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.

Keywords: RNA-binding proteins; TLR signaling; cell signaling; innate immunity; macrophages; protein−RNA interactions; proteomics.

Figure 1.

Schematic of the SILAC labeling (A) The experimental groups (3 biological replicates each) were assigned based on the presence or absence of the LPS and UV irradiation; The SILAC triplex set with UV provided a comparison of RNA-RBP crosslinking upon different LPS treatment durations, while the SILAC duplex sets (+ UV/− UV) served as controls to remove the non-specific RNA-RBP binding at any given LPS exposure. Only the proteins that were observed with at least 5 times or more intensity in the +UV crosslinked samples were retained. (B). UV crosslinking-based RBP extraction workflow.

Figure 2.

RNA-binding proteins detected in the SILAC experiment. A: RNA binding of RBPs reduces significantly at both 30 min and 60 min LPS treatment. B: Overlap between RBPs present at 30 and 60 minutes. C: Hierarchical clustering of the enriched RBPs at 30 and 60 min LPS treatment. The log2 fold change ratios at each time were averaged for 6 biological replicates. Five clusters were outlined based on the changes occurring in the RBP association or dissociation from RNA. D: Overlap between RBPs found in this study and these described by Liepelt et al.

Figure 3.

GO term analysis and pathway enrichment of RBPs at 30 min LPS treatment.

Figure 4.

GO term analysis and pathway enrichment of RBPs at 60 min LPS treatment.

Figure 5.

A and B: Volcano plots depicting unchanging RBPs at the protein level following 30 and 60 min LPS treatment. The log2 fold change ratios at each timepoint were averaged across the 6 biological replicates. C and D: Volcano plots depicting unchanging transcripts following the 30 and 60 min LPS treatment. The log2 fold change ratios at each time were averaged across the 6 biological replicates. E, F, G: The mRNA levels of Il1a (E), Acod1 (F) and MARCKS (G) from IMMs treated with 100 ng/mL of LPS for 30 and 60 minutes were quantified by real-time PCR. Data is representative of 2 independent experiments (at least 3 biological replicates per condition per experiment). Shown as the mean +− SEM. *p < 0.05, **p <0.005 (one-way ANOVA).

Figure 6.

Binding sites at the protein level in unstimulated macrophages (A), and after 30 (C) and 60 (E) minutes of LPS stimulation. Binding sites at the RNA level in unstimulated macrophages (B), and after 30 (D) and 60 (F) minutes of LPS stimulation.