National Institute of Health Dr. Ricardo Jorge
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Regulation of the alternative splicing of RAC1B in tumor cells
No full textDissertação de mestrado em Bioquímica e Biomedicina, apresentada à Faculdade de Ciências da Universidade de Lisboa, 2023.Dissertação orientada por: Doutora Vânia Gonçalves e Doutor Peter Jordan.Trabalho de dissertação de Mestrado realizado no Laboratório de Oncobiologia e Vias de Sinalização do
Instituto Nacional de Saúde Doutor Ricardo Jorge.Cancer is a molecularly heterogeneous disease that presents genetic modifications in different alternative pathways. Namely, a subgroup of colorectal cancer (CRC) is characterized by the simultaneous presence of an oncogenic mutation in BRAF and overexpression of RAC1B, an alternative splicing variant of the small GTPase RAC1. Together, these two changes stimulate signalling pathways that induce the proliferation and survival of CRC cells. Previously, the splicing factors (SFs) ESRP1 and SRSF1 were reported to promote RAC1B overexpression in this type of tumor, while SRSF3 inhibited it. However, the overexpression of RAC1B has also been identified in breast and lung tumors. As such, this work aimed to study the modulatory role of ESRP1, SRSF1 and SRSF3 on RAC1B alternative splicing in breast and lung cancer cells, using CRC cells as an experimental control since the role of these SFs has already been documented in these types of tumor. The SFs were overexpressed in the cells by co-transfection with a RAC1 minigene and the expression levels of the minigene-derived transcripts were analysed by RT-PCR. Next, the SFs endogenous expression was depleted by siRNA transfection and the endogenous levels of RAC1B transcript and protein were assessed through RT-PCR and Western blot, respectively. The obtained results indicate a role for SRSF1 and SRSF3 in positively and negatively modulating RAC1B alternative splicing, respectively, in both breast and lung cancer cells, as previously described for CRC. Intriguingly, ESRP1 inhibited RAC1B alternative splicing in these cells, revealing an opposite role to what was previously observed for CRC. Although future studies should be performed to confirm these results, ESRP1 and SRSF3 act as inhibitors of RAC1B alternative splicing whereas SRSF1 acts as an enhancer, in both breast and lung cancer cells.O foco deste trabalho experimental consistiu em averiguar se os fatores de splicing
ESRP1, SRSF1 e SRSF3 desempenham um efeito modulatório no splicing alternativo de RAC1B em
tumores da mama e do pulmão, nos quais a sua atividade já foi reportada.Funding: Bolsa de Iniciação
à Investigação do Biosystems and Integrative Sciences Institute no âmbito do BioISI Junior Programe
2022 (UIDP/04046/2020) financiada pela Fundação para a Ciência e Tecnologiainfo:eu-repo/semantics/publishedVersio
A network integrative approach to unravel new links between NMD and mRNA processing pathways
No full textNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and
selectively degrades mRNAs carrying premature translation termination codons (PTCs).
This process has been associated with many genetic diseases and some forms of cancer
caused by nonsense or frameshift mutations that introduce PTCs. Moreover, recent
studies have shown that NMD is also involved in the regulation of a large number of
transcripts, suggesting a major role in the control of gene expression. To further
investigate the biological relevance of NMD and how this process can be modulated, we
used a network analysis approach that integrates 1) protein-protein, 2) kinasetarget,
3) phosphatase-target, 4) miRNA-target, 5) transcription factors-target, 6)
gene co-expression, 7) ubiquitination and 8) signaling interactions. The generated
network was 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). Taking in account all information
sources integrated in our network, we have created a scoring algorithm to identify
new potentially important players in NMD, which can be essential to further
understand the interplay between mRNA translation, PTC definition and NMD. 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.N/
An unexpected role for DIS3L2 over human NMD targets
No full textThe 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.FCT/PTDC/BIMONC/4890/2014N/
Integrative network analysis for identification of new proteins involved in NMD or its regulation
No full textBackground: 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.The authors acknowledge the Portuguese Foundation for Science and Technology (FCT) for the PhD
grant: PD/BD/130959/2017N/
Autism Spectrum Disorder: gene variants involved in the nonsense-mediated mRNA decay pathway
No full textGenetic factors account for 50-80% of the familial risk of Autism Spectrum Disorder (ASD), but most of the genetic determinants are still unknown and a role for other regulatory mechanisms is likely. The nonsense-mediated decay (NMD) pathway is essential to control mRNA quality and has an important role in the regulation of the transcriptome. Mutations in genes involved in the NMD pathway, such as the UPF3B gene, a core component of this pathway, were previously linked to ASD. In this study we explored the potential role of NMD factors in ASD.
