Anthropometric Indices and Cardiovascular Risk: A Cross-Sectional Study in Portugal

Introduction: The relationship between abdominal obesity and cardiovascular risk is well established. The objective of this study was to determine the best anthropometric index to assess cardiovascular risk in the Portuguese population aged 40-69 years. Materials and methods: Data from the 1st National Health Examination Survey 2015 were used. The analyzed anthropometric indices included Body Mass Index (BMI), Waist Circumference (WC), Waist-to-Height Ratio (WHtR), Waist-to-Hip Ratio (WHR), and A Body Shape Index (ABSI). The subsample consisted of 2780 individuals who met the inclusion criteria: aged 40-69 years, not pregnant, available information on sex, age, smoking status, systolic blood pressure, total cholesterol, high-density lipoprotein cholesterol, and anthropometric measures (weight, height, WC, hip circumference). Individuals receiving cancer treatment were not included in the study. Those with a previous diagnosis of acute myocardial infarction, stroke, diabetes, chronic kidney disease, or undergoing medication therapy for these conditions were excluded from the analysis due to their already high or very high cardiovascular risk, being the use of SCORE2 inappropriate. The area under the curve (AUC) of the receiver operating characteristic (ROC) was calculated, stratified by sex, to determine the best index for assessing cardiovascular risk. Results: In females, WHR exhibited the highest discriminatory power with an AUC of 0.67 (95% CI: 0.63 to 0.71), closely followed by WHtR with an AUC of 0.66 (95% CI: 0.61 to 0.70) and ABSI with an AUC of 0.65 (95% CI: 0.60 to 0.70). In males, WHtR displayed the highest discriminatory power with an AUC of 0.64 (95% CI: 0.59 to 0.68), closely followed by WHR with an AUC of 0.63 (95% CI: 0.58 to 0.67), and WC had an AUC of 0.62 (95% CI: 0.57 to 0.67). Discussion: Previous research has produced diverse findings regarding the choice of anthropometric indices, with variations across genders. In the present study the AUC values for the analyzed indices encountered for both genders had overlapping confidence intervals, indicating no statistically significant difference in predictive power. Conclusion: In women, the best index was WHR, and in men it was WHtR. However, due to a lack of statistical significance, it was not possible to determine which index had the best predictive ability. Nevertheless, this doesn't invalidate the previously well-established link between abdominal obesity and cardiovascular risk. Cardiovascular disease has a multifactorial etiology, and attempting to find only one variable that predicts the risk of a cardiovascular event can be overly simplistic and limiting.N/

Identification of mTOR and AGO1 IRES trans-acting factors

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 LisboaWork partially supported by UID/MULTI/04046/2019 Research Unit grant from FCT, Portugal (to BioISI)info:eu-repo/semantics/publishedVersio

