Structure-Function Relationships in Kinetoplastid RNA-Editing

动质体 RNA 编辑中的结构与功能关系

• 批准号: 7227761
• 负责人: WILHELMUS G. J. HOL
• 金额: $ 47.49万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7227761
• 关键词: 3' -5' -Exonuclease; African Trypanosomiasis; Amaze; Appendix; Architecture; Blood; Catalytic Domain; Chagas Disease; Cleaved cell; Complex; Dimensions; Enzyme Interaction; Enzymes; Eukaryota; Eukaryotic Cell; Exonuclease; Family; Fingers; Guide RNA; Heterogeneous Nuclear RNA; Instruction; Latin America; Leishmania; Leishmaniasis; Ligase; Messenger RNA; Methods; Mitochondria; Mitochondrial Proteins; Nuclear; Pan Genus; Parasites; Play; Positioning Attribute; Process; Proteins; Protozoa; RNA; RNA Binding; RNA Editing; RNA Ligase (ATP); RNA Sequences; Reaction; Research; Research Personnel; Ribonuclease III; Role; Site; Specificity; Stream; Structure; Structure-Activity Relationship; Substrate Specificity; System; Techniques; Therapeutic; Time; Transferase; Trypanosoma brucei brucei; Water; Work; Zinc Fingers; base; design; endonuclease; helicase; inhibitor/antagonist; insertion/deletion mutation; mRNA Precursor; member; pathogen; programs; size; success; three dimensional structure

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.

描述(申请人提供)：动质体内的RNA编辑，其中几种是全球性病原体，是这些古老的真核生物线粒体中独特的、必不可少的过程。这一过程使用数百个所谓的“引导RNA”，通过在数百个特定编辑位点的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U多得多。在某些情况下，会发生“泛编辑”，这可能会使前消息的大小增加一倍以上。在这种U-插入/删除编辑中，“编辑小体”扮演着中心角色。这是一个由大约20个核编码蛋白组成的集合，其中包括6个不同的RNA编辑酶，执行RNA切割、插入/缺失和宗教反应的精确编排序列。 我们的研究旨在通过结晶学方法在原子水平上解开编辑小体的功能，最终： (I)充分了解其架构； (Ii)揭示每种编辑体酶的底物专一性； (3)阐明引导RNA的关键相互作用：Pre-mRNA与编辑体蛋白的双链作用； (Iv)发现蛋白质和RNA分子在前信息通过六种不同的酶的作用而增长时必须经历的大的构象变化。 我们的建议建立在最近的成功基础上，包括RNA编辑连接酶1催化结构域和编辑3‘-末端尿酰转移酶的晶体结构测定。在后一种情况下，一对精确定位的水分子似乎是U-特异性的关键。已经获得了一个由三种不同的编辑体蛋白组成的亚复合体，它似乎是一个异六聚体。这些初步结果提供了一个很好的平台，可以从这个平台上继续解开这个神奇的U插入/删除机器的许多剩余的机械奥秘。 由于几种编辑蛋白在致病动态体中是必不可少的，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。