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Identification of a regulatory cis-element within the 3 '-untranslated region of the murine inducible nitric oxide synthase (NOS) mRNA: interaction with heterogeneous nuclear ribonucleoproteins I and L and role in the NOS gene expression
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
2007 (English)In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 44, no 4, 434-442 p.Article in journal (Refereed) Published
Abstract [en]

The aim of this study was to investigate the role of heterogeneous nuclear ribonucleoprotein I (hnRNPI) and hnRNPL in the regulation of the murine inducible nitric,oxide synthase (iNOS) gene during inflammation. Treatment of mice with lipopolysaccharide (LPS)/D-galactosamine, or of RAW 264.7 cells with LPS/interferon-gamma (IFN-gamma), strongly increased iNOS expression while reducing hnRNPI levels and complex formation between hnRNPI/hnRNPL and the 3'-untranslated region (3'-UTR) of iNOS mRNA. Introduction of the iNOS 3'-UTR to a luciferase reporter gene reduced its expression in RAW 264.7 cells. However, when hnRNPI and hnRNPL binding sites were deleted, luciferase expression was recovered. LPS/IFN-gamma increased the luciferase activity of the full-length 3'-UTR construct compared to control, while its effects on the deletion constructs were modest. The results indicate that LPS/IFN-gamma induce iNOS through a mechanism involving hnRNPI and hnRNPL binding to iNOS 3'-UTR. Our data suggest that iNOS mRNA degradation is promoted upon binding of hnRNPI and hnRNPL to a destabilizing region within its 3'-UTR, while inflammatory stimuli causing dissociation of the mRNA-protein complex, yield a more stable transcript. This appears to be particularly significant during extended inflammatory stimuli, resulting in sustained nitric oxide production. The critical event launching this process appears to be the degradation of hnRNPI.

Place, publisher, year, edition, pages
2007. Vol. 44, no 4, 434-442 p.
Keyword [en]
iNOS, hnRNP, post-transcriptional regulation, 3'-UTR
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-93760DOI: 10.1016/j.molimm.2006.02.019ISI: 000241460900018PubMedID: 16584775OAI: oai:DiVA.org:uu-93760DiVA: diva2:167339
Available from: 2005-11-25 Created: 2005-11-25 Last updated: 2011-05-19Bibliographically approved
In thesis
1. Post-Transcriptional Regulation of the Murine Inducible Nitric Oxide Synthase Gene
Open this publication in new window or tab >>Post-Transcriptional Regulation of the Murine Inducible Nitric Oxide Synthase Gene
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Large amounts of nitric oxide (NO) are produced by the inducible nitric oxide synthase (iNOS) upon inflammatory stimuli. NO is a multifaceted molecule, which may have beneficial effects as an antimicrobial agent in the immune defense, or cytotoxic effects in chronic inflammations, manifested as e.g. arthritis and asthma. Understanding the mode of regulation of the iNOS gene is a prerequisite for developing intervention strategies in various pathological conditions where detrimental effects of NO need to be prevented.

Transcriptional processes of the iNOS gene regulation are well described, while post-transcriptional events have not been studied in detail. The aim of the present thesis was to investigate post-transcriptional regulatory mechanisms involving the 3’untranslated region (UTR) of the murine iNOS mRNA.

Inflammation-dependent RNA-protein interactions with the iNOS mRNA 3’UTR were characterized by RNA gel shift analysis and UV cross-linking. Trans-acting factors interacting with the 3’UTR were detected in mouse liver and macrophages and identified as heterogeneous nuclear ribonucleoproteins (hnRNP) I and L. Western blot revealed that reduced hnRNPI levels are responsible for the decreased interaction of hnRNPI with iNOS 3’UTR upon induction in inflammatory conditions. This decrease was reversed by the glucocorticoid dexamethasone, concomitant with decreased iNOS mRNA levels and stability. Introduction of the iNOS 3’UTR into a luciferase reporter gene reduced its expression in macrophages. Upon deletions of the binding sites for hnRNPI and hnRNPL, the luciferase expression was recovered. In addition, inflammatory stimuli increased the luciferase activity of the construct with the full-length 3’UTR, while only weak effects of the stimuli were seen on the deletion constructs.

In conclusion, the results suggest that binding of hnRNPI and hnRNPL to the iNOS mRNA 3’UTR promotes degradation of the transcript. Induction of iNOS by inflammatory stimuli dissociates the RNA-protein complex, yielding a more stable mRNA. In addition, post-transcriptional down-regulation of the iNOS gene by the anti-inflammatory glucocorticoid dexamethasone, seems to involve hnRNPI.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 101 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 22
Keyword
Pharmaceutical biochemistry, iNOS, inducible nitric oxide synthase, gene regulation, post-transcriptional, mRNA stability, RNA-protein interactions, dexamethasone, murine, heterogeneous nuclear ribonucleoprotein, hnRNPI, hnRNPL, Farmaceutisk biokemi
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-6137 (URN)91-554-6402-5 (ISBN)
Public defence
2005-12-16, Room C4:305, BMC, Husargatan 3, UPPSALA, 09:00
Opponent
Supervisors
Available from: 2005-11-25 Created: 2005-11-25Bibliographically approved

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