Structure-Function Relationships in Kinetoplastid RNA-Editing

描述（由申请人提供）：着丝质体中的RNA编辑，其中一些是全球性病原体，是这些古老真核生物线粒体中独特且必要的过程。这个过程使用数百个所谓的“引导RNA”，通过u插入和删除数百个特定的编辑位点来编辑不完整的“前信使RNA”。插入的U比删除的U多得多。在某些情况下，“泛编辑”的发生可能会使预消息的大小增加一倍以上。“编辑体”在这种插入/删除编辑中起着核心作用。这是一个大约20个核编码蛋白的组装，包括六种不同的RNA编辑酶，执行精确编排的RNA切割，插入/删除和宗教反应序列。

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.