7SK snRNP调控高等真核细胞转录的结构与分子机理研究

There are a large number of non-coding RNAs in eukaryotic cells. These non-coding RNAs are crucial regulators for physiological and pathological cell function involved in a variety of life events. 7SK RNA is one of typical non-coding RNA and it interacts with LARP7 and MePCE for forming the core of the 7SK ribonucleoprotein (7SK snRNP). The core of 7SK snRNP further interacts with positive transcription elongation factor b (P-TEFb), or heterogeneous ribonucleoproteins (hnRNPs) reversibly, for regulating the elongation stage of the RNA polymerase II indirectly during eukaryotic transcription. It has been reported that 7SK snRNP stays in a highly dynamic state by alternating binding with P-TEFb and hnRNPs in cells. However, the detailed molecular mechanism is still unclear. In this study we attempt to solve the 3D structures of 7SK snRNP protein complexes in two different stages through biochemical and structural biology techniques. The 3D structures of 7SK snRNP are beneficial to understand the detailed interactions between each component. Furthermore, specifical mutants will be designed based on structures of 7SK snRNP to elucidate the molecular basis of 7SK snRNP in gene expression regulation.

真核生物细胞内存在大量非编码RNA，在各类生命活动中发挥重要作用。7SK RNA作为一种典型非编码RNA，与LARP7和MePCE相互作用形成核心7SK核糖核蛋白复合物（7SK snRNP）。核心7SK snRNP复合物通过可逆结合正调控转录延伸因子b（P-TEFb），或核不均一性核糖蛋白（hnRNPs），间接调控真核细胞RNA聚合酶II的转录延伸过程。前期的研究表明7SK snRNP以交替结合P-TEFb和hnRNPs的方式调控RNA聚合酶II的活性，但其分子机制尚未被阐明。本课题拟利用生物化学和结构生物学的技术手段，对两种状态的7SK snRNP复合物进行三维结构解析，获得其组装方式和各组分互作模式。在此基础上，对两种状态7SK snRNPs复合物进行构象分析比对，设计针对性突变体并结合细胞生物学实验，阐明其动态调控真核生物转录的分子机制。

7SK snRNPs作为一种典型的非编码RNA，通过可逆的结合正性转录延伸因子b (positive transcription elongation fractor b, P-TEFb)，对真核细胞RNA聚合酶II参与的转录延伸进行调控,组成7SK snRNPs的7SK RNA由RNA聚合酶III转录。本项目以人源的7SK snRNPs和RNA聚合酶III为研究对象，拟采用分子生物学， 生物化学以及结构生物学等多种技术手段，解析7SK snRNPs/P-TEFb复合物和RNA聚合酶III的组装机制和转录调控机理。通过本项目前期的努力， 研究人员获得了7SK snRNP（HEXIM／P-TEFb）复合物的低分辨率模型，此结果对进一步获得原子分辨率结构做了良好的铺垫，也使研究人员对此复合物有了初步的认知。此外，本研究聚焦了完整的人源RNA聚合酶III。研究人员通过对人源RNA聚合酶III处于不同状态的结构解析，意外了获得了一种处于自抑制状态的RNA聚合酶III构象，完善了人们对RNA聚合酶III转录过程的认知，势必会为人们理解RNA聚合酶III转录调控相关疾病的致病机理提供理论指导。