Developmental regulation of RNA editing in Trypanosoma brucei

项目摘要 布鲁氏锥虫是一种寄生原虫，也是人类非洲锥虫病的病原体。 在36个撒哈拉以南非洲国家。寄生虫经历了几个必需的发育阶段。 在它的整个生命周期中，它在两个宿主-采采蝇和哺乳动物之间过渡。这些主机提供 寄生虫必须能够迅速适应和利用的非常不同的营养环境。至 布氏毛滴虫有效地利用这两种营养环境，调节其线粒体活动。 然而，虽然线粒体基因组中有几个呼吸亚基编码，但大多数 线粒体基因不编码有功能的开放阅读框架。生成可翻译的开放阅读 框架中，寄生虫必须在转录后通过精确插入和删除经常 在一个被称为RNA编辑的过程中，有数百个尿苷残基。伴随着新陈代谢需求的变化， 哺乳动物细长血流形态(BSF)对几种mRNAs的编辑有差异调节 和昆虫媒介原环形式(PCF)寄生虫，这是在 布氏毛虫T.brucei然而，这种监管发生的机制和生命周期中的精确点 它何时被触发是未知的。最近的数据质疑了关于哪些成绩单经历了 发育调节的编辑，以及不同菌株和生长条件的使用也可能导致 差异。我们的假设是，不同mRNAs的编辑在整个布氏锥虫中受到不同的调节 与时间和机制以及编辑附件翻译后修改有关的生命周期 影响这一规律的因素很多。在目标1中，我们将建立一种可以在两者之间过渡的细胞系 增殖期和非增殖期(可传播)生命周期阶段，并使用qRT-PCR和高通量 测序以定义四个生命周期阶段所有12个编辑的mRNA的编辑配置文件。发展上的 编辑的细胞色素b(Cyb)mRNA的调节积累是建立得很好的，辅助因素 在PCF寄生虫中，这种积累需要RBP16和MRP1/2。在目标2中，我们将测试精氨酸 RBP16甲基化调控Cyb mRNA编辑起始及其与编辑的关联 全酶。在目标3中，我们将确定已报道的发育调节的磷酸化是否 MRP1/2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 以史密斯博士在布氏锥虫蛋白质交易博士工作期间获得的技能为基础。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-核酸相互作用方面，以及 生物信息学。史密斯博士将在导师的帮助下，通过 布法罗大学开设的课程。他将在地区和国际会议上介绍他的数据，以及 扩大他潜在的合作者网络。在他的指导委员会的支持下，提出的项目， 将扩大史密斯博士的能力，使他能够放心地建立一个独立的实验室。

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.