What is the mechanism whereby microRNAs cause repression of translation of their target mRNAs in mammalian systems?. (360G-Wellcome-079613_Z_06_Z)

Bioinformatic studies have indicated that the expression of ~30% of all human genes may be regulated by the combinatorial action of several hundred microRNAs, yet the mechanism of action of miRNAs remains obscure and quite controversial. The aim of this proposal is to resolve these controversies: whether miRNAs repress translation of the target mRNA rather than promoting mRNA degradation, and if so, whether they inhibit the initiation rather than the elongation step of mRNA translation. Specific ally, we will use HeLa cell transfection assays and HeLa cell-free extracts to: attempt to recapitulate miRNA-mediated repression in cell-free extracts, and exploit this to analyse the mechanism of repression; identify what proteins are associated with the repressed mRNAs, and use siRNA-mediated knock-down of these and other candidate proteins to test whether they are essential for miRNA-mediated repression. examine possible explanations for the discrepancy between some authors finding inhibition of initiation, while others find the repressed mRNA in polysomes; critically examine the reasons for the reported immunity of some viral IRESs to repression whether this is because they do not require eIF4E, or because no ribosome scanning is involved, or because they were tested as Appp-capped RNAs rather than m7Gppp-capped.