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RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe [Illumina Genome Analyzer]

ABSTRACT: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its role in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe.

ORGANISM(S): Schizosaccharomyces pombe 972h-

PROVIDER: GSE54194 | GEO | 2014/01/23

SECONDARY ACCESSION(S): PRJNA235985

REPOSITORIES: GEO

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RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe [Illumina Genome Analyzer]

Project description:RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its role in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe. Small RNA library from wild type S. pombe.

2014-01-23 | E-GEOD-54194 | biostudies-arrayexpress

RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe [Affymetrix]

2014-01-23 | GSE54190 | GEO

RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe [Affymetrix]

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DamID for Swi6, Rdp1 and Dcr1 in Schizosaccharomyces pombe

Project description:In the fission yeast Schizosaccharomyces pombe, the RNA interference (RNAi) pathway is required to generate small interfering RNAs (siRNAs) that mediate heterochromatic silencing of centromeric repeats. Here we demonstrate that RNAi also functions to repress genomic elements other than constitutive heterochromatin. Using DamID (DNA adenine methyltransferase identification) we show that Dcr1 and Rdp1 physically associate with some euchromatic genes, non-coding RNA (ncRNA) genes, and retrotransposon long terminal repeats (LTRs), and that this association is independent of the Clr4 histone methyltransferase. Physical association of RNAi with chromatin is sufficient to trigger a silencing response but not to assemble heterochromatin. The mode of silencing at the newly identified RNAi targets is consistent with a co-transcriptional gene silencing model as proposed earlier and functions with trace amounts of siRNAs. We anticipate that similar mechanisms could also be operational in other eukaryotes.

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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.

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Nuclear retention of fission yeast dicer is a prerequisite for RNAi-mediated heterochromatin assembly

Project description:RNAseIII ribonucleases act at the heart of RNA silencing pathways by processing precursor RNAs into mature microRNAs and siRNAs. In the fission yeast Schizosaccharomyces pombe, siRNAs are generated by the RNAseIII enzyme Dcr1 and are required for heterochromatin formation. In this study, we have analyzed the subcellular localization of Dcr1 and found that it accumulates in the nucleus and is enriched at the nuclear periphery. Nuclear accumulation of Dcr1 depends on a short motif which constrains nuclear export promoted by the double-stranded RNA binding domain of Dcr1. Absence of this motif renders Dcr1 mainly cytoplasmic and is accompanied by remarkable changes in gene expression and failure to assemble heterochromatin. Our findings suggest that dicer proteins are shuttling proteins and that the steady-state subcellular levels can be shifted towards either compartment. This has implications for the mechanism of RNAi-mediated heterochromatin assembly and the spatial organization of RNA silencing pathways in general. Small RNA libraries from total RNA isolations of wild-type, dcr1Delta and dcr1DeltaC33 cells and subjected to high-throughput sequencing.

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