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Editorial. This article belongs to the Special Issue mRNA Metabolism in Health and Disease. Eukaryotic gene expression involves several interlinked steps, in which messenger RNAs (mRNAs), which code for proteins, are the key intermediates [...]. info:eu-repo/semantics/publishedVersion

As nanoceluloses são nanomateriais inovadores com potencial para uma vasta gama de aplicações industriais e biomédicas. No entanto, a expansão da sua produção tem vindo a suscitar preocupações quanto aos possíveis efeitos, a longo prazo, na saúde humana. Este estudo teve como objetivo avaliar a segurança de algumas nanoceluloses produzidas a partir de matéria-prima nacional, através da caracterização da sua potencial toxicidade em células de mamífero. Para tal, testaram-se duas celuloses nano /microfibrilares (CNF e CMF ) e uma celulose nanocristalina (CNC). Analisou-se a citotoxicidade usando ensaios colorimétricos e o ensaio clonogénico, e a genotoxicidade pelo ensaio do micronúcleo in vitro em células pulmonares de mamífero (A549 e V79 ) e em osteoblastos humanos ( MG-63 ). A indução de espécies reativas de oxigénio (ROS) e a internalização celular foram também estudadas nas células A549. Observou- -se citotoxicidade no ensaio clonogénico, principalmente no caso da CNC, mas não nos restantes ensaios, não havendo também indução de ROS. Todas as nanoceluloses revelaram efeitos genotóxicos nalgumas concentrações, uma vez que induziram micronúcleos e /ou pontes nucleoplásmicas num dos modelos celulares. Para além disso, visualizou-se a internalização da CNF e CMF, mas não da CNC, em células A549. Esta primeira avaliação toxicológica veio contribuir para prevenir a exposição a materiais celulósicos potencialmente perigosos, procurando impulsionar o desenvolvimento de materiais inovadores e mais seguros. Nanocelluloses are innovative nanomaterials with potential for a wide range of industrial and biomedical applications. However, the expansion of its production has raised concerns about their possible long-term effects on human health. This study aimed to evaluate the safety of various nanocelluloses through the characterization of their potential toxicity in mammalian cells. Two samples of nano/microfibrillar celluloses with different pre-treatments (CNF and CMF) and a nanocrystalline cellulose (CNC) were tested. The cytotoxicity of the nanocelluloses was analyzed using colorimetric assays and the clonogenic assay, and genotoxicity by the in vitro micronucleus assay in human alveolar epithelial cells (A549), human osteoblasts (MG-63) and Chinese hamster fibroblasts (V79). Induction of reactive oxygen species (ROS) and cellular internalization were also studied in A549 cells. Cytotoxicity was observed through the clonogenic assay, mainly in the case of CNC, but not in the remaining assays, with no induction of ROS. All nanocelluloses, at some of the concentrations tested, induced micronuclei and/or nucleoplasmic bridges in one of the cellular models. Furthermore, the internalization of CNF and CMF, but not of CNC was visualized in A549 cells. These results aim to contribute to preventing exposure to potentially hazardous cellulosic materials, seeking to boost the development of innovative and safer materials. Projeto ToxApp4NanoCELFI – Uma abordagem de toxicologia preditiva para a caracterização dos potenciais efeitos respiratórios de fibras de nanocelulose funcionalizadas num sistema de co-cultura (PTDC/SAU-PUB/32587/2017). info:eu-repo/semantics/publishedVersion

Human biomonitoring (HBM) data measured in specific contexts or populations provide information for comparing population exposures. There are numerous health-based biomonitoring guidance values, but to locate these values, interested parties need to seek them out individually from publications, governmental reports, websites and other sources. Until now, there has been no central, international repository for this information. Thus, a tool is needed to help researchers, public health professionals, risk assessors, and regulatory decision makers to quickly locate relevant values on numerous environmental chemicals. A free, on-line repository for international health-based guidance values to facilitate the interpretation of HBM data is now available. The repository is referred to as the "Human Biomonitoring Health-Based Guidance Value (HB2GV) Dashboard". The Dashboard represents the efforts of the International Human Biomonitoring Working Group (i-HBM), affiliated with the International Society of Exposure Science. The i-HBM's mission is to promote the use of population-level HBM data to inform public health decision-making by developing harmonized resources to facilitate the interpretation of HBM data in a health-based context. This paper describes the methods used to compile the human biomonitoring health-based guidance values, how the values can be accessed and used, and caveats with using the Dashboard for interpreting HBM data. To our knowledge, the HB2GV Dashboard is the first open-access, curated database of HBM guidance values developed for use in interpreting HBM data. This new resource can assist global HBM data users such as risk assessors, risk managers and biomonitoring programs with a readily available compilation of guidance values. Highlights: An international Human Biomonitoring Working Group (i-HBM) was formed in 2020; i-HBM working group is affiliated with the International Society of Exposure Science (ISES); i-HBM has developed the first curated database of HBM Health-Based Guidance Values or HB2GVs, The guidance values are freely available in the online HB2GV Dashboard. info:eu-repo/semantics/publishedVersion

The tobacco is one of the biggest public health threats, smoking kills more than 7 million people/year worldwide and more than 890,000 are deaths resulting from exposure to Second Hand Smoke (SHS). In adults, SHS is associated to cardiovascular and respiratory diseases, including coronary heart disease and lung cancer, through pathological and molecular mechanisms not yet understood. We aimed to investigate the SHS effects on airway proteome in exposed workers. Nasal epithelium was collected from hospitality workers (non-smokers=40; smokers=12), long-term exposed and non-exposed to SHS at the workplace. Samples were analyzed by shotgun proteomics using an ESI-LQT Orbitrap XL mass spectrometer. The generated MS raw data was submitted to ‘PatternLab for Proteomics’ for peptide identification and relative quantification by label-free - extracted ion chromatograms (XIC). Golden rules were applied to obtain reliable data: in the identification of a protein at least one unique peptide must had to be present in more than 80% of the individuals, and consequently each inferred protein had to be detected in 80% to 100% of the cohort. Two proteins were found to be differentially expressed in the no-smokers exposed to SHS compared with the control: BPI fold-containing family A member 1 (BPIFA1) and Heat shock Protein Beta-1 (HSPB1). The first protein plays a role in the airway inflammatory response after exposure to irritants substances and the second is associated as a regulator of actin filament dynamics. Our findings support the indication that in non-smokers the prolonged exposure to SHS can lead to airway proteome modulation. When validated, the uncovered proteins can be promising candidates to “susceptibility/risk” and/or “predictive” biomarkers for SHS exposure. Gulbenkian Institute of Science; Fundação para a Ciência e a Tecnologia (FCT); National Institute of Health Dr. Ricardo Jorge; Faculty of Sciences of the University of Lisbon; Protein Technology Laboratory - Department of Immunotecnology, Lund University, Sweden; Center of Toxicogenomics and Human Health. N/A

The role of red blood cells (RBC) in the immune system is increasingly recognized. However, RBC-derived molecules with an immunomodulatory role in health and disease, as well as in vaccine immunogenicity are still poorly investigated. Taking as a model the emergent COVID-19 vaccines, we aimed to investigate whether vaccines induce proteome and/or metabolome changes in RBCs able to affect T-cell immune activity, as a mechanistic test for vaccine immunization regulated by RBCs. Our ultimate goal is to identify RBC immunomodulators as potential co-adjuvants in the formulation of next-generation vaccines with bolstered efficacy and duration. A biobank of blood samples collected longitudinally under ‘omics’ quality control from subjects (n=39) that underwent vaccination for COVID-19 between April and September 2021 was created. This biobank is associated with extensive clinical data, including demographic data, COVID-19 PCR diagnosis, hematological and vaccine effectivity data. Linear Mixed Models, were used to evaluate the association between biometrical characteristics, health related habits, vaccine technology and vaccine effectivity and hematological parameters, along the different time-points (t0-t4) under study, i.e, before and after (24-72h or 30 days) of the first and second dose of vaccine. Statistical analyses were performed using R software version 4.1.2. Results showed significant differences (p

Obstructive Sleep Apnea (OSA) syndrome is characterized by recurrent episodes of apneas and hypopneas during sleep, leading to recurrent intermittent hypoxia and sleep fragmentation. No treated OSA can result in metabolic and cardiovascular diseases. By 2D gel-based proteomics approach we have demonstrated that OSA can cause alterations in the red blood cells (RBC) proteome that may be associated with OSA outcomes. OSA induces alterations in the redox/oligomeric states of RBC proteins such as gyceraldehyde-3-phosphate dehydrogenase (GAPDH) and peroxiredoxin-2 (PRDX2) that can be reverted or modulated by PAP treatment. In this study, we applied a shotgun proteomics strategy to further investigate the RBC proteome from patients with OSA before and after PAP treatment to better understand the regulation of RBC homeostasis in the context of OSA and/or under effect of PAP treatment. As a first approach, RBCs samples, corresponding to Snorers patients as control (n=23) and patients with OSA before and after six months of PAP treatment (n=33/condition) were selected from our biobank1. Samples were randomly pooled (n=3 per group/condition) and lysed 1:6 with 5mM sodium phosphate buffer containing 100 mM of N-ethylmaleimide, a reagent that alkylates free sulfhydryl groups, before haemoglobin depletion by using HemovoidTM system. Depleted samples were alkylated, reduced and digested with trypsin and chymotrypsin. The resulting peptides were cleaned with C18 columns and analysed in triplicate by a Nano High Performance Liquid Chromatography (nanoHPLC) on-line coupled to a high-resolution accurate-mass Orbitrap mass spectrometer (Q Exactive, Thermo Scientific) with a nano electrospray ionization source (nanoESI). The acquired mass spectrometry data were analysed by MaxQuant v1.5.8.3 and Perseus v2.0.3.1 software. The preliminary results corroborated our previous findings by showing that proteins associated with stress response and antioxidant regulatory system were the most changed in OSA RBC compared with Snorers ones. The active catalytic cysteine (Cys 51) in the PRDX2 was identified trioxidized –SO3H almost exclusively in OSA RBC before PAP treatment. Further analyses and validation of these data are in progress, which will certainly provide a better understanding of RBC molecular mechanisms and their proteins/PTMs associated with OSA pathology and/or response to PAP therapy. Project partially supported by the Harvard Medical School-Portugal Program (HMSP-ICJ/0022/2011), the ToxOmics (FCT-UID/BIM/00009/2013) and the Portuguese Mass Spectrometry Network (RNEM). CVC is recipient of FCT doctoral scholarship FCT-SFRH/BD/133511/2017. info:eu-repo/semantics/draft

Background: WHO have stated that near 900 000 deaths per year result from exposure to Second- Hand Smoke (SHS). SHS exposure has been linked to cancer, respiratory and cardiovascular diseases and diabetes. However, the associated underlying molecular mechanisms remain to be elucidated. The objective of this proteomics study is to uncover putative key molecules involved in these mechanisms that can be used to predict and monitor diseases risks associated with occupational SHS exposure. Methods: In total, 25 Lisbon restaurants agreed to participate. Nasal epithelium and urine samples were collected from their employees (n=52) for proteomics analysis and cotinine evaluation of SHS exposure, respectively. The subjects were classified as never smoker (N), former smoker (F) and smoker (S); exposed (NE=11; FE=10; SE=4) or non-exposed (N=11; F=8; S=8) to SHS. All subjects were healthy and showed no significant differences in parameters like age, time in the workplace, tobacco smoking habits and spirometry evaluation of pulmonary function. Urine cotinine levels showed significantly elevated in the exposed subjects compared to non-exposed, confirming SHS exposure. Nasal epithelium samples were analyzed by shotgun proteomics using an ESI-LTQOrbitrap mass spectrometer. The “MS raw data” was submitted to “PatternLab for Proteomics” software, with “Comet” search machine algorithm, from where the identified proteins were submitted to a “ClueGO” functional annotation & enrichment analyses in “Cytoscape” software, with the propose to shed some light about the molecular biology involved in the cellular response to the SHS exposition. Results: In NE subjects the SHS is associated with the biologic terms of “Lactate dehydrogenase complex” and “Pentose-Phosphatase Shunt”, also with “Glutathione peroxidase activity” and “Tcell apoptotic process”. At the other end the FE individuals present a specific proteome enriched in biologic information with terms as the “L-Lactate dehydrogenase complex” and the “Peroxisome” as was expected by the results above for the NE cohort; but there were also other different terms as: “Peripheral T cell lymphoma”, “Central carbon metabolism in cancer”, “Myelodysplastic syndrome”, “Monocyte & Granulocyte & Macrophage & Leukocyte Chemotaxis”, Nucleossome, variant H3.1-H2A2-H2B.1&Others” and finally “DNA replication-dependent chromatin assembly”. Conclusions: Proteome of nasal epithelium seems to be modulated by SHS exposure and this is a different and perhaps cumulative process between NE and FE individuals. Acknowledgements: Gulbenkian Foundation and Administração Central do Sistema de Saúde, and ToxOmics Portugal. SN, SP and VMT worked under FCT fellowships. info:eu-repo/semantics/draft

