Tissue specific expression of the retinoic acid receptor-beta 2: regulation by short open reading frames in the 5'-noncoding region.
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, approximately 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-beta 2 (RAR beta 2) 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 RAR beta 2-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 RAR beta 2-ORF, resulting in an altered regulation of RAR beta 2-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.
Unit on Developmental Biology, National Institute of Mental Health, Bethesda, Maryland 20892.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D003062:Codon; D004622:Embryo, Mammalian / Q000502:physiology; D005314:Embryonic and Fetal Development; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D016366:Open Reading Frames; D009928:Organ Specificity; D010442:Peptide Chain Initiation, Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D012315:RNA Caps / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D012270:Ribosomes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis
D003062:Codon; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D012315:RNA Caps; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; D011993:Recombinant Fusion Proteins; C082925:retinoic acid receptor beta; D001616:beta-Galactosidase