Structure and Assembly of Regulatory RNPs

描述(申请人提供)：人7SK小核RNP(SnRNP)是由丰富的非编码7SK RNA和细胞蛋白动态组装而成，调节正转录延伸因子b(P-TEFb)的活性。P-TEFb是一种真核转录因子，调节着从启动子暂停的RNA聚合酶II(RNAPII)到产生性延长的过渡。P-TEFb也是HIV-1Tat反式激活和病毒复制所必需的人类辅因子。人类核心7SK RNP包括331nt RNAPIII转录的非编码7SK RNA，一种不常见的甲基化封帽酶MePCE，它使RNA5‘末端的γ磷酸甲基化，以及与UUU-3’末端相关的La相关蛋白质组7，hLARP7。在活性的7SK SnRNP中，HEXIM和P-TEFb与核心SnRNP结合；P-TEFb在这个复合体中的相互作用通过隔离其活性部位而使其失活。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，但这些相互作用的结构基础尚未建立。我们建议利用核磁共振光谱、X射线结晶学、电子显微镜和生化方法相结合的方法来研究核心7SK SnRNP的结构特征和组装，以及它与HEXIM1和P-TEFb的相互作用，以形成“活性”的SnRNP。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；(2)确定MePCE如何与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对P-TEFb的不正确调控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。本拨款申请中提出的实验结果将有助于理解核心7SK SnRNP是如何组装并最终与P-TEFb结合并使其失活的。这反过来将提供关于转录调控、HIV-1复制、癌症进展机制以及动态非编码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.