Messenger RNA Decay of Immediate Early Genes

描述（由申请人提供）：RNA周转在控制细胞质中mRNA的命运中起关键作用。在哺乳动物细胞中，去腺苷酸化是胞质mRNA降解的主要触发因素，但对其潜在机制及其调控知之甚少。在这个建议中，我们专注于细胞质去腺苷化控制的分子和生化机制。将利用组合策略来剖析mRNA周转的机制步骤，包括1）“表观遗传”方法，例如RNAi敲低和通过多聚（A）核酸酶和其他假定参与酶的催化位点突变体的显性负效应; 2）用于研究蛋白质相互作用的各种方法;和3）用于监测mRNA衰变动力学的两种转录脉冲方法。我们首先集中在两个顺式作用元件的c-fos转录，AU丰富的元素和主要的编码区决定簇，和他们的同源结合蛋白，以了解他们如何介导的mRNA衰变触发初始快速deadenylation步骤，并解决方向性衰变的mRNA体。负责缺省/全局（即，整个poly（A）* mRNA群体）和顺式作用元件介导的去腺苷酸化将通过单独和组合地系统敲低不同类别的poly（A）核酸酶来鉴定。还将评价响应于生理变化的去腺苷化的改变。我们假设主要的胞质多聚腺苷酸结合蛋白PABP 1在去腺苷酸化中起关键作用，并且多聚腺苷酸核酸酶直接或间接与PABP 1/多聚腺苷酸尾复合物的缔合是靶向mRNA的多聚腺苷酸尾以缩短的关键步骤。我们提出的实验将有助于阐明哺乳动物细胞中选择性和差异性mRNA降解的基本原理。

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.