Molecular mechanisms of microRNA mediated regulation

Yale University, New Haven CT

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Abstract

DESCRIPTION (provided by applicant): Summary MicroRNAs are ~22nt small RNAs that regulate gene expression post transcriptionally. They have the potential to regulate a large fraction of the human genes, and have been implicated in multiple aspects of biology from embryonic development, to cellular physiology and disease. Despite their paramount importance in gene regulation, the mechanisms by which miRNAs regulate gene expression remains remain controversial. Three models for miRNA mediated repression have been proposed: repression of translation initiation, repression of translation elongation and mRNA deadenylation. This proposal aims to undertake a systematic analysis of the relative timing of the different events (aim 1, 2) and the molecular effect of disrupting mRNA deadenylation (aim 2) on miRNA mediated gene regulation using an in vivo system. In this proposal we use the zebrafish embryo as an in vivo system to understand the molecular effect of endogenous miRNAs in their targets by comparing wild type embryos with embryos mutants in the miRNA processing pathway. Using high throughput sequencing in this system we propose to i) undertake a temporal genome wide analysis of translation by measuring ribosome density in the mRNA using Ribosome foot- printing (Aim 1), ii) analyze the dynamics of deadenylation in the presence and the absence of endogenous miRNAs (Aim 2), and iii) analyze the effect of blocking the deadenylation machinery on miRNA mediated translational repression and mRNA decay (Aim 2). These experiments will allow us to determine the sequence of events, and the relative contribution of mRNA deadenylation and translational repression during miRNA-mediated gene regulation. MicroRNAs have been implicated in a wide range of developmental defects, neurological disorders, and human disease including tumor formation and metastasis. The results derived from this proposal will provide fundamental insights into the molecular machinery required for miRNAs mediated gene regulation and has the potential to reveal important components in the miRNA pathway that may be used as therapeutic targets to treat human diseases and cancer. PUBLIC HEALTH RELEVANCE: MicroRNAs constitute the tiniest genes in the genome, and have been implicated in human development, cancer and other human diseases. This proposal aims to understand the mechanism by which these microRNAs regulate other genes in the cell, what might help us develop specific ways to modulate their activity during human disease.