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E-GEOD-59300 - A Ribonuclease Coordinates siRNA Amplification and mRNA Cleavage during RNAi

Status
Released on 29 January 2015, last updated on 12 March 2015
Organism
Caenorhabditis elegans
Samples (33)
Protocols (4)
Description
Effective silencing by RNA-interference (RNAi) depends on mechanisms that amplify and propagate the silencing signal. In some organisms, small-interfering (si) RNAs are amplified from target mRNAs by RNA-dependent RNA polymerase (RdRP). Both RdRP recruitment and mRNA silencing require Argonaute proteins, which are generally thought to degrade RNAi targets by directly cleaving them. However in C. elegans, the enzymatic activity of the primary Argonaute, RDE-1, is not required for silencing activity. We show that RDE-1 can instead recruit an endoribonuclease, RDE-8, to target RNA. RDE-8 can cleave RNA in vitro and is needed for the production of 3′ uridylated fragments of target mRNA in vivo. We also find that RDE-8 promotes RdRP activity, thereby ensuring amplification of siRNAs. Together, our findings suggest a model in which RDE-8 cleaves target mRNAs to mediate silencing, while generating 3’ uridylated mRNA fragments to serve as templates for the RdRP-directed amplification of the silencing signal. We examined the role of rde-8 in C. elegans small RNA biogenesis pathways, including endogenous and exogenous RNAi pathways. We performed 3' RACE seq from the sel-1 target mRNA and correlate with small RNAs from wild type, rde-8 and rde-8 transgenic strains after sel-1(RNAi) for different lengths of time.
Experiment type
RNA-seq of non coding RNA 
Contacts
MINSEQE
Exp. designProtocolsVariablesProcessedSeq. reads
Files
Investigation descriptionE-GEOD-59300.idf.txt
Sample and data relationshipE-GEOD-59300.sdrf.txt
Processed data (1)E-GEOD-59300.processed.1.zip
Links