Browsing by Author "Onofre, Claudia"
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- Expression of human Hemojuvelin (HJV) is tightly regulated by two upstream open reading frames in HJV mRNA that respond to iron overload in hepatic cellsPublication . Onofre, Claudia; Tomé, Filipa; Barbosa, Cristina; Silva, Ana Luísa; Romão, LuísaThe 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 2α. 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.
- How mRNA translation is involved in modulating nonsense-mediated decay in transcripts with short open reading framesPublication . Onofre, Claudia; Menezes, Juliane; Peixeiro, Isabel; Costa, Nuno; Barbosa, Cristina; Romão, LuísaBeyond its well-known hematopoietic action, erythropoietin (EPO) has diverse cellular effects in non-hematopoietic tissues. For example, in cases of tissue injury, such as cardiac ischemia or acute myocardial infarct, EPO expression increases locally, providing a cardioprotective effect. Cellular stress activates an integrated stress response, which includes rapid changes in global and gene-specific translation. Translational regulation of specific transcripts mostly occurs at translation initiation and is mediated via different cis-acting elements, including upstream open reading frames (uORFs). The human EPO 5’ untranslated region (5’UTR) has one uORF with 14 codons that is conserved among different species, indicating its potential regulatory role. To test whether EPO expression is translationally regulated in response to ischemia in cardiac tissue, reporter constructs containing the normal or mutant EPO 5’UTR fused to the Firefly luciferase cistron were expressed in H9c2 (heart/myocardium myoblasts) and C2C12 (muscle myoblasts) cell lines. Luminometry assays revealed that the EPO uORF represses translation of the main ORF at about 60-70%, in both cell lines. Under chemical ischemia, EPO uORF-mediated translation repression is specifically released in muscle cells. In response to hypoxia, translational derepression occurs in both cell lines. Although the eIF2-alpha phosphorylation occurs in both conditions, thapsigargin treatment does not affect EPO translation. We are currently exploring additional mechanisms through which EPO cardioprotection effects are regulated at the translational level.
- Regulation of gene expression of human hemojuvelin by two small open reading frames and its relevance in the iron homeostasisPublication . Onofre, Claudia; Romão, Luísa; Crespo, AnaIron is an essential element for many biological reactions carried out by living systems. A tight regulation of the systemic iron homeostasis is crucial to avoid pathological conditions of iron deficiency or overload. Juvenile hemochromatosis, is an early-onset inherited disorder associated to an 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 a class of ligands called bone morphogenetic proteins (BMPs), which trigger a response to the iron increase by activating the hepcidin transcription. 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 (uORFs) 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 assays and normalized to the corresponding mRNA levels, quantified by real-time reverse transcription quantitative polymerase chain reaction (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, translation reinitiation is the main mechanism involved in the production of HJV protein. Aiming to further characterize the mechanism through which the HJV uORFs affect downstream translation, we have observed that the uORF2 encoded peptide seems to cause ribosomal stalling, which also prevents translation of the downstream main ORF. Together, these results produce a framework for understanding how human HJV gene expression is fine-tuned controlled during translation.
- The interplay between mRNA translation and nonsense-mediated decay in transcripts with short open reading framesPublication . Romão, Luísa; Peixeiro, Isabel; Onofre, Claudia; Barbosa, Cristina; Teixeira, AlexandreNMD is one of the better characterized quality control mechanisms which acts as an mRNA surveillance pathway by degrading transcripts harboring premature translation termination codons (PTCs). However, several studies have also implicated NMD in the regulation of steady-state levels of physiological mRNAs, and examples of natural NMD targets are transcripts containing upstream short open reading frames or with long 3’ untranslated regions. Indeed, the strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have reported that human mRNAs with a PTC in close proximity to the translation initiation codon (AUG-proximal PTC), and thus, with a short open reading frame, can substantially escape NMD. Our data support a model in which cytoplasmic poly(A)-binding protein 1 (PABPC1) is brought into close proximity with an AUG-proximal PTC via interactions with the translation initiation complexes. This proximity of PABPC1 to the AUG-proximal PTC allows PABPC1 to interact with eRF3 with a consequent enhancement of the release reaction and repression of the NMD response. Here, we provide strong evidence that the eIF3 is involved in delivering eIF4G-associated PABPC1 into the vicinity of the AUG-proximal PTC. In addition, we dissect the biochemical interactions of the eIF3 subunits in bridging PABPC1/eIF4G complex to the 40S ribosomal subunit. Together, our data provide a framework for understanding the mechanistic details of PTC definition and translation initiation.