We generated a list of 153 genes involved in the NMD pathway using AmiGO, Reactome and a systematic literature review. To identify potentially pathogenic variants in the NMD genes, we analyzed whole exome sequencing data (WES) data from 1338 ASD subjects. We also searched for Copy Number Variants (CNVs) targeting NMD genes in ASD patients (n=3570) and checked their frequency in controls (n=9649).
We identified 43 high impact variants in 28 NMD genes, including the UPF3B and ACE, two genes previously implicated in ASD. Importantly, 11 were novel candidate genes that carry loss-of-function and missense (deleterious and damaging) variants with a frequency of 1 to 5% in this ASD dataset. Additionally, 5 NMD genes were found to be targeted by CNVs in 12 ASD subjects but none of the controls.
The discovery of 33 NMD genes that are intriguing candidates for ASD in large patient genomic datasets provides evidence supporting the involvement of the NMD pathway in ASD pathophysiology.A.R.Marques is recipient of a fellowship from BioSys PhD programme (Ref
PD/BD/113773/2015 from FCT (Portugal).info:eu-repo/semantics/publishedVersio
The tumor suppressor p53 acquires oncogenic functions due to a translational switch during integrated stress response
No full textTo cope with the stress stimuli to which they are often exposed, eukaryotic cells have developed adaptive pathways that restore cellular homeostasis. Under stress conditions there is an overall decrease of protein synthesis, and a concomitant induction of alternative mechanisms of mRNA translation initiation.
The tumour suppressor protein p53 has been considered the guardian of the genome and a master regulator of many cellular functions. However, apart from the full-length p53 (FLp53), several p53 isoforms have been described so far. Some functions of shorter p53 isoforms have already been elucidated and they are different from and complement FLp53 activity, the most mutated gene in cancer.
Here we show that the integrated stress response (ISR) leads to the specific induction of Δ160p53 isoform. Using bicistronic constructs we confirmed the presence of an Internal Ribosome Entry Site (IRES) in p53 mRNA that controls Δ160p53 isoform translation. Subjecting cells to endoplasmic reticulum stress showed that eIF2α phosphorylation is a key event leading to cap-independent expression of Δ160p53 during ISR.
Additionally, cancer-specific mutations in p53 also enhanced cap-independent translation of Δ160p53 via Δ160p53IRES. An antisense morpholino oligo targeting Δ160IRES efficiently reduced Δ160p53 protein levels and significantly impaired oncogenic functions of Δ160p53.
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 PTDC/MED-ONC/32048/2017N/
Transcriptomic screen for DIS3, DIS3L1 and DIS3L2-associated functional networks in colorectal cancer
No full textThe 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/
How mRNA translation is involved in modulating nonsense-mediated decay in transcripts with AUG-proximal nonsense mutations
No full textNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs). In addition, several studies have also implicated NMD in the regulation of steady-state levels of physiological mRNAs, and examples of natural NMD targets are transcripts containing upstream short open reading frames or long 3’ untranslated regions.
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 translation initiation.N/
Analysis of the translatome by ribosome profiling in colorectal cancer
No full textColorectal cancer (CRC) is a serious health problem due to its high incidence and mortality rates despite the major advances in cancer therapeutic approaches. CRC carcinogenesis progression is based in a continuous accumulation of genetic alterations that leads to variations in the overall gene expression profiles. This creates the need for deep analysis of cancer gene expression patterns and, thus, a more reliable understanding of the human proteome to disclose the molecular and cellular pathways as well as the regulatory mechanisms involved in cancer progression.
