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
Project description:The molecular mechanisms for target mRNA degradation in C. elegans undergoing RNA interference (RNAi) are not fully understood. Using a combination of genetic, proteomic and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. The RDE-10/RDE-11 complex acts in parallel of nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a 5-fold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and micro-RNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans. 21-24nt small RNA were purifed from different C. elegans strain populations that underwent sel-1 RNAi
Project description:From a forward genetic screen for C. elegans genes required for RNAi, we identified rde-10 and through proteomic analysis of RDE-10-interacting proteins, we identified a protein complex containing the new RNAi factor RDE-11, the known RNAi factors RSD-2 and ERGO-1, as well as other candidate RNAi factors. The newly identified RNAi defective genes rde-10 and rde-11 encode a novel protein and a RING-type zinc finger domain protein, respectively. Mutations in rde-10 and rde-11 genes cause dosage-sensitive RNAi deficiencies: these mutants are resistant to low dosage, but sensitive to high dosage of double-stranded RNAs. We assessed the roles of rde-10, rde-11, and the dosage-sensitive RNAi defective genes rsd-2, rsd-6 and haf-6 in both exogenous and endogenous small RNA pathways using high-throughput sequencing and qRT-PCR. These genes are required for the accumulation of secondary siRNAs in both exogenous and endogenous RNAi pathways. Small RNA analysis by deep sequencing in various wild type and mutant C. elegans strains.
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.
Project description:The molecular mechanisms for target mRNA degradation in C. elegans undergoing RNA interference (RNAi) are not fully understood. Using a combination of genetic, proteomic and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. The RDE-10/RDE-11 complex acts in parallel of nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a 5-fold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and micro-RNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans. Overall design: 21-24nt small RNA were purifed from different C. elegans strain populations that underwent sel-1 RNAi
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 5’ 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 “trigger” 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.
Project description:Animal germ cells employ small RNA-based mechanisms to recognize and silence DNA that invades their genome. One of these pathways is named the Piwi:piRNA pathway. Biogenesis of piRNAs is poorly understood. In C. elegans, the piRNA (21U-RNA)-binding Argonaute protein PRG-1 is the only known player acting downstream of pre-cursor transcription. From a screen aimed at the isolation of ‘piRNA-induced silencing defective’ mutations we identified, amongst known Piwi-pathway components like MUT-7, RDE-3 and HRDE-1, PID-1 as a novel player. PID-1 is essential for 21U RNA biogenesis and affects an early step in the processing or transport of 21U precursor transcripts. 12 small RNA samples were analyzed as singletons.
Project description:Adenosine deaminases that act on RNA (ADARs) catalyze the conversion of adenosine to inosine in dsRNA. C. elegans ADARs, ADR-1 and ADR-2, promote the expression of genes containing dsRNA structures by preventing their processing into siRNAs and silencing by RNAi. The 26G endogenous siRNA (endo-siRNA) pathway generates a subset of siRNAs distinct from those made in adr-1;adr-2 mutants, but using many of the same factors. We found that adr-1;adr-2;rrf-3 mutants, lacking both ADARs and the RNA-dependent RNA polymerase RRF-3 required for the 26G pathway, display a bursting phenotype rescued by the RNAi factors RDE-1 and RDE-4. To determine what gene expression changes underlie the synthetic phenotype of adr-1;adr-2;rrf-3 mutants, we sequenced poly(A)+ RNA from adr-1;adr-2;rrf-3 embryos, their parent strains, and strains rescued with mutations in rde-1 and rde-4. We found that genes associated with edited structures were robustly downregulated in adr-1;adr-2;rrf-3 mutants in a manner partially dependent on rde-1 and rde-4. Additionally, genes induced during Orsay virus infections were induced in rrf-3 mutants and further upregulated in adr-1;adr-2;rrf-3 mutants, again dependent in part on rde-1 and rde-4. Overall design: RNAseq of poly(A)+ RNA from C.elegans mixed-stage embryos, four biological replicates per genotype, six genotypes: wildtype (Bristol N2), adr-1(uu49);adr-2(uu28), rrf-3(uu56), adr-1(uu49);adr-2(uu28);rrf-3(uu56), adr-1(uu49);adr-2(uu28);rrf-3(uu56);rde-1(uu51), and adr-1(uu49);adr-2(uu28);rrf-3(uu56);rde-4(uu53).
Project description:Freshwater planarians can be used as a model to investigate neuronal function and maintenance in adults in vivo. We knocked down expression for 2 transcription factors, then sequenced resulting animals to examine transcriptional changes that may have occurred. There are 3 triplicated conditions: control(RNAi) using the unc22 gene from C. elegans, lhx1/5-1(RNAi), and pitx(RNAi). Worms were fed RNAi for each gene 5 times over 12 days then were collected 12 days later. Approximately 50 million single-end reads were performed on each sample.
Project description:To determine if an endogenous 22G siRNA sensor transgene is subject to siRNA amplification, small RNAs were deep sequenced from the sensor and from a control transgene that is identical to the sensor but lacks an siRNA target site. Small RNAs were isolated from synchronized young adult C. elegans and subjected to deep sequencing.
Project description:Small RNAs, including piRNAs, miRNAs and endogenous siRNAs, bind Argonaute proteins to form RNA-silencing complexes that target coding genes, transposons and aberrant RNAs. To assess the requirements for endogenous siRNA formation and activity in C. elegans, we developed a GFP-based sensor for the endogenous siRNA 22G siR-1, one of a set of abundant siRNAs processed from a precursor RNA mapping to the X chromosome, the X-cluster. Silencing of the sensor is also dependent on the partially complementary, unlinked 26G siR-O7 siRNA. We show that 26G siR-O7 acts in trans to initiate 22G siRNA formation from the X-cluster. The presence of several mispairs between 26G siR-O7 and the X-cluster mRNA, as well as mutagenesis of the siRNA sensor, indicates that siRNA target recognition is permissive to a degree of mispairing. From a candidate reverse genetic screen, we identified several factors required for 22G siR-1 activity, including the Argonaute ergo-1 and the 3' methyltransferase henn-1. Quantitative RT-PCR of small RNAs in a henn-1 mutant and deep sequencing of methylated small RNAs indicate that siRNAs and piRNAs that associate with PIWI clade Argonautes are methylated by HENN-1, while siRNAs and miRNAs that associate with non-PIWI clade Argonautes are not. Thus, PIWI-class Argonaute proteins are specifically adapted to associate with methylated small RNAs in C. elegans. This SuperSeries is composed of the following subset Series: GSE34320: Analysis of 22G siRNA triggered siRNA amplification in Caenorhabditis elegans GSE34321: Analysis of 3' 2'-O-methylated small RNAs in Caenorhabditis elegans Refer to individual Series