Translational control of ornithine aminotransferase. Modulation by initiation factor eIF-4E.
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.
Fagan
R J
RJ
Departments of Pediatrics, McGill University-Montreal Children's Hospital Research Institute, Quebec, Canada.
Lazaris-Karatzas
A
A
Sonenberg
N
N
Rozen
R
R
D003513:Cycloheximide; D004492:Edetic Acid; D039561:Eukaryotic Initiation Factor-4E; D005091:Exons; D015971:Gene Expression Regulation, Enzymologic; D015345:Oligonucleotide Probes; D009953:Ornithine-Oxo-Acid Transaminase / Q000235:genetics* / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D012175:Retinoblastoma; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D039561:Eukaryotic Initiation Factor-4E; D015345:Oligonucleotide Probes; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D003513:Cycloheximide; D004492:Edetic Acid; D009953:Ornithine-Oxo-Acid Transaminase