IRES-dependent translation of shorter p53 isoforms is affected by mutations in p53

Full-length p53 (FLp53) is a tumour suppressor protein that has been considered a master regulator of many cellular functions. Several isoforms have been described for p53 so far and some of the functions of shorter p53 isoforms have been elucidated and they are different from and complement FLp53 activity. p53 is the most commonly mutated gene in cancer and depending on its mutation status p53 may act as a tumour suppressor or a proto-oncogene. Recently, we have shown that the most common p53 cancer mutants express a larger number and higher levels of shorter p53 protein isoforms that are translated from the mutated FLp53 mRNA (Candeias et al. EMBO R. 2016). Also, we found that cells expressing these shorter p53 isoforms exhibit mutant p53 “gain-of-function” cancer phenotypes, such as enhanced cell survival, proliferation, invasion and adhesion, altered mammary tissue architecture and invasive cell structures. Here, we found that some of these mutations affect the function of an Internal Ribosome Entry Site (IRES) in p53 mRNA. We investigated which mutations influence — by altering IRES structure and function — IRES-dependent translation of shorter p53 isoforms and to what extent this may lead to the onset or progression of some types of tumours.N/

Autism Spectrum Disorder (ASD): genetic, epigenetic and environmental issues

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.Support for this work was provided by Fundação para a Ciência e a Tecnologia (grant PD/BD/113773/2015 to A.R.Marques).N/

The mechanism through which translation-termination codons are recognized as premature

About 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

Regulatory RNAs targeted by Copy Number Variation in Autism Spectrum Disorder

Introduction: Autism Spectrum Disorder (ASD) is a highly heterogeneous neurodevelopmental disorder with an unclear etiology. Genetic factors are estimated to account for ~50-80% of the familial ASD risk but most of the genetic determinants are still not known. Several copy number variants (CNVs) targeting ASD candidate genes explain some ASD cases. Still, further exploration of noncoding RNAs targeted by CNVs is necessary. MicroRNA (miRNA) and long noncoding RNA (lncRNA) are regulatory molecules, abundantly expressed in the brain, that play an important role during early stages of neural development. Thus, they are strong candidates for ASD. The goal of this work is to identify miRNA and lncRNA genes targeted by CNVs in a cohort of ASD patients and examine their target genes and biological pathways. Methods: We compared the frequency of miRNA and lncRNA genes targeted by CNVs in a cohort of 2446 ASD subjects and 9649 ancestry-matched control subjects. Genetic data from ASD patients was obtained from the Autism Genome Project and the control group from the Database of Genomic Variant (DGV). Both cases and controls were quantified using the same detection method. AGP data was transformed to hg19 annotation followed by functional annotation using the most recent dataset from MIRBASE. Statistical analysis was performed using Fisher’s exact test followed by Bonferroni correction (p-value<0.05). Results: We found 9 miRNAs exclusively targeted by CNVs in ASD subjects and 7 miRNAs more frequently targeted by CNVs in ASD subjects, when compared to controls. From these, only 2 were already known to be associated with ASD. Interestingly, we identified 4 novel miRNAs associated with ASD that were previously described to be associated with Schizophrenia, a disorder that presents some phenotypic overlap with ASD. Putative targets of these 16 miRNAs were enriched for ASD risk genes described in SFARI database. Gene enrichment analysis indicates that these genes are involved in neurodevelopmental processes, which is consistent with literature. In addition, we also found 102 novel lncRNAs more frequently targeted by CNVs in ASD. Discussion: These results support our hypothesis that genetic variants targeting noncoding regulatory RNAs are involved in ASD pathophysiology. This innovative approach will allow the identification of novel biomarkers and drug targets in ASD, which can contribute to a better diagnosis and treatment.N/

The interplay between nonsense-mediated mRNA decay and the unfolded protein response: implications for physiology and myocardial infarction