Review Nonsense-mediated decay (NMD) was first described as a quality-control mechanism that targets and rapidly degrades aberrant mRNAs carrying premature termination codons (PTCs). However, it was found that NMD also degrades a significant number of normal transcripts, thus arising as a mechanism of gene expression regulation. Based on these important functions, NMD regulates several biological processes and is involved in the pathophysiology of a plethora of human genetic diseases, including cancer. The present review aims to discuss the paradoxical, pro- and anti-tumorigenic roles of NMD, and how cancer cells have exploited both functions to potentiate the disease. Considering recent genetic and bioinformatic studies, we also provide a comprehensive overview of the present knowledge of the advantages and disadvantages of different NMD modulation-based approaches in cancer therapy, reflecting on the challenges imposed by the complexity of this disease. Furthermore, we discuss significant advances in the recent years providing new perspectives on the implications of aberrant NMD-escaping frameshifted transcripts in personalized immunotherapy design and predictive biomarker optimization. A better understanding of how NMD differentially impacts tumor cells according to their own genetic identity will certainly allow for the application of novel and more effective personalized treatments in the near future. Gonçalo Nogueira, Rafael Fernandes, and Juan Fernandez García-Moreno are recipients of a fellowship from BioSys PhD programme PD65-2012 (SFRH/PD/BD/130959/2017, SFRH/BD/114392/2016 and SFRH/PD/BD/142898/2018, respectively) from FCT. This work was partially supported by UID/MULTI/04046/2019 Research Unit grant (to BioISI). info:eu-repo/semantics/publishedVersion

Review This entry is adapted from 10.3390/biomedicines10081865 (http://repositorio.insa.pt/handle/10400.18/8440) Internal ribosome entry site (IRES)-mediated translation is an alternative mechanism of translation initiation, known for maintaining protein synthesis when canonical translation is impaired. During a stress response, it contributes to cell reprogramming and adaptation to the new environment. info:eu-repo/semantics/publishedVersion

Erratum Front Genet. 2022 Feb 25;13:868306. doi: 10.3389/fgene.2022.868306. eCollection 2022. With the advent of genomic sequencing, a number of balanced and unbalanced structural variants (SVs) can be detected per individual. Mainly due to incompleteness and the scattered nature of the available annotation data of the human genome, manual interpretation of the SV’s clinical significance is laborious and cumbersome. Since bioinformatic tools developed for this task are limited, a comprehensive tool to assist clinical outcome prediction of SVs is warranted. Herein, we present SVInterpreter, a free Web application, which analyzes both balanced and unbalanced SVs using topologically associated domains (TADs) as genome units. Among others, gene-associated data (as function and dosage sensitivity), phenotype similarity scores, and copy number variants (CNVs) scoring metrics are retrieved for an informed SV interpretation. For evaluation, we retrospectively applied SVInterpreter to 97 balanced (translocations and inversions) and 125 unbalanced (deletions, duplications, and insertions) previously published SVs, and 145 SVs identified from 20 clinical samples. Our results showed the ability of SVInterpreter to support the evaluation of SVs by (1) confirming more than half of the predictions of the original studies, (2) decreasing 40% of the variants of uncertain significance, and (3) indicating several potential position effect events. To our knowledge, SVInterpreter is the most comprehensive TAD-based tool to identify the possible disease-causing candidate genes and to assist prediction of the clinical outcome of SVs. SVInterpreter is available at http://dgrctools-insa.min-saude.pt/cgi-bin/SVInterpreter.py. This research was supported by national funds through FCT—Fundação para a Ciência e a Tecnologia, Research Grant HMSP-ICT/0016/2013 of the Harvard Medical School—Portugal Program in Translational Research and Information info:eu-repo/semantics/publishedVersion

Introduction: Autism Spectrum Disorder (ASD) is a neurodevelopmental condition characterized by impairedsocial/communication skills and stereotyped/repetitive behaviors. Genetic factors account for 50-80% of the familialrisk of ASD, but genetic determinants are not fully understood and a role for regulatory processes is plausible. Inthis study, we explored the contribution to ASD etiology of genes involved in an important post-transcriptionalregulatory mechanism implicated in neurodevelopment, the Nonsense-Mediated Decay (NMD). Methods: We first compiled a group of 46 genes encoding NMD factors and regulators. In these genes wesearched for Single Nucleotide Variants (SNVs) and Copy Number Variants (CNVs) in two samples of ASD patients(N=1828 and N=3570, respectively). We observed the frequency of these variants in 60146 controls from gnomADv2.1.1 (for SNVs) and in 10355 controls from the Database of Genomic Variant ( for CNVs). In genes with rarevariants (MAF

Untranslated regions in the messenger RNA (mRNA) are susceptible to the interaction of regulatory elements including proteins or non-coding RNA molecules, being an important hotspot to the study of gene expression regulation. Internal Ribosome Entry Sites (IRES) are secondary structures usually located on the 5’UTR of an mRNA molecule that can recruit the ribosome during initiation of protein synthesis without the involvement of the cap structure. This mechanism tends to appear when the cell is under stress conditions, which might include the presence of oncogenes, growth factors or proteins involved in programmed cell death. This work focuses on the human AGO1, an important key player for RNA-mediated gene silencing, and whether its 5’UTR is capable of driving cap-independent translation initiation. To achieve this goal, a construct containing the 5’UTR of human AGO1 was cloned, taking advantage of a bicistronic vector containing two reporter genes, Renilla luciferase (RLuc) and firefly luciferase (FLuc), the last one cloned downstream from the 5’UTR. We performed luminometry assays to assess the relative translation efficiency of FLuc, which is under the control of AGO1 5’UTR. The results showed that human AGO1 5’UTR mediates a cap-independent eIF4G-dependent mechanism of translation initiation enhanced by a free 5’ end. Also, we saw that this alternative mechanism is maintained, and even enhanced, under stress conditions, such as the knock-down of eukaryotic initiation factor 4E, the protein that directly binds to the cap structure. We are currently investigating what is the minimal sequence required for IRES-mediated translation. Combining these results with emerging RNA-based therapies will be helpful to develop novel strategies to prevent and treat disorders, such as cancer, involving dysregulation of AGO1 translation. info:eu-repo/semantics/publishedVersion

Crucial in several cellular processes, such as nonsense-mediated mRNA decay, cell cycle progression, and telomere maintenance and homeostasis, Up-frameshift 1 (UPF1) has also been considered a tumour suppressor protein in hepatocellular carcinoma and gastric cancer, as it is underexpressed in the latter and negatively correlated to MALAT1 (long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1) expression. UPF1 overexpression inhibits some cancer-specific features, like proliferation, cell cycle progression, cell migration and invasion, and enhances apoptosis, turning the UPF1/MALAT1 pathway a potential therapeutic target for gastric cancer. Here, we investigate the importance of UPF1 internal ribosome entry site (IRES)-dependent translation in tumorigenesis, specifically in colorectal cancer. Thus, we used a bicistronic system with two reporter genes, in which we cloned UPF1 5’ untranslated region (UTR) upstream the second cistron, whose translation will only occur internally. We transcribed the bicistronic mRNA in vitro and transfected HeLa (cervical cancer), NCM460 (normal intestinal-derived colonocytes) and HC116 (pre-metastatic colorectal carcinoma) cells with such mRNA, along with the positive and negative controls. The results show a significant increase in IRES-mediated translation levels compared to those of the negative control, both in normal conditions and under endoplasmic reticulum stress. Also, internal initiation occurs in the absence of the cap structure. Deletional and mutational analysis of UPF1 5’UTR showed that nucleotides 1–100 (stem loop (SL) I) and 151–275 (SL III) — out of 275 nucleotides — are the minimal required sequences for the IRES to work properly. Also, we used RNA antisense oligonucleotides (ASOs) targeting UPF1 IRES SL I and III, and observed a reduced UPF1 expression. Cellular viability increases in HCT116 cells and decreases in NCM460 cells treated with ASOs targeting SL III and SL I, respectively, whereas apoptosis increases in NCM460 cells and decreases in HCT116 cells treated with ASOs targeting SL I. This may be the dawn of a new RNA-based therapeutic approach regulating colorectal cancer development. FCT info:eu-repo/semantics/publishedVersion

The transcription factor p53 is a key cell regulator, having roles in varied cellular processes. Widely known as a tumour suppressor protein, p53 is responsible for signalling the adequate response to DNA damage, oncogenic signalling, or other stress stimuli. The target genes of this protein are involved in cell cycle arrest, senescence, apoptosis, and DNA damage response, among other pathways. Besides the full-length p53 (FLp53), to which these functions are attributed, the TP53 gene encodes for eleven other protein isoforms that result from alternative splicing, internal initiation of translation and transcription from an internal promoter. In striking contrast to FLp53, the N-terminally truncated Δ160p53 exhibits pro-oncogenic traits, although it only differs from FLp53 by the lack of its first 159 amino acids. Δ160p53 promotes cell survival, proliferation, invasion, and adhesion, and it is overexpressed in cancer cells harbouring hotspot p53 mutants. The TP53 gene is frequently mutated in cancer, and hotspot mutants result from single missense mutations that convert p53 in a driver of tumorigenesis. As Δ160p53 presents many of the oncogenic roles attributed to p53 cancer mutants, it is plausible that this isoform could be responsible for the paradoxical mutant p53 functions. However, detailed knowledge on the mode of action of Δ160p53 is still lacking. We have performed additional tests to further characterize the oncogenic traits of this isoform. To evaluate the ability of Δ160p53 to promote anchorage-independent cell growth, we have used the soft agar colony formation assay. Preliminary results show a tendency of Δ160p53 to promote growth, when compared to the control and other isoforms. Our new data complements our previous knowledge on Δ160p53 and reinforces the importance of studying this isoform for therapeutic targeting. PhD Fellowship financed by FCT info:eu-repo/semantics/publishedVersion

This article belongs to the Special Issue p53 in Cancer and beyond—40 Years after Its Discovery p53 is the most commonly mutated gene in human cancers. Two fundamental reasons for this are its long protein isoforms protect from cancer, while its shorter C-terminal isoforms can support cancer and metastasis. Previously, we have shown that the Δ160p53 protein isoform enhances survival and the invasive character of cancer cells. Here, we identified a translation initiation site nine codons downstream of codon 160-the known initiation codon for the translation of Δ160p53-that is recognized by the translation machinery. When translation failed to initiate from AUG160 due to mutation, it initiated from AUG169 instead, producing similar levels of a similar protein, Δ169p53, which promoted cell survival as efficiently as Δ160p53 following DNA damage. Interestingly, almost all mammalian species with an orthologue to human AUG160 also possess one for AUG169, while none of the non-mammalian species lacking AUG160 have AUG169, even if that region of the p53 gene is well conserved. In view of our findings, we do not believe that Δ169p53 acts as a different p53 protein isoform; instead, we propose that the double translation initiation site strengthens the translation of these products with a critical role in cell homeostasis. Future studies will help verify if this is a more general mechanism for the expression of essential proteins in mammals. This research was funded by grants 18K07229 (KAKENHI) from Japan Society for the Promotion of Science (JSPS), PTDC/BIM-ONC/4890/2014 and PTDC/MED-ONC/32048/2017 from Fundação para a Ciência e a Tecnologia of Portugal (FCT) and grants/fellowships from Takeda Science Foundation (2017 Medical Research Fellowship (Oncology, Basic)), Astellas Research Foundation for Pathophysiology and Metabolism (project number 203180600044), AXA Research Fund, Ichiro Kanehara Foundation and Kyoto University (Ishizue, year 2021) attributed to M.M.C. This work was also partially supported by grant UID/MULTI/04046/2013 to BioISI from FCT/MCTES/PIDDAC and by funds from Instituto Nacional de Saúde Doutor Ricardo Jorge. M.J.L.I. and S.N.P. were partially supported by Otsuka Toshimi Scholarship Foundation. ACR is the recipient of a PhD scholarship from the Portuguese Foundation for Science and Technology (2020.06982.BD) info:eu-repo/semantics/publishedVersion

Aims: UPF1 internal ribosome entry site-mediated translation and its importance in tumorigenesis; Confirm UPF1 5’ untranslated region (UTR) can mediate internal ribosome entry site (IRES)-mediated translation; Check whether the IRES-dependent translation is maintained under cap-dependent-impairing conditions (hypoxia); Identify the minimal sequence required for IRES-mediated translation; Investigate the biological role of UPF1 IRES-mediated translation in tumorigenesis. FCT info:eu-repo/semantics/publishedVersion

HBM4EU is a joint effort of 30 countries, the European Environment Agency and the European Commission, co-funded under Horizon 2020. The initiative is coordinating and advancing human biomonitoring in Europe. HBM4EU is generating evidence of the actual exposure of citizens to chemicals and the possible health effects in order to support policy making. The HBM4EU initiative represents a novel collaboration between scientists and chemical risk assessors and risk managers, including several Commission services, EU agencies and representatives for the national level. The project has built bridges between the research and policy worlds in order to deliver benefits to society in terms of enhanced chemical safety. HBM4EU project, Grant Agreement No: 733032 N/A

