The gene encoding human hemojuvelin (HJV) is one of the genes that, when mutated, can cause juvenile hemochromatosis, an early-onset inherited disorder associated with iron overload. The 5= untranslated region of the human HJV mRNA has two upstream open reading frames (uORFs), with 28 and 19 codons formed by two upstream AUGs (uAUGs) sharing the same in-frame stop codon. Here we show that these uORFs decrease the translational efficiency of the downstream main ORF in HeLa and HepG2 cells. Indeed, ribosomal access to the main AUG is conditioned by the strong uAUG context, which results in the first uORF being translated most frequently. The reach of the main ORF is then achieved by ribosomes that resume scanning after uORF translation. Furthermore, the amino acid sequences of the uORF-encoded peptides also reinforce the translational repression of the main ORF. Interestingly, when iron levels increase, translational repression is relieved specifically in hepatic cells. The upregulation of protein levels occurs along with phosphorylation of the eukaryotic initiation factor 2␣. Nevertheless, our results support a model in which the increasing recognition of the main AUG is mediated by a tissue-specific factor that promotes uORF bypass. These results support a tight HJV translational regulation involved in iron homeostasis.

In many eukaryotic messenger ribonucleic acids (mRNAs) one or more short upstream open reading frames (uORFs) precede the initiation codon of the main coding region. For example, in human cells, uORFs are present in about half of the transcripts. Emerging ribosome profiling and peptidomics analyses have recently shown that these uORFs are translated into polypeptides that seem to serve important biological functions. In addition, very interesting examples have shown that these uORFs are cis ‐acting RNA elements that can impact gene expression by repressing translation of the downstream main ORF under control conditions and derepressing it under certain pathophysiological stresses. Furthermore, evidence from genetic and bioinformatic studies implicate disturbed uORF‐mediated translational control in the aetiology of human diseases. Identifying more cases and understanding the aberrant mechanisms of uORF‐mediated translational control, as well as discovering the biological function of the uORF‐encoded polypeptides, is fundamental to advance in diagnosis, prognosis and treatment of many human disorders. Key Concepts: Upstream open reading frames (uORFs) are cis ‐acting RNA elements involved in translational regulation, which precede the initiation codon of the main coding region. For a uORF to function as a translational regulatory element, its initiation codon must be recognised, at least at certain times, by the scanning 40S ribosomal subunit and associated translation initiation factors. uORFs can impact gene expression by repressing translation of the downstream main ORF under control conditions, and derepressing it under certain pathophysiological stresses. The impact the uORFs can have on translation depends on variables, such as (1) the distance between the 5′ cap and the uORF, (2) the context in which the uORF AUG (or non‐AUG) is located, (3) the length of the uORF, (4) the sequence and secondary structure of the uORF, (5) the number of uORFs per transcript, (6) the position of the uORF termination codon and (7) the length of the intercistronic sequence(s). uORF‐encoded polypeptides might serve functional roles in cells. Polymorphisms or mutations that introduce/eliminate uORFs or modify the uORF‐encoded peptide can cause human disease. Understanding the mechanisms through which the uORFs regulate gene expression may lead to innovation in diagnosis, prognosis and treatment of many human disorders.