Pre-mRNA splicing regulation is critical for controlling macrophage activation

前 mRNA 剪接调节对于控制巨噬细胞激活至关重要

• 批准号: 10474615
• 负责人: Kristin Leigh Patrick
• 金额: $ 37.03万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10474615
• 关键词: Affinity Chromatography; Alternative Splicing; Automobile Driving; Biochemical; Cell Nucleus; Cells; Chemicals; Code; Complex; Excision; Exons; Gene Expression; Genetic; Genetic Transcription; Goals; Heterogeneous-Nuclear Ribonucleoprotein Group M; Immune; Inflammation Mediators; Interleukin-6; Introns; Knowledge; Lipopolysaccharides; Macrophage Activation; Mass Spectrum Analysis; Mediating; Modeling; Molecular; Organelles; Outcome; Phosphorylation; Post-Translational Protein Processing; Protein Dephosphorylation; Protein Splicing; RNA; RNA Processing; RNA Splicing; RNA-Binding Proteins; Reading; Regulation; Research; Role; Serine; Signal Transduction; Spliceosomes; Starvation; Stimulus; Stress; Transcript; Viral; carcinogenesis; cohort; combat; crosslinking and immunoprecipitation sequencing; experimental study; immune activation; knock-down; mRNA Precursor; macrophage; pathogen; phosphoproteomics; programs; protein protein interaction; small hairpin RNA; transcriptome sequencing

PROJECT SUMMARY Despite the substantial impact pre-mRNA splicing has on gene expression outcomes, little is known about how the spliceosome itself is modified and regulated during cellular reprogramming. Innate immune cells like macrophages reprogram gene expression when they sense a “danger signal,” such as a pathogen, organelle damage, or chemical signal, to combat the detected threat. While changes that occur transcriptionally during macrophage activation are well characterized, almost nothing is known about how pre- mRNA splicing is regulated following immune stimuli. The long-term goal of this project is to uncover how macrophage activation modifies the spliceosome and to connect these changes with innate immune gene expression outcomes. The spliceosome is a complex and dynamic macromolecular machine. Its ability to recognize introns and catalyze their removal relies on numerous RNA binding proteins that recognize specific sequences in exons and introns to “read” the splicing code. The central hypothesis of this proposal is that during macrophage activation, post-translational modification of splicing factors directs assembly of a specialized spliceosome characterized by a distinct cohort of protein-protein interactions that promotes the innate immune gene expression program. In support of this model, phosphoproteomic experiments reveal that 30+ splicing factors, many with known regulatory roles, are phosphorylated or dephosphorylated at specific serine residues following lipopolysaccharide (LPS)-dependent activation of macrophages. Experiments interrogating one such factor, hnRNP M, show that LPS treatment triggers dephosphorylation concomitant with its redistribution in the nucleus. Loss of hnRNP M by shRNA-mediated knockdown in macrophages alters alternative splicing of a number of pre-mRNAs and leads to hyper-induction of important innate immune transcripts, including the potent inflammatory mediator IL-6 and the key viral restriction factor Mx1. This proposal expands upon these observations, looking globally at changes to the spliceosome following macrophage activation. It will combine high-throughput approaches, including affinity purification-mass spectrometry, phosphoproteomics, RNA-seq, and RNA CLIP-seq with targeted genetic and biochemical experiments to implicate specific splicing factors in driving innate immune gene expression changes. This research program will fill key gaps in our knowledge of how splicing is regulated following macrophage activation and further our understanding of how the spliceosome reads and interprets the splicing code not only during innate immune activation but also during other cellular reprogramming, including differentiation, stress, starvation, and carcinogenesis.

项目摘要 尽管前MRNA剪接对基因表达结果具有重大影响，但知之甚少 关于在细胞重编程过程中如何修饰和调节剪接体本身。先天免疫细胞 像巨噬细胞一样，当他们感觉到“危险信号”（例如病原体）时，重新编程了基因表达， 细胞器损坏或化学信号，以应对检测到的威胁。而发生的变化 巨噬细胞激活期间的转录表征很好，几乎没有任何了解 在免疫刺激后调节mRNA剪接。该项目的长期目标是发现 巨噬细胞激活改变了剪接体，并将这些变化与先天免疫基因联系起来 表达结果。剪接体是一台复杂而动态的大分子机器。它的能力 公认的介绍并催化其去除取决于识别特定的许多RNA结合蛋白 “读取”剪接代码的外显子和内含子中的序列。该提议的核心假设是 在巨噬细胞激活期间，剪接因子的翻译后修饰指导A组装 专门的剪接体，其特征在于独特的蛋白质 - 蛋白质相互作用的队列，该蛋白质相互作用促进 先天免疫基因表达程序。为了支持该模型，磷蛋白质组学实验表明 30多个剪接因子（许多具有已知调节作用的剪接因子）在特定上被磷酸化或去磷酸化 脂多糖（LPS）依赖性巨噬细胞激活后的连续残留物。实验 询问这样一个因素HNRNP M，表明LPS治疗触发了与 它在细胞核中的重新分布。 shRNA介导的巨噬细胞中的HNRNP M损失改变了 许多前MRNA的替代剪接，并导致重要的先天免疫过度诱导 转录本，包括潜在的炎症介质IL-6和关键病毒限制因子MX1。这 提案扩展了这些观察结果，全球介绍了剪接体的变化。 巨噬细胞激活。它将结合高通量方法，包括亲和力纯化质量 具有靶向遗传学和生化的光谱，磷蛋白质组学，RNA-SEQ和RNA夹seq 在驱动先天免疫基因表达变化时进行隐式特定剪接因子的实验。这 研究计划将在我们了解巨噬细胞后如何调节剪接的知识中填补关键空白 激活并进一步了解剪接体如何读取和解释剪接代码不仅 在先天免疫激活期间，以及在其他细胞重编程中，包括分化，应力， 饥饿和癌变。