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

描述(由申请人提供)：本提案的长期目标是通过对3‘加工复合体的全面表征，详细了解哺乳动物mRNA 3’加工的机制。M RNA 3‘端的形成是真核生物基因表达的重要步骤，对RNA代谢的许多方面都有深远的影响，包括m RNA的稳定性、输出和翻译。此外，受调控的mRNA3‘加工已成为基因控制的关键机制。异常的3‘处理信号和突变的3’处理因子会导致许多人类疾病，包括癌症。对于几乎所有的真核生物mRNAs，3‘端的加工包括两个催化步骤，即内切和添加Poly(A)尾巴。这两个步骤都发生在一个大分子机器中，即3‘处理复合体。该复合体的结构-功能关系是理解mRNA3‘加工机制的核心问题。我们最近已经纯化了组装在其mRNA靶上的功能性3‘处理复合体，并开始了蛋白质组、功能和结构分析。在我们的纯化系统的基础上，我们将解决该领域的几个基本和长期存在的问题：如何具体识别mRNAs中的3‘处理信号？3‘加工复合体的蛋白质组成是什么？在3’加工反应过程中它是如何变化的？3‘处理复合体的结构是什么？为了实现我们的目标，我们设计了以下具体目标：1.全面定位纯化的3‘加工复合体内的蛋白质-RNA相互作用。2.表征了3‘加工反应过程中3’加工复合体的组成变化。3.用单粒子冷冻电子显微镜(Cryo-EM)表征了3‘加工反应过程中3’加工复合体的结构变化。这些研究的完成将为mRNA3‘加工复合体的结构-功能关系和动力学提供新的和重要的机制见解，也可能为开发治疗由异常3’加工引起的疾病的新方法提供基础。 公共卫生相关性：信使核糖核酸3‘端的形成是真核基因表达的必要步骤。它在基因调控中起着重要的作用，3‘端的异常形成与多种人类疾病有关。本项目的目标是通过对3‘端加工机制的全面描述来了解mRNA3’加工的机制。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to understand, in detail, the mechanisms of mammalian mRNA 3' processing through comprehensive characterization of the 3' processing complex. mRNA 3'-end formation is an essential step of gene expression in eukaryotes, and has profound influences on many aspects of RNA metabolism, including mRNA stability, export, and translation. Additionally, regulated mRNA 3' processing has emerged as a critical mechanism for gene control. Aberrant 3' processing signals and mutant 3' processing factors cause a number of human diseases, including cancer. For almost all eukaryotic mRNAs, 3' processing involves two catalytic steps, an endonucleolytic cleavage followed by the addition of a poly(A) tail. Both of these steps take place in a macromolecular machinery, the 3' processing complex. The structure-function relationship of this complex is a central question in understanding the mechanism of mRNA 3' processing. We have recently purified the functional 3' processing complex assembled on its mRNA target and initiated proteomic, functional, and structural analyses. Building on our purification system, we will address several fundamental and long-standing questions in the field: How are the 3' processing signals in mRNAs specifically recognized? What is the protein composition of the 3' processing complex and (how) does it change during 3' processing reaction? What is the structure of the 3' processing complex? To achieve our objectives, we have designed the following specific aims: 1. Comprehensively map the protein-RNA interactions within the purified 3' processing complex. 2. Characterize the compositional changes of the 3' processing complex during 3' processing reaction. 3. Characterize the structural changes of the 3' processing complex during 3' processing reaction using single particle cryo-electron microscopy (cryo-EM). Accomplishment of the proposed studies will provide new and significant mechanistic insights into the structure-function relationship and the dynamics of the mRNA 3' processing complex, and may also provide the foundation for development of new therapeutic approaches against diseases caused by aberrant 3' processing. PUBLIC HEALTH RELEVANCE: mRNA 3'-end formation is an essential step of eukaryotic gene expression. It plays an important role in gene regulation, and aberrant 3'-end formation has been implicated in a variety of human diseases. The goal of this project is to understand the mechanism of mRNA 3' processing through comprehensive characterization of the 3' processing machinery.