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Open Access Research article

Functional characterization of two novel 5' untranslated exons reveals a complex regulation of NOD2 protein expression

Philip Rosenstiel1*, Klaus Huse2, Andre Franke1, Jochen Hampe1, Kathrin Reichwald2, Cornelia Platzer3, Roland G Roberts4, Christopher G Mathew4, Matthias Platzer2 and Stefan Schreiber1*

Author Affiliations

1 Institute of Clinical Molecular Biology, University Hospital Schleswig-Holstein, Campus Kiel, Germany

2 Leibniz Institute for Ageing Research – Fritz-Lipmann Institute, Jena, Germany

3 Institut für Laboratoriumsmedizin Berlin, Germany

4 Department of Medical and Molecular Genetics, King's College London, United Kingdom

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BMC Genomics 2007, 8:472  doi:10.1186/1471-2164-8-472

Published: 20 December 2007

Abstract

Background

NOD2 is an innate immune receptor for the bacterial cell wall component muramyl-dipeptide. Mutations in the leucine-rich repeat region of NOD2, which lead to an impaired recognition of muramyl-dipeptide, have been associated with Crohn disease, a human chronic inflammatory bowel disease. Tissue specific constitutive and inducible expression patterns of NOD2 have been described that result from complex regulatory events for which the molecular mechanisms are not yet fully understood.

Results

We have identified two novel exons of the NOD2 gene (designated exon 1a and 1b), which are spliced to the canonical exon 2 and constitute the 5' untranslated region of two alternative transcript isoforms (i.e. exon 1a/1b/2 and exon 1a/2). The two novel transcripts are abundantly expressed and seem to comprise the majority of NOD2 transcripts under physiological conditions. We confirm the expression of the previously known canonical first exon (designated exon 1c) of the gene in unstimulated mononuclear cells. The inclusion of the second alternative exon 1b, which harbours three short upstream open reading frames (uORFs), is downregulated upon stimulation with TNF-α or under pro-inflammatory conditions in the inflamed intestinal mucosa in vivo. Using the different 5' UTR splice forms fused to a firefly luciferase (LUC) reporter we demonstrate a rapamycin-sensitive inhibitory effect of the uORFs on translation efficacy.

Conclusion

The differential usage of two alternative promoters in the NOD2 gene leads to tissue-specific and context-dependent NOD2 transcript isoform patterns. We demonstrate for the first time that context-dependent alternative splicing is linked to uORF-mediated translational repression. The results suggest complex parallel control mechanisms that independently regulate NOD2 expression in the context of inflammatory signaling.