A proteína up-frameshift 1 (UPF1) é uma proteína multifacetada, com um papel fundamental em vários mecanismos celulares. É um fator essencial no mecanismo de decaimento rápido de mRNAs aberrantes com codões nonsense (NMD, do inglês nonsense-mediated mRNA decay). É também uma proteína crucial para a progressão da fase S do ciclo celular e manutenção do tamanho e homeostasia dos telómeros, de um modo totalmente independente das suas funções no NMD. No que respeita à tumorigénese, a UPF1 é frequentemente considerada uma proteína supressora de tumores e um potencial alvo terapêutico para o carcinoma hepatocelular. Além disso, está subexpressa em cancro gástrico e correlacionada negativamente com a expressão de MALAT1 (do inglês long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1). De acordo com o estudo de Karger e colaboradores (colocar entre parenthesis a ref), a sobre-expressão de UPF1 inibe algumas das características específicas do cancro, como sejam a proliferação celular, a progressão do ciclo celular, a migração e a invasão celulares, e aumenta a apoptose. Assim, a UPF1 pode ser considerada um potencial modulador de MALAT1, o que demonstra que a via UPF1/MALAT1 pode ser usada como um alvo terapêutico para o cancro do estômago. Neste trabalho, o nosso grupo, demonstrou que a região 5’ transcrita, mas não traduzida (UTR, do inglês untranslated region) do mRNA que codifica a UPF1 consegue utilizar um mecanismo alternativo de síntese proteica (i.e. tradução do mRNA) mediada por IRES (do inglês, internal ribosome entry sites). Este mecanismo alternativo permite a produção seletiva de proteínas em condições que impedem o normal mecanismo de tradução, funcionando. tanto em condições normais como sob condições de stress, tais como hipóxia, stress do retículo endoplasmático, inibição da via do mTOR, ou inibição da expressão do fator de iniciação eucariótico 4E, a proteína que se liga diretamente à estrutura cap e permite a tradução canónica. Ao fazer uma análise delecional e mutacional da sequência da 5’UTR de UPF1, verificámos que a sequência mínima necessária para a atividade dependente do IRES compreende os nucleótidos 1–100 e 151–275 (do total de 275 nucleótidos que compõem a 5’UTR). Fazendo uma previsão bioinformática da estrutura secundária formada por esta sequência, verificámos que há a formação de três stem loops (SL). O primeiro e o terceiro SL correspondem precisamente às sequências identificadas como essenciais e suficientes para a tradução mediada por IRES. Estes estudos foram conduzidos em células HeLa (cancro do colo do útero), e também em NCM460 (colonócitos derivados de mucosa intestinal normal) e HCT116 (carcinoma colo-rectal em fase pré-metastática), o que nos permite avaliar a importância deste mecanismo no desenvolvimento e progressão do cancro colorretal e, consequentemente, desenvolver novas abordagens terapêuticas, com a utilização de oligonucleótidos antisense de RNA para inibir esta tradução alternativa, o que constituirá uma proof-of-concept de uma potencial terapia para o cancro colorretal baseada em RNA. FCT info:eu-repo/semantics/publishedVersion

This article belongs to the Special Issue mRNA Metabolism in Health and Disease. Familial hypercholesterolemia (FH) is the most common genetic disorder of lipid metabolism, characterized by increased levels of total and LDL plasma cholesterol, which leads to premature atherosclerosis and coronary heart disease. FH phenotype has considerable genetic heterogeneity and phenotypic variability, depending on LDL receptor activity and lifestyle. To improve diagnosis and patient management, here, we characterized two single nucleotide missense substitutions at Methionine 1 of the human LDLR gene (c.1A>T/p.(Met1Leu) and c.1A>C/p.(Met1Leu)). We used a combination of Western blot, flow cytometry, and luciferase assays to determine the effects of both variants on the expression, activity, and synthesis of LDLR. Our data show that both variants can mediate translation initiation, although the expression of variant c.1A>T is very low. Both variants are in the translation initiation codon and codify for the same amino acid p.(Met1Leu), yet they lead to different levels of impairment on LDLR expression and activity, corroborating different efficiencies of the translation initiation at these non-canonical initiation codons. The functional data of these variants allowed for an improved American College of Medical Genetics (ACMG) classification for both variants, which can allow a more personalized choice of the lipid-lowering treatment and dyslipidemia management, ultimately improving patients' prognosis. The authors acknowledge Fundação para a Ciência e a Tecnologia (Ph.D. scholarship no. SFRH/PD/BD/131427/2017 and SFRH/PD/BD/114392/2016). info:eu-repo/semantics/publishedVersion

Up-frameshift 1 (UPF1) is the key factor in nonsense-mediated mRNA decay (NMD, a mechanism that degrades transcripts carrying premature translation termination codons. Besides its role in NMD, UPF1 has also a role in other mechanisms, such as cell cycle progression, being crucial for the G1/S transition, and in telomere maintenance and homeostasis. Furthermore, it has also been identified as having tumour suppressor activity in different cancers, such as hepatocellular carcinoma. Our data supports the existence of an IRES within the human UPF1 5’ untranslated region (UTR). In fact, using a bicistronic reporter system, we observed that UPF1 5’UTR can mediate internal translation in a 5’ cap-independent manner, both in normal and under stress conditions. We concluded that the first 100 nucleotides and the last 125 (out of a total of 275) are the minimal sequences essential for the identified IRES activity. According to the in silico predicted secondary structure, these two segments correspond to two stem-loops. Now, we are experimentally confirming this predicted secondary structure, and understanding the physiological role of this IRES-mediated activity in the NMD process. Also, we are testing its importance for UPF1 function as a tumour suppressor. For that, we are using custom-made antisense oligonucleotides that specifically target those stem-loops and see to what extent they inhibit UPF1 IRES-mediated activity and how such inhibition will play a role in the cell and the functions in which UPF1 participates. Also, we are testing some hallmarks of cancer, such as proliferation, invasion, and apoptosis inhibition when the UPF1 IRES is impaired. With this work, we wish to unravel a new layer of knowledge in gene expression regulation of UPF1. FCT info:eu-repo/semantics/publishedVersion

Introduction: With the advent of genomic sequencing, the identification of structural variants (SVs) is no longer a challenge, being possible to detect an average of 5 K SVs by individual. Contrarily, the annotation of the genome is incomplete, and the data is scattered along different databases, making SV manual evaluation complicated and time-consuming. Also, the available tools are limited on their scope. Thus, to address the need of a comprehensive application to assist evaluation of clinical outcome of SVs, we developed Structural Variant Interpreter (SVInterpreter). Methods: SVInterpreter is a free Python-CGI developed Web application able to analyze SVs using Topologically Associated Domains as genome units, within which genome browsers data, medically actionable genes, virtual gene panels and HPO similarity results, among other information, is retrieved. Results: We started by re-analysing 220 published SVs, of which about 50% were previously classified as VUS. SVInterpreter corroborated the previous classification in about 84% of the SVs. In about 5% of the SVs, SVInterpreter gave indication of possible position effect, through phenotype similarity, disrupted chromatin loops or genome wide association studies. Then, we show the applicability of SVInterpreter on the clinical setting, by inspecting 15 cases analysed by chromosomal microarray or genome sequencing. Conclusions: To our knowledge, SVInterpreter is the most comprehensive TAD based tool to assist prediction of clinical outcome of SVs. Based on gathered information, identification of possible disease-causing candidate genes and SVs is easily achievable. SVInterpreter is available at http://dgrctools-insa.min-saude.pt/cgi-bin/SVInterpreter.py info:eu-repo/semantics/publishedVersion

publicado em: Environ Health Perspect. 2021;1. https://ehp.niehs.nih.gov/doi/abs/10.1289/isee.2021.P-481 BACKGROUND AND AIM:The development of Human Biomonitoring (HBM) surveys is highly dependent on the cooperation and engagement of the individuals being sampled. As such, in the context of the European Human Biomonitoring Initiative (HBM4EU) an online citizen survey was conducted in Europe to gain insights into the perceptions of the population about HBM and exposure to chemicals. METHODS:The survey was undertaken between September 2020 and February 2021 and was available in the languages of all 30 HBM4EU-participating countries (EU, associated countries, Israel) through the HBM4EU website. Dissemination was done by the HBM4EU country representatives (National Hub Contact Points). RESULTS:Responses were received from 5391 participants. Around half were 35-54 years old and the majority were female, with higher education and employed. Concerning the perception of the respondents on HBM, the majority considers it should be done (81.7%), even if the chemicals are considered well managed. Most participants consider it a reliable method (84.0%) that should be performed more often (87.3%) and be more coordinated either at a European (86.2%) or at a national level (83.7%). The large majority of the respondents thinks that HBM results are important to evaluate the chemical exposure of the population (96.2%), to study the health impact of chemical exposure (96.3%) and to support the development of health policies (94.9%). The respondents also consider HBM results highly relevant to raise awareness and promote understanding of the impact of chemical exposure amongst the general population (94.3%), health professionals and policy makers. CONCLUSIONS:The use of HBM as a tool to assess human exposure to chemicals, allowing its management and health protection, was well perceived by respondents across Europe. Furthermore, the need for more studies conducted in a coordinated way was indicated. Future work should comprise population groups less represented here and should also attempt to understand how perceptions change over time. HBM4EU is funded by the European Union’s Horizon 2020 research and innovation programme under grant agreement Nº 733032 info:eu-repo/semantics/publishedVersion

This project was funded by FCT, UIDP/04046/2020 Familial hypercholesterolemia (FH) is the most common genetic disorder conferring an increased cardiovascular risk due to cholesterol accumulation since birth. The majority of patients with FH phenotype have mutations in LDLR, APOB or PCSK9 genes. In about 50% of patients a variant causing disease has not been possible to find. The 5' and 3’ untranslated regions (UTRs) and promoter of these genes is poorly studied. Consequently, few variants were detected in these locations and functional validation is lacking for the ones described. The aim of this project is to perform an in vitro characterization of variants in 5’ UTR and promoter of PCSK9 gene. N/A

This article belongs to the Special Issue mRNA Metabolism in Health and Disease 2.0. Many conditions can benefit from RNA-based therapies, namely, those targeting internal ribosome entry sites (IRESs) and their regulatory proteins, the IRES trans-acting factors (ITAFs). IRES-mediated translation is an alternative mechanism of translation initiation, known for maintaining protein synthesis when canonical translation is impaired. During a stress response, it contributes to cell reprogramming and adaptation to the new environment. The relationship between IRESs and ITAFs with tumorigenesis and resistance to therapy has been studied in recent years, proposing new therapeutic targets and treatments. In addition, IRES-dependent translation initiation dysregulation is also related to neurological and cardiovascular diseases, muscular atrophies, or other syndromes. The participation of these structures in the development of such pathologies has been studied, yet to a far lesser extent than in cancer. Strategies involving the disruption of IRES-ITAF interactions or the modification of ITAF expression levels may be used with great impact in the development of new therapeutics. In this review, we aim to comprehend the current data on groups of human pathologies associated with IRES and/or ITAF dysregulation and their application in the designing of new therapeutic approaches using them as targets or tools. Thus, we wish to summarise the evidence in the field hoping to open new promising lines of investigation toward personalised treatments. This work was partially supported by UID/MULTI/04046/2019 Research Unit grant (to BioISI) info:eu-repo/semantics/publishedVersion

Upstream open reading frames (uORFs) are cis-acting elements located within the 5’ leader sequence (5’UTR) of transcripts, which can regulate translation of the correspondent main open reading frame (mORF). During endoplasmic reticulum (ER) stress, the accumulation of unfolded proteins activates the ER-resident PKR-like ER kinase (PERK), which results in phosphorylation of eIF2α to inhibit global mRNA translation, while allowing the selective uORF-mediated translation of downstream effectors responsible for stress resolution or, ultimately, cell death. The dual role of PERK in regulating cell fate was implicated in human diseases, like diabetes, neurodegenerative disorders and cancer. Moreover, mutations in the EIF2AK3 gene (encoding PERK) were associated to the rare genetic disease, Wolcott-Rallison Syndrome (WRS). In this work, we aimed to study the translational regulatory role of 5 AUG- and 3 non-AUG-uORFs identified in the PERK 5’UTR and assess its biological relevance. While uORF2 and the non-AUG-uORFs 5, 6 and 7 (numbered according to their distance to the 5’ end of the mRNA) do not seem to have a regulatory role, uORF1, uORF3, uORF4 and uORF8 together present a strong repressive effect over mORF translation in basal conditions. Also, we observe that uORF1 is frequently translated allowing low levels of translation re-initiation at the main ORF. Curiously, we found that when PERK is overexpressed, it leads to the spontaneous activation of a portion of PERK in the absence of any stress stimulus, possibly highlighting the biological relevance of its uORF-mediated translational regulation. Conversely, during ER stress, increased bypass of uORF1 results in a modest degree of translational de-repression, which may help to counterbalance the increased rate of PERK protein turnover observed in these conditions. We also observed that alteration of the PERK uORFs by mutations found in WRS patients modify mORF expression, providing a possible link to the disease. Altogether, we highlight the importance of including 5’UTRs in the screening of disease-related mutations and the necessity of functional studies to assess their role in pathogenesis. Partially supported by UID/MULTI/04046/2013 center grant from FCT to BioISI. RF is recipient of a fellowship from BioSys PhD programme (SFRH/BD/114392/2016) from FCT. N/A

