Revised genomic structure of the human ghrelin gene and identification of novel exons, alternative splice variants and natural antisense transcripts

Abstract

Background: Ghrelin 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.

Results: We 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.

Conclusion: The 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.

Figure 1

Initial identification and verification of novel transcribed ghrelin regions using comparative analyses and RT-PCR. A. Mulan sequence conservation profile for the human and murine ghrelin loci. The horizontal axis displays the input sequence. Evolutionary conserved regions (ECRs, > 70% identity; ≥ 99 bp) are depicted as dark red bars above each pairwise alignment. Preproghrelin coding exons 1 to 4 and the non-coding 20 bp exon 0 (blue), intergenic elements (red) and intron sequence (pink) are marked and the vertical axis shows the percent similarity of the murine ghrelin orthologue to the human sequence. Two conserved intergenic regions, 506 and 2.6 kb upstream of exon 1 of the ghrelin gene, can be seen in red. B. Schematic diagram showing the location of RT-PCR primers employed to verify whether the conserved, intergenic regions identified by Mulan were transcribed from the ghrelin gene. A forward primer in the conserved region immediately upstream of the 20 bp exon 0 and a reverse primer in exon 1 amplified a 278 bp PCR fragment (upper panel). A PCR using another exon 1 primer and a forward primer in the conserved region 2.6 kb upstream of exon 1 resulted in a 227 bp PCR fragment (lower panel). The PCRs confirm that the conserved regions predicted by Mulan correspond to ghrelin gene-derived exons. We have termed the conserved regions exon -1 and extended exon 0.

Figure 2

Partial genomic sequence of the human ghrelin gene showing novel exon -1 and extended exon 0 transcription start sites. Exon sequences are boxed and in upper case letters. Extended exon 0 sequence is indicated by dashed boxes. Nucleotide positions are shown on the left and the translation initiation site of preproghrelin is indicated as +1. Intron boundaries are indicated by bold, and intron sequences by lower case letters. Five transcription start sites (TSS) determined by employing an OriGene human Sure-RACE panel, denoted TSS1-5, are underlined and indicated. TSS1 contains a 106 bp exon -1 (exon -1a) and splices into the complete, 736 bp 5' extended exon 0 (exon 0b) [GenBank:EF549561]. TSS2 [GenBank:EF549564] harbours a 92 bp exon -1 (exon -1b) and splices into a 197 bp exon 0 (exon 0c; exon acceptor site is indicated # and bold). TSS3-5 all initiate in exon 0 (exon 0 e-g, respectively). TSS3 was obtained from the ovary [GenBank:EF549563]; TSS4 from testis [GenBank:EF549562] and stomach; TSS5 from adipose tissue [GenBank:EF549565] and leukocytes.

Figure 3

Comparison of novel human and putative murine ghrelin exon sequences. The mouse and human alignments were generated by the ClustalW program and drawn by BOXSHADE . Black shading indicates conserved nucleotides. Transcription start sites determined by 5' RACE (Δ) and CAGE (Cap Analysis of Gene Expression) (*) are indicated, and the exact transcription start site nucleotides are shaded in grey in each species. Exon-intron boundaries are indicated by lowercase letters and bold font. A. Comparison of human and putative mouse transcription start sites (TSSs) in the novel exon -1. Two human TSSs are indicated, corresponding to 106 bp (5' RACE and CAGE tag starting site T03R009D4FFF) and 92 bp exon -1. An initiating methionine (ATG, start codon) which is not followed by a stop codon in exon -1 is underlined. No murine exon -1 CAGE tags were found in the FANTOM3 Basic CAGE viewer (May 2007) B. Comparison of human and mouse extended exon 0 sequences showing several human and murine exon 0 transcription start sites deduced by 5' RACE and CAGE (mouse T06R06CF33DC, T06R06CF32CB and T06R06CF3202). The originally described 20 bp exon 0 transcription start site, present in both human and murine ghrelin transcripts, is indicated by a pound mark (#).

Figure 4

Expression of exon -1 and extended exon 0 mRNAs in stomach and SW1353 cell line. Exons are represented as boxes, PCR primers are indicated as arrows above exons. A. Amplicons spanning the extended exon 0 to exon 4 of the ghrelin gene. A major 1064 base pair amplicon, corresponding to a full-length transcript, is expressed in the human stomach (HS). The SW1353 chondrosarcoma cell line expresses a complex pattern of alternatively-spliced products, which may result from promiscuous splicing of exon 0. B. Exon -1 to 4 amplicons sequenced from the human stomach. Transcripts have been grouped into three major types (I, II and III). Group I amplicons include all preproghrelin exons (exons 1–4), but differ in the length of sequence upstream of exon 1 (the 5' UTR of preproghrelin). Group II transcripts contain exon -1, exon 4 and various combinations of exons downstream of exon 1 (exon 2 to 4), but lack exon 0 and 1. Group III transcripts contain alternative exon -1 and 4 (exon -1* and 4*, respectively) in addition to two novel exons (exon 2* and 2**) in intron 2 of the ghrelin gene.

