Browsing by Issue Date, starting with "2017-10-06"
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- Integrative network analysis for identification of new proteins involved in NMD or its regulationPublication . Nogueira, Gonçalo; Romão, Luísa; Pinto, FranciscoBackground: Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature translation termination codons (PTCs). NMD has been associated with many genetic diseases and some forms of cancer caused by nonsense or frameshift mutations that introduce PTCs. NMD is a complex process that relies on the involvement of numerous players which interact with each other in a highly organized manner. However, the interactions and connectivity among these components is only partly elucidated. Recent studies have shown that NMD is also involved in the regulation of apparently “normal” transcripts, suggesting a greater role in gene expression regulation.
- An unexpected role for DIS3L2 over human NMD targetsPublication . Costa, Paulo; Saramago, Margarida; Viegas, Sandra; Arraiano, Cecília; Santos, Hugo; Gama-Carvalho, Margarida; Romão, LuísaThe final step of cytoplasmic mRNA degradation proceeds in either a 5’-3’ direction, catalyzed by XRN1, or in a 3’-5’ direction catalyzed by the exosome and DIS3L2. In yeast, DIS3/Rrp44 protein is the catalytic subunit of the exosome. In humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and DIS3L2. Important findings over the last years have shed a new light onto the mechanistic details of RNA degradation by these exoribonucleases. In addition, it has been shown that they are involved in growth, mitotic control and important human diseases, including cancer. For example, DIS3L2 inactivation was associated with mitotic abnormalities and altered expression of mitotic checkpoint proteins. In another study, DIS3 was found to be highly expressed in colorectal cancer (CRC), suggesting an oncogenic function. A major challenge in systems biology is to reveal the cellular networks that give rise to specific phenotypes. In this project, we aim to analyze how DIS3, DIS3L1 and DIS3L2 regulate the human transcriptome, and how their functional interactions modulate the transcriptional reprogramming of colorectal cancer cells. In order to unveil the role of these exoribonucleases in general mRNA decay, and/or in cytoplasmic mRNA surveillance mechanisms, such as nonstop- and nonsense-mediated decay (NSD and NMD), we performed their knockdown and measured the mRNA levels of various reporter transcripts (endogenous and exogenous), with emphasis in natural NMD targets. Our results show that DIS3 and DIS3L1 seem to be involved in the normal mRNA turnover, as well as in the NSD and NMD mechanisms. However, some natural NMD targets are resistant to these nucleases. On the other hand, DIS3L2 is not involved in the normal mRNA turnover or in NSD, being specifically involved in the degradation of some NMD targets. Presently, we are interested in identifying the transcript features implicated in the decision-making process of DIS3L2-mediated decay of natural NMD targets, as well as the corresponding mechanism. With this purpose, we performed a bioinformatics analysis of available transcriptomic data from DIS3, DIS3L1, DIS3L2+XRN1, XRN1, or UPF1 (a central player in NMD) knockdown experiments and identified transcripts differentially expressed in each condition. Results show some, but not total, redundancy between the upregulated transcripts, and this supports our experimental data.
- Autism Spectrum Disorder (ASD): genetic, epigenetic and environmental issuesPublication . Marques, Ana Rita; Martiniano, Hugo; Xavier-Santos, João; Asif, M.; Oliveira, Guiomar; Romão, Luísa; Vicente, Astrid M.Autism 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.
- The interplay between nonsense-mediated mRNA decay and the unfolded protein response: implications for physiology and myocardial infarctionPublication . Fernandes, Rafael; Bourbon, Mafalda; Romão, LuísaNonsense-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.
- Gene expression regulation by upstream open reading frames in colorectal cancerPublication . Silva, Joana; Romão, LuísaColorectal 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.