Determining the impact of post-translational modification on Serine Arginine Splicing Factor 7 during macrophage activation

确定巨噬细胞激活过程中翻译后修饰对丝氨酸精氨酸剪接因子 7 的影响

• 批准号: 10462519
• 负责人: Haley Marie Scott
• 金额: $ 3.53万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10462519
• 关键词: Acids; Affinity; Alleles; Ally; Alternative Splicing; Arginine; Automobile Driving; Bacterial Infections; Binding; Binding Proteins; Biochemical; Biological Assay; Categories; Cell Line; Cell Nucleus; Cells; Chemicals; Chromatin; Code; Cues; DNA; Data; Defect; Development; Disease; Event; Excision; Exons; Family; Gene Expression; Genes; Genetic; Genetic Transcription; Germ-Line Mutation; Goals; HDAC6 gene; Heat-Shock Response; Histone Deacetylase; Homeostasis; Immune; Immunoprecipitation; Infection; Innate Immune Response; Interferons; Introns; Knowledge; Ligands; Ligation; Link; Lipopolysaccharides; Macrophage Activation; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Messenger RNA; Metabolism; Modification; Molecular; Movement; Mycobacterium tuberculosis; Nucleoplasm; Outcome; Partner in relationship; Phosphorylation; Phosphotransferases; Play; Polyadenylation; Post-Transcriptional Regulation; Post-Translational Protein Processing; Predisposition; Process; Protein Splicing; Protein-Serine-Threonine Kinases; Proteins; Proteomics; RNA; RNA Processing; RNA Splicing; RNA-Binding Proteins; Regulation; Rest; Role; STY kinase; Series; Serine; Signal Transduction; Site; Somatic Mutation; Spliceosomes; Starvation; Stimulus; Stress; Testing; Therapeutic; Therapeutic Intervention; Transcript; Untranslated RNA; Virus Diseases; Work; base; carcinogenesis; cell type; cohort; design; environmental change; environmental stressor; experimental study; extracellular; follow-up; genetic manipulation; genetic regulatory protein; human disease; human pathogen; inhibitor; insight; knock-down; mRNA Precursor; macrophage; member; novel; pathogen; pathogenic bacteria; phosphoproteomics; programs; protein function; protein protein interaction; response; sensor; small hairpin RNA; transcription regulatory network; transcriptome; virtual

Project Summary There is a fundamental gap in our understanding of how RNA processing factors, specifically splicing proteins, respond to environmental changes and extracellular signals. The overall objective of this proposal is to define how the splicing factor SRSF7 is post-translationally modified and functionally altered in innate immune cells following activation by pathogen sensing. Misregulation of splicing accounts for a multitude of human diseases due to hereditary and somatic mutations in both cis and trans factors. Trans-acting splicing factors include aux- iliary splicing factors such as the Serine Arginine Splicing Factor (SRSF) family, and a recent phosphoproteomics experiment demonstrated that five SRSF proteins are differentially phosphorylated in primary macrophages in- fected with an intracellular bacterial pathogen. Upon stimulation of a macrophage cell line with lipopolysaccharide (LPS), one differentially phosphorylated SRSF protein, SRSF7, moves from the chromatin fraction to the nucle- oplasm and this movement is associated with an apparent gain in phosphorylation. This finding suggests that pathogen sensing may alter the ability of SRSF7 to interact with protein binding partners and/or RNA on chro- matin. Follow-up mass spec immunoprecipitation (MS-IP) of FLAG-SRSF7 in resting macrophages identified novel interactions between SRSF7 and SNRP70, a component of the U1 SNRP, and HDAC6 an important his- tone deacetylase. Additional preliminary data show that shRNA-mediated knockdown of SRSF7 represses levels of interferon stimulated genes (ISGs) in macrophages, suggesting this factor plays an important role in regulating innate immune gene expression. The central hypothesis of this proposal predicts that differential phosphory- lation of SRSF7 impacts its ability to bind proteins and pre-mRNA, which changes how the splicing code is read during macrophage activation. The two main goals of this project are to determine the contribution of phosphorylation of SRSF7 to protein-protein interactions and splicing during macrophage activation and to iden- tify the kinases involved in SRSF7 modification downstream of macrophage activation. To these ends, Aim1 uses immunoprecipitation to elucidate protein-protein interactions as well as RNA immunoprecipitation and RNA affinity assays to link SRSF7 phosphorylation status with protein/transcript binding and gene expression out- comes. Aim 2 employs biochemical inhibitors as well as genetic manipulation to determine the contribution of specific serine-threonine kinases in phosphorylation of SRSF7 during macrophage activation. These experi- ments will provide valuable insight into the activity and regulation of SRSF7 phosphorylation during macrophage activation. Additionally, this project will further our understanding of the mechanisms driving cellular adaptation to dynamic microenvironments. This work has high potential to shift paradigms regarding how a variety of envi- ronmental stresses (e.g. starvation, UV stress, heat shock, osmotic stress, carcinogenesis) functionalize RNA processing machinery and provides potential advancements for possible therapeutic interventions that correct disruption of cellular homeostasis by modulating pre-mRNA splicing.