- The mechanism through which nonsense mutations are recognized as premature translation-termination codonsPublication . Onofre, Claudia; Menezes, Juliane; Peixeiro, Isabel; Barbosa, Cristina; Romão, LuísaAbout one third of the gene mutations found in human genetic disorders, including cancer, result in premature termination codons (PTCs) and the rapid degradation of their mRNAs by nonsense-mediated decay (NMD). NMD controls the quality of eukaryotic gene expression. The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have reported that human mRNAs with a PTC in close proximity to the translation initiation codon (AUG-proximal PTC), and thus, with a short open reading frame, can substantially escape NMD. Our data support a model in which cytoplasmic poly(A)-binding protein 1 (PABPC1) is brought into close proximity with an AUG-proximal PTC via interactions with the translation initiation complexes. This proximity of PABPC1 to the AUG-proximal PTC allows PABPC1 to interact with eRF3 with a consequent enhancement of the release reaction and repression of the NMD response. Here, we provide strong evidence that the eIF3 is involved in delivering eIF4G-associated PABPC1 into the vicinity of the AUG-proximal PTC. In addition, we dissect the biochemical interactions of the eIF3 subunits in bridging PABPC1/eIF4G complex to the 40S ribosomal subunit. Together, our data provide a framework for understanding the mechanistic details of PTC definition and mRNA translation initiation.
- The mechanism through which translation-termination codons are recognized as prematurePublication . Onofre, Claudia; Menezes, Juliane; Peixeiro, Isabel; Barbosa, Cristina; Romão, LuísaAbout one third of the gene mutations found in human genetic disorders, including cancer, result in premature termination codons (PTCs) and the rapid degradation of their mRNAs by nonsense-mediated decay (NMD). NMD controls the quality of eukaryotic gene expression. The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have reported that human mRNAs with a PTC in close proximity to the translation initiation codon (AUG-proximal PTC), and thus, with a short open reading frame, can substantially escape NMD. Our data support a model in which cytoplasmic poly(A)-binding protein 1 (PABPC1) is brought into close proximity with an AUG-proximal PTC via interactions with the translation initiation complexes. This proximity of PABPC1 to the AUG-proximal PTC allows PABPC1 to interact with eRF3 with a consequent enhancement of the release reaction and repression of the NMD response. Here, we provide strong evidence that the eIF3 is involved in delivering eIF4G-associated PABPC1 into the vicinity of the AUG-proximal PTC. In addition, we dissect the biochemical interactions of the eIF3 subunits in bridging PABPC1/eIF4G complex to the 40S ribosomal subunit. Together, our data provide a framework for understanding the mechanistic details of PTC definition and mRNA translation initiation.
- The role of eIF3 subunits in the mechanism of nonsense-mediated mRNA decayPublication . Dias, Patrícia; Onofre, Claudia; Menezes, Juliane; Romão, LuísaPremature translation-termination codons (PTCs) or nonsense codons) can arise from mutations in germ or somatic cells. The introduction of a PTC into an mRNA can trigger nonsense-mediated decay (NMD), an important mRNA surveillance mechanism that typically recognizes and degrades mRNAs containing PTCs to prevent the synthesis of C-terminally truncated proteins potentially toxic for the cell. The physiological importance of NMD is manifested by the fact that about one third of genetic disease-associated mutations generate PTCs. The mammalian translation initiation factor 3 represents the most complex eukaryotic initiation factor (eIF) in mammalian cells. This factor comprises 13 subunits (eIF3a to eIF3m), each one playing an important role in translational control. Disruption of eIF3 initiation factor activity can lead not only to cancer but also neural physiological alterations, and to act as a mediator of infection cascade. Although some eIF3 subunits (for example, e and g) have been implicated in NMD, others were not studied yet. With the aim to identify other eIF3 subunits involved in NMD, we have depleted each one of the eIF3 subunits in HeLa cells and tested its effect in the expression of PTC-free or PTC- containing reporter human β-globin genes. Our data show that eIF3l and eIF3j subunits have an important role in targeting mRNAs for NMD. We will describe the molecular mechanisms underlying these observations.
