Molecular Recognition during pre-mRNA Splicing

描述（由申请人提供）：前体mRNA的剪接为细胞分化和发育提供了转录物多样性的主要来源。已知该过程需要由约100种蛋白质和5种小核（sn）RNA组成的剪接机（剪接体）。前mRNA剪接位点上剪接体组装的早期阶段是人类遗传疾病和癌症中经常出错的关键调控步骤。然而，剪接体究竟如何从数千个相互竞争的前mRNA序列中选择并切除正确的剪接位点，在分子水平上仍然知之甚少。因此，该提议的总体目标是理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。必需剪接因子U2 AF和SF 1的复合物识别邻近3'剪接位点的前mRNA序列，并反过来稳定核心剪接体的结合。前mRNA通过U2 AF的丝氨酸-丝氨酸（RS）区域和U2 AF相关的ATP酶UAP 56的接触是完成这一任务所必需的。本提案的具体目标是解决以下关于前体mRNA剪接的关键早期阶段的核心问题：（1）U2 AF通过什么方式识别不同的剪接位点？(2)SF 1通过什么方式增强U2 AF对剪接位点的识别？(3)U2 AF通过什么方式招募UAP 56，反过来，UAP 56在剪接位点对U2 AF有什么作用？(4)RS区通过什么方式促进剪接体snRNA与前mRNA的结合？我们已经取得了显着的进展，实现这些目标，（i）确定的三维结构的U2 AF结合剪接位点，（ii）评估热力学和结构的贡献SF 1域U2 AF结合，和（iii）表征RNA相互作用的RS域。这些研究将大大推进我们对基因表达这一基本步骤的理解。公共卫生相关性：前体mRNA剪接中的错误导致主要的人类疾病，包括癌症、白血病、强直性肌营养不良、神经和代谢紊乱。通过拟议的工作获得的正常剪接位点识别的调查将作为理解的基础，并在长期开发针对人类疾病的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.