Distinct Functions of Argonaute Slicer in siRNA Maturation and Heterochromatin Formation
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PROVIDER: PRJNA322453 | ENA |
REPOSITORIES: ENA
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Project description:Small-RNA (sRNA)-guided transcriptional gene silencing by Argonaute (Ago)-containing complexes is fundamental to genome integrity and epigenetic inheritance. The RNA cleavage (“Slicer”) activity of Argonaute has been implicated in both sRNA maturation and target RNA cleavage. Typically, Argonaute slices and releases the passenger strand of duplex sRNA to generate active silencing complexes, but it remains unclear whether slicing of target nascent RNAs, or other RNAi components, also contributes to downstream transcriptional silencing. Here, we develop a strategy for loading the fission yeast Ago1 with a single-stranded sRNA guide, which bypasses the requirement for slicer activity in generation of active silencing complexes. We show that slicer-defective Ago1 can mediate secondary sRNA generation, H3K9 methylation, and silencing similar to or better than wild-type and associates with chromatin more efficiently. The results define an ancient and minimal sRNA-mediated chromatin silencing mechanism, which resembles the germline-specific sRNA-dependent transcriptional silencing pathways in Drosophila and mammals. This SuperSeries is composed of the SubSeries listed below.
2016-12-19 | GSE81734 | GEO
Project description:Small-RNA (sRNA)-guided transcriptional gene silencing by Argonaute (Ago)-containing complexes is fundamental to genome integrity and epigenetic inheritance. The RNA cleavage ("Slicer") activity of Argonaute has been implicated in both sRNA maturation and target RNA cleavage. Typically, Argonaute slices and releases the passenger strand of duplex sRNA to generate active silencing complexes, but it remains unclear whether slicing of target nascent RNAs, or other RNAi components, also contributes to downstream transcriptional silencing. Here, we develop a strategy for loading the fission yeast Ago1 with a single-stranded sRNA guide, which bypasses the requirement for slicer activity in generation of active silencing complexes. We show that slicer-defective Ago1 can mediate secondary sRNA generation, H3K9 methylation, and silencing similar to or better than wild-type and associates with chromatin more efficiently. The results define an ancient and minimal sRNA-mediated chromatin silencing mechanism, which resembles the germline-specific sRNA-dependent transcriptional silencing pathways in Drosophila and mammals.
| S-EPMC5576859 | biostudies-literature
Project description:The assembly of fission yeast pericentromeric heterochromatin and generation of small interfering RNAs (siRNAs) from noncoding centromeric transcripts are mutually dependent processes. How this interdependent positive feedback loop is first triggered is a fundamental unanswered question. Here we show that two distinct Argonaute (Ago1)-dependent pathways mediate small RNA generation. RNA-dependent RNA polymerase complex (RDRC) and Dicer act on specific noncoding RNAs to generate siRNAs by a mechanism that requires the slicer activity of Ago1 but is independent of pre-existing heterochromatin. In the absence of RDRC or Dicer, a distinct class of small RNAs, called primal small RNAs (priRNAs), associate with Ago1. priRNAs are degradation products of abundant transcripts, which bind to Ago1 and target antisense transcripts that result from bidirectional transcription of DNA repeats. Our results suggest that a transcriptome surveillance mechanism based on the random association of RNA degradation products with Argonaute triggers siRNA amplification and heterochromatin assembly within DNA repeats. small RNA profiling in wild type S. pombe cells and in 12 mutant cells
2010-02-23 | E-GEOD-19734 | biostudies-arrayexpress
Project description:The assembly of fission yeast pericentromeric heterochromatin and generation of small interfering RNAs (siRNAs) from noncoding centromeric transcripts are mutually dependent processes. How this interdependent positive feedback loop is first triggered is a fundamental unanswered question. Here we show that two distinct Argonaute (Ago1)-dependent pathways mediate small RNA generation. RNA-dependent RNA polymerase complex (RDRC) and Dicer act on specific noncoding RNAs to generate siRNAs by a mechanism that requires the slicer activity of Ago1 but is independent of pre-existing heterochromatin. In the absence of RDRC or Dicer, a distinct class of small RNAs, called primal small RNAs (priRNAs), associate with Ago1. priRNAs are degradation products of abundant transcripts, which bind to Ago1 and target antisense transcripts that result from bidirectional transcription of DNA repeats. Our results suggest that a transcriptome surveillance mechanism based on the random association of RNA degradation products with Argonaute triggers siRNA amplification and heterochromatin assembly within DNA repeats.
