Browsing by Author "Gama-Carvalho, Margarida"
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- Activation of RAC1/PAK1 axis potentiates transcriptional upregulation of DNA damage response genes via the BCL6/STAT5 switchPublication . Barros, Patrícia; Amaral, Andreia; Abrantes, Leonor; Oliveira, Tiago; Lourio, Henriqueta; Silva, Maria João; Jordan, Peter; Gama-Carvalho, Margarida; Matos, PauloColorectal cancer is one of the most prevalent types of cancer worldwide. The GTPase RAC1 and its effector PAK1 have been found overexpressed or hyperactivated in colorectal cancers, particularly those with more aggressive and invasive features, leading to unfavourable clinical prognosis, often resulting from chemoresistance.
- Alternative splicing of tumour-related Rac1b in colorectal cells is regulated by protein phosphorylation of splicing factor SRSF1Publication . Gonçalves, Vânia; Henriques, Andreia; Pereira, Joana; Neves-Costa, Ana; Moyer, Pat; Ferreira Moita, Luís; Gama-Carvalho, Margarida; Matos, Paulo; Jordan, PeterThe pre-messenger RNA of the majority of human genes can generate various transcripts through alternative splicing, and different tissues or disease states show specific patterns of splicing variants. These patterns depend on the relative concentrations of the splicing factors present in the cell nucleus, either as a consequence of their expression levels or of post-translational modifications such as protein phosphorylation, which are determined by signal transduction pathways. Here we analyzed the contribution of protein kinases to the regulation of alternative splicing variant Rac1b that is overexpressed in certain tumor types. In colorectal cells we found that depletion of AKT2, AKT3, GSK3β and SRPK1 significantly decreased endogenous Rac1b levels. Whereas knockdown of AKT2 and AKT3 affected only Rac1b protein levels suggesting a post-splicing effect, the depletion of GSK3β or SRPK1 decreased Rac1b alternative splicing, an effect mediated through changes in splicing factor SRSF1. In particular, the knockdown of SRPK1 or inhibition of its catalytic activity reduced phosphorylation and subsequent translocation of SRSF1 to the nucleus, limiting its availability to promote the inclusion of alternative exon 3b into the Rac1 pre-mRNA. Altogether, the data identify SRSF1 as a prime regulator of Rac1b expression in colorectal cells and provide further mechanistic insights into how the regulation of alternative splicing events by protein kinases can contribute to sustain tumor cell survival.
- 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.
- Analysis of the translatome by ribosome profiling in colorectal cancerPublication . Silva, Joana; Santos, Hugo; Gama-Carvalho, Margarida; Romão, LuísaColorectal 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.
- Analysis of the translatome by ribosome profiling in colorectal cancerPublication . Silva, Joana; Santos, Hugo; Gama-Carvalho, Margarida; Romão, LuísaColorectal 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.
