Prof. Dr. Ostareck-Lederer
University Hospital RWTH Aachen Department of Intensive Care and Intermediate Care
Pauwelsstrasse 30 52074 Aachen
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PROJECT INFORMATION :
Pathogen components, such as bacterial lipopolysaccharides (LPS) that activate Toll-like receptor 4 (TLR4), induce mitogen activated kinases (MAPKs) and NFкB 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.
Focus of the group:
Functional analysis of mRNPs
KEY TECHNOLOGIES :
- in vitro RNA-protein-interaction assays
- in vitro translation assays and sucrose gradient analysis of polysomes and mRNPs
- RBP depletion to study the impact on target mRNA stability, translation and localization
- Fluorescence in situ hybridization (FISH) and confocal laser-scanning microscopy to explore the potential target mRNA localization to cellular structures
- RBP-mediated post-transcriptional control in activated macrophages and erythroid cell maturation
Liepelt A, Naarmann-de Vries IS, Simons N, Eichelbaum K, Foehr S, Archer SK, Castello A, Usadel B, Krijgsveld J, Preiss T, Marx G, Hentze MW, Ostareck DH, Ostareck-Lederer A:
Identification of RNA-binding proteins in macrophages by interactome capture. (2016) Mol Cell Proteomics 15: 2699-2714.
Naarmann-de Vries IS, Brendle A, Bähr-Ivacevic T, Benes V, Ostareck DH, Ostareck-Lederer A: HnRNP K-mediated translational control links NMHC IIA to erythroid enucleation.
(2016) J Cell Sci. 129: 1141-1154.
De Santis R, Liepelt A, Mossanen JC, Dueck A, Simons N, Mohs A, Trautwein C, Meister G, Marx G, Ostareck-Lederer A, Ostareck DH: miR-155 targets Caspase-3 mRNA in activated macrophages.
(2016) RNA Biol. 13: 43-58.
Liepelt A, Mossanen JC, Denecke B, Heymann F, De Santis R, Tacke F, Marx G, Ostareck DH, Ostareck-Lederer A: Translation control of TAK1 mRNA by hnRNP K modulates LPS-induced macrophage activation.
(2014) RNA 20, 899-911.
Naarmann-de Vries IS, Urlaub H, Ostareck DH, Ostareck-Lederer A: Caspase-3 cleaves hnRNP K in erythroid differentiation.
(2013) Cell Death Dis. 4:e548.