Developmental regulation of RNA editing in Trypanosoma brucei

布氏锥虫 RNA 编辑的发育调控

• 批准号: 10468300
• 负责人: Joseph Terrell Smith
• 金额: $ 7.17万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10468300
• 关键词: ABCC1 gene; Academic skills; African; African Trypanosomiasis; Arginine; Binding; Bioenergetics; Bioinformatics; Biological Process; Biology; Blood Circulation; Buffaloes; Cell Line; Cells; Complement; Complex; Country; Data; Development; Doctor of Philosophy; Environment; Exhibits; Genes; Genetic Transcription; Growth; Guide RNA; High-Throughput Nucleotide Sequencing; Holoenzymes; Human; Immunoprecipitation; Insect Vectors; International; Knowledge; Laboratories; Life Cycle Stages; Maintenance; Mammals; Mentors; Messenger RNA; Metabolic; Methylation; Mitochondria; Mitochondrial RNA; Nucleic Acids; Nutritional; Open Reading Frames; Parasites; Pathway interactions; Pattern; Phosphorylation; Physiology; Pleomorphism; Population; Post-Translational Protein Processing; Process; Proteins; Quantitative Reverse Transcriptase PCR; RNA; RNA Editing; Regulation; Reporting; Role; Testing; Transcript; Trypanosoma; Trypanosoma brucei brucei; Tsetse Flies; Universities; Uridine; Virulence; Work; density; environmental change; insertion/deletion mutation; insight; knock-down; meetings; mitochondrial genome; mitochondrial messenger RNA; overexpression; protein transport; respiratory; skills; tool; transmission process

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.

项目概要 布氏锥虫是一种寄生原生动物，是人类非洲锥虫病的病原体 在 36 个撒哈拉以南非洲国家中，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，当它在两个宿主（采采蝇和哺乳动物）之间转换时，这些宿主提供了帮助。 寄生虫必须能够快速适应和利用截然不同的营养环境。 T. brucei 有效地利用这两种营养环境，调节其线粒体活性。 然而，虽然线粒体基因组中编码了几个呼吸亚基，但其中大多数 线粒体基因不编码功能性开放阅读框以产生可翻译的开放阅读。 框架中，寄生虫必须通过精确插入和删除经常修改 mRNA 来进行转录后修饰。 伴随着代谢需求的变化，RNA 编辑过程中存在数百个尿苷残基。 哺乳动物细长血流形式 (BSF) 之间多种 mRNA 的编辑受到差异性调节 和昆虫载体原环形式（PCF）寄生虫，这是RNA编辑已被研究的唯一阶段 然而，这种调节发生的机制以及生命周期中的精确点。 它何时被触发尚不清楚，最近的数据对有关哪些转录本会发生的旧研究提出了质疑。 发育调控的编辑以及不同菌株和生长条件的使用也可能导致 我们的假设是，不同 mRNA 的编辑在整个 T. brucei 中受到差异性调节。 关于时间和机制的生命周期，以及编辑附件的翻译后修改 在目标 1 中，我们将建立一种可以通过这两种途径进行转变的细胞系。 增殖和非增殖（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 个编辑过的 mRNA 在四个生命周期阶段的编辑概况。 编辑的脱辅基细胞色素 b (CYb) mRNA 的调节积累已得到充分证实，并且辅助因素 PCF 寄生虫中的这种积累需要 RBP16 和 MRP1/2 在目标 2 中，我们将测试是否精氨酸。 RBP16 的甲基化调节 CYb mRNA 编辑起始以及 CYb mRNA 与编辑的关联 在目标 3 中，我们将确定所报道的发育调节磷酸化是否存在。 MRP1/2 解释了该因子在 PCF 中支持编辑的 CYb mRNA 水平的能力。 该项目建立在史密斯博士在 T. brucei 蛋白质贩运方面的博士研究期间获得的技能的基础上。 要求他发展新的技能和知识，特别是在RNA生物学、蛋白质-核酸相互作用和 史密斯博士将在导师的帮助下发展专业和学术技能。 他将在布法罗大学提供的课程中展示他的数据，以及 在他的指导委员会的支持下，扩大了他的潜在合作者网络。 将扩展史密斯博士的能力，使他能够轻松地建立一个独立的实验室。