Mammalian Pre-mRNA Splicing Mechanisms

该项目的长期目标是阐明干预的详细分子机制。 通过前-mRNA剪接过程从新生RNA转录本中去除序列或内含子， 真核基因表达中必不可少的一步。人类基因平均有90%的内含子， 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。它有 据估计，大约一半的人类基因另外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 选择性剪接通常是组织特异性的或发育调节的。成绩单在多大程度上 被剪接也受到控制-例如，改变剪接和非剪接病毒RNA的比例 对艾滋病毒复制至关重要。因此，拼接过程的详细工作知识将是必不可少的 如果我们不仅要了解真核基因表达的基本机制，而且要了解它们是如何 与复杂的发育、肿瘤发生、人类遗传疾病和 逆转录病毒感染的进展。 这项提案中的研究将解决三个重要问题：(A)什么是详细的三维 人类剪接体的结构--大分子蛋白质：调节内含子的RNA机器 切除？；(B)外显子连接复合体(EJC)的详细三维结构是什么？ 剪接的信使核糖核酸新陈代谢的调节器；和(C)什么机制稳定地与EJC结合？ 剪接信使核糖核酸？所采用的技术包括：(I)用冷冻-凝胶法测定纯化的络合物的结构。 电子显微镜(EM)；(2)用EM-可见探针标记这些结构以绘制位置图 单个组分；(3)通过以下方法确定相对蛋白质化学计量的新方法 定量质谱学；(Iv)蛋白质-蛋白质相互作用研究；(V)动力学测定 以及与RNA和ATP结合的热力学性质，该蛋白质被认为是 EJC在其绑定伙伴存在或不存在的情况下锚定。

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 RNAis 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.