- Translational control of the human erythropoietin expression via an upstream open reading frame in cardiac tissuePublication . Ferrão, José; Onofre, Claudia; Barbosa, Cristina; Romão, LuísaCellular stress activates an integrated stress response, which includes rapid changes in global and gene-specific translation. Translational regulation of specific transcripts mostly occurs at mRNA translation initiation and is mediated via different cis-acting elements present in the mRNA 5’ untranslated region (5’UTR), such as upstream open reading frames (uORFs). uORFs modulate translation of the main ORF by decreasing the number and/or efficiency of scanning ribosomes to reinitiate at the start codon of the main ORF. Human erythropoietin (EPO) is a glycoprotein synthesized and released mainly from the kidney, which has a key role in hematopoiesis. However, recent studies have revealed that EPO is a multifunctional molecule produced and utilized by many tissues that rapidly responds to different cell stress stimuli and tissue injuries. The 5’UTR sequence of the human EPO mRNA has one uORF with 14 codons, which is conserved among different species, indicating its potential role in translational regulation. To test whether EPO expression is translationally regulated in response to ischemia in cardiac tissue, reporter constructs containing the normal or mutant EPO 5’UTR fused to the Firefly luciferase cistron were expressed in H9c2 (heart myoblasts) and C2C12 (muscle myoblasts) cell lines. Luminometry assays revealed that the EPO uORF represses translation of the main ORF in both cell lines. Under chemical ischemia, EPO uORF-mediated translation repression is specifically released in muscle cells. In response to chemical hypoxia, translational derepression occurs in both cell lines. We are currently exploring additional mechanisms through which EPO cardioprotection effects are regulated at the translational level.
- Translational control of the human erythropoietin via an upstream open reading frame in cardiac and muscle tissuePublication . Onofre, Claudia; Barbosa, Cristina; Romão, LuísaBeyond its well-known hematopoietic action, erythropoietin (EPO) has diverse cellular effects in non-hematopoietic tissues. For example, in cases of tissue injury, such as cardiac ischemia or acute myocardial infarct, EPO expression increases locally, providing a cardioprotective effect. Cellular stress activates an integrated stress response, which includes rapid changes in global and gene-specific translation. Translational regulation of specific transcripts mostly occurs at translation initiation and is mediated via different cis-acting elements, including upstream open reading frames (uORFs). The human EPO 5’ untranslated region (5’UTR) has one uORF with 14 codons that is conserved among different species, indicating its potential regulatory role. To test whether EPO expression is translationally regulated in response to ischemia in cardiac tissue, reporter constructs containing the normal or mutant EPO 5’UTR fused to the Firefly luciferase cistron were expressed in H9c2 (heart/myocardium myoblasts) and C2C12 (muscle myoblasts) cell lines. Luminometry assays revealed that the EPO uORF represses translation of the main ORF at about 60-70%, in both cell lines. Under chemical ischemia, EPO uORF-mediated translation repression is specifically released in muscle cells. In response to hypoxia, translational derepression occurs in both cell lines. Although the eIF2-alpha phosphorylation occurs in both conditions, thapsigargin treatment does not affect EPO translation. We are currently exploring additional mechanisms through which EPO cardioprotection effects are regulated at the translational level.
- Translational control of the human erythropoietin via an upstream open reading frame in cardiac tissuePublication . Onofre, Claudia; Barbosa, Cristina; Romão, LuísaErythropoietin (EPO) is the main hormone that regulates erythropoiesis. Beyond its well-known hematopoietic action, EPO has diverse cellular effects in non-hematopoietic tissues, including cardioprotection. Indeed, in cases of tissue injury the EPO expression increases locally providing a cardioprotective effect supported by numerous experimental data in animal models of ischemia and acute myocardial infarct. Cellular stress activates an integrated stress response, which includes rapid changes in global and gene-specific translation. Translational regulation of specific transcripts mostly occurs at translation initiation and is mediated via different cis-acting elements present in the mRNA 5’ untranslated region (5’UTR); which includes the upstream open reading frames (uORFs). These uORFs modulate translation of the main ORF by decreasing the number and/or efficiency of scanning ribosomes to reinitiate at the start codon of the main ORF. However, in response to abnormal stimuli, they mediate translational derepression of stress-responsive proteins. The 5’ leader sequence of the human EPO mRNA has one uORF with 14 codons that is conserved among different species, indicating its potential regulatory role. In the present work, we aimed to test whether EPO expression is translationally regulated in response to ischemia in cardiac tissue. Reporter constructs containing the normal or mutant EPO 5’ leader sequence fused to the Firefly luciferase cistron were tested in H9C2 (rat heart/myocardium myoblasts) and C2C12 (mouse muscle myoblasts) cell lines. Luciferase activity was measured by luminometry assays and normalized to the corresponding mRNA levels quantified by real-time RT-PCR. Results have revealed that the EPO uORF represses translation of the main ORF at about 60-70%, in both cell lines. The results also show the synthesis of EPO protein mainly occurs by reinitiation after uORF translation demonstrating that this uORF suffers low leaky scanning. In addition, our results show that specifically in C2C12 cells, the EPO 3’-enhancer induces a 4-fold increase in EPO expression, while in H9C2 cells the uORF-mediated translational repression is not affected by the presence of the EPO 3’-enhancer. Nevertheless, in C2C12 cells under chemical ischemia, EPO uORF-mediated translation repression seems to be released. These findings show that cardioprotection effects of EPO might be regulated at the translational level.