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。 序列或内含子通过前mRNA剪接过程从新生RNA转录物中去除， 真核基因表达的一个重要步骤。人类基因平均> 90%是内含子， 很大一部分遗传疾病是由改变剪接位点选择突变引起的。它有 据估计，CA。人类基因的一半还受到选择性剪接， 显著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 选择性剪接通常是组织特异性的或发育调节的。在多大程度上 也受到控制-例如，改变剪接和未剪接的病毒RNAis的比例 对艾滋病毒的复制至关重要。因此，详细的拼接过程的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，还要了解它们是如何表达的， 与发育、肿瘤发生、人类遗传性疾病和遗传性疾病的复杂过程有关 逆转录病毒感染的进展。 本建议中的研究将解决三个重要问题：（A）详细的三维 人类剪接体的结构，大分子蛋白质：介导内含子的RNA机器 切除术？(B)外显子连接复合体（EJC）的详细三维结构是什么？ 剪接的mRNA代谢调节因子？和（C）EJC通过什么机制保持稳定结合 剪接的mRNA利用的技术将包括（i）通过低温冷冻法测定纯化的复合物的结构。 电子显微镜（EM）;（ii）用EM可见探针标记这些结构，以绘制位置 单个组分;（iii）通过以下方法确定相对蛋白质化学计量的新方法： 定量质谱法;（iv）蛋白质-蛋白质相互作用研究;和（v）动力学测定 和热力学性质相对于RNA和ATP结合的蛋白质被认为是作为 EJC在其结合配偶体存在或不存在的情况下锚。

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.