Structure and Function of U6 Spliceosomal RNA

描述（由申请人提供）：剪接体是核糖核蛋白的大型动态组装体，其催化内含子从前信使RNA中去除。剪接体RNA和蛋白质是真核生物基因表达的关键，但对它们的结构和功能知之甚少。这项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，并了解这些结构在整个剪接周期中是如何变化的。目的1研究U6 RNA-Prp 24核糖核蛋白复合物的结构和功能。Prp 24蛋白与游离U6 snRNP中的U6 RNA紧密结合，并且在剪接体组装和激活期间是U6 RNA结构重塑所需的。我们最近解决了Prp 24的N-末端三分之二的晶体结构，并通过NMR定义了一个假定的U6 RNA结合表面。U6 RNA的结构将使用NMR和X-射线研究单独测定和与Prp 24复合测定。这些数据将揭示Prp 24如何重塑U6 RNA结构，以促进在剪接体组装期间与U4 RNA配对，以及在剪接体活化期间与U2 RNA配对。体外生物化学和体内遗传学研究将用于进一步探索Prp 24作为U6 RNA伴侣的机制。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，并与活性位点中的前mRNA底物直接氢键结合。此外，人U2-U6复合物在不存在蛋白质的情况下具有残余催化活性。为了这个目的，我们已经确定了一个良好的折叠片段U2-U6 RNA，保持三级结构，并服从NMR结构测定。作为一个子目标，我们将合作开发新的方法，用于针对小角X射线散射（SAXS）数据和伪CSA约束来细化大型多域RNA NMR结构。我们期望这些方法将是普遍的实用程序的大分子在溶液中的结构测定。Prp 24与U2-U6 RNA构建体结合，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/或分解的真正中间体。这些研究将大大推进我们对驱动剪接体组装、激活、催化和分解的变构级联的理解。Narrative 剪接体是一个复杂的细胞机器，由5个RNA和超过75种蛋白质组成。剪接体对细胞至关重要，即使是其中一个组成部分的微小缺陷也会导致视网膜色素变性，从而导致失明。我们的工作将使我们更好地理解这种非凡的分子机器处理信使RNA的机制，从而使蛋白质能够被细胞正确地制造。

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.