Terapia de supressão da β-talassemia usando Canamicina e Gentamicina

Orientação científica da Doutora Luísa Maria Ferreira Romão Loison, Investigadora Principal do Departamento de Genética Humana do Instituto Nacional de Saúde Doutor Ricardo Jorge e do Doutor Luís Manuel Souto de Miranda, Professor Auxiliar Convidado do Departamento de Biologia da Universidade de Aveiro.Dissertação de mestrado em Biologia Molecular e Celular, apresentada à Universidade de Aveiro, 2016Nonsense mutations are point mutations that originate premature termination codons (PTCs). The expression of PTC-containing genes may lead to the synthesis of truncated proteins. Truncated proteins are shorter proteins that at most times do not have biological function, but may have deleterious functions for the cell. In regular conditions, PTC-containing transcripts are taken to rapid decay, through nonsense mediated mRNA decay (NMD). When a PTC reaches the ribosomal A-site, translation release factors bind it and translation immediately stops. Suppression therapy is a therapeutic approach that aims to suppress PTCs by using low molecular weight compounds to induce the incorporation of near cognate aminoacyl tRNAs when the ribosome reaches a PTC. Thus, translation does not prematurely terminates. Previous studies have shown that some aminoglycosides have the ability to suppress PTCs responsible for diseases like cystic fibrosis and Duchenne muscular dystrophy. Some nonsense mutations are responsible for β-thalassemia disease. In this study two aminoglycoside compounds, kanamycin and gentamicin, were used in order to evaluate their capacity to increase the competition of near cognate aminoacyl tRNAs with translation release factors by the ribosomal A-site, when the ribosome reaches a PTC, therefore avoiding the premature termination of translation.As mutações nonsense são mutações pontuais que originam codões de terminação prematura (PTCs). A expressão de genes portadores de PTCs pode levar à síntese de proteínas truncadas. As proteínas truncadas caracterizam-se por serem menores e, na maioria das vezes, não possuem função biológica, apesar de poderem ter funções deletérias para a célula. Em condições normais, transcritos portadores de PTCs são degradados rapidamente através do processo de nonsense mediated mRNA decay (NMD). Quando um PTC atinge o sítio A ribossomal, os fatores de terminação da tradução ligam-se ao mesmo e a tradução termina imediatamente. A terapia de supressão consiste numa abordagem terapêutica que tem o objetivo de utilizar compostos de baixo peso molecular para induzir a incorporação de aminoaciltRNAs quase cognatos quando o ribossoma atinge um PTC. Assim, a tradução não termina prematuramente. Estudos anteriores mostraram que alguns aminoglicósidos possuem a capacidade de suprimir PTCs responsáveis por doenças, como fibrose quística e distrofia muscular de Duchenne. Algumas mutações nonsense são responsáveis pela β-talassemia. Neste estudo foram utilizados dois aminoglicósidos, canamicina e gentamicina, de modo a avaliar a sua capacidade em aumentar a competitividade de tRNAs quase cognatos com os fatores de terminação da tradução pelo sítio A ribossomal, na presença de um PTC, evitando dessa forma a terminação prematura da tradução.N/

The role of eIF3 subunits in the mechanism of nonsense-mediated mRNA decay

Premature translation-termination codons (PTCs) or nonsense codons) can arise from mutations in germ or somatic cells. The introduction of a PTC into an mRNA can trigger nonsense-mediated decay (NMD), an important mRNA surveillance mechanism that typically recognizes and degrades mRNAs containing PTCs to prevent the synthesis of C-terminally truncated proteins potentially toxic for the cell. The physiological importance of NMD is manifested by the fact that about one third of genetic disease-associated mutations generate PTCs. The mammalian translation initiation factor 3 represents the most complex eukaryotic initiation factor (eIF) in mammalian cells. This factor comprises 13 subunits (eIF3a to eIF3m), each one playing an important role in translational control. Disruption of eIF3 initiation factor activity can lead not only to cancer but also neural physiological alterations, and to act as a mediator of infection cascade. Although some eIF3 subunits (for example, e and g) have been implicated in NMD, others were not studied yet. With the aim to identify other eIF3 subunits involved in NMD, we have depleted each one of the eIF3 subunits in HeLa cells and tested its effect in the expression of PTC-free or PTC- containing reporter human β-globin genes. Our data show that eIF3l and eIF3j subunits have an important role in targeting mRNAs for NMD. We will describe the molecular mechanisms underlying these observations.This work was partially supported by Fundacão para a Ciência e a Tecnologia (PTDC/BIM-MEC/3749/2014 and UID/MULTI/04046/2013 to BioISI from FCT/MCTES/PIDDAC).N/

RNA structure-function analysis of regulatory regions of p53 mRNA

At least half of all tumors exhibit mutations in the tumor suppressor p53 gene. Indeed, the fact that p53 is frequently mutated in cancer led to its identification as an oncogene, when first described in 1979. Later, it was classified as a tumor suppressor, due to the clarification of its wild-type role in maintaining genome integrity and preventing malignant transformation. The p53 gene can encode for many p53 isoforms, by alternative splicing, alternative promoters and internal translation initiation mechanisms. While full-length p53 (FL-p53) protein works as a tumor suppressor by regulating many biological processes such as cell cycle, apoptosis, senescence and DNA repair, shorter p53 protein isoforms seem to play different roles in the cell. Recently, we have shown that the most common p53 mutations induce the expression of shorter p53 isoforms. Furthermore, we found that shorter p53 isoforms are implicated in cancer progression as they promote enhanced cell survival, proliferation, adhesion and formation of invasive cell structures. Here, with a bicistronic system containing two reporter genes (Renilla luciferase and firefly luciferase), we show that expression of shorter p53 isoforms is mediated by a non-canonical translation initiation mechanism regulated by an Internal Ribosome Entry Site (IRES) in the p53 mRNA. By investigating the effect of common p53 missense mutations on the function of this new IRES, through bioluminescence assays and Western blot analysis, we show that some p53 cancer mutations have a preponderant role in IRES-mediated translation induction of shorter p53 isoforms. With the obtained results we identified a new mechanism by which p53 cancer mutations promote tumorigenesis, which may lead to new understandings of the onset and progression of some types of tumors as well as to the development of new cancer therapies.This work is supported by grants PTDC/MED-ONC/32048/2017 and PTDC/BIMONC/4890/2014 from the Fundação para a Ciência e a Tecnologia, 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.info:eu-repo/semantics/publishedVersio

