Regulation of RNA editing in Trypanosoma brucei

描述（由申请人提供）：我们的研究旨在了解引起非洲昏睡病、恰加斯病和利什曼病的动质体寄生虫中RNA编辑的分子机制。这些疾病的药物治疗方法陈旧、昂贵、难以管理，通常毒性很高，并且耐药性正在发展。因此，迫切需要新的化学治疗剂来对抗由动质体寄生虫引起的疾病。新药的开发需要对寄生虫生物学有详细的分子理解。线粒体尿苷插入/缺失RNA编辑是动质体所特有的，在人类宿主中不存在。这一过程对于布氏锥虫的人类感染和昆虫媒介形式的生存都是必不可少的。编辑需要通过转录后插入和缺失尿苷残基来广泛重塑mRNA以产生可翻译的mRNA。用于编辑的序列信息包含在小的反式作用向导RNA（gRNA）中，其顺序地起作用，使得编辑沿着mRNA以3'至5'方向沿着进行。RNA编辑过程由20 S编辑体催化。此外，需要多种辅助因子来对RNA的子集或编辑的RNA的整个池进行生产性编辑。在之前的资助期间，我们报告了对TbRGG 2的研究，TbRGG 2是一种在编辑的起始和3'至5'进展中起关键作用的必需蛋白。TbRGG 2是一种定义不清的大分子复合物，即线粒体RNA结合复合物1（MRB 1）的组成部分。迄今为止分析的耗尽MRB 1组分的细胞在gRNA和mRNA稳定性、编辑受到影响的特定RNA以及编辑受到损害的明显点方面具有显著不同的表型。我们的总体假设是，MRB 1复合物是由亚复合物组成的，其组合作用协调动质体RNA编辑中的多个基本步骤。在本研究中，我们将采用遗传学、基因组学和生物化学相结合的方法来分析MRB 1复合物及其组成蛋白在T.布鲁塞。在目标1中，我们将确定TbRGG 2 RNA结合、退火和解旋活性在编辑起始和3'至5'进展中的作用，并使用深度测序和RNA结构分析来定义TbRGG 2所克服的特异性分子内和分子间相互作用。在目标2中，我们将使用RNAi检查11种以前未研究的MRB 1组分，然后进行一系列测定，以确定在这些细胞中完成编辑的障碍。最后，在目标3中，我们将根据其组分亚复合物及其蛋白质-蛋白质和蛋白质-RNA相互作用来确定MRB 1复合物的组成。目标3的物理数据将在目标1和2中进行的功能研究的背景下进行评价，以建立MRB 1复合体结构和功能的综合模型。总之，这些实验将提供重要的见解，一个重要的基因调控过程，可能被证明是有用的，在治疗致命的疾病引起的动质体寄生虫。

DESCRIPTION (provided by applicant): Our research is aimed at understanding the molecular mechanisms of RNA editing in kinetoplastid parasites, which cause African sleeping sickness, Chagas' disease, and leishmaniasis. Drug treatments for these diseases are antiquated, expensive, difficult to administer, often highly toxic, and resistance is developing. Thus, new chemotherapeutic agents to combat diseases caused by kinetoplastid parasites are urgently needed. Development of new drugs requires a detailed molecular understanding of parasite biology. Mitochondrial uridine insertion/deletion RNA editing is unique to kinetoplastids and absent from their human hosts. This process is essential for survival of both the human infective and insect vector forms of Trypanosoma brucei. Editing entails extensive remodeling of mRNAs by posttranscriptional insertion and deletion of uridine residues to create translatable mRNAs. Sequence information for editing is contained in small, trans acting guide RNAs (gRNAs), which act sequentially such that editing proceeds in a 3' to 5' direction along an mRNA. The RNA editing process is catalyzed by 20S editosomes. In addition, multiple accessory factors are required for productive editing of either subsets of RNAs or the entire pool of edited RNAs. During the previous funding period, we reported studies on TbRGG2, an essential protein that plays a key role in both the initiation and 3' to 5' progression of editing. TbRGG2 is a component of an ill-defined macromolecular complex, the mitochondrial RNA binding complex 1 (MRB1). Cells depleted of MRB1 components analyzed to date have remarkably different phenotypes with regard to gRNA and mRNA stability, the specific RNAs whose editing is affected, and the apparent point at which editing is compromised. Our overarching hypothesis is that the MRB1 complex is composed of subcomplexes whose combinatorial actions coordinate multiple fundamental steps in kinetoplastid RNA editing. In the proposed studies, we will employ a combined genetic, genomic, and biochemical approach to analyze the functions of the MRB1 complex and its constituent proteins in RNA editing in T. brucei. In Aim 1, we will determine the roles of TbRGG2 RNA binding, annealing, and unwinding activities in editing initiation and 3' to 5' progression, and use deep sequencing and RNA structure analysis to define the specific intra- and intermolecular interactions that are overcome by TbRGG2. In Aim 2, we will examine 11 previously unstudied MRB1 components using RNAi, followed by a battery of assays that will define the obstruction to complete editing in these cells. Finally, in Aim 3, we will determine the composition of the MRB1 complex in terms of its component subcomplexes and their protein- protein and protein-RNA interactions. Physical data from Aim 3 will be evaluated in the context of functional studies performed in Aims 1 and 2 to establish a comprehensive model of MRB1 complex structure and function. Together, these experiments will provide important insights into an essential gene regulatory process that may prove useful in the treatment of the deadly diseases caused by kinetoplastid parasites.