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

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. 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-alpha 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. 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.