Molecular Recognition during pre-mRNA Splicing

摘要： 前mRNAs的剪接为细胞分化和转录多样性提供了一个主要来源 发展。众所周知，该过程需要拼接机(剪接体) 由100个蛋白质和5个小核(Sn)RNA组成。剪接体组装的早期阶段 前信使核糖核酸剪接位点是人类遗传病中经常出错的关键调控步骤 和癌症。然而，剪接体到底是如何选择和切除正确的剪接点的 在数千个相互竞争的前信使核糖核酸序列中，人们对 分子水平。因此，这项建议的总体目标是理解顺序的三个-- 剪接体早期3‘端剪接位点选择的空间相互作用 激活。 重要剪接因子U2AF和SF1的复合体识别前-mRNA 与3‘剪接点相邻的序列，进而稳定核心剪接体的结合。 通过U2AF的精氨酸-丝氨酸(RS)区域与U2AF相关的前mRNA接触 ATPase UAP56是完成这一任务所必需的。这项提案的具体目标涉及 以下是关于前信使核糖核酸剪接关键早期阶段的核心问题： (1)U2AF通过什么方式识别不同的剪接位点？ (2)SF1通过什么途径增强U2AF的剪接位点识别？ (3)U2AF通过什么方式招募UAP56，反过来，UAP56的行动是什么 在U2AF的拼接现场？ (4)RS区通过什么方式促进剪接体SnRNAs与 前信使核糖核酸？ 我们已在达致这些目标方面取得重大进展，包括：(I)确定以下三个目标： 与剪接位点结合的U2AF的空间结构，(Ii)热力学和 SF1结构域对U2AF结合的结构贡献，以及(Iii)表征RNA RS域的相互作用。这些研究将大大促进我们对这一点的理解 基因表达的基本步骤。

Abstract: 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.