Functions of mRNA Pseudouridylation

mRNA 假尿苷化的功能

• 批准号: 10442443
• 负责人: Wendy Victoria Gilbert
• 金额: $ 33.5万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10442443
• 关键词: Acute; Affect; Alternative Splicing; Base Pairing; Biochemical; Biological; Biology; Cells; Chromatin; Code; Digestive System Disorders; Disease; Dyskeratosis Congenita; Enzymes; Etiology; Event; Gene Expression; Gene Expression Regulation; Genes; Goals; Health; HepG2; Human; In Vitro; Intellectual functioning disability; Knock-out; Knowledge; Lead; Link; Malignant Neoplasms; Messenger RNA; Mitochondria; Mitochondrial Myopathies; Modification; Molecular; Nucleotides; Production; Proteins; Proteome; Pseudouridine; RNA; RNA Splicing; Reader; Regulatory Element; Reproducibility; Research; Resistance; Resolution; Role; Site; Small Nuclear RNA; Uridine; Viral; Virus Diseases; Work; Yeasts; base; disease-causing mutation; human disease; insight; mRNA Precursor; new therapeutic target; novel; overexpression; stable cell line; transcriptome; transcriptome sequencing

PROJECT SUMMARY Multiple pseudouridine synthases (PUS) are implicated in human disease, but the mechanisms that connect loss of PUS activity to mitochondrial myopathy, digestive disorders, intellectual disability, resistance to viral infection, dyskeratosis congenita, and diverse cancers remain largely unknown. There are several critical gaps in our current knowledge of the functions of PUS proteins. Although the basic biochemical activity of PUS proteins in catalyzing the isomerization of uridine to pseudouridine is well understood, the specific RNA targets of most human PUS proteins are unknown or incompletely known. Our long-term goals include identifying the targets of all PUS proteins and determining the molecular consequences of modification of specific RNAs with pseudouridine in disease-relevant cellular contexts. This will be critical for understanding the etiology of diseases caused by PUS deficiency and may reveal new therapeutic targets for treatment. Our previous studies revealed that pseudouridine is a prevalent modification of nascent pre-messenger RNA. These results, together with quantitative in vitro studies showing that pseudouridine can affect both RNA-protein and RNA-RNA interactions, lead to our central hypothesis that pre-mRNA pseudouridylation controls human gene expression at the level of pre-mRNA splicing. In support of this hypothesis, we have demonstrated that loss of one pre- mRNA modifying pseudouridine synthase, PUS1, causes widespread changes to pre-mRNA splicing in human cells, with more than 3,000 PUS1-sensitive alternative splicing events identified. Our Specific Aims are to (1) Define the splicing-relevant pre-mRNA targets of the predominant pre-mRNA pseudouridylating enzymes: PUS1, PUS7, and RPUSD4; and (2) Elucidate the molecular mechanisms of pseudouridine-sensitive splicing. The proposed work will provide key insight into the molecular functions of pseudouridines in pre-mRNAs and may reveal novel modes of eukaryotic gene regulation. By establishing pre-mRNAs as a broad new class of substrates for PUS enzymes, our work implicates defective splicing as a plausible but understudied mechanism connecting loss of PUS activity to numerous human diseases.

项目摘要 多种假性合酶（PU）与人类疾病有关，但 将脓液活性丧失与线粒体肌病，消化系统疾病联系在一起的机制， 智力残疾，对病毒感染的抵抗力，患者症状，兴奋和多样的癌症 在很大程度上未知。我们当前对 脓蛋白的功能。虽然脓性在催化中的基本生化活性 尿道氨酸与假氨酸的异构化已被充分了解，特定的RNA靶标的 大多数人脓蛋白是未知或未知的。我们的长期目标包括 识别所有脓蛋白的靶标，并确定 在与疾病相关的细胞环境中，用伪嘧啶对特定的RNA进行修饰。这会 对于理解脓缺乏症引起的疾病的病因至关重要，并可能揭示 新的治疗靶标。 我们先前的研究表明，假氨酸是新生的普遍修饰 预上音符RNA。这些结果以及定量的体外研究表明 假氨基氨酸可以影响RNA - 蛋白质和RNA-RNA相互作用，导致我们的中心 假设前mRNA伪苷化控制人类基因在 前mRNA剪接。为了支持这一假设，我们已经证明了一个前的损失 MRNA修饰假氨酸合酶PUS1会导致Pre-MRNA的广泛变化 在人类细胞中的剪接，有3,000多个PUS1敏感的剪接事件 确定。我们的具体目的是（1）定义与剪接相关的前MRNA靶标 主要的mRNA伪源性酶：PUS1，PUS7和RPUSD4； （2） 阐明伪氨酸敏感剪接的分子机制。 拟议的工作将提供有关分子功能的关键见解 peudouridines中的mRNA中的伪胺，并可能揭示了真核基因调节的新型模式。经过 我们的工作将前MRNA作为脓酶的广泛新类底物 暗示有缺陷的剪接是连接脓液损失的合理但研究的机制 许多人类疾病的活动。