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• W1970679013 abstract "•mut-14 and smut-1 are required for germline RNAi and endogenous siRNA formation•MUT-14 and SMUT-1 most likely function at the initiation step of siRNA amplification•MUT-14 containing a point mutation in the DEAD motif is dominant to SMUT-1•siRNA synthesis in vivo, but not in vitro, requires catalytically active MUT-14 More than 2,000 C. elegans genes are targeted for RNA silencing by the mutator complex, a specialized small interfering RNA (siRNA) amplification module which is nucleated by the Q/N-rich protein MUT-16. The mutator complex localizes to Mutator foci adjacent to P granules at the nuclear periphery in germ cells [1Phillips C.M. Montgomery T.A. Breen P.C. Ruvkun G. MUT-16 promotes formation of perinuclear mutator foci required for RNA silencing in the C. elegans germline.Genes Dev. 2012; 26: 1433-1444Crossref PubMed Scopus (95) Google Scholar]. Here, we show that the DEAD box RNA helicase smut-1 functions redundantly in the mutator pathway with its paralog mut-14 during RNAi. Mutations in both smut-1 and mut-14 also cause widespread loss of endogenous siRNAs. The targets of mut-14 and smut-1 largely overlap with the targets of other mutator class genes; however, the mut-14 smut-1 double mutant and the mut-16 mutant display the most dramatic depletion of siRNAs, suggesting that they act at a similarly early step in siRNA formation. mut-14 and smut-1 are predominantly expressed in the germline and, unlike other mutator class genes, are specifically required for RNAi targeting germline genes. A catalytically inactive, dominant-negative missense mutant of MUT-14 is RNAi defective in vivo; however, mutator complexes containing the mutant protein retain the ability to synthesize siRNAs in vitro. The results point to a role for mut-14 and smut-1 in initiating siRNA amplification in germ cell Mutator foci, possibly through the recruitment or retention of target mRNAs. More than 2,000 C. elegans genes are targeted for RNA silencing by the mutator complex, a specialized small interfering RNA (siRNA) amplification module which is nucleated by the Q/N-rich protein MUT-16. The mutator complex localizes to Mutator foci adjacent to P granules at the nuclear periphery in germ cells [1Phillips C.M. Montgomery T.A. Breen P.C. Ruvkun G. MUT-16 promotes formation of perinuclear mutator foci required for RNA silencing in the C. elegans germline.Genes Dev. 2012; 26: 1433-1444Crossref PubMed Scopus (95) Google Scholar]. Here, we show that the DEAD box RNA helicase smut-1 functions redundantly in the mutator pathway with its paralog mut-14 during RNAi. Mutations in both smut-1 and mut-14 also cause widespread loss of endogenous siRNAs. The targets of mut-14 and smut-1 largely overlap with the targets of other mutator class genes; however, the mut-14 smut-1 double mutant and the mut-16 mutant display the most dramatic depletion of siRNAs, suggesting that they act at a similarly early step in siRNA formation. mut-14 and smut-1 are predominantly expressed in the germline and, unlike other mutator class genes, are specifically required for RNAi targeting germline genes. A catalytically inactive, dominant-negative missense mutant of MUT-14 is RNAi defective in vivo; however, mutator complexes containing the mutant protein retain the ability to synthesize siRNAs in vitro. The results point to a role for mut-14 and smut-1 in initiating siRNA amplification in germ cell Mutator foci, possibly through the recruitment or retention of target mRNAs. 22G siR-1 is one of a cluster of secondary 22 nt 5′G-containing small interfering RNAs (siRNAs) (22G-RNAs) produced from the long noncoding RNA linc-22 [2Nam J.-W. Bartel D.P. Long noncoding RNAs in C. elegans.Genome Res. 2012; 22: 2529-2540Crossref PubMed Scopus (158) Google Scholar]. 22G siR-1 formation requires each of the six mutator class genes except the DEAD box RNA helicase mut-14 [1Phillips C.M. Montgomery T.A. Breen P.C. Ruvkun G. MUT-16 promotes formation of perinuclear mutator foci required for RNA silencing in the C. elegans germline.Genes Dev. 2012; 26: 1433-1444Crossref PubMed Scopus (95) Google Scholar]. Consistent with their roles in 22G siR-1 formation, an siR-1 sensor transgene [3Montgomery T.A. Rim Y.