Project description:High throughput sequencing to derive function of cde-1 in endogenous RNAi in C. elegans Small RNAs were cloned from C. elegans adults, following removal of tri-phosphate groups from 5' end. Sequencing was performed using the Illumina 1G platform.
Project description:To genome-widely investigate functions of C. elegans argonautes (AGOs), we applied CRISPR/Cas9 technology to introduce an in-frame GFP::FLAG multiplex tag into the endogenous locus of C. elegans AGOs and performed RNA immunoprecipitation (IP) with anti-GFP or anti-FLAG antibody, followed by unique molecular identifier (UMI)-mediated cDNA library constructions and subjected to high-throughput sequencing. We obtained targets of C. elegans AGOs analyzed from small RNA reads of input and 19 AGO IP samples.
Project description:High-throughput sequencing of mixed-stage Caenorhabditis elegans small RNAs. total RNA, ~18-26nt RNAs isolated using PAGE, ligation to adapters requires 5' monophosphate and 3' OH Keywords: high-throughput 454 sequencing
Project description:RNA interference (RNAi) is a phylogenetically widespread gene silencing process triggered by doublestranded RNA (dsRNA). In plants and C. elegans, two distinct populations of small RNAs have been proposed to participate in RNAi : "Primary siRNAs" (derived from Dicer nuclease-mediated cleavage of the original trigger) and "Secondary siRNAs" (additional small RNAs whose synthesis requires an RNA-directed RNA polymerase [RdRP]). Analyzing small RNAs associated with ongoing RNAi in C. elegans, we found secondary siRNAs to comprise the vast majority. The bulk of secondary siRNAs exhibited structure and sequence indicative of a biosynthetic mode where each molecule derives from an independent de novo initiation by RdRP. Analysis of endogenous small RNAs indicated that a fraction derive from a biosynthetic mechanism that is similar to that of secondary siRNAs formed during RNAi, suggesting that small antisense transcripts derived from cellular mRNAs by RdRP activity may have key roles in cellular regulation. Keywords: C. elegans small RNA sequences from wild type animals fed on sel-1 dsRNA producing bacteria
Project description:RNA interference (RNAi) is a phylogenetically widespread gene silencing process triggered by doublestranded RNA (dsRNA). In plants and C. elegans, two distinct populations of small RNAs have been proposed to participate in RNAi : "Primary siRNAs" (derived from Dicer nuclease-mediated cleavage of the original trigger) and "Secondary siRNAs" (additional small RNAs whose synthesis requires an RNA-directed RNA polymerase [RdRP]). Analyzing small RNAs associated with ongoing RNAi in C. elegans, we found secondary siRNAs to comprise the vast majority. The bulk of secondary siRNAs exhibited structure and sequence indicative of a biosynthetic mode where each molecule derives from an independent de novo initiation by RdRP. Analysis of endogenous small RNAs indicated that a fraction derive from a biosynthetic mechanism that is similar to that of secondary siRNAs formed during RNAi, suggesting that small antisense transcripts derived from cellular mRNAs by RdRP activity may have key roles in cellular regulation. Keywords: C. elegans small RNA sequences from wild type animals fed on sel-1 dsRNA producing bacteria C. elegans small RNA sequences from wild type animals fed on sel-1 dsRNA producing bacteria
Project description:Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes that convert adenosine to inosine in double-stranded RNA (dsRNA). To evaluate effects of ADARs on small RNAs that derive from dsRNA precursors, we performed deep-sequencing, comparing small RNAs from wildtype and ADAR mutant C. elegans. While editing in small RNAs was rare, at least 40% of microRNAs had altered levels in at least one ADAR mutant strain, and miRNAs with significantly altered levels had mRNA targets with correspondingly affected levels. About 40% of siRNAs derived from endogenous genes (endo-siRNAs) also had altered levels in at least one mutant strain, including 63% of Dicer-dependent endo-siRNAs. The 26G class of endo-siRNAs was significantly affected by ADARs, and many altered 26G loci had intronic reads, and histone modifications associated with transcriptional silencing. Our data indicate ADARs, through both direct and indirect mechanisms, are important for maintaining wildtype levels of many small RNAs in C. elegans. Deep sequencing of small RNAs in wild-type (N2), adr-1 null, adr-2 null and adr-1;adr-2 null mixed stage C. elegans
Project description:RNAi, a gene-silencing pathway triggered by double-stranded RNA, is conserved in diverse eukaryotic species but has been lost in the model budding yeast, Saccharomyces cerevisiae. We report that RNAi is present in other budding-yeast species, including Saccharomyces castellii and Candida albicans. These species use noncanonical Dicer proteins to generate siRNAs, which mostly correspond to transposable elements and YM-BM-4 subtelomeric repeats. In S. castellii, RNAi mutants are viable but have excess YM-BM-4 mRNA levels. In S. cerevisiae, introducing Dicer and Argonaute of S. castellii restores RNAi, and the reconstituted pathway silences endogenous retrotransposons. These results identify a novel class of Dicer proteins, bring the tool of RNAi to the study of budding yeasts, and bring the tools of budding yeast to the study of RNAi. Employ high-throughput sequencing of endogenous small RNAs from the budding yeasts Saccharomyces castellii, Kluyveromyces polysporus, Candida albicans, Saccharomyces cerevisiae, and Saccharomyces bayanus.
Project description:From a forward genetic screen for C. elegans genes required for RNAi, we identified rde-10 and through proteomic analysis of RDE-10-interacting proteins, we identified a protein complex containing the new RNAi factor RDE-11, the known RNAi factors RSD-2 and ERGO-1, as well as other candidate RNAi factors. The newly identified RNAi defective genes rde-10 and rde-11 encode a novel protein and a RING-type zinc finger domain protein, respectively. Mutations in rde-10 and rde-11 genes cause dosage-sensitive RNAi deficiencies: these mutants are resistant to low dosage, but sensitive to high dosage of double-stranded RNAs. We assessed the roles of rde-10, rde-11, and the dosage-sensitive RNAi defective genes rsd-2, rsd-6 and haf-6 in both exogenous and endogenous small RNA pathways using high-throughput sequencing and qRT-PCR. These genes are required for the accumulation of secondary siRNAs in both exogenous and endogenous RNAi pathways. Small RNA analysis by deep sequencing in various wild type and mutant C. elegans strains.
Project description:The RNA interference (RNAi) pathway is found in most eukaryotic lineages but curiously is absent in others, including that of Saccharomyces cerevisiae. Here, we show that reconstituting RNAi in S. cerevisiae causes loss of a beneficial dsRNA virus, known as killer virus. Incompatibility between RNAi and killer viruses extends to other fungal species, in that RNAi is absent in all species known to possess dsRNA killer viruses, whereas killer viruses are absent in closely related species that retained RNAi. Thus, the advantage imparted by acquiring and retaining killer viruses explains the persistence of RNAi-deficient species during fungal evolution. Employ high-throughput sequencing of endogenous small RNAs from Saccharomyces cerevisiae wild-type and RNAi-reconstituted strains.