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- Translational control of the human hemojuvelin via upstream open reading framesPublication . Onofre, Claudia; Barbosa, Cristina; Romão, LuísaIron is an essential element for many biological reactions carried out by living systems. A tight regulation of systemic iron homeostasis is crucial to avoid the pathological conditions of iron deficiency or overload. Juvenile hemochromatosis, is an early-onset inherited disorder associated to iron overload caused by mutations on the hepcidin gene or in the gene encoding hemojuvelin (HJV). HJV is a glycosylphosphatidylinositol (GPI)-linked membrane protein shown to be a co-receptor for class of ligands called bone morphogenetic proteins (BMPs). Thus, HJV is involved on iron homeostasis through regulation of hepcidin transcription levels. A better knowledge of the mechanisms implicated in HJV gene expression is crucial to understand its role in the iron homeostasis. The 5’ leader sequence of the human HJV mRNA has two upstream AUGs (uAUGs) that share the same codon stop forming two upstream open reading frames (uORF) with 28 and 19 codons. To evaluate the effect of these uORFs in the translational regulation of HJV, reporter constructs containing several HJV 5’-leader sequences fused to the Firefly luciferase cistron were tested in HeLa and HepG2 cells. Luciferase activity was measured by luminometry and normalized to the corresponding mRNA levels, quantified by real-time RT-PCR, to obtain translation efficiencies. The results revealed that the HJV uORFs decrease the translational efficiency of the main ORF in about 6-fold. Furthermore, we have observed that the HJV mRNA has a low leaky scanning ability that contributes to the translational repression of the main ORF. Thus, reinitiation is the mechanism mainly involved in the production of HJV protein. Aiming to characterize the mechanism through which the HJV uORFs affect downstream translation, we have observed that the amino acid sequences of the uORFs encoded peptides seem to cause ribosomal stalling, which also impede translation of the downstream main ORF.
- An upstream open reading frame regulates de translational efficiency of the human erythropoietin transcriptPublication . Barbosa, Cristina; Romão, LuísaAmong the various cis-acting elements present in the 5’ leader sequence of mRNAs there are upstream open reading frames (uORFs). Although their function is still poorly understood, they are known to downregulate the main ORF expression of several human transcripts that code for key regulatory genes. The human erythropoietin (EPO) is a glycoprotein that was initially characterized has a hormone mainly synthesized and released from the kidney, with a key role in hematopoiesis. However, many recent reports have implicated EPO in several non-hematopoietic functions and have shown its production in several other organs. Consequently, it might be used as a therapeutic target for the treatment of several human disorders. We found a natural occurring 14-codon-uORF on the human EPO transcript. Our belief is that understanding the molecular mechanisms through which the EPO uORF controls translation may be valuable in the determination of these EPO-based therapies. To explore the mechanisms by which EPO uORF controls translational efficiency, HepG2, HEK293 and REPC cells were transfected with several constructs carrying the luciferase reporter gene with the intact or disrupted EPO uORF, with or without the EPO 3’ untranslated region (3’UTR). Luciferase activity was measured by luminometry and normalized to the corresponding mRNA levels to obtain translation efficiencies. The mRNA levels were quantified by real-time RT-PCR. Furthermore, we also analyzed its response to several cell stress stimuli. Results show that the EPO uORF can decrease the main ORF translation efficiency in about 3-fold. In addition, our data support the conclusion that reinitiation, and in less extent leaky scanning, are responsible for the main ORF translation. In addition, the 3’UTR does not affect the role of the uORF, but it increases the luciferase levels, probably by stabilizing the mRNA. Specifically in REPC cells, translational inhibition mediated by the EPO uORF is overridden in response to chemical hypoxia, which is due to less recognition of the uAUG.