Structure and Function of U6 Spliceosomal RNA

描述（由申请人提供）：剪接体是一个巨大的、动态的核糖核蛋白组装体，它催化从前信使rna中去除内含子。剪接体rna和蛋白质对真核生物基因表达至关重要，但对其结构和功能了解甚少。本研究的目的是阐明剪接体中高度保守的RNA复合物和核糖核蛋白的结构，并了解其结构在整个剪接周期中的变化。目的1是研究U6 RNA-Prp24核糖核蛋白复合物的结构和功能。Prp24蛋白在游离的U6 snRNP中与U6 RNA紧密结合，是剪接体组装和激活过程中U6 RNA结构重塑所必需的。我们最近解决了三分之二的Prp24的n端晶体结构，并通过核磁共振确定了一个假定的U6 RNA结合表面。U6 RNA的结构将通过核磁共振和X射线研究单独和与Prp24结合来确定。这些数据将揭示Prp24如何重塑U6 RNA结构，以促进剪接体组装过程中与U4 RNA的配对，以及剪接体激活过程中与U2 RNA的配对。体外生化和体内遗传学研究将进一步探讨Prp24作为U6 RNA伴侣的作用机制。该提案的目的2是确定U2-U6 RNA复合物的结构。U2-U6复合物位于剪接体的催化核心内，并直接与活性位点的pre-mRNA底物形成氢键。此外，人的U2-U6复合物在缺乏蛋白质的情况下具有残余的催化活性。为此，我们鉴定了一个折叠良好的U2-U6 RNA片段，该片段保持三级结构，可用于核磁共振结构测定。作为子目标，我们将合作开发针对小角度x射线散射（SAXS）数据和伪csa约束的大，多域RNA核磁共振结构的新方法。我们期望这些方法对溶液中大分子的结构测定具有普遍的实用价值。Prp24结合到U2-U6 RNA结构，我们将尝试确定这种核糖核蛋白复合物的结构，它可能模拟剪接体激活和/或拆卸的真正中间体。这些研究将极大地促进我们对驱动剪接体组装、激活、催化和拆卸的变构级联的理解。叙述

DESCRIPTION (provided by applicant): The spliceosome is a large, dynamic assembly of ribonucleoproteins that catalyzes intron removal from pre- messenger RNAs. Spliceosomal RNAs and proteins are critical for eukaryotic gene expression, yet little is understood about their structure and function. The goal of the proposed research is to elucidate the structures of highly conserved RNA complexes and ribonucleoproteins from the spliceosome, and understand how the structures change throughout the splicing cycle. Aim 1 is to investigate the structure and function of the U6 RNA-Prp24 ribonucleoprotein complex. Prp24 protein tightly binds to U6 RNA in the free U6 snRNP and is required for U6 RNA structural remodeling during spliceosome assembly and activation. We recently solved the crystal structure of the N-terminal two-thirds of Prp24, and defined a putative U6 RNA binding surface by NMR. The structure of U6 RNA will be determined alone and in complex with Prp24, using both NMR and X- ray studies. These data will reveal how Prp24 remodels the U6 RNA structure to facilitate pairing with U4 RNA during spliceosome assembly, and with U2 RNA during spliceosome activation. In vitro biochemical and in vivo genetic studies will be used to further probe the mechanism by which Prp24 acts as a U6 RNA chaperone. Aim 2 of the proposal is to determine the structure of the U2-U6 RNA complex. The U2-U6 complex resides within the catalytic core of the spliceosome and directly hydrogen bonds to the pre-mRNA substrate in the active site. Furthermore, the human U2-U6 complex has residual catalytic activity in the absence of proteins. For this aim, we have identified a well-folded fragment of U2-U6 RNA that maintains tertiary structure and is amenable to NMR structure determination. As a sub-aim, we will collaboratively develop new methods for refining large, multi-domain RNA NMR structures against small angle X-ray scattering (SAXS) data and pseudo-CSA restraints. We expect these methods will be of general utility for structure determination of large macromolecules in solution. Prp24 binds to the U2-U6 RNA construct and we will attempt to determine the structure of this ribonucleoprotein complex, which may mimic a true intermediate in spliceosome activation and/or disassembly. These studies will significantly advance our understanding of the allosteric cascade that drives spliceosome assembly, activation, catalysis and disassembly.Narrative The spliceosome is an intricate cellular machine composed of 5 RNAs and more than 75 proteins. The spliceosome is so vital to the cell that even a subtle defect in just one of its components results in the disease retinitis pigmentosa, which causes blindness. Our work will result in a better understanding of the mechanism by which this remarkable molecular machine processes messenger RNA so that proteins can be made correctly by cells.