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- 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.
- O mecanismo alternativo de síntese proteica da proteína UPF1 e a sua relevância na tumorigénesePublication . Lacerda, Rafaela; Menezes, Juliane; Romão, LuísaA proteína up-frameshift 1 (UPF1) é uma proteína multifacetada, com um papel fundamental em vários mecanismos celulares. É um fator essencial no mecanismo de decaimento rápido de mRNAs aberrantes com codões nonsense (NMD, do inglês nonsense-mediated mRNA decay). É também uma proteína crucial para a progressão da fase S do ciclo celular e manutenção do tamanho e homeostasia dos telómeros, de um modo totalmente independente das suas funções no NMD. No que respeita à tumorigénese, a UPF1 é frequentemente considerada uma proteína supressora de tumores e um potencial alvo terapêutico para o carcinoma hepatocelular. Além disso, está subexpressa em cancro gástrico e correlacionada negativamente com a expressão de MALAT1 (do inglês long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1). De acordo com o estudo de Karger e colaboradores (colocar entre parenthesis a ref), a sobre-expressão de UPF1 inibe algumas das características específicas do cancro, como sejam a proliferação celular, a progressão do ciclo celular, a migração e a invasão celulares, e aumenta a apoptose. Assim, a UPF1 pode ser considerada um potencial modulador de MALAT1, o que demonstra que a via UPF1/MALAT1 pode ser usada como um alvo terapêutico para o cancro do estômago. Neste trabalho, o nosso grupo, demonstrou que a região 5’ transcrita, mas não traduzida (UTR, do inglês untranslated region) do mRNA que codifica a UPF1 consegue utilizar um mecanismo alternativo de síntese proteica (i.e. tradução do mRNA) mediada por IRES (do inglês, internal ribosome entry sites). Este mecanismo alternativo permite a produção seletiva de proteínas em condições que impedem o normal mecanismo de tradução, funcionando. tanto em condições normais como sob condições de stress, tais como hipóxia, stress do retículo endoplasmático, inibição da via do mTOR, ou inibição da expressão do fator de iniciação eucariótico 4E, a proteína que se liga diretamente à estrutura cap e permite a tradução canónica. Ao fazer uma análise delecional e mutacional da sequência da 5’UTR de UPF1, verificámos que a sequência mínima necessária para a atividade dependente do IRES compreende os nucleótidos 1–100 e 151–275 (do total de 275 nucleótidos que compõem a 5’UTR). Fazendo uma previsão bioinformática da estrutura secundária formada por esta sequência, verificámos que há a formação de três stem loops (SL). O primeiro e o terceiro SL correspondem precisamente às sequências identificadas como essenciais e suficientes para a tradução mediada por IRES. Estes estudos foram conduzidos em células HeLa (cancro do colo do útero), e também em NCM460 (colonócitos derivados de mucosa intestinal normal) e HCT116 (carcinoma colo-rectal em fase pré-metastática), o que nos permite avaliar a importância deste mecanismo no desenvolvimento e progressão do cancro colorretal e, consequentemente, desenvolver novas abordagens terapêuticas, com a utilização de oligonucleótidos antisense de RNA para inibir esta tradução alternativa, o que constituirá uma proof-of-concept de uma potencial terapia para o cancro colorretal baseada em RNA.
- Nonsense suppression therapies in human genetic diseasesPublication . Martins-Dias, Patrícia; Romão, LuísaAbout 11% of all human disease-associated gene lesions are nonsense mutations, resulting in the introduction of an in-frame premature translation-termination codon (PTC) into the protein-coding gene sequence. When translated, PTC-containing mRNAs originate truncated and often dysfunctional proteins that might be non-functional or have gain-of-function or dominant-negative effects. Therapeutic strategies aimed at suppressing PTCs to restore deficient protein function—the so-called nonsense suppression (or PTC readthrough) therapies—have the potential to provide a therapeutic benefit for many patients and in a broad range of genetic disorders, including cancer. These therapeutic approaches comprise the use of translational readthrough-inducing compounds that make the translational machinery recode an in-frame PTC into a sense codon. However, most of the mRNAs carrying a PTC can be rapidly degraded by the surveillance mechanism of nonsense-mediated decay (NMD), thus decreasing the levels of PTC-containing mRNAs in the cell and their availability for PTC readthrough. Accordingly, the use of NMD inhibitors, or readthrough-compound potentiators, may enhance the efficiency of PTC suppression. Here, we review the mechanisms of PTC readthrough and their regulation, as well as the recent advances in the development of novel approaches for PTC suppression, and their role in personalized medicine.
