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J. Biol. Chem., Vol. 266, Issue 4, 2383-2389, Feb, 1991
JM Manzella, W Rychlik, RE Rhoads, JW Hershey and PJ Blackshear
We investigated the possibility that insulin could stimulate translation of
ornithine decarboxylase (ODC) mRNA in a murine fibroblast cell line that
expresses large numbers of human insulin receptors (HIR 3.5 cells). Within
3 h after exposure to 70 nM insulin, ODC enzyme activity increased
approximately 50-fold and mRNA accumulation 3-fold in the HIR 3.5 cells but
not in normal fibroblasts. Pretreatment of cells with cycloheximide
completely inhibited insulin- stimulated ODC expression; actinomycin D
partially inhibited this effect. To determine the influence of the 5'
untranslated region (5'UTR) of ODC mRNA on insulin-regulated ODC
expression, plasmids were constructed which contained sequences from the
5'UTR of a rat ODC mRNA interposed between the ferritin promoter and the
coding region of the human growth hormone gene. These constructions were
then expressed transiently in HIR 3.5 cells. Insulin stimulated a 2-4-fold
change in growth hormone accumulation in the medium of cells transiently
expressing plasmids containing the entire 5'UTR of ODC mRNA or just the
5'-most 115 bases, a G/C-rich conserved sequence predicted to form a
stem-loop structure and shown previously to be responsible for constitutive
inhibition of translation. There was a direct correlation between the
extent of insulin stimulation and the predicted secondary structure of the
added 5'UTR sequences. To determine whether this effect might be due to
insulin activation of initiation factors responsible for melting mRNA
secondary structure, we examined the effect of insulin on the
phosphorylation states of two such factors, eucaryotic initiation factors
eIF-4B and eIF-4E. Insulin stimulated the phosphorylation of both
initiation factors; this stimulation was evident at 15 min and maximal by
60 min. These results suggest a potential general mechanism by which
insulin could preferentially stimulate translation of mRNAs whose 5'UTRs
exhibit significant secondary structure by activating initiation factors
involved in melting such secondary structures.
Insulin induction of ornithine decarboxylase. Importance of mRNA secondary structure and phosphorylation of eucaryotic initiation factors eIF-4B and eIF-4E
Howard Hughes Medical Institute Laboratories, Duke University Medical Center, Durham, North Carolina 27710.
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