Function of the Kinetoplast in the Hemoflagellates

描述（由申请人提供）：U-插入/缺失RNA编辑是锥虫寄生虫线粒体中发生的独特转录后RNA修饰过程。编辑通过在特定位点插入和缺失尿苷来修改从线粒体DNA“隐基因”转录的16种线粒体mRNA中的12种的序列，以使它们可翻译。我们发现了一类新的短RNA（指导RA或gRNA），它们含有通过反式碱基配对进行编辑的序列信息，这使得这种类型的RNA修饰符合中心法则。我们基于gRNA的介导提出的假设编辑模型涉及切割、U-添加或U-缺失和连接的多个3 '-5'循环。我们为该模型提出的负责酶活性的蛋白质被鉴定为1 md多蛋白复合物的组分，RNA编辑核心复合物或RECC。几个额外的多蛋白复合物，可能参与RNA调控与RECC通过RNA接头相互作用。编辑发生在动质体谱系的所有成员中，包括锥虫寄生虫，几种人类疾病的致病因子。如果不了解编辑复合物的分子结构及其与底物RNA的相互作用，就无法理解编辑。我们建议进行高分辨率的结构-功能分析的主要组成部分的编辑反应-1 MD RECC。我们将试图通过以原子分辨率确定这些粒子的3D结构并识别蛋白质组分及其蛋白质-蛋白质和蛋白质-蛋白质来证实和扩展我们假设的编辑反应模型。编辑反应不同功能状态下的RNA相互作用分子结构信息将根据该途径的详细机制进行解释，这些知识将为未来的研究提供基础。研究全酶以及编辑与线粒体RNA调控和蛋白质翻译的可能相互作用。这是重要的，因为锥虫已成为重要的真核生物模型系统，用于研究许多基本的生物学现象，除了RNA编辑，如蛋白质表达，反式剪接表面抗原的变化和鞭毛运动。由于现在看来，线粒体RNA编辑可能与翻译和RNA调控相互作用，对编辑的理解将有助于锥虫作为模型生物系统的发展。此外，U-插入/缺失编辑作为多种其他转录后RNA修饰现象的范例，例如人类大脑中的折返引导的A至I编辑，sno-RNA引导的核糖体RNA的甲基化和假尿苷化，甚至RNAi中的siRNA引导的mRNA切割，并且在该项目中获得的信息可能与理解这些其他生物学重要现象相关。最后但并非最不重要的是，编辑途径对生存能力至关重要，并可能为开发针对这些寄生虫引起的疾病的干预措施提供潜在目标。 公共卫生相关性：本项目旨在获得涉及锥虫线粒体尿苷插入/缺失RNA编辑的核心多蛋白复合物的功能性高分辨率3D结构，其中U在mRNA的编码区内插入和缺失。还将从结构上分析体外编辑反应期间底物RNA的结合和加工。生物医学意义在于，这些寄生虫是许多重要人类疾病的病原体，包括恰加斯病，利什曼病和非洲昏睡病，并且编辑途径在寄生虫生命周期的所有阶段都是必不可少的，并且不存在于人类宿主中，从而使其成为针对这些寄生虫的治疗干预的潜在靶标。

DESCRIPTION (provided by applicant): U-insertion/deletion RNA editing is a unique post-transcriptional RNA modification process occurring in the mitochondria of trypanosomatid parasites. Editing modifies the sequences of 12 of 16 mitochondrial mRNAs transcribed from mitochondrial DNA "cryptogenes" by insertion and deletion of uridines at specific sites to render them translatable. Our discovery of a new class of short RNAs (guide RAs or gRNAs) that contain the sequence information for editing by base pairing in trans brought this type of RNA modification into line with the central dogma. The hypothetical editing model we proposed based on the mediation of gRNAs involved multiple 3'-5' cycles of cleavage, U-addition or U-deletion and ligation. The proteins responsible for the enzymatic activities we proposed for this model were identified as components of a 1 md multiprotein complex, the RNA Editing Core Complex or RECC. Several additional multiprotein complexes which may be involved in RNA regulation interact with the RECC via RNA linkers. Editing occurs in all members of the Kinetoplastid lineage, which includes the trypanosomatid parasites, the causal agents of several human diseases. The understanding of editing cannot proceed without knowledge of the molecular architecture of the editing complexes and their interactions with substrate RNAs. We propose to perform a high resolution structure- function analysis of the main component of the editing reaction - the 1 md RECC. We will attempt to confirm and extend our hypothetical editing reaction model by determining the 3D structure of these particles at an atomic resolution and identifying the protein components and their protein-protein and protein-RNA interactions in different functional states of the editing reaction The molecular structure information will be interpreted in terms of the detailed mechanism of this pathway and this knowledge will provide a basis for future investigations of the holoenzyme and the possible interactions of editing with mitochondrial RNA regulation and protein translation. This is important since the trypanosomatids have become important eukaryotic model systems for studying many basic biological phenomena in addition to RNA editing, such as protein expression, trans-splicing surface antigen variation and flagellar motility. Since it now appears that mitochondrial RNA editing may interact with translation and RNA regulation, an understanding of editing will assist in the development of trypanosomatids as model biological systems. In addition, U-insertion/deletion editing serves as a paradigm for multiple other post-transcriptional RNA modification phenomena such as fold-back guided A to I editing in human brain, sno-RNA guided methylation and pseudouridylation of ribosomal RNAs and even Si RNA-guided cleavage of mRNAs in RNAi, and the information obtained in this project may prove relevant for understanding these other biologically important phenomena. And last but not least, the editing pathway is essential for viability and may provide a potential target for development of interventions against the diseases caused by these parasites. PUBLIC HEALTH RELEVANCE: This project proposes to obtain a functional high resolution 3D structure for the core multiprotein complex involved in trypanosome mitochondrial uridine insertion/deletion RNA editing in which U's are inserted and deleted within coding regions of mRNAs. Binding and processing of substrate RNAs during the in vitro editing reaction will also be analyzed structurally. The biomedical significance is that these parasites are the causal agents of a number of important human diseases including Chagas Disease, Leishmaniasis and African Sleeping Sickness, and that the editing pathway is essential in all stages of the parasite life cycles and not present in the human host, thereby making it a potential target for therapeutic intervention against these parasites.