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

描述（由申请人提供）：布鲁氏锥虫是人类昏睡病和家畜昏睡病的病原体。如果不及时治疗，昏睡病总是致命的。目前的药物是无效和有毒的，而且耐药性正在上升，这突出了确定新的药物靶点的重要性。在布氏体线粒体中，转录后RNA编辑是创建可翻译mrna所必需的。编辑是昆虫原循环媒介期（PF）和血液哺乳动物宿主期（BF）生命周期阶段的重要过程。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RBP16是一种重要的多功能基因调控蛋白，作为CYb mRNA的RNA编辑辅助因子，并稳定几种未编辑的线粒体RNA。RBP16在几个精氨酸残基上被甲基化修饰，这种修饰对其在PF中的大分子相互作用和基因调控功能至关重要。在T. brucei中，线粒体基因表达在PF和BF生命周期阶段受到显著调节。RBP16在感染人类的BF期相对丰富；然而，在寄生虫的这一阶段，它在线粒体基因调控中的作用尚不清楚。在本应用中，我们将通过rnai介导的下调来研究RBP16在BF T. brucei线粒体基因调控中的作用。我们将确定RBP16缺失对BF中RNA编辑和稳定的影响。我们将使用免疫沉淀和梯度沉淀分析BF中RBP16蛋白-蛋白和蛋白- rna相互作用。最后，我们将确定两种主要的蛋白精氨酸甲基转移酶在BF T. brucei RBP16甲基化和大分子相互作用中的作用。我们的假设是RBP16调节BFs中特定线粒体mRNA的编辑和/或丰度，与它在PFs中的影响不同。此外，我们提出RBP16与其他线粒体蛋白一起起作用，并且这些蛋白-蛋白相互作用可能受到蛋白精氨酸甲基化的影响。公共卫生相关性：总的来说，拟议的研究将极大地扩展对线粒体基因调控和精氨酸甲基化在人类感染性布鲁氏体中的功能的理解。

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.