Structure and Function of U6 Spliceosomal RNA

U6 剪接体 RNA 的结构和功能

• 批准号: 8440678
• 负责人: DAVID A BROW
• 金额: $ 33.22万
• 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 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8440678
• 关键词: Base Pairing; Binding; Binding Proteins; Biochemical; Biological; Blindness; Bypass; Calorimetry; Catalysis; Cells; Characteristics; Code; Complex; DNA Sequence Rearrangement; Data; Defect; Diagnostic; Disease; Engineering; Eukaryota; Eukaryotic Cell; Failure; Fluorescence Resonance Energy Transfer; Foundations; Funding; Genes; Genetic; Human; In Vitro; Inborn Genetic Diseases; Individual; Introns; Investigation; Kinetics; Knowledge; Life; Maps; Measures; Messenger RNA; Molecular; Molecular Chaperones; Molecular Machines; Mutation; Organism; Participant; Process; Production; Protein Splicing; Proteins; RNA; RNA Splicing; RNA-Protein Interaction; Reaction; Recombinants; Retinitis Pigmentosa; Ribosomes; Signal Transduction; Site; Small Nuclear Ribonucleoproteins; Spliceosome Assembly Pathway; Spliceosomes; Structure; Testing; Therapeutic Intervention; Thermodynamics; Transcript; U1 small nuclear RNA; U2 small nuclear RNA; U6 small nuclear RNA; Work; Yeasts; human disease; in vivo; mRNA Precursor; mutant; nanomachine; novel; public health relevance; reconstitution; scaffold; stem; tool

DESCRIPTION (provided by applicant): The spliceosome is an intricate biological nanomachine that is unique to the eukaryotic lineage of life. It performs the essential process of removing introns from the primary transcripts of eukaryotic genes, thus creating functional messenger RNAs for protein production. Failure of the spliceosome to accurately and efficiently remove introns, either due to mutations in individual introns or in spliceosome components, results in a range of human diseases including autosomal dominant retinitis pigmentosa (adRP), which causes blindness. We propose to continue our ongoing investigation into the molecular mechanisms of wild-type and mutant spliceosome function. Five small nuclear RNAs, U1, U2, U4, U5 and U6, are central to the splicing process. Each RNA associates with proteins to form a snRNP, and the five snRNPs assemble on an intron to reconstitute a spliceosome. After assembly, a complex activation process ensues that results in expulsion of the U1 and U4 snRNPs, and alignment of the intron splice sites on a U2/U6/U5 scaffold. It is thought that the U2/U6 complex then carries out the two catalytic steps of splicing in conjunction with key protein factors. Our studies will follow the progression of U6 RNA through the splicing cycle. We will determine the mechanism of assembly of the U4/U6 di-snRNP, which is thought to keep U6 in an inactive state during the spliceosome assembly process. In particular, we will test our hypothesis that U2 RNA is actively involved in this process, as well as the proteins Prp24 and Slt11. During the subsequent transition from U4/U6 complex to U2/U6 complex, U6 RNA changes structure to form a remarkably conserved internal stem-loop (ISL), which is thought to be required for catalysis of splicing. Engineered mutations that further stabilize the ISL result i a cold-sensitive block to splicing, providing a genetic and biochemical tool for studying the interconversion of U4/U6 and U2/U6. We will exploit one such mutation to probe the function of the essential U6 RNA-binding protein Cwc2 in spliceosome activation. We will determine the structures of key sub-complexes identified in our studies. The results of these studies will be a deeper understanding of an ancient molecular machine essential to all eukaryotic cells, and possible diagnostic and therapeutic interventions for devastating human diseases.

描述（由申请人提供）：剪接体是一种复杂的生物纳米机器，是真核生物谱系所特有的。它执行从真核基因的初级转录物中去除内含子的基本过程，从而产生用于蛋白质生产的功能性信使RNA。由于单个内含子或剪接体组分中的突变，剪接体不能准确有效地去除内含子，导致一系列人类疾病，包括常染色体显性视网膜色素变性（adRP），其导致失明。我们建议继续我们正在进行的调查野生型和突变体剪接体功能的分子机制。五个小的核RNA，U1，U2，U4，U 5和U6，是剪接过程的核心。每个RNA与蛋白质结合形成snRNP，五个snRNP在内含子上组装以重建剪接体。组装后，一个复杂的激活过程导致U1和U4 snRNP的排出，以及U2/U6/U 5支架上内含子剪接位点的对齐。据认为，U2/U6复合物然后与关键蛋白因子一起进行剪接的两个催化步骤。我们的研究将通过剪接循环跟踪U6 RNA的进展。我们将确定U4/U6 di-snRNP的组装机制，该机制被认为在剪接体组装过程中使U6保持在非活性状态。特别是，我们将测试我们的假设，即U2 RNA积极参与这一过程，以及蛋白质Prp 24和Slt 11。在随后的从U4/U6复合物到U2/U6复合物的转变过程中，U6 RNA改变结构以形成显著保守的内部茎环（ISL），这被认为是催化剪接所必需的。进一步稳定ISL的工程突变导致剪接的冷敏感性阻断，为研究U4/U6和U2/U6的相互转化提供了遗传和生物化学工具。我们将利用一个这样的突变来探测剪接体激活中必需的U6 RNA结合蛋白Cwc 2的功能。我们将确定在我们的研究中确定的关键子复合物的结构。这些研究的结果将是对所有真核细胞所必需的古老分子机器的更深入了解，以及对毁灭性人类疾病的可能诊断和治疗干预。