Structure and Function of U6 Spliceosomal RNA

U6 剪接体 RNA 的结构和功能

• 批准号: 7371205
• 负责人: DAVID A BROW
• 金额: $ 27.36万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7371205
• 关键词: 3' Splice Site; Active Sites; Alternative Splicing; Alzheimer' s Disease; Anisotropy; Base Pairing; Binding; Biochemical; Biochemical Genetics; Biochemistry; Biological Models; Blindness; Catalysis; Catalytic Domain; Cells; Chemicals; Complex; Data; Defect; Disease; Excision; Exons; Funding; Gene Expression; Genes; Genetic; Goals; Health; Heterogeneous Nuclear RNA; Histocompatibility Testing; Human; Human Genome; Hydrogen Bonding; In Vitro; Introns; Left; Life; Messenger RNA; Methods; Modeling; Molecular Chaperones; Molecular Machines; N-terminal; Neoplasm Metastasis; Neurodegenerative Disorders; Nuclear; Nucleotides; Organism; Parkinson Disease; Price; Process; Protein Binding; Proteins; Proteome; RNA; RNA Binding; RNA Splicing; Rate; Research; Residual state; Retinitis Pigmentosa; Ribonucleoproteins; Roentgen Rays; Saccharomyces cerevisiae; Small Nuclear Ribonucleoproteins; Small RNA; Solutions; Source; Spliceosome Assembly Pathway; Spliceosomes; Structure; Surface; Testing; U2 small nuclear RNA; U4 small nuclear RNA; U6 Small Nuclear Ribonucleoproteins; U6 small nuclear RNA; Work; Yeasts; base; in vivo; mRNA Precursor; macromolecule; novel; programs; restraint; size; tumor progression

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.

描述（由申请人提供）：剪接体是核糖核蛋白的大型动态组装，可催化内含子从前Messenger RNA中删除。剪接体RNA和蛋白质对于真核基因表达至关重要，但对其结构和功能几乎没有理解。拟议的研究的目的是阐明从剪接体中高度保守的RNA复合物和核糖核蛋白的结构，并了解整个剪接周期中结构的变化。 AIM 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U6 SNRNP中与U6 RNA紧密结合，在剪接组装和激活过程中，U6 RNA结构重塑所必需。我们最近解决了PRP24的N末端三分之二的晶体结构，并通过NMR定义了假定的U6 RNA结合表面。使用NMR和X-Ray研究，将单独确定U6 RNA的结构，并与PRP24进行复杂。这些数据将揭示PRP24在剪接体组装过程中如何重塑U6 RNA结构，以促进与U4 RNA配对，以及在剪接体激活过程中使用U2 RNA。体外生化和体内遗传研究将用于进一步探测PRP24充当U6 RNA伴侣的机制。该提案的目标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，并直接氢键在活性位点中的前MRNA底物氢键。此外，在没有蛋白质的情况下，人类U2-U6复合物具有残留的催化活性。为此，我们已经确定了一个折叠的U2-U6 RNA片段，该片段维持第三级结构，并且可以根据NMR结构的确定。作为一个子-IAM，我们将协作开发新的方法，用于针对小角度X射线散射（SAXS）数据和伪CSA约束来精炼大型多域RNA NMR结构。我们预计这些方法将是溶液中大型大分子的结构确定的一般效用。 PRP24与U2-U6 RNA构建体结合，我们将尝试确定该核糖核蛋白络合物的结构，该结构可能模仿剪接体激活和/或拆卸时的真实中间体。这些研究将大大提高我们对驱动剪接组装，激活，催化和拆卸的变构级联的理解。 剪接体是由5个RNA和超过75种蛋白质组成的复杂蜂窝机。剪接体对细胞至关重要，即使仅在其成分之一中的细微缺陷也会导致色素性视网膜炎，这会导致失明。我们的工作将更好地理解这种出色的分子机器处理信使RNA的机制，从而可以通过细胞正确制作蛋白质。