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The Vasa homolog RDE-12 engages target mRNA and a secondary-Argonaute WAGO-1 to promote RNAi in C. elegans

ABSTRACT: Argonaute proteins (AGOs) are key nuclease effectors of RNA interference (RNAi) [1]. Although purified AGOs can mediate a single round of target-RNA cleavage in vitro, accessory factors are required for siRNA loading and to achieve multiple-target turnover [2, 3]. To identify AGO co-factors we immunoprecipitated the C. elegans AGO WAGO-1, which engages amplified small RNAs during RNAi [4]. These studies identified a robust association between WAGO-1 and a conserved Vasa ATPase-related protein RDE-12. rde-12 mutants are deficient in RNAi including viral suppression, and fail to produce amplified secondary siRNAs and certain endogenous siRNAs (endo-siRNAs). RDE-12 co-localizes with WAGO-1 in germline P-granules and to peri-nuclear cytoplasmic foci in somatic cells. These findings and our genetic studies suggest that (i) RDE-12 is first recruited to target mRNAs by upstream AGOs (RDE-1 and ERGO-1) where it promotes small-RNA amplification and/or WAGO-1 loading, and that (ii) downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 that may scan for additional target mRNAs.

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE54396 | GEO | 2014/06/24

SECONDARY ACCESSION(S): PRJNA236392

REPOSITORIES: GEO

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The Vasa homolog RDE-12 engages target mRNA and a secondary-Argonaute WAGO-1 to promote RNAi in C. elegans

Project description:Argonaute proteins (AGOs) are key nuclease effectors of RNA interference (RNAi) [1]. Although purified AGOs can mediate a single round of target-RNA cleavage in vitro, accessory factors are required for siRNA loading and to achieve multiple-target turnover [2, 3]. To identify AGO co-factors we immunoprecipitated the C. elegans AGO WAGO-1, which engages amplified small RNAs during RNAi [4]. These studies identified a robust association between WAGO-1 and a conserved Vasa ATPase-related protein RDE-12. rde-12 mutants are deficient in RNAi including viral suppression, and fail to produce amplified secondary siRNAs and certain endogenous siRNAs (endo-siRNAs). RDE-12 co-localizes with WAGO-1 in germline P-granules and to peri-nuclear cytoplasmic foci in somatic cells. These findings and our genetic studies suggest that (i) RDE-12 is first recruited to target mRNAs by upstream AGOs (RDE-1 and ERGO-1) where it promotes small-RNA amplification and/or WAGO-1 loading, and that (ii) downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 that may scan for additional target mRNAs. Examine small RNA population changes in rde-12 mutants

2014-06-24 | E-GEOD-54396 | biostudies-arrayexpress

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Project description:RNA interference (RNAi) is a potent mechanism for down-regulating gene expression. Conserved RNAi pathway components are found in animals, plants, fungi and other eukaryotes. In C. elegans, the RNAi response is greatly amplified by the synthesis of abundant secondary siRNAs. Exogenous double stranded RNA is processed by Dicer and RDE-1/Argonaute into primary siRNA that guides target mRNA recognition. The RDE-10/RDE-11 complex and the RNA dependent RNA polymerase RRF-1 then engage the target mRNA for secondary siRNA synthesis. However, the molecular link between primary siRNA production and secondary siRNA synthesis remains largely unknown. Furthermore, it is unclear if the sub-cellular sites for target mRNA recognition and degradation coincide with sites where siRNA synthesis and amplification occur. In the C. elegans germline, cytoplasmic P granules at the nuclear pores and perinuclear Mutator foci contribute to target mRNA surveillance and siRNA amplification, respectively. We report that RDE-12, a conserved FG domain containing DEAD-box helicase, localizes in P-granules and cytoplasmic foci that are enriched in RSD-6 but are excluded from the Mutator foci. Our results suggest that RDE-12 promotes secondary siRNA synthesis by orchestrating the recruitment of RDE-10 and RRF-1 to primary siRNA targeted mRNA in distinct cytoplasmic compartments.

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