Unknown,Transcriptomics,Genomics,Proteomics

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The DEAD box helicase RDE-12 promotes amplification of RNAi in cytoplasmic foci in C. elegans

ABSTRACT: 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

ORGANISM(S): Caenorhabditis elegans

SUBMITTER: Huan Yang

PROVIDER: E-GEOD-53830 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Deep sequencing was used to detect Flock House Virus (FHV)-derived small RNAs (viRNAs) that were inherited over generations. Small RNA libraries were constructed from 4 different samples: (a) FR1gfp-transgenic worms that can mount an RNAi response and should contain viRNAs (positive control); (b) rde-4(-/-) mutants (negative control as no viRNAs are expected to be produced); (c) FR1gfp; rde-4(-/-) worms that are two generations away from their rde-4(+/-) grandparents, i.e. worms that can themselves not produce viRNAs, but may have inherited viRNA from their grandparents; and (d) the F3 progeny of wild-type animals that contained the FR1gfp transgene (and therefore produced viRNAs), but have lost this transgene through outcrossing with non-transgenic wild-type worms. This tests whether the silencing reagent can exist without its template. The Small RNA libraries were constructed using a protocol that enriches for Dicer products which harbor a single phosphate at the 5M-bM-^@M-^Y end of the RNA (Zamore et al., 2000), such as primary siRNAs, and unlike RdRP products that harbor tri-phosphate ends (Parameswaran et al., 2010). This was the protocol of choice because we were interested in identifying viRNAs that are guaranteed to be derived from the original RNAi-competent parents (Alcazar et al., 2008), and rde-4 animals can not produce primary siRNAs (Grishok et al., 2000; Parrish and Fire, 2001). rde-4 animals are, however, not defective in secondary siRNA production (Blanchard et al., 2011), and can therefore continue to amplify secondary siRNAs de novo; such autonomously-produced siRNAs will not be distinguishable from inherited ones. The detection of rare primary siRNAs is important, as even a single M-bM-^@M-^\triggerM-bM-^@M-^] siRNA can induce a full-blown RNAi-response that is not proportional to the primary trigger (Groenenboom et al., 2005).In agreement with the functional assays we detected different viRNAs complementary to several regions of the viral genome in the positive control (FR1gfp), no viRNAs in the negative control (rde-4(-/-)), and a number of viRNAs in the worms that could not generate their own viRNAs and thus inherited these viRNAs from their grandparents (F3 generation FR1gfp; rde-4(-/-))(Table 2; Figure 4). Moreover, we detect viRNAs in the worms in which the FR1gfp transgene had been crossed out, confirming that the viRNA transmit in a template-independent manner. The inherited viRNA matched the two most abundant types of viRNAs detected in the positive control (Figure 4) and these viRNAs were all of the reverse orientation (negative strand, which typically exists in much lower quantities than the positive strand (Felix et al., 2011)); both observations make it highly unlikely that the detected viRNAs merely represent unspecific break-down products of the viral RNA. In regard to the low number of inherited viRNA reads it needs to be considered that our protocol enriches specifically for rare primary viRNA species. Moreover, these viRNA species are derived from a response mounted in RNAi-competent grandparents, and are therefore possibly diluted over the course of several generations. Taken together, our results support the genetic experiments that argued for the existence of trans-acting factors that are transmitted in a non-Mendelian manner to ensuing generations. 4 samples examined. Small RNA libraries generated from: C. elegans animals.

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The DEAD box helicase RDE-12 promotes amplification of RNAi in cytoplasmic foci in C. elegans

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