Comprehensive analyses of small RNAs associated with C. elegans argonautes
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ABSTRACT:
PROVIDER: PRJNA875268 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR21374053.fastq.gz | Fastqsanger.gz | |||
| SRR21374054.fastq.gz | Fastqsanger.gz | |||
| SRR21374055.fastq.gz | Fastqsanger.gz | |||
| SRR21374056.fastq.gz | Fastqsanger.gz | |||
| SRR21374057.fastq.gz | Fastqsanger.gz |
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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:microRNAs regulate gene expression through interaction with an Argonaute protein family member. While some members of this protein family retain an enzymatic activity capable of cleaving RNA molecules complementary to Argonaute-bound small RNAs, the role of the slicing activity in the canonical microRNA pathway is still unclear in animals. To address the importance of slicing Argonautes in animals, we created Caenorhabditis elegans mutant strains carrying catalytically dead endogenous ALG-1 and ALG-2, the only two slicing Argonautes essential for the miRNA pathway in this animal model. We observe that the loss of ALG-1 and ALG-2 slicing activity affects overall animal fitness and causes phenotypes reminiscent of miRNA defects only when grown and maintained at restrictive temperature. Furthermore, the analysis of global miRNA expression show that the catalytic activity of ALG-1 and ALG-2 differentially regulate the level of specific subsets of miRNAs in young adults. We also demonstrate that altering the slicing activity of those miRNA-specific Argonautes does not result in any defect in the production of canonical miRNAs. Together, these data support that the slicing activity of miRNA-specific Argonautes functions to maintain levels of a set of miRNAs for optimal viability and fitness in animals particularly exposed to specific growing conditions.
2024-03-15 | GSE222733 | 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 SubSeries listed below.
2012-02-03 | GSE35550 | GEO
Project description:Many Argonaute proteins can cleave RNA (“slicing”) as part of the microRNA-induced silencing complex (miRISC), even though miRNA-mediated target repression is generally independent of target cleavage. Here we use C. elegans to examine the role of catalytic residues of miRNA Argonautes in organismal development. In contrast to previous work, mutations in presumed catalytic residues did not interfere with normal development when introduced by CRISPR. We find that unwinding and decay of miRNA star strands is weakly defective in the catalytic residue mutants, with the largest effect observed in embryos. Argonaute-Like Gene 2 (ALG-2) is more dependent on slicing for unwinding than ALG-1. The miRNAs that displayed the greatest (albeit minor) dependence on catalytic residues for unwinding tend to form stable duplexes with their star strand, and in some cases, lowering duplex stability alleviates dependence on catalytic residues. While a few miRNA guide strands are reduced in the mutant background, the basis of this is unclear since changes were not dependent on EBAX-1, an effector of Target-Directed miRNA Degradation (TDMD). Overall, this work defines a role for the catalytic residues of miRNA Argonautes in star strand decay; future work should examine whether this role may contribute to the selection pressure to conserve catalytic activity of miRNA Argonautes across the metazoan phylogeny.
2024-02-26 | GSE256488 | GEO
Project description:Many Argonaute proteins can cleave RNA (“slicing”) as part of the microRNA-induced silencing complex (miRISC), even though miRNA-mediated target repression is generally independent of target cleavage. Here we use C. elegans to examine the role of catalytic residues of miRNA Argonautes in organismal development. In contrast to previous work, mutations in presumed catalytic residues did not interfere with normal development when introduced by CRISPR. We find that unwinding and decay of miRNA star strands is weakly defective in the catalytic residue mutants, with the largest effect observed in embryos. Argonaute-Like Gene 2 (ALG-2) is more dependent on slicing for unwinding than ALG-1. The miRNAs that displayed the greatest (albeit minor) dependence on catalytic residues for unwinding tend to form stable duplexes with their star strand, and in some cases, lowering duplex stability alleviates dependence on catalytic residues. While a few miRNA guide strands are reduced in the mutant background, the basis of this is unclear since changes were not dependent on EBAX-1, an effector of Target-Directed miRNA Degradation (TDMD). Overall, this work defines a role for the catalytic residues of miRNA Argonautes in star strand decay; future work should examine whether this role may contribute to the selection pressure to conserve catalytic activity of miRNA Argonautes across the metazoan phylogeny.
