MicroRNA–Directed siRNA Biogenesis in Caenorhabditis elegans

Abstract

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.

Age-associated changes in expression of small, noncoding RNAs, including microRNAs, in C. elegans

“…A third member, RDE-1, is involved in the double strand RNA-induced siRNA pathway (Tabara et al 1999). Consistent with the previous report (Corrêa et al 2010), most of the reads bound to these Argonaute proteins were found to be miRNAs. Notably, we found that the miRNA reads corresponding to star strands were significantly enriched in the RDE-1 ChIP-Seq library compared to those in ALG-1 or ALG-2 (Supplemental Fig.…”

“…Additionally, the accumulation of star miRNAs as well as mature ones was reduced in rde-1 mutants compared to wild-type animals (Supplemental Fig. S7B; Supplemental Table S7) (sequence data were obtained from GEO, accession: GSE20649) (Corrêa et al 2010). A similar reduction of miRNA star level in rde-1 mutants was also observed in another library obtained from the GEO, GSE19414 (Gent et al 2010) (data not shown).…”

“…The names of gene pathways shown in black represent genes necessary for the normal lifespan, suggesting that these pathways are involved in aging. Detailed results are available in Supplemental Tables S5 and S6. miRNAs in ChIP-Seq (chromatin immunoprecipitation followed by deepsequencing) libraries made for Argonaute proteins, ALG-1, ALG-2, and RDE-1 (Corrêa et al 2010) (sequence data were obtained from the Genome Expression Omnibus [GEO], accession: GSE20649). In addition to ALG-1, another Argonaute protein, ALG-2, is thought to act redundantly with ALG-1 to support it in miRNA processing (Tops et al 2006).…”

Competition between virus-derived and endogenous small RNAs regulates gene expression inCaenorhabditis elegans

“…Again in agreement with previous work (Corrêa et al 2010), we did not observe a direct correlation between expression level of genes in rde-1 mutants and reduced antisense small RNA reads. However, 5% of the genes showing robustly reduced antisense small RNA reads in rde-1 mutants also showed >30% increased expression in rde-1 mutants relative to N2.…”

“…Although most miRNAs are associated with the Argonaute proteins ALG-1 and ALG-2, certain miRNAs have been shown to bind to RDE-1 (Corrêa et al 2010). However, in agreement with this study, we did not find a clear difference in overall read counts of these miRNAs in rde-1 mutants, nor in the expression of predicted target genes of these miRNAs upon infection of N2 (data not shown).…”