Behind the curtain: unveiling DIS3L2 role in NMD and human cancer

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that targets and degrades mRNAs carrying premature translation-termination codons (PTCs), preventing the production of truncated proteins potentially harmful for the cells. In addition to this, several studies have shown that NMD regulates the levels of many physiological mRNAs that encode full-length proteins. Nevertheless, NMD is inhibited in tumor microenvironment and (de)regulates oncogenes and tumor suppressors in several types of cancer. In humans, the mRNA degradation pathways involve exonuclease proteins, such as the DIS3-like protein family (DIS3, DIS3L1 and DIS3L2); however, it is not known whether these proteins are involved in NMD. In order to unveil the role of DIS3L2 in NMD, we performed its knockdown, by RNA interference, in HeLa cells and measured, by RT-qPCR, the mRNA levels and half-lives of various natural NMD targets. Our results show that some NMD targets are highly stabilized in DIS3L2-depleted cells. In addition, mRNA half-life analysis indicate that these NMD targets are in fact direct DIS3L2 substrates. By performing DIS3L2, TUT4 and TUT7 triple knockdown, we also observed that DIS3L2-mediated decay depends on the terminal uridylyl transferases (TUTases) Zcchc6/11 (TUT7/4) activity. Among the NMD targets regulated by DIS3L2, we highlight GADD45A. GADD45A is involved in cell cycle arrest, DNA damage response and apoptotic process. Furthermore, GADD45A deregulation is associated with several types of cancer, such as, esophageal, lung, bladder and pancreatic. Together, our findings establish the role of DIS3L2 and uridylation in NMD and in the regulation of oncogenes and tumor suppressor gene expression. These results might be highly relevant for the advance in diagnosis, prognosis and treatment of many human cancers.FCT/PTDC/BIMONC/4890/2014N/

Establishment of a suppression therapy for beta-thalassemia due to a nonsense mutation