Figure 5

Exon structure of exon -1 to 4 RT-PCR amplicons in human tissues and continuous cell lines. Exons are represented as boxes, PCR primers are indicated as arrows above exons. Transcripts have been grouped into three major types (I, II and III). The tissue where an amplicon was first sequenced from is depicted on the left hand side of each transcript's exon structure. Group I are preproghrelin mRNA variants with different 5' untranslated regions (5' UTRs). Group II lack the coding region 'active core' of ghrelin (in exon 1) and contains one or more downstream exons (2–4). Group III contains the novel exons -1*, 4*, 2* and 2** in various combinations. PBL denotes leukocytes; PC3 is a human prostate carcinoma cell line. Group I: 855 bp, stomach [GenBank:EF549571];777 bp, stomach [GenBank:EF549572];580 bp, stomach [GenBank:EF549573];852 bp, heart [GenBank:EU072085];1316 bp, placenta [GenBank:EU072083];1313 bp, kidney [GenBank:EU072084];1067 bp, kidney [GenBank:EU072087]. Group II: 443 bp, stomach [GenBank:EF549574];326 bp, stomach [GenBank:EF549574];440 bp, PBL [GenBank:EU072086];217 bp, PC3 [GenBank:EF549557]. Group III: 1176 bp, stomach [GenBank:EF549568];921 bp, stomach [GenBank:EF549570];1108 bp, spleen [GenBank:EU072081];950 bp, kidney [GenBank:EU072082].

Figure 6

Structure of ghrelin splice variants containing a putative signal peptide. For each splice variant the exon structure, the predicted protein structure and amino acid sequence is depicted. A putative signal peptide (SP) in exon -1 is indicated by bold letters in the amino acid sequence. Obestatin coding sequence is depicted in red font, while an exon 3-deleted proghrelin peptide is shown in blue font. The splice variant that may encode putative C-ghrelin [GenBank:EF549574] was also expressed in the heart and spleen. Obestatin mRNA variants were obtained from the human stomach [GenBank:EF549575], while a variant that may encode an exon 3-deleted (Δex3) proghrelin peptide [GenBank:EF549557] was found in the PC3 human prostate carcinoma cell line. A mRNA variant obtained from leukocytes with a 5' truncated exon 2 [GenBank:EU072086] may encode a C-ghrelin peptide missing a single glutamine residue at position 24 (des-Gln²⁴-C-ghrelin, underlined in the C-ghrelin amino acid sequence).

Figure 7

Characterisation of ghrelin natural antisense transcripts in the normal human stomach. A. Ethidium bromide stained agarose gel showing the verification of the candidate antisense gene ghrelinOS by orientation-specific RT-PCR in the human stomach. Antisense transcripts were amplified using a primer in exon 4* as the reverse transcription (RT) primer (R), while for the detection of sense transcripts, a primer in exon 2* was used in the RT reaction (F). The RT reactions were subjected to PCR using both the exon 2* and 4* primers (for 30, 35 or 40 cycles). NTC = no template control (water). M = MassRuler Express DNA ladder (Fermentas). B. Alignment of sequences derived from two 5' RLM-RACE products (TSS86 and TSS63) and a CAGE tag starting site corresponding to a 28 bp exon 4* (TSS28, T03F009D342E) with exon 4* and flanking genomic sequence. The lower case letters indicate upstream genomic or downstream intron 4* sequences. The position and sequence of the nested gene-specific 5' RACE primer (5'OS-in-R) is indicated with an arrow and underlined, respectively. For comparison, the (sense) ghrelin gene exon 4 sequence is shaded in grey.

Figure 8

Genomic organisation of sense and natural antisense transcripts associated with the ghrelin gene. A. The natural antisense transcripts are shown in black and the corresponding genomic structure of the ghrelin gene in grey. The previously reported 20 bp exon 0 and extended (188 bp) exon 1 of the ghrelin gene are also shown. Exons are represented as boxes, introns as horizontal lines and sizes (bp) are indicated above each exon. The direction of transcription of each gene is indicated by arrowheads. A tilde symbol (~) in front of the size of some exons indicates that as RT-PCR primers spanned these exons, their exact ends are unknown. The antisense strand ghrelinOS transcripts overlap with the sense exon -1 and 4, but not with the exon 0–3 of the ghrelin gene. The three TSSs resulting in 86, 63 or 28 base bp exon 4* are shown. Note that it has not been determined which TSS(s) the various ghrelinOS variants employ. GhrelinOS1 contains an approximately 880 bp exon -1*a, a 158 bp exon 2*a, a short 68 base pair exon 2** and exon 4*. GhrelinOS1 was demonstrated by experimental procedures (RT-PCR) in this study and is supported by expressed sequence tag (EST) data (EST CF264800). GhrelinOS2 (demonstrated via RT-PCR in our study) is similar to ghrelinOS1, except in this variant exon -1* is ~625 bp long (exon -1*b). GhrelinOS3 (Incyte clone LIFESEQ4072309) is similar to ghrelinOS1, but lacks exon 2** while exon 2* contains a 4 bp 3' extension (exon 2*b). GhrelinOS4 is similar to ghrelinOS3, but contains exon 2*a, while ghrelinOS5 lacks exon 2* and 2** (both splice variants demonstrated in our study). B. Schematic organisation of the intron junctions of ghrelinOS natural antisense mRNA variants ghrelinOS1–5 described in A. All exons are flanked by GT/AG intron junctions (with the exception of ghrelinOS1, 2 and 4 (2*a to -1*a/b which is flanked by GC/AG).