Studying and targeting the non-coding functions of p53 mRNA in carcinogenesis

Funder

Organizational Unit

Publications

Conserved Double Translation Initiation Site for Δ160p53 Protein Hints at Isoform's Key Role in Mammalian Physiology

Publication . López-Iniesta, Maria José; Parker, Shrutee; Ramalho, Ana Catarina; Lacerda, Rafaela; Costa, Inês; Zhao, Jingyuan; Romão, Luísa; Candeias, Marco

p53 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.

2022-12-13Journal article

Open access

Regulation of IRES-mediated translation in p53

Publication . Fonseca Costa, Inês; Lacerda, Rafaela; López-Iniesta, M.; Romão, Luísa; Candeias, Marco M.

The tumor microenvironment is characterized by several stresses impairing canonical translation. However, specific mRNAs harbouring internal ribosome entry sites (IRES), such as several tumour suppressors and oncogenes, can overcome this impairment. The tumor suppressor TP53 gene, an important transcription factor that ensures cellular homeostasis, is frequently mutated in human cancers. Over the years, several p53 isoforms have been identified, which in some cases result from alternative initiation of translation regulated by an IRES. Recently, we have associated mutant p53 “gain-of-function” cancer phenotype, such as enhanced cell survival, invasion, proliferation, and adhesion, with the expression of higher levels of shorter p53 isoforms, such as Δ160p53 isoform.1 Here, we used a bicistronic system containing two reporter luciferases (renilla luciferase and firefly luciferase) to assess IRES-mediated translation. Several p53 mRNA elements were tested in this system and, interestingly, we have found an inhibitory element of IRES-mediated translation. Overall, IRES-regulated translation in malignant cells is used to translate specific proteins that promote cancer progression. Thus, inhibiting translation of oncogenes via IRES could prevent the formation of tumor cells and their adaptation to unfavourable conditions in the tumor microenvironment. 1. Candeias, M. M., Hagiwara, M. & Matsuda, M. Cancer‐specific mutations in p53 induce the translation of Δ160p53 promoting tumorigenesis. EMBO Rep. 17, 1542–1551 (2016).

2020-11-20Conference object

Restricted access

The tumor suppressor p53 acquires oncogenic functions due to a translational switch during integrated stress response

Publication . Lacerda, Rafaela; Fonseca Costa, Inês; López-Iniesta, M.; Romão, Luísa; Candeias, Marco M.

To cope with the stress stimuli to which they are often exposed, eukaryotic cells have developed adaptive pathways that restore cellular homeostasis. Under stress conditions there is an overall decrease of protein synthesis, and a concomitant induction of alternative mechanisms of mRNA translation initiation. The tumour suppressor protein p53 has been considered the guardian of the genome and a master regulator of many cellular functions. However, apart from the full-length p53 (FLp53), several p53 isoforms have been described so far. Some functions of shorter p53 isoforms have already been elucidated and they are different from and complement FLp53 activity, the most mutated gene in cancer. Here we show that the integrated stress response (ISR) leads to the specific induction of Δ160p53 isoform. Using bicistronic constructs we confirmed the presence of an Internal Ribosome Entry Site (IRES) in p53 mRNA that controls Δ160p53 isoform translation. Subjecting cells to endoplasmic reticulum stress showed that eIF2α phosphorylation is a key event leading to cap-independent expression of Δ160p53 during ISR. Additionally, cancer-specific mutations in p53 also enhanced cap-independent translation of Δ160p53 via Δ160p53IRES. An antisense morpholino oligo targeting Δ160IRES efficiently reduced Δ160p53 protein levels and significantly impaired oncogenic functions of Δ160p53. Our data support a model in which an IRES structure in the coding region of p53 is activated under stress conditions, leading to the expression of the oncogenic shorter Δ160p53 isoform, whose structure is affected by cancer-specific mutations in the p53 gene. A better understanding of Δ160p53IRES structure and function may be advantageous for a more efficient therapeutic targeting of p53.

2020-11-20Conference object

Restricted access

Translational Regulation of the Human PERK by Upstream Open Reading Frames

Publication . Fernandes, Rafael; Lopes, Pedro; Romão, Luísa

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 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. 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.

2020-11-20Conference object

Restricted access

uORF-mediated translational regulation of the human PERK mRNA

Publication . Fernandes, Rafael; Lopes, Pedro; Romão, Luísa

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 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. 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.

2020-10-07Conference object

Restricted access