Original Article

Amantadine as a regulator of internal ribosome entry site

Authors: Ying-ju Chen, Shih-jhan Zeng, John TA Hsu, Jim-tong Horng, Hong-ming Yang, Shin-ru Shih, Yu-ting Chu, Tzong-yuan Wu

Abstract

Aim: Studies of eukaryotes have yielded 2 translation initiation mechanisms: a classical cap-dependent mechanism and a cap-independent mechanism proceeding through the internal ribosomal entry site (IRES). We hypothesized that it might be possible to identify compounds that may distinguish between cap-dependent translation and cap-independent IRES-mediated translation.
Methods: To facilitate compound screening, we developed bicistronic reporter constructs containing a β-galactosidase gene (β-gal) and a secreted human placental alkaline phosphatase (SEAP) reporter gene. Following transcription, the β-gal gene is translated by a cap-dependent mechanism, while SEAP expression is controlled by the IRES derived from either enterovirus 71 (EV-71) or encephalomyocardi-tis virus (EMCV). This assay could potentially identify compounds that inhibit SEAP expression (cap-independent) without affecting β-gal activity (cap-dependent).
Results: Using a bicistronic plasmid-based transient transfection assay in the COS-1 cells, we identified amantadine, a compound that inhibited the IRES of EV71- and EMCV-mediated cap-independent translation but did not interfere with cap-dependent translation when the dose of amantadine was lower than 0.25 mg/mL.
Conclusion: These results imply that amantadine may distinguish between cap-dependent translation and cap-independent IRES-mediated translation and can be used to regulate gene expression at a translational level.
Keywords:

Download Citation

Cited times in Scopus