Identification of conformational and dynamic states of pre-microRNA which could potentially be utilised for tumour suppression

MicroRNAs (miRNA) are key regulators of gene expression. Thus, it is not surprising that their deregulation leads to disease. The production of individual miRNA must therefore be strictly controlled. Posttranscriptional regulation of miRNA biogenesis is a multistep process that starts with long precursor RNA molecules which are then processed into mature miRNA (pri-miRNA >> pre-miRNA >> mature miRNA) and involves many protein factors (e.g. Drosha and Dicer). In this project we are interested in how the structure of pre-miRNA regulates its processing by Dicer. As a model RNA I will study pre-let-7f-2 from the let-7 miRNA family that acts as tumor suppressor in cells. My preliminary data show that pre-let-7f-2 adopts two distinct terminal loop structures, which makes it an ideal model for testing how conformational changes in the loop region influences Dicer processing. Combination of classical and recently developed methodologies of NMR spectroscopy will be used to obtain detailed information about RNA structure and dynamics at an atomic level of resolution. To refine determined conformational ensemble of structures based on NMR-derived restraints SAXS data will be used, which will provide the information about the global architecture of pre-let-7f-2. Furthermore, the effects of environmental stimuli on conformational equilibrium of the pre-let-7f-2 will be examined. By designing mutants that will lock individual conformations I will test their impact on efficiency of Dicer processing. Moreover, I will prepare RNAs with modified nucleotides in different positions in the loop. I expect that modifications will change processing efficiency and stability of RNAs compared to native pre-let-7f-2. These data will offer excellent starting points to design pre-micro RNA mimics which could be used as tumor suppressors. Due to the lack of high resolution RNA structures, this work will significantly contribute to the RNA structural database. This project will provide unprecedented information about the structure-function relationship in the biogenesis of miRNAs. Understanding the conformational dynamics happening in the loop of pre-let-7f-2 as a response to environmental stimuli or modifications will importantly contribute to our knowledge on structure-mediated regulation of pre-miRNA processing.