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）：前体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的过程是由RNA和蛋白质的巨道尔顿复合体（称为剪接体）进行的。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上的前体mRNA剪接机制。在指导阶段，将开发方法来位点特异性地荧光标记前mRNA和酵母spliceosomal蛋白，以便通过单分子荧光显微镜观察它们在完整剪接反应期间的动力学。这项工作将集中在解决有关的问题之间的剪接体的酶能够进行lactinase形成和酶的外显子连接，包括直接观察的相互作用的DExD/H盒蛋白质/保真度因子，如Prp 16和Prp 22与剪接体之间的过渡。在独立阶段，将过渡到人类剪接体。这项工作将集中在特定于人类剪接的问题，如在剪接的化学步骤中SR蛋白对前mRNA动力学的影响。此外，将开发和采用化学方法，以提供3'剪接位点识别和外显子连接的高动力学分辨率分析，使用光笼化基团和光在溶液中和在单分子水平上触发这些事件。我在麻省理工学院随JoAnne Stubbe研究生学习嘌呤从头生物合成的同时，接受了广泛的机械酶学和核苷酸合成方面的培训。因此，我希望在我独立的职业生涯中，采用化学和详细的动力学方法来研究复杂的细胞机器，如剪接体。作为美国国立卫生研究院NRSA博士后研究员与梅丽莎摩尔和杰夫盖尔斯，我已经扩大了我的培训，包括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，在这条道路上，我将能够利用化学和物理学来提供对人类生物学的独特和基本的见解。 相关性：前体mRNA剪接是人类基因表达的重要步骤，因此对人类健康和生物学研究具有根本重要性。绝大多数人类基因是选择性剪接的，这一过程中的缺陷可能占人类遗传疾病的15%以上。这里提出的研究将通过阐明基因表达中的这一步骤对人类健康产生重大影响。