National Institute of Health Dr. Ricardo Jorge
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uORF-mediated translational regulation of the human PERK mRNA
No full textUpstream 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 FCTinfo:eu-repo/semantics/publishedVersio
Nonsense-mediated mRNA decay in genetic diseases and cancer: key players, mechanisms, and a novel approach for suppression therapy
No full textReference project: PTDC/BIM-MEC/3749/2014.
Starting date: 01-05-2016. End date: 31-12-2019About 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.FCTN/
Prevalence of abdominal obesity in the Portuguese population
No full textIntroduction: Overweight and obesity cause 1.2 million deaths annually and contribute to 7% of Years Lived with Disability in the World Health Organization (WHO) European Region, increasing the risk of cardiovascular diseases, diabetes, and various cancers. This study aimed to estimate and characterize the prevalence of abdominal obesity (AO) in the Portuguese population aged 25-74 years in 2015, using different anthropometric measures as a proxy of AO: waist circumference (WC), waist-to-hip ratio (WHR), waist-to-height ratio (WHtR), and a body shape index (ABSI).
Materials and methods: Data from the 1st National Health Examination Survey (INSEF) in 2015 were used. The subsample consisted of 4812 individuals who met the inclusion criteria: age 25-74 years, absence of pregnancy and available data on anthropometric measurements (weight, height, WC, hip circumference). Individuals undergoing cancer treatment were excluded from the analysis. The prevalence of abdominal obesity was stratified by sex, age group, marital status, education level, occupational activity, degree of urbanization, health region, and income quintile.
Results: In Portugal, in 2015, the prevalence of abdominal obesity according to each considered anthropometric measure was 40.3% (95% CI: 38.0 - 42.5), 43.6% (95% CI: 40.1 - 47.0), 65.2% (95% CI: 62.8 - 67.5) and 75.5% (95% CI: 74.1 - 76.9) when considering WC, ABSI, WHR and WHtR, respectively. The prevalence was higher in women for WC and ABSI, while it was higher in men for WHR and WHtR. The most affected individuals had 60-74 years old. Regarding education, the highest prevalence was observed among individuals with no formal education or only primary education. In terms of occupation, the prevalence was higher among individuals engaged in low-skilled occupations (e.g., farmers, industrial workers, and construction workers). These findings remain consistent, regardless of the index under consideration.
Discussion: In 2015, INSEF reported 38.9% of Portuguese adults had overweight, and 28.7% were obese. According to the WHO European Regional Obesity Report 2022, Portugal had a 57.5% prevalence of overweight and 20.8% of obesity in 2016. Within the WHO European Region, 58.7% of adults were overweight, and 23.3% were obese. The high prevalence of abdominal obesity in Portugal is a significant public health concern and is in line with European Region obesity trends. To determine the most accurate anthropometric index for assessing abdominal obesity, further research using imaging methods like computed tomography and nuclear magnetic resonance would be essential among the INSEF 2015 participants, as no gold standard index currently exists.
Conclusion: The prevalence of abdominal obesity ranged from 40.3% for WC to 75.5% for WHtR. Nonetheless, irrespective of the index employed, the prevalence remains significantly elevated. The prevalence of abdominal obesity was higher in older age groups, individuals with lower education levels, and those engaged in less skilled occupations. Early identification of abdominal obesity in Primary Healthcare is essential for the most susceptible groups, as well as providing nutritional support, mainly for individuals in socioeconomic vulnerability.N/
Analysis of the translatome by ribosome profiling in colorectal cancer
No full textColorectal cancer (CRC) has a high incidence and mortality rates worldwide. CRC carcinogenesis is a continuous accumulation of genetic alterations with concomitant variations in the gene expression profiles. To study the variations of gene expression profiles involved in cancer progression, the genome-wide analyses of gene expression have so far focused on the abundance of mRNA species as measured either by microarray or RNA sequencing. However, neither approach provides information on protein synthesis, which is the true end-point of gene expression. Ribosome profiling emerges to monitor in vivo translation, providing global and quantitative measurements of translation by deep sequencing of ribosome-protected mRNA fragments (RPFs).
The main goal of this project is to determine the changes between the translatome of CRC and normal colorectal cells and their role in CRC tumorigenesis.
