Browsing by Author "Barbosa, Cristina"
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- Alternative polyadenylation and nonsense-mediated decay coordinately regulate the human HFE mRNA levelsPublication . Martins, Rute; Proença, Daniela; Silva, Bruno; Barbosa, Cristina; Silva, Ana Luísa; Faustino, Paula; Romão, LuísaNonsense-mediated decay (NMD) is an mRNA surveillance pathway that selectively recognizes and degrades defective mRNAs carrying premature translation-termination codons. However, several studies have shown that NMD also targets physiological transcripts that encode full-length proteins, modulating their expression. Indeed, some features of physiological mRNAs can render them NMD-sensitive. Human HFE is a MHC class I protein mainly expressed in the liver that, when mutated, can cause hereditary hemochromatosis, a common genetic disorder of iron metabolism. The HFE gene structure comprises seven exons; although the sixth exon is 1056 base pairs (bp) long, only the first 41 bp encode for amino acids. Thus, the remaining downstream 1015 bp sequence corresponds to the HFE 39 untranslated region (UTR), along with exon seven. Therefore, this 39 UTR encompasses an exon/exon junction, a feature that can make the corresponding physiological transcript NMD-sensitive. Here, we demonstrate that in UPF1-depleted or in cycloheximide-treated HeLa and HepG2 cells the HFE transcripts are clearly upregulated, meaning that the physiological HFE mRNA is in fact an NMD-target. This role of NMD in controlling the HFE expression levels was further confirmed in HeLa cells transiently expressing the HFE human gene. Besides, we show, by 39-RACE analysis in several human tissues that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites – two were previously described and two are novel polyadenylation sites: one located at exon six, which confers NMD-resistance to the corresponding transcripts, and another located at exon seven. In addition, we show that the amount of HFE mRNA isoforms resulting from cleavage and polyadenylation at exon seven, although present in both cell lines, is higher in HepG2 cells. These results reveal that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its protein expression according to the physiological cellular requirements.
- 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.
- An upstream open reading frame regulates the human erythropoietin gene expressionPublication . Barbosa, Cristina; Romão, Luísa
- 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.
- Gene expression regulation by upstream open reading frames and human diseasePublication . Barbosa, Cristina; Peixeiro, Isabel; Romão, LuísaUpstream open reading frames (uORFs) are major gene expression regulatory elements. In many eukaryotic mRNAs, one or more uORFs precede the initiation codon of the main coding region. Indeed, several studies have revealed that almost half of human transcripts present uORFs. Very interesting examples have shown that these uORFs can impact gene expression of the downstream main ORF by triggering mRNA decay or by regulating translation. Also, evidence from recent genetic and bioinformatic studies implicates disturbed uORF-mediated translational control in the etiology of many human diseases, including malignancies, metabolic or neurologic disorders, and inherited syndromes. In this review, we will briefly present the mechanisms through which uORFs regulate gene expression and how they can impact on the organism's response to different cell stress conditions. Then, we will emphasize the importance of these structures by illustrating, with specific examples, how disturbed uORF-mediated translational control can be involved in the etiology of human diseases, giving special importance to genotype-phenotype correlations. Identifying and studying more cases of uORF-altering mutations will help us to understand and establish genotype-phenotype associations, leading to advancements in diagnosis, prognosis, and treatment of many human disorders.
- How mRNA translation is involved in modulating nonsense-mediated decay in transcripts with short open reading framesPublication . Onofre, Cláudia; Menezes, Juliane; Peixeiro, Isabel; Costa, Nuno; Barbosa, Cristina; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs). In addition, 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 long 3’ untranslated regions. 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.
- 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.
- How the interaction of PABPC1 with the translation initiation complex inhibits nonsense-mediated decay of transcripts with an AUG-proximal nonsense codonPublication . Peixeiro, Isabel; Teixeira, Alexandre; Barbosa, Cristina; Romão, Luísa
- Interaction of PABPC1 with the translation initiation complex is critical to the NMD resistance of AUG-proximal nonsense mutations.Publication . Peixeiro, Isabel; Inácio, Ângela; Barbosa, Cristina; Silva, Ana Luísa; Liebhaber, Stephen; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTC). The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have previously reported that mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. Here, we explore the mechanistic basis for this NMD resistance. We demonstrate that translation termination at an AUG-proximal PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. This difference is associated with demonstrated interactions of the cytoplasmic poly(A)-binding protein 1, PABPC1, with the cap-binding complex subunit, eIF4G and the 40S recruitment factor eIF3 as well as the ribosome release factor, eRF3. These interactions, in combination, underlie critical 30–50 linkage of translation initiation with efficient termination at the AUGproximal PTC and contribute to an NMD-resistant PTC definition at an early phase of translation elongation.
- Nonsense-mediated decay resistance of AUG-proximal nonsense-mutated transcripts relies on the interaction of PABPC1 with the translation initiation complexPublication . Peixeiro, Isabel; Barbosa, Cristina; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing a premature termination codon (PTC). The unified model for NMD proposes that the decision of NMD triggering is the outcome of the competition between the cytoplasmatic poly(A)-binding protein 1 (PABPC1) and the NMD effector UPF1 for the termination complex. Consequently, PTCs located far, in a linear sense, from the poly(A) tail and associated PABPC1, in mRNAs containing downstream exon junction complexes (EJCs), are expected to elicit NMD. Nevertheless, we have reported that human b-globin mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. We have reported that translation termination at an AUG-proximal PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. In fact, we have shown that the establishment of an efficient translation termination reaction at the AUG-proximal PTC is dependent on PABPC1 interaction with the initiation factor eIF4G and with the release factor eRF3 at the terminating ribosome. These interactions underlie critical 3’-5’ linkage of translation initiation with efficient termination at the AUG-proximal PTC and contribute to an NMD-resistant PTC definition at an early phase of translation elongation. Furthermore, we provide strong evidence that the eIF3 is involved in delivering eIF4G-associated PABPC1 into the vicinity of the AUG-proximal PTC. This work corroborates a role for PABPC1 on NMD evasion of transcripts carrying an AUG-proximal PTC and provides further insights into the mechanistic details of PTC definition and translation initiation.