Structure-Function Relationships in Kinetoplastid RNA-Editing

描述（由申请人提供）：动质体（其中几种是全球病原体）中的RNA编辑是这些古老真核生物线粒体中独特且必不可少的过程。这个过程使用数百个所谓的“向导RNA”，通过在数百个特定编辑位点上的U插入和删除来编辑不完整的“前信使RNA”。插入的U比删除的多得多。在某些情况下，会发生“平移编辑”，这可能会使前消息的大小增加一倍以上。在这种U-插入/缺失编辑中的核心作用由“编辑体”发挥。这是大约20种核编码蛋白质的组装，包括六种不同的RNA编辑酶，执行RNA切割，插入/缺失和重新连接反应的精确编排序列。 我们的研究旨在通过晶体学方法在原子水平上揭示编辑体的功能，最终： (i)充分了解其架构; (ii)阐明每种编辑体酶的底物特异性; (iii)阐明指导RNA：前mRNA双链体与编辑体蛋白的关键相互作用; (iv)发现蛋白质和RNA分子在六种不同酶的作用下生长时必须经历的巨大构象变化。 我们的提议建立在最近的成功之上，包括RNA编辑连接酶1催化结构域和编辑3 '-末端尿苷酸转移酶的晶体结构测定。在后一种情况下，精确定位的水分子的孤对电子似乎是U特异性的关键。已经获得了三种不同编辑体蛋白质的亚复合物，其似乎是异源六聚体。这些初步的结果提供了一个很好的平台，从那里开始解开这个奇妙的U-插入/缺失机器的许多剩余的机械奥秘。 由于几种编辑蛋白在致病动质体中是必不可少的，因此我们计划确定的结构也是设计关键病原体蛋白的选择性抑制剂的有希望的起点。

DESCRIPTION (provided by applicant): RNA editing in kinetoplastids, several of which are global pathogens, is a unique, and essential, process in the mitochondria of these ancient eukaryotes. This process uses hundreds of so-called "guide RNAs" to edit an incomplete "pre-messenger RNA" by U-insertion and deletion at hundreds of specific editing sites. Many more U's are inserted than deleted. In some cases, "pan-editing" occurs which can more than double the size of the pre-message. A central role in this U-insertion/deletion editing is played by the "editosome". This is an assembly of approximately 20 nuclear-encoded proteins including six different RNA editing enzymes performing a precisely orchestrated sequence of RNA cleavage, insertion/deletion and religation reactions. Our research aims to unravel the functioning of the editosome at the atomic level by crystallographic methods to ultimately: (i) obtain a full understanding of its architecture; (ii) unravel the substrate specificity of each editosomal enzyme; (iii) elucidate key interactions of the guide RNA:pre-mRNA duplex with the editosomal proteins; (iv) discover the large conformational changes the protein and RNA molecules must undergo while the pre-message is growing by the action of the six different enzymes. Our proposal builds on recent successes including the crystal structure determinations of the RNA Editing Ligase 1 catalytic domain and that of the editing 3'-Terminal-Uridylylate Transferase. In the latter case the lone pair of an exquisitely positioned water molecule appears to be the key to U-specificity. A subcomplex of three different editosomal proteins has been obtained which appears to be a heterohexamer. These initial results provide an excellent platform from which to proceed with unraveling the many remaining mechanistic mysteries of this marvelous U-insertion/deletion machinery. Since several editing proteins are essential in pathogenic kinetoplastids, the structures we plan to determine are also promising starting points for the design of selective inhibitors of key pathogen proteins.