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 布氏锥虫的寄生感染。破坏撒哈拉以南非洲的公共卫生和经济。 这些血鞭毛虫的区别是动泡体，它是一种线粒体核蛋白体，含有 连锁的上环和小环DNA。从基础知识的角度来看，多顺反子的发现 锥虫的转录、反式剪接和RNA编辑深刻地影响了概念性结构 核糖核酸生物学。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 关键突破，包括gRNA，编辑体和第一个RNA尿苷酸酶的发现， KRET1 TUTase.PI和其他实验室的持续努力已经将尿苷基化的范围从 一种独特的编辑反应，形成一种在大多数真核生物中突出的复合转录组形成力量。 建立在最近解开的线粒体和胞质mRNA成熟和衰退途径的基础上，以及进展 在TUTase结构生物学中，我们提出了以下具体目标： 目的1研究支持线粒体RNA生物发生的TUTase-3‘-5’核酸外切酶关系。 退化。它的重点是含有KRET1的分子机器识别不同种类的 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。 分子和冷冻-EM方法将定义转录本类型特定的KRET1复合体，并确定其 相互作用、活体RNA靶标和近原子结构。我们将分析一种新的RNA解旋酶KREH3， 作为动泡体DNA大量反义转录和KRET1介导的周转之间的潜在联系。 AIM 2考察了结构决定因素和相互作用，使KRET2和MEAT1能够识别双重 链状RNA底物，如gRNA-mRNA杂交物。这些可编程的线粒体TUTase执行 U-插入编辑，这构成了一种特定于动粒基因组的遗传信息转移方法。 目的3探索TbTUT3和TbTUT4作为胞质mRNA衰变的潜在调节剂，这是一种主要的通道 调控核基因表达以代替缺失的转录调控。通过评估转录组范围的U- 拖尾mRNAs的患病率和衰减率，我们将测试mRNA3‘尿苷基化和定向之间的偶联 通行证。除了寄生虫的RNA新陈代谢，我们正在询问不同的辅助模式是如何赋予 保守的和系统发育广泛的TUTase催化域的正交性和功能。