Understanding the Functions of a key RNA Base Pair in the Catalytic Core of the Spliceosome

了解剪接体催化核心中关键 RNA 碱基对的功能

• 批准号: 9765035
• 负责人: Allyson D Yake
• 金额: $ 2.61万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-13 至 2020-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9765035
• 关键词: Acute Myelocytic Leukemia; Address; Affect; Alleles; Attenuated; Base Pairing; Binding; Biochemical; Biochemical Genetics; Biological Assay; Biological Models; Biological Process; Catalysis; Catalytic Domain; Cell physiology; Complex; Cryoelectron Microscopy; DDX16 Gene; Data; Defect; Disease; Dysmyelopoietic Syndromes; Ensure; Event; GTP-Binding Protein alpha Subunits, Gs; Gene Expression; Genetic; Genome; Grant; Growth; Human; In Vitro; Modeling; Molecular Conformation; Mutagenesis; Mutation; Nucleotides; Pathogenicity; Phenotype; Point Mutation; Process; Proteins; Publishing; Purines; Pyrimidine; RNA; RNA Splicing; RNA-Protein Interaction; Regulation; Research; Resolution; Retinitis Pigmentosa; Ribonucleoproteins; Saccharomyces cerevisiae; Saccharomycetales; Severities; Site; Small Nuclear RNA; Spliceosome Assembly Pathway; Spliceosomes; Structural Models; Structure; Suppressor Mutations; Technology; Testing; U4 small nuclear RNA; U6 small nuclear RNA; Work; Yeasts; base; cold temperature; experimental study; genome-wide; helicase; human disease; in vivo; insight; mRNA Precursor; mutant; novel; protein complex; stem; therapeutic development; tool

Abstract Pre-mRNA splicing, an essential step in human gene expression, is catalyzed by a large and dynamic RNA-protein complex called the spliceosome. An understanding of the structure and function of the spliceosome is critical to the development of therapeutics for splicing-associated diseases such as retinitis pigmentosa and myelodysplastic syndromes, and will provide insight into how RNAs and proteins cooperate to carry out cellular functions. U6 RNA is a core component of the spliceosome and undergoes dramatic rearrangements in conformation and binding partners during each splicing event. We aim to determine how the structure of U6 RNA confers its function throughout the splicing cycle, and will use the genetically tractable budding yeast Saccharomyces cerevisiae as our model system. Based on biochemical and genetic evidence, we hypothesize that the highly conserved U6 nucleotides A62 and C85 participate in important RNA-RNA and/or RNA-protein interactions in the splicing cycle that have not yet been characterized. To determine the function(s) of these residues, we will pursue three specific aims. In Aim 1, we will use biochemical assays to determine the arrest point of the mutations U6-A62U/C85A (U6-UA) and U6-A62C/C85G (U6-CG), and will conduct a genetic experiment to relate the mutants’ defects to the function of a spliceosomal helicase. In Aim 2, we will look for mutations that suppress the defects of U6-UA and U6-CG using genome-wide selections and a selection targeted to the spliceosome’s “master regulator” protein Prp8. In Aim 3, we will use mutagenesis, in vivo photocrosslinking, and pull-down assays to determine how mutations in Prp8 suppress the U6-UA defect. This experimentation will be guided by the wealth of structural information available in published models of spliceosomal complexes solved by cryo-electron microscopy. Our proposed research has the potential to (i) identify a novel cold-sensitive block in the splicing cycle, providing an experimental tool with which to better understand the mechanism of splicing, and (ii) reveal a cascade of molecular interactions important to ensure accurate splicing.

抽象的 前mRNA剪接是人类基因表达的重要步骤，由大而动态的催化 RNA蛋白质复合物称为剪接体。了解的结构和功能 剪接体对于开发用于剪接相关疾病（例如视网膜炎）的治疗剂至关重要 色素和骨髓增生综合征，将提供有关RNA和蛋白质如何合作的见解 执行细胞功能。 U6 RNA是剪接体的核心组成部分，并且经历了戏剧性 在每个剪接事件中，以构象和绑定伙伴进行重新排列。我们旨在确定如何 U6 RNA的结构在整个剪接周期中供认其功能，并将使用一般可拖动 酿酒酵母作为我们的模型系统。基于生化和遗传证据， 我们假设高度保守的U6核苷酸A62和C85参与了重要的RNA-RNA 在剪接周期中尚未表征的剪接周期中的RNA-蛋白质相互作用。确定 这些残基的功能，我们将追求三个具体目标。在AIM 1中，我们将使用生化测定 确定突变U6-A62U/C85A（U6-UA）和U6-A62C/C85G（U6-CG）的逮捕点，并将 进行遗传实验，将突变体的缺陷与剪接体解旋酶的功能联系起来。目标 2，我们将寻找使用全基因组选择和抑制U6-UA和U6-CG缺陷的突变 针对剪接体的“主调节剂”蛋白PRP8的选择。在AIM 3中，我们将在 Vivo Photocrosplink和下拉的刺客，以确定PRP8中的突变如何抑制U6-UA缺陷。 该实验将由已发表模型的大量结构信息指导 通过冷冻电子显微镜求解的剪接体复合物。我们提出的研究有可能（i） 在剪接周期中识别一种新型的冷敏感块，提供了一个实验工具，以改善该工具 了解剪接机制，（ii）揭示了一系列分子相互作用，以确保 准确的剪接。