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Abstract：
RNA-protein interactions within cellular signaling pathways have significant modulatory effects on RNA binding proteins\' (RBPs\') effector functions. During the innate immune response, specific RNA-protein interactions have been reported as a regulatory layer of post-transcriptional control. We investigated changes in the RNA-bound proteome of immortalized mouse macrophages (IMM) following treatment with lipopolysaccharide (LPS). Stable isotope labeling by amino acids in cell culture (SILAC) of cells followed by unbiased purification of RNP complexes at two time points after LPS stimulation and bottom-up proteomic analysis by LC-MS/MS resulted in a set of significantly affected RBPs. Global RNA sequencing and LFQ proteomics were used to characterize the correlation of transcript and protein abundance changes in response to LPS at different time points with changes in protein-RNA binding. Il1α, MARCKS, and ACOD1 were noted as RBP candidates involved in innate immune signaling. The binding sites of the RBP and RNA conjugates at amino acid resolution were investigated by digesting the cross-linked oligonucleotide from peptides remaining after elution using Nuclease P1. The combined data sets provide directions for further studies of innate immune signaling regulation by RBP interactions with different classes of RNA.
摘要：
细胞信号传导途径内的RNA-蛋白质相互作用对RNA结合蛋白(RBPs)效应子功能具有显著的调节作用。在先天免疫反应期间，特异性RNA-蛋白质相互作用已被报道为转录后控制的调节层。我们研究了脂多糖（LPS）处理后永生化小鼠巨噬细胞（IMM）的RNA结合蛋白质组的变化。在细胞的细胞培养物（SILAC）中通过氨基酸进行稳定同位素标记，然后在LPS刺激后的两个时间点对RNP复合物进行无偏倚的纯化，并通过LC-MS/MS进行自下而上的蛋白质组学分析，产生了一组明显受影响的RBP。全局RNA测序和LFQ蛋白质组学用于表征在不同时间点响应于LPS的转录物和蛋白质丰度变化与蛋白质-RNA结合变化的相关性。Il1α,MARCKS,和ACOD1被标记为参与先天免疫信号传导的RBP候选物。通过使用核酸酶P1从洗脱后剩余的肽中消化交联的寡核苷酸来研究RBP和RNA缀合物在氨基酸分辨率时的结合位点。组合的数据集为通过RBP与不同类型的RNA相互作用的先天免疫信号调节的进一步研究提供了方向。
