Factors affecting mRNA deadenylation

描述(申请人提供)：这项研究的目的是阐明控制信使核糖核酸降解的机制。酵母中信使核糖核酸降解的主要途径包括最初的聚(A)去烯化，然后是5‘末端去甲基化，以及5’-3‘核糖核酸酶的消化。不同mRNA的去烯化速率有很大差异，对RNA的降解速率也有相应的影响。酵母中主要的胞质去烯基酶是CCR4-NOT复合体，而CCR4的S去烯化能力取决于mRNP结构的状态。MRNP由与Poly(A)尾部结合的Poly(A)结合蛋白(PAB1)、连接mRNA 5‘端和RNA 3’端的翻译起始因子(TIF)以及其他与mRNA的3‘UTR结合的因子如PUF3组成。PAB1-mRNP在调节去烯基化中起着关键作用。富含脯氨酸(P)和RRM1结构域是CCR4去烯化和介导PAB1自结合所必需的；P结构域似乎也影响PAB1与Poly(A)的S脱落率。PAB1的自结合反过来又抑制了PAB1与Poly(A)的结合能力，这表明影响PAB1的S关闭速率和/或自结合的因素将控制CCR4的作用。我们还建立了TIF缺陷依赖于PAB1的这两个结构域来加速去烯化作用，而TIF eIF4G与RRM1和P结构域相联系。此外，我们还发现，UPF1蛋白可以促进无义介导的死烯基化，它特异性地与PAB1的RRM1结合，是该结构域加速CCR4作用所必需的。同样，PUF3依赖于PAB1的RRM1结构域加速去烯化。在这个提案中，我们将讨论PAB1是如何抑制死烯基化的，以及哪些因素参与了将PAB1从mRNA中移除以允许CCR4功能的因素。RRM1 PAB1突变对PAB1脱落率的影响将被确定，并与它们对体内CCR4去烯化和PAB1自结合的影响相关。将开发体外系统来检测RRM1突变对CCR4去烯化的影响，并将使用分析超速离心法研究PAB1的自结合。将评估PUF3/UPF1/TIFs在控制PAB1与聚(A)尾部的相互作用、对PAB1自结合以及在体外对CCR4去烯化的影响。 与公共健康相关：这项建议通过研究蛋白质表达是如何控制的，与公共健康相关。对调节蛋白质合成的时间和程度的因素的表征将阐明蛋白质产生异常导致特定疾病状态的过程。

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.