Structure and Assembly of Regulatory RNPs

描述（由申请人提供）：人7 SK小核RNP（snRNP）是丰富的长非编码7 SK RNA和细胞蛋白的动态组装体，其调节正转录延伸因子B（P-TEF B）的活性。P-TEFb是真核生物中mRNA转录延伸过程中的一个重要转录因子，它调节启动子暂停RNA聚合酶II（RNAPII）向生产性延伸的转变。P-TEFb也是HIV-1达特反式激活和病毒复制的必需人辅因子。人核心7SK RNP包含331 nt RNAPIII转录的非编码7SK RNA、一种称为MePCE的不寻常的甲基加帽酶，其使RNA 5'末端的γ磷酸甲基化，以及La相关蛋白7组，hLARP 7，其与末端UUU-3'缔合。在活性7SK snRNP中，HEXIM和P-TEFb结合核心snRNP;该复合物中P-TEFb的相互作用通过螯合其活性位点使其失活。最近的研究强调了hLARP 7和MePCE在7SK RNA稳定性和P-TEFb组装中的重要性;然而，这些相互作用的结构基础尚未建立。我们建议调查的结构特征和组装的核心7SK snRNP及其与HEXIM 1和P-TEFb的相互作用，形成“活性”snRNP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。我们的具体目标是：（1）确定La相关蛋白hLARP 7如何与7SK RNA相互作用;（2）确定MePCE如何与7SK RNA和hLARP 7相互作用;（3）确定核心和“活性”7SK RNP组装的要求;和（4）确定“活性”7SK RNP的结构。7SK snRNP对P-TEFb的不当调节在包括心脏肥大、白血病、淋巴瘤、宫颈癌、乳腺癌和胃癌在内的无数疾病中起作用。在这项资助申请中提出的实验结果将导致对核心7SK snRNP如何组装并最终结合到P-TEFb并使其失活的理解。这反过来将提供转录调控，HIV-1复制，癌症进展机制和动态非编码RNA指导的细胞功能的基本信息。

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.