Open AccessAmantadine as a regulator of internal ribosome entry site 1
Author(s) -
CHEN Yingju,
ZENG Shihjhan,
HSU John TA,
HORNG Jimtong,
YANG Hongming,
SHIH Shinru,
CHU Yuting,
WU Tzongyuan
Publication year - 2008
Publication title -
acta pharmacologica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.514
H-Index - 90
eISSN - 1745-7254
pISSN - 1671-4083
DOI - 10.1111/j.1745-7254.2008.00876.x
Subject(s) - internal ribosome entry site , biology , translation (biology) , reporter gene , microbiology and biotechnology , eukaryotic translation , messenger rna , gene expression , gene , genetics
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.