Browsing by Issue Date, starting with "2018-11-15"
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- Common p53 mutations induce IRES-mediated translation of oncogenic shorter p53 isoformsPublication . Pereira, Bruna; Lacerda, Rafaela; Maria López-Iniesta, M; Romão, Luísa; Candeias, MarcoAt least half of all tumors exhibit mutations in the tumor suppressor p53 gene. Indeed, the fact that p53 is frequently mutated in cancer led to its identification as an oncogene, when first described in 1979. Later, it was classified as a tumor suppressor, due to the clarification of its wild-type role in maintaining genome integrity and preventing malignant transformation. The p53 gene can encode for many p53 isoforms, by alternative splicing, alternative promoters and internal translation initiation mechanisms. While full-length p53 (FL-p53) protein works as a tumor suppressor by regulating many biological processes such as cell cycle, apoptosis, senescence and DNA repair, shorter p53 protein isoforms seem to play different roles in the cell. Recently, we have shown that the most common p53 mutations induce the expression of shorter p53 isoforms. Furthermore, we found that shorter p53 isoforms are implicated in cancer progression as they promote enhanced cell survival, proliferation, adhesion and formation of invasive cell structures. Here, with a bicistronic system containing two reporter genes (Renilla luciferase and firefly luciferase), we show that expression of shorter p53 isoforms is mediated by a non-canonical translation initiation mechanism regulated by an Internal Ribosome Entry Site (IRES) in the p53 mRNA. By investigating the effect of common p53 missense mutations on the function of this new IRES, through bioluminescence assays and Western blot analysis, we show that some p53 cancer mutations have a preponderant role in IRES-mediated translation induction of shorter p53 isoforms. With the obtained results we identified a new mechanism by which p53 cancer mutations promote tumorigenesis, which may lead to new understandings of the onset and progression of some types of tumors as well as to the development of new cancer therapies.
- 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.
- Fanconi's anemia - Retrospective study over a period of 37 yearsPublication . Ambrósio, Ana Paula; Silva, Maria do Céu; Silva, Neuza; Viegas, Mónica; Furtado, José; Correia, HildebertoFanconi anemia (FA) is a rare disease, with an estimated frequency of 1 to 5 per 1,000,000 births, which may increase in some ethnic group (like Ashkenazi Jewish and Gypsy). It’s an autosomal recessive disease that may have an X-linked transmission. Patients with FA may have congenital malformations, bone marrow failure, hypersensitivity to clastogenic agents, chromosomal fragility, and increased susceptibility to oncological diseases. Due to the great complexity of this pathology, the first approach to diagnosis consists of the detection of chromosomal aberrations (breaks, structural rearrangements, rings) in peripheral blood cells in culture with clastogenic agent such as diepoxybutane (DEB) or mitomycin C (MMC). We intend to present the results of chromosome instability studies induced by DEB and MMC performed in our institution. A retrospective 37 years series (1980-2017) of 274 samples sent to the cytogenetic laboratory with suspicion of FA and 28 samples of relatives of patients with FA were perform. The samples were process according with the protocol established by the International Fanconi Anemia Registry (IFAR). In the 274 analysed samples, 39 cases with AF were identify. In the cytogenetic studies of relatives with AF, 2 positive cases were identified for FA. Abnormal karyotypes were also observed in 8 samples suspected of AF. In this study, 41 new cases of AF were identify, mainly from the Lisbon and Tagus Valley regions and some specific cases from Azores, the central region and from the Portuguese speaking African countries (PALOP). This study evidences that the majority of the presented cases are underdiagnosed. These results do not allow to estimate a frequency of patients with AF in Portugal, since it does not include individuals from all Portuguese regions, and are include two individuals of PALOP origin. It would be interesting to carry out Next generation sequencing on the Fanconi positive samples in order to obtain in a single assay the analysis of the various genes involved in the pathology thus identifying the genetic change causing the disease. The authors have no conflict of interest.
- Modulation of protein translation mediated by upstream open reading frames (uORFs) in PERK mRNAPublication . Fernandes, Rafael; Romão, LuísaObjective: PERK is one of the three sensor proteins from the ER that are responsible for inducing the Unfolded Protein Response (UPR) when stress occurs (4). It is also the kinase responsible for phosphorylating eIF2α in these conditions (4). In this work we intended to determine if PERK is regulated at the translational level by uORFs in normal and ER stress conditions, and understand the impact of this regulation for the cell-stress response.
