gms | German Medical Science

Angiotensinogen is the natural substrate for renin required for the production of all active peptides of the renin-angiotensin system. The gene coding for angiotensinogen is composed of 5 exons. Exon 2 (Ex2) is with 854 nucleotides (nt) the longest exon and contains the translational start codon, the signal peptide, and the angiotensin I encoding sequence. However, the length of Ex2 by far exceeds the normal length of internal exons in mammalian genes (150-250 nt). Thus, we hypothesized that a splicing enhancer may be necessary to include this exon into mature mRNA. Indeed, by studying the expression of the mouse angiotensinogen (mAOG) gene by RT-PCR and RNase protection assay (RPA) in different tissues, we detected a small portion (<10%) of mAOG-mRNA which lacked Ex2 confirming alternative splicing of this gene. By expressing a full-length mAOG-gene with a viral promoter in COS7 cells followed by RPA analysis, we could detect the appearance of both mRNA isoforms in these cells (70% full-length, 30% truncated). This cell culture system allowed us to localize the putative splicing enhancer within Ex2 responsible for exon inclusion. To this purpose, the mAOG gene was linearized with Sfi I and the exo-endonuclease Bal-31 was used to generate internal deletions of different lengths in Ex2. These truncated mAOG gene clones were again transfected into COS7 cells and expression analysis was made by RPA. Clones containing an Ex2 of less than 200 nt included it into the mature mRNA probably due to the small size. Clones with Ex2 longer than 340 nt excluded it (30% full-length, 70% truncated) as long as nt 102 to 174 were missing in the construct. We conclude that this region contains an exonic splicing enhancer necessary to avoid skipping of the Ex2. Since Ex2 is essential for angiotensinogen function, interfering with this splicing process might be of therapeutic interest.