Genome wide analyses of gene expression have so far focused on the abundance of mRNA species as measured either by microarray or, more recently, by RNA deep sequencing. However, neither approach provides information on protein synthesis, an important end point of gene expression. Ribosome profiling is an emerging technique that uses deep sequencing to monitor in vivo translation and provide global and quantitative measurements of translation. It can also reveal unexpected complexity in translation, including the presence of ribosomes outside of classical protein-coding regions of the transcriptome. In this approach, translation is profiled by nuclease footprinting of ribosomes on RNA templates and high-throughput sequencing in order to determine the precise positions of ribosomes on a transcript and its overall density. Ribosome profiling studies have been performed in cancer cell lines, where they showed an increase in overall protein identification and new proteins not yet annotated that possibly were originated from N-terminal extensions or upstream open reading frames (uORFs).
The main goal of this project is to determine the changes between the translatome of CRC and normal colorectal cells and the role of such alterations in the tumorigenesis process of CRC cells. For this purpose, we will perform ribosome profiling in normal (NCM460) and CRC (HCT116) cell lines. Bioinformatics and gene ontology analysis of the translated mRNAs will elucidate the main cellular pathways through which the corresponding proteins are involved in CRC progression. Then, we will dissect which of these proteins can interfere and induce cell survival of CRC cells. Furthermore, we aim to analyze the potential contribution of translatable short alternative ORFs (AltORFs) and/or the corresponding peptides towards CRC progression. This information will be crucial to the development of new therapeutic strategies for CRC.N/
EVITA – Epidemiologia e Vigilância dos Traumatismos e Acidentes: relatório 2023
No full textRelatório EVITA – Epidemiologia e Vigilância dos Traumatismos e Acidentes relativo ao ano de 2023. O sistema EVITA é um sistema de recolha e análise de dados sobre os acidentes domésticos e de lazer que implicaram recurso às urgências das unidades do Serviço Nacional de Saúde (SNS).
Em Portugal, a notificação dos acidentes domésticos e de lazer (ADL) realiza-se através do sistema de vigilância EVITA, criado em 2000 na continuidade do sistema ADELIA (Acidentes Domésticos e de Lazer – Informação Adequada), sob a coordenação do Departamento de Epidemiologia do INSA. Este sistema conta com a colaboração da entidade Serviços Partilhados do Ministério da Saúde.
Os objetivos do sistema EVITA são: a) fornecer informação essencial sobre a epidemiologia dos ADL em Portugal; b) manter um sistema de vigilância que permita a caracterização dos ADL, a identificação das situações de risco, os agentes envolvidos, bem como os produtos perigosos que propiciem a ocorrência de ADL; c) manter uma base de dados disponível para a comunidade cientifica, a partir da qual seja possível realizar estudos epidemiológicos na área dos ADL, e avaliar políticas de prevenção baseadas na evidência.
O presente relatório apresenta a análise descritiva dos dados recolhidos pelo sistema EVITA no decurso do ano de 2023. Evidenciam-se, dessa análise, os seguintes resultados: - Uma proporção de ADL superior no sexo feminino nos grupos etários acima dos 55 anos; - Uma frequência mais elevada de ADL ocorridos na habitação, com destaque para o sexo masculino dos 0-44 anos e para o sexo feminino a partir dos 75 anos; - A proporção de ADL ocorridos em “Escola, área institucional e recintos públicos” foi mais elevada no sexo masculino entre os 0 e os 54 anos, e a partir dos 55 anos no sexo feminino; - O “Mecanismo de lesão” mais frequente foi a “Queda”; - Cerca de 57% de todos os ADL envolveram uma “Contusão, hematoma”; - A parte do corpo lesada referida com maior frequência foram os “Membros”