- How DIS3L2 meets NMD-targets: I’m really into “U”!Publication . da Costa, Paulo J.; Saramago, Margarida; Viegas, Sancra C.; Arraiano, Cecília M.; Romão, LuísaThe nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs carrying a premature translation-termination codon but also regulates the abundance of a large number of physiological RNAs that encode full-length proteins. Also, NMD regulates the levels of many physiological PTC-free mRNAs that encode full-length proteins. In human cells, NMD-targeted mRNAs are degraded by endonucleolytic cleavage and exonucleolytic degradation from both 5’ and 3’ ends. This is achieved by a process not yet completely understood that promotes the decay of the mRNAs in 5’-to-3’ and 3’-to-5’ by the XRN1 and exosome, respectively. In yeast, Dis3/Rrp44 protein is the catalytic subunit of the exosome, but in humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and the Perlman syndrome-associated exoribonuclease DIS3L2. Conversely, to its counterparts, DIS3L2 activity is independent of the exosome. In order to unveil the role of DIS3L2 in NMD, we performed its knockdown in HeLa cells and measured the mRNA levels of various natural NMD-targets. Our results show that DIS3L2 is involved in NMD-targets decay. Besides that, DIS3L2 acts directly on NMD-targets and interacts with the key NMD factor UPF1. We also show that DIS3L2-mediated decay depends on the activity of the terminal uridylyl transferases (TUTases) 4 and 7, which adds non-templated uridines to the mRNAs 3’ end, marking these mRNAs for DIS3L2 degradation. Together, our findings establish a direct role of DIS3L2 in NMD in an uridylation-dependent manner.
- Correlation between peripheral cytopenias and cytogenetic changes in the bone marrow in a paediatric population. Experience of 22 yearsPublication . Silva, Maria do Céu; Ambrósio, Ana Paula; Silva, Neuza; Viegas, Mónica; Correia, HildebertoThe haemogram is the most frequent request and an essential tool in the diagnosis of different pathologies in paediatric age, especially in haematological diseases. Peripheral cytopenias is the first laboratory finding suggestive of haematological disease, such as myelodysplastic syndrome, idiopathic thrombocytopenic purpura, among others. Confirmation of these pathologies should include the study of bone marrow, with analysis by different methodologies, including conventional cytogenetic karyotype analysis. In this work, we intend to present and establish a correlation between the results obtained by conventional cytogenetics in bone marrow samples and observation of peripheral cytopenias in a paediatric population over 22 years. A retrospective 22-year series (1995-2017) of 154 bone marrow samples from a paediatric population was analysed, which at the initial diagnosis presented peripheral cytopenia. The samples were process according to the established protocol for chromosome analysis in bone marrow, including cell culture, for each biological product, followed by a cytogenetic study to identify the karyotype. In the 154 samples analysed with peripheral cytopenias, 31 were bicytopenias, 33 pancytopenias, 21 neutropenias, 11 anaemias and 58 thrombocytopenias, of which 22 were of idiopathic origin. We identify 15 samples with abnormal karyotype, some of which presented a complex karyotype. Samples with abnormal karyotypes had pancytopenia or bicytopenia at the same time. Peripheral cytopenias are extremely important for suspicion of paediatric haematological diseases, especially in myelodysplastic syndrome. Conventional cytogenetic analysis of the bone marrow plays a fundamental role in the confirmation of these pathologies, their clinical evolution and the choice of proper therapy, However, micro-arrays should be performed with the aim of identifying micro-deletions / duplications or loss of heterozygosity that are characteristic in this group of pathologies. The author’s don´t have conflict of interest.
- Genomic Pitt_falls: Inglorious Ring(16)Publication . Marques, BárbaraPrenatal diagnosis of chromosomal disorders
- Evidence for a role of nonsense-mediated mRNA decay pathway genes in Autism Spectrum DisorderPublication . Marques, Ana; Martiniano, Hugo; Santos, J.X.; Vilela, J.; Asif, M.; Oliveira, G.; Romão, Luísa; Vicente, AstridIntroduction: Autism Spectrum Disorder (ASD) is a highly heterogeneous neurodevelopmental disorder with an unclear etiology. Genetic factors are estimated to account for 50 to 80% of the familial ASD risk, but most of the genetic determinants are still not known and a role for other regulatory mechanisms is likely. The nonsense-mediated decay (NMD) pathway controls mRNA quality and plays an important role in the regulation of the transcriptome. Mutations in genes involved in the NMD pathway have been linked to neurodevelopmental disorders, with intriguing evidence for an involvement of mutations in the UPF3B gene, a core component of the NMD pathway, in ASD.