The overall goal of this proposal is to understand the mechanism of how microRNA and microRNA associated proteins interact with the ribosome to inhibit protein synthesis. It is through these interactions that gene expression is regulated. Athough microRNA research is a brand new field, current data has shown that these small biomolecules are linked to many critical cellular processes such as cellular differentiation, growth and proliferation; all of which, if not tightly controlled, can lead to diseases and cancer. The specific goal of this proposal is to determine the stage at which translation is repressed by miRNAs through developing an in vitro assay using rabbit reticulocyte lysate, a robust translational system. Previous studies have been performed in vivo and results obtained regarding the stage at which microRNA represses translation are conflicting. A biochemical approach will be taken, such that protein fractionation, protein purification, and target mRNA will be assembled in rabbit reticulocyte lysate in order to recapitulate the mechanism in vitro under tightly controlled conditions. Experiments to be conducted are in vitro translation and toeprinting assays, and results will be analyzed through PAGE, autoradiography, and polysome profile. In humans, the process of protein biosynthesis is regulated by microRNAs during many stages of development. Defects in this regulation may contribute significantly to the onset of many diseases, including cancer. Understanding the molecular mechanism of microRNA function will greatly aid in future development of therapies for diseases affecting development and leading to cancer. ? ? ?
