Post-transcriptional control of gene expression in inflammation and cell differentiation
Project management
Prof. Dr. rer. nat. Antje Ostareck-Lederer
Tel.: 0241-80-36567
Prof. Dr. rer. nat. Dirk Ostareck
Tel.: 0241-80-36568
1) Optimization of small molecules to modulate HNRNPK-controlled TAK1 synthesis in macrophages
Summary
Macrophage activation through bacterial lipopolysaccharide (LPS) mediated TLR4 signaling induces inflammatory cytokine expression. Persisting macrophage activity and excessive cytokine secretion lead to hyperinflammation and subsequent immune paralysis, which trigger systemic inflammation, sepsis and ultimate organ failure. The control of the central TLR4-downstream kinase TAK1 can be employed to regulate macrophage activity.
We identified HNRNPK as regulator of TAK1 mRNA, which does not affect TAK1 gene activity, but directly modulates TAK1 protein synthesis and finally macrophage LPS response in vitro and in vivo.
Our innovative approach takes advantage of small molecules to control HNRNPK-TAK1 mRNA binding and TAK1 expression. To optimize suitable molecular components structural analyses and functional group modifications are currently applied.
The design of active and specific compounds not only provides potential for sepsis therapies, but also opportunities to program tumor-associated macrophages to exploit specific antitumor activities.
Patent
EP 2855519 B1, US 9745369 B2
Modulation of the TLR4-signaling pathway
Ostareck-Lederer, A.,Ostareck, D.H., Marx, G., Liepelt, A., Mossanen, J.
Department of Intensive Care Medicine, University Hospital RWTH Aachen
Reference
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.
RNA. 2014 Jun;20(6):899-911. doi: 10.1261/rna.042788.113. PMID: 24751651
2) Differential METTL3/14-dependent RNA m6A methylation in LPS activated macrophages
Collaboration
Kathi Zarnack
Buchmann Institute for Molecular Life Sciences (BMLS) & Faculty of Biological Sciences, Goethe University Frankfurt, Frankfurt, Germany
Julian König
Institute of Molecular Biology (IMB), Mainz, Germany
Summary
To restore immune cell homeostasis in-depth understanding of molecular mechanisms underlying sepsis development is required. The RNA N6-methyladenosine (m6A) modification has been recognized as a major post-transcriptional regulator of inflammation-related gene expression. M6A, the most abundant internal RNA modification is an essential regulator of almost all aspects of RNA metabolism. As dynamic modification it is specifically deposited by methyltransferases (writer), recognized by reader proteins and removed by demethylases (erasers). Its reversibility enables cells to respond quickly to external signals critical for immune recognition and activation of the innate and adaptive immune system.
To address the impact of bacterial infections on the macrophage m6A epitrascriptome, we recently performed deep mRNA m6A profiling at single nucleotide resolution. Our analysis of m6A coverage in non-induced and LPS-activated murine RAW264.7 macrophages revealed differential methylated m6A sites in mRNAs encoding inflammation-related proteins. We will investigate the m6A methylation of confirmed mRNA targets by SELECT-PCR and encoded protein expression in vitro and in vivo. To characterize reader protein interaction with m6A sites in selected mRNAs and their impact on macrophage activation, METTL3/14 knockdowns will be performed. Differential methylated target mRNAs will be examined in mouse models (CLP and LPS) and further validated in cells of septic patients.
The studies will provide insight in the role of m6A modifications in sepsis and path the way for new and personalized sepsis treatment concepts.
Reference
Körtel N, Rücklé C, Zhou Y, Busch A, Hoch-Kraft P, Sutandy FXR, Haase J, Pradhan M, Musheev M, Ostareck D, Ostareck-Lederer A, Dieterich C, Hüttelmaier S, Niehrs C, Rausch O, Dominissini D, König J, Zarnack K.: Deep and accurate detection of m6A RNA modifications using miCLIP2 and m6Aboost machine learning.
Nucleic Acids Res. 2021 Sep 20;49(16):e92. doi: 10.1093/nar/gkab485. PMID: 34157120
3) Post-transcriptional control of macrophage gene expression mediated by RBPs
Summary
The regulation of macrophage migration, phagocytotic activity and cytokine expression requires timely gene transcription and post-transcriptional gene expression control. As trans-acting factors RNA binding proteins (RBPs) regulate mRNA processing at all levels. Thereby they enable rapid cellular responses to inflammatory mediators and facilitate a coordinated systemic immune response.
Previously, we identified the mRNA interactome of RAW264.7 macrophages. Among the newly classified RBPs, we characterized P23, a HSP90 co-chaperone, also known as cytosolic prostaglandin E2 synthase (PTGES3), which exhibited differential mRNA binding upon LPS induction. RNAseq of the P23 RNA interactome revealed that 44 mRNAs were reduced in response to LPS. We specifically analyzed Kif15 mRNA encoding kinesin family member 15, a motor protein implicated in cytoskeletal reorganization and cell mobility.
Noteworthy, the unexpected P23 RBP function affects macrophage activity. P23 depletion enhances macrophage phagocytosis and accelerates migration of untreated macrophages. Mechanistically P23 reduction mediates Kif15 mRNA destabilization and diminished KIF15 expression.
This novel insight sheds light on inflammatory response modulation and provides indication for molecular interventions. Currently we investigate how other post-transcriptionally controlled mRNAs affected by newly identified RBPs contribute to inflammation responses.
References
[1] Barton H, Zechendorf E, Ostareck D, Ostareck-Lederer A, Stoppe C, Zayat R, Simon PT, Marx G, Bickenbach J.: Prognostic Value of GDF-15 in Predicting Prolonged Intensive Care Stay following Cardiac Surgery: A Pilot Study.
