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）：这项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聚（A）deadenylation，然后进行5'末端脱甲和5'-3'RNase消化。不同mRNA的去甲基化速率发生了很大的变化，对RNA降解速率的相应影响。酵母中的主要细胞质denenylase是CCR4-不复合物，CCR4的降苯基酸酯能力取决于MRNP结构的状态。 MRNP由与聚（a）尾巴结合的poly（a）结合蛋白（PAB1）组成，将mRNA的5'端连接到RNA的3'末端的翻译起始因子（TIFS）以及其他因子，例如PUF3，例如与mRNA 3'UTR结合的PUF3。 PAB1-MRNP在调节deadenylation中起关键作用。富含脯氨酸的（P）和RRM1结构域是CCR4去苯基化并介导PAB1自我关联所必需的。 P结构域似乎还会影响Pol（a）的PAB1的OFF速率。 PAB1自我关联反过来抑制了PAB1结合聚（A）的能力，这表明影响PAB1的OFF率和/或自我关联的因素将控制CCR4的作用。我们还确定，TIF缺陷会加速deadenylation，取决于PAB1的这两个域，而TIF EIF4G与RRM1和P结构域建立了接触。此外，我们发现，加速无义介导的deadenylation的UPF1蛋白与PAB1的RRM1特别结合，该域要求加速CCR4作用。同样，PUF3加速了取决于PAB1的RRM1结构域。在此提案中，我们将解决PAB1如何抑制deadenylation以及从mRNA中去除PAB1以允许CCR4功能的哪些因素。 RRM1 PAB1突变对PAB1外率的影响将确定并与它们对CCR4体内deadenylation和PAB1自相关的影响相关。将开发体外系统来检查RRM1突变对CCR4 deadenylation的影响，并将使用分析性超速离心来研究PAB1自我关联。 PUF3/UPF1/TIFS对PAB1与Poly（A）尾巴的相互作用，PAB1自我关联以及在体外CCR4 Deadenylation的影响。公共卫生相关性：通过研究如何控制蛋白质表达，该提案与公共卫生有关。调节何时及在多大程度上合成蛋白质的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。