Pre-mRNA Splicing Mechanisms

前体 mRNA 剪接机制

• 批准号: 8399727
• 负责人: Melissa J. Moore
• 金额: $ 37.51万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8399727
• 关键词: 3-Dimensional; Biological; Boxing; Cells; Chemicals; Collaborations; Complex; Complex Mixtures; Computers; Cryoelectron Microscopy; Deposition; Development; Disease; Dissociation; Excision; Exons; Fluorescence Microscopy; Funding; Gene Expression; Gene Expression Process; Genes; Glass; Goals; Grant; Health; Human; Image; In Vitro; Individual; Introns; Kinetics; Laboratories; Learning; Macromolecular Complexes; Mediating; Messenger RNA; Metabolism; Methodology; Microscope; Modeling; Molecular; Monitor; Nature; Organism; Pathway interactions; Preparation; Procedures; Process; Protein Family; Proteins; RNA; RNA Splicing; RNA chemical synthesis; RNase protection assay; Reporting; Set protein; Small Nuclear RNA; Small Nuclear Ribonucleoproteins; Spectrum Analysis; Spliceosome Assembly Pathway; Spliceosomes; Staging; Structure; Study models; System; Talents; Technology; Time; Transcript; U2 Small Nuclear Ribonucleoprotein; Universities; Virginia; Work; Yeasts; anti-cancer therapeutic; crosslink; design; fluorescence microscope; fluorophore; high throughput screening; human disease; inhibitor/antagonist; interdisciplinary approach; mRNA Decay; mRNA Precursor; member; novel; particle; polypeptide; public health relevance; reconstitution; reconstruction; research study; single molecule; small molecule; stoichiometry; three dimensional structure

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the detailed molecular mechanisms by which intervening sequences or introns are removed from nascent RNA transcripts through the process of pre-mRNA splicing. Alterations in this essential step in eukaryotic gene expression are known to underly many human diseases, so detailed understanding of the mechanisms involved is important for the betterment of human health. Pre- mRNA splicing is carried out by the spliceosome, a ~3 MDa macromolecular complex consisting of 5 small nuclear RNAs (snRNAs: U1, U2, U4, U5 and U6) and >100 polypeptides. While much progress has been made in defining these component parts, much remains to be learned about how these myriad pieces function together to mediate precise and timely intron removal. This proposal describes development of methodologies for application of single molecule total internal reflection fluorescence (SM-TIRF) microscopy to complex macromolecular systems such as the spliceosome. A novel SM-TIRF microscope with multi-wavelength capabilities designed and implemented in Jeff Gelles' laboratory at Brandeis University allows for simultaneous monitoring of multiple fluorophores interacting with single fluorescent pre-mRNA molecules tethered to a glass coverslip. Using this Colocalization Single Molecule Spectroscopy (CoSMoS) approach, one can directly observe the detailed kinetics of snRNP and splicing factor association/dissociation with pre-mRNA over the full course of spliceosome assembly and splicing. As there currently exists no fully-reconstituted system that faithfully replicates all stages of the splicing pathway, all experiments are carried out in crude cell lysates using fluorescently-tagged endogenous proteins. This approach will be used to: (1) Establish the first complete kinetic framework for spliceosome assembly on a model pre-mRNA; (2) Determine to what extent spliceosome assembly proceeds via the same or different dynamic pathway(s) on other transcripts; (3) Investigate the nature of spliceosomal discard pathways; and (4) Investigate the dynamics of DExH/D-box proteins with the spliceosome. Because CoSMoS analysis does not require purified components and can be carried out in highly complex mixtures, this technology should be broadly applicable to the study of many other macromolecular machines that, like the spliceosome, cannot be reassembled from component parts in vitro. )

描述(由申请人提供)：该项目的长期目标是阐明通过前信使核糖核酸剪接过程从新生RNA转录本中移除插入序列或内含子的详细分子机制。真核基因表达的这一重要步骤的改变是已知的许多人类疾病的基础，因此详细了解其中涉及的机制对于改善人类健康非常重要。剪接体是由U1、U2、U4、U5和U6 5个小核RNA和&gt；100多肽组成的~3MDA大分子复合体，通过剪接体进行mRNA的剪接。虽然在确定这些组成部分方面已经取得了很大进展，但关于这些无数片段如何共同发挥作用以调节准确和及时的内含子去除，仍有许多有待了解。这项建议描述了单分子全内反射荧光(SM-TIRF)显微镜应用于复杂大分子系统(如剪接体)的方法的发展。杰夫·盖尔斯在布兰迪斯大学的实验室设计并实施了一种具有多波长能力的新型SM-TIRF显微镜，它允许同时监测多个荧光团与绑在玻璃盖片上的单个荧光前mRNA分子相互作用。利用这种共定位单分子光谱(COSMOS)方法，可以直接观察剪接体组装和剪接的全过程中SnRNP和剪接因子与前mRNA的结合/解离的详细动力学。由于目前还没有完全重组的系统来忠实地复制剪接途径的所有阶段，所有的实验都是在使用荧光标记的内源蛋白的粗细胞裂解液中进行的。这一方法将用于：(1)建立第一个完整的剪接体组装动力学框架；(2)确定剪接体组装通过其他转录本上相同或不同的动态途径(S)进行到什么程度；(3)研究剪接体丢弃途径的性质；以及(4)研究剪接体与剪接体之间的动力学。由于COSMOS分析不需要纯化的成分，而且可以在高度复杂的混合物中进行，这项技术应该广泛适用于许多其他大分子机器的研究，这些机器像剪接体一样，不能在体外从组成成分重新组装。)