Characterization of the mammalian mRNA 3'-end processing complex

项目概要： 本提案的长期目标是详细了解 哺乳动物mRNA的3'加工及其调控。mRNA 3 '-末端形成，通常 包括内切核酸裂解，随后是多聚腺苷酸化，是一个重要步骤 真核基因表达的影响，它显着影响RNA的许多方面， 代谢，包括mRNA的稳定性，亚细胞定位和翻译。在 此外，大多数真核基因产生多种mRNA同种型， 3'末端通过交替聚腺苷酸化（阿帕）。最近的研究表明， 阿帕在发育过程中受到高度调控，在后发育过程中发挥着重要作用。 转录基因调控异常的阿帕模式与广泛的 从癌症到神经系统疾病。两个关键突出 mRNA 3'加工领域的问题是：1）什么是mRNA 3'加工的分子机制？ mRNA 3'加工机制（包括多聚腺苷酸位点（PAS）机制） 切割和多聚腺苷酸化的识别和催化）？2)PAS怎么样 选择还是阿帕监管？为了解决这些基本问题，必须 了解mRNA 3'加工因子的结构与功能关系。在 在过去的资助期间发表的研究，我们已经重建了关键 哺乳动物mRNA 3'加工复合物的模块，其特征在于AAUAAA如何 和富含铀/铀的下游元素（两个关键元素，定义了大多数 哺乳动物PAS）的识别，并揭示mRNA 3'加工和剪接， 也可以通过类似的机制进行调节。基于这些发现，我们 建议定义完全重组人体的结构-功能关系 mRNA 3'加工复合物，并系统地表征RNA结合的作用 RBP在调节PAS选择和阿帕中的作用。

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, subcellular localization 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 neurological disorders. Two key outstanding questions in the mRNA 3' processing field have been: 1) what is the molecular mechanism of mRNA 3' processing (including mechanisms for poly(A) site (PAS) recognition and catalysis of cleavage and polyadenylation)? 2) how is PAS selection or APA regulated? To address these fundamental questions, it is essential to understand the structure-function relationship of mRNA 3' processing factors. In published studies during the previous funding periods, we have reconstituted key modules of the mammalian mRNA 3' processing complex, characterized how AAUAAA and the U/GU-rich downstream element (two key elements that define the majority of mammalian PAS) are recognized, and revealed that mRNA 3' processing and splicing can be regulated through a similar mechanism. Building on these findings, here we propose to define the structure-function relationship of the fully reconstituted human mRNA 3' processing complex and systematically characterize the role of RNA-binding proteins (RBPs) in regulating PAS selection and APA.