Structural Analyses of Fission Yeast Splicing Complexes

裂变酵母剪接复合物的结构分析

• 批准号: 6932393
• 负责人: Kathleen L Gould
• 金额: $ 35.77万
• 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 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6932393
• 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械，可催化从预上音符RNA（前MRNA）中切除非编码内含子的切除。它由五个小的核核糖核蛋白亚基（SNRNP）和许多非SNRNP剪接因子形成。但是，尚不清楚如何在较大的单元内组织SNRNP来执行前MRNA剪接的催化步骤。通过靶向剪接因子CDC5P进行串联亲和力纯化，我们从裂变酵母schizosacachomyces pombe纯化了稳定的37S剪接样粒子。该复合物包含最著名的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遗传操作来增强我们对这种粒子的理解，该粒子的组成表明它类似于在剪接的第二步中捕获的人类剪接体。总体而言，我们的目标是使用冷冻电子显微镜和单个粒子重建技术来确定该剪接体复合物的三维结构，以提供U2，U5，U6，U6，U6-SNRNP的拼接复合物的整体组织。我们还提出了旨在进一步理解裂变酵母中剪接体组装的实验和剪接体复合物中蛋白-RNA相互作用的理解。这些研究构成了对核心剪接体结构和功能更全面理解的重要一步，如果我们要阐明影响剪接场所选择的机制以及影响生长和发展的蛋白质多样性，这是必要的。