Browsing by Author "Neves, Ana Rita"
Now showing 1 - 7 of 7
Results Per Page
Sort Options
- Brand New World of TranslationPublication . Neves, Ana Rita; Lacerda, Rafaela; Romão, Luísa; Candeias, MarcoIn recent years, non-canonical translation initiation mechanisms have been recognized as key factors in the development of different diseases such as cancer, as they present a survival answer during stress conditions by ensuring the expression of vital proteins. Internal Ribosome Entry Sites (IRESes) were first discovered on viruses, and later in eukaryotes, as mRNA secondary structures capable of recruiting the ribosome to the vicinities of an initiation codon. One of the most studied cancer-related genes, the p53 tumor suppressor gene, was found to possess on its mRNA an IRES capable of regulating the expression of the full length isoform, p53FL, and one of its isoforms, Δ40p53 differently by the interaction with MDM2 protein, an IRES trans-acting factor (ITAF) of p53. Our aim is to study a shorter p53 protein isoform that lacks tumor suppressor behaviour acting instead as a cancer promoter (Candeias et al., 2016). One of our goals is to characterize the IRES associated with its expression. For that we will try to perform a sequencing reaction variant where the fragments to be sequenced result from uncomplete reverse transcription due to nucleotide-specific modification. Additionally, we will try to unveil new ITAFs by pulling-down the IRES and, consequently, associated factors, using two different methods: IRES biotinylation and MS2 tagging. Furthermore, we intend to find new IRESes by pulling-down MDM2 and possible bounded mRNAs followed by RNA-sequencing in order to identify them.
- Characterization of an Internal Ribosome Entry Site (IRES) in p53 mRNAPublication . Neves, Ana Rita; Lacerda, Rafaela; Marques-Ramos, Ana; Romão, Luísa; Matsuda, M.; Candeias, MarcoThe tumour suppressor p53 gene is one of the most studied cancer-related genes. So far, many p53 isoforms have been identified either resulting from alternative splicing or from non-canonical translation mechanisms. It is known that cap-dependent translation is repressed under stress conditions to preserve energy. Therefore, other translational mechanisms are required to keep the synthesis of vital proteins. Internal Ribosome Entry Sites (IRESes) were first discovered in viruses, and then observed in eukaryotes, as secondary structures present in RNA that were capable of recruiting ribosomes to the vicinity of an initiation codon inserted in an optimal environment allowing cap-independent translation of mRNAs. Translation of Δ40p53, a p53 isoform, is one example of this non-canonical mechanism due to the presence of an IRES near an alternative initiation codon (AUG40). Here, we will present and characterize a new IRES in p53 mRNA. We present details on the localization, structure, function and regulation of this IRES under normal and stress conditions. Importantly, our data reveals that the function of this IRES is required for cell survival and proliferation under certain cell conditions. This finding can have grave implications for understanding p53 function dynamics and cancer progression in specific environments.
- Genetics of personalized medicine: cancer and rare diseasesPublication . Siefers Alves, Inês; Condinho, Manuel; Custódio, Sónia; Pereira, Bruna; Fernandes, Rafael; Gonçalves, Vânia; da Costa, Paulo J.; Lacerda, Rafaela; Marques, Ana Rita; Martins-Dias, Patrícia; Nogueira, Gonçalo; Neves, Ana Rita; Pinho, Patrícia; Rodrigues, Raquel; Rolo, Eva; Silva, Joana; Travessa, André; Pinto-Leite, Rosário; Sousa, Ana; Romão, LuísaThe 21st annual meeting of the Portuguese Society of Human Genetics (SPGH), organized by Luísa Romão, Ana Sousa and Rosário Pinto Leite, was held in Caparica, Portugal, from the 16th to the 18th of November 2017. Having entered an era in which personalized medicine is emerging as a paradigm for disease diagnosis, treatment and prevention, the program of this meeting intended to include lectures by leading national and international scientists presenting exceptional findings on the genetics of personalized medicine. Various topics were discussed, including cancer genetics, transcriptome dynamics and novel therapeutics for cancers and rare disorders that are designed to specifically target molecular alterations in individual patients. Several panel discussions were held to emphasize (ethical) issues associated with personalized medicine, including genetic cancer counseling.
