Identification of a Novel Regulatory Element in the Assembly of the Spliceosome

剪接体组装中新型调控元件的鉴定

• 批准号: 9171059
• 负责人: Corina Maeder
• 金额: $ 40.18万
• 依托单位: TRINITY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9171059
• 关键词: Active Sites; Address; Affect; Animal Model; Binding; Biochemistry; Biophysics; Burn injury; Collaborations; Collection; Complex; Cryoelectron Microscopy; Disease; Fluorescence; Future; Gene Expression; Gene Expression Process; Genes; Genetic; Genetic Transcription; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; Heart; Housing; In Vitro; Label; Link; Mass Spectrum Analysis; Measures; Messenger RNA; Mitosis; Modeling; Molecular; Molecular Biology; Monitor; Mutate; Pathway interactions; Process; Protein Splicing; Proteins; RNA; RNA Helicase; RNA Splicing; RNA, Messenger, Splicing; Regulation; Regulatory Element; Regulatory Pathway; Research; Retinitis Pigmentosa; Role; Saccharomyces cerevisiae; Science; Small Nuclear Ribonucleoproteins; Spliceosome Assembly Pathway; Spliceosomes; Students; Syndrome; TXN gene; Techniques; Testing; Therapeutic Agents; Thermodynamics; Time; Training; U5 small nuclear RNA; U6 small nuclear RNA; Universities; Work; career; cell growth; experience; human disease; in vitro Assay; in vivo; insight; mRNA Precursor; mutant; novel; premature; prevent; protein function; research study; single molecule; single-molecule FRET; thioredoxin-like protein; undergraduate student

PROJECT SUMMARY Pre-messenger RNA splicing regulates the maturation of a nascent mRNA, an essential process in gene expression. Pre-mRNA splicing is facilitated and regulated by the spliceosome, a large RNA/protein macromolecular machine. The spliceosome is assembled through a complex, multi-step pathway from 5 small nuclear ribonucleoprotein complexes (snRNPS), however the molecular interactions regulating each step is still unknown. The goal of this proposal is to define the roles of an essential splicing protein Dib1 in the regulation of spliceosome assembly. The work in this proposal addresses the proposed model that the presence of a small thioredoxin-like protein Dib1 in the U4/U6-U5 triple snRNP restricts successful incorporation of the pre- messenger RNA into the active site of the spliceosome, therefore acting as a regulator of spliceosome assembly. The proteins of the U4/U6-U5 triple snRNP complex being studied in this project have been directly linked to the human diseases retinitis pigmentosa and Burn-McKeown syndrome. Thus, understanding the function of Dib1 will provide direct insight into the cause of these diseases. Using the model organism, Saccharomyces cerevisiae, the first aim will determine whether Dib1 is a regulatory element of splicing by characterizing the effects of a collection of Dib1 mutants on protein stability, spliceosome assembly, splicing and cell growth. The second aim will determine the temporal relationship and distance between Dib1 and pre- mRNA on the assembling spliceosome using single molecule fluorescence techniques. Successful completion of these studies will identify a novel regulatory element in spliceosome assembly. Moreover, this research will contribute a new understanding of Dib1, which has been linked to other aspects of gene expression including mitosis and transcription, but about which little is known.

项目摘要 前信使RNA剪接调节新生mRNA的成熟，这是基因表达的重要过程。 表情前体mRNA剪接是由剪接体（一种大的RNA/蛋白质）促进和调节的。 高分子机器剪接体是通过一个复杂的，多步骤的途径从5个小的 核核糖核蛋白复合物（snRNPS），然而，调节每个步骤的分子相互作用仍然是 未知该提案的目标是确定一个重要的剪接蛋白Dib 1在调控中的作用， spliceosome组装。本提案中的工作涉及所提议的模型， U4/U6-U 5三重snRNP中的小硫氧还蛋白样蛋白Dib 1限制了前- 信使RNA进入剪接体的活性位点，因此充当剪接体的调节剂 组装件.在本项目中研究的U4/U6-U 5三重snRNP复合物的蛋白质已被直接 与人类疾病视网膜色素变性和Burn-McKeown综合征有关。因此，理解 Dib 1的功能将为这些疾病的病因提供直接的见解。利用模式生物， 酿酒酵母，第一个目标将确定Dib 1是否是剪接的调控元件， 表征Dib 1突变体集合对蛋白质稳定性、剪接体组装、剪接 和细胞生长。第二个目标将确定Dib 1和pre-dib之间的时间关系和距离。 用单分子荧光技术检测mRNA在剪接体上的表达。成功完成 这些研究将确定一种新的剪接体组装调控元件。此外，这项研究将 贡献了对Dib 1的新理解，Dib 1与基因表达的其他方面有关，包括 有丝分裂和转录，但关于这一点知之甚少。