Browsing by Issue Date, starting with "2023-11-23"
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- Non-canonical synthesis of UPF1 protein contributes to its oncogenic role in colorectal cancerPublication . Lacerda, Rafaela; Menezes, Juliane; Antunes Elias, Adriana; Romão, LuísaColorectal cancer (CRC) is the third leading cause worldwide and projections point towards an increase over the next two decades. Gene expression dysregulation of several genes involved in CRC contribute to disease development. The up-frameshift 1 (UPF1) protein plays important roles in several cellular mechanisms and acts as a tumour suppressor in most cancers. However, in CRC, this protein has been described as working as an oncogenic protein. In order to understand the molecular mechanisms underlying the oncogenic role of UPF1 in CRC, we have analysed mRNA and protein levels in different types of cancer. In silico analyses have shown that UPF1 is overexpressed in CRC and lung cancer compared to the other analysed cancers. Also, UPF1 expression is significantly greater in CRC than in normal tissues. Experimentally, we observed that UPF1 expression is maintained under stress conditions that compromise global protein synthesis. In this regard, we tested whether UPF1 translation initiation can be mediated through an alternative cap-independent mechanism. We showed that the 5’ untranslated region (UTR) of UPF1 transcript allows cap-independent translation initiation and mapped the minimal sequence required for this mechanism to work. This region also mediates translation initiation in transcripts lacking a cap structure and under stress conditions like endoplasmic reticulum stress, hypoxia and mTOR pathway inhibition. Then, we designed antisense RNA oligonucleotides (ASOs) that target the minimal region and observed a reduced expression of UPF1 in CRC cells treated with those ASOs compared to cells treated with control ASOs. All in all, these results show that alternative translation initiation mediated through UPF1 5’UTR allows UPF1 expression levels to be maintained under conditions observed in the tumour microenvironment, which globally repress protein synthesis. Thus, ASOs targeting the minimal region responsible for allowing UPF1 expression can be the beginning of a new RNA-based therapy to prevent CRC development.
- Translational Control of Δ160p53 Keeps the Dark Side of TP53 in CheckPublication . Ramalho, Ana Catarina; Lacerda, Rafaela; Romão, Luísa; Candeias, Marco M.The TP53 tumour suppressor gene was discovered over 40 years ago, but to this day some aspects of its regulation and function remain a mystery. It encodes the full-length p53 protein (FLp53), a transcription factor with a key role in stress response in multicellular organisms, that can either direct cells towards apoptosis or recovery of homeostasis. With such a decisive role, its expression and activity are tightly regulated. A vast set of RNA-binding proteins (RBPs) have been described to affect the translation of FLp53 or the stability of p53 mRNA in response to different perturbations. But in addition to FLp53, there is a group of shorter protein isoforms lacking the N-terminal region, which have well-described functions, and are translated from the same mRNA. The shorter and less studied isoform is Δ160p53, which promotes cell survival, proliferation, and invasion. Despite its usual low levels, it is commonly overexpressed in tumours. However, the detailed mechanisms and factors involved in the regulation of Δ160p53 are still unknown. In this work, a mass spectrometry was performed to identify the proteins in an RNA-protein co-immunoprecipitation of the p53 mRNA using the MS2 system in the p53-null cell line H1299. The validation of the hits was undertaken by western blot with specific antibodies after immunoprecipitation. The effect of the binding proteins on the translation of Δ160p53 was assessed by overexpression or knockdown, and the expression levels were verified by western blot or luminescence assays. The mass spectrometry allowed the identification of potential new binding partners of the p53 mRNA. Resorting to the literature and to computational tools available online to predict protein-RNA interactions, a few hits were selected for follow-up and their interactions confirmed. Simultaneously, the modulation of Δ160p53 expression by some of these proteins was verified. Considering the importance of TP53 in deciding the fate of the cell, the observation of abnormal levels of the oncoprotein Δ160p53 in cancer is intriguing. Understanding the control of its translation could uncover strategies to block it and pave way for new cancer therapies.
