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. Overall design: 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.