Inhibition of CCAAT/enhancer-binding protein α and β translation by upstream open reading frames

CCAAT/enhancer-binding protein (C/EBP) alpha is a bZIP transcription factor whose expression is restricted to specific cell types. Analysis of C/EBP alpha mRNA and protein levels in various mammalian cells indicates that expression of this gene is controlled both transcriptionally and post-transcriptionally. We report here that C/EBP alpha translation is repressed in several cell lines by an evolutionarily conserved upstream open reading frame (uORF), which acts in cis to inhibit C/EBP alpha translation. Mutations that disrupt the uORF completely abolished translational repression of C/EBP alpha. The related c/ebp beta gene also contains an uORF that suppresses translation. The length of the spacer sequence between the uORF terminator and the ORF initiator codon (7 bases in all c/ebp alpha genes and 4 bases in c/ebp beta homologs) is precisely conserved. The effects of insertions, deletions, and base substitutions in the C/EBP alpha spacer showed that both the length and nucleotide sequence of the spacer are important for efficient translational repression. Our data indicate that the uORFs regulate translation of full-length C/EBP alpha and C/EBP beta and do not play a role in generating truncated forms of these proteins, as has been suggested by start site multiplicity models.