Project description:Diverse naturally-occurring small RNA species interact with Argonaute proteins to mediate sequence-specific regulation in animals. In addition to micro-RNAs (miRNAs), which collectively regulate thousands of target mRNAs, other endogenous small RNA species include the Piwi-associated piRNAs that are important for fertility and a less well-characterized class of small RNAs often referred to simply as endo-siRNAs. Here we have utilized deep-sequencing technology and C. elegans genetics to explore the biogenesis and function of endo-siRNAs. We describe conditional alleles of the dicer-related helicase, drh-3, that implicate DRH-3 in both the response to foreign dsRNA as well as the RNA-dependent RNA Polymerase (RdRP)-dependent biogenesis of a diverse class of endogenous small RNAs, termed 22G-RNAs. We show that 22G-RNAs are abundantly expressed in the germline and maternally inherited and are the products of at least two distinct 22G-RNA systems. One system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germline nuage-related structures termed P-granules. The WAGO 22G-RNA system silences transposons, pseudogenes and cryptic loci as well as a number of genes. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one of the multiple, distinct WAGO surveillance pathways. These findings broaden our understanding of the biogenesis and diversity of 22G-RNA species and suggest potential novel regulatory functions for these small RNAs. 18 samples examined. Small RNA libraries generated from: C. elegans animals with mutations in the WAGO pathway and a WAGO-1 immunopercipitate.
Project description:To explore the roles of piRNAs and WAGO-class 22G-RNAs in regulating gene expression and transposon silencing in Caenorhabditis elegans, we used RNA-seq to assess changes in small RNA and mRNA levels in prg-1 and mut-16 mutants, which disable the piRNA and WAGO-class 22G-RNA pathways respectively. We identified numerous roles for piRNAs and WAGO-class 22G-RNAs in regulating germline genes, including transposons, histones, and spermatogenic and oogenic transcripts.
Project description:Diverse naturally-occurring small RNA species interact with Argonaute proteins to mediate sequence-specific regulation in animals. In addition to micro-RNAs (miRNAs), which collectively regulate thousands of target mRNAs, other endogenous small RNA species include the Piwi-associated piRNAs that are important for fertility and a less well-characterized class of small RNAs often referred to simply as endo-siRNAs. Here we have utilized deep-sequencing technology and C. elegans genetics to explore the biogenesis and function of endo-siRNAs. We describe conditional alleles of the dicer-related helicase, drh-3, that implicate DRH-3 in both the response to foreign dsRNA as well as the RNA-dependent RNA Polymerase (RdRP)-dependent biogenesis of a diverse class of endogenous small RNAs, termed 22G-RNAs. We show that 22G-RNAs are abundantly expressed in the germline and maternally inherited and are the products of at least two distinct 22G-RNA systems. One system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germline nuage-related structures termed P-granules. The WAGO 22G-RNA system silences transposons, pseudogenes and cryptic loci as well as a number of genes. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one of the multiple, distinct WAGO surveillance pathways. These findings broaden our understanding of the biogenesis and diversity of 22G-RNA species and suggest potential novel regulatory functions for these small RNAs.
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
Project description:Post-transcriptional control of mRNAs by RNA-binding proteins (RBPs) has a prominent role in the regulation of gene expression. RBPs interact with mRNAs to control their biogenesis, splicing, transport, localization, translation, and stability. Defects in such regulation can lead to a wide range of human diseases from neurological disorders to cancer. Many RBPs are conserved between Caenorhabditis elegans and humans, and several are known to regulate apoptosis in the adult C. elegans germ line. How these RBPs control apoptosis is, however, largely unknown. Here, we identify mina-1(C41G7.3) in a RNA interference-based screen as a novel regulator of apoptosis, which is exclusively expressed in the adult germ line. The absence of MINA-1 causes a dramatic increase in germ cell apoptosis, a reduction in brood size, and an impaired P granules organization and structure. In vivo crosslinking immunoprecipitation experiments revealed that MINA-1 binds a set of mRNAs coding for RBPs associated with germ cell development. Additionally, a system-wide analysis of a mina-1 deletion mutant compared to wild type, including quantitative proteome and transcriptome data, hints to a post-transcriptional regulatory RBP network driven by MINA-1 during germ cell development in C. elegans. In particular, we found that the germline-specific Argonaute WAGO-4 protein levels are increased in the mina-1 mutant background. Phenotypic analysis of double mutant mina-1;wago-4 revealed that contemporary loss of MINA-1 and WAGO-4 strongly rescues the phenotypes observed in the mina-1 mutant background. To strengthen this functional interaction, we found that upregulation of WAGO-4 in mina-1 mutant animals causes hypersensitivity to exogenous RNAi. Our comprehensive experimental approach allowed us to describe a phenocritical interaction between two RBPs controlling germ cell apoptosis and exogenous RNAi. These findings broaden our understanding of how RBPs can orchestrate different cellular events such as differentiation and death 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.