Eukaryotic cells have developed different mechanisms and adaptive pathways that allow them to cope with external stress stimuli. Under stress conditions, global protein synthesis is shut down, and some alternative mechanisms of mRNA translation initiation are induced. Although the tumour suppressor protein p53 — the most mutated gene in cancer — has been considered the guardian of the genome and a master regulator of several cellular functions, the truth is it is not just one isoform, the full-length (FLp53), but also many other p53 isoforms that have been described so far. Based on our previous results, some functions of the shorter isoforms are different from and complement FLp53 activity. Here we show the specific induction of Δ160p53 isoform during integrated stress response (ISR). We confirmed the presence of an Internal Ribosome Entry Site (IRES) in p53 mRNA that controls Δ160p53 isoform translation, using a bicistronic reporter construct. When subjecting cells to endoplasmic reticulum stress, we showed that eIF2α phosphorylation is a key event leading to cap-independent expression of Δ160p53 during ISR. Also, some cancer-specific mutations in the DNA-binding domain of p53 enhance cap-independent translation of Δ160p53 via Δ160p53IRES. Using an antisense morpholino oligo targeting Δ160IRES significantly reduces Δ160p53 protein levels and impaired its oncogenic functions. Additionally, we found the 5’untranslated region of Δ160p53 inhibits the IRES activity. Our data support a model in which an IRES structure in the coding region of p53 is activated under stress conditions, leading to the expression of the oncogenic shorter Δ160p53 isoform, whose structure is affected by cancer-specific mutations in the p53 gene. A better understanding of Δ160p53IRES structure and function may be advantageous for a more efficient therapeutic targeting of p53. FCT info:eu-repo/semantics/publishedVersion

The European Human Biomonitoring Initiative (HBM4EU) is a project gathering 30 countries, funded under Horizon 2020 and running from 2017 until 2021. The goal of HBM4EU is to generate evidence on the current exposure of European citizens to chemicals and on their possible health effects to assess the associated risks. Following a systematic prioritization exercise, the mycotoxins Deoxynivalenol (DON) and Fumonisin B1 (FB1) were considered as priority substances around which the HBM4EU research programme was developed. As part of the HBM4EU project, several policy questions are being addressed for these mycotoxins, concerning analytical methods, exposure levels and high exposure population groups in Europe (including workers), associated time trends, risk characterization, exposure models and toxicokinetic data, human biomonitoring guidance values, key events that determine the health effects of the target mycotoxins, effect biomarkers, data gaps and research needs. Key outputs from HBM4EU achieved until now for DON and FB1, include: i) a biomarker selected to assess human exposure to DON (total urinary DON) that will be used in the aligned studies, ii) several European laboratories selected to perform DON analysis after passing an interlaboratory study, ii) a research protocol on human exposure and geographic variations in Europe, iv) a risk assessment plan to assess DON and FB1 exposure in Europe, v) a review of available toxicokinetics models, vi) a draft on the possible mechanisms of FB1-induced adverse health effects and vii) a specific effect biomarker for FB1. Work co-funded by the HBM4EU project, Grant Agreement No: 733032 and by Portuguese national funds: FCT/MCTES through CESAM (UIDP/50017/2020 + UIDB/50017/2020) and ToxOmics (UIDB/00009/2020 & UIDP/00009/2020). N/A

Review About 11% of all human disease-associated gene lesions are nonsense mutations, resulting in the introduction of an in-frame premature translation-termination codon (PTC) into the protein-coding gene sequence. When translated, PTC-containing mRNAs originate truncated and often dysfunctional proteins that might be non-functional or have gain-of-function or dominant-negative effects. Therapeutic strategies aimed at suppressing PTCs to restore deficient protein function—the so-called nonsense suppression (or PTC readthrough) therapies—have the potential to provide a therapeutic benefit for many patients and in a broad range of genetic disorders, including cancer. These therapeutic approaches comprise the use of translational readthrough-inducing compounds that make the translational machinery recode an in-frame PTC into a sense codon. However, most of the mRNAs carrying a PTC can be rapidly degraded by the surveillance mechanism of nonsense-mediated decay (NMD), thus decreasing the levels of PTC-containing mRNAs in the cell and their availability for PTC readthrough. Accordingly, the use of NMD inhibitors, or readthrough-compound potentiators, may enhance the efficiency of PTC suppression. Here, we review the mechanisms of PTC readthrough and their regulation, as well as the recent advances in the development of novel approaches for PTC suppression, and their role in personalized medicine. This work was partially supported by UID/MULTI/04046/2019 Research Unit Grant (to BioISI) and by PTFC/BIM-MEC/3749/2014 research Grant (to LR) from Fundação para a Ciência e a Tecnologia, Portugal. info:eu-repo/semantics/publishedVersion

[Abstract] The alkaline comet assay, or single cell gel electrophoresis, is one of the most popular methods for assessing DNA damage in human population. One of the open issues concerning this assay is the identification of those factors that can explain the large inter-individual and inter-laboratory variation. International collaborative initiatives such as the hCOMET project - a COST Action launched in 2016 - represent a valuable tool to meet this challenge. The aims of hCOMET were to establish reference values for the level of DNA damage in humans, to investigate the effect of host factors, lifestyle and exposure to genotoxic agents, and to compare different sources of assay variability. A database of 19,320 subjects was generated, pooling data from 105 studies run by 44 laboratories in 26 countries between 1999 and 2019. A mixed random effect log-linear model, in parallel with a classic meta-analysis, was applied to take into account the extensive heterogeneity of data, due to descriptor, specimen and protocol variability. As a result of this analysis interquartile intervals of DNA strand breaks (which includes alkali-labile sites) were reported for tail intensity, tail length, and tail moment (comet assay descriptors). A small variation by age was reported in some datasets, suggesting higher DNA damage in oldest age-classes, while no effect could be shown for sex or smoking habit, although the lack of data on heavy smokers has still to be considered. Finally, highly significant differences in DNA damage were found for most exposures investigated in specific studies. In conclusion, these data, which confirm that DNA damage measured by the comet assay is an excellent biomarker of exposure in several conditions, may contribute to improving the quality of study design and to the standardization of results of the comet assay in human populations. This article is based upon work from COST Action hCOMET CA15132, supported by COST (European Cooperation in Science and Technology www.cost.eu) - STSM fellowships for Mirta Milić (IMROH, EU 19); IMROH, Zagreb, Croatia, Institute for Medical Research and Occupational Health (IMROH), Zagreb, Croatia, and the Ministry of Science, Education and Sports of the Republic of Croatia (Grant No. 022-0222148-2125) (EU4); Cancer Plan for PestiBG; Grant number: no ENV201401(EU 8, EU9); Italian Ministry of Education, University and Research PRIN 2005, prot. 2005058197 and Cariplo Foundation (Milan, Italy), Rif. Pratica 2007-5810 and Rif. Pratica 2010.2303 (EU 18); Associazione Italiana per la Ricerca sul Cancro (AIRC) (IG 2015/17564). (EU19); European Union Integrated Projects New Generis, 6th Framework Programme, Priority 5: Food Quality and Safety; Newborns and Genotoxic Exposure Risks, FOOD-CT-2005-016320 (EU22); ACT project No. 036APy/09 and No. 005DBB/12 (EU 24); FCT-SFRH/BPD/96196/2013, SFRH/BPD/100948/2014, Portugal (EU 26); MZ 2012/8-UKBA-8; VEGA 1/0703/13, APVV 15-0063 (EU30); Xunta de Galicia (XUGA 10605B98; INCITE08PXIB106155PR; ED481B2016/190-0; Grants ED431B2019/02), Spain (EU 32); Grant 01 173034, Ministry of Education, Science and Technological Development of the Republic of Serbia (EU 42); The Centre for Industrial and Technological Development within National Strategic Consortia for Techical Research (Industrial Research diets and food with specific characteristics for elderly, SENIFOOD); University of Navarra LE/97; Physiopathology of Obesity and Nutrition (CIBER Obn); Carlos III Health Research Institute (CB12/03/30002); Ministerio de Economia y Compatitividad (‘Ramón y Cajal’ Programme, RYC-2013-14370) of the Spanish Government for personal support (EU 45); the Ministry of Education, Youth and Sports of the Czech Republic project Healthy Aging in Industrial Environment HAIE (CZ.02.1.01/0.0/0.0/16_019/0000798) which is co-financed by the European Union (European Structural and Investment funds; Operation Programme Research, Development and Education); MYES LO 1508 (EU 46); MICRODIAB Study; ClinicalTrials.org (#NCT02231736) (EU 52); The study was funded by the Italian Ministry for Education, University and Scientific Research (MIUR) - Research No. 2005-062547 (EU14, EU53); Projects financed from Serbian Ministry of Education, Science and Technological Development #11146002, #175035, #173034 (EU 54); Mehr foundation organisation, UK (EU 55); MCTI/CNPQ No. 01/2016-Universal; FAPESC No. 09/2015; MEC/MCTI/CAPES/CNPQ/FAPS/ No. 09/2014, Brazil (CSA 6); the National Nuclear Energy Agency of Indonesia (Badan Tenaga Nuklir Nasional) with contract number 080.01.06 3447.001 001.052.A (AS4); Slovak Grant Agency (APVT-21 013202, APVT-21- 017704); Ministry of Health, Slovak Republic (2005/43-SZU-21, 2006/07- SZU-02 MZ SR, 2005/42-SZU-20

The DIS3-like 3′-5′ exoribonuclease 2 (DIS3L2) triggers decay in an exosome-independent manner and preferentially degrades RNA species possessing a non-templated oligo-uridine 3’-end tail. It is capable of inducing decay over a variety of RNAs, including mRNAs, rRNAs, miRNAs and other non-coding RNAs. It has been shown that DIS3L2 is involved in cancer-related cellular processes. Nevertheless, its function in tumorigenesis remains largely unexplored. Recently, we and others showed that DIS3L2-mediated decay together with uridylation also participate in nonsense-mediated mRNA decay (NMD), thus revealing a new NMD branch. NMD is a surveillance pathway that recognizes and degrades mRNAs harboring premature translation-termination codons, protecting the cell from potentially harmful truncated proteins. However, NMD also regulates the level of normal and fully functional mRNAs, arising as a mechanism of gene expression regulation. Here, we aim to analyze how DIS3L2 and uridylation regulate the human transcriptome, in order to shed light on how this ribonuclease is related to NMD and how its deregulation contributes to tumorigenesis. For this purpose, high-throughput mRNA sequencing has been performed in the SW480 colorectal cancer cell line depleted of DIS3L2 or DIS3L2 plus terminal uridylyl transferases 4 and 7. Gene ontology analysis over the set of genes up-regulated under those two conditions, show enrichment in molecular functions and biological processes related with cancer, and cell events directly implicated in RNA processing and RNA degradation. Preliminary results on the features of the deregulated transcripts also show significant differences between conditions, an important aspect that is guiding us to determine grades of sensitivities in the decay of DIS3L2-subtrates. Currently, we are unveiling the role of DIS3L2 in oncogenesis and analyzing its substrate specificity. N/A

Upstream open reading frames (uORFs) are cis-acting elements located within the 5’ leader sequence (5’UTR) of transcripts, which can regulate translation of the correspondent main open reading frame (mORF). During endoplasmic reticulum (ER) stress, the accumulation of unfolded proteins activates the ER-resident PKR-like ER kinase (PERK), which results in phosphorylation of eIF2α to inhibit global mRNA translation, while allowing the selective uORF-mediated translation of downstream effectors responsible for stress resolution or, ultimately, cell death. The dual role of PERK in regulating cell fate was implicated in human diseases, like diabetes, neurodegenerative disorders and cancer. Moreover, mutations in the EIF2AK3 gene (encoding PERK) were associated to the rare genetic disease, Wolcott-Rallison Syndrome (WRS). In this work, we aimed to study the translational regulatory role of 5 AUG- and 3 non-AUG-uORFs identified in the PERK 5’UTR and assess its biological relevance. While uORF2 and the non-AUG-uORFs 5, 6 and 7 (numbered according to their distance to the 5’ end of the mRNA) do not seem to have a regulatory role, uORF1, uORF3, uORF4 and uORF8 together present a strong repressive effect over mORF translation in basal conditions. Curiously, we found that when PERK is overexpressed, it leads to the spontaneous activation of a portion of PERK in the absence of any stress stimulus, possibly highlighting the biological relevance of its uORF-mediated translational regulation. Conversely, during ER stress, increased bypass of uORF1 results in a modest degree of translational de-repression, which may help to counterbalance the increased rate of PERK protein turnover observed in these conditions. We also observed that alteration of the PERK uORFs by mutations found in WRS patients modify mORF expression, providing a possible link to the disease. Altogether, we highlight the importance of including 5’UTRs in the screening of disease-related mutations and the necessity of functional studies to assess their role in pathogenesis. work partially supported by UID/MULTI/04046/2013 center grant to BioISI and PTDC/MED-ONC/32048/2017 to LR from FCT. RF is recipient of a fellowship from BioSys PhD programme (SFRH/BD/114392/2016) from FCT. N/A

The tumor microenvironment is characterized by several stresses impairing canonical translation. However, specific mRNAs harbouring internal ribosome entry sites (IRES), such as several tumour suppressors and oncogenes, can overcome this impairment. The tumor suppressor TP53 gene, an important transcription factor that ensures cellular homeostasis, is frequently mutated in human cancers. Over the years, several p53 isoforms have been identified, which in some cases result from alternative initiation of translation regulated by an IRES. Recently, we have associated mutant p53 “gain-of-function” cancer phenotype, such as enhanced cell survival, invasion, proliferation, and adhesion, with the expression of higher levels of shorter p53 isoforms, such as Δ160p53 isoform.1 Here, we used a bicistronic system containing two reporter luciferases (renilla luciferase and firefly luciferase) to assess IRES-mediated translation. Several p53 mRNA elements were tested in this system and, interestingly, we have found an inhibitory element of IRES-mediated translation. Overall, IRES-regulated translation in malignant cells is used to translate specific proteins that promote cancer progression. Thus, inhibiting translation of oncogenes via IRES could prevent the formation of tumor cells and their adaptation to unfavourable conditions in the tumor microenvironment. 1. Candeias, M. M., Hagiwara, M. & Matsuda, M. Cancer‐specific mutations in p53 induce the translation of Δ160p53 promoting tumorigenesis. EMBO Rep. 17, 1542–1551 (2016). The project is funded by FCT grant PTDC/MED-ONC/32048/2017 N/A

