INT复合物调节U snRNA 3'加工的结构基础

U snRNA是剪接体对前体信使RNA进行剪接的核心催化组分，其转录的3‘加工成熟依赖于整合因子(INT)复合物。INT复合物是一个至少由14个亚基组成、分子量超过1MDa的蛋白质复合物，近期研究展示出INT复合物及组分在个体发育中的重要作用，其组分突变能够引起发育障碍、神经系统疾病乃至肿瘤生成，但是基于该复合物空间结构的组装机制和功能执行等问题仍处于未知状态。据此，本项目计划对INT复合物进行结构解析和机制研究。计划通过标签蛋白质组分过表达和内源性提取的方法，综合多种纯化分离技术手段，获取性质均一且良好的完整INT复合物或组分蛋白，随后借助冷冻电镜和X射线晶体学技术进一步捕获其结构，搭建原子模型。在结构分析的基础上，本项目还希望明确各组分在INT复合物组装时发挥的作用和关键突变对复合物功能的影响，最终在分子水平阐述INT复合物调控U snRNA 3’加工和转录终止的作用机制。

U-rich small nuclear RNAs (U snRNAs) are key catalytic components of spliceosome during splicing of precursor messager RNA (pre-mRNA). The transcriptional termination and maturation of the snRNAs relies on its 3'-end processing, which is mediated by the integrator (INT) complex. As a protein complex composed of at least 14 subunits, INT has a molecular weight of over than 1 MDa. Despite its role in mediating small nuclear RNA maturation, disrupted/mutated INT has been recently identified closely associated with embryogenesis difficulties, neuro disorders, and even cancer. Nevertheless, little is known about INT complex’s mechanisms of assembling and functioning. Therefore, this project is designed to investigate the functions of integrator complex from a structure study-based view...In order to obtain homogenous and well-qualified multi-complex or sub-complex structural information of INT, we plan to comprehensively integrate multiple extraction methods for purification of tag-based merged proteins, then applying cryonic electron microscopy and X-ray crystallography analysis for capture of images and construction of molecular models. We also plan to explore INT complex’s molecular functions as well as the impact of components mutation on INT complex, so as to explain the regulation of INT-mediated U snRNA transcription on a sub-atomic scale.