Regulation of RNA editing in Trypanosoma brucei

描述(申请人提供)：我们的研究旨在了解动体寄生虫中RNA编辑的分子机制，这些寄生虫会导致非洲昏睡病、恰加斯病和利什曼病。这些疾病的药物治疗方法陈旧、昂贵、难以管理、往往毒性很高，而且耐药性正在形成。因此，迫切需要新的化疗药物来对抗动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编辑是动体所特有的，在人类宿主中是不存在的。这一过程对于人类感染性和昆虫媒介形式的布氏锥虫都是至关重要的。编辑需要通过转录后插入和删除尿苷残基来广泛重塑mRNAs，以创建可翻译的mRNAs。用于编辑的序列信息包含在小的反式作用引导RNA(GRNAs)中，其顺序地作用，使得编辑沿着mRNA在3‘到5’方向进行。RNA编辑过程是由20S编辑小体催化的。此外，对RNA子集或整个编辑的RNA池进行高效编辑需要多个辅助因素。在之前的资助期间，我们报告了对TbRGG2的研究，TbRGG2是一种在编辑的启动和3‘到5’进展中发挥关键作用的重要蛋白质。TbRGG2是一种定义不明确的大分子复合体--线粒体RNA结合复合体1(MRB1)的组成部分。到目前为止，被分析的缺失MRB1组分的细胞在gRNA和mRNA稳定性、其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。我们的主要假设是，MRB1复合体由子复合体组成，这些子复合体的组合行为协调了动质RNA编辑的多个基本步骤。在拟议的研究中，我们将使用遗传、基因组和生化相结合的方法来分析MRB1复合体及其组成蛋白在布鲁氏锥虫RNA编辑中的功能。在目标1中，我们将确定TbRGG2 RNA结合、退火和解离活动在编辑起始和3‘到5’进程中的作用，并使用深度测序和RNA结构分析来定义TbRGG2克服的特定的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个以前未研究过的MRB1组分，然后进行一系列分析，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MRB1复合体的组成亚复合体及其蛋白质-蛋白质和蛋白质-RNA相互作用来确定MRB1复合体的组成。来自目标3的物理数据将在目标1和目标2中进行的功能研究的背景下进行评估，以建立MRB1复合体结构和功能的综合模型。总之，这些实验将为一个基本的基因调控过程提供重要的见解，该过程可能被证明对治疗由动体寄生虫引起的致命疾病有用。

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.