Dis Markers. 2021 Jun 15;2021:5564334. doi: 10.1155/2021/5564334. PMID: 34221186
[2] de Vries S, Benes V, Naarmann-de Vries IS, Rücklé C, Zarnack K, Marx G, Ostareck DH, Ostareck-Lederer A.: P23 Acts as Functional RBP in the Macrophage Inflammation Response.
Front Mol Biosci. 2021 Jun 11;8:625608. doi: 10.3389/fmolb.2021.625608. eCollection 2021. PMID: 34179071
[3] Ostareck DH, Ostareck-Lederer A.: RNA-Binding Proteins in the Control of LPS-Induced Macrophage Response.
Front Genet. 2019 Feb 4;10:31. doi: 10.3389/fgene.2019.00031. PMID: 30778370
[4] Liepelt A, Naarmann-de Vries IS, Simons N, Eichelbaum K, Föhr 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.
Mol Cell Proteomics. 2016 Aug;15(8):2699-714. doi: 10.1074/mcp.M115.056564. PMID: 27281784
4) HNRNPK as regulator in erythroid differentiation
Collaboration
Dierk Niessing
Institute of Pharmaceutical Biotechnology, Ulm University, Ulm, Germany
Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany
Summary
Post-transcriptional control is essential to safeguard structural and metabolic changes in enucleated reticulocytes during their terminal maturation to functional erythrocytes. HNRNPK regulates the timely synthesis of arachidonate 15-lipoxygenase (ALOX15), which initiates mitochondria degradation at the final stage of reticulocyte maturation. It constitutes a silencing complex at the ALOX15 mRNA 3’ UTR that inhibits translation initiation at the AUG by impeding 60S and 40S ribosomal subunits joining. We elucidated how HNRNPK interferes with 80S ribosome assembly. Three independent interaction screens consistently demonstrated differential HNRNPK binding to RPS19 of the 40S subunit. Dimethylation of HNRNPK R256, R258 and R268 affects its RPS19 interaction. Decreasing HNRNPK arginine dimethylation is linked to diminishing interaction with RPS19 during K562 cell erythroid maturation in vitro and in vivo. RPS19 depletion from premature K562 cells results in ALOX15 synthesis induction and mitochondria degradation.
Interestingly, RPS19 W52, frequently mutated in Diamond-Blackfan Anemia (DBA), participates in HNRNPK binding. Currently we evaluate the impact of HNRNPK-RPS19 interaction in normal erythropoiesis and anemia.
Selected references
[1] Naarmann-de Vries IS, Senatore R, Moritz B, Marx G, Urlaub H, Niessing D, Ostareck DH, Ostareck-Lederer A.: Methylated HNRNPK acts on RPS19 to regulate ALOX15 synthesis in erythropoiesis.
Nucleic Acids Res. 2021 Apr 6;49(6):3507-3523. doi: 10.1093/nar/gkab116. PMID: 33660773
[2] Naarmann-de Vries IS, Brendle A, Bähr-Ivacevic T, Benes V, Ostareck DH, Ostareck-Lederer A.: Translational control mediated by hnRNP K links NMHC IIA to erythroid enucleation.
J Cell Sci. 2016 Mar 15;129(6):1141-54. doi: 10.1242/jcs.174995. PMID: 26823606
[3] Moritz B, Lilie H, Naarmann-de Vries IS, Urlaub H, Wahle E, Ostareck-Lederer A, Ostareck DH.: Biophysical and biochemical analysis of hnRNP K: arginine methylation, reversible aggregation and combinatorial binding to nucleic acids.
Biol Chem. 2014 Jul;395(7-8):837-53. doi: 10.1515/hsz-2014-0146. PMID: 25003387
[4] Naarmann IS, Harnisch C, Müller-Newen G, Urlaub H, Ostareck-Lederer A, Ostareck DH.: DDX6 recruits translational silenced human reticulocyte 15-lipoxygenase mRNA to RNP granules.
RNA. 2010 Nov;16(11):2189-204. doi: 10.1261/rna.2211110. PMID: 20884783
[5] Naarmann IS, Harnisch C, Flach N, Kremmer E, Kühn H, Ostareck DH, Ostareck-Lederer A.: mRNA silencing in human erythroid cell maturation: heterogeneous nuclear ribonucleoprotein K controls the expression of its regulator c-Src.
J Biol Chem. 2008 Jun 27;283(26):18461-72. doi: 10.1074/jbc.M710328200. PMID: 18441016
[6] Messias AC, Harnisch C, Ostareck-Lederer A, Sattler M, Ostareck DH.: The DICE-binding activity of KH domain 3 of hnRNP K is affected by c-Src-mediated tyrosine phosphorylation.
J Mol Biol. 2006 Aug 18;361(3):470-81. doi: 10.1016/j.jmb.2006.06.025. PMID: 16854432
[7] Ostareck-Lederer A, Ostareck DH, Rucknagel KP, Schierhorn A, Moritz B, Huttelmaier S, Flach N, Handoko L, Wahle E.: Asymmetric arginine dimethylation of heterogeneous nuclear ribonucleoprotein K by protein-arginine methyltransferase 1 inhibits its interaction with c-Src.
J Biol Chem. 2006 Apr 21;281(16):11115-25. doi: 10.1074/jbc.M513053200. PMID: 16492668
[8] Ostareck DH, Ostareck-Lederer A, Shatsky IN, Hentze MW.: Lipoxygenase mRNA silencing in erythroid differentiation: The 3'UTR regulatory complex controls 60S ribosomal subunit joining.
Cell. 2001 Jan 26;104(2):281-90. doi: 10.1016/s0092-8674(01)00212-4. PMID: 11207
