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Hairpin RNA induces secondary small interfering RNA synthesis and silencing in trans in fission yeast

ABSTRACT: 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.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE20330 | GEO | 2010/02/15

SECONDARY ACCESSION(S): PRJNA125397

REPOSITORIES: GEO

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Hairpin RNA induces secondary small interfering RNA synthesis and silencing in trans in fission yeast

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

Hairpin RNA induces secondary small interfering RNA synthesis and silencing in trans in fission yeast.

Project description:RNA interference (RNAi) is widespread in eukaryotes and regulates gene expression transcriptionally or post-transcriptionally. In fission yeast, RNAi is tightly coupled to template transcription and chromatin modifications that establish heterochromatin in cis. Exogenous double-stranded RNA (dsRNA) triggers seem to induce heterochromatin formation in trans only when certain silencing proteins are overexpressed. Here, we show that green fluorescent protein (GFP) hairpin dsRNA allows production of high levels of Argonaute-associated small interfering RNAs (siRNAs), which can induce heterochromatin formation at a remote locus. This silencing does not require any manipulation apart from hairpin expression. In cells expressing a ura4(+)-GFP fusion gene, production of GFP siRNAs causes the appearance of ura4 siRNAs from the target gene. Production of these secondary siRNAs depends on RNA-dependent RNA polymerase Rdp1 (RDRP(Rdp1)) function and other RNAi pathway components. This demonstrates that transitivity occurs in fission yeast and implies that RDRP(Rdp1) can synthesize RNA from targeted RNA templates in vivo, generating siRNAs not homologous to the hairpin.

| S-EPMC2828748 | biostudies-literature

Effects of RNAi protein overexpression in mutant cells on H3K9 dimethylation in fission yeast cells

Project description:We test the effects of overexpressing RNAi proteins Dcr1, Rdp1, or Ago1 in wildtype, dcr1∆, ago1∆, or rdp1∆ cells. We find that overexpression does not generally affect H3K9me2 at euchromatic loci, but there are varying effects in H3K9me2 on centromeric heterochromatin.

2013-12-26 | GSE52444 | GEO

Effects of RNAi protein overexpression in mutant cells on H3K9 dimethylation in fission yeast cells

Project description:We test the effects of overexpressing RNAi proteins Dcr1, Rdp1, or Ago1 in wildtype, dcr1M-bM-^HM-^F, ago1M-bM-^HM-^F, or rdp1M-bM-^HM-^F cells. We find that overexpression does not generally affect H3K9me2 at euchromatic loci, but there are varying effects in H3K9me2 on centromeric heterochromatin. Examination of genome-wide enrichment in H3K9 dimethylation in fission yeast haploid cells

2013-12-26 | E-GEOD-52444 | biostudies-arrayexpress

FIERY1 promotes microRNA accumulation by suppressing rRNA-derived small interfering RNAs in Arabidopsis [RNA-seq]

Project description:Plant microRNAs (miRNAs) associate with ARGONAUTE1 (AGO1) to direct post-transcriptional gene silencing and regulate numerous biological processes. Although AGO1 predominantly binds miRNAs in vivo, AGO1 also associates with endogenous small interfering RNAs (siRNAs). It is unclear whether the miRNA/siRNA balance affects miRNA activities. Here we report that FIERY1 (FRY1), which is involved in 5’-3’ RNA degradation, regulates miRNA abundance and function by suppressing the biogenesis of ribosomal RNA-derived siRNAs (risiRNAs). In mutants of FRY1 and the nuclear 5’-3’ exonuclease genes XRN2 and XRN3, we found that a large number of 21-nt risiRNAs were generated through an endogenous siRNA biogenesis pathway. The production of risiRNAs correlated with pre-rRNA processing defects in these mutants. We also show that these risiRNAs were loaded into AGO1, causing reduced loading of miRNAs. This study reveals a previously unknown link between rRNA processing and miRNA accumulation.

