Structural Analyses of Fission Yeast Splicing Complexes

裂变酵母剪接复合物的结构分析

• 批准号: 7118219
• 负责人: Kathleen L Gould
• 金额: $ 35.89万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7118219
• 关键词: RNA splicing; Schizosaccharomyces pombe; cryoelectron microscopy; fungal genetics; gene mutation; intermolecular interaction; molecular assembly /self assembly; nucleic acid purification; precursor mRNA; protein localization; protein structure function; small nuclear ribonucleoproteins; spliceosomes

DESCRIPTION (provided by applicant): The spliceosome is a complex macromolecular machinery that catalyses the excision of non-coding introns from a pre-messenger RNA (pre-mRNA). It is formed from five small nuclear ribonucleoprotein subunits (snRNPs) and numerous non-snRNP splicing factors. However, how the snRNPs are organized within a larger unit to execute the catalytic steps of pre-mRNA splicing is not known. By targeting the splicing factor Cdc5p for tandem affinity purification, we have purified a stable 37S spliceosomal-like particle from the fission yeast, Schizosaccharomyces pombe. This complex contains most known U2 and U5 snRNP proteins, the U2, U5, and U6 snRNAs, and numerous uncharacterized splicing factors. Our project proposes to utilize the facile genetic manipulations available in yeast to enhance our understanding of this particle whose composition suggests that it is similar to the human spliceosome arrested at the second step of splicing. Overall, our goal is to determine the three-dimensional structure of this spliceosomal complex using cryo-electron microscopy and single particle reconstruction techniques to provide a first view of the overall organization of a U2, U5, U6-snRNP-containing splicing complex. We also propose experiments designed to further our understanding of spliceosome assembly in fission yeast and protein-RNA interaction within spliceosomal complexes. These studies constitute a significant step towards achieving a more comprehensive understanding of core spliceosome structure and function, which is necessary if we are to elucidate the mechanisms governing splice site selection and protein diversity that impact growth and development.

描述（由申请人提供）：剪接体是一种复杂的大分子机制，可催化前信使 RNA（pre-mRNA）中非编码内含子的切除。它由五个小核核糖核蛋白亚基 (snRNP) 和许多非 snRNP 剪接因子形成。然而，snRNP 如何组织在更大的单元内以执行前 mRNA 剪接的催化步骤尚不清楚。通过针对剪接因子 Cdc5p 进行串联亲和纯化，我们从裂殖酵母裂殖酵母中纯化了稳定的 37S 剪接体样颗粒。该复合物包含大多数已知的 U2 和 U5 snRNP 蛋白、U2、U5 和 U6 snRNA，以及许多未表征的剪接因子。我们的项目建议利用酵母中可用的简便遗传操作来增强我们对这种颗粒的理解，该颗粒的组成表明它与剪接第二步中捕获的人类剪接体相似。总体而言，我们的目标是使用冷冻电子显微镜和单粒子重建技术确定该剪接体复合物的三维结构，以提供包含 U2、U5、U6-snRNP 剪接复合物的整体组织的第一视角。我们还提出了一些实验，旨在进一步了解裂殖酵母中的剪接体组装以及剪接体复合物中蛋白质-RNA 相互作用。这些研究是朝着更全面地了解核心剪接体结构和功能迈出的重要一步，如果我们要阐明影响生长和发育的剪接位点选择和蛋白质多样性的控制机制，这是必要的。