Cancer is the second leading cause of death globally; therefore, its study is crucial to discover new therapies. Under stress, the regular process of protein synthesis (canonical translation) is impaired, while a back-up mechanism mediated by internal ribosome entry sites (IRES) continues to function, allowing the synthesis of proteins that maintain cellular viability. This also happens in cancer cells, contributing for their survival and consequent tumorigenesis. IRES-mediated translation and its regulation by IRES trans-acting factors (ITAFs) has been correlated to metastasis and chemotherapeutic drug resistance. Therefore, our main goal was to validate ITAFs and assess their significance in cancer onset, thus becoming candidates as novel therapeutic targets. A bicistronic reporter system, which contains a first cistron translated via canonical translation and a second one translated by IRES of mTOR1 and AGO12 was used to test IRES-driven translation initiation activity. Experiments were carried out in which several proteins (hnRNPs) were silenced by specific siRNAs to analyse their function as ITAFs of mTOR and AGO1 IRESs. Also, distinct drugs were applied to simulate endoplasmic reticulum (ER) or hypoxia stress, to evaluate their effect on IRES activity. The relative IRES activity was assessed by luminescence tests and the protein levels by Western blot. In general, knockdown of hnRNPK and hnRNPU seems to decrease the IRES activity by ~60% and ~30% respectively, while hnRNPC knockdown does not show a significant effect. Regarding the ER stress, hnRNPK knockdown seems to decrease even more the IRES activity, while hnRNPU depletion induces a significant increase. On the other hand, in hypoxia, the hnRNPs knockdowns do not significantly affect IRES activity. These results indicate that hnRNPK and hnRNPU may function as ITAFs of mTOR and AGO1 IRES activity in cells under ER stress. Our data can be decisive for a better understanding of carcinogenesis and suggest new therapeutic targets for cancer treatment. 1. Marques-Ramos, A., et.al. 2017. RNA. 23, 1712-1728 2. Lacerda, R. 2016. Faculdade de Ciências e Tecnologia da Universidade NOVA de Lisboa Work partially supported by UID/MULTI/04046/2019 Research Unit grant from FCT, Portugal (to BioISI) info:eu-repo/semantics/publishedVersion

This article belongs to the Special Issue mRNA Metabolism in Health and Disease Autism Spectrum Disorder (ASD) is a heterogeneous neurodevelopmental condition with unclear etiology. Many genes have been associated with ASD risk, but the underlying mechanisms are still poorly understood. An important post-transcriptional regulatory mechanism that plays an essential role during neurodevelopment, the Nonsense-Mediated mRNA Decay (NMD) pathway, may contribute to ASD risk. In this study, we gathered a list of 46 NMD factors and regulators and investigated the role of genetic variants in these genes in ASD. By conducting a comprehensive search for Single Nucleotide Variants (SNVs) in NMD genes using Whole Exome Sequencing data from 1828 ASD patients, we identified 270 SNVs predicted to be damaging in 28.7% of the population. We also analyzed Copy Number Variants (CNVs) from two cohorts of ASD patients (N = 3570) and discovered 38 CNVs in 1% of cases. Importantly, we discovered 136 genetic variants (125 SNVs and 11 CNVs) in 258 ASD patients that were located within protein domains required for NMD. These gene variants are classified as damaging using in silico prediction tools, and therefore may interfere with proper NMD function in ASD. The discovery of NMD genes as candidates for ASD in large patient genomic datasets provides evidence supporting the involvement of the NMD pathway in ASD pathophysiology. This research was supported by Fundação para a Ciência e a Tecnologia (UIDB/04046/2020 and UIDP/04046/2020 Centre grants to BioISI and PAC-POCI-01-0145-FEDER-016428 MEDPERSYST to A.M.V.) and by National Institute of Health Doutor Ricardo Jorge. A.R.M., J.V. and J.X.S. are recipients of a fellowship from BioSys PhD programme PD65-2012 (A.R.M. Ref: PD/BD/113773/2015; J.X.S. Ref: PD/BD/114386/2016; J.V. Ref: PD/BD/131390/2017) from Fundação para a Ciência e a Tecnologia (Portugal). info:eu-repo/semantics/publishedVersion

Upstream open reading frames (uORFs) are cis-acting elements located within the 5’ leader sequence (5’UTR) of transcripts, which can regulate translation of the correspondent main open reading frame (mORF). During endoplasmic reticulum (ER) stress, the accumulation of unfolded proteins activates the ER-resident PKR-like ER kinase (PERK), which results in phosphorylation of eIF2α to inhibit global mRNA translation, while allowing the selective uORF-mediated translation of downstream effectors responsible for stress resolution or, ultimately, cell death. The dual role of PERK in regulating cell fate was implicated in human diseases, like diabetes, neurodegenerative disorders and cancer. Moreover, mutations in the EIF2AK3 gene (encoding PERK) were associated to the rare genetic disease, Wolcott-Rallison Syndrome (WRS). In this work, we aimed to study the translational regulatory role of 5 AUG- and 3 non-AUG-uORFs identified in the PERK 5’UTR and assess its biological relevance. While uORF2 and the non-AUG-uORFs 5, 6 and 7 (numbered according to their distance to the 5’ end of the mRNA) do not seem to have a regulatory role, uORF1, uORF3, uORF4 and uORF8 together present a strong repressive effect over mORF translation in basal conditions. Curiously, we found that when PERK is overexpressed, it leads to the spontaneous activation of a portion of PERK in the absence of any stress stimulus, possibly highlighting the biological relevance of its uORF-mediated translational regulation. Conversely, during ER stress, increased bypass of uORF1 results in a modest degree of translational de-repression, which may help to counterbalance the increased rate of PERK protein turnover observed in these conditions. We also observed that alteration of the PERK uORFs by mutations found in WRS patients modify mORF expression, providing a possible link to the disease. Altogether, we highlight the importance of including 5’UTRs in the screening of disease-related mutations and the necessity of functional studies to assess their role in pathogenesis. Work partially supported by UID/MULTI/04046/2013 center grant to BioISI and PTDC/MED-ONC/32048/2017 to LR from FCT. RF is recipient of a fellowship from BioSys PhD programme (SFRH/BD/114392/2016) from FCT info:eu-repo/semantics/publishedVersion

Based in part on the previous version of this eLS article “ Upstream Open Reading Frames and Human Genetic Disease” (2014) by Cristina Barbosa, Cláudia Onofre and Luísa Romão. Upstream open reading frames (uORFs) are cis‐acting elements, located before or overlapped with the main coding ORF (mORF), that regulate cap‐dependent translation efficiency in a transcript‐specific manner. More than half of the human transcripts bear at least one uORF. In addition, it has been recently revealed that many of these uORFs initiate at non‐AUG codons, which significantly increases the complexity and diversity of the human translatome. These regulons are considered repressors of downstream translation but, in some biological contexts, they induce mORF expression. There are several the mechanisms by which AUG and non‐AUG uORFs regulate gene expression, allowing the cell to control transcript‐specific translation according to its needs. Also, we describe several examples of uORF genetic variants associated with human genetic diseases. Studying these cases and understanding the resultant abnormal mechanisms of uORF‐mediated translational control is of extreme importance for the development of new therapeutic strategies. Key Concepts: Upstream open reading frames (uORFs) are cis‐acting translational regulatory elements present within the 5′ leader sequence of mRNAs. uORFs can regulate gene expression by repressing or promoting translation of the downstream main ORF (mORF), according to the cellular environment. The number of uORFs, the intercistronic distance, the overlap with the mORF and the context of the initiation codon are the uORF‐related structural features that most influence their translational regulatory capacity: uORF‐mediated repression of mORF translation is usually achieved by ribosome dissociation, ribosome stalling, induction of nonsense‐mediated mRNA decay (NMD) or production of inhibitory peptides; uORF‐mediated induction of mORF translation is usually achieved by ribosome bypass or translation reinitiation; uORFs initiated by non‐AUG codons are more frequent than previously appreciated, having important biological functions; uORF‐altering polymorphisms and mutations, which create, disrupt or change a uORF, can cause human genetic diseases. Studying and understanding the uORF‐mediated mechanisms of gene expression regulation may provide knowledge to develop novel therapies for several human diseases. info:eu-repo/semantics/publishedVersion

Review Alternative splicing (AS) of precursor mRNA (pre-mRNA) is a cellular post-transcriptional process that generates protein isoform diversity. Nonsense-mediated RNA decay (NMD) is an mRNA surveillance pathway that recognizes and selectively degrades transcripts containing premature translation-termination codons (PTCs), thereby preventing the production of truncated proteins. Nevertheless, NMD also fine-tunes the gene expression of physiological mRNAs encoding full-length proteins. Interestingly, around one third of all AS events results in PTC-containing transcripts that undergo NMD. Numerous studies have reported a coordinated action between AS and NMD, in order to regulate the expression of several genes, especially those coding for RNA-binding proteins (RBPs). This coupling of AS to NMD (AS-NMD) is considered a gene expression tool that controls the ratio of productive to unproductive mRNA isoforms, ultimately degrading PTC-containing non-functional mRNAs. In this review, we focus on the mechanisms underlying AS-NMD, and how this regulatory process is able to control the homeostatic expression of numerous RBPs, including splicing factors, through auto- and cross-regulatory feedback loops. Furthermore, we discuss the importance of AS-NMD in the regulation of biological processes, such as cell differentiation. Finally, we analyze interesting recent data on the relevance of AS-NMD to human health, covering its potential roles in cancer and other disorders. This work was partially supported by UID/MULTI/04046/2019 Research Unit grant from Fundação para a Ciência e a Tecnologia (FCT), Portugal (to BioISI). J.F.G.-M. is recipient of a fellowship from BioSys PhD programme PD65-2012 (Ref SFRH/PD/BD/142898/2018) from FCT. info:eu-repo/semantics/publishedVersion

The scanning model for eukaryotic mRNA translation initiation states that the small ribosomal subunit, along with initiation factors, binds to the cap structure at the 5’ end of the mRNA and scans the 5’ untranslated region (5’UTR) until an initiation codon is found. However, when cells are exposed to stress stimuli, cap-dependent translation is inhibited, while the synthesis of some proteins is maintained by alternative mechanisms of translation initiation, which are vital for cell survival and stress recovery. Here we show two examples in which a translational switch occurs during integrated stress response (ISR). In the first case, tumor suppressor p53, we show that the ISR leads to the specific induction of a shorter p53 isoform (Δ160p53 isoform). This induction is dependent on translation elongation but does not require the eIF4E-eIF4G interaction. Studies using bicistronic constructs with wild-type Δ160p53 or reporter genes confirmed the presence of an Internal Ribosome Entry Site (IRES) in p53 mRNA, being eIF2α phosphorylation a key event leading to cap-independent expression of Δ160p53 during ISR. Interestingly, cancer-specific mutations in p53 also enhance cap-independent translation of Δ160p53 via Δ160p53IRES. Our data support a model in which an IRES structure in the coding region of p53, and the cancer-specific mutations that affect this structure, control p53 oncogenic functions by regulating Δ160p53 protein expression. A better understanding of Δ160p53IRES structure and function may be advantageous for a more efficient therapeutic targeting of p53. Human up-frameshift 1 (UPF1) is a key-protein involved in nonsense-mediated mRNA decay, telomere replication and homeostasis, and cell cycle progression. These crucial UPF1 functions suggest its tight gene expression regulation. Indeed, our results show that UPF1 5’UTR is able to mediate cap-independent translation in a bicistronic luciferase vector expressed in cervical and colorectal cancer cell lines. Such activity is maintained under endoplasmic reticulum stress. Interestingly, we found that the UPF1 5’UTR IRES function is inhibited when the first 100 nucleotides, or the last 125, are absent or altered. Understanding these IRESs mechanism of function and their biological relevance might provide tools for developing new therapies for human diseases such as cancer. This work was supported by grants PTDC/MED-ONC/32048/2017 and PTDC/BIM-ONC/4890/2014 from the Fundação para a Ciência e a Tecnologia (FCT), by Grants-in-Aid 16K21111 and 18K07229 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, by Takeda Foundation and Astellas Grant, by Instituto Nacional de Saúde Doutor Ricardo Jorge, and by UIDB/04046/2020 and UIDP/04046/2020 Centre grants from FCT, Portugal (to BioISI). info:eu-repo/semantics/publishedVersion

