[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: The importance of the melanin-concentrating hormone (MCH) system for regulation of energy homeostasis and body weight has been demonstrated in rodents. We analysed the human MCH receptor 1 gene (MCHR1) with respect to human obesity.
This consisted of genomic screening of 13.4 kb encompassing the MCHR1 in extremely obese German children and adolescents and association analyses for two coding single nucleotide polymorphisms (SNPs). To confirm initial positive association results, additional association studies and transmission disequilibrium tests in further German, Danish, French and American samples were conducted. Selected SNPs were investigated using functional in vitro studies and reporter gene assays.
Single-stranded conformation polymorphism analysis, re-sequencing, PCR-restriction fragment length polymorphism analyses, tetra-primer amplification refractory mutation systems, matrix-assisted laser desorption/ionization time of flight mass spectrometry and reporter gene assays were carried out as well as measuring inositol phosphate formation, inhibition of cAMP formation and activation of p42/44 MAP kinase.
We identified 11 infrequent variations and two SNPs in the MCHR1 coding sequence and 18 SNPs (eight novel) in the flanking sequence. Association and transmission disequilibrium with obesity were detected for several SNPs in independent study groups of German obese children and adolescents and controls. In two German samples, encompassing 4056 and 295 individuals, trends towards association with obesity were detected. Findings in a second epidemiological German sample and in Danish, French and American samples were negative. Functional in vitro studies as well as reporter gene assays revealed no significant results.
Our initial association of MCHR1 alleles/haplotype detected might be related to juvenile-onset obesity, conditional on a particular genetic and/or environmental background. Alternatively, we could not exclude the possibility that the initially detected association represented a false positive finding.
European Journal of Endocrinology 07/2005; 152(6):851-62. · 3.14 Impact Factor