2019-10-08 | GSE133411 | GEO

FIERY1 promotes microRNA accumulation by suppressing rRNA-derived small interfering RNAs in Arabidopsis [miRNA-seq]

2019-10-08 | GSE126482 | GEO

Schizosaccharomyces pombe

Project description:Generation of Rdp1-independent primary siRNAs

| PRJNA229166 | ENA

Arabidopsis AGO1 N-terminal Poly-Q domain promotes phase separation and association with stress granules during heat stress

Project description:In Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing. Nuclear AGO1 is loaded with miRNAs and exported to the cytosol where it associates to the rough ER to conduct miRNA-mediated translational repression, mRNA cleavage and biogenesis of phased siRNAs. These latter, as well as other cytosolic siRNAs, are loaded into cytosolic AGO1, but in which compartment this happens is not known. Moreover, the effect of stress on AGO1 localization is still unclear. Here, we show that heat stress (HS) promotes AGO1 protein accumulation, which co-localize with components of the siRNA bodies and of stress granules (SGs). AGO1 does not need SGS3, a key component of siRNA bodies, to efficiently form condensates during HS. Instead, we found that the still poorly characterized N-terminal Poly-Q domain of AGO1, which contains a prion-like domain, is sufficient to undergo phase separation. Moreover, an exposure of 1 hour to HS only moderately affected AGO1 loading by miRNAs and target cleavage, suggesting that its localization in condensates protects AGO1 rather than promote its activity in reprograming gene expressing during stress. Collectively, our work shed new light on the impact of high temperature on a main effector of RNA silencing in plants.

2024-06-22 | PXD048594 | Pride

Generation of Rdp1-independent primary siRNAs

Project description:We tested whether we could increase the population of Rdp1-independent primary siRNAs in fission yeast by overexpressing the Dcr1 ribonuclease. We found that in addition to generation of centromeric siRNAs, which was expected, Dcr1 overexpression also resulted in generation of genome-wide primary siRNAs, mapping to many open reading frames.

2013-12-26 | GSE52532 | GEO

A non-Dicer RNase III and four other novel factors required for RNAi-mediated transposon suppression in the human pathogenic yeast C. neoformans

Project description:The human pathogenic yeast Cryptococcus neoformans silences transposable elements using endo-siRNAs and an Argonaute, Ago1. Endo-siRNAs production requires the RNA-dependent RNA polymerase, Rdp1, and two partially redundant Dicer enzymes, Dcr1 and Dcr2, but is independent of histone H3 lysine 9 methylation. We describe here an insertional mutagenesis screen for factors required to suppress the mobilization of the C. neoformans HARBINGER family DNA transposon HAR1. Validation experiments uncovered five novel genes (RDE1-5) required for HAR1 suppression and global production of suppressive endo-siRNAs. The RDE genes impact global siRNA levels and transposon mobilization but not global transcript levels, suggesting the endo-siRNAs do not act by impacting target transcript synthesis or turnover. RDE3 encodes a non-Dicer RNase III related to S. cerevisiae Rnt1, RDE4 encodes a predicted terminal nucleotidyltransferase, while RDE5 has no strongly predicted encoded domains. Affinity purification-mass spectrometry studies reveal that Rde3 and Rde5 are physically associated. RDE1 encodes a G-patch protein homologous to the S. cerevisiae Sqs1/Pfa1, a nucleolar protein that directly activates the essential helicase Prp43 during rRNA biogenesis. Rde1 copurifies Rde2, another novel protein obtained in the screen, as well as Ago1, a homolog of Prp43, and numerous predicted nucleolar proteins. We also describe the isolation of conditional alleles of PRP43, which are defective in RNAi. This work reveals unanticipated requirements for a non-Dicer RNase III and presumptive nucleolar factors for endo-siRNA biogenesis and transposon mobilization suppression in C. neoformans.

2020-07-13 | GSE128009 | GEO

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