Abstract
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.
Highlights
Drial enzyme expressed at high levels in liver, kidney, Rat liver, kidney, andretina express high levels of the and retina
OATmRNA of the RB355 strain was present in the lation initiates at exon 3
Ornithine aminotransferase (OAT) mRNA levels are similar in thetwo and protein were reduced in RB355, suggesting that strains, Y79 contains 3-fold greater levels of immunoreactive eIF-4E might be rate-limiting for OAT translation
Summary
264,20513-20517) indicated similar OAT mRNA levels in the two strains with %fold greater immunoreactive OAT protein and enzyme activity in. 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 enzyme in vivo [1].OAT in rat is regulated in atissue-specific manner. Estrogen and thyroid hormone increase OAT mRNA and enzyme activity [6]. OAT mRNA levels are similar in thetwo and protein were reduced in RB355, suggesting that strains, Y79 contains 3-fold greater levels of immunoreactive eIF-4E might be rate-limiting for OAT translation. OAT protein and enzyme activity [6].In thiscommunication, Overexpression of awild-typeIF-4Ein. RB355 shifted the OAT mRNA into denser fractions of the gradient and increased the amount of OAT protein to the level observed in Y79; overexpressionof a mutant eIF-4E had nosuch effect. We show that theexon 2-containing transcript is translated less efficiently than theprimary transcript and that its translationis not affected by the overexpression of eIF-4E
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