Mechanisms for Rearranging RNA during Pre-mRNA Splicing

Pre-mRNA 剪接过程中 RNA 重排机制

• 批准号: 7197759
• 负责人: JONATHAN P STALEY
• 金额: $ 29.08万
• 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 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7197759
• 关键词: 3' Splice Site; 5' Splice Site; ATP phosphohydrolase; Address; Animal Model; Binding; Biochemical; Biochemistry; Biological Assay; Boxing; Catalysis; Cells; Complement; Defect; Disease; Engineering; Exons; Family; Genetic; Goals; Guanosine Diphosphate; Guanosine Triphosphate Phosphohydrolases; Helix (Snails); Heterogeneous Nuclear RNA; Human; Hydrolysis; In Vitro; Lead; Life; Ligation; Link; Mediating; Modeling; Molecular; Molecular Genetics; Play; Prevalence; RNA; RNA Splicing; Regulation; Research Personnel; Ribonucleoproteins; Role; Saccharomyces cerevisiae; Site; Small Nuclear RNA; Specificity; Spliced Genes; Spliceosome Assembly Pathway; Spliceosomes; Staging; Structure; Testing; Thinking; Time; Yeasts; crosslink; ear helix; human disease; in vitro Assay; in vivo; insight; mRNA Precursor; mouse Gdi2 protein; mutant; novel; prevent; stem; tool

DESCRIPTION (provided by applicant): Pre-mRNA splicing must occur with high fidelity to prevent catastrophic errors. Yet, the molecular mechanisms of fidelity in splicing are understood poorly. Splicing is catalyzed by the spliceosome, a dynamic ribonucleoprotein machine in which small nuclear RNA (snRNA) components play key roles in substrate recognition and catalysis. The long-term goals of this proposal are to understand how fidelity is established in splicing and how spliceosome dynamics promote fidelity. We aim to understand how the spliceosome promotes splicing of a genuine substrate and antagonizes splicing of an aberrant substrate. Further, we aim to understand how splicing of a genuine substrate and stalling of an aberrant substrate each lead to spliceosome disassembly. Recently, we have made significant breakthroughs in understanding how the U2 and U6 snRNAs promote splicing of a genuine substrate, how the DExD/H box ATPase Prp22 antagonizes splicing of an aberrant substrate and how spliceosome disassembly is regulated. Specifically, we aim (i) to determine the role of Prp22 in antagonizing aberrant intermediates, (ii) to determine the roles of the DExD/H box ATPases Prp43 and Brr2 and the EF-2-like GTPase Snu114 in spliceosome disassembly, (iii) to investigate the role of U2/U6 helix I sequences in promoting splicing and (iv) to determine the role of the U2 loop Ha in remodeling the spliceosome for exon ligation. We will pursue these aims using a combined approach of molecular genetics and biochemistry. These studies will likely have broad implications for understanding (i) the function and regulation of DExD/H box ATPases, (ii) the mechanisms for establishing fidelity in splicing and (iii) the mechanisms for regulating the activity of the spliceosome. Further, as fidelity is intimately linked to splice site choice, these studies promise to impact our understanding of the mechanisms for regulating splicing. To investigate splicing, we will utilize the model organism baker's yeast, which resembles a simplified human cell and which has served as a long-standing model for human disease. Significantly, as life requires the faithful expression of genes, splicing errors commonly lead to human disease. Thus, these studies will lead to an understanding of how fidelity is established in human cells, how compromised fidelity may contribute to disease and how fidelity might be engineered to treat disease.

描述（由申请人提供）：Pre-mRNA 剪接必须以高保真度进行，以防止灾难性错误。然而，人们对剪接保真度的分子机制知之甚少。剪接由剪接体催化，剪接体是一种动态核糖核蛋白机器，其中小核 RNA (snRNA) 成分在底物识别和催化中发挥关键作用。该提案的长期目标是了解剪接中如何建立保真度以及剪接体动力学如何促进保真度。我们的目标是了解剪接体如何促进真正底物的剪接并拮抗异常底物的剪接。此外，我们的目标是了解真实底物的剪接和异常底物的停滞如何导致剪接体分解。最近，我们在理解U2和U6 snRNA如何促进真正底物的剪接、DExD/H盒ATP酶Prp22如何拮抗异常底物的剪接以及如何调节剪接体解体方面取得了重大突破。具体来说，我们的目标是 (i) 确定 Prp22 在拮抗异常中间体中的作用，(ii) 确定 DExD/H 盒 ATP 酶 Prp43 和 Brr2 以及 EF-2 样 GTP 酶 Snu114 在剪接体拆卸中的作用，(iii) ) 研究 U2/U6 螺旋 I 序列在促进剪接中的作用以及 (iv) 确定 U2 环 Ha 在促进剪接中的作用重塑剪接体以进行外显子连接。我们将利用分子遗传学和生物化学的结合方法来实现这些目标。这些研究可能对理解 (i) DExD/H 盒 ATP 酶的功能和调节、(ii) 建立剪接保真度的机制和 (iii) 调节剪接体活性的机制产生广泛的影响。此外，由于保真度与剪接位点的选择密切相关，这些研究有望影响我们对剪接调节机制的理解。为了研究剪接，我们将利用模型生物面包酵母，它类似于简化的人类细胞，并且长期以来一直作为人类疾病的模型。值得注意的是，由于生命需要基因的忠实表达，剪接错误通常会导致人类疾病。因此，这些研究将有助于了解人类细胞中保真度是如何建立的、保真度受损如何导致疾病以及如何设计保真度来治疗疾病。