Project description:Comprehensive discovery of endogenous Argonaute binding sites in C. elegans Experiment Overall Design: Total RNA were extracted from whole alg-1(gk214) (alg-1(-)) and N2 (wildtype) worms and used to compare the expression profiles of genes that were targeted by ALG-1 protein.
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
Project description:Argonaute-associated siRNAs and Piwi-associated piRNAs have overlapping roles in silencing mobile genetic elements in animals. In C. elegans, mutator-class (mut) genes mediate siRNA-guided repression of transposons as well as exogenous RNA-directed gene silencing (RNAi), but their roles in endogenous RNA silencing pathways are not well understood. To characterize the endogenous small RNAs dependent on mutator-class genes, small RNA populations from a null allele of mut-16, as well as a regulatory mut-16(mg461) allele that disables only somatic RNAi, were subjected to deep sequencing.
Project description:Argonaute-associated siRNAs and Piwi-associated piRNAs have overlapping roles in silencing mobile genetic elements in animals. In C. elegans, mutator-class (mut) genes mediate siRNA-guided repression of transposons as well as exogenous RNA-directed gene silencing (RNAi), but their roles in endogenous RNA silencing pathways are not well understood. To characterize the endogenous small RNAs dependent on mutator-class genes, small RNA populations from a null allele of mut-16, as well as a regulatory mut-16(mg461) allele that disables only somatic RNAi, were subjected to deep sequencing. Small RNA analysis in wild type and mut-16 mutant C. elegans strains
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 SubSeries listed below.
Project description:piRNAs are required to maintain germline integrity and fertility but their mechanism of action is poorly understood. Here we demonstrate that C. elegans piRNAs silence transcripts in trans through imperfectly complementary sites. We find that target silencing is independent of Piwi endonuclease activity or “slicing”. Instead, we show that piRNAs initiate a localized secondary endogenous small interfering RNA (endo-siRNA) response. Endogenous protein-coding gene, pseudogene and transposon transcripts exhibit Piwi-dependent endo-siRNAs at sites complementary to piRNAs and are derepressed in Piwi mutants. Genomic loci of piRNA biogenesis are depleted of protein-coding genes but not pseudogenes or transposons. Our data suggest that nematode piRNA clusters are evolving to generate piRNAs against active mobile elements. Thus, piRNAs provide heritable, sequence-specific triggers for RNAi in C. elegans.
Project description:piRNAs are required to maintain germline integrity and fertility but their mechanism of action is poorly understood. Here we demonstrate that C. elegans piRNAs silence transcripts in trans through imperfectly complementary sites. We find that target silencing is independent of Piwi endonuclease activity or “slicing”. Instead, we show that piRNAs initiate a localized secondary endogenous small interfering RNA (endo-siRNA) response. Endogenous protein-coding gene, pseudogene and transposon transcripts exhibit Piwi-dependent endo-siRNAs at sites complementary to piRNAs and are derepressed in Piwi mutants. Genomic loci of piRNA biogenesis are depleted of protein-coding genes but not pseudogenes or transposons. Our data suggest that nematode piRNA clusters are evolving to generate piRNAs against active mobile elements. Thus, piRNAs provide heritable, sequence-specific triggers for RNAi in C. elegans. Affymetrix mRNA expression data from wild-type and two independent prg-1;prg-2 double mutant C. elegans strains (mRNA)