ABSTRACT
Ornithine-delta-aminotransferase (OAT) catalyzes the reversible transamination of ornithine to glutamate semialdehyde. OAT is abundant in liver, kidney and retina; hereditary deficiency of the enzyme leads to chorioretinal degeneration. Studies of OAT regulation in retinoblastomas have revealed an alternatively spliced OAT mRNA, which contains an additional exon (exon 2) in the 5' untranslated region. Estrogen and thyroid hormone were previously shown to increase OAT mRNA levels approximately 3-fold and 5-fold, respectively, in these cells. To determine the mechanism of hormonal action in retinoblastomas, we performed nuclear transcription assays and analyzed the distribution of OAT mRNAs in individual fractions of a polysome gradient. Thyroid hormone increased the rate of transcription of the OAT mRNA in these cells. Estrogen did not stimulate transcription; it was associated with increased translation, since it resulted in a shift of the major (spliced) OAT mRNA species into denser fractions of the polysome gradient. Cycloheximide treatment suggested that the latter effect was due to increased initiation of translation. The unspliced OAT mRNA, which is inefficiently compared to the spliced mRNA, was insensitive to estrogen in these experiments.
Subject(s)
Estradiol/pharmacology , Ornithine-Oxo-Acid Transaminase/biosynthesis , Protein Biosynthesis/drug effects , Alternative Splicing , Cycloheximide/pharmacology , Gene Expression Regulation, Enzymologic , Humans , Ornithine-Oxo-Acid Transaminase/genetics , Polymerase Chain Reaction , Polyribosomes/metabolism , RNA, Messenger/biosynthesis , Retinoblastoma/metabolism , Transcription, Genetic/drug effects , Triiodothyronine/pharmacology , Tumor Cells, CulturedABSTRACT
Ornithine aminotransferase (OAT) is a mitochondrial enzyme expressed at high levels in liver, kidney, and retina. To characterize OAT regulation in retinal lines, we have been studying OAT synthesis in retinoblastomas RB355 and Y79. Our previous data (Fagan, R.J., Sheffield, W.P., and Rozen, R. (1989) J. Biol. Chem. 264, 20513-20517) indicated similar OAT mRNA levels in the two strains with 3-fold greater immunoreactive OAT protein and enzyme activity in Y79. To examine the regulatory mechanisms in these cell lines, we performed nuclear runoff experiments and characterized polysome-associated OAT mRNAs. The nuclear runoff data did not reveal any differences in transcription between the two strains. However, OAT mRNA of the RB355 strain was present in the lighter polysome fractions as compared with Y79. Treatment with cycloheximide, which slows the rate of elongation, indicated that initiation was decreased in RB355. Eukaryotic initiation factor eIF-4E mRNA and protein were reduced in RB355, suggesting that eIF-4E might be rate-limiting for OAT translation. Overexpression of a wild-type eIF-4E in RB355 shifted the OAT mRNA into denser fractions of the gradient and increased the amount of OAT protein to the level observed in Y79; overexpression of a mutant eIF-4E had no such effect. We previously identified an alternatively spliced OAT mRNA (containing exon 2) in these cells. This mRNA appeared in the lightest fractions of the gradient in both strains and was not affected by eIF-4E overexpression.
Subject(s)
Gene Expression Regulation, Enzymologic , Ornithine-Oxo-Acid Transaminase/genetics , Peptide Initiation Factors/metabolism , Protein Biosynthesis , Cycloheximide , Edetic Acid , Eukaryotic Initiation Factor-4E , Exons , Oligonucleotide Probes , Ornithine-Oxo-Acid Transaminase/metabolism , Polymerase Chain Reaction , Polyribosomes/metabolism , RNA Splicing , RNA, Messenger/metabolism , Retinoblastoma , Transcription, Genetic , Tumor Cells, CulturedABSTRACT
Ornithine-delta-aminotransferase (OAT) is a nuclear-encoded, mitochondrial enzyme that converts ornithine to glutamate semialdehyde. Although OAT is expressed in most tissues of the rat, liver, kidney, and retina have the highest levels of OAT activity. Studies of OAT regulation in liver and kidney have indicated transcriptional and translational controls for the enzyme in a tissue-specific manner. Little is known about OAT modulation in retinal tissue, although chorio-retinal degeneration is the predominant feature in a hereditary disorder of OAT deficiency, gyrate atrophy. To characterize OAT regulation in retinal lines, we studied its synthesis in two retinoblastoma strains, Y79 and RB355. Baseline OAT mRNA levels were similar in the two cell lines, yet Y79 expressed 3-fold more immunoreactive OAT protein and enzyme activity than RB355; this finding suggested the presence of a post-transcriptional mechanism for the regulation of steady-state OAT levels. Treatment of the two strains with estradiol or thyroid hormone for 24 h resulted in approximately 5-fold increases in OAT protein and activity. Since similar increases in OAT mRNA levels were observed in both strains after Northern blotting, it is likely that these hormones exert their effects at the transcriptional level. Finally, primer extension analysis revealed two OAT mRNA species in both strains, due to the presence of an additional exon (exon 2) in one of the transcripts. The absence of this exon in other tissues reflects the unique mechanisms which govern OAT in retinoblastomas.