-S. Zhang C. Dowen R.H. Phillips C.M. Fischer S.E.J. Ruvkun G. PIWI associated siRNAs and piRNAs specifically require the Caenorhabditis elegans HEN1 ortholog henn-1.PLoS Genet. 2012; 8: e1002616Crossref PubMed Scopus (89) Google Scholar] is desilenced in each mutator mutant except mut-14(pk738) (Figure 1A). Each mutant assayed is presumed null, containing early stop codons or large deletions, except mut-14(pk738), which encodes a protein bearing an amino acid substitution in the conserved catalytic core of the DEAD motif. To generate a null allele of mut-14, mut-14(mg464), hereafter referred to simply as mut-14, we deleted the mut-14 coding sequence [4Frøkjaer-Jensen C. Davis M.W. Hollopeter G. Taylor J. Harris T.W. Nix P. Lofgren R. Prestgard-Duke M. Bastiani M. Moerman D.G. Jorgensen E.M. Targeted gene deletions in C. elegans using transposon excision.Nat. Methods. 2010; 7: 451-453Crossref PubMed Scopus (74) Google Scholar]. Animals containing the mut-14(pk738) point mutation were deficient in their ability to inactivate germline mRNAs by RNAi but competent to inactivate somatic mRNAs by RNAi (Figure 1B) [1Phillips C.M. Montgomery T.A. Breen P.C. Ruvkun G. MUT-16 promotes formation of perinuclear mutator foci required for RNA silencing in the C. elegans germline.Genes Dev. 2012; 26: 1433-1444Crossref PubMed Scopus (95) Google Scholar]. In contrast, animals containing the mut-14 deletion were competent for both germline and somatic gene inactivations by RNAi, similar to the wild-type (Figure 1B). Y38A10A.6, hereafter referred to as synthetic mutator-1 (smut-1), is one of two closely related paralogs of mut-14, although unlike MUT-14, smut-1 contains a serine instead of an alanine within its DEAD motif (DESD) (Figures S1A and S1B available online). Similar to the mut-14 deletion and to the wild-type, smut-1(tm1301), a strain containing a large deletion in smut-1, was susceptible to both germline and somatic RNAi. In contrast, a strain carrying deletions in both mut-14 and smut-1 was defective for germline RNAi but normal for somatic RNAi, similar to mut-14(pk738) (Figure 1B). ZC317.1, the other closely related paralog of mut-14 (Figure S1A), is predicted by RNA sequencing [5Gerstein M.B. Lu Z.J. Van Nostrand E.L. Cheng C. Arshinoff B.I. Liu T. Yip K.Y. Robilotto R. Rechtsteiner A. Ikegami K. et al.modENCODE ConsortiumIntegrative analysis of the Caenorhabditis elegans genome by the modENCODE project.Science. 2010; 330: 1775-1787Crossref PubMed Scopus (722) Google Scholar] to contain an early stop codon that truncates the C-terminal helicase domain (Figures S1B and S1C). We did not observe RNAi defects in a ZC317.1 deletion mutant, nor did we observe somatic RNAi defects in animals containing mutations in all three related helicases (Figure S1D). GFP expression from the siR-1 sensor was strongly elevated in both the mut-14(pk738) smut-1 and mut-14 smut-1 double mutants, but not in mut-14 or smut-1 single-deletion mutants (Figures 1C, S1E, and S1F). 22G siR-1 levels were moderately reduced in smut-1 (p = 0.026) and to a greater degree in the mut-14 smut-1 double mutant (p < 0.001), but not in the mut-14 single mutant (Figure S1G). The levels of each of two ERGO-1 class 26G-RNAs, which act upstream of the production of certain 22G-RNAs, were also significantly reduced in the mut-14 smut-1 double mutant (p < 0.05), but not in either single mutant (Figure S1G). Although 22G siR-1 is somatic, its formation is initiated by an ERGO-1 class 26G-RNA during oogenesis and/or embryogenesis [3Montgomery T.A. Rim Y.-S. Zhang C. Dowen R.H. Phillips C.M. Fischer S.E.J. Ruvkun G. PIWI associated siRNAs and piRNAs specifically require the Caenorhabditis elegans HEN1 ortholog henn-1.PLoS Genet. 