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.

项目摘要/摘要 蛋白质后翻译修饰（PTM）和替代剪接使A的有限基因组能够 细胞通过多样化其蛋白质库来动态地应对环境变化。先前的研究 已经揭示了环境提示通过大量RNA结合的PTM影响替代剪接 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）的残留物形成s-硝基硫醇（SNO）修饰的蛋白质， 已被证明会改变蛋白质功能，以在细胞生理学上发挥着重要作用，包括调节 基因表达。具体而言，已知S-亚硝基化也直接调节关键转录因子以及 为了修饰改变表观基因组的酶。有趣的是，异源的RNA结合蛋白 核核糖核蛋白（HNRNP）家族，包括息肉嘧啶片结合蛋白（PTB）是 在多种条件下和许多细胞中，在SNO修饰蛋白的蛋白质组学鉴定中常见 类型。因为PTB是替代剪接的主要调节剂，所以我们正在探索一个中心假设，即 NO可以通过PTB的SNO修饰来动态调节RNA剪接。 我们的实验室最近将S-亚硝基化映射到PTB中的单一半胱氨酸残基。戏剧性 当这种半胱氨酸突变时，当细胞暴露于NO时，基因表达的交替是不存在的 支持SNO在调节PTB活性具有范围内的含义中的作用。这 提案将通过三个独立目标询问这些发现：AIM 1将发展生物信息 基于RNA测序分析的管道，以确定由SNO-PTB调节的特定替代转录本， 包括那些可能对全球基因表达具有巨大影响的人； AIM 2将阐明如何SNO 影响PTB构象及其与剪接体的蛋白质和RNA成分的关联，提供A NO对替代剪接的影响的分子机制；目标3需要使用条件 PTB无法进行SNO修饰的敲入突变小鼠，允许确定 通过观察NO信号传导失调的后果，SNO-PTB的生理作用。 该项目将促进我们对无信号作为作为关键机制的作用的理解 通过对替代方案的中央调节剂进行SNO修饰对环境提示的蜂窝响应 剪接，PTB。迄今为止，NO对蜂窝功能的全局影响已归因于广泛 蛋白质的修饰。 NO在调节替代剪接方面的作用以前是未接受的，并且 提供了有关健康和疾病中细胞功能动态调节的新观点。这项研究会 因此，第一次定义了NO在替代剪接中的作用，有可能打开新的研究领域。