Structure and Assembly of Regulatory RNPs

监管 RNP 的结构和组装

基本信息

批准号：
8915719
负责人：
JULI FEIGON
金额：
$ 29.26万
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：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8915719
关键词：
Active Sites Affinity Labels Applications Grants Architecture Binding Binding Sites Biochemical Biogenesis C-terminal Cancer Etiology Cell physiology Characteristics Complex Core Assembly Cryoelectron Microscopy DNA-Directed RNA Polymerase Disease Docking Dwarfism Electron Microscopy Electrons Elongation Factor Enzymes Epithelial Frameshift Mutation Genetic Transcription Goals HIV HIV-1 Health Heart Hypertrophy Holoenzymes Human In Vitro Investigation Lead Length Malignant Neoplasms Malignant neoplasm of cervix uteri Mammary gland Messenger RNA Methods Modeling NMR Spectroscopy Negative Staining Play Positive Transcriptional Elongation Factor B Protein Binding Proteins RNA RNA Binding RNA Polymerase II RNA Recognition Motif RNA Stability RRM1 gene Regulation Resolution Ribonucleoproteins Role Sampling Site Small Nuclear Ribonucleoproteins Structure Tail Telomerase Telomerase RNA Component Tetrahymena Transactivation Transcript Transcription Elongation Transcriptional Regulation Untranslated RNA Viral X-Ray Crystallography affinity labeling base cofactor in vivo inorganic phosphate insight leukemia/lymphoma malignant breast neoplasm malignant stomach neoplasm methyl group promoter reconstitution research study synaptotagmin I transcription factor tumor progression

项目摘要

DESCRIPTION (provided by applicant): The human 7SK small nuclear RNP (snRNP) is a dynamic assembly of the abundant 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 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 core 7SK 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 group 7, hLARP7, that associates with the terminal UUU-3'. In the active 7SK snRNP, HEXIM and P-TEFb bind the core snRNP; interaction of P-TEFb in this complex inactivates it by sequestering its active site. Recent studies have highlighted the importance of hLARP7 and MePCE in 7SK RNA stability and P-TEFb assembly; however, the structural basis of these interactions has not been established. We propose to investigate the structural characteristics and assembly of the core 7SK snRNP and its interactions with HEXIM1 and P-TEFb to form the "active" snRNP using a combination of NMR spectroscopy, X-ray crystallography, electron microscopy, and biochemical methods. Our specific aims are: (1) Determine how the La related protein hLARP7 interacts with 7SK RNA; (2) Determine how MePCE interacts with 7SK RNA and hLARP7; (3) Determine the requirements for assembly of the core and "active" 7SK RNPs; and (4) Determine the architecture of the "active" 7SK RNP. Improper P-TEFb regulation by the 7SK snRNP plays a role in myriad diseases including cardiac hypertrophy, leukemia, lymphoma, cervical cancer, breast cancer, and gastric cancer. The results of the experiments proposed in this grant application will lead to an understanding of how the core 7SK snRNP assembles and ultimately binds to and inactivates P-TEFb. This in turn will provide fundamental information on transcription regulation, HIV-1 replication, mechanism of cancer progression, and dynamic non-coding RNA-directed cellular function.

描述（由申请人提供）：人类 7SK 小核 RNP (snRNP) 是丰富的长非编码 7SK RNA 和细胞蛋白的动态组装，调节正转录延伸因子 b (P-TEFb) 的活性。 TEFb 是 mRNA 转录延伸的重要真核转录因子，调节从启动子暂停的 RNA 聚合酶 II (RNAPII) 到有效延伸的转变。也是 HIV-1 Tat 反式激活的重要人类辅助因子，因此人类核心 7SK RNP 包含 331 nt RNAPIII 转录的非编码 7SK RNA，这是一种称为 MePCE 的不寻常甲基加帽酶，可甲基化 RNA 上的 γ 磷酸盐。 5' 末端，以及与活性 7SK 中末端 UUU-3' 相关的 La 相关蛋白组 7 hLARP7。 snRNP、HEXIM 和 P-TEFb 与核心 snRNP 结合；该复合物中 P-TEFb 的相互作用通过隔离其活性位点使其失活，然而，最近的研究强调了 hLARP7 和 MePCE 在 7SK RNA 稳定性和 P-TEFb 组装中的重要性；这些相互作用的结构基础尚未确定。我们建议研究核心 7SK snRNP 的结构特征和组装及其与 HEXIM1 和 P-TEFb 的相互作用以形成。我们的具体目标是： (1) 确定 La 相关蛋白 hLARP7 如何与 7SK RNA 相互作用；与 7SK RNA 和 hLARP7 相互作用；(3) 确定核心和“活性”7SK RNP 的组装要求；以及 (4) 确定“活性”7SK RNP 的结构。7SK snRNP 的不当 P-TEFb 调节在多种疾病中发挥作用，包括心脏肥大、白血病、淋巴瘤、宫颈癌、乳腺癌和胃癌。拨款申请将有助于了解核心 7SK snRNP 如何组装并最终结合并灭活 P-TEFb，这反过来将提供有关转录调控、HIV-1 复制、癌症进展机制和动态非编码RNA指导的细胞功能。