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Factors affecting mRNA deadenylation

影响 mRNA 去腺苷化的因素

基本信息

批准号：
8005169
负责人：
CLYDE L DENIS
金额：
$ 8.49万
依托单位：
UNIVERSITY OF NEW HAMPSHIRE
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-01-20 至 2011-12-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The goal of this research is to elucidate the mechanisms that govern mRNA degradation. The principal pathway for mRNA degradation in yeast involves an initial poly(A) deadenylation, followed by 5' end decapping, and 5'-3' RNase digestion. The rate of deadenylation of different mRNA varies considerably with corresponding effects on the rate of degradation of the RNA. The major cytoplasmic deadenylase in yeast is the CCR4-NOT complex, and CCR4's ability to deadenylate is dependent on the state of the mRNP structure. The mRNP consists of the poly(A) binding protein (PAB1) bound to the poly(A) tail, translation initiation factors (TIFs) linking the 5' end of the mRNA to the 3' end of the RNA, and other factors, such as PUF3, that bind to the 3' UTR of mRNA. The PAB1-mRNP plays a key role in regulating deadenylation. The proline-rich (P) and RRM1 domains are required for CCR4 deadenylation and mediate PAB1 self-association; the P domain also appears to affect PAB1's off rate with the poly(A). PAB1 self-association, in turn, inhibits the ability of PAB1 to bind poly(A), suggesting that factors affecting PAB1's off rate and/or self-association will control CCR4 action. We have also established that TIF defects accelerate deadenylation dependent on these two domains of PAB1, and the TIF, eIF4G, make contacts to the RRM1 and P domain. Moreover, we have found that the UPF1 protein, which accelerates nonsense-mediated deadenylation, binds specifically to RRM1 of PAB1 and is required by this domain to accelerate CCR4 action. Similarly, PUF3 accelerates deadenylation dependent on the RRM1 domain of PAB1. In this proposal, we will address how PAB1 inhibits deadenylation and what factors are involved in removing PAB1 from the mRNA to allow CCR4 function. The effects of RRM1 PAB1 mutations on the off-rate of PAB1 will be determined and correlated to their effects on CCR4 in vivo deadenylation and PAB1 self- association. In vitro systems will be developed to examine the effects of RRM1 mutations on CCR4 deadenylation, and analytical ultracentrifugation will be used to study PAB1 self-association. The effects of PUF3/UPF1/TIFs on controlling PAB1 interaction with the poly(A) tail, on PAB1 self-association, and on CCR4 deadenylation in vitro will be assessed. PUBLIC HEALTH RELEVANCE: This proposal is relevant to public health by studying how protein expression is controlled. The characterization of the factors that regulate when and to what extent proteins are synthesized will illuminate the processes by which aberrant protein production leads to particular disease states.

描述（由申请人提供）：本研究的目的是阐明控制mRNA降解的机制。酵母中mRNA降解的主要途径涉及初始的聚（A）去腺苷酸化，随后是5'端去帽和5'-3 'RNA酶消化。不同mRNA的去腺苷化速率随着对RNA降解速率的相应影响而显著变化。酵母中主要的细胞质去腺苷酶是CCR 4-NOT复合物，并且CCR 4的去腺苷化能力取决于mRNP结构的状态。mRNP由与poly（A）尾结合的poly（A）结合蛋白（PAB 1）、连接mRNA 5'末端与RNA 3'末端的翻译起始因子（TIF）以及其他因子（例如PUF 3）组成，这些因子与mRNA的3' UTR结合。PAB 1-mRNP在调节去腺苷化中起关键作用。富含脯氨酸（P）和RRM 1结构域是CCR 4去腺苷化和介导PAB 1自缔合所必需的; P结构域似乎也影响PAB 1与poly（A）的解离速率。PAB 1的自缔合反过来又抑制PAB 1结合poly（A）的能力，这表明影响PAB 1解离速率和/或自缔合的因素将控制CCR 4的作用。我们还确定TIF缺陷依赖于PAB 1的这两个结构域加速去腺苷化，并且TIF eIF 4G与RRM 1和P结构域接触。此外，我们发现，UPF 1蛋白，加速无义介导的deadenylation，特异性结合到PAB 1的RRM 1，并需要由这个域加速CCR 4的行动。类似地，PUF 3依赖于PAB 1的RRM 1结构域加速去腺苷化。在这个提议中，我们将讨论PAB 1如何抑制去腺苷化，以及哪些因素参与了从mRNA中去除PAB 1以允许CCR 4功能。将确定RRM 1 PAB 1突变对PAB 1解离速率的影响，并将其与其对CCR 4体内去腺苷化和PAB 1自缔合的影响相关联。将开发体外系统来检查RRM 1突变对CCR 4去腺苷化的影响，并将使用分析性超离心来研究PAB 1自缔合。将评估PUF 3/UPF 1/TIF对控制PAB 1与poly（A）尾的相互作用、对PAB 1自缔合和对CCR 4脱腺苷化的体外作用。公共卫生相关性：该提案通过研究如何控制蛋白质表达与公共卫生相关。对蛋白质合成时间和程度的调控因素的表征将阐明异常蛋白质产生导致特定疾病状态的过程。