We will analyze ribosome profiling data already available for the CRC HCT116 cell line, as well as for other cancer and non-neoplasic cell lines. Gene ontology and network interaction analysis of the differentially translated mRNAs will elucidate the main molecular pathways through which the corresponding proteins are involved in CRC progression. Furthermore, we aim to analyze the potential of translatable short open reading frames (ORFs) and/or the corresponding peptides to regulate CRC progression.
Our computational analysis of ribosome profiling data from HCT116 and non-neoplasic mammary gland (MCF-10A) cell lines identified 1666 5’ untranslated regions (5’UTRs) differentially expressing RPFs. Among these, 702 5’UTRs showed an increased accumulation of RPFs in HCT116/MCF-10A and were enriched in cell cycle regulatory genes. The remaining had a decreased RPFs accumulation and was enriched in genes involved in cell adhesion, migration, and angiogenesis. Based on these analysis and others previously published, ABCE1, ABCF1, ABCF2 and ABCF3 mRNAs were chosen for further studies. Semi-quantitative RT-PCR has shown a down-regulation of these transcripts in HCT116 cells in comparison to the non-neoplasic colorectal cell line (NCM460) and two CRC cell lines (CaCo-2 and SW480). In addition, we are testing the potential function of several upstream ORFs (uORFs) present in the ABCE1 and ABCF3 5’UTRs. For this purpose, we are first mapping the exact 5’-end of these 5’UTRs by cRACE.N/
The mechanism through which translation-termination codons are recognized as premature
No full textAbout one third of the gene mutations found in human genetic disorders, including cancer, result in premature termination codons (PTCs) and the rapid degradation of their mRNAs by nonsense-mediated decay (NMD). NMD controls the quality of eukaryotic gene expression. The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have reported that human mRNAs with a PTC in close proximity to the translation initiation codon (AUG-proximal PTC), and thus, with a short open reading frame, can substantially escape NMD. Our data support a model in which cytoplasmic poly(A)-binding protein 1 (PABPC1) is brought into close proximity with an AUG-proximal PTC via interactions with the translation initiation complexes. This proximity of PABPC1 to the AUG-proximal PTC allows PABPC1 to interact with eRF3 with a consequent enhancement of the release reaction and repression of the NMD response. Here, we provide strong evidence that the eIF3 is involved in delivering eIF4G-associated PABPC1 into the vicinity of the AUG-proximal PTC. In addition, we dissect the biochemical interactions of the eIF3 subunits in bridging PABPC1/eIF4G complex to the 40S ribosomal subunit. Together, our data provide a framework for understanding the mechanistic details of PTC definition and mRNA translation initiation.FCT/PTDC/BIMONC/4890/2014info:eu-repo/semantics/publishedVersio
The interplay between nonsense-mediated mRNA decay and the unfolded protein response: implications for physiology and myocardial infarction
No full textNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and degrades mRNAs carrying premature translation-termination codons (PTCs), protecting the cell from potentially harmful truncated proteins. Furthermore, recent studies have demonstrated that NMD is also a mechanism of gene expression regulation. This feature is reflected on its ability to regulate the cell response to many stress conditions, such as endoplasmic reticulum (ER) stress, hypoxia, reactive oxygen species, and nutrient deprivation [3,4,5]. Stress conditions, specifically ER stress, has been related to myocardial infarction, a pathological state that occurs during ischemia, where nutrient and oxygen deprivation in the heart causes aggregation of proteins in the ER and the activation of the the three arms (ATF6, IRE1α and PERK) of the unfolded protein response (UPR) to mitigate the stress and avoid cell death. Given that NMD was seen to be able to regulate the UPR and to protect cells from death during ER stress, in this work we intend to study the impact of NMD in the PERK-mediated response to ER stress induced by ischemia during myocardial infarction, and its impact to the pathophysiology of this disease. For this purpose, differentiated H9c2 cells will be used as a model of cardiomyocytes, which will help us to dissect the crosstalk between NMD and UPR in myocardial infarction-mimicking conditions. By now, we have already established the differentiation protocol for the H9c2 cell line in order to obtain mature cardiac-like cells, and we are now optimizing and establishing the experimental conditions to further develop this project.This project is partially supported by Fundação para a Ciência e Tecnologia (UID/Multi/04046/2013 to BioISI from FCT/MCTES/PIDDAC).N/
