Browsing by Author "Lacerda, Rafaela"
Now showing 1 - 10 of 56
Results Per Page
Sort Options
- Alternative mechanisms of mRNA translationiInitiation in cellular stress response and cancerPublication . Lacerda, Rafaela; Menezes, Juliane; Candeias, Marco M.Throughout evolution, eukaryotic cells have devised different mechanisms to cope with stressful environments. When eukaryotic cells are exposed to stress stimuli, they activate adaptive pathways that allow them to restore cellular homeostasis. Most types of stress stimuli have been reported to induce a decrease in overall protein synthesis accompanied by induction of alternative mechanisms of mRNA translation initiation. Here, we present well-studied and recent examples of such stress responses and the alternative translation initiation mechanisms they induce, and discuss the consequences of such regulation for cell homeostasis and oncogenic transformation.
- Analysis of human Argonaute 1 5’ untranslated region shows internal ribosome entry site activityPublication . Lacerda, Rafaela; Teixeira, Alexandre; Marques-Ramos, Ana; Romão, Luísa
- Analysis of the 5’ untranslated region of human UPF1 mRNA indicates both cryptic promoter and internal ribosome entry site activityPublication . Lacerda, Rafaela; Marques-Ramos, Ana; Teixeira, Alexandre; Romão, LuísaApart from its role in nonsense-mediated mRNA decay, a mechanism that promotes rapid degradation of transcripts carrying premature translation termination codons, the human up-frameshift 1 (UPF1) DNA and RNA helicase protein plays a crucial role in telomere replication and homeostasis, and in cell cycle progression. Due to its relevance for several physiological roles, and to the fact that it is expressed during G2/M phase, in which overall protein synthesis is reduced, we hypothesized that its translation may occur via an internal ribosome entry site (IRES). IRESs can occur at the 5’ untranslated region (UTR) of transcripts and allow the direct recruitment of the ribosome to the vicinity of the main AUG, therefore bypassing the need of scanning the entire UTR. To test this hypothesis, we cloned the human UPF1 5’UTR in the dicistronic vector p_Renilla_Firefly and transfected HeLa cells with either this construct or the control counterparts. We observed a 15- to 25-fold increase in relative luciferase activity of the UPF1 5’UTR-containing construct compared to the levels obtained from the empty counterpart, which suggests the presence of an IRES. However, these levels of luciferase activity could be due to the presence of a cryptic promoter. Hence, we transfected cells with promoterless plasmids and observed a 20-fold increase in relative luciferase activity levels. These data demonstrate that UPF1 5’UTR contains a cryptic promoter, whose activity may be masking IRES activity. To check the IRES activity alone, we have transfected cells with in vitro transcribed, capped and polyadenylated mRNAs and observed a 2-fold increase in protein levels. This is also observed in two other cell lines. Besides, UPF1 IRES activity is maintained under conditions of global protein synthesis inhibition. Deletional analysis of UPF1 5’UTR revealed that the first 50 nucleotides at the 5’ end of this region are essential for both cryptic promoter and IRES activity. These results evidence, for the first time, the existence of both a cryptic promoter and an IRES element within UPF1 5’UTR and provide new insights on the regulation of UPF1 expression in human cells.
- 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.
- 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.
- Cap-independent translation regulation of mammalian target of rapamycin (mTOR)Publication . Marques-Ramos, Ana; Teixeira, Alexandre; Lacerda, Rafaela; Romão, Luísa
- 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.
- Characterization of an oncogenic isoform of TP53: Δ160p53Publication . Ramalho, Ana Catarina; Rita, Filipa; López-Iniesta, M.; Lacerda, Rafaela; Romão, Luísa; Candeias, Marco M.The transcription factor p53 is a key cell regulator, having roles in varied cellular processes. Widely known as a tumour suppressor protein, p53 is responsible for signalling the adequate response to DNA damage, oncogenic signalling, or other stress stimuli. The target genes of this protein are involved in cell cycle arrest, senescence, apoptosis, and DNA damage response, among other pathways. Besides the full-length p53 (FLp53), to which these functions are attributed, the TP53 gene encodes for eleven other protein isoforms that result from alternative splicing, internal initiation of translation and transcription from an internal promoter. In striking contrast to FLp53, the N-terminally truncated Δ160p53 exhibits pro-oncogenic traits, although it only differs from FLp53 by the lack of its first 159 amino acids. Δ160p53 promotes cell survival, proliferation, invasion, and adhesion, and it is overexpressed in cancer cells harbouring hotspot p53 mutants. The TP53 gene is frequently mutated in cancer, and hotspot mutants result from single missense mutations that convert p53 in a driver of tumorigenesis. As Δ160p53 presents many of the oncogenic roles attributed to p53 cancer mutants, it is plausible that this isoform could be responsible for the paradoxical mutant p53 functions. However, detailed knowledge on the mode of action of Δ160p53 is still lacking. We have performed additional tests to further characterize the oncogenic traits of this isoform. To evaluate the ability of Δ160p53 to promote anchorage-independent cell growth, we have used the soft agar colony formation assay. Preliminary results show a tendency of Δ160p53 to promote growth, when compared to the control and other isoforms. Our new data complements our previous knowledge on Δ160p53 and reinforces the importance of studying this isoform for therapeutic targeting.
- 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.