Studies of the structural rearrangements associated with the dynamic spliceosome

 描述(申请人提供)：剪接体是一种动态的大分子机器，它催化从前信使RNA(前信使RNA)中切除非编码内含子形成成熟信息(MRNA)，这一过程被称为前信使RNA剪接。~3MDA复合体由RNA和蛋白质组成，由四个小的核核糖核蛋白亚基(SNRNPs；U1、U2、U5、U4/U6)和许多非SnRNP剪接因子以一系列调控步骤组装而成。虽然剪接体是一种基本的细胞机器，但其复杂和动态的性质使得获取结构信息具有挑战性。不存在分辨率高于30？的多核小核糖核(SnRNP)复合体的结构。剪接体中RNA和蛋白质在剪接反应的任何阶段的分子组织尚不清楚，在从剪接前复合体到剪接后复合体的转变过程中发生的整体构象变化也尚未被表征。这项提议的目的是定义从激活前的剪接体到激活后的剪接体的转变所需的构象变化。在目标1中，我们将比较激活前、激活和激活后剪接体的结构，以绘制前mRNA剪接所需的整体构象变化。在目标2中，我们将定义预激活、激活和激活后剪接体的分子组织并绘制近端蛋白质-蛋白质相互作用网络图。在目标3中，我们将使用结构/功能研究来确定SF3复合体在催化激活过程中的作用，以及在具有该亚复合体突变的骨髓增生异常综合征(MDS)和慢性淋巴细胞白血病(CLL)患者中这一功能是如何改变的。这项工作将提供对激活所需的构象变化的直接洞察，并定义从激活前的复合体到激活后的复合体的转变过程中的剪接体组织。这些目标的完成将极大地促进我们对剪接体在健康和疾病条件下的结构和组织的理解。

 DESCRIPTION (provided by applicant): The spliceosome is a dynamic macromolecular machine that catalyzes the excision of non-coding introns from pre-messenger RNAs (pre-mRNA) to form mature messages (mRNA), a process called pre- mRNA splicing. The ~3 MDa complex, composed of RNAs and proteins, assembles in a series of regulated steps from four small nuclear ribonucleoprotein subunits (snRNPs; U1, U2, U5, U4/U6) and numerous non- snRNP splicing factors. While the spliceosome is a fundamental cellular machine, its complex and dynamic nature has made obtaining structural information challenging. There are no structures of multi-snRNP (small nuclear ribonucleic particle) complexes at resolutions better than 30Å. The molecular organization of RNA and proteins within the spliceosome at any stage of the splicing reaction is not known and the global conformational changes that occur during the transitions from a pre- to post-splicing complex have not been characterized. The goal of this proposal is to define the conformational changes required for the transition from a pre- to post- activated spliceosome. In Aim 1 we will compare the structures of pre-activated, activated, and post-activated spliceosomes to map the global conformational changes required for pre-mRNA splicing. In Aim 2 we will define the molecular organization and map proximal protein-protein interaction networks of pre-activated, activated, and post-activated spliceosomes. In Aim 3 we will use structure/function studies to determine the role of the SF3 complex during catalytic activation and how this function is altered in Myelodysplastic Syndromes (MDS) and Chronic Lymphocytic Leukemia (CLL) patients that have mutations in this sub-complex. This work will provide direct insight into the conformational changes required for activation and define spliceosome organization during the transitions from a pre- to post-activated complex. Completion of these aims will significantly advance our understanding of the structure and organization of the spliceosome under conditions of both health and disease.