Browsing by Author "Teixeira, Alexandre"
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- Analysis of human Argonaute 1 5’ untranslated region shows internal ribosome entry site activityPublication . Lacerda, Rafaela; Teixeira, Alexandre; Marques-Ramos, Ana; Romão, Luísa
- Analysis of Pelagia noctiluca proteome Reveals a Red Fluorescent Protein, a Zinc Metalloproteinase and a PeroxiredoxinPublication . Frazão, Bárbara; Campos, Alexandre; Osório, Hugo; Thomas, Benjamin; Leandro, Sérgio; Teixeira, Alexandre; Vasconcelos, Vitor; Antunes, AgostinhoPelagia noctiluca is the most venomous jellyfish in the Mediterranean Sea where it forms dense blooms. Although there is several published research on this species, until now none of the works has been focused on a complete protein profile of the all body constituents of this organism. Here, we have performed a detailed proteomics characterization of the major protein components expressed by P. noctiluca. With that aim, we have considered the study of jellyfish proteins involved in defense, body constituents and metabolism, and furthered explore the significance and potential application of such bioactive molecules. P. noctiluca body proteins were separated by1D SDS-PAGE and 2DE followed by characterization by nanoLC-MS/MS and MALDI-TOF/TOF techniques. Altogether, both methods revealed 68 different proteins, including a Zinc Metalloproteinase, a Red Fluorescent Protein (RFP) and a Peroxiredoxin. These three proteins were identified for the first time in P. noctiluca. Zinc Metalloproteinase was previously reported in the venom of other jellyfish species. Besides the proteins described above, the other 65 proteins found in P. noctiluca body content were identified and associated with its clinical significance. Among all the proteins identified in this work we highlight: Zinc metalloproteinase, which has a ShK toxin domain and therefore should be implicated in the sting toxicity of P. noctiluca.; the RFP which are a very important family of proteins due to its possible application as molecular markers; and last but not least the discovery of a Peroxiredoxin in this organism makes it a new natural resource of antioxidant and anti-UV radiation agents.
- Analysis of the 5’ untranslated region of human UPF1 mRNA indicates both cryptic promoter and internal ribosome entry site activityPublication . Lacerda, Rafaela; Marques-Ramos, Ana; Teixeira, Alexandre; Romão, LuísaApart from its role in nonsense-mediated mRNA decay, a mechanism that promotes rapid degradation of transcripts carrying premature translation termination codons, the human up-frameshift 1 (UPF1) DNA and RNA helicase protein plays a crucial role in telomere replication and homeostasis, and in cell cycle progression. Due to its relevance for several physiological roles, and to the fact that it is expressed during G2/M phase, in which overall protein synthesis is reduced, we hypothesized that its translation may occur via an internal ribosome entry site (IRES). IRESs can occur at the 5’ untranslated region (UTR) of transcripts and allow the direct recruitment of the ribosome to the vicinity of the main AUG, therefore bypassing the need of scanning the entire UTR. To test this hypothesis, we cloned the human UPF1 5’UTR in the dicistronic vector p_Renilla_Firefly and transfected HeLa cells with either this construct or the control counterparts. We observed a 15- to 25-fold increase in relative luciferase activity of the UPF1 5’UTR-containing construct compared to the levels obtained from the empty counterpart, which suggests the presence of an IRES. However, these levels of luciferase activity could be due to the presence of a cryptic promoter. Hence, we transfected cells with promoterless plasmids and observed a 20-fold increase in relative luciferase activity levels. These data demonstrate that UPF1 5’UTR contains a cryptic promoter, whose activity may be masking IRES activity. To check the IRES activity alone, we have transfected cells with in vitro transcribed, capped and polyadenylated mRNAs and observed a 2-fold increase in protein levels. This is also observed in two other cell lines. Besides, UPF1 IRES activity is maintained under conditions of global protein synthesis inhibition. Deletional analysis of UPF1 5’UTR revealed that the first 50 nucleotides at the 5’ end of this region are essential for both cryptic promoter and IRES activity. These results evidence, for the first time, the existence of both a cryptic promoter and an IRES element within UPF1 5’UTR and provide new insights on the regulation of UPF1 expression in human cells.
- Assessing the involvement of Dis3L1 in mammalian quality control pathwaysPublication . Reis, Filipa Pereira; Teixeira, Alexandre; Cruz, David; Morgado, Ana; Arraiano, Cecília Maria; Romão, LuísaThe exosome is an evolutionarily conserved protein complex that is involved in all aspects of RNA metabolism, namely, RNA decay, processing and quality control. The only catalytic subunit of the core exosome is a 3´end exoribonuclease from the RNase II family of enzymes. In humans, two different homologues of this protein were identified, Dis3 and Dis3L1. While Dis3 mainly localizes in the nucleoplasm and has endonucleolytic activity, Dis3L1 is strictly cytoplasmic and has no endonucleolytic activity. The rapid decay of aberrant transcripts is not completely understood, but it is known that involves both 5’ to 3’ and 3’ to 5’ degradation. Despite that it localizes in the same compartment where NMD generally occurs, nothing is known about the role of Dis3L1 in quality control processes. In this work, we assessed the involvement of Dis3L1 in the 3’ to 5’ degradation of reporter human b-globin transcripts with premature termination and nonstop codons.
