RNA Uridylation in Trypanosomes

锥虫中的 RNA 尿苷化

• 批准号: 10591650
• 负责人: Ruslan Afasizhev
• 金额: $ 64.18万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-05 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10591650
• 关键词: Ablation; Adaptor Signaling Protein; Affect; Africa South of the Sahara; Antisense RNA; Biogenesis; Biology; Catalytic Domain; Cell Cycle Regulation; Cells; Code; Complex; Coupling; Cryoelectron Microscopy; Crystallization; Crystallography; DNA; Degradation Pathway; Development; Disease; Double-Stranded RNA; Elements; Enzymatic Biochemistry; Enzymes; Eukaryota; Exhibits; Exonuclease; Gene Expression; Genes; Genetic; Genetic Transcription; Genetic Translation; Goals; Guide RNA; Half-Life; Hybrids; Hydrolysis; Insect Vectors; Kinetoplast DNA; Knowledge; Laboratories; Link; Maternal Messenger RNA; Mediating; Messenger RNA; Metabolic; Methods; MicroRNAs; Mitochondria; Mitochondrial RNA; Modality; Modeling; Modification; Molecular; Molecular Machines; Nuclear; Nucleoproteins; Parasites; Parasitic infection; Pathway interactions; Perception; Phosphodiesterase I; Phylogenetic Analysis; Polyadenylation; Prevalence; Process; Protein Isoforms; Proteins; Public Health; RNA; RNA Cap-Binding Proteins; RNA Decay; RNA Degradation; RNA Editing; RNA Helicase; RNA Processing; RNA metabolism; Reaction; Resolution; Ribonucleoproteins; Roentgen Rays; Role; Route; Shapes; Site; Specificity; Structure; Tail; Testing; Textiles; Trans-Splicing; Transcript; Transcriptional Regulation; Trypanosoma; Trypanosoma brucei brucei; Variant; Work; crosslink; epitranscriptomics; genetic information; helicase; hemoflagellate; in vivo; insertion/deletion mutation; instrument; mRNA Decay; mRNA Transcript Degradation; marginalized population; mitochondrial messenger RNA; novel; operation; particle; pathogen; programs; protein complex; spleen exonuclease; structural biology; synergism; transcriptome; uridylate

ABSTRACT Parasitic infections by Trypanosoma brucei spp. undermine public health and economy in Sub-Saharan Africa. These hemoflagellates are distinguished by the kinetoplast, a mitochondrial nucleoprotein body containing interlinked maxicircle and minicircle DNA. From a basic knowledge perspective, findings of polycistronic transcription, trans-splicing and RNA editing in trypanosomatids have profoundly influenced the conceptual fabric of RNA biology. Accordingly, ongoing work on U-insertion/deletion mitochondrial mRNA editing has produced pivotal breakthroughs, including discoveries of gRNA, the editosome, and the first RNA uridylating enzyme, KRET1 TUTase. Sustained efforts by the PI and other laboratories have expanded the uridylation’s breadth from a unique editing reaction into a compound transcriptome-shaping force prominent in most eukaryotes. Building on recently unraveled mitochondrial and cytosolic mRNA maturation and decay pathways, and progress in TUTase structural biology, we submit these Specific Aims: Aim 1 investigates the TUTase–3′-5′ exonuclease nexus underpinning mitochondrial RNA biogenesis and degradation. It focuses on mechanisms by which KRET1-containing molecular machines recognize diverse ribonucleoprotein particles, thus governing processing, functions, and metabolic fates of their cargo RNA. Molecular and Cryo-EM approaches will define transcript type-specific KRET1 complexes and determine their interactomes, in vivo RNA targets and near-atomic structures. We will analyze a novel RNA helicase, KREH3, as the potential link between massive antisense transcription of kinetoplast DNA and KRET1-mediated turnover. Aim 2 examines structural determinants and interactions enabling KRET2 and MEAT1 to recognize double- stranded RNA substrates, such as gRNA–mRNA hybrids. These programable mitochondrial TUTases execute U-insertion editing, which constitutes a Kinetoplastea-specific method of genetic information transfer. Aim 3 explores TbTUT3 and TbTUT4 as potential modulators of cytosolic mRNA decay, a principal conduit regulating nuclear gene expression in lieu of missing transcriptional control. By assessing transcriptome-wide U- tailed mRNAs prevalence and decay rates, we will test the coupling between mRNA 3′ uridylation and directional clearance. Beyond the parasite RNA metabolism, we are inquiring how the divergent auxiliary modalities confer orthogonal specificities and functions to a conserved and phylogenetically pervasive TUTase catalytic domain.

摘要 布氏锥虫寄生虫感染。破坏撒哈拉以南非洲的公共卫生和经济。 这些血鞭毛虫的特征是动质体，一种线粒体核蛋白体， 相互连接的大环和小环DNA。从基础知识的角度来看，多顺反子的发现 锥虫中的转录、反式剪接和RNA编辑深刻地影响了 RNA生物学因此，正在进行的关于U-插入/缺失线粒体mRNA编辑的工作已经产生了 关键性突破，包括gRNA、编辑体和第一个RNA尿苷化酶的发现， KRET1 TUTase。PI和其他实验室的持续努力已经扩大了尿苷化的广度， 一种独特的编辑反应，转化为在大多数真核生物中突出的复合转录组形成力。 基于最近解开的线粒体和胞质mRNA成熟和衰变途径， 在TUTase结构生物学中，我们提出了这些具体目标： 目的1研究TUTase-3′-5′外切核酸酶与线粒体RNA生物合成的关系， 降解它的重点是含有KRET 1的分子机器识别多种 核糖核蛋白颗粒，从而控制其货物RNA的加工，功能和代谢命运。 分子和Cryo-EM方法将定义转录本类型特异性KRET 1复合物，并确定其 相互作用体、体内RNA靶标和近原子结构。我们将分析一种新的RNA解旋酶KREH 3， 作为动基体DNA的大量反义转录和KRET 1介导的周转之间的潜在联系。 目的2研究结构决定因素和相互作用，使KRET 2和MEAT 1识别双- 链RNA底物，如gRNA-mRNA杂合体。这些可编程的线粒体TUTases执行 U-插入编辑，其构成动质体特异性的遗传信息传递方法。 目的3探索TbTUT 3和TbTUT 4作为细胞溶质mRNA衰变的潜在调节剂， 调节核基因表达代替缺失的转录控制。通过评估转录组范围的U- 尾mRNAs的流行率和衰减率，我们将测试mRNA 3′尿苷化和定向 间隙除了寄生虫的RNA代谢，我们正在研究不同的辅助模式如何赋予 与保守且系统发育上普遍存在的TUTase催化结构域具有正交的特异性和功能。