- Internal ribosome entry site (IRES)-mediated translation as a putative candidate mechanism to mRNA-based therapiesPublication . Neto, Maria Inês; Lacerda, Rafaela; Romão, LuísaUntranslated regions in the messenger RNA (mRNA) are susceptible to the interaction of regulatory elements including proteins or non-coding RNA molecules, being an important hotspot to the study of gene expression regulation. Internal Ribosome Entry Sites (IRES) are secondary structures usually located on the 5’UTR of an mRNA molecule that can recruit the ribosome during initiation of protein synthesis without the involvement of the cap structure. This mechanism tends to appear when the cell is under stress conditions, which might include the presence of oncogenes, growth factors or proteins involved in programmed cell death. This work focuses on the human AGO1, an important key player for RNA-mediated gene silencing, and whether its 5’UTR is capable of driving cap-independent translation initiation. To achieve this goal, a construct containing the 5’UTR of human AGO1 was cloned, taking advantage of a bicistronic vector containing two reporter genes, Renilla luciferase (RLuc) and firefly luciferase (FLuc), the last one cloned downstream from the 5’UTR. We performed luminometry assays to assess the relative translation efficiency of FLuc, which is under the control of AGO1 5’UTR. The results showed that human AGO1 5’UTR mediates a cap-independent eIF4G-dependent mechanism of translation initiation enhanced by a free 5’ end. Also, we saw that this alternative mechanism is maintained, and even enhanced, under stress conditions, such as the knock-down of eukaryotic initiation factor 4E, the protein that directly binds to the cap structure. We are currently investigating what is the minimal sequence required for IRES-mediated translation. Combining these results with emerging RNA-based therapies will be helpful to develop novel strategies to prevent and treat disorders, such as cancer, involving dysregulation of AGO1 translation.
- Learning from mRNA: the relevance of the tumour suppressor protein UPF1’s internal ribosome entry site-mediated translation in tumorigenesisPublication . Lacerda, Rafaela; Menezes, Juliane; Romão, LuísaCrucial in several cellular processes, such as nonsense-mediated mRNA decay, cell cycle progression, and telomere maintenance and homeostasis, Up-frameshift 1 (UPF1) has also been considered a tumour suppressor protein in hepatocellular carcinoma and gastric cancer, as it is underexpressed in the latter and negatively correlated to MALAT1 (long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1) expression. UPF1 overexpression inhibits some cancer-specific features, like proliferation, cell cycle progression, cell migration and invasion, and enhances apoptosis, turning the UPF1/MALAT1 pathway a potential therapeutic target for gastric cancer. Here, we investigate the importance of UPF1 internal ribosome entry site (IRES)-dependent translation in tumorigenesis, specifically in colorectal cancer. Thus, we used a bicistronic system with two reporter genes, in which we cloned UPF1 5’ untranslated region (UTR) upstream the second cistron, whose translation will only occur internally. We transcribed the bicistronic mRNA in vitro and transfected HeLa (cervical cancer), NCM460 (normal intestinal-derived colonocytes) and HC116 (pre-metastatic colorectal carcinoma) cells with such mRNA, along with the positive and negative controls. The results show a significant increase in IRES-mediated translation levels compared to those of the negative control, both in normal conditions and under endoplasmic reticulum stress. Also, internal initiation occurs in the absence of the cap structure. Deletional and mutational analysis of UPF1 5’UTR showed that nucleotides 1–100 (stem loop (SL) I) and 151–275 (SL III) — out of 275 nucleotides — are the minimal required sequences for the IRES to work properly. Also, we used RNA antisense oligonucleotides (ASOs) targeting UPF1 IRES SL I and III, and observed a reduced UPF1 expression. Cellular viability increases in HCT116 cells and decreases in NCM460 cells treated with ASOs targeting SL III and SL I, respectively, whereas apoptosis increases in NCM460 cells and decreases in HCT116 cells treated with ASOs targeting SL I. This may be the dawn of a new RNA-based therapeutic approach regulating colorectal cancer development.
- Internal Ribosome Entry Site-Dependent Translation Dysregulation-Related DiseasesPublication . Marques, Rita; Lacerda, Rafaela; Romão, LuísaInternal ribosome entry site (IRES)-mediated translation is an alternative mechanism of translation initiation, known for maintaining protein synthesis when canonical translation is impaired. During a stress response, it contributes to cell reprogramming and adaptation to the new environment.
