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: 7082425
• 负责人: WILHELMUS G. J. HOL
• 金额: $ 43.62万
• 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/7082425
• 关键词: Mastigophora; X ray crystallography; chemical structure function; conformation; enzyme complex; enzyme mechanism; nucleic acid structure; posttranscriptional RNA processing; precursor mRNA; structural biology

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（由申请人提供）：动力质体中的RNA编辑，其中几种是全球病原体，是这些古代真核生物的线粒体中的独特而必不可少的过程。此过程使用数百个所谓的“指南RNA”来编辑不完整的“预选前RNA”，并在数百个特定的编辑站点上通过u-insertion和删除。插入更多的u比已删除更多。在某些情况下，“泛编辑”会发生，可能是预见前的两倍以上。 “ editosome”在此U插入/删除编辑中的核心角色。这是大约20种核编码蛋白的组装，其中包括六种不同的RNA编辑酶，这些酶进行精确精心策划的RNA裂解，插入/缺失和宗教反应。 我们的研究旨在通过晶体学方法在原子水平上阐明编辑体的功能，以最终： （i）对其建筑有充分的理解； （ii）阐明每个编辑体酶的底物特异性； （iii）阐明指导RNA的键相互作用：前mRNA双链体与编辑体蛋白； （iv）发现蛋白质和RNA分子必须通过六种不同酶的作用而生长的大构象变化。 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催化结构域的晶体结构确定以及编辑3'-末端 - 尿液转移酶的催化域。在后一种情况下，孤独位置的水分子似乎是U特异性的关键。已经获得了三种不同的编辑体蛋白的子复合物，这似乎是杂己。这些最初的结果提供了一个很好的平台，可以从该平台上揭示这种奇妙的U插入/删除机械的剩余机械谜团。 由于几种编辑蛋白在致病性动力质体中至关重要，因此我们计划确定的结构也是有希望的起点，用于设计关键病原体蛋白的选择性抑制剂。