Structural Analyses of Fission Yeast Splicing Complexes

描述(申请人提供)：剪接体是一种复杂的大分子机器，催化从前信使RNA(前信使RNA)中切除非编码内含子。它由5个小的核核糖核蛋白亚基和许多非核糖核蛋白剪接因子组成。然而，SnRNP如何在一个更大的单位内组织以执行前mRNA剪接的催化步骤尚不清楚。通过将剪接因子CDC5p用于串联亲和纯化，我们从裂殖酵母中纯化了一个稳定的37S剪接体样颗粒。这个复合体包含最已知的U2和U5SnRNP蛋白，U2，U5和U6 SnRNAs，以及许多未知的剪接因子。我们的项目建议利用酵母中可用的简便遗传操作来加强我们对这种颗粒的理解，这种颗粒的组成表明它类似于在剪接的第二步阻止的人类剪接体。总体而言，我们的目标是使用冷冻电子显微镜和单粒子重建技术来确定这个剪接体复合体的三维结构，以提供包含U2、U5、U6-SnRNP的剪接复合体的整体组织的第一视图。我们还提出了旨在加深我们对裂解酵母中剪接体组装和剪接体复合体中蛋白质-RNA相互作用的理解的实验。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发育的剪接位点选择和蛋白质多样性的机制，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.