Percorrer por autor "Silva, Joana"
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- Analysis of the translatome by ribosome profiling in colorectal cancerPublication . Silva, Joana; Romão, Luísa; Luchessi, AugustoColorectal cancer (CRC) is a serious health problem due to its high incidence and mortality rates despite the major advances in cancer therapeutic approaches [1]. CRC carcinogenesis progression is based in a continuous accumulation of genetic alterations that leads to variations in the overall gene expression profiles [2]. 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 [2-4]. 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 [5,6]. However, neither approach provides information on protein synthesis, an important end point of gene expression [5,7]. Ribosome profiling is an emerging technique that uses deep sequencing to monitor in vivo translation and provide global and quantitative measurements of translation [7,8]. It can also reveal unexpected complexity in translation, including the presence of ribosomes outside of classical protein-coding regions of the transcriptome [5]. 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 [8]. 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) [9-12]. 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.
- Analysis of the translatome by ribosome profiling in colorectal cancerPublication . Silva, JoanaColorectal cancer (CRC) is a serious health problem due to its high incidence and mortality rates despite the major advances in cancer therapeutic approaches [1]. CRC carcinogenesis progression is based in a continuous accumulation of genetic alterations that leads to variations in the overall gene expression profiles [2]. 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 [2-4]. 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 [5,6]. However, neither approach provides information on protein synthesis, an important end point of gene expression [5,7]. Ribosome profiling is an emerging technique that uses deep sequencing to monitor in vivo translation and provide global and quantitative measurements of translation [7,8]. It can also reveal unexpected complexity in translation, including the presence of ribosomes outside of classical protein-coding regions of the transcriptome [5]. 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 [8]. 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) [9-12]. 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.
- 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.
- Analysis of translation of 5’ untranslated regions in cancerPublication . Silva, Joana; Romão, Luísa; Luchessi, AugustoShort upstream open reading frames (uORFs) are cis-acting elements located within the 5'- leader sequence of transcripts. Recent genome-wide ribosome profiling (RiboSeq) studies have demonstrated the widespread presence of uORFs in the transcriptome and have shown that many uORFs can initiate with non-AUG codons. uORFs can impact gene expression of the downstream main open reading frame (mORF) by triggering messenger RNA (mRNA) decay or by regulating translation. Thus, disruption, elimination or creation of uORFs can elicit the development of several genetic diseases, such as cancer. The ATP-binding cassette subfamily E member 1 (ABCE1) gene, belongs to the ABC gene family of transporters. However, it does not behave as a drug transporter like the other members of this family. ABCE1 actively participates in the different stages of the translation process and is involved in cell proliferation and anti-apoptotic signaling processes, associating ABCE1 to a potential oncogenic function. RiboSeq occupancy profiles of the ABCE1 mRNA 5’-leader sequence indicate an active translation associated with the presence of uORFs, which is suggestive of a high translational regulation. Our aim was to study the translational regulation mediated by the five AUG and five non- AUG uORFs present in the human ABCE1 5’-leader sequence in colorectal cancer. With this purpose, we constructed a set of Firefly luciferase (FLuc) reporter vectors derived from the wild-type one containing the native configuration of the human ABCE1 5’-leader sequence upstream of the FLuc ORF, and transiently transfected colorectal cancer HCT116 cells. Here we show that ABCE1 mORF expression is regulated by its uORFs. Our results are consistent with a model wherein uORF1 recruits ribosomes onto the mRNA, behaving like a ribosomal barrier. The ribosomes that efficiently bypass uORF1 and/or uORF2, must probably reinitiate at uORF3 and/or uORF5, while uORF4 is greatly bypassed. uORF3 and uORF5 function as repressive uORFs that may cooperate to reach a maximum repression of the mORF. Thus, both bypass and reinitiation events of the AUG uORFs within ABCE1 5’-leader sequence contribute for the translational control of the mORF. In constrast, the non-AUG uORFs seem to be devoided of a significant inhibitory activity. The AUG uORF-mediated translational control is maintained in normal and in endoplasmic reticulum (ER) stress conditions, which keeps the expression level of ABCE1 at a minimum, showing that ABCE1 is a resistant transcript whose functions are equally essential in normal and in coditions of global translation impairment. In addition, we show that ABCE1 uORF-mediated translational regulation is preserved in non-tumorigenic and cancerous cells, which is consistent with a lack of an oncogenic function by the uORFs, as well as ABCE1 himself, in the colorectal cancer cell line tested. This study contributes with an additional example of how uORF-mediated translational regulation can occur. In addition, it reveals how important is to screen the 5’-leader sequence of the transcripts in search for potential disease-related variants. This information might be relevant for the implementation of new diagnostic and/or therapeutic tools for diseases associated with the deregulation of uORF-mediated translational control.
- Analysis of translation of 5’ untranslated regions in colorectal cancerPublication . Silva, Joana; Romão, LuísaCarcinogenesis is characterized by a continuous accumulation of genetic alterations that changes the overall gene expression profiles. Those alterations have been stuied by microarray or RNA sequencing that measure the abundance of mRNA but do not provide information on protein synthesis, which is a step closer to end-point of gene expression. Ribosome profiling (Ribo-seq) emerges to monitor in vivo translation by deep sequencing of ribosome-protected mRNA fragments. This technique reveals the presence of ribosomes outside of known protein-coding regions, identifying translation of upstream open reading frames (uORFs) within 5’ untranslated regions (5’UTRs). Our aim is to determine the role of specific uORFs in cancer tumorigenesis, mainly in colorectal cancer (CRC). Thus, we will use already available Ribo-seq data from different cancer cell lines to get the 5’UTR translation profiles to choose potential uORFs-containing targets. 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. Based in 5’UTR ribosome occupancy profiles from Ribo-seq analysis we chose ABCE1, PAIP2, eIF4G2 and eIF2A as our uORFs-containing mRNAs. By semi-quantitative RT-PCR ABCE1 transcript is shown down- and up-regulated in HCT116 and SW480 cells, respectively, in comparison to the non-neoplasic colorectal cell line (NCM460). To test the potential function of uORFs of our transcripts, we are now mapping the exact 5’-end of each 5’UTRs by circular rapid amplification of cDNA ends to finally clone them in a reporter plasmid and study their function in translational control.
- 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.
- 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. Its carcinogenesis process is characterized by a continuous accumulation of genetic alterations that changes the overall gene expression profiles. 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. Ribosome profiling (RiboSeq) emerges to monitor in vivo translation by deep sequencing of ribosome-protected mRNA fragments (RPFs). This technique detects ribosomes outside of known protein-coding regions, identifying translation of upstream open reading frames (uORFs) within 5’ untranslated regions (5’UTRs). 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.
- Gene expression regulation by upstream open reading frames in rare diseasesPublication . Silva, Joana; Fernandes, Rafael; Romão, LuísaUpstream open reading frames (uORFs) constitute a class of cis-acting elements that regulate translation initiation. Mutations or polymorphisms that alter, create or disrupt a uORF have been widely associated with several human disorders, including rare diseases. In this mini-review, we intend to highlight the mechanisms associated with the uORF-mediated translational regulation and describe recent examples of their deregulation in the etiology of human rare diseases. Additionally, we discuss new insights arising from ribosome profiling studies and reporter assays regarding uORF features and their intrinsic role in translational regulation. This type of knowledge is of most importance to design and implement new or improved diagnostic and/or treatment strategies for uORF-related human disorders.