Structure and Assembly of Regulatory RNPs

监管 RNP 的结构和组装

基本信息

批准号：
9131803
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/9131803
关键词：
Active Sites Affinity Labels Applications Grants Architecture Binding Binding Proteins 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 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 Virus Replication X-Ray Crystallography affinity labeling base cofactor in vivo inorganic phosphate insight knock-down 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（由适用提供）：人类7SK小核RNP（SNRNP）是大量长的非编码7SK RNA和细胞蛋白的动态组装，可调节阳性转录伸长因子B（P-TEFB）的活性。 P-TEFB是mRNA转录伸长的必不可少的真核转录因子，它调节从启动子暂停的RNA聚合酶II（RNAPII）转变为产物伸长。 P-TEFB也是HIV-1 TAT反式激活的必不可少的人辅助因子，因此是病毒复制。人核7SK RNP包括331 NT RNAPIII-TRANSED非编码7SK RNA，这是一种称为MEPCE的不寻常的甲基帽酶，该酶称为MEPCE，该酶在RNA 5'末端上甲基甲基化的γ磷酸盐甲基甲基，LA相关的蛋白质组7，HLARP7，HLARP7，与该阶段的usiatial usiatical uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuue-3。在活跃的7SK SNRNP中，Hexim和P-TEFB结合了核心SNRNP； P-TEFB在该复合物中的相互作用通过隔离其活性位点使其失活。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。但是，尚未建立这些相互作用的结构基础。我们建议使用NMR光谱，X射线晶体学，电子显微镜和生物化学方法的组合研究Core 7SK SNRNP及其与Hexim1和P-Tefb的相互作用的结构特征和组装。我们的具体目的是：（1）确定LA相关蛋白HLARP7如何与7SK RNA相互作用；（2）确定MEPCE如何与7SK RNA和HLARP7相互作用；（3）确定组装核心和“ Active” 7SK RNP的要求；（4）确定“活动” 7SK RNP的体系结构。 7SK SNRNP的P-TEFB调节不当在无数疾病中发挥作用，包括心脏肥大，白血病，淋巴瘤，宫颈癌，乳腺癌和胃癌。本赠款应用程序中提出的实验结果将导致对Core 7SK SNRNP的组合方式并最终与P-TEFB结合并失活的理解。反过来，这将提供有关转录调控，HIV-1复制，癌症进展机理和动态非编码RNA指导的细胞功能的基本信息。