Molecular Recognition during pre-mRNA Splicing

描述(申请人提供)：前mRNAs的剪接为细胞分化和发育提供了转录多样性的主要来源。众所周知，这个过程需要一个由~100个蛋白质和5个小核(Sn)RNA组成的剪接机(剪接体)。剪接体在前信使核糖核酸剪接位点上组装的早期阶段是关键的调控步骤，在人类遗传性疾病和癌症中经常出错。然而，剪接体如何从数千个相互竞争的Pre-mRNA序列中选择和切除正确的剪接位点，在分子水平上仍然知之甚少。因此，这项提议的总体目标是了解在剪接体激活的早期阶段指导3‘剪接位点选择的顺序三维相互作用。重要的剪接因子U2AF和SF1的复合体识别3‘剪接位点附近的前mRNA序列，进而稳定核心剪接体的结合。完成这一任务需要通过U2AF的精氨酸-丝氨酸(RS)区域和U2AF相关的ATPase UAP56的前mRNA接触。本提案的具体目的是解决关于Pre-mRNA剪接的关键早期阶段的以下核心问题：(1)U2AF通过什么途径识别不同的剪接位点？(2)SF1通过什么途径增强U2AF对剪接位点的识别？(3)U2AF通过什么途径招募UAP56，反过来，UAP56在剪接位点对U2AF有什么作用？(4)RS区域通过什么途径促进剪接体SnRNA与Pre-mRNA的联系？我们已经在以下方面取得了重大进展：(I)确定U2AF与剪接位点结合的三维结构，(Ii)评估SF1结构域对U2AF结合的热力学和结构贡献，以及(Iii)通过RS结构域表征RNA相互作用。这些研究将极大地促进我们对基因表达这一基本步骤的理解。公共卫生相关性：前信使核糖核酸剪接错误导致人类重大疾病，包括癌症、白血病、强直性肌营养不良、神经和代谢紊乱。通过拟议的工作将获得的正常剪接位点识别的调查将作为了解人类疾病的有害剪接变体的基础，并在长期内开发针对有害剪接变体的治疗方法。

DESCRIPTION (provided by applicant): Splicing of pre-mRNAs provides a major source of transcript diversity for cell differentiation and development. It is known that this process requires a splicing machine (spliceosome) composed of ~100 proteins and 5 small nuclear (sn)RNAs. The early stages of spliceosome assembly on pre-mRNA splice sites are key regulated steps that often go awry in human genetic diseases and cancers. Yet, exactly how the spliceosome selects and excises the correct splice sites from amidst thousands of competing pre-mRNA sequences remains poorly understood at the molecular level. Hence, the overall goal of this proposal is to understand the sequential three- dimensional interactions that guide 3' splice site selection in the early stages of spliceosome activation. A complex of the essential splicing factors U2AF and SF1 recognizes pre-mRNA sequences adjacent the 3' splice site, and in turn stabilizes association of the core spliceosome. Pre-mRNA contacts by an arginine-serine (RS) region of U2AF, and the U2AF-associated ATPase UAP56, are required to accomplish this task. Specific aims of this proposal address the following central questions concerning the critical early stages of pre-mRNA splicing: (1) By what means does U2AF recognize diverse splice sites? (2) By what means does SF1 enhance splice site recognition by U2AF? (3) By what means does U2AF recruit UAP56, and in turn, what is the action of UAP56 on U2AF at the splice site? (4) By what means does an RS region promote association of spliceosomal snRNAs with the pre-mRNA? We have already made significant advances towards these aims by (i) determining three-dimensional structures of U2AF bound to splice sites, (ii) evaluating thermodynamic and structural contributions of SF1 domains to U2AF binding, and (iii) characterizing RNA interactions by RS domains. These studies will significantly advance our understanding of this fundamental step of gene expression. PUBLIC HEALTH RELEVANCE: Errors in pre-mRNA splicing contribute to major human diseases, including cancers, leukemias, myotonic dystrophies, neurological and metabolic disorders. The investigation of normal splice site recognition to be gained by the proposed work would serve as a basis for understanding, and in the long term developing treatments against, harmful splice variants of human disease.