Dissertação de mestrado em Bioquímica, especialização em Bioquímica Médica, apresentada à Faculdade de Ciências da Universidade de Lisboa, 2017Orientadora Luísa Romão, Departamento de Genética Humana do INSA.Beta-thalassemia is a genetic disorder caused by the absence or reduction of human beta-globin protein levels, which causes a reduction on hemoglobin synthesis. This reduction can be caused by a premature termination codon (PTC) in the human beta-globin gene. The messenger ribonucleic acid (mRNA) containing a PTC can be recognized and degraded by the nonsense-mediated decay mechanism (NMD), if the PTC is located 50-54 nucleotides upstream of the last exon-exon junction. If the PTC is located near to the initiation codon (generally an AUG) or downstream of the last exon-exon junction, then this mRNA is not degraded by NMD. In the first case a small peptide is degraded by proteolysis and in the second case a long truncated protein is synthesised. These proteins aggregate and precipitate, leading to a severe damage of erythroid precursors. The conventional therapies for beta-thalassemia temporarily restore the levels of human beta-globin protein, however, these therapies have secondary effects that lead to the deterioration of the patient health. Given this, a therapy that could restore the levels of human beta-globin protein by inducing the readthrough of the PTC in human beta-globin gene, would be an advantage to these patients. Suppression therapy has been studied in detail for genetic diseases caused by PTCs. Salvatori et al. demonstrated the capability of G418 to induce the readthrough of a PTC at codon 39 of human beta-globin gene, however, the capability of suppression by this aminoglycoside at other codons of human beta-globin gene, and the use of other suppression compounds, have not yet been investigated. Suppression therapy consists in the readthrough of a nonsense codon into a sense codon, which allows the mRNA to be totally translated, resulting in a complete protein with partial or complete function. There are some compounds, including aminoglycosides and non-aminoglycosides, that bind to the decoding center of the ribosome and allow the near-cognate aminoacyl-tRNA binding, resuming translation. The main aims of this project were to understand if G418, an aminoglycoside, or PTC124, a non-aminoglycoside, produce efficient levels of readthrough of PTCs in human beta-globin mRNA, and to understand how different PTCs on beta-globin mRNA respond to the suppression therapy. Therefore, HeLa and HEK293 cells were transiently transfected with constructs containing the first 55 codons of human beta-globin gene fused to firefly luciferase gene (betaWT-FLUC, beta15-FLUC or beta39-FLUC). The results were obtained by Western blot and bioluminescence assays. The results obtained by Western blot seem to indicate that G418 induce the readthrough of PTCs at codon 15 or 39 of human beta-globin gene in HEK293 treated with concentrations higher than 400 microg/mL. Additionally, a PTC at codon 15 of human beta-globin gene seems to respond more efficiently to the readthrough than a PTC at codon 39. The results obtained for PTC124 are not conclusive, however, PTC124 might be inducing the readthrough of a PTC at codons 15 and 39 of human beta-globin gene in HEK293 cells treated with 5 microM, or 5, 10 and 15 microM of PTC124, respectively. Since G418 revealed to be able to restore the full-length human beta-globin protein in constructs containing a PTC at codon 15 and 39 of human beta-globin gene, it arises as a promising compound to be used in a future therapy for beta-thalassemia. Before that, it is important to study the effect of NMD in suppression therapy and how its inhibition can enhance the suppressive effect.A célula necessita de manter os níveis de proteína regulados, uma vez que estas possuem funções que determinam a sobrevivência e adaptação da célula em resposta a diferentes estímulos. De forma a regular estes níveis a célula altera os padrões de expressão génica, controlando vários processos celulares, nomeadamente, a transcrição e a tradução. Uma desregulação nestes processos pode levar a inúmeras doenças, como as doenças genéticas. A β-talassemia é um exemplo de uma doença genética, caraterizada pela redução ou ausência de β-globina, a qual leva consequentemente a anemia. A redução dos níveis de proteína pode ser causada pela presença de codões prematuros da tradução (PTCs) no seu mRNA. O mRNA que contenha estes PTCs pode ser envidado para o mecanismo de degradação mediado por mutações nonsense (NMD) caso cumpra os requisitos necessários. O NMD é um mecanismo de controlo de qualidade da célula, uma vez que tem um papel protetor contra erros genéticos. Durante o splicing são adicionados complexos exão-exão (EJCs) a 20-24 nucleótidos a montante das junções exão-exão. Durante a ronda pioneira da tradução todos os EJCs adicionados são removidos. No entanto, na presença de um PTC localizado a 50-54 nucleótidos a montante da junção exão-exão, o EJC não é removido e interage com outros complexos proteicos de forma a recrutar endonucleases e exonucleases, os quais levam à degradação do mRNA. Os mRNAs que contenham PTCs próximos do codão de iniciação são uma exceção a esta regra, assim como mRNAs que contenham PTCs a jusante da ultima junção exão-exão. Quando o mRNA que contenha o PTC não é enviado para NMD pode ocorrer a produção de pequenos péptidos, que são degradados por proteólise, ou de longas proteínas truncadas, que agregam e precipitam, levando à destruição dos percursores eritroides e ao agravamento do fenótipo do doente. Sendo assim, tanto o tipo de mutação com a sua posição no gene influenciam o fenótipo e a severidade da β-talassemia. Esta doença apresenta três fenótipos, a β-talassemia suave, a intermédia e a grave. Os pacientes que apresentam β-talassemia suave são assintomáticos, no entanto aqueles cujo fenótipo é mais grave, apresentam anemias severas com necessidade de transfusões sanguíneas recorrentes. A β-talassemia intermédia apresenta sintomas com grau de severidade entre a β-talassemia suave e β-talassemia grave. Apesar de não existir uma cura para