Single Molecule Analysis of Spliceosome Catalysis and Fidelity

剪接体催化和保真度的单分子分析

• 批准号: 7570401
• 负责人: Aaron Andrew Hoskins
• 金额: $ 8.91万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7570401
• 关键词: 3' Splice Site; ATP phosphohydrolase; Accounting; Address; Anabolism; Award; Biochemistry; Biological Assay; Biology; Boxing; Catalysis; Cell Extracts; Chemicals; Chemistry; Complex; Defect; Environment; Enzymatic Biochemistry; Enzymes; Event; Excision; Exons; Fluorescence; Fluorescence Microscopy; Gene Expression; Genes; Goals; Health; Hela Cells; Hereditary Disease; Heterogeneity; Human; Human Biology; Human Genetics; Introns; Kinetics; Label; Ligation; Light; Measurement; Melissa; Mentors; Messenger RNA; Methods; National Research Service Awards; Nuclear Extract; Pathway interactions; Phase; Play; Process; Protein Splicing; Proteins; Purines; RNA; RNA Biochemistry; RNA Splicing; Reaction; Research; Resolution; Role; Sampling; Site; Solutions; Spliceosome Assembly Pathway; Spliceosomes; Synthesis Chemistry; System; Techniques; Training; United States National Institutes of Health; Work; Yeasts; career; chemical addition; dalton; graduate student; insight; mRNA Precursor; nucleotide metabolism; protein complex; purine; single molecule; single-molecule FRET; tool; yeast genetics

DESCRIPTION (provided by applicant): Pre-mRNA splicing is an essential step in eukaryotic gene expression. The processes of intron excision and exon ligation are carried out by a mega-Dalton complex of RNAs and proteins called the spliceosome. This proposal seeks to develop and utilize new chemical and biophysical methods to study the mechanism of pre-mRNA splicing at the single molecule level. During the mentored phase, methods will be developed to site-specifically fluorescently label pre-mRNAs and yeast spliceosomal proteins in order to observe their dynamics during a complete splicing reaction by single molecule fluorescence microscopy. This work will focus on addressing questions relevent to the transition of the spliceosome between an enzyme capable of carrying out lariat formation and an enzyme competent for exon ligation, including direct observation of the interaction of DExD/H-box proteins/fidelity factors such as Prp16 and Prp22 with the spliceosome. During the independent phase, a transition will be made to the human spliceosome. This work will focus on questions specific to human splicing such as the influence of SR proteins on pre-mRNA dynamics during the chemical steps of splicing. In addition, chemical methods will be developed and employed to provide a high kinetic resolution analysis of 3' splice site recognition and exon ligation using photocaging groups and light to trigger these events in solution and at the single molecule level. I have received extensive training in mechanistic enzymology and nucleotide synthesis while studying de novo purine biosynthesis as a graduate student with JoAnne Stubbe at MIT. As a result, I wish to employ chemical and detailed kinetic methods to the study of complex cellular machines such as the spliceosome during my independent career. As a NIH NRSA postdoctoral research fellow with Melissa Moore and Jeff Gelles, I have expanded my training to include RNA biochemistry, yeast genetics, and single molecule fluorescence microscopy. This enivronment has allowed me to formulate a career path in which I will be able to use chemistry and phyiscs to provide unique and fundamental insight into human biology. RELEVANCE: Pre-mRNA splicing is an essential step in human gene expression and is therefore of fundamental importance to the study of human health and biology. The vast majority of human genes are alternatively spliced and defects in this process may account for >15% of human genetic diseases. The research proposed here will have a significant impact on human health by elucidating this step in gene expression.

描述（由申请人提供）：Pre-mRNA 剪接是真核基因表达的重要步骤。内含子切除和外显子连接过程是由称为剪接体的 RNA 和蛋白质的兆道尔顿复合物执行的。该提案旨在开发和利用新的化学和生物物理方法来研究单分子水平的前体mRNA剪接机制。在指导阶段，将开发对前 mRNA 和酵母剪接体蛋白进行位点特异性荧光标记的方法，以便通过单分子荧光显微镜观察它们在完整剪接反应过程中的动态。这项工作将重点解决与能够进行套索形成的酶和能够进行外显子连接的酶之间的剪接体转变相关的问题，包括直接观察 DExD/H-box 蛋白/保真因子（例如 Prp16 和 Prp22）与剪接体的相互作用。在独立阶段，将转变为人类剪接体。这项工作将重点关注人类剪接的具体问题，例如剪接化学步骤中 SR 蛋白对前 mRNA 动态的影响。此外，将开发和采用化学方法，使用光笼基团和光在溶液中和单分子水平上触发这些事件，对 3' 剪接位点识别和外显子连接进行高动力学分辨率分析。作为一名研究生，我在麻省理工学院与 Jo​​Anne Stubbe 一起研究从头嘌呤生物合成，期间接受了机械酶学和核苷酸合成方面的广泛培训。因此，在我的独立职业生涯中，我希望采用化学和详细的动力学方法来研究复杂的细胞机器，例如剪接体。作为 NIH NRSA 博士后研究员，我与梅丽莎·摩尔 (Melissa Moore) 和杰夫·盖尔斯 (Jeff Gelles) 一起，将我的培训范围扩大到了 RNA 生物化学、酵母遗传学和单分子荧光显微镜。这种环境使我能够制定一条职业道路，使我能够利用化学和物理学为人类生物学提供独特而基本的见解。 相关性：mRNA 前体剪接是人类基因表达的重要步骤，因此对于人类健康和生物学研究具有根本重要性。绝大多数人类基因都是选择性剪接的，这一过程中的缺陷可能导致超过 15% 的人类遗传疾病。这里提出的研究将通过阐明基因表达的这一步骤对人类健康产生重大影响。