MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression of complementary mRNAs. They have diverse unique expression patterns and have been implicate in a number of biological processes. It has been shown that in many physiological and pathological conditions individual miRNAs are subjected to post-transcriptional regulation, both at the level of Drosha and/or Dicer processing. The host lab has recently found that hnRNP A1, a protein implicated in many aspects of RNA processing, act as an auxiliary factor for the Drosha-mediated processing of a microRNA precursor, pri-miR-18a. hnRNP A1 binds to the loop of this pri-miRNA and induces a relaxation at the stem creating a more favourable cleavage site for Drosha. Our first objective is to define the functional domains in hnRNP A1 that are required for miR-18a processing. Thus, we will determine whether RNA binding and/or the unwinding activities of hnRNP A1 are required for miR-18a processing. The host lab also found that approximately 14% of all pri-miRNAs have highly conserved loops suggesting that these conserved loops act as landing pads for trans-acting factors influencing miRNA processing. In order to address the question of how widespread is the contribution of hnRNP A1 and other auxiliary factors to pri-miRNA processing I propose to identify other microRNAs that require hnRNP A1 for their processing. Secondly, I plan to identify different auxiliary factors required for the processing of microRNAs containing a conserved terminal loop by affinity pull-down assays followed by peptide mass fingerprint analysis. In summary, experiments carried out in this proposal will reveal the level of complexity for the regulation of microRNA biogenesis and their contribution to physiological states and pathological conditions.
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