Characterization of the mammalian mRNA-3'-end processing complex

 描述（由申请人提供）： 项目概述：本项目的长期目标是详细了解哺乳动物mRNA 3'加工及其调控的机制。mRNA 3 '端的形成，通常涉及内切核酸裂解，然后是多聚腺苷酸化，是真核基因表达的重要步骤，它显著影响RNA代谢的许多方面，包括mRNA的稳定性和翻译。此外，大多数真核基因通过交替多聚腺苷酸化（阿帕）产生具有不同3'末端的多种mRNA同种型。近年来的研究表明，阿帕在发育过程中受到高度调控，并在转录后基因调控中发挥重要作用。异常的阿帕模式与从癌症到神经肌肉疾病的多种疾病有关。因此，mRNA 3'加工领域的一个中心问题是如何识别多聚腺苷酸化位点（PAS）以及如何调节PAS选择。大多数哺乳动物PAS含有AAUAAA六聚体和富含U/UG的下游元件（DSE）。根据该领域目前的模型，这些关键的顺式元件分别被CPSF复合物的CPSF 160和CstF复合物的CstF 64特异性识别。然而，我们发表的和初步的数据在几个关键方面挑战了这个模型：1）我们最近表明CPSF亚基CPSF 30和Wdr 33，而不是通常认为的CPSF 160，直接与AAUAAA结合; 2）我们提供了证据，证明CPSF-RNA相互作用特异性的维持需要一个新的校正因子。（见初步数据）; 3）我们已经证明，一般的3'加工因子CstF 64实际上仅结合哺乳动物PAS的一个子集，并且我们提供了证据，即识别具有不同序列特征的PAS需要不同的RNA结合蛋白（见初步数据）。这些观察结果不仅显著改变了PAS识别的当前模型，而且还揭示了mRNA 3'加工的复杂性比以前认识到的要大得多。在这里，我们建议更好地定义哺乳动物PAS识别的分子机制，通过深入表征所涉及的关键蛋白质-RNA相互作用。完成拟议的研究将提供开创性的见解哺乳动物mRNA 3'加工及其调控的基本机制。由于异常的PAS选择与广泛的人类疾病有关，因此更好地了解PAS识别的机制可能为开发这些疾病的新疗法提供基础。

 DESCRIPTION (provided by applicant): Project Summary: The long-term goal of this proposal is to understand, in detail, the mechanisms of mammalian mRNA 3' processing and its regulation. mRNA 3'-end formation, typically involving an endonucleolytic cleavage followed by polyadenylation, is an essential step of eukaryotic gene expression and it significantly impacts many aspects of RNA metabolism, including mRNA stability and translation. In addition, the majority of eukaryotic genes produce multiple mRNA isoforms with distinct 3' ends through alternative polyadenylation (APA). Recent studies have revealed that APA is highly regulated in development and plays an important role in post-transcriptional gene regulation. Aberrant APA patterns have been associated with a wide range of diseases, from cancer to neuromuscular disorders. As such, a central question in the mRNA 3' processing field has been how polyadenylation sites (PAS) are recognized and how PAS selection can be regulated. The majority of mammalian PAS contain an AAUAAA hexamer and a U/UG-rich downstream element (DSE). According to the current model in the field, these key cis-elements are specifically recognized by CPSF160 of the CPSF complex and CstF64 of the CstF complex respectively. However, our published and preliminary data have challenged this model in several key aspects: 1) we have recently shown that the CPSF subunits CPSF30 and Wdr33, but not CPSF160 as was generally believed, directly bind to AAUAAA; 2) we have provided evidence that maintenance of the CPSF-RNA interaction specificity requires a novel proofreading factor(s) (see preliminary data); 3) we have demonstrated that the general 3' processing factor CstF64 in fact only binds to a subset of mammalian PAS, and we provided evidence that recognition of PAS with different sequence features requires distinct RNA-binding protein(s) (see preliminary data). These observations have not only significantly changed the current model for PAS recognition, but also revealed much greater complexity in mRNA 3' processing than previously appreciated. Here we propose to better define the molecular mechanisms of mammalian PAS recognition through an in-depth characterization of the key protein-RNA interactions involved. Accomplishing the proposed research will provide seminal insights into the fundamental mechanisms of mammalian mRNA 3' processing and its regulation. As aberrant PAS selection has been associated with a broad spectrum of human diseases, a better understanding of the mechanisms for PAS recognition may provide the foundation for the development of new therapeutics for these diseases.