- 15q11.2q13.1 interstitial gain in a fetus with an increased risk for T21: When classification and clinical outcome are divergentPublication . Serafim, Sílvia; Pedro, Sónia; Marques, Bárbara; Tarelho, Ana; Viegas, Mónica; Simão, Laurentino; Ferreira, Cristina; Carvalho, Inês; Cohen, Álvaro; Correia, HildebertoIntroduction: Copy number variants (CNV) of the 15q11.2q13.1 region are associated to recurrent microdeletion/microduplication syndromes in which the phenotype is dependent on the parental origin of the CNV. We report the case of a fetus from a healthy 39-year-old G6P3A2 woman, with an increased risk for trisomy 21 in the 1st trimester prenatal screening. Chromosomal microarray analysis (CMA) was requested and revealed a pathogenic duplication in which the outcome was dependent of the parental origin of the affected allele. Methods: DNA was extracted from a chorionic villus sample and CMA was performed using Cytoscan™ 750K. Parental follow-up studies to assess the origin of the CNV were performed. Results: The CMA profile revealed a male fetus with a 4,89 Mb interstitial gain in 15q11.2q13.1. CMA of the parents showed that the duplication was paternally inherited. Discussion: The detected CNV is a recurrent known microduplication and according to the American College of Medical Genetics and Genomics guidelines is classified as pathogenic. However the phenotype is dependent on the parental origin of the duplication. When it arises in the maternally allele it has a severe outcome with hypotonia, cognitive deficit, seizures, among others. If the CNV occurs in the paternal allele although some patients might show developmental delays and behavioral disturbances most cases are rarely symptomatic. In this case, CMA of both parents showed that the CNV identified in the fetus was paternally inherited. Although the CMA result did not explained the increased risk for T21 after determining the duplication had been inherited from the father it allowed the prediction of the most likely resulting phenotype for the fetus as a milder or even asymptomatic. Follow-up ultrasounds at gestation age of 17w+6d and echocardiogram at 21w+3d showed no structural abnormalities. The baby was born at 37w+5d with an Apgar index of 10/10/10, with no dysmorphic features or malformations, and a normal physical exam. This case illustrates that although the use of genetic tools using artificial intelligence and following determined guidelines can be helpful for the purpose of consistency and standardization on the classification we always need careful evaluation from a clinical laboratory geneticist on the context of each case. Additionally it also shows how critical parental testing can be, not only to assess recurrence risk but to provide the best possible tool to ascertain the outcoming phenotype and allow the best choices to the couple after genetic counselling.
- Morte súbita neonatal num recém-nascido com perturbação do desenvolvimento intelectual ligada ao X tipo Nascimento por delecção do gene UBE2APublication . Furtado Gomes, Inês; Martins, Marta; Marques, Bárbara; Correia, Hildeberto; Sanchez, BrunoIntrodução: A perturbação do desenvolvimento intelectual ligada ao X (PDIX) tipo Nascimento é uma síndrome genética rara caracterizada por défice intelectual, dismorfismos craniofaciais característicos e anomalias congénitas, nomeadamente defeitos cardíacos e do tracto génito-urinário. Descrição do caso: Recém-nascido do sexo masculino, admitido com 12 horas de vida na Unidade de Cuidados Intensivos Neonatais por hipoglicémia e dificuldade alimentar. Gestação vigiada, com diagnóstico pré-natal de agenésia renal esquerda. Parto eutócico às 36 semanas sem necessidade de manobras de reanimação e com somatometria ao nascimento adequada à idade gestacional. No exame objectivo destacava-se hipotonia axial e dismorfismos craniofaciais e dos membros (Figura 1): fendas palpebrais amendoadas, inclinadas para cima, hipertelorismo; dorso nasal côncavo, narinas antevertidas; boca larga com cantos voltados para baixo e lábios finos; pavilhões auriculares hipoplásicos; inserção proximal dos polegares; pés com primeiros dedos largos e sobreposição do segundo e terceiro dedos. Durante as primeiras duas semanas de vida apresentou quadro de hipertensão pulmonar, colestase, dificuldade alimentar e má progressão ponderal. Identificada comunicação interventricular perimembranosa, sem sinais de disfunção cardíaca. Sem outras malformações identificadas. Paragem cardiorrespiratória súbita ao 20º dia de vida, sem resposta a medidas de suporte avançado de vida. Exame anatomopatológico post-mortem não conclusivo quanto à causa de morte. O estudo genético (Array-CGH) identificou uma delecção de 386 kb na região Xq24, englobando o gene UBE2A e 6 genes contíguos, compatível com PDIX tipo Nascimento. Conclusão: Actualmente, encontram-se descritos 58 casos de PDIX tipo Nascimento, dos quais apenas um com diagnóstico no período neonatal. Apesar de constituir uma entidade rara, apresenta um fenótipo característico, passível de ser identificado em idade precoce. O caso descrito corresponde ao primeiro relato de morte súbita neonatal num indivíduo com este síndrome, realçando a importância do reconhecimento clínico desta entidade. Um diagnóstico atempado permitirá uma abordagem clínica dirigida, o estabelecimento do prognóstico e o aconselhamento genético da família.