Molecular Recognition during pre-mRNA Splicing

摘要： 前体mRNA的剪接为细胞分化提供了转录物多样性的主要来源， 发展众所周知，该过程需要拼接机（spliceosome）组成， 约100种蛋白质和5种小核（sn）RNA。剪接体组装的早期阶段 前mRNA剪接位点是人类遗传疾病中经常出错的关键调控步骤 和癌症。然而，剪接体究竟如何选择和切除正确的剪接位点， 在数以千计的竞争前mRNA序列中， 分子水平。因此，本提案的总体目标是了解顺序的三个- 在剪接体的早期阶段指导3'剪接位点选择的三维相互作用 activation. 必需剪接因子U2 AF和SF 1的复合物识别前mRNA 序列邻近3'剪接位点，并反过来稳定核心剪接体的缔合。 前体mRNA通过U2 AF的丝氨酸-丝氨酸（RS）区域接触，以及U2 AF相关的 ATP酶UAP 56是完成这一任务所必需的。该提案的具体目标是解决 以下是关于前体mRNA剪接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的行动是什么 在U2 AF的剪接位点上 (4)RS区通过什么方式促进剪接体snRNAs与 前体mRNA 我们已在实现这些目标方面取得重大进展，包括：（i）确定三项目标： 结合剪接位点的U2 AF的三维结构，（ii）评估热力学和 SF 1结构域对U2 AF结合的结构贡献，以及（iii）表征RNA RS域的相互作用。这些研究将大大推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.