Production of rat soluble and membrane-bound catechol O-methyltransferase forms from bifunctional mRNAs.
In the rat, the catechol O-methyltransferase (COMT) gene has been found to contain two promoters, P1 and P2. This organization enables the gene to produce a soluble (S-COMT) and a membrane-associated (MB-COMT) protein by using two in-frame ATG initiation codons (S- and MB-ATG). The P1 promoter expresses a 1.6 kb transcript (S-mRNA) which codes for the S-COMT polypeptide only. Here we demonstrate that the P2 promoter controls the expression of alternatively spliced 1.9 kb transcripts (MB-mRNA) which differ by a 27-nucleotide region immediately upstream of the MB-AUG codon. The presence of the 27-base sequence alters the nucleotide at position -3 from G to C, thereby changing the translation initiation context of the MB-AUG codon. Expression experiments in COS-7 cells using full-length COMT cDNAs showed that this alteration affected the initiation of the translation of the MB-AUG and consequently changed the relative amounts of MB- and S-COMT polypeptides produced. No proteolytic cleavage of the MB-COMT form to S-COMT was detected in in vitro or in vivo pulse-chase experiments. We conclude that the bifunctional 1.9 kb mRNAs are able to produce both S-COMT and MB-COMT polypeptide by the leaky scanning mechanism of translation initiation.
Tenhunen
J
J
Orion-Farmos Pharmaceuticals, Research Center, Helsinki, Finland.
Ulmanen
I
I
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D002394:Catechol O-Methyltransferase / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D004247:DNA; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D010455:Peptides / Q000378:metabolism; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats
D010455:Peptides; D012333:RNA, Messenger; D004247:DNA; D002394:Catechol O-Methyltransferase