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A Comprehensive Survey of C. elegans Argonaute Proteins Reveals Organism-wide Gene Regulatory Networks and Functions [mutants]

ABSTRACT: Argonaute (AGO) proteins associate with small RNAs to direct their effector function on complementary transcripts. The nematode C. elegans contains an expanded family of 19 functional AGO proteins, many of which have not been fully characterized. In this work we systematically analyzed every C. elegans AGO, using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG tags. We have characterized the expression patterns of each AGO throughout development, identified small RNA binding complements, and determined the effects of ago loss on small RNA populations and developmental phenotypes. Our analysis indicates stratification of subsets of AGOs into distinct regulatory modules, and integration of our data led us to uncover novel stress-induced fertility and pathogen response phenotypes due to ago loss.

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE208701 | GEO | 2022/07/22

REPOSITORIES: GEO

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A Comprehensive Survey of C. elegans Argonaute Proteins Reveals Organism-wide Gene Regulatory Networks and Functions [Argonaute IP]

Project description:Argonaute (AGO) proteins associate with small RNAs to direct their effector function on complementary transcripts. The nematode C. elegans contains an expanded family of 19 functional AGO proteins, many of which have not been fully characterized. In this work we systematically analyzed every C. elegans AGO, using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG tags. We have characterized the expression patterns of each AGO throughout development, identified small RNA binding complements, and determined the effects of ago loss on small RNA populations and developmental phenotypes. Our analysis indicates stratification of subsets of AGOs into distinct regulatory modules, and integration of our data led us to uncover novel stress-induced fertility and pathogen response phenotypes due to ago loss.

2022-07-22 | GSE208700 | GEO

The Vasa homolog RDE-12 engages target mRNA and a secondary-Argonaute WAGO-1 to promote RNAi 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. Examine small RNA population changes in rde-12 mutants

2014-06-24 | E-GEOD-54396 | biostudies-arrayexpress

Small RNAs in four Argonaute complexes in Arabidopsis

2008-04-06 | GSE10036 | GEO

Sequential rounds of RNA-dependent RNA transcription drive endogenous small-RNA biogenesis in ERGO-1/Argonaute pathway

Project description:Argonaute (AGO) proteins interact with distinct classes of small RNAs to direct multiple regulatory outcomes. In many organisms, including plants, fungi and nematodes, cellular RNAdependent RNA polymerases (RdRPs) utilize AGO targets as templates for amplification of silencing signals. Here, we show that distinct RdRPs function sequentially to produce small RNAs that target endogenous loci in C. elegans. We show that DCR-1, the RdRP RRF-3, and the dsRNA-binding protein RDE-4, are required for the biogenesis of 26nt RNAs, termed 26GRNAs, and that 26G-RNAs engage the Piwi-clade AGO, ERGO-1. Our findings support a model in which targeting by ERGO-1 recruits a second RdRP (RRF-1 or EGO-1), which in turn transcribes 22G-RNAs that interact with worm-specific AGOs (WAGOs) to direct gene silencing. ERGO-1 targets exhibit a non-random distribution in the genome and appear to include many gene duplications, suggesting that this pathway may control over-expression resulting from gene expansion. 8 samples examined. Small RNA libraries generated from: C. elegans animals.

2010-02-25 | E-GEOD-18714 | biostudies-arrayexpress

Slicing-Independent RISC Activation Requires the Argonaute PAZ Domain

Project description:Small RNAs regulate the genetic networks through a ribonucleoprotein complex called the RNA induced silencing complexes (RISC), which in mammals contains at its center one of four Argonaute proteins (Ago1-4). A key regulatory event in the RNAi and miRNA pathways is Ago loading, where double stranded small RNA duplexes are incorporated into RISC (pre-RISC) and then become single stranded (mature-RISC), a process that is not well understood. The Agos contain an evolutionary conserved PAZ (Piwi/Argonaute/Zwille) domain whose primary function is to bind the 3’-end of small RNAs. We created multiple Paz domain disrupted Ago mutant proteins and studied their biochemical properties and biological functionality in cells. We found that the Paz domain is dispensable for Ago loading of slicing-competent RISC. In contrast, in the absence of slicer activity or slicer substrate duplex RNAs, Paz-disrupted Agos bound duplex siRNAs but were unable to unwind/eject the passenger strand and form functional RISC complexes. We have discovered that the highly conserved Paz domain plays an important role in RISC activation, providing new mechanistic insights into how miRNAs regulate genes, as well as new insights for future design of miRNA and RNAi-based therapeutics. Various Argonautes associated small RNA profiles were generated by deep sequencing the Agos-IP samples in HEK293 Cells transfected with corresponding Argonaute.

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Comprehensive analyses of small RNAs associated with C. elegans argonautes

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

Small RNAs in four Argonaute complexes in Arabidopsis

Project description:The core components of RNA silencing effector complexes include small RNAs and members of the Argonaute (AGO) protein family. Arabidopsis encodes ten AGOs and complex population of small RNAs. It remains largely unknown how these small RNAs are recognized and recruited by each AGO complex. Here we purified four AGO complexes and identified small RNAs in each complex by deep sequencing. Keywords: Small RNA sorting Small RNAs were prepared from Arabidopsis AGO1, AGO2, AGO4 and AGO5 complexes, ligated to a 3' adaptor and a 5' acceptor sequentially, and then RT-PCR amplified. PCR products were reamplified using a pair of solexa cloning primers and then provided for sequencing. For technical details, see Qi, Y., He, X., Wang, X.J., Kohany, O., Jurka, J., and Hannon, G.J. 2006. Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443(7114): 1008-1012.

2008-04-05 | E-GEOD-10036 | biostudies-arrayexpress

The Vasa homolog RDE-12 engages target mRNA and a secondary-Argonaute WAGO-1 to promote RNAi in C. elegans

2014-06-24 | GSE54396 | GEO

Deciphering the cell-specific role of Arabidopsis Argonaute 3 [smallRNA-Seq]

Project description:ARGONAUTE (AGO) proteins are the major effectors of RNA silencing. In Arabidopsis, loading of 21-22-nt long small RNAs into AGOs results in post-transcriptional gene silencing (PTGS) by mRNA cleavage and/or translational repression. On the other hand, loading of 24-nt sRNAs results in transcriptional gene silencing (TGS) by RNA directed DNA methylation (RdDM). The Arabidopsis genome encodes 10 AGOs, which are known, for most, to belong to either pathway. Here we characterized the cell specific role of Arabidopsis AGO3. It is specifically expressed in tissue close to vascular termination and strongly accumulates in chalazal seed integuments. AGO3 encodes a functional AGO able to bind sRNAs with a preference for 24nt in length with a 5’ nucleotide bias for Adenosine. Down-regulation of AGO3 affects gene expression in siliques and its expression is strongly induced in the plant vasculature upon proteasome inhibition. Our results suggest that AGO3 might act to regulate gene expression by a novel RNA silencing pathway involving 24nt sRNA-directed PTGS.

2021-08-30 | GSE74722 | GEO

Deciphering the cell-specific role of Arabidopsis Argonaute 3 [RNA-seq]

2021-08-30 | GSE74721 | GEO

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