Small RNA loading licenses Argonaute for assembly into a transcriptional silencing complex
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ABSTRACT:
ORGANISM(S): Schizosaccharomyces pombe
PROVIDER: GSE65223 | GEO | 2015/03/01
SECONDARY ACCESSION(S): PRJNA273446
REPOSITORIES: GEO
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Project description:We analyzed the small RNAs bound to the fission yeast RITS and ARC complexes, and to Ago1 in wild-type and ARC mutant cells, to gain insight into the molecular role of ARC. We found that ARC contains longer heterochromatic siRNAs than RITS, and that ARC subunit Arb1 is required for loading small RNAs onto Ago1. Sequencing of 18-28 nt small RNAs from Tas3, Arb1 and Ago1 purifications.
2015-03-01 | E-GEOD-65223 | biostudies-arrayexpress
Project description:Argonautes and their small-RNA cofactors form the core effectors of ancient and diverse gene-silencing mechanisms whose roles include regulation of gene expression and defense against foreign genetic elements. Although Argonautes generally act within multisubunit complexes, what governs their assembly into these machineries is not well defined. Here, we show that loading of small RNAs onto Argonaute is a checkpoint for Argonaute's association with conserved GW-protein components of silencing complexes. We demonstrate that the Argonaute small interfering RNA chaperone (ARC) complex mediates loading of small RNAs onto Ago1 in Schizosaccharomyces pombe and that deletion of its subunits, or mutations in Ago1 that prevent small-RNA loading, abolish the assembly of the GW protein-containing RNA-induced transcriptional silencing (RITS) complex. Our studies uncover a mechanism that ensures that Argonaute loading precedes RITS assembly and thereby averts the formation of inert and potentially deleterious complexes.
| S-EPMC4385408 | 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:We provide evidence of the generation of Rdp1-mediated secondary siRNAs in vivo in fission yeast. Secondly, we show that the presence of Ago1-associated siRNAs does not guarantee robust silencing. We obtained sequences from gel purified small RNAs of wt and rdp1delta fission yeast cells and FLAG-ago1 associated small RNAs.
2010-02-15 | E-GEOD-20330 | biostudies-arrayexpress
Project description:Drosophila Argonaute-1 and Argonaute-2 differ in function and small RNA content. AGO2 binds to siRNAs, whereas AGO1 is almost exclusively occupied by microRNAs. microRNA duplexes are intrinsically asymmetric, with one strand, the miR strand, preferentially entering AGO1 to recognize and regulate the expression of target mRNAs. The other strand, miR*, has been viewed as a by-product of microRNA biogenesis. Here, we show that miR*s are often loaded as functional species into AGO2. This indicates that each microRNA precursor can potentially produce two mature small RNA strands that are differentially sorted within the RNAi pathway. miR* biogenesis depends upon the canonical microRNA pathway, but loading into AGO2 is mediated by factors traditionally dedicated to siRNAs. By inferring and validating hierarchical rules that predict differential AGO loading, we find that intrinsic determinants, including structural and thermodynamic properties of the processed duplex, regulate the fate of each RNA strand within the RNAi pathway. These were independently processed and sequenced using the Illumina GAII platform. In total, five libraries were analyzed.
2009-11-15 | E-GEOD-17734 | biostudies-arrayexpress
Project description:Drosophila Argonaute-1 and Argonaute-2 differ in function and small RNA content. AGO2 binds to siRNAs, whereas AGO1 is almost exclusively occupied by microRNAs. microRNA duplexes are intrinsically asymmetric, with one strand, the miR strand, preferentially entering AGO1 to recognize and regulate the expression of target mRNAs. The other strand, miR*, has been viewed as a by-product of microRNA biogenesis. Here, we show that miR*s are often loaded as functional species into AGO2. This indicates that each microRNA precursor can potentially produce two mature small RNA strands that are differentially sorted within the RNAi pathway. miR* biogenesis depends upon the canonical microRNA pathway, but loading into AGO2 is mediated by factors traditionally dedicated to siRNAs. By inferring and validating hierarchical rules that predict differential AGO loading, we find that intrinsic determinants, including structural and thermodynamic properties of the processed duplex, regulate the fate of each RNA strand within the RNAi pathway.
2009-11-15 | GSE17734 | 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. This SuperSeries is composed of the SubSeries listed below.
2016-12-19 | GSE81734 | GEO
Project description:Leishmania donovani, an intracellular protozoan parasite, is the causative agent of visceral leishmaniasis or kala-azar, the most severe form of leishmaniasis in humans. To date, our understanding of the molecular mechanisms associated with the pathogenicity of Leishmania infection is still limited. RNA interference—collectively RNA silencing pathways—participates in the regulation of various biological processes in most eukaryotic cells. Complexes of Argonaute proteins with small RNAs are core components of the RNA interference system and play a key role in silencing gene expression. It is becoming increasingly clear that several intracellular pathogens target host cell RNA interference pathways to promote their survival. In this study, we investigated the potential role of host macrophage Argonautes in Leishmania pathogenesis. Western blot analysis showed that protein abundance of infected macrophage Argonaute 1 (Ago1) was selectively and significantly higher than that of non-infected control at 24 h post-infection, suggesting that Ago1 plays a role in pathogenicity. In fact, siRNA-mediated downregulation of Ago1 enhanced Leishmania clearance from infected host cells, linking macrophage Ago1 to Leishmania virulence. To investigate the mechanisms of host Ago1 in Leishmania pathogenesis, a stable isotope labeling by amino acids in cell culture (SILAC)-based whole proteome approach was employed, which showed that expression of several previously reported Leishmania pathogenesis-related proteins were dependent on the level of macrophage Ago1. Moreover, the proteomic-based detailed biochemical analysis showed that Leishmania modulated host RNA-induced silencing complex (RISC) composition during infection, strongly suggesting macrophage RISC targeting. Strikingly, Leishmania proteins were detected as part of host RISC in infected cells. Together, our results demonstrate that Leishmania targets host RNA interference machinery to promote its survival inside the host macrophage.
2024-01-26 | PXD037042 | Pride
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