RNA Uridylation in Trypanosomes

锥虫中的 RNA 尿苷化

基本信息

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
项目类别：
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

项目摘要

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.

抽象的布氏锥虫的寄生虫感染损害了撒哈拉以南非洲地区的公共卫生和经济。这些血鞭毛虫以动质体为特征，动质体是一种线粒体核蛋白体，含有从基础知识的角度来看，多顺反子的发现是相互连接的大环和小环DNA。锥虫的转录、反式剪接和 RNA 编辑深刻地影响了概念结构因此，正在进行的 U 插入/缺失线粒体 mRNA 编辑工作已经产生。突破，包括 gRNA、编辑体和第一个 RNA 尿苷酸化酶的发现， KRET1 TUTase。PI 和其他实验室的持续努力扩大了尿苷化的广度。对大多数真核生物中突出的复合转录组塑造力的独特编辑反应。以最近解开的线粒体和胞浆 mRNA 成熟和衰变途径及进展为基础在 TUTase 结构生物学中，我们提出了以下具体目标：目标 1 研究支持线粒体 RNA 生物发生的 TUTase–3'-5' 核酸外切酶关系它专注于含有 KRET1 的分子机器识别不同的机制。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。分子和冷冻电镜方法将定义转录本类型特异性 KRET1 复合物并确定其我们将分析一种新型 RNA 解旋酶 KREH3，作为动质体 DNA 的大量反义转录与 KRET1 介导的周转之间的潜在联系。目标 2 检查结构决定因素和相互作用，使 KRET2 和 MEAT1 能够识别双链状 RNA 底物，例如 gRNA-mRNA 杂合体，这些可编程线粒体 TUTase 执行。 U-插入编辑，构成了 Kinetoplastea 特有的遗传信息转移方法。目标 3 探索 TbTUT3 和 TbTUT4 作为胞质 mRNA 衰减的潜在调节剂，胞质 mRNA 衰减是主要管道通过评估转录组范围内的 U- 来调节核基因表达。带尾 mRNA 的流行率和衰减率，我们将测试 mRNA 3' 尿苷化和方向性之间的耦合除了寄生虫 RNA 代谢之外，我们还在探究不同的辅助模式如何发挥作用。与保守且系统发育普遍的 TUTase 催化结构域正交的特异性和功能。