Mammalian Pre-mRNA Splicing Mechanisms

描述(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转录本中去除插入序列或内含子的详细分子机制，这是真核基因表达的重要步骤。人类基因平均有90%的内含子，而相当大比例的遗传病是由改变剪接位点选择的突变引起的。据估计，大约一半的人类基因还受到选择性剪接的影响，这显著增加了由我们令人惊讶的小基因组编码的蛋白质组的复杂性。这种选择性剪接通常是组织特异性的或发育调节的。转录本的剪接程度也受到控制--例如，改变剪接和非剪接病毒RNA的比例对艾滋病毒的复制至关重要。因此，如果我们不仅要了解真核基因表达的基本机制，而且要了解它们与发育、肿瘤发生、人类遗传疾病和逆转录病毒感染进展的复杂过程，详细的剪接过程的工作知识将是必不可少的。这项建议中的研究将解决三个重要问题：(A)人类剪接体的详细三维结构是什么，大分子蛋白质：介导内含子切除的RNA机器？(B)外显子连接复合体(EJC)的详细三维结构是什么，它是剪接mRNA代谢的关键调节因素？(C)是什么机制使EJC稳定地与剪接的mRNA结合？所使用的技术将包括：(I)用冷冻电子显微镜(EM)确定纯化的络合物的结构；(Ii)用EM可见的探针标记这些结构，以绘制单个组分的位置；(Iii)通过定量质谱学确定相对蛋白质化学计量的新方法；(Iv)蛋白质-蛋白质相互作用研究；以及(V)在存在或不存在结合伙伴的情况下，确定被认为作为EJC锚的蛋白质与RNA和ATP结合的动力学和热力学性质。

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, an essential step in eukaryotic gene expression. Human genes are on average >90% intron, and a significant percentage of genetic diseases arise from mutations that that alter splice site choice. It has been estimated that ca. half of of human genes are additionally subject to alternative splicing, which significantly increases the complexity of the proteome encoded by our surprisingly small genome. This alternative splicing is often tissue-specifically or developmentally regulated. The extent to which a transcript is spliced is also subject to control - for example, altering the ratio of spliced and unspliced viral RNA is critical to the replication of HIV. Thus a detailed working knowledge of the splicing process will be essential if we are to understand not only the basic mechanisms of eukaryotic gene expression, but also how they relate to the complex processes of development, oncogenesis, human genetic disorders and the progression of retroviral infection. Studies in this proposal will address three important questions: (A) What is the detailed three-dimensional architecture of the human spliceosome, the macromolecular protein: RNA machine that mediates intron excision?; (B) What is the detailed three-dimensional architecture of the exon junction complex (EJC), a key regulator of spliced mRNA metabolism?; and (C) By what mechanism does the EJC remain stably bound to spliced mRNA? Techniques utilized will include (i) structure determination of purified complexes by cryo- electron microscopy (EM); (ii) labeling of those structures with EM-visible probes to map the locations individual components; (iii) a new methodology for determining relative protein stoichiometries by quantitative mass spectrometry; (iv) protein-protein interaction studies; and (v) determination of the kinetic and thermodynamic properties with respect to RNA and ATP binding by the protein thought to serve as the EJC anchor in the presence or absence of its binding partners.