On the basis of the in vitro properties of Dcr2, we propose that the RDRC product end bearing the RDRC initiation site is most efficiently processed into a 23- or 24-nt sRNA duplex. Any subsequent dicing of the same RDRC product would be expected to yield sRNA duplexes of a broader size distribution. We did not detect any evidence of processive dicing on dsRNA substrates in vitro: substrates with 5¢-triphosphates produced detectable amounts of only 23- and 24-nt sRNAs and none of the longer sRNAs that would be expected from a second cycle of dicing (Fig. 4). In addition, because longer sRNAs are not readily detectable in T. thermophila, any such sRNAs must be produced with an abundance or stability lower than that of 23- and 24-nt sRNAs in vivo. Dicer enzymes from other organisms associate with dsRBPs that influence substrate selection, cleavage specificity and dicing efficiency29–31. We did not find similar proteins associated with T. thermophila Dcr2, perhaps because RDRC serves as a substitute. Dicer-associated dsRBPs can also contribute to the process of sRNA strand selection for effector RNP assembly that respects the relative thermodynamic stability of sRNA duplex ends32. We would not expect to find a Dcr2-associated protein with this role because the 23- and 24-nt sRNAs have a strand bias that does not obey stability rules9. Instead, strand selection may be determined by Dcr2 processing polarity, which in turn derives from coupling to RDRC. Dcr2 could be directed to the dsRNA end created by Rdr1 initiation solely through physical and functional coupling to RDRC. Alternatively, other RDRC
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activities (such as 3¢ tailing of the Rdr1 product) could direct the initiation of Dcr2 processing to the 5¢-triphosphate end. Dicer processing polarity has been proposed to influence sRNA strand selection in human cells33, but not in fly embryo extract34. In addition, an sRNA duplex produced by terminal dicing of an RDRC product would have inherent strand asymmetry owing to the presence of a single 5¢-triphosphate that could be recognized in downstream RNP assembly steps. Evidence of RdRP and Dicer coupling in other organisms The physical and functional coupling of T. thermophila RdRP and Dicer is likely to have relevance to other organisms. In A. thaliana proper localization of the Dicer DCL3 depends on the RdRP RDR2, suggesting that these two enzymes may interact35. In wheat-germ extract, functional coupling between RdRP and Dicer has been detected: dsRNA added directly to extract was diced into sRNAs of B21 nt and B24 nt, whereas ssRNA copied into dsRNA in extract was preferentially diced into sRNAs of B24 nt (ref. 23). In C. elegans, the RdRP RRF-3 and the Dicer DCR-1 are required for accumulation of strand-asymmetric X-cluster sRNAs and some endo-siRNAs36,37. RRF-3 is among B20 proteins found to be robustly associated with immunopurified DCR-1 (ref. 37). By contrast, an RdRP-dependent but Dicer-independent pathway of siRNA biogenesis has been proposed for secondary siRNAs and endosiRNAs in C. elegans12–14. At least a few endo-siRNAs and secondary siRNAs have triphosphate or diphosphate 5¢ ends, and secondary siRNAs accumulate with an absolute strand bias14. In the Dicerindependent model, short siRNA-sized RdRP products are synthesized by a specialized RdRP such as RRF-1, which is required for secondary siRNA accumulation. An alternative model of secondary siRNA production has been proposed that has more similarity to our model of T. thermophila sRNA biogenesis. In this model, RRF-1 initiates dsRNA synthesis at the 3¢ end of a template generated by siRNA-guided endonucleolytic cleavage or at an internal site, and this dsRNA substrate is then processed by ‘nonprogressive’ dicing14. This coupling of RdRP and Dicer activities would yield a short RNA duplex in which only the RdRP product strand has a 5¢-triphosphate. Assembly of this strand into an effector RNP could require removal of the g and b phosphate groups by an RNA phosphatase such as C. elegans PIR-1, which has been identified in association with immunopurified DCR-1 (ref. 37). Most endogenous T. thermophila 23- and 24-nt sRNAs lack a 5¢-triphosphate (M.T. Couvillion and K.C., unpublished data), suggesting the possible involvement of a PIR-1 homolog (Fig. 7). Lastly, some features of our biogenesis model could have relevance for understanding the biogenesis of endogenous siRNAs in organisms thought to lack an RdRP. Piwi-family proteins in Drosophila melanogaster and mammals associate with strand-asymmetric sRNAs8,10. These sRNA classes could gain their bias in polarity from directional processing of precursor transcripts, asymmetric sRNA duplex end structures or both. Substrate selection and RNA synthesis by eukaryotic RdRP In vivo, RdRP enzymes must be highly specific in template selection to prevent downregulation of essential RNAs. This specificity could derive from structural features in the templates, the absence of mRNA-binding proteins or mRNA modifications, or the activity of upstream sRNAs or protein interactions22,38–42. We observed that RDRC could catalyze ssRNA extension of both the template and product strand in vitro. If these activities occur in vivo, they could contribute to Rdr1 template specificity, influence the polarity of