2024-02-26 | GSE256489 | GEO
Project description:Caenorhabditis elegans contains twenty-five Argonautes, of which, only ALG-1 and ALG-2 are known to interact with miRNAs. ALG-5 belongs to the AGO subfamily of Argonautes that includes ALG-1 and ALG-2, but its role in small RNA pathways is unknown. We analyzed by high-throughput sequencing the small RNAs associated with ALG-5, ALG-1, and ALG-2, as well as changes in mRNA expression in alg-5, alg-1, and alg-2 mutants.
2017-06-09 | GSE98935 | GEO
Project description:Histone H3 lysine 9 methylation (H3K9me) and nuclear RNAi are two processes with roles in repression of repetitive elements and regulation of gene expression. Recently, they were also implicated in dosage compensation, a mechanism that accommodates for the differences in gene dosage between the biological sexes. In Caenorhabditis elegans, dosage compensation is achieved via epigenetic modifications on both X chromosomes of hermaphrodites, effectively dampening their expression by half to match the single X chromosome in males. This chromosome-wide process also relies on the anchoring and compaction of the X chromosomes near the nuclear lamina, mediated by the H3K9 histone methyltransferases MET-2, SET-25 and SET-32. H3K9me is also involved in gene silencing during nuclear RNAi, and recent evidence suggests that mutations in the nuclear RNAi machinery impact X chromosome compaction and dosage compensation. The nuclear Argonautes HRDE-1 and NRDE-3 are responsible for the recruitment of the H3K9 histone methyltransferases to targets in the germline and the soma, respectively, during nuclear RNAi. We investigated the impact of the loss of nuclear Argonautes and/or H3K9 methyltransferases on both dosage compensation and global gene expression during somatic development. Our immunofluorescence and bioinformatic analyses suggest that the nuclear RNAi and H3K9me pathways are involved in the packaging of the X chromosomes during dosage compensation, without consequence to their transcriptional output. Instead, mutant RNAi mutants exhibit global transcriptional differences, in which HRDE-1 and NRDE-3 affect expression of their native targets through different modes of regulation and different relationships between each nuclear Argonaute and H3K9 methylation.
2024-10-01 | GSE277005 | GEO
Project description:The Argonautes (AGOs) are widely expressed, evolutionarily conserved RNA binding proteins that play an important role in gene expression regulation. The AGOs bind to small regulatory noncoding RNAs such as micro RNAs (miRNAs), short interfering RNAs (siRNAs), Piwi-interacting RNAs (piRNAs) etc. The small regulatory noncoding RNAs serve the function of guiding the AGOs to the right target RNAs by complementary base pairing. Additionally, the AGOs interact with GW182 (TNRC6A/-B/-C) proteins and together with small RNAs, they form an effector ribonucleo protein complex named, RNA Induced Silencing Complex (RISC) that regulates several aspects of transcriptional and post-transcriptional gene expression. ALG-1 (Argonaute Like Gene) and ALG-2 are the AGO proteins in C. elegans that are required for miRNA mediated gene expression regulation. Our efforts towards the characterization of the protein complexes comprised of ALG-1 led to the identification of DPF-3, a conserved protease belonging to clinically relevant Di Peptidyl Peptidase IV family, as the novel interacting partner of ALG-1. We have further explored the role of DPF-3 in AGO regulation.
2025-03-06 | PXD060454 | Pride
Project description:Canonical small interfering RNAs (siRNAs) are generated by the cleavage of double-stranded RNA (dsRNA) by the ribonuclease Dicer. siRNAs are found in plants, animals, and some fungi where they bind to Argonautes to direct RNA silencing. In this study, we characterized the canonical Dicer-dependent siRNAs of C. elegans. We identified thousands of endogenous loci, representing dozens of unique elements, that give rise to low to moderate levels of siRNAs, called 23H-RNAs. These loci include repetitive elements, alleged coding genes, pseudogenes, non-coding RNAs, and unannotated features, many of which adopt hairpin structures. Using protein-small RNA co-immunoprecipitation, we identified the Argonautes that associate with 23H-RNAs and using mRNA-sequencing we explored their roles in gene regulation.
2024-01-29 | GSE254442 | GEO