Insulin induction of ornithine decarboxylase. Importance of mRNA secondary structure and phosphorylation of eucaryotic initiation factors eIF-4B and eIF-4E.
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.
Manzella
J M
JM
Howard Hughes Medical Institute Laboratories, Duke University Medical Center, Durham, North Carolina 27710.
Rychlik
W
W
Rhoads
R E
RE
Hershey
J W
JW
Blackshear
P J
PJ
D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction; D039561:Eukaryotic Initiation Factor-4E; D039642:Eukaryotic Initiation Factors; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D009690:Nucleic Acid Conformation; D009955:Ornithine Decarboxylase / Q000096:biosynthesis* / Q000235:genetics; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010766:Phosphorylation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics; D011972:Receptor, Insulin / Q000378:metabolism
D039561:Eukaryotic Initiation Factor-4E; D039642:Eukaryotic Initiation Factors; D007328:Insulin; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; C031799:eIF-4B; D003609:Dactinomycin; D003513:Cycloheximide; D011972:Receptor, Insulin; D009955:Ornithine Decarboxylase