Ingested double-stranded RNA engages multiple silencing mechanisms in C. elegans
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ABSTRACT:
ORGANISM(S): Caenorhabditis elegans
PROVIDER: GSE76424 | GEO | 2017/01/01
SECONDARY ACCESSION(S): PRJNA307258
REPOSITORIES: GEO
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Project description:Effective silencing by RNA-interference (RNAi) depends on mechanisms that amplify and propagate the silencing signal. In some organisms, small-interfering (si) RNAs are amplified from target mRNAs by RNA-dependent RNA polymerase (RdRP). Both RdRP recruitment and mRNA silencing require Argonaute proteins, which are generally thought to degrade RNAi targets by directly cleaving them. However in C. elegans, the enzymatic activity of the primary Argonaute, RDE-1, is not required for silencing activity. We show that RDE-1 can instead recruit an endoribonuclease, RDE-8, to target RNA. RDE-8 can cleave RNA in vitro and is needed for the production of 3′ uridylated fragments of target mRNA in vivo. We also find that RDE-8 promotes RdRP activity, thereby ensuring amplification of siRNAs. Together, our findings suggest a model in which RDE-8 cleaves target mRNAs to mediate silencing, while generating 3’ uridylated mRNA fragments to serve as templates for the RdRP-directed amplification of the silencing signal. We examined the role of rde-8 in C. elegans small RNA biogenesis pathways, including endogenous and exogenous RNAi pathways. We performed 3' RACE seq from the sel-1 target mRNA and correlate with small RNAs from wild type, rde-8 and rde-8 transgenic strains after sel-1(RNAi) for different lengths of time.
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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. Examination of exogenous dsRNA trigger derived siRNA in wildtype and rde-12 mutant animals
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Project description:Mobile genetic elements threaten genome integrity in all organisms. MUT-2/RDE-3 is a ribonucleotidyltransferase required for transposon silencing and RNA interference (RNAi) in C. elegans. When tethered to RNAs in heterologous expression systems, RDE-3 can add long stretches of alternating non-templated uridine (U) and guanosine (G) ribonucleotides to the 3’ termini of these RNAs (polyUG or pUG tails). Here, we show that, in its natural context in C. elegans, RDE-3 adds pUG tails to transposon RNAs, as well as to targets of RNAi. pUG tails with more than 16 perfectly alternating 3’ U and G nucleotides convert RNA fragments into agents of gene silencing. pUG tails promote gene silencing by recruiting RNA-dependent RNA Polymerases (RdRPs), which use pUG-tailed RNAs (pUG RNAs) as templates to synthesize small interfering RNAs (siRNAs). Our results show that cycles of pUG RNA-templated siRNA synthesis and siRNA-directed mRNA pUGylation underlie dsRNA-directed transgenerational epigenetic inheritance in the C. elegans germline. We speculate that this pUG RNA/siRNA silencing loop allows parents to inoculate progeny against the expression of unwanted or parasitic genetic elements.
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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.
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