Kinetoplastid RNA editing

动质体寄生虫的RNA编辑需要线粒体mRNAs的大规模重塑 转录后插入和缺失尿苷(U)残基以产生编码蛋白质的mRNAs 参与寄生虫生物能量学的研究。U插入/缺失RNA编辑是动质体所特有的，对 布鲁氏毛滴虫的存活和毒力。RNA编辑全酶由两个亚复合体组成：RECC(RNA 编辑核心复合体)和RESC(RNA编辑底物结合复合体)。反式作用引导RNA (GRNAs)指导U插入/缺失，按顺序作用，使编辑沿着mRNA进行3‘到5’。 RECC包含编辑酶，以及RECC催化U插入/缺失的机制 对单个编辑站点进行了广泛的研究。最近，RESC已成为编辑的脚手架 以及mRNAs、gRNAs和recc之间相互作用的协调者，尽管它所通过的机制 完成这些任务基本上是未知的。更复杂的是，编辑特定的mRNAs 人类血流型(BF)和昆虫媒介原环型(PF)寄生虫之间的差异调节。 发育调节的机制(S)和生命周期中的精确点(S) 受到影响，仍然是完全神秘的。本应用程序的目标是(1)描述不同的角色 RESC的组成和一种新的RESC相关复合体在gRNA的运输和利用中，以及(2)定义 在布氏锥虫生活中，作为阶段特定调节控制点的编辑过程的几个方面 周而复始。为了实现这些目标，我们开发了一种创新而强大的生物信息学工具(HTS/Treat) 这使得我们现在可以在单核苷酸分辨率下检查线粒体转录组，并组成 部分编辑的信使核糖核酸中间体以阐明gRNA使用的特定方面的方式。将这一点结合起来 生物信息学方法结合RNA免疫沉淀和蛋白质-蛋白质相互作用的研究，我们将定义 特定RESC蛋白的功能机制与RESC的异质性和动态相关 交互作用，并阐明编辑过程中受发育控制的步骤。我们还将 利用允许产生大量昆虫变环形式的最新技术 (Mf)T.brucei，一种在Pf和Bf之间基本上未被开发的中间体。使用HTS/Treat，我们将定义 MFS中的RNA编辑模式，并揭示部分或所有mRNA的BF RNA编辑模式是否出现在 MFS，提供了对布氏锥虫发育过程中RNA编辑的时间控制的洞察。建议数 研究解决了关于基本动质RNA编辑方面的几个重要知识空白 进程。此外，这项工作可能有助于为未来的药物开发奠定基础，并提供重要的 对高等生物体中核糖核蛋白复合体功能的洞察。

RNA editing in kinetoplastid parasites entails massive remodeling of mitochondrial mRNAs by posttranscriptional insertion and deletion of uridine (U) residues to generate mRNAs encoding proteins involved in parasite bioenergetics. U insertion/deletion RNA editing is unique to kinetoplastids and essential for T. brucei survival and virulence. The RNA editing holoenzyme comprises two subcomplexes: RECC (RNA Editing Core Complex) and RESC (RNA Editing Substrate Binding Complex). Trans-acting guide RNAs (gRNAs) direct U insertion/deletion, acting sequentially such that editing proceeds 3' to 5' along an mRNA. RECC contains the editing enzymes, and the mechanisms by which RECC catalyzes U insertion/deletion at a single editing site have been extensively studied. More recently, RESC has emerged as the scaffold for editing and a coordinator of interactions between mRNAs, gRNAs, and RECC, although the mechanisms by which it accomplishes these tasks are essentially unknown. Adding even more complexity, editing of specific mRNAs is differentially regulated between human bloodstream form (BF) and insect vector procyclic form (PF) parasites. The mechanism(s) of developmental regulation, and the precise point(s) in the life cycle at which they are effected, remain entirely mysterious. The goals of this application are to (1) delineate the roles of distinct components of RESC and a novel RESC-related complex in gRNA trafficking and utilization, and (2) to define aspects of the editing process that serve as control points for stage specific regulation during the T. brucei life cycle. To accomplish these goals, we developed an innovative and powerful bioinformatic tool (HTS/TREAT) that permits us to now examine the mitochondrial transcriptome at single nucleotide resolution, and group partially edited mRNA intermediates in ways that illuminate specific aspects of gRNA usage. Combining this bioinformatic approach with RNA immunoprecipitation and protein-protein interaction studies, we will define the mechanisms by which specific RESC proteins function, correlate these to RESC heterogeneity and dynamic interactions, and elucidate the steps in the editing process that are developmentally controlled. We will also take advantage of recent technology that permits generation of substantial numbers of insect metacyclic form (MF) T. brucei, a largely unexplored intermediate between PF and BF. Using HTS/TREAT, we will define the patterns of RNA editing in MFs, and reveal whether BF RNA editing patterns for some or all mRNAs emerge in MFs, providing insight into the temporal control of RNA editing during T. brucei development. The proposed studies address several important gaps in knowledge regarding the essential kinetoplastid RNA editing process. Moreover, this work may help lay the foundation for future drug development and provide important insights into ribonucleoprotein complex function in higher organisms.