Structural Biology of Regulatory RNPs

项目摘要/摘要 大多数非编码RNA与蛋白质组装，调节不同的细胞过程，包括DNA复制， RNA转录、RNA加工和翻译。这些RNA-蛋白质复合体(RNP)通常是动态的 包括核心RNP和其他蛋白质的组件，这些蛋白质瞬时结合或与不同的 功能。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 结晶学、电子显微镜以及严格的生物化学和分子生物学研究 端粒酶和7SK RNP两种调控RNP的结构、组装、动力学和功能。端粒酶 通过重复合成短端粒重复序列延长线性染色体的3‘端 (人类中的TTAGGG)使用其整合的端粒酶RNA(TER)的RNA模板和其 特化端粒酶逆转录酶(TERT)。它是一种高度受监管的衰老决定因素， 肿瘤发生和干细胞更新。我们实验室开创了端粒酶结构研究的先河，大多数 最近用DNA测定了四膜虫端粒酶活性的4.8°低温电子显微镜结构。 为了阐明端粒重复合成的完整机制以及端粒酶是如何被招募到和 在端粒调控下，我们建议获得(1)四氢线虫端粒酶的原子分辨结构 催化循环中的每一步以及与人类端粒酶活性和疾病突变相关的结构 以及(2)研究端粒酶和AT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本的见解， 与疾病相关的TERT和TER突变会影响活性，也是设计靶向药物的结构基础 端粒酶活性。人类7SK是一种丰富的核长非编码RNA，调节RNA聚合酶II (RNAPII)转录，主要是通过与蛋白质组装形成RNP，从而隔离和失活 阳性转录延伸因子b(P-TEFb)。P-TEFb是超级 磷酸化负转录伸长因子的伸长复合体和RNAPII CTD 刺激mRNA转录本的生产性延伸。7SK还调节小鼠的RNAPII转录 通过与hnRNP R的相互作用来理解核RNA、增强子RNA和轴突维持 7SK调节P-TEFb活性的结构基础，我们建议：(1)确定P-TEFb活性的调控机制 7SK抗压核心7SK RNP的组装和结构，包括7SK，甲基磷酸盐封盖 酶(MePCE)和La相关蛋白7(Larp7)，以及(2)决定Hexim和P-TEFb如何相互作用 以7SK为核心的RNP与对方共同组成“主动”的7SK RNP。与P-TEFb调控不当有关的疾病 包括心脏肥大、癌症和原始侏儒症，而P-TEFb是HIV宿主辅助因子 复制。这些研究将提供有关结构、相互作用和机制的原子级信息 这对于了解端粒酶和7SK RNPs对健康和疾病的各种影响至关重要。

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.