Tissue specific expression of the retinoic acid receptor-β2: Regulation by short open reading frames in the 5′-noncoding region

Abstract

The 40-S subunit of eukaryotic ribosomes binds to the capped 5′-end of mRNA and scans for the first AUG in a favorable sequence context to initiate translation. Most eukaryotic mRNAs therefore have a short 5′-untranslated region (5′-UTR) and no AUGs upstream of the translational start site; features that seem to assure efficient translation. However, ∼5-10% of all eukaryotic mRNAs, particularly those encoding for regulatory proteins, have complex leader sequences that seem to compromise translational initiation. The retinoic-acid-receptor-β2 (RARβ2) mRNA is such a transcript with a long (461 nucleotides) 5′-UTR that contains five, partially overlapping, upstream open reading frames (uORFs) that precede the major ORF. We have begun to investigate the function of this complex 5′-UTR in transgenic mice, by introducing mutations in the start/stop codons of the uORFs in RARβ2-lacZ reporter constructs. When we compared the expression patterns of mutant and wild-type constructs we found that these mutations affected expression of the downstream RARβ2-ORF, resulting in an altered regulation of RARβ2-lacZ expression in heart and brain. Other tissues were unaffected. RNA analysis of adult tissues demonstrated that the uORFs act at the level of translation; adult brains and hearts of transgenic mice carrying a construct with either the wild-type or a mutant UTR, had the same levels of mRNA, but only the mutant produced protein. Our study outlines an unexpected role for uORFs: control of tissue-specific and developmentally regulated gene expression.