Messenger RNA Decay of Immediate Early Genes

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DESCRIPTION (provided by applicant): RNA turnover plays a critical role in controlling the fate of mRNA in the cytoplasm. In mammalian cells, deadenylation is the major trigger of cytoplasmic mRNA degradation, yet little is known about the underlying mechanism and its regulation. In this proposal, we focus on the molecular and biochemical mechanisms for control of cytoplasmic deadenylation. A combined strategy will be exploited to dissect the mechanistic steps of mRNA turnover, including 1)"epigenetic" approaches such as RNAi knock-down and dominant-negative effect by catalytic site mutants of poly(A) nuclease and other putative participating enzymes; 2) various methodologies for investigating protein-interactions; and 3) two transcriptional pulsing approaches for monitoring mRNA decay kinetics. We first concentrate on two cis-acting elements in the c-fos transcript, the AU-rich element and the major coding-region determinant, and their cognate binding proteins to learn how they mediate mRNA decay by triggering the initial rapid deadenylation step, and also to address the directionality of decay of the mRNA body. Poly(A) nucleases responsible for default/global (i.e., entire poly(A)* mRNA population) and cis-acting element-mediated deadenylation will be identified by systematic knock-down of different classes of poly(A) nuclease individually and in combination. Alterations of deadenylation in response to physiological changes will be evaluated as well. We hypothesize that the major cytoplasmic poly(A)-binding protein, PABP1, plays a key role in deadenylation and that association of poly(A) nuclease(s), either directly or indirectly, with the PABP1/poly(A) tail complex is a critical step in targeting the poly(A) tail of an mRNA for shortening. Our proposed experiments will help elucidate fundamental principles that govern selective and differential mRNA degradation in mammalian cells.