Project description:C.elegans small RNAs from HA::ALG-1, HA::ALG-2 and HA::RDE-1 IP and rde-1 mutants Small RNAs were cloned from transgenic or mutant C. elegans adults. Sequencing was performed using 454 and Illumina platforms.
Project description:RNA interference (RNAi) is a post-transcriptional silencing process, triggered by double-stranded RNA (dsRNA), leading to the destabilization of homologous mRNAs. A distinction has been made between endogenous RNAi-related pathways and the exogenous RNAi pathway, the latter being essential for the experimental use of RNAi. Previous studies have shown that, in Caenorhabditis elegans, a complex containing the enzymes Dicer and the Argonaute RDE-1 process dsRNA. Dicer is responsible for cleaving dsRNA into short interfering RNAs (siRNAs) while RDE-1 acts as the siRNA acceptor. RDE-1 then guides a multi-protein complex to homologous targets to trigger mRNA destabilization. However, endogenous role(s) for RDE-1, if any, have remained unexplored. We here show that RDE-1 functions as a scavenger protein, taking up small RNA molecules from many different sources, including the microRNA (miRNA) pathway. This is in striking contrast to Argonaute proteins functioning directly in the miRNA pathway, ALG-1 and ALG-2: these proteins exclusively bind miRNAs. While playing no significant role in the biogenesis of the main pool of miRNAs, RDE-1 binds endogenous miRNAs and triggers RdRP activity on at least one perfectly matching, endogenous miRNA target. The resulting secondary siRNAs are taken up by a set of Argonaute proteins known to act as siRNA acceptors in exogenous RNAi, resulting in strong mRNA destabilization. Our results show that RDE-1 in an endogenous setting is actively screening the transcriptome using many different small RNAs, including miRNAs, as a guide, with implications for the evolution of transcripts with a potential to be recognized by Dicer.
Project description:We aimed to characterize the cell-type specific loading patterns of miRNAs in c.elegans. We expressed cell-type specific HA-epitope tagged versions of Argonaute-like 1 (ALG-1) and ALG-2 from three major tissue types i.e. intestine, nervous system and body wall mucle. We found that most miRNAs display highly cell-type specific loading patterns. ALG-1 is more ubiquitously loaded whereas ALG-2 is eniriched for miRNA loading within the nervous system. Addtionally we show that there is flexibility in ALG loading which changes during the aging process.
Project description:Investigated the specific miRNAs that are assocaited with ALG-1 and ALG-2 in adult (D5) C. elegans. ALG-1 and ALG-2 were IP-ed from D5 collected C.elegans, RNA was isolated and used for small RNA sequencing. Two biological replicates were used.
Project description:Expession data from L1-L2 stage nematodes (C. elegans), wild type and four mutants (alg-1, zfp-1, rde-4, lin-35). In C. elegans, a vast number of endogenous short RNAs corresponding to thousands of genes have been discovered recently. This finding suggests that these short interfering RNAs may contribute to regulation of many developmental and other signaling pathways in addition to silencing viruses and transposons. Based on this microarray analysis of gene expression in RNA interference (RNAi)-related mutants rde-4, zfp-1 and alg-1 and the Retinoblastoma (Rb) mutant lin-35, we found that a component of Dicer complex RDE-4 and a chromatin-related zinc finger protein ZFP-1, not implicated previously in endogenous RNAi, regulate overlapping sets of genes. Notably, genes a) upregulated in the rde-4 and zfp-1 mutants and b) upregulated in the lin-35(Rb) mutant, but not the downregulated genes are highly represented in the set of genes with corresponding endogenous short interfering RNAs (endo-siRNAs). Keywords: wildtype-mutant comparison, RNAi, Rb, endo-siRNA
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.
Project description:We report the the 'translatome' of the nervous system in c.elegans by immunoprecipitating FLAG- epitope tagged RPL-18 expressed under a nervous system specific promoter. In addition we compared this to the nervous system specific 'translatome' in an alg-2 mutant background. As such we were able to determine the specifc effects of ALG-2 on the nervous sytem gene expression landscape.
Project description:In order to examine the misregulation of genes in loss of function alg-1 and alg-2 versus WT, RNA-seq was performed on D5 C.elegans in WT(N2), alg-1(gk214), alg-2(ok304)
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.