ABSTRACT: 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. sequencing of 20-30nt sRNAs from total RNA
Project description:We use small RNA sequencing to look for spreading of secondary and tertiary siRNAs along transcripts targeted by ectopic hairpin-derived primary siRNAs. We find that signals within the 3' UTR inhibit siRNA spreading. sequencing of 20-30nt sRNAs from Argonaute immunoprecipitation
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.
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. Examination of genome-wide enrichment in H3K9 dimethylation in fission yeast haploid cells
Project description:By surveying miRNA populations in each sex, we identified sets of miRNAs differentially expressed in male and female tissues across various stages of development. Small RNAs cloning of dissected male and female tissues from Drosophila melanogaster at various stages
Project description:Argonaute proteins (AGOs) are key nuclease effectors of RNA interference (RNAi) . 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 . 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
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. Overall design: Methylation levels in strains expressing Dam-Swi6, Dam-Rdp1 and Dam-Dcr1 were compared to those in a Dam only expressing strain. Three biological replicates for each experiment were performed.
Project description:We report that rice endosperm shows a specific hypomethylation of DNA in the maternal genome, preferring regions of high DNA accessibility. Maternally expressed imprinted genes are enriched for hypomethylation at putative promoter regions and transcriptional termini, and paternally expressed genes at promoters and gene bodies, mirroring our recent results in A. thaliana. However, unlike in A. thaliana, rice endosperm sRNA populations are dominated by specific strong sRNA-producing loci, and imprinted 24-nt sRNAs are expressed from both parental genomes and correlate with hypomethylation. Overlaps between imprinted sRNA loci and imprinted genes expressed from opposite alleles suggest that sRNAs may regulate genomic imprinting. Whereas sRNAs in seedling tissues primarily originate from small Class II (cut and paste) transposable elements, those in endosperm are much more uniformly derived, including sequences from other TE classes, as well as genic and intergenic regions. Our data indicate that the endosperm exhibits a unique pattern of sRNA expression and suggest that demethylation of maternal endosperm DNA is conserved in flowering plants. Examination of DNA methylation and small RNA expression in the seeds of two cultivars from the japonica subspecies of Oryza sativa L.
Project description:Diverse naturally-occurring small RNA species interact with Argonaute proteins to mediate sequence-specific regulation in animals. In addition to micro-RNAs (miRNAs), which collectively regulate thousands of target mRNAs, other endogenous small RNA species include the Piwi-associated piRNAs that are important for fertility and a less well-characterized class of small RNAs often referred to simply as endo-siRNAs. Here we have utilized deep-sequencing technology and C. elegans genetics to explore the biogenesis and function of endo-siRNAs. We describe conditional alleles of the dicer-related helicase, drh-3, that implicate DRH-3 in both the response to foreign dsRNA as well as the RNA-dependent RNA Polymerase (RdRP)-dependent biogenesis of a diverse class of endogenous small RNAs, termed 22G-RNAs. We show that 22G-RNAs are abundantly expressed in the germline and maternally inherited and are the products of at least two distinct 22G-RNA systems. One system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germline nuage-related structures termed P-granules. The WAGO 22G-RNA system silences transposons, pseudogenes and cryptic loci as well as a number of genes. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one of the multiple, distinct WAGO surveillance pathways. These findings broaden our understanding of the biogenesis and diversity of 22G-RNA species and suggest potential novel regulatory functions for these small RNAs. 18 samples examined. Small RNA libraries generated from: C. elegans animals with mutations in the WAGO pathway and a WAGO-1 immunopercipitate.
Project description:Small interfering RNAs (siRNAs) are known to be involved in both transposon silencing and centromere function, leading us to investigate the interplay between these two roles in the Schizosaccharomyces lineage. In S. pombe, the centromeric repeats produce dicer-dependent siRNAs that are required for maintenance of centromeric structure, function and transcriptional silencing via Argonaute-dependent heterochromatin formation13. However, transposons are silenced in S. pombe by RNAi-independent mechanisms and do not produce abundant siRNAs. To investigate whether centromere-directed siRNA production is conserved within the transposon-rich centromeres of S. japonicus, we isolated and sequenced small RNAs from log-phase S. japonicus cultures. The small RNAs have a modal size of 23 nucleotides and 94% map to transposons, both telomeric and centromeric. Isolation and computational analysis of small RNAs from wild-type S. japonicus