2012; 8: e1002616Crossref PubMed Scopus (89) Google Scholar]; thus, it is possible that mut-14 and smut-1 are indirectly involved in 22G siR-1 formation in the soma via their role in 26G-RNA formation in the germline. Consistent with a requirement for mut-14 and smut-1 specifically in germline RNAi, mut-14 and smut-1 promoters drive expression of mCherry predominantly in germ cells (Figure 1D). mCherry expression from the smut-1 promoter, but not the mut-14 promoter, was also relatively strong in developing embryos (Figure S1H). We sequenced small RNAs from the wild-type and from mut-14 and smut-1 single and double mutants, each of which also contained the siR-1 sensor transgene (Table S1). smut-1 displayed very little change in siRNA levels across each of the six C. elegans chromosomes, relative to the wild-type, whereas mut-14 displayed widespread but modest loss of siRNAs, which was strongly enhanced in the mut-14 smut-1 double mutant (Figure 2A). siRNAs depleted in mut-14 and mut-14 smut-1 were predominantly 22G-RNAs derived from coding genes, pseudogenes, and transposons (Figure 2B). We identified 2,335 coding genes, pseudogenes, and transposons that were depleted of siRNAs by >3-fold in mut-14 smut-1 (Figure 2C). To determine which classes of siRNAs are dependent on mut-14 and smut-1, we examined 22G-RNA levels from mRNA targets of the Argonautes WAGO-1, CSR-1, ERGO-1, and ALG-3/ALG-4, which represent each of the C. elegans endogenous siRNA pathways [6Vasale J.J. Gu W. Thivierge C. Batista P.J. Claycomb J.M. Youngman E.M. Duchaine T.F. Mello C.C. Conte Jr., D. Sequential rounds of RNA-dependent RNA transcription drive endogenous small-RNA biogenesis in the ERGO-1/Argonaute pathway.Proc. Natl. Acad. Sci. USA. 2010; 107: 3582-3587Crossref PubMed Scopus (124) Google Scholar, 7Gu W. Shirayama M. Conte Jr., D. Vasale J. Batista P.J. Claycomb J.M. Moresco J.J. Youngman E.M. Keys J. Stoltz M.J. et al.Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline.Mol. Cell. 2009; 36: 231-244Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 8Claycomb J.M. Batista P.J. Pang K.M. Gu W. Vasale J.J. van Wolfswinkel J.C. Chaves D.A. Shirayama M. Mitani S. Ketting R.F. et al.The Argonaute CSR-1 and its 22G-RNA cofactors are required for holocentric chromosome segregation.Cell. 2009; 139: 123-134Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 9Conine C.C. Batista P.J. Gu W. Claycomb J.M. Chaves D.A. Shirayama M. Mello C.C. Argonautes ALG-3 and ALG-4 are required for spermatogenesis-specific 26G-RNAs and thermotolerant sperm in Caenorhabditis elegans.Proc. Natl. Acad. Sci. USA. 2010; 107: 3588-3593Crossref PubMed Scopus (141) Google Scholar, 10Han T. Manoharan A.P. Harkins T.T. Bouffard P. Fitzpatrick C. Chu D.S. Thierry-Mieg D. Thierry-Mieg J. Kim J.K. 26G endo-siRNAs regulate spermatogenic and zygotic gene expression in Caenorhabditis elegans.Proc. Natl. Acad. Sci. USA. 2009; 106: 18674-18679Crossref PubMed Scopus (115) Google Scholar]. ERGO-1 and ALG-3/ALG-4 bind 26G-RNAs but trigger formation of 22G-RNAs from target mRNAs [6Vasale J.J. Gu W. Thivierge C. Batista P.J. Claycomb J.M. Youngman E.M. Duchaine T.F. Mello C.C. Conte Jr., D. Sequential rounds of RNA-dependent RNA transcription drive endogenous small-RNA biogenesis in the ERGO-1/Argonaute pathway.Proc. Natl. Acad. Sci. USA. 2010; 107: 3582-3587Crossref PubMed Scopus (124) Google Scholar, 9Conine C.C. Batista P.J. Gu W. Claycomb J.M. Chaves D.A. Shirayama M. Mello C.C. Argonautes ALG-3 and ALG-4 are required for spermatogenesis-specific 26G-RNAs and thermotolerant sperm in Caenorhabditis elegans.Proc. Natl. Acad. Sci. USA. 2010; 107: 3588-3593Crossref PubMed Scopus (141) Google Scholar, 11Gent J.I. Lamm A.T. Pavelec D.M. Maniar J.M. Parameswaran P. Tao L. Kennedy S. Fire A.Z. Distinct phases of siRNA synthesis in an endogenous RNAi pathway in C. elegans soma.Mol. Cell. 2010; 37: 679-689Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar]. 22G-RNAs derived from WAGO and ERGO-1 targets were strongly depleted in the mut-14 smut-1 double mutant but only modestly or not at all in the single mutants (Figure 2D). In contrast, the levels of 22G-RNAs derived from ALG-3/ALG-4 targets were not substantially affected in any of the mut-14 and smut-1 mutants, nor were the levels of primary ALG-3/ALG-4 class 26G-RNAs (Figures 2D and S1G). CSR-1 class siRNA levels appeared to be elevated in the mut-14 smut-1 double mutant, possibly a normalization artifact caused by reduced levels of WAGO and ERGO-1 class siRNAs, as a CSR-1 siRNA that we examined by qRT-PCR was unaffected (Figures 2D and S1G). The siR-1 sensor is subject to transgene silencing in the germline, independent of 22G siR-1 [3Montgomery T.A. Rim Y.-S. Zhang C. Dowen R.H. Phillips C.M. Fischer S.E.J. Ruvkun G. PIWI associated siRNAs and piRNAs specifically require the Caenorhabditis elegans HEN1 ortholog henn-1.PLoS Genet. 