Identification of RNA-binding Proteins in Macrophages by Interactome Capture.
Pathogen components, such as lipopolysaccharides of Gram-negative bacteria that activate Toll-like receptor 4, induce mitogen activated protein kinases and NFκB through different downstream pathways to stimulate pro- and anti-inflammatory cytokine expression. Importantly, post-transcriptional control of the expression of Toll-like receptor 4 downstream signaling molecules 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. To systematically identify macrophage RBPs and their response to LPS stimulation, we employed RNA interactome capture in LPS-induced and untreated murine RAW 264.7 macrophages. This combines RBP-crosslinking to RNA, cell lysis, oligo(dT) capture of polyadenylated RNAs and mass spectrometry analysis of associated proteins. Our data revealed 402 proteins of the macrophage RNA interactome including 91 previously not annotated as RBPs. A comparison with published RNA interactomes classified 32 RBPs uniquely identified in RAW 264.7 macrophages. Of these, 19 proteins are linked to biochemical activities not directly related to RNA. From this group, we validated the HSP90 cochaperone 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 cell-specific adapter molecule, as novel macrophage RBPs. Our study expands the mammalian RBP repertoire, and identifies macrophage RBPs that respond to LPS. These RBPs are prime candidates for the post-transcriptional regulation and execution of LPS-induced signaling pathways and the innate immune response. Macrophage RBP data have been deposited to ProteomeXchange with identifier PXD002890.
From the ‡Department of Intensive Care and Intermediate Care, University Hospital RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany;
Anke
A
Liepelt
From the ‡Department of Intensive Care and Intermediate Care, University Hospital RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany;
Isabel S
IS
Naarmann-de Vries
From the ‡Department of Intensive Care and Intermediate Care, University Hospital RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany;
Nadine
N
Simons
§European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany;
Katrin
K
Eichelbaum
§European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany;
Sophia
S
Föhr
¶EMBL-Australia Collaborating Group, Department of Genome Sciences, The John Curtin School of Medical Research, The Australian National University, Garran Rd, Acton (Canberra) ACT 2601, Australia;
Stuart K
SK
Archer
§European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany;
Alfredo
A
Castello
‖Institute of Biology I, RWTH Aachen, Worringer Weg 2, 52074 Aachen, Germany;
Björn
B
Usadel
§European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany;
Jeroen
J
Krijgsveld
¶EMBL-Australia Collaborating Group, Department of Genome Sciences, The John Curtin School of Medical Research, The Australian National University, Garran Rd, Acton (Canberra) ACT 2601, Australia; **Victor Chang Cardiac Research Institute, Darlinghurst (Sydney), New South Wales 2010, Australia.
Thomas
T
Preiss
From the ‡Department of Intensive Care and Intermediate Care, University Hospital RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany;
Gernot
G
Marx
§European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany;
Matthias W
MW
Hentze
From the ‡Department of Intensive Care and Intermediate Care, University Hospital RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany; aostareck@ukaachen.de dostareck@ukaachen.de.
Dirk H
DH
Ostareck
From the ‡Department of Intensive Care and Intermediate Care, University Hospital RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany; aostareck@ukaachen.de dostareck@ukaachen.de.
Antje
A
Ostareck-Lederer
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D004268:DNA-Binding Proteins / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D000071876:Prostaglandin-E Synthases / Q000378:metabolism*; D060066:Protein Interaction Maps / Q000187:drug effects; D040901:Proteomics / Q000379:methods*; D000067996:RAW 264.7 Cells; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000032:analysis*; D017423:Sequence Analysis, RNA / Q000379:methods
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C094983:Hcls1 protein, mouse; D008070:Lipopolysaccharides; D016601:RNA-Binding Proteins; D012313:RNA; D000071876:Prostaglandin-E Synthases; C000605868:Ptges3 protein, mouse