Translational Regulation of the Human PERK by Upstream Open Reading Frames

Upstream 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 with 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.Work supported by INSA and UIDB/04046/2020 (DOI: 10.54499/UIDB/04046/2020 ) and UIDP/04046/2020 (DOI: 10.54499/UIDP/04046/2020) Centre grants from FCT, Portugal (to BioISI).N/

A iniciação não-canónica da síntese da proteína UPF1 contribui para a sua função oncogénica no cancro colo-rectal

O cancro colo-rectal (CCR) é a terceira causa de morte ao nível mundial e as projecções apontam para um aumento durante as próximas duas décadas. A desregulação da expressão de vários genes no CCR contribui para o desenvolvimento da doença. A proteína up-frameshift 1 (UPF1) está envolvida em vários mecanismos celulares e, ao contrário da maioria dos cancros, em que a UPF1 desempenha um papel suppressor de tumor, no CCR, esta proteína actua como oncogene. Tendo como objectivo perceber que mecanismos moleculares podem contribuir para o papel oncogénico desta proteína no CCR, analisámos os níveis de expressão do RNA mensageiro (mRNA) e da proteína em vários tipos de cancro e nos respectivos tecidos normais. As análises in silico revelaram que há uma sobrexpressão de UPF1 (mRNA e proteína) no CCR comparativamente à maioria dos restantes cancros analizados. Estes níveis também são significativamente mais elevados no CCR do que no respectivo tecido normal. Experimentalmente, verificámos que a expressão de UPF1 é mantida em condições de stresse celular. Para perceber qual o mecanismo responsável pela manutenção da expressão de UPF1, testámos se a região 5’ não-traduzida do seu mRNA consegue mediar a síntese proteica por um mecanismo alternativo. Verificámos que esta região é capaz de mediar a tradução de UPF1 através da um local interno de entrada do ribossoma, mesmo em condições em que a síntese proteica canónica está inibida. Mapeámos também a sequência mínima necessária para o funcionamento desta região. Posteriormente, usámos oligonucleótidos de RNA antissense que hibridam na sequência mínima e observámos uma redução da expressão de UPF1 em células de CCR. Globalmente, estes resultados mostram que os níveis de expressão da UPF1 são mantidos por um mecanismo alternativo de tradução que permite a síntese proteica em condições inerentes ao microambiente tumoral. Assim, estes oligonucleótidos de RNA antissense poderão ser os precursores de uma nova terapia baseada em RNA para prevenir o desenvolvimento do CCR.Work supported by INSA and UIDB/04046/2020 (DOI: 10.54499/UIDB/04046/2020 ) and UIDP/04046/2020 (DOI: 10.54499/UIDP/04046/2020) Centre grants from FCT, Portugal (to BioISI).N/

The p53 short isoforms are activated by the integrated stress response to enhance survival in wild-type p53 cancer cell lines

Introduction: The full-length p53 (FLp53) protein is a transcriptional factor that mediates cellular stress responses. The appropriate response is modulated according to the nature and extent of the damage, which means that p53 may promote cell cycle arrest and DNA repair or apoptosis in different situations. In all cases, p53 manages the response by modulating the expression of a wide range of target genes, as it contains two N-terminal transactivation domains. However, this gene also encodes for shorter isoforms that lack the N-terminal region and display oncogenic functions: Δ133p53 and Δ160p53. In contrast to the protective nature of the full-length protein, these two isoforms promote cell survival, proliferation and invasion. Both are typically overexpressed in tumours, while in normal tissues their levels are low or undetectable. Data from this study indicate that the elevated levels of these isoforms may be owed to the Integrated Stress Response (ISR), which is typically a pro-survival programme, and it is commonly activated in cancer cells. The ISR is initiated by a group of kinases in response to different stress stimuli, converging in the phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α). This blocks global translation, while promoting the expression of activating transcription factor 4 (ATF4), the main effector of ISR. Materials and Methods: In this work, cancer cell lines with endogenous p53 were used to verify the expression of its isoforms during ISR, which was induced by treatment with thapsigargin and tunicamycin. To investigate if they were translated internally, bicistronic construct systems were employed. The interaction of the isoforms with FLp53 was verified by co-immunoprecipitation and their effect on the mRNA levels of p53 target genes was evaluated. Results and discussions: The translation of Δ160p53 and Δ133p53 was promoted by ISR induction in the cell lines tested via internal translation from FLp53 mRNA. Interaction of both isoforms with FLp53 was also confirmed, and it was demonstrated that Δ160p53 selectively acts as a regulator of p53 transcriptional activity on some genes. Conclusion: These results hint at a physiological role for p53 short isoforms in the modulation of p53 target gene expression during ISR. On the other hand, their activation by abnormal ISR induction in cancer cells supports oncogenicity. The uncovering of an intersection between p53 isoforms and ISR could open a new path for future cancer therapies.This research was funded by grants 18K07229 (KAKENHI) from Japan Society for the Promotion of Science (JSPS) and PTDC/MED-ONC/32048/2017 from Fundação para a Ciência e a Tecnologia of Portugal (FCT). This work was supported by FCT through the individual research grant 2020.06982.BDN/