Gene expression regulation by upstream open reading frames in colorectal cancer
No full textColorectal cancer (CRC) has a high incidence and mortality rates worldwide [1]. Its carcinogenesis process is characterized by a continuous accumulation of genetic alterations that changes the overall gene expression profiles [2]. Those alterations have been studied by microarray and RNA sequencing that measure the abundance of mRNA but do not provide information on protein synthesis, a step closer to the end-point of gene expression [3-5]. Ribosome profiling (RiboSeq) emerges to monitor in vivo translation by deep sequencing of ribosome-protected mRNA fragments (RPFs) [5,6]. This technique detects ribosomes outside of known protein-coding regions, identifying translation of upstream open reading frames (uORFs) within 5’ untranslated regions (5’UTRs) [3,4]. The aim of this work is to determine the role of specific uORFs in CRC tumorigenesis. For that, we looked for potential uORFs-containing targets based in the 5’UTR RPFs occupancy from RiboSeq data from different cancer cell lines already available. We chose ABCE1, PAIP2, eIF4G2 and eIF2A as uORFs-containing mRNAs. Gene ontology analyses revealed an important role in translational control for the proteins encoded by these transcripts. By semi-quantitative RT-PCR, ABCE1 transcript is shown down-regulated in HCT116 cells in comparison to the non-neoplasic colorectal cell line (NCM460). To analyze the role of such uORFs in translational regulation and their biological function at the level of cell viability and proliferation, and acquisition of CRC features, a reporter plasmid was constructed carrying the ABCE1 5’UTR fused to the Firefly luciferase (Fluc) ORF (pGL2-ABCE1). Each one of the five upstream AUGs in ABCE15’UTR was mutated to obtained constructs with non-functinal uORFs and only one functional uORF. HCT116 cells were transiently transfected with pGL2-ABCE1 or each one of the above mentioned constructs. Fluc expression and activity was assessed by Western blot and luminometry assays, respectively. Results show a decrease in the translational efficiency of Fluc by pGL2-ABCE1. Moreover, the construct carrying only the uORF3 functional exhibits a stronger repression efficacy compared to pGL2-ABCE1 and the other constructs.This work was partially supported by Fundação para a Ciência e a Tecnologia (UID/MULTI/04046/2013 to BioISI from FCT/MCTES/PIDDAC). Joana Silva is supported by a fellowship from Fundação para a Ciência e a Tecnologia (SFRH/BD/106081/2015)N/
Autism Spectrum Disorder (ASD): genetic, epigenetic and environmental issues
No full textAutism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social communication/interaction and by unusual repetitive and restricted behaviors and interests. ASD often co-occurs in the same families with other neuropsychiatric diseases (NPD), such as intellectual disability, schizophrenia, depression and attention deficit hyperactivity disorder.
Genetic factors have an important role in ASD etiology. Multiple copy number variants (CNVs) and single nucleotide variants (SNVs) in candidate genes have been associated with an increased risk to develop ASD [8-10]. Nevertheless, recent heritability estimates and the high genotypic and phenotypic heterogeneity characteristic of ASD indicate a role of environmental and epigenetic factors, such as long noncoding RNA (lncRNA) and microRNA (miRNA), as modulators of genetic expression and clinical presentation.
The aim of this project is to understand the role of lncRNA, miRNA and other epigenetic factors in ASD. For this purpose we are, in a first approach, screening for CNVs and SNVs encompassing lncRNA and miRNA loci in two large datasets: the Autism Genome Project (AGP), with CNV data from 2611 autism trios and the ARRA Autism Sequencing Collaboration, with whole exome sequencing data (WES) from 3056 autism trios. These datasets include data from Portuguese ASD probands recruited by our team. Thus far we have explored the variant call format files that contain all WES variants called by GATK. We started by testing different annotation tools and databases to obtain the best subset of variants that will be filtered according to their genomic coordinates and their pathogenic status. We are also selecting the CNVs from the AGP file that contain lncRNA and miRNA loci. The goal is to identify individuals with potential mutations in lncRNA and miRNA loci that may be disrupting their function upon target genes. Experimental validation will be carried out by measuring gene expression in these patients.