a β-talassemia existem algumas terapias convencionais, como por exemplo, as transfusões sanguíneas que permitem restaurar temporariamente os níveis de β-globina no sangue. No entanto, estas terapias possuem efeitos secundários, nomeadamente a deposição de elevados níveis de ferro em diferentes órgãos e tecidos que prejudicam a saúde do doente. Desta forma, é importante estudar uma terapia que permita restaurar os níveis da proteína β-globina, assim como a sua função. Existem diversos artigos que relatam uma terapia de supressão onde são restaurados os níveis e a função de proteína em doenças causadas pela presença de um PTC, como na fibrose cística ou na distrofia muscular Duchenne. Esta terapia consiste na releitura de um codão que levaria à terminação prematura da tradução. Este processo permite que o mRNA seja totalmente traduzido, resultando na síntese de uma proteína completa e com pelo menos alguma da sua função restaurada. De forma a ser possível esta releitura, alguns compostos aminoglicósidos e não-aminoglicósidos permitem a ligação de um aminoacil-tRNA com duas bases compatíveis com o codão stop prematuro, anulando a ligação dos fatores de libertação da tradução ao PTC. Assim sendo, ocorre a incorporação de um novo aminoácido e a tradução continua. A terapia de supressão tem sido estudada em diversas doenças genéticas causadas pela presença de PTCs em genes, que levam à terminação prematura da tradução. Salvatori et al. demonstrou a capacidade do composto aminoglicósido G418 de induzir a releitura de um PTC no codão 39 no mRNA da β-globina humana. No entanto, a capacidade de supressão deste composto, assim como de outros agentes supressores, ainda não foi estudada na releitura de PTCs em outros locais do mRNA da β-globina humana. Assim, o objetivo principal deste projeto foi estudar que compostos aminoglicósidos e não-aminoglicósidos permitiam obter níveis eficientes de supressão de codões prematuros da tradução no mRNA da β-globina humana. Um segundo objetivo foi compreender como diferentes PTCs em diferentes posições do mRNA respondem a esta terapia de supressão. De forma a ser possível observar a proteína β-globina humana por Western blot, delineou-se uma estratégia que consistiu em clonar a open reading frame (ORF) do enhanced green fluorescent protein (EGFP), sem os codões de iniciação e terminação, no exão 3 do gene da β-globina. Apesar de inúmeras tentativas, não foi possível obter nenhum clone positivo. Dado que a estratégia anterior se mostrou difícil de alcançar, foi delineada uma segunda estratégia. Utilizaram-se construtos que continham 55 codões da β-globina humana em fusão com o gene da firefly luciferase. Estes construtos apesar de não sensíveis ao NMD por não conterem junções exão-exão, permitiram no entanto, compreender as características da terapia de supressão na releitura dos PTCs no mRNA da β-globina humana. De forma a alcançar os objetivos propostos, as células HeLa ou HEK293 foram transfectadas com os constructos referidos, sendo que o gene da β-globina não apresentava mutações (βWT-FLUC) ou possuía uma mutação nonsense no codão 15 (TGA) (β15-FLUC) ou 39 (TAG) (β39-FLUC). As células foram tratadas com G418 (aminoglicósido) ou PTC124 (não-aminoglicósido) e analisou-se a ocorrência de supressão por ensaios de bioluminescência e por Western blot. Após a análise dos resultados obtidos para as células HeLa transfectadas com constructos contendo um PTC no codão 39 no gene da β-globina humana, observou-se que o G418 levou ao aumento da atividade relativa da firefly luciferase na maioria das concentrações testadas. Os resultados obtidos para as células HEK293 aparentam indicar a presença de supressão de um PTC localizado no codão 15 ou 39 no mRNA da β-globina humana, quando estas células são tratadas com G418. No Western blot observou-se a presença de uma banda com tamanho similar à βWT-FLUC quando as células HEK293 transfetadas com construtos contendo um PTC localizado no codão 39 são tratadas com 500, 750, 1000 e 1250 μg/mL. Relativamente às células HEK293 transfetadas com constructos contendo uma mutação no codão 15 pudemos observar a presença de uma banda com tamanho similar ao βWT-FLUC para as células tratadas com G418 a 400, 500, 750, 1000 e 1250 μg/mL. Adicionalmente, foi observado um aumento da atividade relativa da firefly luciferase nestas condições. Este composto não parece influenciar a expressão do constructo βWT-FLUC em nenhuma concentração testada, em ambas as linhas celulares. Relativamente à eficiência de supressão, o composto G418 parece ser mais eficiente na releitura do PTC no codão 15 no mRNA da β-globina humana, relativamente à releitura do PTC no codão 39. Os resultados apresentados para o não-aminoglicósido PTC124 não são claros devido ao facto de as bandas presentes nos Western blots serem difusas. No entanto, é possível que o PTC124 induza a releitura do PTC no codão 15 do mRNA da β-globina humana, quando as células HEK293 são tratadas com PTC124 a 5 μM. Pareceu ser possível a releitura do PTC no codão 39 do mRNA da β-globina humana, quando as células HEK293 são tratadas com PTC124 a 5, 10 ou 15 μM. A eficiência da terapia de supressão pode ser influenciada pelo tripleto que compõe o codão prematuro da tradução ou pela sequência flanqueadora deste codão. Os resultados obtidos permitiram compreender que ambas as sequências flanqueadoras da mutação no codão 15 e no codão 39 permitem que ocorra a releitura do PTC, embora haja uma maior eficiência na releitura do PTC no codão 15 do mRNA da β-globina humana utilizando o composto G418. Apesar de estes resultados positivos indicarem um avanço para estabelecer uma terapia de supressão para a β-talassemia, existem várias vertentes desta terapia que ainda necessitam de ser exploradas. Dado que o NMD pode limitar os níveis de mRNA disponíveis para a releitura do codão, é importante testar a sua inibição (parcial) neste tipo de terapia, utilizando compostos que alteram a eficiência do NMD. Adicionalmente, é importante estudar a função da proteína sintetizada, dado que, após a reinserção de um novo aminácido, é possível que a sua função não seja completa.FCT/PTDC/BIMONC/4890/2014N/

