Studies of the structural rearrangements associated with the dynamic spliceosome

 描述（由申请人提供）：剪接体是一种动态大分子机器，其催化从前信使RNA（前mRNA）切除非编码内含子以形成成熟信使（mRNA），该过程称为前mRNA剪接。由RNA和蛋白质组成的~3 MDa复合物在一系列受调控的步骤中由四个小的核核糖核蛋白亚基（snRNP; U1、U2、U 5、U4/U6）和许多非snRNP剪接因子组装。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得结构信息具有挑战性。在30 μ m以上的分辨率下，没有发现多snRNP（小核核糖核酸颗粒）复合物的结构。在剪接反应的任何阶段，剪接体内RNA和蛋白质的分子组织尚不清楚，并且在从剪接前复合物到剪接后复合物的过渡期间发生的全局构象变化尚未被表征。该提议的目标是定义从激活前到激活后剪接体转变所需的构象变化。在目的1中，我们将比较预激活，激活，和激活后剪接体的结构，以映射前mRNA剪接所需的全局构象变化。在目标2中，我们将定义的分子组织和地图近端蛋白质-蛋白质相互作用网络的预激活，激活和激活后剪接体。在目标3中，我们将使用结构/功能研究来确定SF 3复合物在催化活化过程中的作用，以及这种功能在骨髓增生异常综合征（MDS）和慢性淋巴细胞白血病（CLL）患者中如何改变，这些患者在这种亚复合物中有突变。这项工作将提供直接洞察激活所需的构象变化，并定义剪接体组织从激活前到激活后的复杂过渡过程中。这些目标的完成将大大推进我们对健康和疾病条件下剪接体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.