Single Molecule Study of Spliceosomal RNAs

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。异常的前mrna剪接对细胞具有致命的影响，并且与许多人类疾病如癌症和神经退行性疾病有关。剪接体是由五种小核仁rna （snRNA）和大量催化剪接的蛋白质组成的动态组装体。两个snrna， U2和U6，形成剪接体的活性位点。近年来，U2/U6络合物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，但具体的结构动力学信息仍然缺乏支持这一假设。研究U2/U6复合体的结构动力学对于理解剪接体的活化和催化是至关重要的，因为这种酶在细胞生长、分化和疾病中起着关键作用。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。我们之前已经证明了我们的方法特别适合于阐明RNA酶的结构动力学，并揭示了在集成平均实验中隐藏的重要信息。我们的目标是(1)通过单分子荧光揭示U2/U6的构象动力学，(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，(5)阐明剪接体蛋白Prp24在剪接体激活中的作用。公共卫生相关性：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。异常剪接可以对细胞产生致命的影响，并且与许多人类疾病，如癌症和神经退行性疾病有关。剪接体是一种催化剪接的大rna -蛋白复合物。剪接体催化中心的结构由于其动力学性质，近年来一直是一个有争议的问题。研究剪接体催化核的结构动力学是了解其生物学功能的必要条件，因为这种酶在细胞生长、分化和疾病中起着关键作用。我们建议使用强大的单分子荧光技术来解析这些结构动力学，并以前所未有的细节表征其机制。

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.