Browsing by Author "Peixeiro, Isabel"
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- Control of human beta-globin mRNA stability and its impact on beta-thalassemia phenotypePublication . Peixeiro, Isabel; Silva, Ana Luísa; Romão, LuísaMessenger RNA (mRNA) stability is a critical determinant that affects gene expression. Many pathways have evolved to modulate mRNA stability in response to developmental, physiological and/or environmental stimuli. Eukaryotic mRNAs have a considerable range of half-lives, from as short as a few minutes to as long as several days. Human globin mRNAs constitute an example of highly stable mRNAs. However, a wide variety of naturally occurring mutations that result in the clinical syndrome of thalassemia can trigger accelerated mRNA decay thus controlling mRNA quality prior to translation. Distinct surveillance mechanisms have been described as being targeted for specific defective globin mRNAs. Here, we review mRNA stability mechanisms implicated in the control of beta-globin gene expression and the surveillance pathways that prevent translation of aberrant beta-globin mRNAs. In addition, we emphasize the importance of these pathways in modulating the severity of the beta-thalassemia phenotype.
- 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.
- Nonsense-mediated decay resistance of AUG-proximal nonsense-mutated transcripts relies on the link between translation initiation and premature stop codon definitionPublication . Peixeiro, Isabel; Barbosa, Cristina; Silva, Ana Luísa; Romão, LuísaThe nonsense-mediated decay (NMD) is a surveillance mechanism that recognizes and rapidly degrades transcripts bearing a premature termination codon (PTC). Although in mammals the location of a PTC more than 50 nucleotides upstream the terminal exonexon junction has been pointed as a mark for NMD, it is now known that the physical distance between the PTC and cytoplasmic poly(A)-binding protein 1 (PABPC1) is a crucial determinant in PTC definition. We have previously reported that mRNAs carrying an AUG-proximal PTC evade NMD although apparently fulfilling the defined criteria for PTC definition. This unexpected NMD-evasion reflects an AUG-proximity effect. To test the hypothesis that the AUG-proximity effect on NMD resistance could be the result of a positional effect of PABPC1 relative to the PTC due to the inherent nature of the process of short ORF translation process, the PABPC1/eRF3 interaction was inhibited by RNA interference. Under conditions of overexpression of a PABPC1 mutant lacking the eRF3-interaction domain (PABPC1DelC), levels of beta15 transcripts were quantified by RT-qPCR and showed to be significantly decreased. The reciprocal experiment, i.e. conditions of overexpression of an eRF3 mutant lacking the PABPC1-interaction domain (eRF3DelN) was also performed. Results showed that the NMD-resistance of an AUG-proximal nonsense-mutated mRNA can be converted to NMD-sensitive when PABPC1/eRF3 interaction is impaired. Moreover, inhibition of PABPC1-eIF4G interaction by overexpression of PAIP2 protein, as well as the knockdown of the initiation factor eIF3h and eIF3f subunit, targets these transcripts for NMD. Our findings support a role for PABPC1 and associated initiation factors during translation in NMD-resistance of AUG-proximal nonsense-mutated transcripts, providing evidence for a link between translation initiation and PTC definition.
- The interaction between mRNA translation and nonsense-mediated decay in AUG-proximal nonsense-mutated transcriptsPublication . Onofre, Cláudia; Menezes, Juliane; Peixeiro, Isabel; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTCs). Although NMD has been intensively studied, it is still poorly understood how NMD discriminates between PTCs and normal stop codons. The unified model for NMD proposes that the decision of NMD triggering is the outcome of the competition between the cytoplasmic 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 residual downstream exon junction complexes (EJCs), are expected to elicit NMD. Nevertheless, we have reported that human mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD through a mechanism independent of translation re-initiation. Here, we will present the mechanistic basis for this NMD resistance and how it involves the step of mRNA translation initiation.
- The interaction between mRNA translation and nonsense-mediated decay in AUG-proximal nonsense-mutated transcriptsPublication . Onofre, Cláudia; Menezes, Juliane; Peixeiro, Isabel; Barbosa, Cristina; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTCs). Although NMD has been intensively studied, it is still poorly understood how NMD discriminates between PTCs and normal stop codons. The unified model for NMD proposes that the decision of NMD triggering is the outcome of the competition between the cytoplasmic 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 residual downstream exon junction complexes (EJCs), are expected to elicit NMD. Nevertheless, we have reported that human mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD through a mechanism independent of translation re-initiation. Here, we will present the mechanistic basis for this NMD resistance and how it involves the step of mRNA translation initiation.