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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要尽管前 mRNA 剪接对基因表达结果具有重大影响，但人们知之甚少关于剪接体本身在细胞重编程过程中如何被修饰和调节。就像巨噬细胞在感知到“危险信号”（例如病原体）时重新编程基因表达一样，细胞器损伤或化学信号，以对抗检测到的威胁而发生的变化。巨噬细胞激活期间的转录已得到很好的表征，但几乎不知道如何预 mRNA 剪接在免疫刺激后受到调节该项目的长期目标是揭示如何调节。巨噬细胞激活会修饰剪接体并将这些变化与先天免疫基因联系起来剪接体是一种复杂且动态的大分子机器。识别内含子并催化其去除依赖于许多识别特定的RNA结合蛋白外显子和内含子中的序列来“读取”剪接代码该提案的中心假设是：在巨噬细胞激活过程中，剪接因子的翻译后修饰指导巨噬细胞的组装专门的剪接体，其特征是一组独特的蛋白质-蛋白质相互作用，可促进为了支持这一模型，磷酸化蛋白质组学实验表明： 30 多个剪接因子，其中许多具有已知的调节作用，在特定的位置被磷酸化或去磷酸化脂多糖（LPS）依赖性巨噬细胞激活实验后的丝氨酸残基。研究其中一个因子 hnRNP M 表明，LPS 治疗会引发去磷酸化，同时 shRNA介导的巨噬细胞敲低导致hnRNP M在细胞核中的重新分布发生改变。许多前体 mRNA 的选择性剪接并导致重要先天免疫的过度诱导转录物，包括有效的炎症介质 IL-6 和关键的病毒限制因子 Mx1。提案扩展了这些观察结果，从全局角度观察剪接体的变化如下它将结合高通量方法，包括亲和纯化质量。光谱分析、磷酸蛋白质组学、RNA-seq 和 RNA CLIP-seq 以及靶向遗传和生物化学涉及驱动先天免疫基因表达变化的特定剪接因子的实验。研究计划将填补我们关于巨噬细胞如何调控剪接的知识空白激活并进一步我们对剪接体如何读取和解释剪接代码的理解在先天免疫激活期间以及其他细胞重编程期间，包括分化、应激、饥饿和致癌。