- DIS3L2 knockdown impairs key oncogenic properties of colorectal cancer cells via the mTOR signaling pathwayPublication . García-Moreno, Juan F.; Lacerda, Rafaela; da Costa, Paulo J.; Pereira, Marcelo; Gama-Carvalho, Margarida; Matos, Paulo; Romão, LuísaDIS3L2 degrades different types of RNAs in an exosome-independent manner including mRNAs and several types of non-coding RNAs. DIS3L2-mediated degradation is preceded by the addition of nontemplated uridines at the 3’end of its targets by the terminal uridylyl transferases 4 and 7. Most of the literature that concerns DIS3L2 characterizes its involvement in several RNA degradation pathways, however, there is some evidence that its dysregulated activity may contribute to cancer development. In the present study, we characterize the role of DIS3L2 in human colorectal cancer (CRC). Using the public RNA datasets from The Cancer Genome Atlas (TCGA), we found higher DIS3L2 mRNA levels in CRC tissues versus normal colonic samples as well as worse prognosis in patients with high DIS3L2 expression. In addition, our RNA deep-sequencing data revealed that knockdown (KD) of DIS3L2 induces a strong transcriptomic disturbance in SW480 CRC cells. Moreover, gene ontology (GO) analysis of significant upregulated transcripts displays enrichment in mRNAs encoding proteins involved in cell cycle regulation and cancer-related pathways, which guided us to evaluate which specific hallmarks of cancer are differentially regulated by DIS3L2. To do so, we employed four CRC cell lines (HCT116, SW480, Caco-2 and HT-29) differing in their mutational background and oncogenicity. We demonstrate that lack depletion of DIS3L2 results in reduced cell viability of highly oncogenic SW480 and HCT116 CRC cells, but had little or no impact in the more differentiated Caco-2 and HT-29 cells. Remarkably, the mTOR signaling pathway, crucial for cell survival and growth, is downregulated after DIS3L2 KD, whereas AZGP1, an mTOR pathway inhibitor, is upregulated. Furthermore, our results indicate that depletion of DIS3L2 disturbs metastasis-associated properties, such as cell migration and invasion, only in highly oncogenic CRC cells. Our work reveals for the first time a role for DIS3L2 in sustaining CRC cell proliferation and provides evidence that this ribonuclease is required to support the viability and invasive behavior of dedifferentiated CRC cells.
- Experimental supporting data on DIS3L2 over nonsense-mediated mRNA decay targets in human cellsPublication . da Costa, Paulo J.; Menezes, Juliane; Saramago, Margarida; García-Moreno, Juan F.; Santos, Hugo A.; Gama-Carvalho, Margarida; Arraiano, Cecília M.; Viegas, Sandra C.; Romão, LuísaIn this article, we present supportive data related to the research article “A role for DIS3L2 over natural nonsense-mediated mRNA decay targets in human cells” [1], where interpretation of the data presented here is available. Indeed, here we analyze the impact of the DIS3L2 exoribonuclease over nonsense-mediated mRNA decay (NMD)-targets. Specifically, we present data on: a) the expression of various reporter human β-globin mRNAs, monitored by Northern blot and RT-qPCR, before and after altering DIS3L2 levels in HeLa cells, and b) the gene expression levels of deregulated transcripts generated by re-analyzing publicly available data from UPF1-depleted HeLa cells that were further cross-referenced with a dataset of transcripts upregulated in DIS3L2-depleted cells. These analyses revealed that DIS3L2 regulates the levels of a subset of NMD-targets. These data can be valuable for researchers interested in the NMD mechanism.
- Functional networks of DIS3L2 in cancer and NMDPublication . Garcia-Moreno, Juan; Costa, Paulo J. da; Gama-Carvalho, Margarida; Matos, Paulo; Romão, LuísaIn the flow of information from DNA to mRNA to proteins, mRNAs undergo a number of processing steps, since they are synthesized in the nucleus, until they are translated in the cytoplasm. Eukaryotic cells tightly control the fidelity of this process, via quality control pathways, among them, the nonsense-mediated mRNA decay (NMD). NMD recognizes and degrades mRNAs harboring premature translation-termination codons (PTCs) and regulates normal and fully functional mRNA levels. A new branch of the NMD pathway is starting to be revealed, which is characterized by the involvement of the DIS3L2 3’ to 5’ exoribonuclease. This protein has special relevance, given its exosome-independent action and its uridylation-mediated decay. Interestingly, mutations on this ribonuclease induce deregulation of cell-cycle genes leading to a faster cell growth and decreased chromosome stability. In this project, we aim to analyze how DIS3L2 and uridylation regulate the human transcriptome, in order to shed light on how this ribonuclease is related to NMD and how its deregulation contributes to tumorigenesis. For this purpose, high-throughput mRNA sequencing has been performed in the SW480 colorectal cancer cell line depleted of DIS3L2 or DIS3L2 plus terminal uridylyl transferases (TUTases), TUT4 and TUT7. Gene ontology analysis over the set of genes up-regulated under those two conditions, show enrichment in molecular functions and biological processes related with cancer, and cell events directly implicated RNA processing and RNA degradation. Preliminary results on genetic features in the pool of deregulated transcripts also show significant differences between conditions, an important aspect that will guide us to determine grades of sensitivities in the decay of DIS3L2-subtrates. Currently, we are setting various approaches in order to unveil the role of DIS3L2 in oncogenesis and go deeper in its substrate preference.
