Mechanisms of microRNA mediated gene silencing in C. elegans

Abstract

Mechanisms of microRNA mediated gene silencing in C. elegans Xavier Ding Dissertation abstract microRNAs (miRNAs) are a large family of small non-coding RNAs, which post-transcriptionally repress numerous genes; a type of regulation which is important for countless physiological processes of multicellular organisms in health and disease. miRNAs are genomically encoded and transcribed as long precursors, which undergo a refined and tightly regulated maturation process giving rise to ~22 nucleotide-long RNAs. These small RNAs function as part of an RNA-protein complex termed miRNA induced silencing complex (miRISC). miRNAs, and by extension miRISCs, typically bind partially complementary elements in the 3' untranslated region (UTR) of target messenger RNAs (mRNAs), which become consequently repressed. A large set of studies clearly indicates that miRISC-mediated repression is achieved in metazoans by a variable combination of target mRNA degradation and translational repression. However, the phase of translation that is inhibited is a controversial subject. Various models have been proposed, based mostly on the study of artificial target reporters, which support either an initiation or a post-initiation block model. Argonaute proteins are core component of the miRISC and directly bind miRNAs. The GW182 protein, another miRISC component, has recently emerged as an essential mediator of miRNA-mediated repression action. However, its precise molecular function is still unclear. The work presented here aimed at understanding the in vivo mechanistic aspects of miRNA-mediated repression in more details, using the nematode Caenorhabditis elegans as a model organism and focusing on the well characterized let-7 miRNA. Initial results from a large-scale genetic screen revealed a strong genetic interaction between let-7 and various translation initiation factors, leading me to propose that C. elegans miRNAs inhibit the initiation of translation on their target mRNAs. Additional genetic experiments uncovered an unsuspected widespread genetic interaction between let-7 and the translation machinery and suggested that let-7 might function by inhibiting the eukaryotic translation initiation factor 3 (eIF3) activity. Biochemical experiments demonstrated that a large set of C. elegans miRNA targets are translationally repressed at the initiation step, sometimes in combination with mRNA degradation, and that the C. elegans GW182 homologs AIN-1 and AIN-2 are essential for these mechanisms. Additional unpublished data revealed that translational repression is specifically mediated by AIN-1, whereas depletion of both AIN-1 and AIN-2 is necessary to prevent miRNA target degradation. Collectively, these results show that C. elegans miRNAs employ at least two mechanisms in vivo, i.e. target degradation and inhibition of translation initiation, which are likely to be independent pathways.