Cytosolic mRNA Uridylation in Trypanosoma brucei

摘要 最近，末端尿苷酰转移酶（TUTases）引起的RNA尿苷化已成为主要的转录组- 原生生物、真菌、植物和后生动物有机体的塑造力。线粒体酶的开创性工作 从动质体寄生虫布氏锥虫引入了TUTases的生物学特性的第一个例子， 在RNA编辑和小RNA生物合成中的功能，并建立了它们的基本结构域结构和原子结构域。 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， 增殖期和非增殖期。由于大多数蛋白质缺乏转录控制- 编码基因，转录后调节机制对这些转换的主要贡献是很好的 接纳的话然而，目前的努力主要集中在识别序列的RNA结合蛋白上， 元件在3个非翻译区，并作为mRNA稳定性的正或负调节剂。这 一项提案侧重于胞质3-脱氧尿苷化，并试图评估mRNA在转录组范围内的发生， 发育开关上的U形加尾，以及mRNA尿苷酸化和衰变之间的潜在偶联。我们 先前鉴定了两种胞质TUTases，TbTUT 3和TbTUT 4，现在提供了初步数据支持 TbTUT 3参与mRNA 3 ′-5 ′端降解。Aim 1将建立基于深度测序的技术（Tail- Seq），并开发一个生物信息学管道，用于在实验室适应的环境中对mRNA末端进行无偏分析。 单形昆虫和血流增殖形式。调查将扩大到一个多形细胞系 能够在体外从血流增殖细长型分化为非增殖型， 有传播能力的矮桩形式，并进一步进入早期昆虫形式。目的2将连接mRNA尿苷化活性 与特定的TUTase结合，并检验尿苷酸化的mRNA被3 ′胞外体靶向的假设。这项工作 将成为研究锥虫基因在发育和发病机制中表达的有力工具， 将产生一个全面的看法mRNA的3个碱基的修改。通过研究早期分化的真核生物， 解决了尿苷化诱导的RNA衰变的进化保守性，并可能验证TUTases作为 可下药的目标

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.