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

项目摘要 在我们对RNA处理因素，特别是剪接蛋白质是如何影响的理解上，存在着根本的差距， 对环境变化和细胞外信号做出反应。这项提案的总体目标是界定 剪接因子SRSF7在先天免疫细胞中的翻译后修饰和功能改变 在被病原体感应激活之后。剪接调控不当是导致多种人类疾病的原因 由于顺式和反式因子的遗传性和体细胞突变。反式剪接因子包括AUX- 辅助性剪接因子，如丝氨酸精氨酸剪接因子(SRSF)家族和最近的磷酸蛋白质组学 实验证明，5种SRSF蛋白在原代巨噬细胞中有不同的磷酸化。 感染了细胞内的细菌病原体。脂多糖刺激巨噬细胞系的实验研究 (LPs)是一种差异磷酸化的SRSF蛋白，SRSF7从染色质部分移动到核内-- 卵质，这种运动与明显的磷酸化增加有关。这一发现表明 病原体感应可能改变SRSF7与蛋白质结合伙伴和/或Chro上的RNA相互作用的能力。 马丁。静息巨噬细胞中FLAG-SRSF7的MS-IP追踪研究 SRSF7和SNRP70之间的新的相互作用，SNRP70是U1 SNRP的一个组成部分，而HDAC6是一个重要的His- 内酯脱乙酰酶。更多的初步数据显示，shRNA介导的SRSF7基因敲除抑制了基因水平 巨噬细胞中干扰素刺激基因(ISGs)的表达，提示该因子在巨噬细胞中起着重要的调节作用 先天免疫基因的表达。这一提议的中心假设预测，差异磷光- SRSF7的突变影响其结合蛋白质和前-mRNA的能力，从而改变剪接密码的方式 是在巨噬细胞激活期间读取的。该项目的两个主要目标是确定 巨噬细胞活化过程中SRSF7的磷酸化对蛋白质相互作用和剪接的影响 确定巨噬细胞激活下游参与SRSF7修饰的激酶。为此，Aim1 使用免疫沉淀来阐明蛋白质-蛋白质的相互作用以及RNA免疫沉淀和RNA 亲和力分析将SRSF7的磷酸化状态与蛋白质/转录结合和基因表达联系起来。 来了。Aim 2使用生化抑制剂以及基因操作来确定 巨噬细胞激活过程中SRSF7磷酸化的丝氨酸-苏氨酸激酶。这些经验- 对巨噬细胞中SRSF7磷酸化的活性和调节提供了有价值的见解 激活。此外，这个项目将加深我们对驱动细胞适应的机制的理解。 到动态的微环境。这项工作具有很高的潜力，可以改变关于各种环境如何- 心理应激(如饥饿、紫外线应激、热休克、渗透应激、致癌)使RNA发挥功能 并为可能的治疗干预提供潜在的进步，以纠正 通过调节前-信使核糖核酸剪接来破坏细胞的动态平衡。

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.