MicroRNAs (miRNAs) are small RNAs that play a pivotal role in post-transcriptional gene regulation. Most commonly in animals, these regulatory RNAs target mRNAs by imperfectly binding to complementary sites in 3'' untranslated regions (3''UTR), thereby affecting the translation of the targets, or reducing their stability. Despite the significant roles miRNAs play in various biological processes, the mechanistic details of how they regulate gene expression remain unclear. We present here the first in vitro translation system derived from C.elegans embryos that recapitulates cap- and polyA-dependent translation as well as miRNA-dependent silencing. Using luciferase reporters fused to an artificial 3''UTR containing sites complementary to the maternally contributed
mir-35 miRNA family, we observed up to 70% inhibition by
mir-35 over a 3-hour translation time-course. Upon addition of 2''-O-methyl oligonucleotide inhibitors for the endogenous
mir-35 family, translation of our reporters was restored. To understand how this inhibition occurred, we examined the fate of our reporter mRNAs. Inhibited mRNA reporters became fully deadenylated within the first 60 minutes of incubation with our cell-free extract, while a reporter bearing a mutation in the region complementary to the miRNA seed was not susceptible to inhibition or deadenylation. MiRNA-mediated deadenylation is independent of the translation of the targets as neither ApppN capping of the transcripts, nor the presence of the translation inhibitor cycloheximide inhibited deadenylation. We will describe our efforts to screen for endogenous miR-35 targets via deadenylation and translation inhibition assays. We will further define the genetic requirements for translation repression and processing of target mRNAs by testing extracts that are RNAi-depleted. Our findings indicate that deadenylation plays a prominent role in miRNA-mediated control of gene expression in the C.elegans embryo.