RNA helicases, the largest class of enzymes in eukaryotic RNA metabolism, are highly conserved and essential players in almost all cellular RNA transactions. Mutations and deregulation of numerous RNA helicases have been linked to disease states including cancer, viral infections, and neurodegenerative disorders. Despite the pivotal biological roles of these enzymes, it is not known how the vast majority of RNA helicases exerts physiological reactions at the molecular level. Two major problems prevent clear definitions of biochemical mechanisms for physiological reactions of RNA helicases. First, it is not well understood how other proteins and co-factors affect biochemical functions of RNA helicases. Second, it is not known where almost all RNA helicases bind their biological substrates. The proteins, most of which are not inherently sequence or structure specific, thus cannot be physically linked to their substrates. In this proposal, we directly address both of these longstanding problems in the RNA helicase field. We focus on the DEAD-box RNA helicase Ded1p from Saccharomyces cerevisiae and its function in translation initiation.
In Aim 1 we will determine how biochemical characteristics of Ded1p and the interaction with the co-factor eIF4G1 affect Ded1p's function in translation initiation, using a combination of molecular biological and quantitative biochemical and biophysical approaches.
In Aim 2 we will define physiological RNA binding sites of Ded1p, using a systematic approach based on crosslinking and next generation sequencing. We plan to further analyze the functional significance of Ded1p binding to its cognate sites on mRNAs using molecular biology and biochemical approaches. We expect our studies to provide fundamental new insight into molecular functions of RNA helicases.
(no changes from the original application) Defects in RNA metabolism have been linked to many diseases including cancer, viral infections, and neurodegenerative disorders. To examine the molecular basis for these diseases and to guide the development of potential therapeutic agents, we propose to delineate critical aspects of the molecular function for RNA helicases, the largest class of enzymes in eukaryotic RNA metabolism.
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