Identification of RNA-binding proteins in macrophages by interactome capture

通过相互作用组捕获鉴定巨噬细胞中的 RNA 结合蛋白

• 批准号: 313418556
• 负责人: Professorin Dr. Antje Ostareck-Lederer
• 金额: --
• 依托单位: Klinik für Operative Intensivmedizin und Intermediate Care
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/313418556?language=en
• 关键词: Identification; RNA; binding; proteins; macrophages

Pathogen components, such as bacterial lipopolysaccharides (LPS) that activate Toll-like receptor 4 (TLR4), induce mitogen activated kinases (MAPKs) and NFkappaB through different downstream pathways to stimulate inflammatory cytokine expression. Although these mediators are essential to combat and coordinate the cellular infection response, their excessive expression causes systemic inflammation and sepsis, which are, besides cardiovascular diseases and cancer, the third leading cause of death worldwide. Importantly, post-transcriptional control of TLR4 downstream signaling molecule expression contributes to the tight regulation of inflammatory cytokine synthesis in macrophages. Emerging evidence highlights the role of RNA binding proteins (RBPs) in the post-transcriptional control of the innate immune response. By employing RNA interactome capture, which combines RBP-crosslinking to RNA in LPS-induced and untreated murine RAW 264.7 macrophages, cell lysis, oligo(dT) capture of polyadenylated RNAs and mass spectrometry analysis, macrophage RBPs were systematically identified and their response to LPS stimulation was characterized. Our data revealed 402 proteins of the macrophage RNA interactome including 91 previously unknown RBPs. A comparison with the published RNA interactomes identified 32 RBPs so far unique to RAW 264.7 macrophages. Of these, 19 proteins are linked to biochemical activities not directly related to RNA. From this group, HSP90 co-chaperone P23 that was demonstrated to exhibit cytosolic prostaglandin E2 synthase 3 (PTGES3) activity, and the hematopoietic cell-specific Lyn substrate 1 (HCLS1 or HS1), a hematopoietic specific adapter molecule, were validated as novel macrophage RBPs. We initiated UV-crosslinking and immunoprecipitation (CLIP) combined with deep sequencing of mRNAs that co-purify with novel identified macrophage RBPs. Based on that, mRNA-protein interaction maps will be generated to identify specific mRNAs encoding TLR4 downstream signaling molecules or their modulators and we will analyze how identified target mRNAs are post-transcriptionally regulated by RBPs in vitro and in vivo. Within the SPP 1935, the project will help to expand the mammalian RBP repertoire and will identify macrophage mRNPs that are prime candidates for the regulation and execution of LPS-induced TLR4 signaling pathways and the innate immune response. Information about underlying molecular mechanisms of RBP function will advance the understanding of their roles in inflammatory response modulation and will provide knowledge about their potential as therapeutic targets, to prevent systemic inflammation and sepsis.

病原体组分，如激活Toll样受体4（TLR 4）的细菌脂多糖（LPS），通过不同的下游途径诱导促分裂原激活激酶（MAPK）和NF κ B，以刺激炎性细胞因子表达。尽管这些介质对于对抗和协调细胞感染反应是必不可少的，但它们的过度表达引起全身性炎症和脓毒症，这是除心血管疾病和癌症之外的全球第三大死亡原因。重要的是，TLR 4下游信号分子表达的转录后控制有助于巨噬细胞中炎性细胞因子合成的严格调节。新出现的证据强调了RNA结合蛋白（RBP）在先天免疫应答的转录后控制中的作用。通过采用RNA相互作用组捕获，其将LPS诱导的和未处理的鼠RAW 264.7巨噬细胞中的RBP交联与RNA结合，细胞裂解，聚腺苷酸化RNA的寡聚（dT）捕获和质谱分析，系统地鉴定巨噬细胞RBP，并表征其对LPS刺激的响应。我们的数据揭示了402种巨噬细胞RNA相互作用体蛋白，包括91种以前未知的RBP。与已发表的RNA相互作用组的比较鉴定了RAW 264.7巨噬细胞特有的32种RBP。其中，19种蛋白质与生物化学活动有关，而与RNA没有直接关系。从该组中，证明表现出胞质前列腺素E2合酶3（PTGES 3）活性的HSP 90共伴侣P23和造血细胞特异性林恩底物1（HCLS 1或HS 1）（造血特异性衔接分子）被验证为新的巨噬细胞RBP。我们启动了紫外线交联和免疫沉淀（CLIP），结合与新鉴定的巨噬细胞RBP共纯化的mRNA的深度测序。在此基础上，将生成mRNA-蛋白质相互作用图谱，以识别编码TLR 4下游信号分子或其调节剂的特定mRNA，并分析所识别的靶mRNA如何在体外和体内受RBP的转录后调控。在SPP 1935中，该项目将有助于扩大哺乳动物RBP库，并将鉴定巨噬细胞mRNP，它们是调节和执行LPS诱导的TLR 4信号通路和先天免疫反应的主要候选者。有关RBP功能的潜在分子机制的信息将促进对其在炎症反应调节中的作用的理解，并将提供有关其作为治疗靶点的潜力的知识，以预防全身性炎症和脓毒症。