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RDRC product processing by Dicer, and/or influence Dicer-independent RNA degradation by the exosome or other nucleases43. The biochemical and genetic tools available in T. thermophila make this organism an excellent system for investigating the template specificity and activities of eukaryotic RdRP. METHODS Purifications. Extracts were prepared from cells starved for at least 12 h in 10 mM Tris-HCl (pH 7.5) before lysis with 0.2% (v/v) Nonidet P40 (NP-40) in 3–4 cell pellet volumes of T2MGN50 buffer (20 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 10% (v/v) glycerol and 50 mM NaCl) containing b-mercaptoethanol and protease inhibitors (PMSF, aprotinin, leupeptin and pepstatin A). The lysate was centrifuged at 100,000g for 1 h before application to batch binding to IgG agarose (Sigma) for 1–2 h at 4 1C. The resin was rinsed twice with cold T2MGN50 buffer containing PMSF, 0.1% (v/v) Tween-20 and 0.1% (v/v) NP-40. For low-stringency washes, the same buffer was used for six washes (5 min each) at 4 1C, omitting PMSF in the last three washes. High-stringency washes were performed with the sample buffer plus 200 mM NaCl at room temperature. For elution, all samples were rinsed into low-stringency wash buffer and TEV protease was added for 1 h at 4 1C. We carried out mass spectrometry as described44. Assays. RdRP assays were similar to described assays23. Reactions contained B3.0–7.5 ng (B0.02–0.04 pmol) of Rdr1, 5 mM dithiothreitol (DTT), 2 units of RNasin (Invitrogen), 20 mCi [a-32P]CTP (PerkinElmer) and 50 nM ssRNA, unless indicated otherwise. We estimated protein concentrations by SyproRuby staining (Invitrogen) using a titration of standards. Reactions containing gelpurified, in vitro–transcribed RNA were incubated at 30 1C for 1–2 h and stopped with either TE (10 mM Tris-HCl (pH 7.5) and 1 mM EDTA) for analysis on denaturing gels, or TE plus 0.5 M NaCl for nuclease digestion or nondenaturing gels. Products were purified by phenol/chloroform/isoamyl alcohol (25:24:1) extraction and precipitated in ethanol with linear polyacrylamide. We also added sheared yeast tRNA (1 mg; Sigma) unless samples were being used for nuclease treatment. For nuclease treatment, samples were resuspended in 10 mM Tris-HCl (pH 7.5) plus 50 mM NaCl. Digestion with S1 nuclease (Fermentas) or RNase I (Ambion) was performed at 37 1C in recommended buffers containing 300 mM NaCl. Samples were analyzed on denaturing gels (7%–12% (w/v) bis/acrylamide (19:1), 7 M urea, 45% (v/v) formamide, 1 TBE) after resuspension in 94% (v/v) formamide containing 30 mM EDTA and boiling at 100 1C. Denaturing gels were run at B40–45 1C and fixed in 20% (v/v) methanol and 10% (v/v) acetic acid. Samples for analysis on nondenaturing gels (10% (w/v) bis/acrylamide (19:1), 0.5 TBE) were resuspended in 5% (v/v) glycerol. Dicing assays were performed similarly to RdRP assays except that the reactions contained only ATP or no NTPs, B1.5–5 ng (B0.006–0.02 pmol) of Dcr2 with or without B1–3.75 ng (B0.006–0.02 pmol) of purified RDRC and B80–100 nM dsRNA. The duplexes had blunt ends or 3¢ 2-nt overhangs of CA nucleotides, as preferred by human Dicer45. For internally labeled dsRNA substrates, [a-32P]UTP (Amersham) was incorporated into one strand. Complementary RNAs were annealed by heating to 65 1C and cooling to room temperature. We prepared duplexes with 5¢-hydroxyl or 5¢-monophosphate groups by treatment with alkaline phosphatase (NEB) for 5¢-hydroxyl ends followed by T4 polynucleotide kinase (Ambion) to add 5¢-monophosphates. Dicing reactions were performed for 1–3 h at 30 1C. For internally labeled dsRNA substrate, B10,000 c.p.m. of radioactive RNA was combined with nonradioactive RNA to achieve a final RNA concentration of B80–100 nM. We analyzed products on denaturing gels. Gene cloning and strain construction. See Supplementary Methods online. Mass spectrometry. See Supplementary Methods. Note: Supplementary information is available on the Nature Structural & Molecular Biology website. ACKNOWLEDGMENTS We thank the Collins laboratory for discussions and comments on this manuscript, and I. Macrae and J. Doudna for technical suggestions. This
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ARTICLES research was supported by a Howard Hughes Medical Institute Predoctoral Fellowship to S.R.L. AUTHOR CONTRIBUTIONS S.R.L. performed the experiments, and K.C. and S.R.L. wrote the manuscript. COMPETING INTERESTS STATEMENT The authors declare no competing financial interests.
