Browsing by Author "Santos, Hugo"
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- 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; Romão, LuísaColorectal cancer (CRC) has a high incidence and mortality rates worldwide [1]. CRC carcinogenesis is a continuous accumulation of genetic alterations with concomitant variations in gene expression profiles [2]. To study the variations of 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 [3,4]. However, neither approach provides information on protein synthesis, which is the true end-point of gene expression [3-5]. 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) [5,6]. This technique has revealed unexpected complexity in translation, including the presence of ribosomes outside of classical protein-coding regions of the transcriptome [3]. 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. For that, we aim to analyze ribosome profiling data already available for the CRC cell line HCT116, and eventually data from non-neoplasic colorectal cells (if available). 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 function of translatable small open reading frames (sORFs), such as the upstream ORFs (uORFs), and/or the corresponding encoded peptides in the regulation of CRC progression. We have performed a computational analysis of HCT116 Ribo-Seq data to detect potential translatable uORFs. For that we are currently determining the number of RPFs in the 5’UTR of transcripts. Meanwhile, and based on previously published data about the prediction/detection of translatable alternative ORFs (AltORFs) in CRC cells [7], ABCE1, ABCF1, ABCF2 and ABCF3 mRNAs were chosen for further studies. To analyze their mRNA expression levels, we performed semi-quantitative RT-PCR analysis using RNA from HCT116, Caco-2 and SW480 CRC cells, as well as from non-neoplasic colorectal NCM460 cells. Our results show that these mRNAs are down-regulated in HCT116 cells comparing to their expression in the other three cell lines. In addition, we have been involved in mapping, by circular rapid amplification of cDNA ends (cRACE), cloning and sequencing, the exact 5’ end of the ABCE1 5’UTR. After getting this information, we will clone this 5’UTRs in a reporter construct that will allow us to test the ABCE1 uORF potential function in CRC progression.
- 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.
- Bioinformatics study of expression from genomes of epidemiologically related MRSA CC398 isolates from human and wild animal samplesPublication . Ribeiro, Miguel; Sousa, Margarida; Borges, Vítor; Gomes, João Paulo; Duarte, Sílvia; Isidro, Joana; Vieira, Luís; Torres, Carmen; Santos, Hugo; Capelo, José Luís; Poeta, Patrícia; Igrejas, GilbertoOne of the most important livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) genetic lineages is the clonal complex (CC) 398, which can cause typical S. aureus-associated infections in people. In this work, whole-genome sequencing, RNA-sequencing, and gel-based comparative proteomics were applied to study the genetic characteristics of three MRSA CC398 isolates recovered from humans (strains C5621 and C9017), and from an animal (strain OR418). Of the three strains, C9017 presented the broadest resistance genotype, including resistance to fluroquinolone, clindamycin, tiamulin, macrolide and aminoglycoside antimicrobial classes. The scn, sak, and chp genes of the immune evasion cluster system were solely detected in OR418. Pangenome analysis showed a total of 288 strain-specific genes, most of which are hypothetical or phage-related proteins. OR418 had the most pronounced genetic differences. RNAIII (δ-hemolysin) gene was clearly the most expressed gene in OR418 and C5621, but it was not detected in C9017. Significant differences in the proteome profiles were found between strains. For example, the immunoglobulin-binding protein Sbi was more abundant in OR418. Considering that Sbi is a multifunctional immune evasion factor in S. aureus, the results point to OR418 strain having high zoonotic potential. Overall, multiomics biomarker signatures can assume an important role to advance precision medicine in the years to come. SIGNIFICANCE: MRSA is one of the most representative drug-resistant pathogens and its dissemination is increasing due to MRSA capability of establishing new reservoirs. LA-MRSA is considered an emerging problem worldwide and CC398 is one of the most important genetic lineages. In this study, three MRSA CC398 isolates recovered from humans and from a wild animal were analyzed through whole-genome sequencing, RNA-sequencing, and gel-based comparative proteomics in order to gather systems-wide omics data and better understand the genetic characteristics of this lineage to identify distinctive markers and genomic features of relevance to public health.
