Multiple transcripts coding for the menkes gene: evidence for alternative splicing of Menkes mRNA

Biochem J. 1998 Aug 15;334 ( Pt 1)(Pt 1):71-7. doi: 10.1042/bj3340071.

Abstract

We isolated cDNA fragments from four human cell lines that had sequences 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 analysis 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 start codons were also present. More striking, however, was an unusual union between exons 2 and 16 that retained an in-frame reference to exon 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 specific for exon 23 that avoided sequences in the exon 3-15 region hybridized to a 5.5 kb band on Northern blot analysis. Western blotting provided immunochemical evidence for the presence of both a 170 kDa and a 57 kDa protein with ATP7A sequences in detergent extracts of Caco-2 and induced BeWo cells. Extracts from non-induced BeWo cells, which lack the capacity to express the Menkes gene (MNK), showed neither protein. 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 cells have the capacity to synthesize more than one MNK mRNA. The one characterized in this report codes for a 57-59 kDa protein that lacks the core structure of the ATP7A protein. The smaller variant could be an alternative spliced form of MNK mRNA.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphatases / biosynthesis
  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / genetics*
  • Alternative Splicing*
  • Amino Acid Sequence
  • Base Sequence
  • Carrier Proteins / biosynthesis
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics*
  • Cation Transport Proteins*
  • Cell Line
  • Cloning, Molecular
  • Copper-Transporting ATPases
  • DNA, Complementary
  • Exons
  • Fibroblasts / enzymology
  • Humans
  • Menkes Kinky Hair Syndrome / enzymology
  • Menkes Kinky Hair Syndrome / genetics*
  • Models, Molecular
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Protein Biosynthesis
  • Protein Structure, Secondary
  • RNA, Messenger / biosynthesis
  • RNA, Messenger / genetics
  • Recombinant Fusion Proteins*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Sequence Alignment
  • Sequence Homology, Nucleic Acid
  • Transcription, Genetic*

Substances

  • Carrier Proteins
  • Cation Transport Proteins
  • DNA, Complementary
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Adenosine Triphosphatases
  • ATP7A protein, human
  • Copper-Transporting ATPases