Cytosolic mRNA Uridylation in Trypanosoma brucei

布氏锥虫胞浆 mRNA 尿苷化

基本信息

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
项目类别：
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

项目摘要

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关于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，并继续进行鉴于大多数蛋白质缺乏转录控制，每个宿主都处于增殖和非增殖阶段。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。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。先前鉴定了两种胞质 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性昆虫和血流增殖形式的研究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， Aim 2 将连接 mRNA 尿苷化活性。这项工作针对特定的 TUTase 并测试尿苷化 mRNA 是 3ʹ 外泌体靶向的假设。将建立一个强大的工具来研究锥虫基因表达在发育和发病机制中的作用，以及通过早期研究分化的真核生物，它还将产生 mRNA 3ʹ 修饰的全面视图。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。