2012; 8: e1002616Crossref PubMed Scopus (89) Google Scholar]. siRNAs derived from the siR-1 sensor were depleted in mut-14 and to a greater extent in the mut-14 smut-1 double, but not in the smut-1 single, mutant (Figures 2D–2E). We conclude that mut-14 and smut-1 have overlapping roles in WAGO and ERGO-1 pathways, transgene silencing, and exogenous RNAi, although their roles in these pathways may be restricted to the germline and early embryos. mut-14(pk738) encodes a protein bearing a point mutation that alters an amino acid in the DEAD motif and, unlike the mut-14 deletion allele, is germline RNAi defective (Figure 1B). We sequenced small RNAs from the wild-type and from mut-14(pk738) and smut-1 single and double mutants (Table S1). The proportion of features depleted of siRNAs was similar between mut-14(pk738) and the mut-14(pk738) smut-1 double mutant (Figure 2F). The vast majority of genes depleted of siRNAs in the mut-14(pk738) smut-1 double mutant were also affected in the mut-14(pk738) single mutant (Figure 2G). WAGO class 22G-RNAs were depleted to similar levels in mut-14(pk738) and the mut-14(pk738) smut-1 double mutant (Figure 2H), both of which resemble the depletion seen in the mut-14 smut-1 double deletion mutant (Figure 2D). In contrast, 22G-RNA levels from ERGO-1 targets were somewhat elevated in mut-14(pk738) but depleted by ∼80% in mut-14(pk738) smut-1, similar to what was observed in the mut-14 smut-1 deletion mutant (Figures 2D and 2H). When fused to mCherry or GFP, wild-type MUT-14 and MUT-14 containing the pk738 mutation (MUT-14DKAD) colocalized at the nuclear periphery, indicating that mut-14(pk738) produces a stable protein that localizes to the proper compartment (Figure 2I). These results suggest that mut-14(pk738) is antagonistic to smut-1 during WAGO class 22G-RNA formation. That mut-14(pk738) is not antagonistic to smut-1 in ERGO-1 class 26G-RNA and ergo-1-dependent 22G-RNA formation may be related to differential expression of smut-1 and mut-14 during oogenesis and embryogenesis, the stages during which 26G-RNAs are produced (Figure S1H) [6Vasale J.J. Gu W. Thivierge C. Batista P.J. Claycomb J.M. Youngman E.M. Duchaine T.F. Mello C.C. Conte Jr., D. Sequential rounds of RNA-dependent RNA transcription drive endogenous small-RNA biogenesis in the ERGO-1/Argonaute pathway.Proc. Natl. Acad. Sci. USA. 2010; 107: 3582-3587Crossref PubMed Scopus (124) Google Scholar, 10Han T. Manoharan A.P. Harkins T.T. Bouffard P. Fitzpatrick C. Chu D.S. Thierry-Mieg D. Thierry-Mieg J. Kim J.K. 26G endo-siRNAs regulate spermatogenic and zygotic gene expression in Caenorhabditis elegans.Proc. Natl. Acad. Sci. USA. 2009; 106: 18674-18679Crossref PubMed Scopus (115) Google Scholar]. MUT-14 is part of an siRNA amplification module that includes the mutators MUT-2, MUT-7, RDE-2, MUT-15, and MUT-16 and which colocalizes with the RNA-dependent RNA polymerase RRF-1 [1Phillips C.M. Montgomery T.A. Breen P.C. Ruvkun G. MUT-16 promotes formation of perinuclear mutator foci required for RNA silencing in the C. elegans germline.Genes Dev. 2012; 26: 1433-1444Crossref PubMed Scopus (95) Google Scholar]. To determine whether mut-14, smut-1, and the other mutators converge on a common set of targets, we subjected wild-type and mutant animals to small RNA sequencing (Table S1). Each of the mutator mutants was depleted of 22G-RNAs derived from WAGO and ERGO-1 targets, but not substantially from CSR-1 or ALG-3/ALG-4 targets (Figure 3A). Although the genes depleted of siRNAs in each of the mutator mutants largely overlapped, mut-16 displayed the greatest loss of siRNAs (Figures 3A and S2A). Thus, we defined mutator targets as the ∼2,300 genes depleted of siRNAs by >3-fold in mut-16 (Table S2). Of the six mutator mutants, mut-16 and the mut-14 smut-1 double mutant showed the strongest depletion of siRNAs from mutator targets (Figure 3B and Table S2). When clustered by depletion in mutator target siRNAs, mut-16 and the mut-14 smut-1 double mutant assembled more closely with one another than with any of the other mutator mutants (Figure 3C). There was a genome-wide enrichment, particularly from mutator target genes, for 21 nt small RNA species in mut-16 and mut-14 smut-1 mutants, but not in any of the other mutator mutants (Figures 3D and S2B–S2D). We examined residual siRNAs in each mutator mutant to determine whether they contained