The integrated stress response releases the oncoprotein in TP53

Introduction: The TP53 gene is surrounded by a great duality: it is a critical tumour suppressor gene, however, its protective nature is frequently lost in tumours, where it becomes a powerful oncogene. Numerous studies attribute the oncogenic profile of TP53 to the missense mutations that commonly occur in cancer. We propose that this duality is intrinsic to TP53 instead of a consequence of mere somatic mutations, which could not have been evolutionarily selected for. This gene encodes for a set of protein isoforms with distinct and complementary functions, from which the full-length p53 (FLp53) is the best characterized. FLp53 is a transcription factor that mediates stress responses by promoting cell cycle arrest, DNA repair or apoptosis. In stark contrast to FLp53, the shorter isoform Δ160p53 promotes cell survival, proliferation, and invasion, and it is commonly overexpressed in tumours. Here we identify a disruption in the normal balance of p53 isoforms upon induction of the Integrated Stress Response (ISR), with the translation of Δ160p53 being favoured. Materials and Methods: Different cell lines were used to verify the expression of endogenous p53 isoforms during ISR, and the internal translation of Δ160p53 was tested with bicistronic constructs. The interaction of Δ160p53 with FLp53 was assessed by co-immunoprecipitation followed by western blot, and its effect in the expression of target genes was measured by RT-qPCR. Cells were treated with thapsigargin and tunicamycin to induce ISR when required. Results: The induction of ISR in a group of cell lines led to increased levels of endogenous Δ160p53 protein, as well as increased luminescence signal in the bicistronic system. The FLp53-Δ160 interaction was confirmed, and the role of Δ160p53 in the selective regulation of p53 target genes was uncovered. Conclusions: These data uncover a mode of activation of the oncogenic Δ160p53 and how this isoform can work together with FLp53. In the future, we aim to explore the clinical potential of these discoveries.This research was funded by grants 18K07229 (KAKENHI) from Japan Society for the Promotion of Science (JSPS) and PTDC/MED-ONC/32048/2017 from Fundação para a Ciência e a Tecnologia of Portugal (FCT). This work was supported by FCT through the individual research grant 2020.06982.BD.N/

Non-canonical synthesis of UPF1 protein contributes to its oncogenic role in colorectal cancer

Colorectal cancer (CRC) is the third leading cause worldwide and projections point towards an increase over the next two decades. Gene expression dysregulation of several genes involved in CRC contribute to disease development. The up-frameshift 1 (UPF1) protein plays important roles in several cellular mechanisms and acts as a tumour suppressor in most cancers. However, in CRC, this protein has been described as working as an oncogenic protein. In order to understand the molecular mechanisms underlying the oncogenic role of UPF1 in CRC, we have analysed mRNA and protein levels in different types of cancer. In silico analyses have shown that UPF1 is overexpressed in CRC and lung cancer compared to the other analysed cancers. Also, UPF1 expression is significantly greater in CRC than in normal tissues. Experimentally, we observed that UPF1 expression is maintained under stress conditions that compromise global protein synthesis. In this regard, we tested whether UPF1 translation initiation can be mediated through an alternative cap-independent mechanism. We showed that the 5’ untranslated region (UTR) of UPF1 transcript allows cap-independent translation initiation and mapped the minimal sequence required for this mechanism to work. This region also mediates translation initiation in transcripts lacking a cap structure and under stress conditions like endoplasmic reticulum stress, hypoxia and mTOR pathway inhibition. Then, we designed antisense RNA oligonucleotides (ASOs) that target the minimal region and observed a reduced expression of UPF1 in CRC cells treated with those ASOs compared to cells treated with control ASOs. All in all, these results show that alternative translation initiation mediated through UPF1 5’UTR allows UPF1 expression levels to be maintained under conditions observed in the tumour microenvironment, which globally repress protein synthesis. Thus, ASOs targeting the minimal region responsible for allowing UPF1 expression can be the beginning of a new RNA-based therapy to prevent CRC development.Work supported by INSA and UIDB/04046/2020 (DOI: 10.54499/UIDB/04046/2020 ) and UIDP/04046/2020 (DOI: 10.54499/UIDP/04046/2020) Centre grants from FCT, Portugal (to BioISI).N/