2010-02-23 | GSE19734 | GEO
Project description:In the Caenorhabditis elegans germline, thousands of mRNAs are concomitantly expressed with antisense 22G-RNAs, which are loaded into the Argonaute CSR-1. Despite their essential functions for animal fertility and embryonic development, how CSR-1 22G-RNAs are produced remains unknown. Here, we show that CSR-1 slicer activity is primarily involved in triggering the synthesis of small RNAs on the coding sequences of germline mRNAs and post-transcriptionally regulates a fraction of targets. CSR-1-cleaved mRNAs prime the RNA-dependent RNA polymerase, EGO-1, to synthesize 22G-RNAs in phase with ribosome translation in the cytoplasm, in contrast to other 22G-RNAs mostly synthesized in germ granules. Moreover, codon optimality and efficient translation antagonize CSR-1 slicing and 22G-RNAs biogenesis. We propose that codon usage differences encoded into mRNA sequences might be a conserved strategy in eukaryotes to regulate small RNA biogenesis and Argonaute targeting
2021-04-23 | PXD020293 | Pride
Project description:The Argonautes are the central protein component in small RNA silencing pathways. Of the four human Argonautes (hAgo1-4) only hAgo2 is an active slicer. We determined the structure of hAgo1 bound to endogenous copurified RNAs to 1.75 Å resolution and hAgo1 loaded with let-7 miRNA to 2.1 Å. Both structures are strikingly similar to the structures of hAgo2. A conserved DEDH catalytic tetrad within the PIWI domain of hAgo2 is required for its slicing activity. hAgo3, with an intact tetrad, could be activated by swapping in the N domain of hAgo2, without additional PIWI domain mutations. An additional mutation on a loop adjacent to the active site of hAgo1 results in slicer activity that is substantially enhanced by swapping in the N domain of hAgo2. Intriguingly, the elements that make Argonaute an active slicer involve a sophisticated interplay between the active site and more distant regions of the enzyme. RNAsprofiling of small RNAs associated with recombinant protein used for chrystallographic studies
2013-05-20 | E-GEOD-46870 | biostudies-arrayexpress
Project description:The Argonautes are the central protein component in small RNA silencing pathways. Of the four human Argonautes (hAgo1-4) only hAgo2 is an active slicer. We determined the structure of hAgo1 bound to endogenous copurified RNAs to 1.75 Å resolution and hAgo1 loaded with let-7 miRNA to 2.1 Å. Both structures are strikingly similar to the structures of hAgo2. A conserved DEDH catalytic tetrad within the PIWI domain of hAgo2 is required for its slicing activity. hAgo3, with an intact tetrad, could be activated by swapping in the N domain of hAgo2, without additional PIWI domain mutations. An additional mutation on a loop adjacent to the active site of hAgo1 results in slicer activity that is substantially enhanced by swapping in the N domain of hAgo2. Intriguingly, the elements that make Argonaute an active slicer involve a sophisticated interplay between the active site and more distant regions of the enzyme.
2013-05-20 | GSE46870 | GEO
Project description:The Arabidopsis ARGONAUTE (AGO) protein AGO1 associates with microRNA (miRNA) and specific classes of short-interfering RNA (siRNA). AGO1-small RNA complexes recognize target RNA transcripts through base-pairing interactions and inhibit translation of target RNAs through endonucleolytic cleavage (slicing) or non-degradative mechanisms. The PIWI domain of AGO1 contains a metal-coordinating triad [Asp-Asp-His] (DDH) that is required for slicer activity. Here, we compared the activities of wild type (DDH) and slicer active-site defective (DAH) forms of AGO1 by sequencing small RNA and target transcript RNAs that co-immunoprecipitated with hemagglutinin (HA)-tagged AGO1 proteins. We found that the population of miRNA that associated with both AGO1-DDH and AGO1-DAH proteins largely overlapped, suggesting that cleavage activity does not affect miRNA maturation. In contrast, slicer-defective AGO1-miRNA complexes associated with target RNA more effectively than did wild type AGO1-miRNA. These data indicate that slicer-defective AGO proteins can be used as an approach to capture AGO-small RNA-target RNA ternary complexes more efficiently for genome-wide analyses.
2012-09-28 | GSE40256 | GEO
Project description:The Arabidopsis ARGONAUTE (AGO) protein AGO1 associates with microRNA (miRNA) and specific classes of short-interfering RNA (siRNA). AGO1-small RNA complexes recognize target RNA transcripts through base-pairing interactions and inhibit translation of target RNAs through endonucleolytic cleavage (slicing) or non-degradative mechanisms. The PIWI domain of AGO1 contains a metal-coordinating triad [Asp-Asp-His] (DDH) that is required for slicer activity. Here, we compared the activities of wild type (DDH) and slicer active-site defective (DAH) forms of AGO1 by sequencing small RNA and target transcript RNAs that co-immunoprecipitated with hemagglutinin (HA)-tagged AGO1 proteins. We found that the population of miRNA that associated with both AGO1-DDH and AGO1-DAH proteins largely overlapped, suggesting that cleavage activity does not affect miRNA maturation. In contrast, slicer-defective AGO1-miRNA complexes associated with target RNA more effectively than did wild type AGO1-miRNA. These data indicate that slicer-defective AGO proteins can be used as an approach to capture AGO-small RNA-target RNA ternary complexes more efficiently for genome-wide analyses.
2012-09-28 | GSE40258 | GEO
Project description:Distinct Functions of Argonaute Slicer in siRNA Maturation and Heterochromatin Formation [ChIP-Seq]
| PRJNA322452 | ENA