Browsing by Author "Candeias, Marco"
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- 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.
- Cancer-specific mutations in p53 induce the translation of Δ160p53 promoting tumorigenesisPublication . Candeias, Marco; Hagiwara, Masatoshi; Matsuda, MichiyukiWild-type p53 functions as a tumour suppressor while mutant p53 possesses oncogenic potential. Until now it remains unclear how a single mutation can transform p53 into a functionally distinct gene harbouring a new set of original cellular roles. Here we show 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 full-length p53 mRNA. Cells expressing mutant p53 exhibit "gain-of-function" cancer phenotypes, such as enhanced cell survival, proliferation, invasion and adhesion, altered mammary tissue architecture and invasive cell structures. Interestingly, Δ160p53-overexpressing cells behave in a similar manner. In contrast, an exogenous or endogenous mutant p53 that fails to express Δ160p53 due to specific mutations or antisense knock-down loses pro-oncogenic potential. Our data support a model in which "gain-of-function" phenotypes induced by p53 mutations depend on the shorter p53 isoforms. As a conserved wild-type isoform, Δ160p53 has evolved during millions of years. We thus provide a rational explanation for the origin of the tumour-promoting functions of p53 mutations.
- Cap-independent translation ensures mTOR expression and function upon protein synthesis inhibitionPublication . Marques-Ramos, Ana; Romão, Luísa; Candeias, Marco; Menezes, Juliane; Lacerda, Rafaela; Willcocks, M.; Teixeira, Alexandre; Locker, NicolasThe mechanistic/mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that integrates cellular signals from the nutrient and energy status to act, namely, on the protein synthesis machinery. While major advances have emerged regarding the regulators and effects of the mTOR signaling pathway, little is known about the regulation of mTOR gene expression. Here, we show that the human mTOR transcript can be translated in a cap-independent manner, and that its 5' untranslated region (UTR) is a highly folded RNA scaffold capable of binding directly to the 40S ribosomal subunit. We further demonstrate that mTOR is able to bypass the cap requirement for translation both in normal and hypoxic conditions. Moreover, our data reveal that the cap-independent translation of mTOR is necessary for its ability to induce cell-cycle progression into S phase. These results suggest a novel regulatory mechanism for mTOR gene expression that integrates the global protein synthesis changes induced by translational inhibitory conditions.
- 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.
- Common p53 mutations induce IRES-mediated translation of oncogenic shorter p53 isoformsPublication . Pereira, Bruna; Lacerda, Rafaela; Maria López-Iniesta, M; Romão, Luísa; Candeias, MarcoAt least half of all tumors exhibit mutations in the tumor suppressor p53 gene. Indeed, the fact that p53 is frequently mutated in cancer led to its identification as an oncogene, when first described in 1979. Later, it was classified as a tumor suppressor, due to the clarification of its wild-type role in maintaining genome integrity and preventing malignant transformation. The p53 gene can encode for many p53 isoforms, by alternative splicing, alternative promoters and internal translation initiation mechanisms. While full-length p53 (FL-p53) protein works as a tumor suppressor by regulating many biological processes such as cell cycle, apoptosis, senescence and DNA repair, shorter p53 protein isoforms seem to play different roles in the cell. Recently, we have shown that the most common p53 mutations induce the expression of shorter p53 isoforms. Furthermore, we found that shorter p53 isoforms are implicated in cancer progression as they promote enhanced cell survival, proliferation, adhesion and formation of invasive cell structures. Here, with a bicistronic system containing two reporter genes (Renilla luciferase and firefly luciferase), we show that expression of shorter p53 isoforms is mediated by a non-canonical translation initiation mechanism regulated by an Internal Ribosome Entry Site (IRES) in the p53 mRNA. By investigating the effect of common p53 missense mutations on the function of this new IRES, through bioluminescence assays and Western blot analysis, we show that some p53 cancer mutations have a preponderant role in IRES-mediated translation induction of shorter p53 isoforms. With the obtained results we identified a new mechanism by which p53 cancer mutations promote tumorigenesis, which may lead to new understandings of the onset and progression of some types of tumors as well as to the development of new cancer therapies.
- Conserved Double Translation Initiation Site for Δ160p53 Protein Hints at Isoform's Key Role in Mammalian PhysiologyPublication . López-Iniesta, Maria José; Parker, Shrutee; Ramalho, Ana Catarina; Lacerda, Rafaela; Costa, Inês; Zhao, Jingyuan; Romão, Luísa; Candeias, Marcop53 is the most commonly mutated gene in human cancers. Two fundamental reasons for this are its long protein isoforms protect from cancer, while its shorter C-terminal isoforms can support cancer and metastasis. Previously, we have shown that the Δ160p53 protein isoform enhances survival and the invasive character of cancer cells. Here, we identified a translation initiation site nine codons downstream of codon 160-the known initiation codon for the translation of Δ160p53-that is recognized by the translation machinery. When translation failed to initiate from AUG160 due to mutation, it initiated from AUG169 instead, producing similar levels of a similar protein, Δ169p53, which promoted cell survival as efficiently as Δ160p53 following DNA damage. Interestingly, almost all mammalian species with an orthologue to human AUG160 also possess one for AUG169, while none of the non-mammalian species lacking AUG160 have AUG169, even if that region of the p53 gene is well conserved. In view of our findings, we do not believe that Δ169p53 acts as a different p53 protein isoform; instead, we propose that the double translation initiation site strengthens the translation of these products with a critical role in cell homeostasis. Future studies will help verify if this is a more general mechanism for the expression of essential proteins in mammals.
- Human mTOR mRNA allows cap-independent translation that insures its expression and function during inhibition of global translationPublication . Marques-Ramos, Ana; Candeias, Marco; Menezes, Juliane; Lacerda, Rafaela; Willcocks, M.; Locker, Nicolas; Romão, LuísaThe mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that integrates signals from the cellular nutrient- and energy-status, acting namely on the protein synthesis machinery. Deregulation of mTOR signaling is implicated in major diseases, such as cancer, mainly due to its role in regulating protein synthesis. Major advances are emerging regarding the regulators and effects of mTOR signaling pathway; however, regulation of mTOR gene expression is not well known. Here, we show that the 5’ untranslated region of the human mTOR transcript forms a highly folded RNA scaffold capable of binding directly to the 40S ribosomal subunit. We further demonstrate that this cis-acting RNA regulon is active both in normal and stress conditions, and that its activation status in response to translational adverse conditions parallels mTOR protein levels. Moreover, our data reveal that the cap-independent translation of mTOR is necessary for its ability to induce cell cycle progression into S-phase. These results suggest a novel regulatory mechanism of mTOR gene expression that integrates the protein profile rearrangement triggered by global translation inhibitory conditions.
- 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.
- Non-coding functions of p53 mRNAPublication . Candeias, MarcoWe now want to test the relevance of the non-coding functions of p53 mRNA in carcinogenesis.
- 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.