Translational Control of Δ160p53 Keeps the Dark Side of TP53 in Check

The TP53 tumour suppressor gene was discovered over 40 years ago, but to this day some aspects of its regulation and function remain a mystery. It encodes the full-length p53 protein (FLp53), a transcription factor with a key role in stress response in multicellular organisms, that can either direct cells towards apoptosis or recovery of homeostasis. With such a decisive role, its expression and activity are tightly regulated. A vast set of RNA-binding proteins (RBPs) have been described to affect the translation of FLp53 or the stability of p53 mRNA in response to different perturbations. But in addition to FLp53, there is a group of shorter protein isoforms lacking the N-terminal region, which have well-described functions, and are translated from the same mRNA. The shorter and less studied isoform is Δ160p53, which promotes cell survival, proliferation, and invasion. Despite its usual low levels, it is commonly overexpressed in tumours. However, the detailed mechanisms and factors involved in the regulation of Δ160p53 are still unknown. In this work, a mass spectrometry was performed to identify the proteins in an RNA-protein co-immunoprecipitation of the p53 mRNA using the MS2 system in the p53-null cell line H1299. The validation of the hits was undertaken by western blot with specific antibodies after immunoprecipitation. The effect of the binding proteins on the translation of Δ160p53 was assessed by overexpression or knockdown, and the expression levels were verified by western blot or luminescence assays. The mass spectrometry allowed the identification of potential new binding partners of the p53 mRNA. Resorting to the literature and to computational tools available online to predict protein-RNA interactions, a few hits were selected for follow-up and their interactions confirmed. Simultaneously, the modulation of Δ160p53 expression by some of these proteins was verified. Considering the importance of TP53 in deciding the fate of the cell, the observation of abnormal levels of the oncoprotein Δ160p53 in cancer is intriguing. Understanding the control of its translation could uncover strategies to block it and pave way for new cancer therapies.Work supported by INSA and UIDB/04046/2020 (DOI: 10.54499/UIDB/04046/2020 ) and UIDP/04046/2020 (DOI: 10.54499/UIDP/04046/2020) Centre grants from FCT, Portugal (to BioISI).N/

Internal ribosome entry site-mediated translation of UPF1 contributes to its oncogenic role in colorectal cancer

Colorectal cancer (CRC) is the third cause of death worldwide and projections point towards an increase for the next two decades. Many genes are misregulated in CRC, contributing to the development of the disease. Up-frameshift 1 (UPF1) is involved in many cellular mechanisms such as nonsense-mediated mRNA decay, cell cycle progression, or telomere maintenance and homeostasis. It also works as a tumour suppressor protein in most cancers but not in CRC, in which UPF1 plays an oncogenic role. We used the Xena platform to perform in silico analyses that revealed UPF1 is overexpressed in CRC contrary to several other analysed cancers. Besides, UPF1 expression levels are increased in CRC compared to the counterpart normal tissues. Experimentally, we confirmed these data and observed that endogenous UPF1 expression is maintained in different CRC cell lines under endoplasmic reticulum (ER) stress. To understand the mechanism underlying such maintenance, we used a bicistronic reporter construct to test whether UPF1 5’UTR can mediate alternative translation initiation and we concluded that such sequence contains an internal ribosome entry site (IRES) that maintains UPF1 expression in both normal and stress conditions in a 5’ cap-independent way. 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. Using RNA antisense oligonucleotides (ASOs) targeting UPF1 IRES SL I and III, we observed a reduced UPF1 expression in HCT116 (CRC) cells. Altogether, these results suggest that UPF1 expression levels are maintained by the IRES-mediated translation mechanisms under conditions impairing canonical translation, such as those that mimic the tumour microenvironment. Thus, ASOs may be an upcoming therapy to target such alternative mechanism of translation initiation and prevent CRC development.Work supported by INSA and UIDB/04046/2020 (DOI: 10.54499/UIDB/04046/2020 ) and UIDP/04046/2020 (DOI: 10.54499/UIDP/04046/2020) Centre grants from FCT, Portugal (to BioISI).N/

Regulation of Δ160p53: a p53 short isoform with oncogenic functions

p53 is a transcription factor that activates tumor suppressor genes in response to various cellular stresses.Work supported by INSA and UIDB/04046/2020 (DOI: 10.54499/UIDB/04046/2020 ) and UIDP/04046/2020 (DOI: 10.54499/UIDP/04046/2020) Centre grants from FCT, Portugal (to BioISI).N/

Characterization of an oncogenic isoform of TP53: Δ160p53

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.PhD Fellowship financed by FCTinfo:eu-repo/semantics/publishedVersio

O mecanismo alternativo de síntese proteica da proteína UPF1 e a sua relevância na tumorigénese

A 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.FCTinfo:eu-repo/semantics/publishedVersio