modifications that were absent in wild-type animals that might to point to a specific role in siRNA maturation. We observed an ∼3-fold increase in the proportions of siRNAs containing nontemplated 3′ uracil (U) additions in mut-16 and mut-14 smut-1 but not in any of the other mutator mutants (Figure S2E). The proportion of siRNAs containing 3′ nontemplated Us was elevated ∼8.5-fold in the mut-14(pk738) smut-1 double mutant, and nearly every siRNA-yielding gene had elevated levels of nontemplated Us, including CSR-1 targets (Figures S2E–S2F). The reason for this is unclear; however, the size distribution and elevated levels of nontemplated Us observed in residual siRNAs in mut-16 and mut-14 smut-1 are features more common to CSR-1 class siRNAs than to WAGO class (Figure S2G) [3Montgomery T.A. Rim Y.-S. Zhang C. Dowen R.H. Phillips C.M. Fischer S.E.J. Ruvkun G. PIWI associated siRNAs and piRNAs specifically require the Caenorhabditis elegans HEN1 ortholog henn-1.PLoS Genet. 2012; 8: e1002616Crossref PubMed Scopus (89) Google Scholar, 8Claycomb J.M. Batista P.J. Pang K.M. Gu W. Vasale J.J. van Wolfswinkel J.C. Chaves D.A. Shirayama M. Mitani S. Ketting R.F. et al.The Argonaute CSR-1 and its 22G-RNA cofactors are required for holocentric chromosome segregation.Cell. 2009; 139: 123-134Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 12van Wolfswinkel J.C. Claycomb J.M. Batista P.J. Mello C.C. Berezikov E. Ketting R.F. CDE-1 affects chromosome segregation through uridylation of CSR-1-bound siRNAs.Cell. 2009; 139: 135-148Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar]. It is possible that some mutator targets also produce CSR-1 class siRNAs or that in the absence of the mutator pathway some mutator targets are misrouted into the CSR-1 pathway. We did not find evidence for siRNA precursors in any of our libraries, although our analysis was limited to sequences <30 nt long, which may indicate that the mutators function to facilitate RNA-dependent RNA polymerase activity on target mRNAs and are not involved in processing siRNAs into their 22 nt mature form. mut-16, but not mut-14 or smut-1, is required for formation and proper localization of the mutator complex (Figure S2H) [1Phillips C.M. Montgomery T.A. Breen P.C. Ruvkun G. MUT-16 promotes formation of perinuclear mutator foci required for RNA silencing in the C. elegans germline.Genes Dev. 2012; 26: 1433-1444Crossref PubMed Scopus (95) Google Scholar], indicating that mut-16 acts upstream of other mutators during siRNA biogenesis. Because mut-16 and the mut-14 smut-1 double mutant have the most dramatic effects on siRNA levels and show similar anomalies in residual siRNAs, we propose that they act at a similarly early step in siRNA formation. Although mut-14 and smut-1 are not required for mutator protein localization, they could be required to recruit mRNAs into Mutator foci to initiate siRNA amplification. Each of the mutators except mut-14 and smut-1 are required for both somatic and germline RNAi [1Phillips C.M. Montgomery T.A. Breen P.C. Ruvkun G. MUT-16 promotes formation of perinuclear mutator foci required for RNA silencing in the C. elegans germline.Genes Dev. 2012; 26: 1433-1444Crossref PubMed Scopus (95) Google Scholar]. It is possible that another gene fulfills the function in somatic cells that mut-14 and smut-1 serve in the germline. Alternatively, mut-14 and smut-1 could have a role in RNAi that is specifically required in germ cells. What distinguishes germline RNAi from somatic RNAi? In germ cells, each of the mutator proteins localize to perinuclear compartments, called Mutator foci, adjacent to P granules [1Phillips C.M. Montgomery T.A. Breen P.C. Ruvkun G. MUT-16 promotes formation of perinuclear mutator foci required for RNA silencing in the C. elegans germline.Genes Dev. 2012; 26: 1433-1444Crossref PubMed Scopus (95) Google Scholar]. To determine whether the mutators are also compartmentalized in the soma, we examined GFP expression in animals containing either the mut-16 promoter (mut-16prom::GFP) or the mut-16 promoter and coding sequence fused to GFP (mut-16::GFP). mut-16 is required for mutator complex formation in both the soma and the germline and for its localization to the nuclear periphery in germ cells [1Phillips C.M. Montgomery T.A. Breen P.C. Ruvkun G. MUT-16 