Identification of RNA-binding proteins in macrophages by interactome capture

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Pathogen components, such as bacterial lipopolysaccharides (LPS) that activate Toll-like receptor 4 (TLR4), induce mitogen activated kinases (MAPKs) and NFkappaB through different downstream pathways to stimulate inflammatory cytokine expression. Although these mediators are essential to combat and coordinate the cellular infection response, their excessive expression causes systemic inflammation and sepsis, which are, besides cardiovascular diseases and cancer, the third leading cause of death worldwide. Importantly, post-transcriptional control of TLR4 downstream signaling molecule expression contributes to the tight regulation of inflammatory cytokine synthesis in macrophages. Emerging evidence highlights the role of RNA binding proteins (RBPs) in the post-transcriptional control of the innate immune response. By employing RNA interactome capture, which combines RBP-crosslinking to RNA in LPS-induced and untreated murine RAW 264.7 macrophages, cell lysis, oligo(dT) capture of polyadenylated RNAs and mass spectrometry analysis, macrophage RBPs were systematically identified and their response to LPS stimulation was characterized. Our data revealed 402 proteins of the macrophage RNA interactome including 91 previously unknown RBPs. A comparison with the published RNA interactomes identified 32 RBPs so far unique to RAW 264.7 macrophages. Of these, 19 proteins are linked to biochemical activities not directly related to RNA. From this group, HSP90 co-chaperone P23 that was demonstrated to exhibit cytosolic prostaglandin E2 synthase 3 (PTGES3) activity, and the hematopoietic cell-specific Lyn substrate 1 (HCLS1 or HS1), a hematopoietic specific adapter molecule, were validated as novel macrophage RBPs. We initiated UV-crosslinking and immunoprecipitation (CLIP) combined with deep sequencing of mRNAs that co-purify with novel identified macrophage RBPs. Based on that, mRNA-protein interaction maps will be generated to identify specific mRNAs encoding TLR4 downstream signaling molecules or their modulators and we will analyze how identified target mRNAs are post-transcriptionally regulated by RBPs in vitro and in vivo. Within the SPP 1935, the project will help to expand the mammalian RBP repertoire and will identify macrophage mRNPs that are prime candidates for the regulation and execution of LPS-induced TLR4 signaling pathways and the innate immune response. Information about underlying molecular mechanisms of RBP function will advance the understanding of their roles in inflammatory response modulation and will provide knowledge about their potential as therapeutic targets, to prevent systemic inflammation and sepsis.