Structural Biology of Regulatory RNPs

项目概要/摘要 大多数非编码RNA与蛋白质组装以调节包括DNA复制在内的多种细胞过程， RNA转录、RNA加工和翻译。这些RNA-蛋白质复合物（RNP）通常是动态的 包括核心RNP加上其他蛋白质的组装体，所述其他蛋白质瞬时结合或与不同的蛋白质形成复合物。 功能协调发展的我们将使用一种综合的结构生物学方法，结合核磁共振光谱，X射线， 晶体学和电子显微镜沿着严格的生物化学和分子生物学来研究 两种调节性RNP，端粒酶和7SK RNP的结构、装配、动力学和功能。端粒酶 通过重复合成短端粒重复序列延伸线性染色体的3 '端 （人类中的TTAGGG）使用RNA模板，该RNA模板是其整合的端粒酶RNA（TER）的一部分， 端粒酶逆转录酶（TERT）。它是一个高度调节的衰老决定因素， 肿瘤发生和干细胞更新。我们的实验室是端粒酶结构研究的先驱， 最近用DNA测定了活性四膜虫端粒酶的4.8kb冷冻电镜结构。 阐明端粒重复序列合成的完整机制，以及端粒酶如何被募集到 调节在端粒，我们建议获得（1）原子分辨率结构的四膜虫端粒酶在 催化循环中的每个步骤以及人类端粒酶的结构与活性和疾病突变的相关性 （2）研究端粒酶和端粒酶中端粒DNA相关蛋白的结构和功能， 端粒这些研究将为端粒酶的机制和调控提供基础性的见解， 与疾病相关的TERT和TER突变影响活性，以及设计靶向药物的结构基础 端粒酶活性人7SK是一种丰富的核长非编码RNA，调节RNA聚合酶II RNAPII转录，主要通过与蛋白质组装形成RNP， 正转录延伸因子B（P-TEF B）。P-TEFb是超级晶体管的组成部分， 延伸复合物，其磷酸化负转录延伸因子和RNAPII CTD， 刺激mRNA转录物的生产性延伸。7SK还调节RNAPII转录的小 核RNA、增强子RNA和轴突维持通过其与hnRNP R的相互作用。了解 7SK调节P-TEFb活性的结构基础，我们建议（1）确定 包括7SK、磷酸甲酯封端的抗应力芯7SK RNP的组件和结构 酶（MePCE）和La相关蛋白组7（Larp7），以及（2）确定Hexim和P-TEFb如何相互作用 与7SK核心RNP和彼此形成"有源" 7SK RNP。与P-TEFb失调有关的疾病 包括心脏肥大、癌症和原始侏儒症，而P-TEFb是HIV宿主辅因子 复制的这些研究将提供有关结构、相互作用和机制的原子级信息 端粒酶和7SK RNP的研究对于理解它们对健康和疾病的无数影响至关重要。

Project Summary/Abstract Most noncoding RNAs assemble with proteins to regulate diverse cellular processes including DNA replication, RNA transcription, RNA processing, and translation. These RNA-protein complexes (RNP) are often dynamic assemblies that include a core RNP plus other proteins that bind transiently or form complexes with different functions. We will use an integrative structural biology approach combining NMR spectroscopy, X-ray crystallography, and electron microscopy along with rigorous biochemistry and molecular biology to investigate structure, assembly, dynamics, and function of two regulatory RNPs, telomerase and 7SK RNP. Telomerase extends the 3'-ends of linear chromosomes by repetitively synthesizing the short telomere repeat sequence (TTAGGG in humans) using an RNA template that is part of its integral telomerase RNA (TER) and its specialized telomerase reverse transcriptase (TERT). It is a highly-regulated determinant of aging, tumorigenesis, and stem cell renewal. Our laboratory has pioneered structural studies of telomerase, most recently determining a 4.8Å cryo-electron microscopy structure of active Tetrahymena telomerase with DNA. To elucidate the complete mechanism of telomere repeat synthesis and how telomerase is recruited to and regulated at telomeres, we propose to obtain (1) atomic resolution structures of Tetrahymana telomerase at each step in the catalytic cycle and correlate structure with activity and disease mutations in human telomerase and (2) investigate the structure and function of telomeric DNA-associated proteins in telomerase and at telomeres. These studies will provide fundamental insights into telomerase mechanism and regulation, how TERT and TER mutations linked to disease affect activity, and a structural basis for designing drugs to target telomerase activity. Human 7SK is an abundant nuclear long noncoding RNA that regulates RNA polymerase II (RNAPII) transcription, primarily by assembling with proteins to form an RNP that sequesters and inactivates the positive transcription elongation factor b (P-TEFb). P-TEFb is an integral component of the super elongation complex that phosphorylates negative transcription elongation factors and the RNAPII CTD to stimulate productive elongation of mRNA transcripts. 7SK also regulates the RNAPII transcription of small nuclear RNAs, enhancer RNAs, and axon maintenance through its interaction with hnRNP R. To understand the structural basis of 7SK regulation of P-TEFb activity, we propose to (1) determine the mechanism of assembly and structure of the stress-resistant core 7SK RNP, comprising 7SK, methylphosphate capping enzyme (MePCE), and La related protein group 7 (Larp7), and (2) determine how Hexim and P-TEFb interact with 7SK core RNP and each other to form the “active” 7SK RNP. Diseases linked to P-TEFb misregulation include cardiac hypertrophy, cancers, and primordial dwarfism, and P-TEFb is a host cofactor for HIV replication. These studies will provide atomic-level information on the structures, interactions, and mechanisms of telomerase and 7SK RNPs that is critical to understanding their myriad effects on health and disease.