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Functions of mRNA Pseudouridylation

mRNA 假尿苷化的功能

基本信息

批准号：
10659705
负责人：
Wendy Victoria Gilbert
金额：
$ 34.51万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2027-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10659705
关键词：
Affect Base Pairing Binding Binding Proteins Binding Sites Biochemical Biological Biological Process Biology Birth Cells Cellular Stress Cessation of life Chemicals Data Deposition Digestive System Disorders Disease Dyskeratosis Congenita Enzymes Etiology Eukaryotic Cell Gene Expression Gene Expression Regulation Genetic Transcription Goals Health HepG2 Human In Vitro Intellectual functioning disability Isomerism Knowledge Link Malignant Neoplasms Measurement Mediating Messenger RNA Metabolism Methods MicroRNAs Mitochondrial Myopathies Modification Molecular Molecular Conformation Mutation Nucleosides Physiological Proteins Pseudouridine RNA RNA-Binding Proteins RNA-Protein Interaction Reader Regulation Research Resistance Role Site Testing Translations Untranslated Regions Uridine Vertebral column Virus Diseases Work cell type chemical property defined contribution disease-causing mutation gene correction genetic manipulation human disease mRNA Export mRNA Precursor new therapeutic target novel stoichiometry transcriptome

项目摘要

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 recent work (Martinez et al. Mol Cell 2022) established that pseudouridine (Ψ) is deposited co-transcriptionally in nascent human pre-mRNA at thousands of sites where it is poised to affect every step of mRNA metabolism from birth to death. This renewal application will rigorously investigate the biochemical basis for mRNA regulation by pseudouridylation. The biological effects of Ψ must originate in chemical differences between U and Ψ, which directly affect RNA backbone conformation, the stability of base pairs, and the binding of proteins. Our preliminary data establish that hundreds of pseudouridines are present within the experimentally determined binding sites of regulatory RNA-binding proteins (RBPs) and micro RNAs (miRNAs) in human HepG2 cells. Widespread occurrence of Ψ in RBP and miRNA binding sites—together with biochemical studies of artificially pseudouridylated RNAs that demonstrate the capacity of Ψ to determine whether, and to what extent, an mRNA will be bound—establish a strong premise for the proposed work. We will (1) Determine impact of mRNA Ψ on functional interactions with regulatory RNA-binding proteins, (2) Identify the roles of pseudouridylation in miRNA-mediated gene regulation, and (3) Define the contributions of regulated RNA pseudouridylation to the control of gene expression. Together, the proposed work will elucidate the molecular consequences of mRNA pseudouridylation in human cells and define functionally relevant mRNA targets of human pseudouridine synthases. This will fill a critical knowledge gap for understanding how RNA modifying enzymes that are essential for human health control gene expression.

项目概要多种假尿苷合酶（PUS）与人类疾病有关，但其机制将 PUS 活性丧失与线粒体肌病、消化系统疾病、智力障碍联系起来残疾、对病毒感染的抵抗力、先天性角化不良和多种癌症仍然存在很大程度上不为人知。我们目前对功能的了解存在几个关键差距 PUS 蛋白。尽管 PUS 蛋白的基本生化活性是催化尿苷异构化为假尿苷是众所周知的，大多数的特定 RNA 靶标人类 PUS 蛋白未知或不完全已知。我们的长期目标包括识别所有 PUS 蛋白的靶标并确定其分子后果在疾病相关的细胞环境中用假尿苷修饰特定的 RNA。这将对于理解 PUS 缺乏引起的疾病的病因学至关重要，并可能揭示治疗的新治疗靶点。我们最近的工作（Martinez 等人 Mol Cell 2022）证实假尿苷 (Ψ) 沉积在新生人类前体 mRNA 的数千个位点上进行共转录影响从出生到死亡的mRNA代谢的每一步。此续签申请将严格研究假尿苷化调节 mRNA 的生化基础。这 Ψ 的生物效应必定源自 U 和 Ψ 之间的化学差异，这直接影响RNA主链构象、碱基对的稳定性和蛋白质的结合。我们的初步数据表明，实验中存在数百个假尿苷。确定调节性 RNA 结合蛋白 (RBP) 和微小 RNA (miRNA) 的结合位点在人类 HepG2 细胞中。 RBP 和 miRNA 结合位点中广泛存在 Ψ 对人工假尿苷化 RNA 的生化研究证明了 Ψ 来确定 mRNA 是否会结合以及结合到什么程度——建立一个强大的拟议工作的前提。我们将 (1) 确定 mRNA Ψ 对功能的影响与调节性 RNA 结合蛋白的相互作用，(2) 鉴定假尿苷化在 miRNA 介导的基因调控，以及 (3) 定义受调控 RNA 的贡献假尿苷化来控制基因表达。总之，拟议的工作将阐明 mRNA 的分子后果人类细胞中的假尿苷化并定义人类功能相关的 mRNA 靶标假尿苷合酶。这将填补理解 RNA 如何运作的关键知识空白修饰对人类健康控制基因表达至关重要的酶。

项目成果

期刊论文数量（11）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Pseudo-Seq: Genome-Wide Detection of Pseudouridine Modifications in RNA.

DOI：
10.1016/bs.mie.2015.03.011
发表时间：
2015
期刊：
Methods in enzymology
影响因子：
0
作者：
Carlile TM;Rojas-Duran MF;Gilbert WV
通讯作者：
Gilbert WV

Identification of new branch points and unconventional introns in Saccharomyces cerevisiae.

DOI：
10.1261/rna.057216.116
发表时间：
2016-10
期刊：
RNA (New York, N.Y.)
影响因子：
0
作者：
Gould GM;Paggi JM;Guo Y;Phizicky DV;Zinshteyn B;Wang ET;Gilbert WV;Gifford DK;Burge CB
通讯作者：
Burge CB

Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells.

DOI：
10.1038/nature13802
发表时间：
2014-11-06
期刊：
NATURE
影响因子：
64.8
作者：
Carlile, Thomas M.;Rojas-Duran, Maria F.;Zinshteyn, Boris;Shin, Hakyung;Bartoli, Kristen M.;Gilbert, Wendy V.
通讯作者：
Gilbert, Wendy V.

Pseudouridine site assignment by high-throughput in vitro RNA pseudouridylation and sequencing.