MicroRNAs (miRNAs) are an evolutionarily conserved class of short ( 22 nucleotide) RNAs known to play critical roles in a variety of cellular processes. MicroRNAs basepair primarily to sites of partial complementarity in the 3 untranslated region (UTR) of target messenger RNAs (mRNAs) and down-regulate gene expression through their association with Argonaute proteins in the RNA-induced Silencing Complex (RISC). The molecular mechanism through which RISC mediates post-transcriptional gene regulation has been the subject of many studies, which variously implicate translation initiation, elongation, mRNA deadenylation, or cellular compartmentalization as the root causes of this regulation. To elucidate the mechanisms of repression of gene expression, our lab has focused on how RISC recognizes target mRNAs.
One approach we have used to explore these mechanisms is to study miRNA binding sites in mRNAs. These binding sites are also called miRNA response elements (MREs). MREs are sequences in the 3 UTR of mRNAs and typically have a conserved stretch of 7 nucleotides that are able to base pair with the 5 region of corresponding miRNAs. The matching sequences in miRNAs are also conserved and are termed seed regions. Notably, many conserved MREs exhibit sequence conservation exclusively in regions that correspond to miRNA seeds while the remainder of the MRE sequence is diverse. Our work on seed sequences has shown that the seed is somewhat flexible within a miRNA, allowing an mRNA to be repressed following base pairing to either nucleotides 2-8 or 3-9 of a miRNA (Nahvi et al., RNA, 2009).
Another approach we have used to understand target recognition is to examine the structure of Argonaute proteins. We have characterized an allosteric relationship between the known binding site for the 5 end of a miRNA and a novel site which binds to the cap structure of mature mRNAs. We have used in vitro biochemistry and in vivo cell culture assays to probe the function and interaction of these two sites. Our experiments have indicated that binding at either site dramatically enhances binding at the other site, and that both sites must be intact for Argonaute to mediate translational repression (Djuranovic et al., NSMB, 2010). While we have called the novel binding site a cap-binding site, it is possible that this site could alternatively or conditionally bind other cap-like moieties such as free nucleotides in the cells (e.g., as a sensor of cell cycle stages). We are currently exploring the importance of other players, such as GW182, in translational repression, as well as clarifying the contribution of particular Argonaute domains.
Finally, we have used a Drosophila S2 cell-based expression system to investigate the kinetics of miRNA mediated translational repression and decay. For many years it was known the miRNAs could silence mRNAs by two pathways: 1) Repressing translation of the mRNA or 2) Degrading the mRNA via the RISC complex pathway. Our work with this reporter-based system has indicated that translational repression kinetically precedes mRNA deadenylation and decay during miRNA mediated silencing (Djuranovic et al., Science 2012).