Lipopolysaccharide Regulates the Macrophage RNA-Binding Proteome

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.