Nitric oxide as a novel regulator of alternative splicing

一氧化氮作为选择性剪接的新型调节剂

• 批准号: 10673458
• 负责人: Joseph Christian Schindler
• 金额: $ 5.27万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10673458
• 关键词: Abbreviations; Address; Affect; Alternative Splicing; Area; Biochemical; Biology; Cardiovascular system; Cell physiology; Cells; Co-Immunoprecipitations; Code; Cues; Cultured Cells; Cysteine; Data; Defect; Development; Disease; Environment; Environmental Exposure; Environmental Science; Enzymes; Epigenetic Process; Exposure to; Family; Foundations; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Proteins; Genes; Genetic Transcription; Genome; Goals; Health; Histones; Immunologics; Immunoprecipitation; In Vitro; Knock-in; Laboratories; Maps; Mass Spectrum Analysis; Mediating; Messenger RNA; MicroRNAs; Modification; Molecular; Mus; Mutant Strains Mice; Mutate; Mutation; National Institute of Environmental Health Sciences; Neurologic; Nitric Oxide; Nuclear; Nuclear Magnetic Resonance; Pathologic; Pathway interactions; Physical condensation; Physiological; Play; Polypyrimidine Tract-Binding Protein; Post-Translational Protein Processing; Production; Property; Protein Conformation; Proteins; Proteomics; Publishing; RNA; RNA Binding; RNA Processing; RNA Splicing; RNA metabolism; RNA-Binding Proteins; RNA-Protein Interaction; Regulation; Reporting; Research; Ribonucleoproteins; Role; S-Nitrosothiols; Signal Transduction; Signal Transduction Pathway; Signaling Molecule; Site; Spliceosomes; Structure; Sulfhydryl Compounds; System; Testing; Time; Tissues; Transcript; bioinformatics pipeline; cell growth regulation; cell type; cofactor; combinatorial; crosslink; environmental change; epigenome; fluorescence imaging; genetic regulatory protein; hypoxia inducible factor 1; in utero; mouse model; novel; novel strategies; prevent; protein function; response; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

PROJECT SUMMARY/ABSTRACT Protein post-translational modification (PTM) and alternative splicing enable the limited genome of a cell to dynamically respond to environmental changes by diversifying its protein repertoire. Previous studies have revealed that environmental cues impact alternative splicing through numerous PTMs of RNA-binding proteins associated with the spliceosome. S-nitrosylation, the reversible covalent PTM of a protein cysteine residue by the gaseous signaling molecule nitric oxide (NO) to form an S-nitrosothiol (SNO)-modified protein, has been shown to alter protein function to play profound roles on cellular physiology, including the regulation of gene expression. Specifically, S-nitrosylation is known to directly regulate key transcription factors as well as to modify enzymes that alter the epigenome. Intriguingly, RNA-binding proteins of the heterogenous nuclear ribonucleoprotein (hnRNP) family, including the polypyrimidine tract-binding protein (PTB), are common in proteomic identifications of SNO-modified proteins under multiple conditions and in many cell types. Because PTB is a master regulator of alternative splicing, we are exploring the central hypothesis that NO enables a cell to dynamically regulate RNA splicing through SNO modification of PTB. Our laboratory recently mapped S-nitrosylation to a single cysteine residue in PTB. Dramatic alternations in gene expression when cells are exposed to NO are absent when this cysteine is mutated, strongly supporting a role for SNO in regulating the activity of PTB with transcriptome-wide implications. This proposal will interrogate these findings through three independent aims: Aim 1 will develop a bioinformatic pipeline based on RNA sequencing analyses to identify specific alternative transcripts regulated by SNO-PTB, including those that may have an outsized effect on global gene expression; Aim 2 will elucidate how SNO affects PTB conformation and its association with protein and RNA components of the spliceosome, offering a molecular mechanism for the influence of NO on alternative splicing; and Aim 3 entails the use of a conditional knock-in mutant mouse in which PTB cannot undergo the SNO modification, allowing determination of the physiological role of SNO-PTB by observing the consequences of dysregulated NO signaling. This project will advance our understanding of the role of NO signaling as a crucial mechanism of the cellular response to environmental cues through SNO-modification of a central regulator of alternative splicing, PTB. Heretofore, global effects of NO on cellular function have been attributed to widespread modification of proteins. The role of NO in regulating alternative splicing is previously unappreciated, and provides new perspectives on dynamic regulation of cellular function in health and disease. This study will thus define the role of NO in alternative splicing for the first time, potentially opening new areas of research.

项目总结/摘要 蛋白质翻译后修饰（PTM）和可变剪接使有限的基因组， 细胞通过多样化其蛋白质库来动态响应环境变化。以前的研究 已经揭示了环境因素通过许多RNA结合的PTM影响选择性剪接， 与剪接体相关的蛋白质。S-亚硝基化，蛋白质半胱氨酸的可逆共价PTM 通过气态信号分子一氧化氮（NO）残基形成S-亚硝基硫醇（SNO）修饰的蛋白质， 已被证明可以改变蛋白质的功能，在细胞生理学上发挥深远的作用，包括调节 的基因表达。具体来说，S-亚硝基化已知也直接调节关键转录因子 来修饰改变表观基因组的酶。有趣的是，异源细胞的RNA结合蛋白 核核糖核蛋白（hnRNP）家族，包括多聚嘧啶片段结合蛋白（PTB），是 在多种条件下和许多细胞中，SNO修饰蛋白的蛋白质组学鉴定中常见 类型因为PTB是选择性剪接的主要调节因子，我们正在探索中心假设， NO使细胞能够通过PTB的SNO修饰来动态调节RNA剪接。 我们的实验室最近映射的S-亚硝基化的一个单一的半胱氨酸残基在PTB。戏剧性 当细胞暴露于NO时，当这种半胱氨酸突变时，基因表达的改变不存在， 支持SNO在调节PTB活性中的作用，具有转录组范围的影响。这 该提案将通过三个独立的目标来询问这些发现：目标1将开发生物信息学 基于RNA测序分析的管道，以鉴定由SNO-PTB调节的特定替代转录物， 包括那些可能对全球基因表达产生巨大影响的基因; Aim 2将阐明SNO是如何 影响PTB构象及其与剪接体的蛋白质和RNA组分的结合， NO对选择性剪接影响的分子机制; Aim 3需要使用条件 敲入突变小鼠，其中PTB不能经历SNO修饰，从而允许测定PTB的表达。 通过观察NO信号失调的后果来研究SNO-PTB的生理作用。 该项目将促进我们对NO信号转导作为一种重要机制的作用的理解， 通过SNO修饰的替代性中枢调节因子对环境信号的细胞反应 剪接，PTB。迄今为止，一氧化氮对细胞功能的全球影响已被广泛归因于 蛋白质的修饰。NO在调节可变剪接中的作用以前未被认识， 为健康和疾病中细胞功能的动态调节提供了新的视角。本研究将 因此，首次定义了NO在选择性剪接中的作用，可能开辟新的研究领域。