权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Single Molecule Study of Spliceosomal RNAs

剪接体 RNA 的单分子研究

基本信息

批准号：
8266517
负责人：
Christine S Chow
金额：
$ 26.5万
依托单位：
WAYNE STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-07-01 至 2014-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8266517
关键词：
Active Sites Affect Biological Process Breast Catalysis Catalytic Domain Cells Colorectal Comparative Study Complex DNA Sequence Rearrangement Data Disease Elements Environment Enzymes Fluorescence Fluorescence Resonance Energy Transfer Goals Health Human Ions Laboratories Lead Link Magnesium Malignant Neoplasms Malignant neoplasm of ovary Messenger RNA Modification Molecular Molecular Conformation Molecular Machines Monitor Nature Neurodegenerative Disorders Outcome Parkinson Disease Play Post-Transcriptional RNA Processing Post-Translational Protein Processing Process Proteins RNA RNA Folding RNA Splicing RNA, Messenger, Splicing RNA-Protein Interaction Research Research Personnel Resources Role Small Nucleolar RNA Solutions Spectrum Analysis Spliceosomes Structure System Techniques Testing Work Yeasts base cell growth cofactor experience human disease in vivo innovation mRNA Precursor magnesium ion member multidisciplinary protein complex research study single molecule tool

项目摘要

DESCRIPTION (provided by applicant): From yeast to humans, splicing is an essential step in the maturation of precursor messenger RNA (pre-mRNA). Anomalous pre-mRNA splicing can have lethal effects for the cell and has been linked to numerous human diseases such as cancer and neurodegenerative disorders. The spliceosome is a dynamic assembly of five small nucleolar RNAs (snRNA) and a large number of proteins that catalyzes splicing. Two snRNAs, U2 and U6, form the active site of the spliceosome. The structure of the U2/U6 complex has been the focus of much debate in recent years, because evidence has been presented for alternative conformations. We hypothesize that these conformations reflect different states of spliceosome activation, but specific structural dynamics information to support this hypothesis is still lacking. It is essential to study the structural dynamics of the U2/U6 complex to understand spliceosomal activation and catalysis, because this enzyme plays key roles in cell growth, differentiation and disease. We propose to use the powerful single molecule fluorescence technique to investigate these structural dynamics. We have previously demonstrated our approach to be particularly suited to elucidate the structural dynamics of RNA enzymes and reveal important information otherwise hidden in ensemble-averaged experiments. We aim at (1) revealing the U2/U6 conformational dynamics by single molecule fluorescence, (2) elucidating the role of Mg2+ ions in these dynamics, (3) linking these dynamics to spliceosomal activation in vivo, (4) comparing the structural dynamics of the U2/U6 complex from humans and yeast and (5) elucidating the role of spliceosomal protein Prp24 in spliceosomal activation. PUBLIC HEALTH RELEVANCE: From yeast to humans, splicing is an essential step in the maturation of messenger RNAs that are used to synthesize functional proteins. Anomalous splicing can have lethal effects for the cell and has been linked to numerous human diseases such as cancer and neurodegenerative disorders. The spliceosome is a large RNA-protein complex that catalyzes splicing. The structure of the catalytic center of the spliceosome has been a matter of debate in recent years because of its dynamic nature. It is essential to investigate the structural dynamics of the catalytic core of the spliceosome to understand its biological function, because this enzyme plays key roles in cell growth, differentiation and disease. We propose to use the powerful single molecule fluorescence technique to resolve these structural dynamics and characterize their mechanism in unprecedented detail.

描述（由申请人提供）：从酵母到人类，剪接是前体信使 RNA (pre-mRNA) 成熟的重要步骤。异常的 mRNA 前体剪接可能对细胞产生致命影响，并与癌症和神经退行性疾病等多种人类疾病有关。剪接体是五个小核仁 RNA (snRNA) 和大量催化剪接的蛋白质的动态组装体。两个 snRNA U2 和 U6 形成剪接体的活性位点。近年来，U2/U6 复合体的结构一直是争论的焦点，因为已经提出了其他构象的证据。我们假设这些构象反映了剪接体激活的不同状态，但仍然缺乏支持这一假设的具体结构动力学信息。研究 U2/U6 复合物的结构动力学对于了解剪接体激活和催化作用至关重要，因为这种酶在细胞生长、分化和疾病中发挥着关键作用。我们建议使用强大的单分子荧光技术来研究这些结构动力学。我们之前已经证明我们的方法特别适合阐明 RNA 酶的结构动力学，并揭示隐藏在整体平均实验中的重要信息。我们的目标是 (1) 通过单分子荧光揭示 U2/U6 构象动力学，(2) 阐明 Mg2+ 离子在这些动力学中的作用，(3) 将这些动力学与体内剪接体激活联系起来，(4) 比较人类和酵母的 U2/U6 复合物的结构动力学，以及 (5) 阐明剪接体蛋白 Prp24 在剪接体激活。公共健康相关性：从酵母到人类，剪接是用于合成功能蛋白的信使 RNA 成熟的重要步骤。异常剪接可能对细胞产生致命影响，并与癌症和神经退行性疾病等多种人类疾病有关。剪接体是一种催化剪接的大型 RNA-蛋白质复合物。剪接体催化中心的结构由于其动态性质近年来一直是一个争论的问题。研究剪接体催化核心的结构动力学对于了解其生物学功能至关重要，因为这种酶在细胞生长、分化和疾病中发挥着关键作用。我们建议使用强大的单分子荧光技术来解决这些结构动力学问题，并以前所未有的细节表征其机制。