promotes formation of perinuclear mutator foci required for RNA silencing in the C. elegans germline.Genes Dev. 2012; 26: 1433-1444Crossref PubMed Scopus (95) Google Scholar]. Free GFP expressed from the mut-16 promoter was present throughout the germline and soma, in contrast to mut-14 and smut-1, which are predominantly expressed in the germline (Figures 1D and 4A ). The MUT-16::GFP translational fusion protein formed distinct perinuclear foci in the germline but appeared to be diffuse throughout the cytoplasm in somatic cells (Figures 4A and S3A). MUT-7::GFP also appeared to be diffuse in somatic cells, indicating that the mutators are not compartmentalized in the soma, consistent with our previous observations (Figure S3B) [1Phillips C.M. Montgomery T.A. Breen P.C. Ruvkun G. MUT-16 promotes formation of perinuclear mutator foci required for RNA silencing in the C. elegans germline.Genes Dev. 2012; 26: 1433-1444Crossref PubMed Scopus (95) Google Scholar]. Thus, there are at least two features that distinguish germline RNAi from somatic RNAi: (1) the requirement for either mut-14 or smut-1 and (2) compartmentalization of the mutator complex. Therefore, it is possible that the role of mut-14 and smut-1 is related to compartmentalization of the RNAi pathway. We performed a yeast two-hybrid screen to test mutator protein interactions with other validated and predicted small RNA factors, including SMUT-1, which was identified in a screen for gene inactivations that desilence an RNAi sensor (Table S3) [13Kim J.K. Gabel H.W. Kamath R.S. Tewari M. Pasquinelli A.E. Rual J.-F. Kennedy S. Dybbs M. Bertin N. Kaplan J.M. et al.Functional genomic analysis of RNA interference in C. elegans.Science. 2005; 308: 1164-1167Crossref PubMed Scopus (236) Google Scholar, 14Robert V.J. Sijen T. van Wolfswinkel J. Plasterk R.H.A. Chromatin and RNAi factors protect the C. elegans germline against repetitive sequences.Genes Dev. 2005; 19: 782-787Crossref PubMed Scopus (112) Google Scholar]. As predicted by its role in nucleating the mutator complex and its prion-like Q/N-rich region, MUT-16 was the most promiscuous factor, binding to 12 of the 58 proteins assayed. MUT-16 interactors included the mutators SMUT-1 and RDE-2, the Argonautes WAGO-1 and ERGO-1, the RNA-dependent RNA polymerase EGO-1, and several chromatin factors, including MES-4 and GFL-1 (Figure S3C and Table S4). These results are consistent with the requirement for mut-16 in siRNA formation from WAGO and ERGO-1 targets and point to a direct interaction between MUT-16 and SMUT-1. EGO-1 may not associate with MUT-16 in vivo in wild-type animals, as we did not observe EGO-1 at Mutator foci [1Phillips C.M. Montgomery T.A. Breen P.C. Ruvkun G. MUT-16 promotes formation of perinuclear mutator foci required for RNA silencing in the C. elegans germline.Genes Dev. 2012; 26: 1433-1444Crossref PubMed Scopus (95) Google Scholar]. Furthermore, in MUT-16::GFP immunoprecipitates we detected FLAG::RRF-1, but not HA::EGO-1 (Figure 4B). ego-1 functions redundantly with rrf-1 [7Gu W. Shirayama M. Conte Jr., D. Vasale J. Batista P.J. Claycomb J.M. Moresco J.J. Youngman E.M. Keys J. Stoltz M.J. et al.Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline.Mol. Cell. 2009; 36: 231-244Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar], suggesting that in the absence of rrf-1, EGO-1 might bind to MUT-16. In coimmunoprecipitation assays of mCherry::EGO-1 and MUT-16::GFP, we detected only a very weak interaction between EGO-1 and MUT-16 in rrf-1 mutants (Figure S3D). EGO-1 also functions in the CSR-1 pathway in P granules and therefore may have a stronger affinity for factors involved in CSR-1 class siRNA formation than for the mutators [7Gu W. Shirayama M. Conte Jr., D. Vasale J. Batista P.J. Claycomb J.M. Moresco J.J. Youngman E.M. Keys J. Stoltz M.J. et al.Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline.Mol. Cell. 2009; 36: 231-244Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 8Claycomb J.M. Batista P.J. Pang K.M. Gu W. Vasale J.J. van Wolfswinkel J.C. Chaves D.A. Shirayama M. Mitani S. Ketting R.F. et al.The Argonaute CSR-1 and its 22G-RNA cofactors are required for holocentric chromosome segregation.Cell. 2009; 139: 123-134Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar]. Because RRF-1 coimmunoprecipitates with MUT-16 and is therefore associated with the mutator complex, we reasoned that we could pull down any factor in the complex and perform siRNA synthesis in vitro. This would allow us to test whether or not complexes containing wild-type or catalytically dead MUT-14 are competent for siRNA synthesis in an in vitro context in which the pathway is not compartmentalized. We immunoprecipitated wild-type MUT-14 (MUT-14::GFP) and catalytically dead MUT-14 (MUT-14DKAD::GFP) and incubated the purified complexes with a mixture of radiolabeled and nonlabeled ribonucleotides. Both MUT-14::GFP and MUT-14DKAD::GFP coimmunoprecipitated mCherry::RRF-1 (Figure 4C). Complexes containing either wild-type or catalytically dead MUT-14 immunoprecipitated from mut-14 or the mut-14 smut-1 double mutant were proficient for siRNA synthesis (Figures 4C and S3E). In contrast, a control immunoprecipitate containing free GFP (mut-16prom::GFP) failed to produce siRNAs, as did FLAG::RRF-1 complex immunoprecipitated from mut-16 mutants in which the mutator complex does not form (Figures 4C and S3E) [1Phillips C.M. Montgomery T.A. Breen P.C. Ruvkun G. MUT-16 promotes formation of perinuclear mutator foci required for RNA silencing in the C. elegans germline.Genes Dev. 2012; 26: 1433-1444Crossref PubMed Scopus (95) Google Scholar]. Addition of an in vitro-transcribed B0250.8 mRNA, an endogenous target of mut-14 and smut-1 (Table S2), was not necessary and was actually inhibitory to siRNA formation, indicating that the complex coimmunoprecipitated RNA (Figure 4C). It is unclear whether the complex contained RNA in vivo or whether it bound RNA during incubation of the cell lysate with GFP antibody. Our results demonstrate that mut-14 and smut-1 are not required for RNAi in the soma or for siRNA formation in vitro, two distinct contexts in which the mutator complex is not compartmentalized. Nor are they required for localization of other mutator proteins. Yet the data point to an essential role for mut-14 and smut-1 at an early step in siRNA formation in the germline, in which the mutator complex is compartmentalized. DEAD box helicases have numerous roles in RNA processing, such as localized unwinding of RNA duplexes and as RNA clamps [15Linder P. Jankowsky E. From unwinding to clamping - the DEAD box RNA helicase family.Nat. Rev. Mol. Cell Biol. 2011; 12: 505-516Crossref PubMed Scopus (687) Google Scholar]. Vasa, which is closely related to MUT-14 and SMUT-1 (blastp p values ≤ 5 × 10−19, 24% identity), functions along with UAP56 in the Drosophila germline to transport RNA from the site of transcription in the nucleus to perinuclear piRNA processing compartments called nuage [16Zhang F. Wang J. Xu J. Zhang Z. Koppetsch B.S. Schultz N. Vreven T. Meignin C. Davis I. Zamore P.D. et al.UAP56 couples piRNA clusters to the perinuclear transposon silencing machinery.Cell. 2012; 151: 871-884Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar]. It is possible that MUT-14 and SMUT-1 are involved in transporting mRNAs from P granules, sites of mRNA surveillance, into Mutator foci nucleated by MUT-16 to initiate siRNA amplification. The more modest reduction in siRNA levels in the other mutators suggests that they serve accessory roles in siRNA biogenesis (Figure 4D). mut-14(mg464) and ZC317.1(mgDf465) deletion alleles were generated using MosDEL [4Frøkjaer-Jensen C. Davis M.W. Hollopeter G. Taylor J. Harris T.W. Nix P. Lofgren R. Prestgard-Duke M. Bastiani M. Moerman D.G. Jorgensen E.M. Targeted gene deletions in C. elegans using transposon excision.Nat. Methods. 2010; 7: 451-453Crossref PubMed Scopus (74) Google Scholar]. Transgenes were generated using Life Technologies Multisite Gateway Technology and introduced into C. elegans using MosSCI (Table S5) [17Frøkjaer-Jensen C. Davis M.W. Hopkins C.E. Newman B.J. Thummel J.M. Olesen S.-P. Grunnet M. Jorgensen E.M. Single-copy insertion of transgenes in Caenorhabditis elegans.Nat. Genet. 2008; 40: 1375-1383Crossref PubMed Scopus (714) Google Scholar]. C. elegans were cultured at 20°C [18Brenner S. The genetics of Caenorhabditis elegans.Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar]. Immunofluorescence assays were done as described [1Phillips C.M. Montgomery T.A. Breen P.C. Ruvkun G. MUT-16 promotes formation of perinuclear mutator foci required for RNA silencing in the C. elegans germline.Genes Dev. 2012; 26: 1433-1444Crossref PubMed Scopus (95) Google Scholar]. For yeast two-hybrid assays, candidate gene sequences were cloned into bait and prey vectors using Gateway Technology and then transformed into Saccharomyces cerevisiae GAL4 and GAL80 deletion strains Y8800 (prey, MATa) and Y8930 (bait, MATα). For assessment of RNAi defects in mutator mutants, animals were fed E. coli expressing double-stranded RNA with sequence homology to germline or somatic genes. Taqman quantitative RT-PCR assays were done as described (Table S5) [10Han T. Manoharan A.P. Harkins T.T. Bouffard P. Fitzpatrick C. Chu D.S. Thierry-Mieg D. Thierry-Mieg J. Kim J.K. 26G endo-siRNAs regulate spermatogenic and zygotic gene expression in Caenorhabditis elegans.Proc. Natl. Acad. Sci. USA. 2009; 106: 18674-18679Crossref PubMed Scopus (115) Google Scholar]. Small RNA sequencing was done as described [3Montgomery T.A. Rim Y.-S. Zhang C. Dowen R.H. Phillips C.M. Fischer S.E.J. Ruvkun G. PIWI associated siRNAs and piRNAs specifically require the Caenorhabditis elegans HEN1 ortholog henn-1.PLoS Genet. 2012; 8: e1002616Crossref PubMed Scopus (89) Google Scholar]. Sequences were aligned to the C. elegans reference genome WS204 using CASHX v. 2.0 [19Fahlgren N. Sullivan C.M. Kasschau K.D. Chapman E.J. Cumbie J.S. Montgomery T.A. Gilbert S.D. Dasenko M. Backman T.W.H. Givan S.A. Carrington J.C. Computational and analytical framework for small RNA profiling by high-throughput sequencing.RNA. 2009; 15: 992-1002Crossref PubMed Scopus (106) Google Scholar]. Coimmunoprecipitation and western blot assays were done as described [1Phillips C.M. Montgomery T.A. Breen P.C. Ruvkun G. MUT-16 promotes formation of perinuclear mutator foci required for RNA silencing in the C. elegans germline.Genes Dev. 2012; 26: 1433-1444Crossref PubMed Scopus (95) Google Scholar]. RdRP assays were done as described [20Tang G. Reinhart B.J. Bartel D.P. Zamore P.D. A biochemical framework for RNA silencing in plants.Genes Dev. 2003; 17: 49-63Crossref PubMed Scopus (795) Google Scholar]. Thanks to Ulandt Kim and Yanqun Wang for Illumina sequencing. Strains were provided by the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440), and Shohei Mitani of the Japanese National Bioresources Project. Mos strains were generated by the Ewbank and Segalat labs as part of the NEMAGENETAG project funded by the European Community and distributed by M. Carre-Pierrat at the UMS 3421, supported by the CNRS. This work was supported by the NIH (GM44619 to G.R.), Colorado State University (T.A.M.), the Massachusetts General Hospital Executive Committee of Research (C.M.P. and T.A.M.), the Damon Runyon Cancer Research Foundation (C.M.P. and T.A.M.), and an ERC Starting Grant (202819) from the Ideas Program of the European Union Seventh Framework Program (R.F.K.). The NCBI Gene Expression Omnibus accession number for the small RNA high-throughput sequencing data reported in this paper is GSE54320. Download .pdf (3.23 MB) Help with pdf files Document S1. Supplemental Experimental Procedures, Figures S1–S3, and Tables S1 and S3–S5 Download .xlsx (.38 MB) Help with xlsx files Table S2. Normalized siRNA Reads from mutator Targets in Each of the mutator Mutants, Related to Figure 3 The Vasa Homolog RDE-12 Engages Target mRNA and Multiple Argonaute Proteins to Promote RNAi in C. elegansShirayama et al.Current BiologyMarch 27, 2014In BriefShirayama et al. identify the Vasa homolog RDE-12 as a protein that promotes RNAi and virus suppression in C. elegans. RDE-12 is recruited to target mRNA by primary AGO RDE-1 to promote small RNA amplification. RDE-12 forms a complex with secondary AGO WAGO-1 in P granules and may have additional functions in target mRNA surveillance and silencing. Full-Text PDF Open ArchiveThe DEAD Box Helicase RDE-12 Promotes Amplification of RNAi in Cytoplasmic Foci in C. elegansYang et al.Current BiologyMarch 27, 2014In BriefYang et al. reports a new RNAi effector in C. elegans, RDE-12, which is required for the amplification of secondary siRNA. RDE-12 localizes to cytoplasmic foci to coordinate the recruitment of target mRNA and other RNAi effectors, such as RDE-10 and RRF-1/RdRP. Full-Text PDF Open Archive" @default.
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