- IRES-dependent translation of shorter p53 isoforms is affected by mutations in p53Publication . Lacerda, Rafaela; Neves, Ana Rita; Maruo, M; Romão, Luísa; Matsuda, M; Candeias, MarcoFull-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.
- p53 mutations influence IRES-dependent expression of p53 isoformsPublication . Lacerda, Rafaela; Neves, Ana Rita; Maruo, M.; Romão, Luísa; Matsuda, M.; Candeias, MarcoFull-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. Using bicistronic constructs, primer extension, FACS and other techniques , 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.
- Shorter p53 isoform expression through na Internal Ribosome Entry Site (IRES) in p53 mRNAPublication . Neves, Ana Rita; Lacerda, Rafaela; Marques-Ramos, Ana; Romão, Luísa; Matsuda, M; Candeias, MarcoThe tumour suppressor p53 gene is one of the most studied cancer-related genes. So far, many p53 isoforms have been identified either resulting from alternative splicing, alternative translation or alternative promoter usage. It is known that cap-dependent translation is repressed under stress conditions to preserve energy. Therefore, other translational mechanisms are required to keep the synthesis of stress-response proteins. Internal Ribosome Entry Sites (IRESes) were first discovered in viruses, and then observed in eukaryotes, as secondary structures present in RNA that were capable of recruiting ribosomes to the vicinity of an initiation codon inserted in an optimal environment allowing cap-independent translation of mRNAs. Translation of Δ40p53, a p53 isoform, is one example of this non-canonical mechanism due to the presence of an IRES near an alternative initiation codon (AUG40). Here, we will present a new IRES in p53 mRNA, including details on the localization and regulation of this IRES under normal and stress conditions.
- Translational switch during integrated stress response: the examples of p53 and UPF1Publication . Lacerda, Rafaela; Pereira, Bruna; Menezes, Juliane; Ramos, Ana; Neves, Ana Rita; Candeias, Marco M; Romão, LuísaThe scanning model for eukaryotic mRNA translation initiation states that the small ribosomal subunit, along with initiation factors, binds to the cap structure at the 5’ end of the mRNA and scans the 5’ untranslated region (5’UTR) until an initiation codon is found. However, when cells are exposed to stress stimuli, cap-dependent translation is inhibited, while the synthesis of some proteins is maintained by alternative mechanisms of translation initiation, which are vital for cell survival and stress recovery. Here we show two examples in which a translational switch occurs during integrated stress response (ISR). In the first case, tumor suppressor p53, we show that the ISR leads to the specific induction of a shorter p53 isoform (Δ160p53 isoform). This induction is dependent on translation elongation but does not require the eIF4E-eIF4G interaction. Studies using bicistronic constructs with wild-type Δ160p53 or reporter genes confirmed the presence of an Internal Ribosome Entry Site (IRES) in p53 mRNA, being eIF2α phosphorylation a key event leading to cap-independent expression of Δ160p53 during ISR. Interestingly, cancer-specific mutations in p53 also enhance cap-independent translation of Δ160p53 via Δ160p53IRES. Our data support a model in which an IRES structure in the coding region of p53, and the cancer-specific mutations that affect this structure, control p53 oncogenic functions by regulating Δ160p53 protein expression. A better understanding of Δ160p53IRES structure and function may be advantageous for a more efficient therapeutic targeting of p53. Human up-frameshift 1 (UPF1) is a key-protein involved in nonsense-mediated mRNA decay, telomere replication and homeostasis, and cell cycle progression. These crucial UPF1 functions suggest its tight gene expression regulation. Indeed, our results show that UPF1 5’UTR is able to mediate cap-independent translation in a bicistronic luciferase vector expressed in cervical and colorectal cancer cell lines. Such activity is maintained under endoplasmic reticulum stress. Interestingly, we found that the UPF1 5’UTR IRES function is inhibited when the first 100 nucleotides, or the last 125, are absent or altered. Understanding these IRESs mechanism of function and their biological relevance might provide tools for developing new therapies for human diseases such as cancer.