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1. Zamore, P.D. & Haley, B. Ribo-gnome: the big world of small RNAs. Science 309, 1519–1524 (2005). 2. Parker, J.S. & Barford, D. Argonaute: a scaffold for the function of short regulatory RNAs. Trends Biochem. Sci. 31, 622–630 (2006). 3. Tomari, Y. & Zamore, P.D. Perspective: machines for RNAi. Genes Dev. 19, 517–529 (2005). 4. Gregory, R.I., Chendrimada, T.P., Cooch, N. & Shiekhattar, R. Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell 123, 631–640 (2005). 5. Maniataki, E. & Mourelatos, Z. A human, ATP-independent, RISC assembly machine fueled by pre-miRNA. Genes Dev. 19, 2979–2990 (2005). 6. Khvorova, A., Reynolds, A. & Jayasena, S.D. Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216 (2003). 7. Vazquez, F. et al. Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol. Cell 16, 69–79 (2004). 8. Kim, V.N. Small RNAs just got bigger: Piwi-interacting RNAs (piRNAs) in mammalian testes. Genes Dev. 20, 1993–1997 (2006). 9. Lee, S.R. & Collins, K. Two classes of endogenous small RNAs in Tetrahymena thermophila. Genes Dev. 20, 28–33 (2006). 10. Vagin, V.V. et al. A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313, 320–324 (2006). 11. Ambros, V., Lee, R.C., Lavanway, A., Williams, P.T. & Jewell, D. MicroRNAs and other tiny endogenous RNAs in C. elegans. Curr. Biol. 13, 807–818 (2003). 12. Ruby, J.G. et al. Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell 127, 1193–1207 (2006). 13. Pak, J. & Fire, A. Distinct populations of primary and secondary effectors during RNAi in C. elegans. Science 315, 241–244 (2007). 14. Sijen, T., Steiner, F.A., Thijssen, K.L. & Plasterk, R.H. Secondary siRNAs result from unprimed RNA synthesis and form a distinct class. Science 315, 244–247 (2007). 15. Cerutti, H. & Casas-Mollano, J.A. On the origin and functions of RNA-mediated silencing: from protists to man. Curr. Genet. 50, 81–99 (2006). 16. Vaucheret, H. Post-transcriptional small RNA pathways in plants: mechanisms and regulations. Genes Dev. 20, 759–771 (2006). 17. Mochizuki, K. & Gorovsky, M.A.A. Dicer-like protein in Tetrahymena has distinct functions in genome rearrangement, chromosome segregation, and meiotic prophase. Genes Dev. 19, 77–89 (2005). 18. Malone, C.D., Anderson, A.M., Motl, J.A., Rexer, C.H. & Chalker, D.L. Germ line transcripts are processed by a Dicer-like protein that is essential for developmentally programmed genome rearrangements of Tetrahymena thermophila. Mol. Cell. Biol. 25, 9151–9164 (2005). 19. Mochizuki, K. & Gorovsky, M.A. Small RNAs in genome rearrangement in Tetrahymena. Curr. Opin. Genet. Dev. 14, 181–187 (2004).
