Cytosolic mRNA Uridylation in Trypanosoma brucei

布氏锥虫胞浆 mRNA 尿苷化

• 批准号: 9226222
• 负责人: Ruslan Afasizhev
• 金额: $ 20.56万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-23 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9226222
• 关键词: Accounting; Address; Affect; Affinity; Affinity Chromatography; African; African Trypanosomiasis; Architecture; Binding Sites; Biogenesis; Bioinformatics; Biological Process; Biology; Blood Circulation; Catalytic Domain; Cell Line; Cell Nucleus; Cell Survival; Code; Complex; Coupling; DNA Probes; Data; Degradation Pathway; Development; Elements; Enzymes; Eukaryota; Excision; Exonuclease; Exoribonucleases; Gene Expression; Genes; Genetic; Genetic Transcription; Goals; Human; In Vitro; Individual; Insecta; Institutes; Knock-in; Knowledge; Laboratories; Libraries; Link; Mammals; Mass Spectrum Analysis; Medical; Messenger RNA; Metabolism; Methods; Mitochondria; Modeling; Modification; Mono-S; Nature; Nuclear; Organism; Parasites; Pathogenesis; Pathway interactions; Pattern; Plants; Play; Pleomorphism; Poly(A) Tail; Poly(A)-Binding Proteins; Polyadenylation; Population; Process; Proliferating; Proteins; Proteomics; Protocols documentation; RNA; RNA Decay; RNA Editing; RNA Polymerase II; RNA-Binding Proteins; Recruitment Activity; Regulator Genes; Repression; Role; Shapes; Small RNA; Specificity; Structure; Tail; Technology; Testing; Time; Trans-Splicing; Transcript; Transcriptional Regulation; Transferase; Translational Repression; Translations; Trypanosoma; Trypanosoma brucei brucei; Untranslated Regions; Work; adenylate; base; crosslink; deep sequencing; design; exosome; experimental study; fungus; genetic approach; in vivo; insight; mRNA Decay; mRNA Stability; mRNA Transcript Degradation; neglected tropical diseases; polyadenylated messenger RNA; thermostability; tool; transcriptome; transcriptome sequencing; transmission process; uridylate

ABSTRACT RNA uridylation by terminal uridylyl transferases (TUTases) has recently emerged as the major transcriptome- shaping force in protists, fungi, plants and metazoan organisms. Pioneering work on mitochondrial enzymes from the kinetoplastid parasite Trypanosoma brucei introduced the first examples of TUTases’ biological functions in RNA editing and small RNA biogenesis, and established their basic domain architecture and atomic details of UTP selectivity. African trypanosomes cycle between mammals and insects, and proceed through proliferating and non-proliferating stages in each host. Given the lack of transcriptional control for most protein- coding genes, the major contribution of post-transcriptional regulatory mechanisms to these transitions is well accepted. However, current efforts chiefly concentrate on RNA binding proteins that recognize sequence elements in 3ʹ untranslated regions and function as positive or negative modulators of mRNA stability. This proposal focuses on cytosolic 3ʹ uridylation and seeks to assess the transcriptome-wide occurrence of mRNA U-tailing upon developmental switches, and the potential coupling between mRNA uridylation and decay. We previously identified two cytosolic TUTases, TbTUT3 and TbTUT4, and now provide preliminary data supporting TbTUT3’s involvement in mRNA 3ʹ-5ʹ degradation. Aim 1 will establish deep sequencing-based technology (Tail- Seq) and develop a bioinformatics pipeline for unbiased analysis of mRNA termini in laboratory-adapted monomorphic insect and bloodstream proliferative forms. The inquiry will be extended to a pleomorphic cell line capable of differentiating in vitro from the bloodstream proliferative slender form to the non-proliferative, transmission-competent stumpy form, and further into early insect form. Aim 2 will link mRNA uridylation activity to a specific TUTase and test the hypothesis that uridylated mRNAs are targeted by the 3ʹ exosome. This work will institute a powerful tool for studying trypanosomal gene expression in development and pathogenesis, and will yield a comprehensive view of mRNA 3ʹ modifications. By investigating early diverged eukaryotes, it will also address the evolutionary conservation of uridylation-induced RNA decay and may validate TUTases as druggable targets.

抽象的 末端尿苷酰转移酶 (TUTases) 引起的 RNA 尿苷化最近已成为主要的转录组。 原生生物、真菌、植物和后生动物的塑造力。线粒体酶的开创性工作 来自动质体寄生虫布氏锥虫 (Trypanosoma brucei) 引入了 TUTase 生物活性的第一个例子 在RNA编辑和小RNA生物合成中发挥作用，并建立了它们的基本结构域和原子结构 UTP 选择性的详细信息。非洲锥虫在哺乳动物和昆虫之间循环，并通过 每个宿主的增殖和非增殖阶段。鉴于大多数蛋白质缺乏转录控制 编码基因，转录后调控机制对这些转变的主要贡献是众所周知的 公认。然而，目前的努力主要集中在识别序列的RNA结合蛋白上。 3′非翻译区的元件，作为 mRNA 稳定性的正向或负向调节剂。这 提案重点关注细胞质 3ʹ 尿苷化并寻求评估 mRNA 在转录组范围内的发生情况 发育开关上的 U 尾部，以及 mRNA 尿苷化和衰变之间的潜在耦合。我们 之前鉴定了两种胞质 TUTase，TbTUT3 和 TbTUT4，现在提供初步数据支持 TbTUT3 参与 mRNA 3ʹ-5ʹ 降解。目标1将建立基于深度测序的技术（Tail- Seq）并开发生物信息学管道，用于实验室适应的 mRNA 末端的无偏差分析 单形昆虫和血流增殖形式。调查将扩展到多形性细胞系 能够在体外区分血流增殖细长形式和非增殖细长形式， 具有传播能力的树桩形态，并进一步转变为早期昆虫形态。目标 2 将连接 mRNA 尿苷化活性 特定的 TUTase 并测试尿苷化 mRNA 是 3ʹ 外泌体靶向的假设。这部作品 将建立一个强大的工具来研究发育和发病机制中锥虫基因表达，以及 将产生 mRNA 3ʹ 修饰的全面视图。通过研究早期分化的真核生物，它还将 解决尿苷化诱导的 RNA 衰减的进化保守性，并可能验证 TUTases 可药物靶标。