Regulation of IRES-mediated translation under physiological and cellular stress conditions

Licursi, Maria (2012) Regulation of IRES-mediated translation under physiological and cellular stress conditions. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Cap-dependent and IRES-mediated translation initiation mechanisms are regulated differently by cellular machinery. Under conditions such as viral infection, apoptosis and cellular stress, cap-dependent protein synthesis becomes impaired and translation driven by some IRES elements becomes upregulated. The objective of this study was to investigate how cells regulate cap-dependent and IRES-mediated translation in different cellular environments. -- To determine whether efficiency of translation initiation by IRES elements differs under normal physiological conditions, we tested 13 bicistronic reporter constructs containing different viral and cellular IRES elements. Bicistronic vectors harboring IRES elements are essential to achieve efficient expression of multiple genes in gene therapy protocols and biomedical applications. The IRES element commonly used in current bicistronic vectors originates from the EMCV and, therefore, was used as standard in this study. The in vitro screening in human and mouse fibroblast and hepatocarcinoma cells revealed that the VCIP IRES was the only IRES element that directed translation more efficiently than the EMCV IRES in all cell lines. Furthermore, the VCIP IRES initiated greater reporter expression levels than the EMCV IRES in transfected mouse livers. These results demonstrate that IRES-mediated translation efficiency in physiological conditions is dependent on IRES elements as well as cell types. In addition, this data suggests that VCIP-IRES containing vectors have great potential to improve gene expression and could increase the benefits of bicistronic vectors for experimental and therapeutic purposes. -- Viral IRES-mediated translation often remains active when cellular cap-dependent translation is severely impaired under cellular stresses induced by virus infection. Next, we used bicistronic reporter constructs harboring viral IRES elements to investigate how cellular stresses influence the efficiency of viral IRES-mediated translation. Mouse cell line NIH3T3 cells transfected with these bicistronic reporter constructs were subjected to different cellular stresses. Increased translation initiation was observed under amino acid starvation only when EMCV or FMDV IRES elements were present. To identify cellular mechanisms that promoted viral IRES-mediated translation, we investigated the involvement of eukaryotic initiation factor 4E binding protein (4E-BP), general control nondepressed 2 (GCN2) and eukaryotic initiation factor 2B (eiF2B), since these are known to be modulated under amino acid starvation. Knockdown of 4E-BP1 impaired the promotion of EMCV and FMDV IRES-mediated translation under amino acid starvation while GCN2 and eiF2B were not involved. To further investigate how 4E-BP1 regulates translation initiated by EMCV and FMDV IRES elements, we used a phosphoinositide-3 kinase inhibitor (LY294002), an mTOR inhibitor (Torin1) or leucine starvation to mimic 4E-BP1 dephosphorylation induced by amino acid starvation. 4E-BP1 dephosphorylation induced by these treatments was not sufficient to promote the viral IRES-mediated translation. These results suggest that 4E-BP1 regulates EMCV and FMDV IRES mediated translation under amino acid starvation, but not via its dephosphorylation.

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