- Functional networks of DIS3L2 in cancer and NMDPublication . García-Moreno, Juan; da Costa, Paulo J; Menezes, Juliane; Pereira, Marcelo; Gama-Carvalho, Margarida; Matos, Paulo; Romão, LuísaIn the flow of information from DNA to mRNA to proteins, mRNAs undergo a number of processing steps, since they are synthesized in the nucleus, until they are translated in the cytoplasm. Eukaryotic cells tightly control the fidelity of this process, via quality control pathways, among them, the nonsense-mediated mRNA decay (NMD). NMD recognizes and degrades mRNAs harboring premature translation-termination codons (PTCs), protecting the cell from potentially harmful truncated proteins. However, NMD can also regulate normal and fully functional mRNA levels, arising as a surveillance and a gene expression regulation pathway. A new branch of the NMD pathway is starting to be revealed, which is characterized by the involvement of the DIS3L2 3’ to 5’ exoribonuclease. This protein, has special relevance, given its exosome-independent action and its uridylation-mediated decay. In addition, mutations on this ribonuclease, induce deregulation of cell-cycle genes leading to a faster cell growth and decreased chromosome stability, while DIS3L2 downregulation enhances cancer stem cell properties. Several lines of evidence point to an oncogenic role of DIS3L2 and its mediated decay over a number of NMD targets, however further research is needed to unveil the mechanism by which this nuclease is involved in NMD and how it mediates cancer related processes. In this project, we aim to analyze how DIS3L2 and uridylation regulate the human transcriptome, in order to shed light on how this ribonuclease is related to NMD and how its deregulation contributes to tumorigenesis. For this purpose, high-throughput mRNA sequencing has been performed in the SW480 colorectal cancer cell line depleted of DIS3L2 or DIS3L2 plus terminal uridylyl transferases (TUTases), TUT4 and TUT7. Preliminary results show gene ontology enrichment in molecular functions and biological processes related with cancer.
- Identification of novel biomarkers to distinguish polygenic and monogenic dyslipidemiaPublication . Correia, Marta; Gama-Carvalho, Margarida; Bourbon, MafaldaObjectives: This project aims to identify novel biomarkers to distinguish polygenic and monogenic dyslipidaemia, by an integrative approach, based on the detailed characterization of the lipid profile ID of patients with severe dyslipidaemia. This is expected to contribute to an early and accurate diagnosis, allowing the implementation of a successful therapeutic plan that can be applied still in childhood to effectively abolish/decrease the patient’s cardiovascular risk. Background: Dyslipidaemia is one of the major cardiovascular risk factors. It can be due to primary causes (i.e. monogenic, characterized by a single gene mutation, or dyslipidaemia of polygenic/environmental causes), or secondary to specific disorders such as obesity, diabetes mellitus or hypothyroidism. Monogenic patients present the most severe phenotype and so they need to be identified at an early age, allowing the implementation of pharmacologic treatment able to decrease cardiovascular risk. However, the majority of hyperlipidemic patients most likely have a polygenic disease that can be controlled just by the implementation of a healthy lifestyle. Thus, the distinction between monogenic and polygenic dyslipidaemia is important for a prompt diagnosis, cardiovascular risk assessment, counselling and treatment. In spit of this, none of the current standard biomarkers (i.e. total cholesterol, LDL-c, HDL-c, triglycerides), can effectively make that discrimination between patients.
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