The bovine mimecan gene - Molecular cloning and characterization of two major RNA transcripts generated by alternative use of two splice acceptor sites in the third exon

Mimecan is a proteoglycan expressed by many connective tissues. It was orig inally isolated in a truncated form as a bone-associated glycoprotein, oste oglycin, and was considered an osteoinductive factor. Recently, we demonstr ated that the full-length translation product of the cDNA encoding mimecan is a corneal keratan sulfate proteoglycan present in other tissues without keratan sulfate chains. We also described multiple mimecan mRNA transcripts generated by differential splicing and alternative polyadenylation, In thi s study, we isolated genomic clones and determined the genomic organization of the bovine mimecan gene. The gene is spread over >33 kilobases of conti nuous DNA sequence and contains eight exons, The newly discovered first exo n, identified by 5'-rapid amplification of cDNA ends, consists of a 5'-untr anslated region and is enriched in C + G nucleotides. Two transcription ini tiation sites starting at the first and at the second exons were determined by primer extension. Molecular characterization shows that alternatively s pliced RNA isoforms are generated by the use of two distinct splice accepto r sites in the third exon situated 278 base pairs apart. We determined a pa rtial genomic structure of the human mimecan gene and demonstrated two alte rnatively spliced RNA transcripts that are generated likewise. Despite the diversity of mimecan transcripts, the primary structure of the core protein is encoded from exons 3 to 8 and remains unchanged, indicating its functio nal importance. Using ribonuclease protection assay, we analyzed the patter ns of spliced RNA expressed in cultured bovine keratocytes. We demonstrated that their expression is differentially modulated in a temporal manner by basic fibroblast growth factor.