Tese de doutoramento em Biologia (Biologia de Sistemas), apresentada à Faculdade de Ciências da Universidade de Lisboa, 2020. Tese orientada por: Doutora Luísa Romão (orientadora) e Doutora Mafalda Bourbon (co-orientadora). Upstream open reading frames (uORFs) are cis-acting elements located within the 5’ leader sequence (or 5’ untranslated region; 5’UTR) of transcripts that can regulate translation of the correspondent main open reading frame (mORF). During basal conditions, uORFs are typically considered to be repressors of downstream translation, as they can impede the ribosomes to access the mORF or even induce mRNA degradation by the nonsense-mediated mRNA decay (NMD) pathway. However, during stress conditions, phosphorylation of the eukaryotic initiation factor 2α (eIF2α) allows the expression of several stress-responsive proteins through uORF-mediated mechanisms, while global mRNA translation is inhibited. During endoplasmic reticulum (ER) stress, for instance, the accumulation of unfolded proteins leads to activation of the ER-resident PKR-like ER kinase (PERK) that phosphorylates eIF2α as part of the stress-protective mechanisms of the unfolded protein response (UPR) and the integrated stress response (ISR). This results in the selective uORF-mediated translation of downstream effectors, like the activating transcription factor 4 (ATF4), the CCAAT-enhancer-binding protein homologous protein (CHOP) and the growth arrest and DNA damage-inducible protein 34 (GADD34), which drive stress resolution or, in the case of a prolonged stress, cell death. The dual role of PERK in regulating cell fate has been reported to be involved in the outcome of several human diseases, including diabetes, neurodegenerative disorders and cancer. Moreover, mutations in the EIF2AK3 gene that encodes PERK have been implicated in the development of the rare genetic disease, Wolcott-Rallison Syndrome (WRS). Interestingly, data from ribosome-profiling (Ribo-seq) studies suggested the existence of uORFs within PERK 5’UTR, which could be involved in the regulation of PERK expression. In this work, we aimed to study the translational regulatory role of five AUG- and three non-AUG-uORFs identified in the PERK 5’UTR and assess its impact in cell homeostasis and human disease. While uORF2 and the non-AUG-uORFs 5, 6 and 7 do not seem to have a significant regulatory role, uORF1, uORF3, uORF4 and uORF8 together present a strong repressive effect over mORF translation in basal conditions, possibly by providing a barrier to the scanning ribosomes and precluding translation reinitiation at the mORF, without affecting the PERK mRNA levels. Curiously, we found that when we induce PERK overexpression, it leads to the spontaneous activation of a portion of PERK in the absence of any stress stimulus, possibly highlighting the biological relevance of its uORF-mediated translational regulation in maintaining its physiological basal levels. Conversely, during stress conditions, increased bypass of uORF1 results in a modest degree of translational de-repression, which may help to counterbalance the increased rate of PERK protein turnover observed in these conditions. We also found that alteration of the PERK uORFs by mutations found in WRS patients modify PERK expression, providing a possible link with the disease phenotype. Finally, we tested the impact of PERK unbalanced expression in the viability of HCT116 cells but, at least in our experimental conditions, no differences were found. Altogether, we provide a new example of a transcript containing uORFs that fine-tune mORF translation. Moreover, we highlight the importance of including 5’UTRs, like the one of PERK, in the screening of stress-related mutations and the necessity of functional studies to assess their relevance in the pathogenesis of human diseases. This may provide vital information for the development of new therapeutic strategies. A expressão génica em eucariotas é um processo complexo que compreende vários passos altamente regulados. Em particular, a tradução do RNA mensageiro (mRNA) representa um passo chave da expressão génica, cuja regulação permite à célula rapidamente alterar a síntese de proteínas de uma forma espácio-temporal em resposta a diferentes estímulos. O processo de tradução divide-se em quatro etapas: iniciação, alongamento, terminação e reciclagem de ribossomas. A etapa de iniciação representa o passo limitante e, como tal, é o mais regulado. De entre os múltiplos fatores regulatórios que podem atuar nesta etapa, encontram-se as pequenas grelhas de leitura a montante (do inglês, upstream open reading frames, uORFs), que correspondem a pequenas regiões potencialmente traduzidas definidas por um codão de iniciação na região 5’ não traduzida (do inglês, 5’ untranslated region, 5’UTR) dos mRNAs, em fase com um codão de terminação a montante ou sobreposto com a grelha de leitura principal (do inglês, main open reading frame, mORF) que codifica a proteína. As uORFs são tipicamente consideradas repressoras da tradução em condições fisiológicas normais, uma vez que funcionam como “barreiras” aos ribossomas que, ao traduzirem-nas, podem não chegar a traduzir a mORF. Outra característica regulatória importante das uORFs é a possibilidade de induzirem a degradação do mRNA pelo processo de decaimento do RNA mensageiro mediado por mutações sem sentido (do inglês, nonsense-mediated mRNA decay, NMD), uma vez que os seus codões de terminação podem ser reconhecidos como prematuros aquando da terminação da tradução. Contudo, em condições de stress as uORFs são muitas vezes responsáveis por permitir a tradução da mORF. Isto acontece porque nestas condições ocorre a fosforilação do fator eucariótico de iniciação da tradução 2α (do inglês, eukaryotic initiation factor 2α, eIF2α), o que favorece o não reconhecimento de codões de iniciação de algumas uORFs normalmente com contextos Kozak fracos (do inglês, leaky scanning ou ribossome bypass), ou a ocorrência de reiniciação da tradução na mORF após tradução da uORF. A proteína cinase tipo PKR residente no retículo endoplasmático (do inglês, PKR-like ER kinase, PERK) é uma das responsáveis por fosforilar o eIF2α em condições de stress. Estruturalmente, a PERK é uma proteína transmembranar do ER que possui três domínios: (i) um domínio lumenal regulatório; (ii) um domínio transmembranar; e (iii) um domínio catalítico citoplasmático. Em condições de homeostasia, a PERK encontra-se num estado monomérico inativo. No entanto, quando ocorre stress do ER, a acumulação de proteínas mal enoveladas promove a oligomerização de domínios lumenais de PERK que aproximam os domínios citoplasmáticos de forma a promover a sua auto fosforilação e consequente ativação. Uma vez ativada, a PERK fosforila o eIF2α de forma a inibir globalmente a tradução do mRNA como mecanismo de proteção, enquanto favorece a tradução mediada por uORFs de proteínas responsáveis por aumentar a capacidade processadora do ER como parte dos mecanismos da resposta a proteínas mal enoveladas (do inglês, unfolded protein response, UPR) e da resposta integrada ao stress (do inglês, integrated stress response, ISR). Algumas destas proteínas são a ATF4 (do inglês, activating transcritpion factor 4), a CHOP (do inglês, CCAAT-enhancer-binding protein homologous protein) e a GADD34 (do inglês, growth arrest and DNA damageinducible protein 34) que, no caso de um estímulo prolongado ou intenso, podem também desencadear vias pró-apoptóticas. O papel da PERK na regulação da homeostasia da célula e da sua sobrevivência foi implicado em várias doenças, como a diabetes mellitus, as doenças neurodegenerativas e o cancro. Além disso, mutações no gene que codifica a PERK (o gene EIF2AK3) foram associadas ao desenvolvimento da doença rara, síndrome de Wolcott-Rallison (do inglês, WolcottRallison syndrome, WRS), caracterizada por diabetes mellitus neonatal permanente e displasia epifisária múltipla. Isto sugere que a expressão e a atividade da PERK são essenciais para o normal funcionamento da célula, devendo ser corretamente reguladas. Curiosamente, dados de perfil ribossomal (do inglês, ribosome profiling) mostram a existência de vários eventos de iniciação de tradução na 5’UTR do mRNA da PERK, até mesmo em codões de iniciação não canónicos, o que sugere a existência de uORFs com potencial regulatório. Tendo em conta a inexistência de estudos sobre as uORFs do mRNA da PERK, o objetivo do presente trabalho foi estudar o papel regulatório de cinco uORFs iniciadas por AUG e três uORFs iniciadas por codões não canónicos identificadas no transcrito da PERK, bem como avaliar a sua relevância para a homeostasia da célula e a saúde humana. Para o efeito, a sequência de DNA complementar da 5’UTR do mRNA da PERK foi clonada a montante da ORF da luciferase do pirilampo para obter um plasmídeo que permite quantificar, através de ensaios de luminometria, o efeito das uORFs na tradução da mORF. Por mutagénese dirigida foram obtidas várias construções que permitiram estudar o efeito individual ou combinado dessas uORFs e explorar o seu mecanismo de regulação da tradução. De acordo com os nossos resultados, as uORFs da PERK inibem fortemente a tradução da mORF, tendo-se registado uma redução de 92% e de 89% na atividade relativa da luciferase promovida pela 5’UTR da PERK na linha celular de cancro colorretal, HCT116, e na linha celular embrionária de rim, HEK293, respetivamente. Este efeito repressor é mediado principalmente pela uORF1 iniciada por um codão AUG com contexto Kozak forte, e é maximizado pelas uORFs 3, 4 e 8, também iniciadas por AUG mas com contextos intermédios. As restantes uORFs da PERK, a uORF2 iniciada por AUG, e as uORFs iniciadas por codões não canónicos, as uORFs 5, 6 e 7, parecem não contribuir significativamente para este processo regulatório, provavelmente por serem iniciadas por codões de iniciação com contextos Kozak fracos que favorecem o leaky scanning. Tendo em conta o tamanho das uORFs, é possível que o efeito repressor das uORFs 1, 3 e 4 seja devido ao impedimento de reiniciação da tradução na mORF após a sua tradução. Já a uORF8, que é mais pequena e poderia permitir reiniciação, poderá dever o seu papel inibitório ao facto de ter o codão de terminação sobreposto ao codão de iniciação da PERK. Em concordância com um papel regulatório ao nível da tradução, estas uORFs não parecem induzir a degradação do mRNA da PERK por NMD. Tendo em conta que verificámos que a PERK é uma proteína estável em condições normais e que, quando em excesso, pode ser espontaneamente ativada e promover inadvertidamente a inibição da tradução global, sugerimos que o efeito repressor das suas uORFs contribui para manter a expressão da PERK apenas a um nível basal. Em oposição, verificámos que em células tratadas com o indutor de stress do ER, tapsigargina, a degradação da PERK é favorecida. Curiosamente, a fosforilação do eIF2α que ocorre nestas condições permite algum leaky scanning da uORF1, resultando numa ligeira de-repressão ao nível da tradução da mORF, que poderá, desta forma, ajudar a contrabalançar a perda de PERK. Tendo em conta o papel notório das uORFs na regulação dos níveis da PERK e a relação desta cinase com o desenvolvimento de WRS, estudámos o efeito de mutações identificadas em doentes com WRS, que eliminam ou alteram as uORFs, na regulação da expressão da PERK. Os nossos resultados mostram que algumas destas mutações podem alterar a taxa de produção de PERK, o que poderá explicar o fenótipo da doença, necessitando, no entanto, de confirmação experimental adicional. Finalmente estudámos o impacto que a expressão excessiva ou deficiente de PERK poderia ter na viabilidade de células HCT116, não tendo verificado qualquer alteração significativa, para além da ativação espontânea de uma porção da PERK quando sobre expressa. No entanto, existem outros estudos usando linhas celulares e condições experimentais diferentes nos quais a produção desequilibrada de PERK afeta a viabilidade ou o normal funcionamento da célula, sugerindo que o contexto biológico pode influenciar as consequências da desregulação da PERK. Em suma, neste estudo é apresentado um novo exemplo de um transcrito cuja tradução da mORF é regulada por uORFs. Adicionalmente, é destacada a importância de se incluírem as 5’UTRs dos genes, como o da PERK, no rastreio de mutações que possam estar associadas ao desenvolvimento de doenças genéticas, bem como o estudo detalhado das suas consequências biológicas. Este tipo de informações é imprescindível para o desenvolvimento de novas terapias ou para a aplicação adequada das já existentes. Rafael Queirós Fernandes foi bolseiro de doutoramento no âmbito do Programa doutoral BioSys em Sistemas Biológicos, Genómica Funcional & Integrativa (FCT/PD/00065/2012) da Faculdade de Ciências da Universidade de Lisboa. Este projeto foi financiado pela Fundação para a Ciência e a Tecnologia do Ministério da Ciência, Tecnologia e Ensino Superior com a bolsa de doutoramento PD/BD/114392/2016. N/A

Antibiotic resistance and hospital acquired infections are on the rise worldwide. Vancomycin-resistant enterococci have been reported in clinical settings in recent decades. In this multiomics study, we provide comprehensive proteomic and transcriptomic analyses of a vancomycin-resistant Enterococcus faecalis clinical isolate from a patient with a urinary tract infection. The previous genotypic profile of the strain C2620 indicated the presence of antibiotic resistance genes characteristic of the vanB cluster. To further investigate the transcriptome of this pathogenic strain, we used whole genome sequencing and RNA-sequencing to detect and quantify the genes expressed. In parallel, we used two-dimensional gel electrophoresis followed by MALDI-TOF/MS (Matrix-assisted laser desorption/ionization-Time-of-flight/Mass spectrometry) to identify the proteins in the proteome. We studied the membrane and cytoplasm subproteomes separately. From a total of 207 analysis spots, we identified 118 proteins. The protein list was compared to the results obtained from the full transcriptome assay. Several genes and proteins related to stress and cellular response were identified, as well as some linked to antibiotic and drug responses, which is consistent with the known state of multiresistance. Even though the correlation between transcriptome and proteome data is not yet fully understood, the use of multiomics approaches has proven to be increasingly relevant to achieve deeper insights into the survival ability of pathogenic bacteria found in health care facilities. L.P. was granted a PhD fellowship by Fundação para a Ciência e a Tecnologia and European Social Fund (SFRH/BD/81307/2011). This work was supported by the Associate Laboratory for Green Chemistry-LAQV, which is financed by national funds from Fundação para a Ciência e a Tecnologia/ MCTES (UID/QUI/50006/2019). This research is also a result of the GenomePT project (POCI-01-0145-FEDER- 022184), supported by COMPETE 2020—Operational Programme for Competitiveness and Internationalisation, Lisboa Portugal Regional Operational Programme (Lisboa, 2020), Algarve Portugal Regional Operational Programme (CRESC Algarve, 2020), under the PORTUGAL 2020 Partnership Agreement, through the EuropeanRegionalDevelopment Fund, andbyFundação para a Ciência e a Tecnologia.Work performed in University of La Rioja was supported by project SAF2016- 76571-R of Agencia Estatal de Investigatio´n and FEDER. info:eu-repo/semantics/publishedVersion