项目概要 我们对 RNA 加工因子（特别是剪接蛋白）如何理解存在根本性的差距。 对环境变化和细胞外信号做出反应。该提案的总体目标是定义 剪接因子 SRSF7 如何在先天免疫细胞中进行翻译后修饰和功能改变 通过病原体感应激活后。剪接错误调控导致多种人类疾病 由于顺式和反式因子的遗传和体细胞突变。反式作用剪接因子包括辅助 纤毛剪接因子，例如丝氨酸精氨酸剪接因子 (SRSF) 家族，以及最近的磷酸化蛋白质组学 实验表明，五种 SRSF 蛋白在初级巨噬细胞中被差异磷酸化， 感染细胞内细菌病原体。用脂多糖刺激巨噬细胞系 (LPS)，一种差异磷酸化的 SRSF 蛋白 SRSF7，从染色质部分移动到细胞核 oplasm 和这种运动与磷酸化的明显增益有关。这一发现表明 病原体感应可能会改变 SRSF7 与蛋白质结合伴侣和/或染色质 RNA 相互作用的能力 晨祷。静息巨噬细胞中 FLAG-SRSF7 的后续质谱免疫沉淀 (MS-IP) 鉴定 SRSF7 和 SNRP70（U1 SNRP 的一个组成部分）和 HDAC6 之间的新相互作用是一个重要的组 音脱乙酰酶。其他初步数据表明 shRNA 介导的 SRSF7 敲低可抑制水平 巨噬细胞中干扰素刺激基因（ISG）的存在，表明该因子在调节中发挥重要作用 先天免疫基因表达。该提案的中心假设预测差异磷酸化 SRSF7 的连接会影响其结合蛋白质和前 mRNA 的能力，从而改变剪接代码的方式 在巨噬细胞激活期间读取。该项目的两个主要目标是确定 SRSF7 的磷酸化对巨噬细胞激活过程中蛋白质-蛋白质相互作用和剪接的影响，并确定 验证参与巨噬细胞激活下游 SRSF7 修饰的激酶。为此，Aim1 使用免疫沉淀法阐明蛋白质-蛋白质相互作用以及 RNA 免疫沉淀法和 RNA 亲和力测定将 SRSF7 磷酸化状态与蛋白质/转录本结合和基因表达输出联系起来 来了。目标 2 采用生化抑制剂以及基因操作来确定 巨噬细胞激活期间 SRSF7 磷酸化中的特异性丝氨酸-苏氨酸激酶。这些经验 这些文章将为巨噬细胞过程中 SRSF7 磷酸化的活性和调节提供有价值的见解 激活。此外，该项目将进一步了解驱动细胞适应的机制 动态微环境。这项工作具有很大的潜力来改变关于各种环境如何 环境应激（例如饥饿、紫外线应激、热休克、渗透应激、致癌）使 RNA 功能化 加工机械，并为可能的治疗干预措施提供潜在的进步，以纠正 通过调节前 mRNA 剪接破坏细胞稳态。