The human mTOR transcript allows cap-independent translation that insures its expression and function during inhibition of global translation

The mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that integrates signals from the cellular nutrient- and energy-status, acting namely on the protein synthesis machinery. Deregulation of mTOR signaling is implicated in major diseases, such as cancer, mainly due to its role in regulating protein synthesis. Major advances are emerging regarding the regulators and effects of mTOR signaling pathway; however, regulation of mTOR gene expression is not well known. Here, we show that the 5’ untranslated region of the human mTOR transcript forms a highly folded RNA scaffold capable of binding directly to the 40S ribosomal subunit. We further demonstrate that this cis-acting RNA regulon is active both in normal and stress conditions, and that its activation status in response to translational adverse conditions parallels mTOR protein levels. Moreover, our data reveal that the cap-independent translation of mTOR is necessary for its ability to induce cell cycle progression into S-phase. These results suggest a novel regulatory mechanism of mTOR gene expression that integrates the protein profile rearrangement triggered by global translation inhibitory conditions.This work was partially supported by Fundação Merck Sharp and Dohme and Fundação para a Ciência e a Tecnologia (FCT) through center grant UID/MULTI/04046/2013 (to BioISI) and research grant PTDC/BIM-ONC/4890/2014. Ana Marques-Ramos, Juliane Menezes, and Rafaela Lacerda were supported by Fellowships from FCT [SFRH/BD/33462/2008 to A.M.-R., FCT/SFRH/BPD/98360/2013 to J.M., and SFRH/BD/74778/2010 to R.L.]. Marco Marques Candeias was supported by fellowships from the Japan Society for the Promotion of Science (JSPS/FF1/184) and AXA Research Fund and JSPS Grant-in-Aid for Young Scientists (B) (16K21111).N/

The interaction between mRNA translation and nonsense-mediated decay in AUG-proximal nonsense-mutated transcripts

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTCs). Although NMD has been intensively studied, it is still poorly understood how NMD discriminates between PTCs and normal stop codons. The unified model for NMD proposes that the decision of NMD triggering is the outcome of the competition between the cytoplasmic poly(A)-binding protein 1 (PABPC1) and the NMD effector UPF1 for the termination complex. Consequently, PTCs located far, in a linear sense, from the poly(A) tail and associated PABPC1, in mRNAs containing residual downstream exon junction complexes (EJCs), are expected to elicit NMD. Nevertheless, we have reported that human mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD through a mechanism independent of translation re-initiation. Here, we will present the mechanistic basis for this NMD resistance and how it involves the step of mRNA translation initiation.This work was partially supported by Fundação para a Ciência e a Tecnologia (PEst-OE/BIA/UI4046/2011 and FCT/PTDC/BIM-MED/0352/2012).N/

Non-canonical translation initiation of proteins with potential relevance in colorectal cancer

About non-canonical translation initiation of proteins with potential relevance in colorectal cancer.FCTN/