Structural Analyses of Fission Yeast Splicing Complexes

描述（由申请人提供）：剪接体是一种复杂的大分子机制，可以催化从前信使RNA （pre-mRNA）中切除非编码内含子。它由5个小核核糖核蛋白亚基（snRNPs）和许多非snrnp剪接因子组成。然而，snRNPs如何在一个更大的单元内组织以执行pre-mRNA剪接的催化步骤尚不清楚。通过靶向剪接因子Cdc5p进行串联亲和纯化，我们从裂糖酵母（Schizosaccharomyces pombe）中纯化出一个稳定的37S剪接体样颗粒。该复合体包含最著名的U2和U5 snRNP蛋白，U2、U5和U6 snRNAs，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单遗传操作来增强我们对这种颗粒的理解，其组成表明它类似于在剪接的第二步中捕获的人类剪接体。总的来说，我们的目标是利用低温电子显微镜和单粒子重建技术确定这种剪接体复合体的三维结构，以提供含有U2， U5， u6 - snrnp的剪接复合体的整体组织的第一个视图。我们还提出了旨在进一步了解裂变酵母剪接体组装和剪接体复合体内蛋白质- rna相互作用的实验。这些研究是实现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。

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.