Mechanisms for Rearranging RNA during Pre-mRNA Splicing

Pre-mRNA 剪接过程中 RNA 重排机制

• 批准号: 8446368
• 负责人: JONATHAN P STALEY
• 金额: $ 30.01万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8446368
• 关键词: ATP phosphohydrolase; Biochemistry; Biogenesis; Biological Assay; Blindness; Boxing; Catalysis; Catalytic Domain; Cell Nucleus; Cells; Chemicals; Chemistry; Couples; Cytoplasm; DNA Sequence Rearrangement; Data; Defect; Discrimination; Disease; Docking; Etiology; Exons; Family; Gene Expression; Genes; Genetic; Goals; Guanosine Triphosphate Phosphohydrolases; Human; In Vitro; Introns; Kinetics; Ligation; Measures; Molecular Conformation; Molecular Genetics; Mutation; Pathway interactions; Play; Process; Proteins; Proteomics; RNA; RNA Splicing; Reaction; Recycling; Regulation; Retinitis Pigmentosa; Ribonucleoproteins; Role; Saccharomycetales; Site; Small Nuclear RNA; Specificity; Spliceosomes; Staging; Structure; Technology; Telomerase RNA Component; Testing; Time; U1 small nuclear RNA; U2 Small Nuclear Ribonucleoprotein; Ubiquitin; Untranslated RNA; Work; Yeasts; cost effective; deep sequencing; human disease; in vitro Assay; insight; mRNA Precursor; meetings; member; next generation sequencing; novel strategies; prevent; public health relevance; research study; single molecule; single-molecule FRET; stem; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): Splicing is essential and prevalent in humans. Human genes in are interrupted on average by eight introns. Moreover, nearly 95% of human genes are alternatively spliced at least 15% of the time, providing a versatile mechanism to regulate gene expression quantitatively and qualitatively. Not surprisingly then, at least 15% of human diseases derive from defects in pre-mRNA splicing. Yet, our mechanistic understanding of the spliceosome remains primitive. In the spliceosome, a ribonucleoprotein machine composed of five small nuclear RNAs (snRNAs) and 80 conserved proteins, the snRNAs play key roles in both substrate recognition and likely catalysis. Throughout the splicing cycle the snRNA components undergo dramatic rearrangements thought to require eight conserved members of the DExD/H box family of ATPases. Our long-term goal is to understanding the function and mechanism of the DExD/H box ATPases and the snRNA rearrangements they catalyze. In this proposal, we aim to capitalize on our recent advances. First, by establishing unique assays for the critical but poorly understood fidelity activities in splicing, we have found evidence in vitro that each chemical step in splicing is proofread by a specific DExD/H box ATPase to reject suboptimal substrates and further that a distinct DExD/H box ATPase functions at both chemical steps to discard rejected substrates. However, it remains unclear how these DExD/H box ATPases permit discrimination between optimal and suboptimal substrates and whether kinetic proofreading plays a role. Second, we have also implicated a dual role for the DExD/H box ATPase Brr2 and regulation of Brr2 by a GTPase and ubiquitin, but the mechanism and utility of these switches in the splicing cycle remains to be investigated. Third, the DExD/H box ATPase Prp2 is the last ATPase required in spliceosome activation, but the function of this ATPase in promoting the catalytic conformation of the spliceosome remains enigmatic. Through our studies of the role of the DExD/H box ATPase Prp16 in rearranging the catalytic conformation of the spliceosome, we have gained insight into the snRNA configuration of the catalytic conformation and potential consequences of Prp2-dependent RNA rearrangements. Thus, the aims of this proposal are i) to define the ATP dependent mechanism for establishing fidelity during exon ligation, ii) to investigate a role for ubiquitin in regulating Brr2-dependent spliceosome activation and disassembly and iii) to investigate the role of Prp2 in spliceosome activation. We will pursue these aims using a combined approach of genetics and biochemistry in budding yeast, while exploiting emerging single molecule and next generation sequencing technologies. Through these studies, we expect to advance our understanding of the role of DExD/H box ATPases in fidelity, the mechanisms for regulating these ATPases, and the function of these ATPases in rearranging noncoding RNA. Further, given our recent implication of hBrr2-defective RNA unwinding in the etiology of autosomal dominant retinitis pigmentosa, this work will also have important implications for this common form of blindness.

描述（由申请人提供）：剪接在人类中至关重要且普遍存在。人类基因平均被八个内含子打断。此外，将近95％的人类基因至少在15％的时间内被剪接，提供了一种多功能机制来定量和质量地调节基因表达。毫不奇怪，至少有15％的人类疾病来自前MRNA剪接中的缺陷。然而，我们对剪接体的机械理解仍然是原始的。在剪接体中，由五个小核RNA（SNRNA）和80个保守蛋白组成的核糖核蛋白机器在底物识别和可能的催化中起关键作用。在整个剪接周期中，SNRNA组件经历了戏剧性的重排，被认为需要八个ATPases的DEXD/H Box家族的保守成员。我们的长期目标是了解DEXD/H Box ATPases的功能和机制以及它们催化的SNRNA重排。在此提案中，我们旨在利用我们最近的进步。首先，通过建立对剪接中关键但知之甚少的保真度活动的独特测定，我们发现了体外的证据表明，剪​​接中的每个化学步骤都是由特定的DEXD/H盒ATPase校对的，以拒绝次优基质，进一步拒绝脱落的DEXD/H BOX ATPase函数在两种化学上拒绝拒绝的底物。但是，尚不清楚这些DEXD/H框ATPases如何允许歧视最佳和次优基质以及动力学校对是否起作用。其次，我们还暗示了DEXD/H Box ATPase BRR2的双重作用以及通过GTPase和泛素对BRR2的调节，但是在剪接周期中，这些切换的机制和效用仍有待研究。第三，DEXD/H盒ATPase PRP2是剪接体激活中所需的最后一个ATPase，但是该ATPase在促进剪接体的催化构象中的功能仍然神秘。通过研究DEXD/H Box ATPase PRP16在重新排列剪接体催化构象中的作用，我们已经深入了解了PRP2依赖性RNA重排的催化构象和潜在后果的SnRNA构型。因此，该提案的目的是i）定义依赖于ATP的机制，以在外显子结扎期间建立忠诚度，ii）研究泛素在调节BRR2依赖性剪接体激活中的作用，并拆卸和III）研究PRP2在夹层体激活中的作用。我们将利用新兴的酵母中的遗传学和生物化学方法来追求这些目标，同时利用新兴的单分子和下一代测序技术。通过这些研究，我们期望我们对DEXD/H框ATPases在忠诚度的作用，调节这些ATP酶的机制以及这些ATPase在重新排列非编码RNA中的功能的理解。此外，鉴于我们最近在常染色体显性视网膜炎色素炎的病因中对HBRR2缺陷RNA的含义，这项工作也将对这种常见的失明形式具有重要意义。