Role of the guide RNA binding protein RBP16 in the bloodstream form of T. brucei

描述（由申请方提供）：寄生原生动物布氏锥虫是人类昏睡病和家畜长角线虫病的病原体。昏睡病如果不治疗，一定是致命的。目前的药物是无效和有毒的，耐药性正在上升，突出了确定新的药物靶点的重要性。于T.在布鲁氏菌线粒体中，转录后RNA编辑是产生可翻译mRNA所必需的。编辑是前循环昆虫媒介阶段（PF）和血流哺乳动物宿主阶段（BF）生命周期阶段中的必要过程。因此，参与RNA编辑的蛋白质是化疗攻击的有希望的靶标。RNA结合蛋白RBP 16是一种必需的多功能基因调控蛋白，在PF T中作为CYb mRNA的RNA编辑辅助因子，以及稳定几种从未编辑过的线粒体RNA。布鲁塞。RBP 16通过几个精氨酸残基的甲基化修饰，这种修饰对于其大分子相互作用和PF中的基因调控功能是重要的。在布氏杆菌中，线粒体基因表达在PF和BF生命周期阶段之间受到显著调节。RBP 16在感染人类的BF阶段相对丰富;然而，对于其在寄生虫的该阶段中的线粒体基因调控中的作用一无所知。在本申请中，我们将研究RBP 16在BF T中的作用。使用RNAi介导的下调进行布鲁氏菌线粒体基因调控。我们将确定RBP 16缺失对BF中RNA编辑和稳定化的影响。我们将使用免疫沉淀和梯度沉降分析来分析BF中RBP 16蛋白质-蛋白质和蛋白质-RNA相互作用。最后，我们将确定两个主要的蛋白质精氨酸甲基转移酶在RBP 16甲基化和BF T中大分子相互作用中的作用。布鲁塞。我们的假设是RBP 16调节BF中特定线粒体mRNA的编辑和/或丰度，与它在PF中影响的那些不同。此外，我们认为RBP 16与其他线粒体蛋白结合，这些蛋白质-蛋白质相互作用可能受到蛋白质精氨酸甲基化的影响。公共卫生相关性：总之，拟议的研究将大大扩展对线粒体基因调控和精氨酸甲基化在人类感染性T细胞中的功能的理解。布鲁塞。

DESCRIPTION (provided by applicant): The parasitic protozoan, Trypanosoma brucei, is the causative agent of sleeping sickness in humans and nagana in domestic livestock. Sleeping sickness is invariably fatal if left untreated. Current drugs are ineffective and toxic, and drug resistance is on the rise, highlighting the importance of identifying novel drug targets. In T. brucei mitochondria, post-transcriptional RNA editing is required for the creation of translatable mRNAs. Editing is an essential process in both the procyclic insect vector stage (PF) and the bloodstream mammalian host stage (BF) life cycle stages. Thus, proteins involved in RNA editing are promising targets for chemotherapeutic attack. The RNA binding protein, RBP16, is an essential multi-functional gene regulatory protein that acts as an RNA editing accessory factor for CYb mRNA, as well as stabilizing several never-edited mitochondrial RNAs, in PF T. brucei. RBP16 is modified by methylation on several arginine residues, and this modification is important for its macromolecular interactions and gene regulatory functions in PF. In T. brucei, mitochondrial gene expression is dramatically regulated between PF and BF life cycle stages. RBP16 is relatively abundant in BF stage that infects humans; however, nothing is known about its role in mitochondrial gene regulation in this stage of the parasite. In this application, we will examine the role of RBP16 in BF T. brucei mitochondrial gene regulation using RNAi-mediated down-regulation. We will determine the effect of RBP16 depletion on both RNA editing and stabilization in BF. We will analyze RBP16 protein-protein and protein-RNA interactions in BF using immunoprecipitation and gradient sedimentation analyses. Finally, we will determine the roles of two major protein arginine methyltransferases in RBP16 methylation and macromolecular interactions in BF T. brucei. Our hypothesis is that RBP16 regulates the editing and/or abundance of specific mitochondrial mRNA in BFs, distinct from those it affects in PFs. Furthermore, we propose that RBP16 does so in conjunction with additional mitochondrial proteins and that these protein-protein interactions may be impacted by protein arginine methylation. PUBLIC HEALTH RELEVANCE: Together, the proposed studies will greatly expand the understanding of both mitochondrial gene regulation and the functions of arginine methylation in the human infectious form of T. brucei.