- Regulation of the Human PERK mRNA Translation by Upstream Open Reading FramesPublication . Fernandes, Rafael; Rodrigues, Rosário; Lopes, Pedro; Romão, LuísaUpstream open reading frames (uORFs) are cis-acting elements located within the 5’ leader sequence (5’UTR) of transcripts, which can regulate translation of the correspondent main open reading frame (mORF). During endoplasmic reticulum (ER) stress, the accumulation of unfolded proteins activates the ER-resident PKR-like ER kinase (PERK), which results in phosphorylation of eIF2α to inhibit global mRNA translation, while allowing the selective uORF-mediated translation of downstream effectors responsible for stress resolution or, ultimately, cell death. The dual role of PERK in regulating cell fate was implicated in human diseases, like diabetes, neurodegenerative disorders and cancer. Moreover, mutations in the EIF2AK3 gene (encoding PERK) were associated to the rare genetic disease, Wolcott-Rallison Syndrome (WRS). In this work, we aimed to study the translational regulatory role of 5 AUG- and 3 non-AUG-uORFs identified in the PERK 5’UTR and assess its biological relevance. While uORF2 and the non-AUG-uORFs 5, 6 and 7 (numbered according to their distance to the 5’ end of the mRNA) do not seem to have a regulatory role, uORF1, uORF3, uORF4 and uORF8 together present a strong repressive effect over mORF translation in basal conditions. Also, we observe that uORF1 is frequently translated allowing low levels of translation re-initiation at the main ORF. Curiously, we found that when PERK is overexpressed, it leads to the spontaneous activation of a portion of PERK in the absence of any stress stimulus, possibly highlighting the biological relevance of its uORF-mediated translational regulation. Conversely, during ER stress, increased bypass of uORF1 results in a modest degree of translational de-repression, which may help to counterbalance the increased rate of PERK protein turnover observed in these conditions. We also observed that alteration of the PERK uORFs by mutations found in WRS patients modify mORF expression, providing a possible link to the disease. Altogether, we highlight the importance of including 5’UTRs in the screening of disease-related mutations and the necessity of functional studies to assess their role in pathogenesis.
- 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.
- Unraveling the role of internal ribosome entry site (IRES) mediated translation in human UPF1 expression and functionPublication . Lacerda, Rafaela; Menezes, Juliane; Romão, LuísaUp-frameshift 1 (UPF1) is the key factor in nonsense-mediated mRNA decay (NMD, a mechanism that degrades transcripts carrying premature translation termination codons. Besides its role in NMD, UPF1 has also a role in other mechanisms, such as cell cycle progression, being crucial for the G1/S transition, and in telomere maintenance and homeostasis. Furthermore, it has also been identified as having tumour suppressor activity in different cancers, such as hepatocellular carcinoma. Our data supports the existence of an IRES within the human UPF1 5’ untranslated region (UTR). In fact, using a bicistronic reporter system, we observed that UPF1 5’UTR can mediate internal translation in a 5’ cap-independent manner, both in normal and under stress conditions. We concluded that the first 100 nucleotides and the last 125 (out of a total of 275) are the minimal sequences essential for the identified IRES activity. According to the in silico predicted secondary structure, these two segments correspond to two stem-loops. Now, we are experimentally confirming this predicted secondary structure, and understanding the physiological role of this IRES-mediated activity in the NMD process. Also, we are testing its importance for UPF1 function as a tumour suppressor. For that, we are using custom-made antisense oligonucleotides that specifically target those stem-loops and see to what extent they inhibit UPF1 IRES-mediated activity and how such inhibition will play a role in the cell and the functions in which UPF1 participates. Also, we are testing some hallmarks of cancer, such as proliferation, invasion, and apoptosis inhibition when the UPF1 IRES is impaired. With this work, we wish to unravel a new layer of knowledge in gene expression regulation of UPF1.
- UPF1 internal ribosome entry site-mediated translation and its importance in tumorigenesisPublication . Lacerda, Rafaela; Menezes, Juliane; Romão, LuísaAims: UPF1 internal ribosome entry site-mediated translation and its importance in tumorigenesis; Confirm UPF1 5’ untranslated region (UTR) can mediate internal ribosome entry site (IRES)-mediated translation; Check whether the IRES-dependent translation is maintained under cap-dependent-impairing conditions (hypoxia); Identify the minimal sequence required for IRES-mediated translation; Investigate the biological role of UPF1 IRES-mediated translation in tumorigenesis.