- Cap-independent translation ensures mTOR expression and function upon protein synthesis inhibitionPublication . Marques-Ramos, Ana; Romão, Luísa; Candeias, Marco; Menezes, Juliane; Lacerda, Rafaela; Willcocks, M.; Teixeira, Alexandre; Locker, NicolasThe mechanistic/mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that integrates cellular signals from the nutrient and energy status to act, namely, on the protein synthesis machinery. While major advances have emerged regarding the regulators and effects of the mTOR signaling pathway, little is known about the regulation of mTOR gene expression. Here, we show that the human mTOR transcript can be translated in a cap-independent manner, and that its 5' untranslated region (UTR) is a highly folded RNA scaffold capable of binding directly to the 40S ribosomal subunit. We further demonstrate that mTOR is able to bypass the cap requirement for translation both in normal and hypoxic conditions. Moreover, our data reveal that the cap-independent translation of mTOR is necessary for its ability to induce cell-cycle progression into S phase. These results suggest a novel regulatory mechanism for mTOR gene expression that integrates the global protein synthesis changes induced by translational inhibitory conditions.
- Cap-independent translation regulation of mammalian target of rapamycin (mTOR)Publication . Marques-Ramos, Ana; Teixeira, Alexandre; Lacerda, Rafaela; Romão, Luísa
- A Comparative Overview of the Role of Human Ribonucleases in Nonsense-Mediated mRNA DecayPublication . da Costa, Paulo J.; Menezes, Juliane; Guedes, Raquel; Reis, Filipa P.; Teixeira, Alexandre; Saramago, Margarida; Viegas, Sandra C.; Arraiano, Cecília M.; Romão, LuísaEukaryotic cells possess surveillance mechanisms that detect and degrade defective transcripts. Aberrant transcripts include mRNAs with a premature termination codon (PTC), targeted by the nonsense-mediated decay (NMD) pathway, and mRNAs lacking a termination codon, targeted by the nonstop decay (NSD) pathway. The eukaryotic exosome, a ribonucleolytic complex, plays a crucial role in mRNA processing and turnover through its catalytic subunits PM/Scl100 (Rrp6 in yeast), DIS3 (Rrp44 in yeast), and DIS3L1. Additionally, eukaryotic cells have other ribonucleases, such as SMG6 and XRN1, that participate in RNA surveillance. However, the specific pathways through which ribonucleases recognize and degrade mRNAs remain elusive. In this study, we characterized the involvement of human ribonucleases, both nuclear and cytoplasmic, in the mRNA surveillance mechanisms of NMD and NSD. We performed knockdowns of SMG6, PM/Scl100, XRN1, DIS3, and DIS3L1, analyzing the resulting changes in mRNA levels of selected natural NMD targets by RT-qPCR. Additionally, we examined the levels of different human β-globin variants under the same conditions: wild-type, NMD-resistant, NMD-sensitive, and NSD-sensitive. Our results demonstrate that all the studied ribonucleases are involved in the decay of certain endogenous NMD targets. Furthermore, we observed that the ribonucleases SMG6 and DIS3 contribute to the degradation of all β-globin variants, with an exception for βNS in the former case. This is also the case for PM/Scl100, which affects all β-globin variants except the NMD-sensitive variants. In contrast, DIS3L1 and XRN1 show specificity for β-globin WT and NMD-resistant variants. These findings suggest that eukaryotic ribonucleases are target-specific rather than pathway-specific. In addition, our data suggest that ribonucleases play broader roles in mRNA surveillance and degradation mechanisms beyond just NMD and NSD.
- Different Bcl2 transcripts coding the same protein have different posttranscriptional control mechanismsPublication . Marques-Ramos, Ana; Teixeira, Alexandre; Romão, Luísa
- How far ‘AUG-proximity effect’ goes?Publication . Pereira, Francisco J.C.; Teixeira, Alexandre; Kong, Jian; Silva, Ana Luísa; Liebhaber, Stephen A.; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs). We have previously shown that mRNAs carrying a PTC located in close proximity to the translation initiation AUG codon escape NMD. This was called the “AUG-proximity effect”. The present work illustrates that the extension of the AUG-proximity effect, i.e. to what position in the open reading frame (ORF) an AUG-proximal PTC does not trigger NMD, is different between human a- and b-globin mRNAs. Remarkably, our data also demonstrate that, contrary to what occurs in the b-globin transcripts, a-globin mRNAs carrying an AUG-proximal PTC allow for efficient translation re-initiation, although it only partially explains their NMD resistance. In addition, our results reveal that in the a- and b-globin transcripts, the extension of the AUG-proximity effect is determined by the ORF sequence. Furthermore, we show how the mRNA secondary structure, which is affected by the ORF sequence, determines the AUG-proximity effect extension. Our data point out that the time taken to translate the short ORF, affected by its sequence and stability, besides being involved in modulating translation re-initiation, also plays an important role in establishing the extension of the AUG-proximity effect.
- 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
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