Structure and Function of U6 Spliceosomal RNA

描述(申请人提供)：剪接体是核糖核蛋白的一个大的，动态的组装，催化从前信使RNA中去除内含子。剪接体RNA和蛋白质是真核基因表达的关键，但人们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复合体和核糖核蛋白的结构，并了解这些结构在剪接周期中是如何变化的。目的1研究U6RNA-Prp24核糖核蛋白复合体的结构和功能。Prp24蛋白与U6 RNA紧密结合，在剪接体组装和激活过程中，Prp24蛋白是U6 RNA结构重塑所必需的。我们最近解决了Prp24的N端三分之二的晶体结构，并通过核磁共振定义了一个可能的U6 RNA结合面。U6 RNA的结构将通过核磁共振和X射线研究单独确定，也将与Prp24形成络合物。这些数据将揭示Prp24如何重塑U6 RNA结构，以促进在剪接体组装过程中与U4 RNA配对，并在剪接体激活过程中与U2 RNA配对。体外生化和体内遗传学研究将被用来进一步探索Prp24作为U6 RNA伴侣的机制。该提案的目标2是确定U2-U6 RNA复合体的结构。U2-U6复合体位于剪接体的催化核心内，并直接与活性部位的前mRNA底物氢键。此外，人的U2-U6复合体在没有蛋白质的情况下仍具有催化活性。为此，我们鉴定了一个折叠良好的U2-U6 RNA片段，它保持了三级结构，并适合于核磁共振结构测定。作为一个子目标，我们将合作开发新的方法来针对小角X射线散射(SAXS)数据和伪CSA约束提纯大的多域RNA核磁共振结构。我们希望这些方法将对溶液中大分子的结构测定具有普遍的实用价值。Prp24与U2-U6 RNA结构结合，我们将试图确定这个核糖核蛋白复合体的结构，它可能模拟剪接体激活和/或拆卸中的真实中间体。这些研究将极大地促进我们对驱动剪接体组装、激活、催化和分解的变构级联的理解。 剪接体是一个复杂的细胞机器，由5个RNA和超过75个蛋白质组成。剪接体对细胞是如此重要，即使它的一个组成部分存在微小的缺陷，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，从而使细胞能够正确地制造蛋白质。

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.