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，催化前信使RNA（pre-mRNA）中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。然而，snRNP如何组织在一个更大的单元内以执行前体mRNA剪接的催化步骤尚不清楚。通过靶向剪接因子Cdc 5 p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37 S剪接体样颗粒。这种复合物包含大多数已知的U2和U 5 snRNP蛋白，U2，U 5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单遗传操作来增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被捕的人类剪接体。总的来说，我们的目标是使用冷冻电子显微镜和单颗粒重建技术来确定这种剪接体复合物的三维结构，以提供U2，U 5，U6-snRNP包含剪接复合物的整体组织的第一视图。我们还提出了实验设计，以进一步了解剪接体组装在裂变酵母和蛋白质-RNA相互作用内剪接体复合物。这些研究构成了一个重要的一步，实现更全面的了解核心剪接体的结构和功能，这是必要的，如果我们要阐明剪接位点选择和蛋白质多样性的影响生长和发育的机制。

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.