Structural biology of 7SK RNP and its interaction with HIV-1 Tat

7SK RNP 的结构生物学及其与 HIV-1 Tat 的相互作用

基本信息

批准号：
10170271
负责人：
JULI FEIGON
金额：
$ 47.94万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-01 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10170271
关键词：
Active Sites Binding Binding Proteins Binding Sites Biochemical C-terminal CDK9 Protein Kinase Cell Maintenance Complement Factor B Complex Cryoelectron Microscopy Cyclin-Dependent Kinases Disease Drug Targeting Elongation Factor Enzymes Genetic Transcription Goals HIV HIV-1 Health Heart Hypertrophy Human In Vitro Laboratories Lead Link Messenger RNA Methods Methylation Molecular Molecular Conformation NMR Spectroscopy Phosphotransferases Play Positive Transcriptional Elongation Factor B Proteins RNA RNA Binding RNA Conformation RNA Polymerase II RNA Recognition Motif RNA-Protein Interaction Regulation Reporting Resistance Role Small Nuclear Ribonucleoproteins Stress Structure Trans-Activators Transactivation Transcription Elongation Transcription Initiation Transcriptional Regulation U6 small nuclear RNA Untranslated RNA Viral Virus Replication Work X-Ray Crystallography circular RNA cofactor cyclin T1 experimental study in vivo inorganic phosphate insight leukemia/lymphoma promoter protein complex recruit stoichiometry structural biology three dimensional structure transcription factor

项目摘要

PROJECT SUMMARY/ABSTRACT The human 7SK RNP is a dynamic assembly of the long non-coding 7SK RNA and cellular proteins that regulates the activity of positive transcription elongation factor b (P-TEFb). P-TEFb is an essential eukaryotic transcription factor for mRNA transcription elongation, which regulates the transition from promoter proximal paused RNA polymerase II (RNAPII) into productive elongation. P-TEFb is also an essential human cofactor for HIV-1 Tat transactivation and therefore viral replication. The human 7SK core RNP comprises the 331 nt RNAPIII-transcribed non-coding 7SK RNA, an unusual methyl capping enzyme called MePCE that methylates the γ phosphate on the RNA 5' terminus, and the La related protein 7, Larp7, that associates with the terminal hairpin and UUU-3'OH. In the active 7SK snRNP, Hexim and P-TEFb, a heterodimer of Cyclin T1 and the kinase Cdk9, bind the 7SK core RNP; interaction of P-TEFb in this complex inactivates it by sequestering its active site. Despite the central role of 7SK in transcription regulation of mRNA, other RNAPII RNAs, and HIV-1 transcription, relatively little is known at a structural or mechanistic level about how cellular proteins assemble with 7SK RNA to form a functional 7SK RNP or how Tat interacts with it to ultimately release P-TEFb. We will employ a combination of NMR spectroscopy, X-ray crystallography, and cryo electron microscopy along with biochemical methods to investigate the structures and assembly of the 7SK core RNP (MePCE–7SK–Larp7) and 7SK core RNP plus Hexim and P-TEFb (the `active' 7SK RNP) in order to achieve an atomic-level understanding of this important host RNP for HIV-1 viral replication. These structural studies will lay the groundwork for elucidating the molecular mechanisms of Tat-Hexim competition in the context of 7SK RNP. We aim to dissect the potential intermediate steps (i.e. Tat-bound 7SK RNP) that lead to P-TEFb hijacking from 7SK RNP into the HIV-1 viral super-elongation complex. The results of these experiments will provide fundamental molecular insights into and a structural basis for drug targeting of this largely structurally uncharacterized RNP that is essential for HIV-1 transcription and therefore escape from latency.

项目概要/摘要人类 7SK RNP 是长非编码 7SK RNA 和细胞蛋白的动态组装体，调节正转录延伸因子 b (P-TEFb) 的活性，P-TEFb 是真核生物必需的。 mRNA 转录延伸的转录因子，调节从启动子近端的转变暂停 RNA 聚合酶 II (RNAPII) 进入生产性延伸，P-TEFb 也是一种重要的人类辅助因子。 HIV-1 Tat 反式激活和病毒复制人类 7SK 核心 RNP 包含 331 nt。 RNAPIII 转录的非编码 7SK RNA，一种不寻常的甲基加帽酶，称为 MePCE，可甲基化 RNA 5' 末端的 γ 磷酸盐以及与末端结合的 La 相关蛋白 7 Larp7 活性 7SK snRNP、Hexim 和 P-TEFb（Cyclin T1 的异二聚体）中的发夹和 UUU-3'OH。激酶 Cdk9，与该复合物中的 P-TEFb 相互作用，结合 7SK 核心 RNP，通过隔离其使其失活；尽管 7SK 在 mRNA、其他 RNAPII RNA 和 HIV-1 的转录调控中发挥着核心作用。转录，在结构或机制水平上对细胞蛋白质如何组装知之甚少与 7SK RNA 形成功能性 7SK RNP 或 Tat 如何与其相互作用以最终释放 P-TEFb。结合使用 NMR 光谱学、X 射线晶体学和冷冻电子显微镜生化方法研究 7SK 核心 RNP (MePCE–7SK–Larp7) 的结构和组装和 7SK 核心 RNP 加上 Hexim 和 P-TEFb（“主动”7SK RNP），以实现原子级对 HIV-1 病毒复制的这一重要宿主 RNP 的了解将为这些结构研究奠定基础。为阐明 7SK RNP 背景下 Tat-Hexim 竞争的分子机制奠定了基础。我们的目标是剖析导致 P-TEFb 劫持的潜在中间步骤（即 Tat 结合的 7SK RNP）这些实验的结果将提供从 7SK RNP 到 HIV-1 病毒超伸长复合物的过程。基本的分子见解和药物靶向的结构基础未表征的 RNP 对 HIV-1 转录至关重要，因此可以逃避潜伏期。