With the advance of genome sequencing technologies, it is currently possible to identify a large number of chromosomal or genomic structural variants in a single individual. Therefore, the validation and manual assessment of structural variants clinical significance becomes unpractical and time consuming when performed with previous methodologies. In order to assist the validation process, we developed two clinically inspired bioinformatics tools - TADGConTool and CNV-ConTool. They were developed in python with a Common Gateway Interface that allows easy and user-friendly access through any standards compliant web browser (available at: http://dgrctools.insa.min- saude.pt/). TAD-GConTool collects genomic information of breakpoint regions, using topological associated domains (TADs) as reference. It then accesses public databases to retrieve elements found inside TADs, and the associated clinical phenotypes, highlighting those causing dominant disorders. CNV-ConTool searches for overlaps between patient-specific breakpoints and CNVs, and those reported in several public databases. These tools were already successfully applied to about 40 cases studied under the project “Next-gen cytogenetics enters clinical care and annotates the human genome” (HMSPICT/0016/2013) and are now being made available to the broader scientific community. These tools allowed a faster and more informed evaluation of the genomic structural variants, helping select potential pathogenic variants, either by identifying phenotype- associated genes, or by overlapping deletions and duplications with already described benign or pathogenic CNVs. As genome sequencing is becoming more and more a routine method for identification of chromosomal and genomic structural variants, such clinically oriented bioinformatics tools are crucial and represent the first level of analysis toward personalized genomic medicine. This research was supported by national funds through FCT - Fundação para a Ciência e a Tecnologia, Research Grant HMSP-ICT/0016/2013. Study supported by Fundação para a Ciência e Tecnologia project HMSP-ICT/0016/2013. info:eu-repo/semantics/publishedVersion

In this study, we examined the effect of six months of positive airway pressure (PAP) therapy on Obstructive Sleep Apnea (OSA) red blood cell (RBC) proteome by two dimensional difference gel electrophoresis (2D-DIGE) - based proteomics followed by Western blotting (WB) validation. The discovered dysregulated proteins/proteoforms are associated with cell death, H2O2 catabolic/metabolic process, stress response, and protein oligomerization. Validation by nonreducing WB was performed for peroxiredoxin-2 (PRDX2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by using antibodies against the sulfinylated/sulfonylated cysteine of these proteins to better evaluate their redox&ndash, oligomeric states under OSA and/or in response to PAP therapy. The results indicated that the redox&ndash, oligomeric state of GAPDH and PRDX2 involving overoxidation by sulfinic/sulfonic acids were differentially modulated in OSA RBC, which might be compromising RBC homeostasis. PAP therapy by restoring this modulation induced a higher oligomerization of overoxidized GAPDH and PRDX2 in some patients that could be associated with eryptosis and the chaperone &ldquo, gain&rdquo, of function, respectively. This varied response following PAP may result from the complex interplay between OSA and OSA metabolic comorbidity. Hence, information on the redox status of PRDX2 and GAPDH in RBC will help to better recognize OSA subtypes and predict the therapeutic response in these patients. GAPDH monomer combined with body mass index (BMI) and PRDX2 S-S dimer combined with homeostatic model assessment for insulin resistance (HOMA-IR) showed to be very promising biomarkers to predict OSA and OSA severity, respectively.

© 2020 The Author(s)., [Introduction]: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. [Areas covered]: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. [Expert commentary]: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine., This paper was funded by the European cooperation in science and technology - COST action No. CA16113 - CliniMARK: ‘good biomarker practice’ to increase the number of clinically validated biomarkers.

Tumor suppressor p53 is mutated in half of all human cancers and for that reason it became one of the most studied genes in the history of medical research. Its functions as a transcription factor and its posttranslational regulation were subjects of intense studies for decades. But it was not until more recently that the importance of its translational regulation became clear. p53 is involved in many cellular processes, stress response pathways, tumor suppressing signals, developmental checkpoints and even aging. In order to diversify and differentiate its action, p53 mRNA is translated into several different isoforms that harbor different functions and act independently or together as heterotetramers. The possibility of expressing each isoform one by one through different regulatory structures in its DNA and mRNA gives p53 the ability to be activated in many different ways leading to specific and adequate cellular outcomes. p53 mRNAs can be translated into four different isoforms, each with its respective splice variations, from four diferente translation initiation codons present in codons 1, 40, 133 and 160, and named full length (FL) p53, delta40p53 (deltaNp53 or p53/47), delta133p53 and delta160p53, respectively. The shorter delta133p53 and delta160p53 isoforms are the products of an alternative transcript (delta133p53 mRNA) originating from an internal promoter in intron four of the p53 gene. Most of the p53 functions and regulatory mechanisms described so far are related to FLp53, because for many years, this was the only known p53 isoform. In this study, we want to investigate the translational regulation of other p53 isoforms and its impact on the p53 pathway, stress response and carcinogenesis. FCT N/A

We now want to test the relevance of the non-coding functions of p53 mRNA in carcinogenesis. FCT N/A

Reference project: PTDC/BIM-MEC/3749/2014. Starting date: 01-05-2016. End date: 31-12-2019 About one third of all genetic diseases and many forms of cancer are caused by nonsense or frameshift mutations that introduce premature translation-termination codons (PTCs) (1,2). Indeed, PTCs contribute significantly to the spectrum of inherited human diseases such as cystic fibrosis, Duchenne muscular dystrophy, beta-thalassemia, and many forms of cancer. Generally, the presence of a PTC results in premature termination of mRNA translation and in rapid degradation of the PTC-containing mRNAs through the mechanism of nonsense-mediated decay (NMD). Eukaryotic mRNA translation initiates with the recruitment of the cap-binding eukaryotic initiation factor 4F (eIF4F), which comprises eIF4E, eIF4A and eIF4G, to the mRNA 5’ end (3). eIF4G has a binding site for eIF4E and the cytoplasmic poly(A)-binding protein 1 (PABPC1), which in turn is bound to the poly(A) tail, resulting in mRNA circularization (4). The unwinding of the 5’UTR by the helicase eIF4A, enables binding of the 40S ribosomal subunit. The association of eIF1, eIF1A and eIF3 to the 40S subunit facilitates binding of the ternary complex eIF2-GTP-Met-tRNAi (3). The resulting 43S preinitiation complex can land next to the cap and scans in a 5’ to 3’ direction until it recognizes an AUG codon in a consensus sequence, base-pairing with Met-tRNAi (3). Then, there is joining of 60S subunit to form an 80S ribosome, and elongation can start and the polypeptide is synthesized (3). The termination event occurs when an elongating ribosome encounters an in-frame stop codon. The eukaryotic release factor 1 (eRF1) recognizes stop codons within the ribosomal A site and triggers the hydrolysis of the ester bond, stimulated by eRF3 (3). Interactions of the eRFs with cellular proteins playing key roles in other gene expression processes may be the means by which termination is adjusted and linked to mRNA translation and NMD. NMD controls the quality of eukaryotic gene expression and also degrades and controls the levels of physiologic mRNAs (1,2,5,6). The NMD pathway is found in all eukaryotes. Several NMD key factors are highly conserved among diverse species, including UPF1, UPF2, and UPF3 (1,2). Translating ribosomes normally displace the UPF2/UPF3 containing exon junction protein complexes (EJCs) from the open reading frame (ORF) during the pioneer round of translation (1,2). However, if an mRNA contains a PTC located more than 50-54 nucleotides upstream the last exon-exon junction, the ribosome will fail to displace distal EJC(s). If a PTC prohibits removal of distal EJCs from an mRNA during the initial round of translation, UPF1 and the SMG1 kinase associate with the eRF1 and eRF3 release factors on the ribosomal termination complex at the PTC. UPF1 then interacts with the UPF2/UPF3 proteins at the downstream EJC complex. This interaction induces UPF1 phosphorylation by SMG1 and marks the mRNA as PTC-containing (2). A complex composed of SMG5, SMG6, SMG7, and the PP2A phosphatase then dephosphorylates UPF1, and the mRNA is subsequently triggered to rapid decay by SMG6 endonucleolytic attack and exonucleolytic degradation from both 5’ and 3’ ends by a not yet completely understood process that recruits decapping and 5’-to-3’ exonuclease activities, as well as deadenylating and 3’-to-5’ exonuclease exosome activities (2,7). It has been shown that the catalytic subunits of the RNA exosome are the RNaseII-family exoribonucleases DIS3 and DIS3L1 (8,9). Interestingly, DIS3L1 is mainly cytoplasmic, whereas DIS3 is mainly localized in the nucleoplasm (8,9). More recently, another RNaseII homologue (DIS3L2) has been characterized (10), which is active in 3’-5’ cytoplasmic RNA decay, independently of the exosome (11). Despite the fact that DIS3L1 and DIS3L2 localize in the same compartment where NMD occurs, little is known about their role in this process. Nevertheless, it has been shown that mutations in the DIS3 locus are associated with aberrant accumulation of processing intermediates and aberrant forms of some RNAs (8), which evidences its essential role in RNA surveillance processes. In addition, significant findings over the last years have shown that human DIS3 paralogous are involved in growth, mitotic control, and important diseases such as cancer (8-10). For example, DIS3L2 inactivation was associated with mitotic abnormalities and altered expression of mitotic checkpoint proteins (10). Genetic diseases and cancer attributable to PTCs affect millions of patients worldwide. Thus, the high incidence of PTCs suggests that therapeutic strategies aimed at suppressing PTCs to restore deficient protein function – so-called suppression therapies – have the potential to provide a therapeutic benefit for many patients and with a broad range of genetic disorders (12,13). This therapeutic approach uses readthrough drugs, such as aminoglycosides, that induce the translational machinery to recode an in-frame PTC into a sense codon (12,13). Suppression therapy increases the frequency that near-cognate aminoacyl-tRNAs bind at a PTC and subsequently transfer their amino acid to the nascent polypeptide (12). However, there are several obstacles that must be overcome before aminoglycosides can be used long term in the suppression of nonsense mutations. First, the efficiency of suppressing PTCs is greatly influenced by the identity of the stop codon (TAA, TAG or TGA) and the surrounding mRNA sequence. Second, the long-term use of aminoglycosides is limited due to side effects (12,13). New strategies developed to overcome this issue include the discovery of non-aminoglycoside agents such as PTC124 (Ataluren®) (14), among others (12). While the most promising drug, PTC124, was found to be safe and offers a therapeutic benefit to many patients, not all patients respond equally well to its administration. One factor that possibly affects the response to suppression therapy in many patients is the high efficiency of NMD. Based on these data, this project included the following aims: (A) To study the role of DIS3-like proteins in the mRNA decay pathway inherent to NMD; (B) To analyze how DIS3L1 regulates the human transcriptome and how its functional interactions modulate the transcriptional reprogramming of colorectal cancer (CRC) cells; (C) To study the interplay between the mechanisms of PTC definition, mRNA translation, and NMD; (D) To establish an efficient PTC suppression therapy for beta-thalassemia. FCT N/A

In this article, we present supportive data related to the research article “A role for DIS3L2 over natural nonsense-mediated mRNA decay targets in human cells” [1], where interpretation of the data presented here is available. Indeed, here we analyze the impact of the DIS3L2 exoribonuclease over nonsense-mediated mRNA decay (NMD)-targets. Specifically, we present data on: a) the expression of various reporter human β-globin mRNAs, monitored by Northern blot and RT-qPCR, before and after altering DIS3L2 levels in HeLa cells, and b) the gene expression levels of deregulated transcripts generated by re-analyzing publicly available data from UPF1-depleted HeLa cells that were further cross-referenced with a dataset of transcripts upregulated in DIS3L2-depleted cells. These analyses revealed that DIS3L2 regulates the levels of a subset of NMD-targets. These data can be valuable for researchers interested in the NMD mechanism. This work was partially supported by Fundação para a Ciencia e a Tecnologia (FCT) (PTFC/BIM-MEC/3749/2014 to LR and UID/MULTI/04046/2013 to BioISI). PJdC, HAS and JFG-M are recipients of a fellowship from BioSys PhD programme (SFRH/BD/52495/2014, SFRH/BD/52492/2014, and PD/BD/ 142898/2018, respectively) and JM is a postdoctoral fellow (SFRH/BPD/98360/2013) from FCT. Work at ITQB-NOVA was financially supported by: Project LISBOA-01-0145-FEDER-007660 funded by the European Regional Development Fund (FEDER) through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) and by FCT funds: PTDC/BIA-MIC/1399/2014 to CMA and PTFC/BIM-MEC/3749/2014 to SCV. SCV was financed by program IF of FCT (IF/00217/2015). MS was financed by an FCT contract according to DL57/2016 [SFRH/BPD/109464/2015] info:eu-repo/semantics/publishedVersion