Nonsense-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 (1). Recent studies demonstrated that NMD also targets mRNAs transcribed from a large subset of wild-type genes, arising as a mechanism of gene expression regulation (2,3). This raised the possibility that NMD is a controlled mechanism, an idea that was confirmed by recent studies, where NMD activity was seen to be modulated in specific cell types (4) and auto regulated through its intrinsic mechanism of mRNA degradation (5). Cellular stress, such as endoplasmic reticulum (ER) stress, hypoxia, reactive oxygen species, and nutrient deprivation were also seen to modulate the magnitude of NMD by mechanisms that are beginning to be understood (6). For example, the activation of kinases, as part of the cell-stress corrective pathways, induces the phosphorylation of the eukaryotic initiation factor 2 alpha (eIF2α), reducing protein translation and thus impairing NMD activity (7,8). In contrast, this eIF2α phosphorylation-dependent inhibition of NMD in stress conditions is responsible for the upregulation of many stress-related transcripts that are responsible for allowing the cell to cope with stress (7,9–11). There is currently great interest in decoding the mechanisms that couple stress signaling to human pathology. Only recently has ER stress been considered a potential contributor to cardiac and vascular diseases (12). Myocardial infarction is a pathological state that occurs during ischemia, where there is nutrient and oxygen deprivation in the heart, causing aggregation of proteins in the ER. This aggregation triggers ER stress and the three arms (ATF6, IRE1α and PERK) of the unfolded protein response (UPR), to mitigate or eliminate the stress (12). Ultimately, if the stimulus is continued, cell death is activated (13,14). NMD plays a role in the regulation of the UPR, establishing a threshold for its activation and its time-dependent attenuation, that is accomplished, in part, through degradation of the IRE1α mRNA (9). NMD also protects the cell from death in response to stress, but the mechanism for this remains unclear (9). Despite being a very well-studied mechanism, NMD and its role in cell physiology needs to be further explored. Given this and the all above-mentioned, the main goal of this project is to understand the role 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, H9C2 cell line will be used as a model of cardiomyocytes, which will help us to dissect the crosstalk communication between NMD and UPR, through the PERK pathway, in myocardial infarction-mimicking conditions.N/

Transcriptomic screen for DIS3, DIS3L1 and DIS3L2-associated functional networks in colorectal cancer

The 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. 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 (Astuti et al., 2012). In another study, DIS3 was found to be highly expressed in colorectal cancer (CRC), suggesting an oncogenic function (Camps et al., 2013). A major challenge in systems biology is to reveal the cellular networks that give rise to specific phenotypes (Lan et al., 2013). In this project, we aim to analyze how DIS3 and DIS3L1 regulate the human transcriptome, and how their functional interactions modulate the transcriptional reprogramming of colorectal cancer cells. We will perform an extensive characterization of the DIS3 and DIS3L1 mRNA targets, using DIS3 and DIS3L1 knockdown and microarray analysis, in normal colorectal cells, and in different CRC cell lines, in the presence and absence of stress stimuli, such as hypoxia. Differential expression and gene set enrichment analyses of collected data will elucidate new cellular pathways regulated by DIS3 and DIS3L1 and/or by their targets, as well as how they can be involved in CRC. In addition, this analysis may reveal novel functional networks through which the RNA exosome modulates the eukaryotic transcriptome.N/

Analysis of the translatome by ribosome profiling in colorectal cancer

Colorectal 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/

Study of the function of translating ribosomes within mRNA 5’ untranslated regions in colorectal cancer tumorigenesis

Colorectal cancer (CRC) has a high incidence and mortality rates worldwide. Its carcinogenenic process is based in a continuous accumulation of genetic alterations with concomitant variations in the profiles of gene expression. In order to study the variations in the gene expression profiles involved in cancer progression, genome-wide analyses have so far focused on the abundance of mRNA as measured either by microarray or RNA sequencing. However, neither approach provides information on the rate of protein synthesis, a step closer to the end-point of gene expression. Furthermore, the correlation between transcript and protein abundance is underestimated, as it does not account for the translational regulation mechanisms, thus limiting and masking the analysis of gene expression. To this end, ribosome profiling (Ribo-seq) emerges to monitor in vivo translation, providing global and quantitative measurements of translation by deep sequencing of ribosome-protected mRNA fragments (RPFs). The advent of this technique led to the identification of translation beyond the known annotated coding sequences. For instance, Ribo-seq analysis is informative about other start sites relative to the annotated canonical start codon leading to alternative open reading frames (AltORFs). In addition, this approach detects translating ribosomes within the 5’ untranslated regions (5’UTRs) consistent with the translation of upstream ORFs (uORFs), as well as ribosomes at the 3’UTR. Our aim is to determine the biological role of specific uORFs in the process of CRC tumorigenesis. For that, we will use already available Ribo-seq data from different cancer cell lines to get the 5’UTR translation profiles and choose potential uORFs-containing targets for further study. Then, we will analyze the role of such uORFs in translational regulation and study the biological function of those translatable uORFs at the level of cell viability and proliferation, and acquisition of malignant features to understand their involvement in CRC development. We analyzed the 5’UTR ribosome occupancy profiles obtained by available Ribo-seq data using a selection criteria based on a higher number of RPFs at the 5’UTR of cancer cells compared to the non-neoplasic cell lines as a proxy of uORFs translation. Then, those targets were characterized in terms of molecular function and biological process by gene ontology analysis in order to choose the ones with a cancer-related function. We are currently mapping the exact 5’-end of each transcript 5’UTR by circular rapid amplification of cDNA ends (cRACE) to finally clone them in a reporter plasmid and study their function in translational control.info:eu-repo/semantics/publishedVersio

The interplay between nonsense-mediated mRNA decay (NMD) and the unfolded protein response (UPR) in response to myocardial infarction

Nonsense-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. 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/

The mechanism through which nonsense mutations are recognized as premature translation-termination codons

About 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/4890/2014N/