- How the DIS3 proteins shape the human transcritpomePublication . Costa, Paulo; Santos, Hugo; Gama Carvalho, Margarida; Romão, LuísaThe final step of eukaryotic mRNA degradation proceeds in either a 5’-3’ direction, catalyzed by XRN1, or in a 3’-5’ direction catalyzed by DIS3, DIS3L1 (the catalytic subunits of the exosome) and/or DIS3L2 (exosome-independent). 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. With the aim of analyzing how DIS3, DIS3L1 and DIS3L2 regulate the human transcriptome, each one of these nucleases was depleted by RNA interference in HeLa cells and levels of several reporter mRNAs was monitored by RT-qPCR. Our results show that these exoribonucleases are target specific and not directly involved in a particular mRNA surveillance mechanism. In parallel, our bioinformatics analysis of available transcriptomic data from cells depleted of DIS3L1, DIS3L2, XRN1, or UPF1 (which has a central role in nonsense-mediated mRNA decay) has shown some, but not full, redundancy among the transcripts regulated by these nucleases, which supports our experimental data. Presently, we are exploring the molecular mechanisms underlying our observations.
- The DIS3 proteins family: role in the human transcriptome regulation and CRC tumorigenesisPublication . Costa, Paulo; Santos, Hugo; Gama-Carvalho, Margarida; Romão, LuísaThe final step of eukaryotic mRNA degradation proceeds in either a 5’-3’ direction, catalyzed by XRN1, or in a 3’-5’ direction catalyzed by DIS3, DIS3L1 (the catalytic subunits of the exosome) and/or DIS3L2 (exosome-independent). 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. With the aim of analyzing how DIS3, DIS3L1 and DIS3L2 regulate the human transcriptome, each one of these nucleases was depleted by RNA interference in HeLa cells and levels of several reporter mRNAs was monitored by RT-qPCR. Our results show that these exoribonucleases are target specific and not directly involved in a particular mRNA surveillance mechanism. In parallel, our bioinformatics analysis of available transcriptomic data from cells depleted of DIS3L1, DIS3L2, XRN1, or UPF1 (which has a central role in nonsense-mediated mRNA decay) has shown some, but not full, redundancy among the transcripts regulated by these nucleases, which supports our experimental data. Presently, we are exploring the molecular mechanisms underlying our observations and looking for relationships between their specific targets and their potential role in tumorigenesis.
- Transcriptomic screen for DIS3, DIS3L1 and DIS3L2-associated functional networks in colorectal cancerPublication . Costa, Paulo; 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 (1). In another study, DIS3 was found to be highly expressed in colorectal cancer (CRC), suggesting an oncogenic function (2). A major challenge in systems biology is to reveal the cellular networks that give rise to specific phenotypes (3). 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.
- Transcriptomic screen for DIS3, DIS3L1 and DIS3L2-associated functional networks in colorectal cancerPublication . Costa, Paulo J. da; Saramago, Margarida; Viegas, Sandra C.; Arraiano, Cecília; Santos, Hugo; Gama-Carvalho, Margarida; Romão, LuísaThe final step of eukaryotic mRNA degradation proceeds in either a 5’-3’ direction, catalyzed by XRN1, or in a 3’-5’ direction catalyzed by DIS3, DIS3L1 (the catalytic subunits of the exosome) and/or DIS3L2 (exosome-independent). 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. In this work, 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. Each one of these nucleases was depleted by RNA interference in HeLa cells and levels of several endogenous targets was monitored by RT-qPCR. Our results show that these exoribonucleases are target specific and not directly involved in any known mRNA decay mechanisms such as nonsense-mediated decay (NMD). However, we do not know yet what defines such target preference. In parallel, our bioinformatics analysis of available transcriptomic data from cells depleted of DIS3L1, DIS3L2, XRN1 or UPF1 (which has a central role in NMD) has shown some, but not full, redundancy among the transcripts regulated by these nucleases, which supports our experimental data. Presently, we are exploring the mechanism through which DIS3L2 is involved in NMD and how it modulates the expression of NMD targets.