Abstract
BackgroundGhrelin is a multifunctional peptide hormone expressed in a range of normal tissues and pathologies. It has been reported that the human ghrelin gene consists of five exons which span 5 kb of genomic DNA on chromosome 3 and includes a 20 bp non-coding first exon (20 bp exon 0). The availability of bioinformatic tools enabling comparative analysis and the finalisation of the human genome prompted us to re-examine the genomic structure of the ghrelin locus.ResultsWe have demonstrated the presence of an additional novel exon (exon -1) and 5' extensions to exon 0 and 1 using comparative in silico analysis and have demonstrated their existence experimentally using RT-PCR and 5' RACE. A revised exon-intron structure demonstrates that the human ghrelin gene spans 7.2 kb and consists of six rather than five exons. Several ghrelin gene-derived splice forms were detected in a range of human tissues and cell lines. We have demonstrated ghrelin gene-derived mRNA transcripts that do not code for ghrelin, but instead may encode the C-terminal region of full-length preproghrelin (C-ghrelin, which contains the coding region for obestatin) and a transcript encoding obestatin-only. Splice variants that differed in their 5' untranslated regions were also found, suggesting a role of these regions in the post-transcriptional regulation of preproghrelin translation. Finally, several natural antisense transcripts, termed ghrelinOS (ghrelin opposite strand) transcripts, were demonstrated via orientation-specific RT-PCR, 5' RACE and in silico analysis of ESTs and cloned amplicons.ConclusionThe sense and antisense alternative transcripts demonstrated in this study may function as non-coding regulatory RNA, or code for novel protein isoforms. This is the first demonstration of putative obestatin and C-ghrelin specific transcripts and these findings suggest that these ghrelin gene-derived peptides may also be produced independently of preproghrelin. This study reveals several novel aspects of the ghrelin gene and suggests that the ghrelin locus is far more complex than previously recognised.
Highlights
Ghrelin is a multifunctional peptide hormone expressed in a range of normal tissues and pathologies
It is well established that ghrelin is a multifunctional peptide with roles in growth hormone release, appetite regulation and gut motility [2] and we have demonstrated that it plays a role in cancer cell proliferation [3,4,5]
The conserved regions upstream of exon 1 are ghrelin exons To examine whether the conserved regions identified using Mulan were transcribed, PCRs from human stomach (using cDNA reverse transcribed with oligo(dT)18 primers) were performed with antisense primers in exon 1 of ghrelin and sense primers in the conserved regions
Summary
Ghrelin is a multifunctional peptide hormone expressed in a range of normal tissues and pathologies. The ghrelin gene (GHRL) spans 5 kb on chromosome 3 [6,7,8] and exons 1 to 4 encode an 117 amino acid preprohormone, preproghrelin. Exon 3 codes for obestatin, a recently identified 23 amino acid ghrelin gene-derived peptide hormone [9]. C-ghrelin, encoded by exons 2, 3 and 4, is a 66 amino acid peptide that contains the 23 amino acid obestatin peptide within its sequence [10,11]. It is currently not known if obestatin is cleaved from the large preproghrelin peptide, or whether distinct human obestatin-only and C-ghrelin-only transcripts exist. The transcript lacks the coding sequence of obestatin, but contains a putative peptide containing the first five amino acids of ghrelin and a novel 19 amino acid sequence
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