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

项目摘要 我们对RNA加工因子，特别是剪接蛋白质， 对环境变化和细胞外信号作出反应。本提案的总体目标是确定 剪接因子SRSF 7在先天免疫细胞中是如何在免疫后被修饰和功能改变的 在病原体感应激活之后。剪接的失调导致了许多人类疾病 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- 纤毛剪接因子，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差异磷酸化， 感染了细胞内的细菌病原体在用脂多糖刺激巨噬细胞系后， (LPS)一种差异磷酸化的SRSF蛋白SRSF 7从染色质部分移动到核中， 这种运动与磷酸化的明显增加有关。这一发现表明 病原体感知可能改变SRSF 7与蛋白质结合伴侣和/或染色体上RNA相互作用的能力， 马丁。静息巨噬细胞中FLAG-SRSF 7的后续质谱免疫沉淀（MS-IP）鉴定 SRSF 7和SNRP 70（U1 SNRP的一个组分）与HDAC 6之间的新相互作用是一个重要的his-factors。 音调脱乙酰酶另外的初步数据显示，shRNA介导的SRSF 7敲低抑制了 干扰素刺激基因（ISGs）在巨噬细胞中的表达，这表明该因子在调节巨噬细胞中的免疫反应中起重要作用。 先天免疫基因表达该建议的中心假设预测，差异性磷酸化- SRSF 7的表达影响其结合蛋白质和前mRNA的能力，这改变了剪接密码 在巨噬细胞激活期间读取。该项目的两个主要目标是确定 SRSF 7的磷酸化对巨噬细胞活化过程中蛋白质-蛋白质相互作用和剪接的影响， 激活参与巨噬细胞活化下游SRSF 7修饰的激酶。为此，AIM 1 使用免疫沉淀来阐明蛋白质-蛋白质相互作用以及RNA免疫沉淀和RNA 将SRSF 7磷酸化状态与蛋白质/转录本结合和基因表达联系起来的亲和力测定 来了目的2采用生化抑制剂以及遗传操作，以确定贡献 特异性丝氨酸-苏氨酸激酶在巨噬细胞活化期间SRSF 7磷酸化中的作用。这些经验- 这些结果将为研究SRSF 7磷酸化在巨噬细胞吞噬过程中的活性和调节提供有价值的见解。 activation.此外，该项目将进一步加深我们对细胞适应机制的理解 to dynamic动态microenvironments微环境.这项工作有很大的潜力，以改变范式，关于如何各种环境， 环境胁迫（例如饥饿、UV胁迫、热休克、渗透胁迫、致癌作用）使RNA功能化 加工机械，并为可能的治疗干预提供了潜在的进步， 通过调节前体mRNA剪接破坏细胞内稳态。

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.