Comprehensive analyses of small RNAs associated with C. elegans argonautes
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ABSTRACT:
PROVIDER: PRJNA875268 | ENA |
REPOSITORIES: ENA
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Project description:To genome-widely investigate functions of C. elegans argonautes (AGOs), we applied CRISPR/Cas9 technology to introduce an in-frame GFP::FLAG multiplex tag into the endogenous locus of C. elegans AGOs and performed RNA immunoprecipitation (IP) with anti-GFP or anti-FLAG antibody, followed by unique molecular identifier (UMI)-mediated cDNA library constructions and subjected to high-throughput sequencing. We obtained targets of C. elegans AGOs analyzed from small RNA reads of input and 19 AGO IP samples.
2023-02-13 | GSE212382 | GEO
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
2012-02-03 | E-GEOD-35550 | biostudies-arrayexpress
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.
2012-02-03 | GSE35550 | GEO
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2009-10-06 | E-GEOD-17787 | biostudies-arrayexpress
Project description:To identify methylated small RNAs in C. elegans, we deep sequenced both β-eliminated and untreated small RNAs isolated from wild type C. elegans.
2011-12-10 | GSE34321 | GEO
Project description:High-throughput sequencing of mixed-stage Caenorhabditis elegans small RNAs. total RNA, ~18-26nt RNAs isolated using PAGE, ligation to adapters requires 5' monophosphate and 3' OH Keywords: high-throughput 454 sequencing
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Project description:To identify methylated small RNAs in C. elegans, we deep sequenced both β-eliminated and untreated small RNAs isolated from wild type C. elegans. Small RNAs were isolated from larval and adult stage C. elegans and either subjected to β-elimination or no treatment. Small RNA cDNA libraries were sequenced on an Illumina HiSeq instrument, and enrichment or depletion of small RNAs by β-elimination was assessed after library size normalization based on the number of mappable reads in each library.
2011-12-10 | E-GEOD-34321 | biostudies-arrayexpress
Project description:Small RNA pathways play diverse regulatory roles in the nematode C. elegans. However, our understanding of small RNA pathways, their conservation, and their roles in other nematodes is limited. Here, we analyzed small RNA pathways in the parasitic nematode Ascaris. Ascaris has ten Argonautes with five worm-specific Argonautes (WAGOs) that are associated with secondary 5’-triphosphate small RNAs (22-24G-RNAs). These Ascaris WAGOs and their small RNAs target repetitive sequences (WAGO-1, WAGO-2, WAGO-3, and NRDE-3) or mature mRNAs (CSR-1, NRDE-3, and WAGO-3) and are similar to the C. elegans mutator, nuclear, and CSR-1 small RNA pathways. Ascaris CSR-1 likely functions to “license” gene expression in the absence of an Ascaris piRNA pathway. Ascaris ALG-4 and its associated 26G-RNAs target and appear to repress specific mRNAs during meiosis in the testes. Notably, Ascaris WAGOs (WAGO-3 and NRDE-3) small RNAs change their targets between repetitive sequences and mRNAs during spermatogenesis or in early embryos illustrating target plasticity of these WAGOs. We provide a unique and comprehensive view of mRNA and small RNA expression throughout nematode spermatogenesis that illustrates the dynamics and flexibility of small RNA pathways. Overall, our study provides key insights into the conservation and divergence of nematode small RNA pathways.
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Project description:Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes that convert adenosine to inosine in double-stranded RNA (dsRNA). To evaluate effects of ADARs on small RNAs that derive from dsRNA precursors, we performed deep-sequencing, comparing small RNAs from wildtype and ADAR mutant C. elegans. While editing in small RNAs was rare, at least 40% of microRNAs had altered levels in at least one ADAR mutant strain, and miRNAs with significantly altered levels had mRNA targets with correspondingly affected levels. About 40% of siRNAs derived from endogenous genes (endo-siRNAs) also had altered levels in at least one mutant strain, including 63% of Dicer-dependent endo-siRNAs. The 26G class of endo-siRNAs was significantly affected by ADARs, and many altered 26G loci had intronic reads, and histone modifications associated with transcriptional silencing. Our data indicate ADARs, through both direct and indirect mechanisms, are important for maintaining wildtype levels of many small RNAs in C. elegans. Deep sequencing of small RNAs in wild-type (N2), adr-1 null, adr-2 null and adr-1;adr-2 null mixed stage C. elegans
2012-04-04 | E-GEOD-28888 | biostudies-arrayexpress
Project description:High throughput sequencing to derive function of cde-1 in endogenous RNAi in C. elegans
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