Effect of stimulation with lipopolysaccharide (LPS) at 0,30, and 60 minutes on gene expression in immortalized mouse macrophages (IMM).

The interactions between RNA and protein within cellular signaling pathways, known as the interactome, have modulatory effects on RNA binding proteins (RBP’s) effector functions, which contribute to post-transcriptional control of inflammatory cytokine release in the innate immune response. Post-translational modifications (PTMs) have a regulatory effect on a protein’s effector function. Thus, investigating the effect of RNA-protein interactions can answer questions central to our understanding of cellular signaling. Global RNA sequencing was used to investigate the differentially expressed (DE) genes from baseline following induction with LPS. Shotgun proteomics along with SILAC labeling was used to quantify relative abundance and differential protein expression in response to LPS. To assess the proteins interacting with RNA after LPS induction, total RNA associated protein purification was utilized. Using the tandem MS approach and methodology mentioned above, we were able to identify a list of candidate RNA binding proteins. Global RNA sequencing provided a baseline transcriptomic profile as well as quantification of DE genes across a stimulation time course (0, 30, 60 minutes). We then handpicked potential RBPs of immunological interest (Il1a, MARCKS, and Acod1). Interestingly, Il1a, MARCKS, and Acod1 were all highly DE compared to baseline in the RNA Seq results and displayed a tendency to disassociate from RNA as the LPS time course pursued. Il1a, MARCKS, and Acod1 can all directly regulate pro-inflammatory gene expression in macrophages. These RBPs could offer a new regulatory mechanism to tune their effector function, which could lead to new therapeutic innovations for disease pathologies these proteins are implicated in. This research was supported by the Intramural Research Program of NIAID, NIH. To invstigate changes in the transcriptome following stimulation with LPS, we examined 3 biological replicates per time point, where 0 minute was the control and 30 and 60 minutes correspond to the treated samples.

细胞信号通路内RNA与蛋白质的相互作用，即相互作用组（interactome），可对RNA结合蛋白（RNA Binding Protein，RBP）的效应功能产生调控作用，进而参与固有免疫应答中炎性细胞因子释放的转录后调控过程。翻译后修饰（Post-translational Modifications，PTMs）对蛋白质的效应功能具有调控效应。因此，解析RNA-蛋白质相互作用的效应，可解答我们理解细胞信号通路的核心科学问题。研究人员采用全局RNA测序技术，分析经脂多糖（Lipopolysaccharide，LPS）诱导后相较于基线的差异表达基因（Differentially Expressed，DE）。结合鸟枪法蛋白质组学（Shotgun proteomics）与稳定同位素标记氨基酸细胞培养（Stable Isotope Labeling by Amino acids in Cell culture，SILAC）标记技术，可定量分析脂多糖刺激下蛋白质的相对丰度与差异表达情况。为评估脂多糖诱导后与RNA结合的蛋白质，研究人员采用了总RNA结合蛋白纯化方法。借助上述串联质谱（Tandem Mass Spectrometry，Tandem MS）技术与实验流程，我们成功鉴定出一批候选RNA结合蛋白。全局RNA测序不仅提供了基线转录组谱，还可定量分析刺激时间进程（0、30、60分钟）下的差异表达基因。随后我们手动筛选出具有免疫学研究价值的潜在RNA结合蛋白：Il1a、MARCKS与Acod1。有趣的是，在RNA测序结果中，这三个基因对应的转录本均相较于基线呈现显著差异表达，且随着脂多糖刺激时间进程的推进，它们表现出与RNA解离的趋势。Il1a、MARCKS与Acod1均可直接调控巨噬细胞内的促炎基因表达。这些RNA结合蛋白或许可提供一种全新的调控机制，以调节其效应功能，进而为这些蛋白所参与的疾病病理过程带来新的治疗创新思路。本研究由美国国立卫生研究院（National Institutes of Health，NIH）下属的国家过敏和传染病研究所（National Institute of Allergy and Infectious Diseases，NIAID）的院内研究计划资助。为探究脂多糖刺激后转录组的变化，我们为每个时间点设置了3次生物学重复：0分钟为对照组，30分钟与60分钟为处理组样本。