MULTIPLE TRANSCRIPTS CODING FOR THE MENKES GENE - EVIDENCE FOR ALTERNATIVE SPLICING OF MENKES MESSENGER-RNA

We isolated cDNA fragments from four human cell lines that had sequenc es for the Menkes Cu-transporting ATPase (ATP7A). Primers designed to generate a 4.8 kb cDNA with the complete open reading frame generated a 1.9 kb cDNA in addition to the expected 4.8 kb product. Sequence ana lysis revealed that the 1.9 kb cDNA encoded one of the six Cu-binding sites and two of the eight transmembrane domains of ATP7A. Stop and st art codons were also present. More striking, however, was an unusual u nion between exons 2 and 16 that retained an in-frame reference to exo n 23. The 1.9 kb cDNA thus appeared to be a truncated Menkes mRNA that coded for an ATP7A variant that lacked exons 3-15, A 530 bp probe spe cific for exon 23 that avoided sequences in the exon 3-15 region hybri dized to a 5.5 kb band on Northern blot analysis. Western blotting pro vided immunochemical evidence for the presence of both a 170 kDa and a 57 kDa protein with ATP7A sequences in detergent extracts of Caco-2 a nd induced BeWo cells. Extracts from non-induced BeWo cells, which lac k the capacity to express the Menkes gene (MNK), showed neither protei n. In a cell-free reticulocyte lysate, a plasmid containing the 1.9 kb cDNA insert directed the synthesis of a 59 kDa protein with antigenic properties of ATP7A. These studies provide evidence that non-Menkes c ells have the capacity to synthesize more than one MNK mRNA. The one c haracterized in this report codes for a 57-59 kDa protein that lacks t he core structure of the ATP7A protein. The smaller variant could be a n alternative spliced form of MNK mRNA.