Mammalian Pre-mRNA Splicing Mechanisms

描述（由申请人提供）：本项目的长期目标是阐明通过前体mRNA剪接过程（真核基因表达的重要步骤）从新生RNA转录物中去除间插序列或内含子的详细分子机制。人类基因平均>90%是内含子，并且显著百分比的遗传疾病是由改变剪接位点选择的突变引起的。据估计，CA。人类基因的一半还受到选择性剪接的影响，这大大增加了由我们惊人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。转录本剪接的程度也受到控制-例如，改变剪接和未剪接的病毒RNA的比例对HIV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等复杂过程的关系，那么对剪接过程的详细工作知识将是必不可少的。这项研究计划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？剪接体是介导内含子切除的大分子蛋白质：RNA机器。(B)剪接mRNA代谢的关键调节因子外显子连接复合体（EJC）的详细三维结构是什么？和（C）EJC通过什么机制保持与剪接mRNA的稳定结合？利用的技术将包括（i）通过冷冻电子显微镜（EM）确定纯化的复合物的结构;（ii）用EM可见探针标记这些结构以映射各个组分的位置;（iii）通过定量质谱法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;和（v）确定在存在或不存在其结合配偶体的情况下，被认为用作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.