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: 7923646
• 负责人: WILHELMUS G. J. HOL
• 金额: $ 22.84万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7923646
• 关键词: 3' -5' -Exonuclease; African Trypanosomiasis; Amaze; Architecture; Blood; Catalytic Domain; Chagas Disease; Cleaved cell; Complex; Dimensions; Enzyme Interaction; Enzymes; Eukaryota; Exonuclease; Family; Fingers; Guide 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; protein complex; 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（由申请人提供）：动质体（其中几种是全球性病原体）中的 RNA 编辑是这些古老真核生物线粒体中独特且重要的过程。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进行U-插入和删除来编辑不完整的“前信使RNA”。插入的 U 数量多于删除的 U 数量。在某些情况下，会发生“泛编辑”，这可能会使前置消息的大小增加一倍以上。在这种 U 插入/删除编辑中，“编辑体”发挥着核心作用。这是大约 20 种核编码蛋白质的组合，包括六种不同的 RNA 编辑酶，执行精确编排的 RNA 切割、插入/删除和再连接反应序列。 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) 揭示每种编辑体酶的底物特异性； (iii) 阐明引导 RNA:pre-mRNA 双链体与编辑体蛋白的关键相互作用； (iv) 发现当前信息在六种不同酶的作用下生长时，蛋白质和 RNA 分子必须经历大的构象变化。 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'-末端尿苷酸转移酶的晶体结构测定。在后一种情况下，精确定位的孤对水分子似乎是 U 特异性的关键。已经获得了三种不同编辑体蛋白的亚复合物，其似乎是异六聚体。这些初步结果提供了一个极好的平台，可用于解开这种奇妙的 U 型插入/删除机制的许多剩余机制之谜。 由于几种编辑蛋白在致病动质体中至关重要，因此我们计划确定的结构也是设计关键病原体蛋白选择性抑制剂的有希望的起点。

项目成果

KREPA6 is an RNA-binding protein essential for editosome integrity and survival of Trypanosoma brucei.

KREPA6 是一种 RNA 结合蛋白，对于布氏锥虫的编辑体完整性和生存至关重要。

• DOI: 10.1261/rna.763308
• 发表时间: 2008
• 期刊: RNA (New York, N.Y.)
• 作者: TarunJr,SalvadorZipagan;Schnaufer,Achim;Ernst,NancyLewis;Proff,Rosemary;Deng,Junpeng;Hol,Wim;Stuart,Kenneth
• 通讯作者: Stuart,Kenneth

Explorations of linked editosome domains leading to the discovery of motifs defining conserved pockets in editosome OB-folds.

• DOI: 10.1016/j.jsb.2012.07.012
• 发表时间: 2012-11
• 期刊: JOURNAL OF STRUCTURAL BIOLOGY
• 影响因子: 3
• 作者: Park, Young-Jun;Hol, Wim G. J.
• 通讯作者: Hol, Wim G. J.

The structure of the C-terminal domain of the largest editosome interaction protein and its role in promoting RNA binding by RNA-editing ligase L2.

• DOI: 10.1093/nar/gks369
• 发表时间: 2012-08
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Park YJ;Budiarto T;Wu M;Pardon E;Steyaert J;Hol WG
• 通讯作者: Hol WG

Structures of a key interaction protein from the Trypanosoma brucei editosome in complex with single domain antibodies.

来自布氏锥虫编辑体的关键相互作用蛋白与单域抗体的复合物的结构。

• DOI: 10.1016/j.jsb.2010.10.007
• 发表时间: 2011
• 期刊: Journal of structural biology
• 影响因子: 3
• 作者: Wu,Meiting;Park,Young-Jun;Pardon,Els;Turley,Stewart;Hayhurst,Andrew;Deng,Junpeng;Steyaert,Jan;Hol,WimGJ
• 通讯作者: Hol,WimGJ