Lipopolysaccharide Regulates the Macrophage RNA-Binding Proteome

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.

Celebrating Women in Proteomics and Metabolomics

“…Protein interaction studies performed by Hui-Su Kim and Yong-In Kim from the group of Je-Yoel Cho uncovered the regulation of ARID3C subcellular localization via its interaction with NPM1, which controls the function of ARID3C in immune response by promoting gene expression for monocyte-to-macrophage differentiation . Another application to characterizing mechanisms underlying innate immune responses comes from the group of Aleksandra Nita-Lazar through the use of RNA sequencing and proteome analyses for the identification of the RNA-bound proteome in immortalized mouse macrophages upon exposure to lipopolysaccharide stimulation . Investigating immune signaling in response to type I and II interferons and virus infections, Krystal Lum from the Cristea group demonstrated the induction of distinct protein complexes and ISG profiles that coordinate antiviral defenses .…”