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）：本研究的目标是阐明控制 mRNA 降解的机制。酵母中 mRNA 降解的主要途径涉及最初的聚 (A) 脱腺苷化，然后是 5' 末端脱帽和 5'-3' RNase 消化。不同 mRNA 的去腺苷酸化率差异很大，并对 RNA 的降解率产生相应的影响。酵母中主要的细胞质去腺苷酸酶是 CCR4-NOT 复合物，CCR4 去腺苷酸化的能力取决于 mRNP 结构的状态。 mRNP由与poly(A)尾结合的poly(A)结合蛋白(PAB1)、连接mRNA 5'端和RNA 3'端的翻译起始因子(TIF)和其他因子组成，例如 PUF3，它与 mRNA 的 3' UTR 结合。 PAB1-mRNP 在调节去腺苷酸化中发挥着关键作用。富含脯氨酸 (P) 和 RRM1 结构域是 CCR4 去腺苷化和介导 PAB1 自关联所必需的； P 结构域似乎也会影响 PAB1 与 Poly(A) 的解离速率。 PAB1 自缔合反过来又抑制 PAB1 结合 Poly(A) 的能力，表明影响 PAB1 解离速率和/或自缔合的因素将控制 CCR4 的作用。我们还确定，TIF 缺陷加速依赖于 PAB1 的这两个结构域的去腺苷化，并且 TIF、eIF4G 与 RRM1 和 P 结构域接触。此外，我们发现加速无义介导的去腺苷化的 UPF1 蛋白与 PAB1 的 RRM1 特异性结合，并且是该结构域加速 CCR4 作用所必需的。同样，PUF3 加速依赖于 PAB1 的 RRM1 结构域的去腺苷化。在本提案中，我们将讨论 PAB1 如何抑制去腺苷化，以及哪些因素参与从 mRNA 中去除 PAB1 以允许 CCR4 发挥功能。将确定 RRM1 PAB1 突变对 PAB1 解离速率的影响，并将其与它们对 CCR4 体内脱腺苷化和 PAB1 自关联的影响相关联。将开发体外系统来检查 RRM1 突变对 CCR4 去腺苷化的影响，并使用分析超速离心来研究 PAB1 自关联。将评估 PUF3/UPF1/TIF 对控制 PAB1 与 Poly(A) 尾相互作用、PAB1 自缔合以及体外 CCR4 去腺苷化的影响。公共健康相关性：该提案通过研究蛋白质表达的控制方式与公共健康相关。调节蛋白质合成时间和程度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。