610
VOLUME 14
20. Yao, M.C. & Chao, J.L. RNA-guided DNA deletion in Tetrahymena: an RNAi-based mechanism for programmed genome rearrangements. Annu. Rev. Genet. 39, 537–559 (2005). 21. Howard-Till, R.A. & Yao, M.C. Induction of gene silencing by hairpin RNA expression in Tetrahymena thermophila reveals a second small RNA pathway. Mol. Cell. Biol. 26, 8731–8742 (2006). 22. Motamedi, M.R. et al. Two RNAi complexes, RITS and RDRC, physically interact and localize to noncoding centromeric RNAs. Cell 119, 789–802 (2004). 23. Tang, G., Reinhart, B.J., Bartel, D.P. & Zamore, P.D. A biochemical framework for RNA silencing in plants. Genes Dev. 17, 49–63 (2003). 24. Zhang, H., Kolb, F.A., Brondani, V., Billy, E. & Filipowicz, W. Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J. 21, 5875–5885 (2002). 25. Macrae, I.J. & Doudna, J.A. Ribonuclease revisited: structural insights into ribonuclease III family enzymes. Curr. Opin. Struct. Biol. 17, 138–145 (2006). 26. Kurihara, Y. & Watanabe, Y. Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc. Natl. Acad. Sci. USA 101, 12753–12758 (2004). 27. Dlakic, M. DUF283 domain of Dicer proteins has a double-stranded RNA-binding fold. Bioinformatics 22, 2711–2714 (2006). 28. Makeyev, E.V. & Bamford, D.H. Cellular RNA-dependent RNA polymerase involved in posttranscriptional gene silencing has two distinct activity modes. Mol. Cell 10, 1417–1427 (2002). 29. Rossi, J.J. Mammalian Dicer finds a partner. EMBO Rep. 6, 927–929 (2005). 30. Leuschner, P.J., Obernosterer, G. & Martinez, J. MicroRNAs: Loquacious speaks out. Curr. Biol. 15, R603–R605 (2005). 31. Kurihara, Y., Takashi, Y. & Watanabe, Y. The interaction between DCL1 and HYL1 is important for efficient and precise processing of pri-miRNA in plant microRNA biogenesis. RNA 12, 206–212 (2006). 32. Tomari, Y., Matranga, C., Haley, B., Martinez, N. & Zamore, P.D. A protein sensor for siRNA asymmetry. Science 306, 1377–1380 (2004). 33. Rose, S.D. et al. Functional polarity is introduced by Dicer processing of short substrate RNAs. Nucleic Acids Res. 33, 4140–4156 (2005). 34. Preall, J.B., He, Z., Gorra, J.M. & Sontheimer, E.J. Short interfering RNA strand selection is independent of dsRNA processing polarity during RNAi in Drosophila. Curr. Biol. 16, 530–535 (2006). 35. Pontes, O. et al. The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center. Cell 126, 79–92 (2006). 36. Lee, R.C., Hammell, C.M. & Ambros, V. Interacting endogenous and exogenous RNAi pathways in Caenorhabditis elegans. RNA 12, 589–597 (2006). 37. Duchaine, T.F. et al. Functional proteomics reveals the biochemical niche of C. elegans DCR-1 in multiple small-RNA-mediated pathways. Cell 124, 343–354 (2006). 38. Gazzani, S., Lawrenson, T., Woodward, C., Headon, D. & Sablowski, R. A link between mRNA turnover and RNA interference in Arabidopsis. Science 306, 1046–1048 (2004). 39. Axtell, M.J., Jan, C., Rajagopalan, R. & Bartel, D.P. A two-hit trigger for siRNA biogenesis in plants. Cell 127, 565–577 (2006). 40. Allen, E., Xie, Z., Gustafson, A.M. & Carrington, J.C. microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121, 207–221 (2005). 41. Yoshikawa, M., Peragine, A., Park, M.Y. & Poethig, R.S. A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev. 19, 2164–2175 (2005). 42. Schramke, V. et al. RNA-interference-directed chromatin modification coupled to RNA polymerase II transcription. Nature 435, 1275–1279 (2005). 43. Jensen, T.H. & Moore, C. Reviving the exosome. Cell 121, 660–662 (2005). 44. Witkin, K.L., Prathapam, R. & Collins, K. Positive and negative regulation of Tetrahymena telomerase holoenzyme. Mol. Cell. Biol. 27, 2074–2083 (2007). 45. Vermeulen, A. et al. The contributions of dsRNA structure to Dicer specificity and efficiency. RNA 11, 674–682 (2005).
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