A second approach will involve exploring available multiplex families in which ASD co-occurs with other NPDs. Segregation analysis will allow us to define patterns of NPD transmission, identify common gene variants and explore the role of modulating epigenetic factors that lead to differential disease expression.Support for this work was provided by Fundação para a Ciência e a Tecnologia (grant PD/BD/113773/2015 to A.R.Marques).N/
Human AGO1 5’UTR mediates an elF4G-enhanced but cap-independent mechanism of translation initiation
No full textArgonaute 1 (AGO1) is an essential effector in RNA-mediated gene silencing pathways. It regulates developmental control and stem cell maintenance, and is related to tumourigenesis. Such functions suggest its expression must be tightly regulated and, most likely, at protein synthesis level. Thus, we investigated whether AGO1 expression is controlled by alternative mechanisms of translation initiation. For that, we cloned the AGO1 5’UTR in a bicistronic luciferase vector upstream the downstream cistron (Firefly luciferase [FLuc]), and transfected HeLa cells with this construct. We observed a significant increase in FLuc expression levels compared to those from Renilla luciferase (upstream cistron) in cells transfected with the AGO1 5’UTR-containing constructs compared to those transfected with the empty transcript. Under cap-dependent translation initiation-impairing conditions, we saw that the identified cap-independent translation activity was enhanced upon knock-down of eukaryotic initiation factor (eIF) 4E, the cap-binding protein. However, inhibiting the eIF4G–eIF4E interaction significantly reduces such activity, suggesting AGO1 5’UTR-mediated translation may be dependent on eIF4G. Furthermore, in cells transfected with in vitro transcribed, capped and polyadenylated bicistronic AGO1 5’UTR-containing mRNA, the relative FLuc expression levels did not increase significantly, indicating that AGO1 5’UTR cannot mediate internal cap-independent translation initiation when it does not go through a nuclear experience. Nonetheless, in cells transfected with cap-lacking monocistronic transcripts, relative FLuc expression levels mediated by the AGO1 5’UTR were significantly increased. These results indicate that AGO1 5’UTR sequence mediates a non-canonical cap-independent eIF4G-dependent mechanism of translation initiation that seems to be enhanced by a free 5’ end.This work was partially financed by Fundação para a Ciência e a Tecnologia (SFRH/BD/74778/2010).N/
Integrative network approach to identify new players involved in NMD or its regulation
No full textNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and
selectively degrades mRNAs carrying premature translation-termination codons (PTCs). The
physiological importance of NMD is manifested by the fact that about one third of all genetic
diseases and some forms of cancer are caused by nonsense or frameshift mutations that
introduce PTCs, and NMD can modulate the clinical phenotype of these diseases. Noteworthy,
in total, genetic diseases attributable to PTCs affect millions of patients worldwide. Recent
studies have shown that NMD also targets mRNAs transcribed from a large subset of wild-type
genes, shaping their levels. NMD is a complex process where several proteins interact with
each other and cooperate to induce degradation of a given transcript. Although this pathway
has been extensively studied, the interactions and connectivity among these components is only
partly elucidated. Aiming to expand the knowledge about the NMD pathway, we are combining
bioinformatics, network analysis and experimental work to identify new proteins involved in NMD
or its regulation. Our work, begins with a network analysis approach that integrates publicly
available data regarding different types of interactions: 1) protein-protein, 2) kinase-target, 3)
phosphatase-target, 4) miRNA-target, 5) transcription factor-target, 6) gene co-expression and
7) ubiquitination-target. Additionally, our network include data regarding known NMD-targets
and NMD-triggering features. The generated network will be used to find novel NMD-associated
proteins, prioritizing candidates with simultaneous interactions with different mRNA processing
pathways (mRNA splicing, mRNA transport, mRNA translation and mRNA decay). Following
data integration, we will develop a scoring algorithm to select the most central proteins in the
generated network, which can be essential to further understand NMD and its regulation. The
predicted candidates will be experimentally validated and their role in NMD will be tested. Due
to the diversity of regulatory links integrated in this workflow, we propose it can be applied to
find molecular bridges between related biological processes and generate novel hypotheses
about the molecular mechanisms co-regulating these phenomena.The authors acknowledge the Portuguese Foundation for Science and Technology (FCT) for the PhD
grant: PD/BD/130959/2017.N/