Mechanisms for Rearranging RNA during Pre-mRNA Splicing

Pre-mRNA 剪接过程中 RNA 重排机制

• 批准号: 7907190
• 负责人: JONATHAN P STALEY
• 金额: $ 22.41万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-18 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7907190
• 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; Gene Expression; Genetic; Goals; Guanosine Triphosphate Phosphohydrolases; 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; Time; Yeasts; crosslink; human disease; in vitro Assay; in vitro testing; in vivo; insight; mRNA Precursor; 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）：必须以高保真度进行前MRNA剪接，以防止灾难性错误。然而，在剪接中的忠诚度的分子机制差不多。剪接由剪接体催化，这是一种动态的核糖核蛋白机器，其中小核RNA（SNRNA）成分在底物识别和催化中起关键作用。该提案的长期目标是了解如何在剪接中建立忠诚度以及剪接动力学如何促进忠诚度。我们旨在了解剪接体如何促进真正的底物的剪接并拮抗异常底物的剪接。此外，我们旨在了解真正的基材的拼接和对异常底物的失速如何导致剪接体拆卸。最近，我们在了解U2和U6 SNRNA中如何促进真正的底物的剪接，DEXD/H BOX ATPase PRP22如何拮抗异常底物的剪接以及如何调节剪接组合体的剪接。具体而言，我们的目的是确定PRP22在拮抗异常中间体中的作用，（ii）确定DEXD/H Box ATPases PRP43和BRR2以及EF-2样的GTPase SNU114在SpliceOsome脱离的（III）中，以确定U2/U6 Thelix I semit semiv semiv se semiv semiv i6 helix I se sequiv se ef i2/u2 helix I se semiv s if i6 helix I semiv s if brr2和ef-2样的gtpase snu114的作用（ii）。 U2循环HA在重塑外显子连接的剪接体时。我们将使用分子遗传学和生物化学的综合方法来追求这些目标。这些研究可能对理解（i）DEXD/H Box ATPases的功能和调节具有广泛的影响，（ii）建立在剪接方面的保真度的机制以及（iii）调节剪接体活性的机制。此外，由于忠诚度与剪接站点的选择密切相关，因此这些研究有望影响我们对调节剪接机制的理解。为了研究剪接，我们将利用模型有机体面包师的酵母，该酵母类似于简化的人类细胞，并成为人类疾病的长期模型。值得注意的是，由于生活需要忠实的基因表达，因此剪接错误通常会导致人类疾病。因此，这些研究将导致人们了解人类细胞中如何建立忠诚度，如何损害忠诚可能会导致疾病以及如何设计忠诚度以治疗疾病。