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）：剪接在人类中是必需且普遍的。人类基因平均被八个内含子打断。此外，近 95% 的人类基因至少在 15% 的时间内进行选择性剪接，这提供了一种定量和定性调节基因表达的通用机制。毫不奇怪，至少 15% 的人类疾病源自前体 mRNA 剪接缺陷。然而，我们对剪接体的机械理解仍然很原始。在剪接体中，一种由 5 个小核 RNA (snRNA) 和 80 个保守蛋白组成的核糖核蛋白机器，snRNA 在底物识别和可能的催化中发挥着关键作用。在整个剪接周期中，snRNA 成分经历剧烈的重排，被认为需要 ATP 酶 DExD/H 盒家族的八个保守成员。我们的长期目标是了解 DExD/H 盒 ATP 酶的功能和机制以及它们催化的 snRNA 重排。在这项提案中，我们的目标是利用我们最近的进展。首先，通过对剪接中关键但知之甚少的保真度活性建立独特的测定，我们在体外发现了证据，表明剪接中的每个化学步骤都由特定的 DExD/H 盒 ATP 酶校对，以拒绝次优底物，并且进一步证明，不同的 DExD/ H 盒 ATP 酶在两个化学步骤中均发挥作用，以丢弃被拒绝的底物。然而，目前尚不清楚这些 DExD/H 盒 ATP 酶如何区分最佳和次优底物以及动力学校对是否发挥作用。其次，我们还暗示了 DExD/H 盒 ATP 酶 Brr2 的双重作用以及 GTP 酶和泛素对 Brr2 的调节，但这些开关在剪接周期中的机制和效用仍有待研究。第三，DExD/H盒ATP酶Prp2是剪接体激活所需的最后一个ATP酶，但该ATP酶在促进剪接体催化构象中的功能仍然是个谜。通过我们对 DExD/H 盒 ATP 酶 Prp16 在重排剪接体催化构象中的作用的研究，我们深入了解了催化构象的 snRNA 构象以及 Prp2 依赖性 RNA 重排的潜在后果。因此，本提案的目的是 i) 定义在外显子连接过程中建立保真度的 ATP 依赖性机制，ii) 研究泛素在调节 Brr2 依赖性剪接体激活和分解中的作用，以及 iii) 研究 Prp2 在剪接体激活。我们将在芽殖酵母中使用遗传学和生物化学的组合方法来实现这些目标，同时利用新兴的单分子和下一代测序技术。通过这些研究，我们希望加深对 DExD/H 盒 ATP 酶在保真度中的作用、调节这些 ATP 酶的机制以及这些 ATP 酶在重排非编码 RNA 中的功能的理解。此外，鉴于我们最近发现 hBrr2 缺陷性 RNA 解旋在常染色体显性遗传性色素性视网膜炎的病因学中的意义，这项工作也将对这种常见的失明形式产生重要影响。