Tese de doutoramento em Biologia (Biologia de Sistemas), apresentada à Faculdade de Ciências da Universidade de Lisboa, 2019. Orientadora: Doutora Luísa Romão (INSA) e Co-Orientador: Doutor Augusto Luchessi Short upstream open reading frames (uORFs) are cis-acting elements located within the 5'- leader sequence of transcripts. Recent genome-wide ribosome profiling (RiboSeq) studies have demonstrated the widespread presence of uORFs in the transcriptome and have shown that many uORFs can initiate with non-AUG codons. uORFs can impact gene expression of the downstream main open reading frame (mORF) by triggering messenger RNA (mRNA) decay or by regulating translation. Thus, disruption, elimination or creation of uORFs can elicit the development of several genetic diseases, such as cancer. The ATP-binding cassette subfamily E member 1 (ABCE1) gene, belongs to the ABC gene family of transporters. However, it does not behave as a drug transporter like the other members of this family. ABCE1 actively participates in the different stages of the translation process and is involved in cell proliferation and anti-apoptotic signaling processes, associating ABCE1 to a potential oncogenic function. RiboSeq occupancy profiles of the ABCE1 mRNA 5’-leader sequence indicate an active translation associated with the presence of uORFs, which is suggestive of a high translational regulation. Our aim was to study the translational regulation mediated by the five AUG and five non- AUG uORFs present in the human ABCE1 5’-leader sequence in colorectal cancer. With this purpose, we constructed a set of Firefly luciferase (FLuc) reporter vectors derived from the wild-type one containing the native configuration of the human ABCE1 5’-leader sequence upstream of the FLuc ORF, and transiently transfected colorectal cancer HCT116 cells. Here we show that ABCE1 mORF expression is regulated by its uORFs. Our results are consistent with a model wherein uORF1 recruits ribosomes onto the mRNA, behaving like a ribosomal barrier. The ribosomes that efficiently bypass uORF1 and/or uORF2, must probably reinitiate at uORF3 and/or uORF5, while uORF4 is greatly bypassed. uORF3 and uORF5 function as repressive uORFs that may cooperate to reach a maximum repression of the mORF. Thus, both bypass and reinitiation events of the AUG uORFs within ABCE1 5’-leader sequence contribute for the translational control of the mORF. In constrast, the non-AUG uORFs seem to be devoided of a significant inhibitory activity. The AUG uORF-mediated translational control is maintained in normal and in endoplasmic reticulum (ER) stress conditions, which keeps the expression level of ABCE1 at a minimum, showing that ABCE1 is a resistant transcript whose functions are equally essential in normal and in coditions of global translation impairment. In addition, we show that ABCE1 uORF-mediated translational regulation is preserved in non-tumorigenic and cancerous cells, which is consistent with a lack of an oncogenic function by the uORFs, as well as ABCE1 himself, in the colorectal cancer cell line tested. This study contributes with an additional example of how uORF-mediated translational regulation can occur. In addition, it reveals how important is to screen the 5’-leader sequence of the transcripts in search for potential disease-related variants. This information might be relevant for the implementation of new diagnostic and/or therapeutic tools for diseases associated with the deregulation of uORF-mediated translational control. A expressão génica é extremamente regulada ao nível da tradução do RNA mensageiro (mRNA), sendo que por sua vez, este processo é dividido em diferentes fases: iniciação, alongamento, terminação e reciclagem dos ribossomas. A etapa da iniciação é vista como o passo limitante do processo de tradução, e é por isso altamente regulada pela existência de vários elementos regulatórios localizados na região 5’-não traduzida dos transcritos. De entre estes elementos que actuam numa configuração em cis, salientamos as pequenas grelhas de leitura a montante (do inglês, upstream open reading frames, uORFs). Estes elementos reguladores são potencialmente traduzidos e são definidos por um codão de iniciação na região 5’-não traduzida do mRNA e um codão de terminação localizado a montante ou sobreposto com a grelha de leitura principal (do inglês, main open reading frame, mORF). Estudos rescentes do perfil de ribossomas (do inglês, ribosome profiling, RiboSeq) monstraram a presença generalizada de uORFs no transcritoma, revelando ainda que uma grande maioria das uORFs inicia a sua sequência com codões não-canónicos diferindo em um nucléotido do codão de iniciação comummente usado, o AUG. É de facto estimado que aproximadamente metade dos transcritos têm pelo menos uma uORF. Estes elementos surgem em determinadas classes de transcritos, como é o caso dos fatores de transcrição, recetores celulares, oncogenes e genes envolvidos no crescimento e diferenciação celular. As uORFs regulam a expressão génica da sua mORF ao desencadearem a degradação do mRNA ou por regularem a tradução. Em condições fisiológicas normais, estes pequenos elementos regulatórios inibem a tradução da respetiva mORF, obtendo-se uma repressão da expressão de 30 a 80%. A inibição da expressão da mORF depende em grande parte de um contexto de Kozak forte envolvendo o codão de iniciação da uORF, bem como de outras características, como por exemplo, uma longa distância entre a extremidade 5’ do transcrito e o início da sequência da uORF, a presença de múltiplas uORFs, uma longa uORF e/ou uma pequena distância entre o codão de terminação da uORF e o início da mORF. A repressão da tradução induzida por uma uORF pode ser devida a um bloqueio dos ribossomas aquando da tradução da uORF ou por dissociação e reciclagem dos mesmos após terminação da tradução da uORF. Em condições de stress, as uORFs podem ser vistas como elementos que permitem a tradução da mORF. Isto pode acontecer através de mecanismos de reiniciação da tradução após tradução da uORF ou por não reconhecimento do codão de iniciação da uORF (do inglês, leaky scanning ou ribosomal bypass), levando a que os ribosomas iniciem a tradução no codão de iniciação da mORF. Desta forma, compreende-se que a disrupção, eliminação ou criação de uORFs pode levar ao desenvolvimento de várias doenças genéticas, como é o caso do cancro. O gene ABCE1 (do inglês, ATP-binding cassette subfamily E member 1) pertence à família de genes transportadores ABC. Contudo não se comporta como um transportador de drogas como os outros membros desta família pelo facto de não apresentar domínios transmembranares. A proteína ABCE1 participa ativamente nas diferentes fases do processo de tradução, bem como, nos processos de proliferação celular e evasão da morte celular por apoptose, sugerindo que esta proteína pode ter funções oncogénicas. A ocupação ribosomal da sequência 5’-não traduzida do mRNA do gene ABCE1 obtida por estudos de RiboSeq, é indicativa de uma tradução activa nesta região que por sua vez parece estar associada com a presença de uORFs, as quais poderão estra envolvidas no controlo traducional da expressão da mORF. Para além disso, dados de RiboSeq numa linha celular de cancro coloretal, HCT116, mostraram a existência de pequenas ORFs que iniciam com codões de iniciação não-canónicos na região 5’-não traduzida do transcrito ABCE1. Paralelamente, foram também identificadas bioinfomaticamente uORFs iniciadas em AUG para este transcrito. Contudo, não foi realizado nenhum estudo experimental para investigar a função biológica das uORFs no mRNA do ABCE1 humano. Tendo em conta a falta de informação relativamente às uORFs presentes na região 5’-não traduzida do mRNA do ABCE1, o objectivo deste projecto foi o estudo da função biológica destas dez uORFs, tanto iniciadas em AUGs como em codões não-canónicos, em células do cancro coloretal. Para tal estabeleceram-se as seguintes tarefas: (i) determinar o impato destas uORFs na expressão da respectiva mORF; (ii) identificar os mecanismos pelos quais estas uORFs regulam a tradução da mORF; (iii) determinar o impacto de condições de stress, nomeadamente condições de stress do retículo endoplasmático (do inglês, endoplasmic reticulum, ER), na regulação da tradução mediada por estas uORFs; e (iv) verificar se a regulação da tradução promovida por estas uORFs tem um papel no desenvolvimento tumoral do cancro coloretal. Para tal, a sequência de DNA complementar (cDNA) da região 5’-não traduzida do mRNA do ABCE1 humano foi clonada a montante da ORF da luciferase do pirilampo (do inglês, Firefly luciferase, FLuc), num vetor plasmídico, obtendo-se assim a contrução ABCE1_5’UTR. Foi realizada mutagénese dirigida neste vetor de forma a obter todas os construções necessárias. Cada uma destas construções foi transfetada transitóriamente na linha celular HCT116, simultaneamente com um vector que expressa a luciferase da Renilla como controlo. Após lise celular, os lisados proteicos foram analisados através de ensaios de luminometria. Os nossos resultados mostram que as uORFs presentes na região 5’-não traduzida do mRNA do ABCE1 humano têm impacto na tradução da respetiva mORF, como demonstrado pela redução de 70% da actividade relativa da luciferase após transfeção com a contrução ABCE1_5’UTR. Esta repressão da mORF do mRNA do ABCE1 é promovida principalmente por duas uORFs iniciadas em AUG, a uORF3 e a uORF5, as quais parecem ter um efeito aditivo de forma a alcançar um máximo de repressão da mORF. As restantes uORFs iniciadas em AUG do transcrito ABCE1 (uORF1, uORF2 e uORF4) não apresentam capacidade inibitória significativa. Embora todos os AUGs das cinco uORFs tenham o potencial de serem reconhecidas pela maquinaria de tradução, destas uORFs, a uORF1 e uORF5 são eficientemente reconhecidas devido aos seus codões de iniciação terem uma sequência de Kozak mais forte. Após a sua tradução, ocorre reiniciação da tradução no codão de iniciação seguinte que se encontre nas condições mais adequadas à tradução. Contrariamene, a uORF2, uORF3 e uORF4 são menos eficientemente reconhecidas. No conjunto, as cinco uORFs iniciadas em AUG regulam a tradução da sua mORF através de mecanismos de reiniciação da tradução e leaky scanning. Em contraste, as uORFs iniciadas em codões não-canónicos não apresentam uma capacidade inibitória significativa. A regulação da tradução do mRNA do ABCE1 promovida pelas uORFs iniciadas em AUG foi mantida quer em condições normais ou de stress do ER, indicando que a expressão da proteína ABCE1 deve ser mantida num determinado nível para exercer as suas funções na célula mesmo em condições de inibição global da tradução. O padrão de regulação por estas uORFs é igualmente mantido em células do cancro coloretal bem como em células não tumorais do cólon (linha celular NCM460), mostrando que as mesmas não têm impacto no processo tumorigénico, o que por sua vez é concordante com um papel não-tumorigénico da proteína ABCE1 na linha de cancro coloretal testada. Este estudo constitui um exemplo adicional sobre os mecanismos subjacentes à regulação da tradução do mRNA mediada por uORF(s). Além disso, revela o quão importante é a análise da região 5’-não traduzida dos transcritos de modo a identificar potenciais variantes associadas a doenças genéticas. Esta informação poderá ser relevante para a implementação de novas ferramentas de diagnóstico e/ou terapêuticas para doenças associadas à desregulação do controlo traducional mediado por uORF(s). Joana Filipa Pires Silva foi bolseira de doutoramento no âmbito do Programa doutoral BioSys em Sistemas Biológicos, Genómica Funcional & Integrativa (FCT/PD/00065/2012) da Faculdade de Ciências da Universidade de Lisboa. Este projecto foi financiado pela Fundação para a Ciência e Tecnologia do Ministério da Ciência, Tecnologia e Ensino Superior com a bolsa de doutoramento SFRH/BD/106081/2015 e pelo BioISI – Biosystems & Integrative Sciences Institute da Faculdade de Ciências da Universidade de Lisboa (UID/MULTI/04046/2013) N/A

The 21st annual meeting of the Portuguese Society of Human Genetics (SPGH), organized by Luísa Romão, Ana Sousa and Rosário Pinto Leite, was held in Caparica, Portugal, from the 16th to the 18th of November 2017. Having entered an era in which personalized medicine is emerging as a paradigm for disease diagnosis, treatment and prevention, the program of this meeting intended to include lectures by leading national and international scientists presenting exceptional findings on the genetics of personalized medicine. Various topics were discussed, including cancer genetics, transcriptome dynamics and novel therapeutics for cancers and rare disorders that are designed to specifically target molecular alterations in individual patients. Several panel discussions were held to emphasize (ethical) issues associated with personalized medicine, including genetic cancer counseling. info:eu-repo/semantics/publishedVersion

Upstream open reading frames (uORFs) constitute a class of cis-acting elements that regulate translation initiation. Mutations or polymorphisms that alter, create or disrupt a uORF have been widely associated with several human disorders, including rare diseases. In this mini-review, we intend to highlight the mechanisms associated with the uORF-mediated translational regulation and describe recent examples of their deregulation in the etiology of human rare diseases. Additionally, we discuss new insights arising from ribosome profiling studies and reporter assays regarding uORF features and their intrinsic role in translational regulation. This type of knowledge is of most importance to design and implement new or improved diagnostic and/or treatment strategies for uORF-related human disorders. This work was partially supported by Fundação para a Ciência e a Tecnologia (UID/MULTI/04046/2013 to BioISI from FCT/MCTES/PIDDAC). JS and RF are supported by fellowships from Fundação para a Ciência e a Tecnologia (SFRH/BD/106081/2015 and SFRH/BD/114392/2016, respectively). info:eu-repo/semantics/publishedVersion