Developmental regulation of RNA editing in Trypanosoma brucei

项目摘要 布氏锥虫是一种寄生性原生动物，是非洲人类锥虫病的病原体 在36个撒哈拉以南非洲国家。寄生虫的进展，通过几个必要的发展阶段 在它的整个生命周期中，它在两个宿主之间转换，采采蝇和哺乳动物。这些主机提供 非常不同的营养环境，寄生虫必须能够迅速适应和利用。到 有效地利用这两种营养环境，T.布氏杆菌调节其线粒体活性。 然而，虽然线粒体基因组中编码了几种呼吸亚基，但大多数这些亚基都是由线粒体基因组编码的。 线粒体基因不编码功能性开放阅读框。生成可翻译的开放式阅读 因此，寄生虫必须通过精确插入和删除通常的转录后修饰mRNAs。 在一个称为RNA编辑的过程中产生数百个尿苷残基。随着代谢需求的变化， 几种mRNA的编辑在哺乳动物细长血流形式（BSF） 和昆虫载体原环形式（PCF）寄生虫，其中RNA编辑已被研究的唯一阶段， T.布鲁塞。然而，这种调节发生的机制和生命周期中的精确点 什么时候被触发是未知的。最近的数据质疑旧的研究，关于哪些成绩单经历 发育调节编辑，以及使用不同的菌株和生长条件也可能导致 差异我们的假设是，不同mRNA的编辑在T.布氏 关于时间和机制生命周期，以及编辑附件的翻译后修饰 这些因素促成了这一规定。在目标1中，我们将建立一个细胞系， 增殖性和非增殖性（可传播）生命周期阶段，并使用qRT-PCR和高通量 测序以定义所有12种编辑的mRNA在四个生命周期阶段中的编辑谱。发育 编辑的脱辅基细胞色素B（CYB）mRNA的调节积累已经确立，辅助因子 在PCF寄生虫中的这种蓄积需要RBP 16和MRP 1/2。在目标2中，我们将测试精氨酸是否 RBP 16的甲基化调节CYb mRNA编辑的起始以及CYb mRNA与编辑的关联 全酶在目标3中，我们将确定是否报告的发育调节磷酸化的 MRP 1/2解释了该因子支持编辑的CYb mRNA水平的能力，特别是在PCF中。这个项目 建立在史密斯博士在博士期间获得的技能之上。T.布鲁塞。该项目将 要求他发展新的技能和知识，特别是在RNA生物学，蛋白质-核酸相互作用， 生物信息学史密斯博士将在导师的帮助下发展专业和学术技能，并通过 布法罗大学提供的课程。他将在区域和国际会议上展示他的数据， 扩大他的潜在合作者网络。在他的指导委员会的支持下， 将扩大史密斯博士的能力，使他能够舒适地开始一个独立的实验室。

Project Summary Trypanosoma brucei is a parasitic protozoan and the causative agent of human African trypanosomiasis in 36 sub-Saharan African countries. The parasite progresses through several, required developmental stages throughout its life cycle as it transitions between its two hosts, the tsetse fly and mammals. These hosts offer very different nutritional environments which the parasite must be able to rapidly adapt to and exploit. To efficiently take advantage of both nutritional environments, T. brucei modulates its mitochondrial activity. However, while several respiratory subunits are encoded in the mitochondrial genome, most of these mitochondrial genes do not encode functional open reading frames. To generate translatable open reading frames, the parasite must post-transcriptionally modify the mRNAs by precise insertion and deletion of often hundreds of uridine residues in a process termed RNA editing. Concomitant with changing metabolic demands, editing of several mRNAs is differentially regulated between mammalian long slender bloodstream form (BSF) and insect vector procyclic form (PCF) parasites, the only stages in which RNA editing has been investigated in T. brucei. However, the mechanisms by which this regulation takes place and the precise points in the life cycle when it is triggered are unknown. Recent data call into question older studies regarding which transcripts undergo developmentally regulated editing, and use of different strains and growth conditions has likely also caused discrepancies. Our hypothesis is that editing of distinct mRNAs is differentially regulated throughout the T. brucei life cycle with regard to both timing and mechanism, and that post-translational modification of editing accessory factors contributes to this regulation. In Aim 1, we will establish a cell line that can transition through both proliferative and non-proliferative (transmissible) life cycle stages and use qRT-PCR and high throughput sequencing to define the editing profile of all 12 edited mRNAs in four life cycle stages. Developmentally regulated accumulation of edited apocytochrome b (CYb) mRNA is well established, and accessory factors RBP16 and MRP1/2 are required for this accumulation in PCF parasites. In Aim 2, we will test whether arginine methylation of RBP16 regulates CYb mRNA editing initiation and association of CYb mRNA with the editing holoenzyme. In Aim 3, we will determine whether the reported developmentally regulated phosphorylation of MRP1/2 accounts for the ability of this factor to support edited CYb mRNA levels specifically in PCF. This project builds upon the skills Dr. Smith acquired during his Ph.D. work on protein trafficking in T. brucei. The project will require him to develop new skills and knowledge, notably in RNA biology, protein-nucleic acid interactions, and bioinformatics. Dr. Smith will develop professional and academic skills with the help of mentors, and through courses offered at the University at Buffalo. He will present his data at regional and international meetings, and broaden his network of potential collaborators. The proposed project, with support from his mentoring committee, will expand Dr. Smith’s capabilities to a point where he will be comfortable starting an independent laboratory.