Identification of mTOR and AGO1 IRES trans-acting factors

Marques, Rita; Lacerda, Rafaela; Romão, Luísa

http://hdl.handle.net/10400.18/7466

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Authors

Marques, Rita

Lacerda, Rafaela

Romão, Luísa

Abstract(s)

Cancer is the second leading cause of death globally; therefore, its study is crucial to discover new therapies. Under stress, the regular process of protein synthesis (canonical translation) is impaired, while a back-up mechanism mediated by internal ribosome entry sites (IRES) continues to function, allowing the synthesis of proteins that maintain cellular viability. This also happens in cancer cells, contributing for their survival and consequent tumorigenesis. IRES-mediated translation and its regulation by IRES trans-acting factors (ITAFs) has been correlated to metastasis and chemotherapeutic drug resistance. Therefore, our main goal was to validate ITAFs and assess their significance in cancer onset, thus becoming candidates as novel therapeutic targets. A bicistronic reporter system, which contains a first cistron translated via canonical translation and a second one translated by IRES of mTOR1 and AGO12 was used to test IRES-driven translation initiation activity. Experiments were carried out in which several proteins (hnRNPs) were silenced by specific siRNAs to analyse their function as ITAFs of mTOR and AGO1 IRESs. Also, distinct drugs were applied to simulate endoplasmic reticulum (ER) or hypoxia stress, to evaluate their effect on IRES activity. The relative IRES activity was assessed by luminescence tests and the protein levels by Western blot. In general, knockdown of hnRNPK and hnRNPU seems to decrease the IRES activity by ~60% and ~30% respectively, while hnRNPC knockdown does not show a significant effect. Regarding the ER stress, hnRNPK knockdown seems to decrease even more the IRES activity, while hnRNPU depletion induces a significant increase. On the other hand, in hypoxia, the hnRNPs knockdowns do not significantly affect IRES activity. These results indicate that hnRNPK and hnRNPU may function as ITAFs of mTOR and AGO1 IRES activity in cells under ER stress. Our data can be decisive for a better understanding of carcinogenesis and suggest new therapeutic targets for cancer treatment. 1. Marques-Ramos, A., et.al. 2017. RNA. 23, 1712-1728 2. Lacerda, R. 2016. Faculdade de Ciências e Tecnologia da Universidade NOVA de Lisboa