Received for publication January 19, 2005.
Revised March 21, 2005.
Accepted for publication March 22, 2005.

Chemical-based translational induction of luciferase expression: an efficient tool for the in vivo screening of protein farnesylation inhibitors

Abstract

We describe the development of a cell system for the in vivo screening of inhibitors of the mevalonate pathway. To this aim we have constructed a bicistronic mRNA, transcribed from a constitutive CMV promoter, containing the Renilla luciferase RNA open reading frame sequence as first cistron and the Firefly luciferase RNA sequence as a second cistron. The inter cistronic space is made of the R17 binding sequence of the bacteriophage R17 protein. Translation of the second cistron is switched on by a chimeric protein able to bind to a specific sequence in the hairpin and to induce internal ribosome entry in the RNA. This chimeric protein is made up of the bacteriophage RNA binding domain (R17) fused to the ribosome recruitment core of the eIF-4G1 eukaryotic translation initiation factor and to the CAAX box of H-Ras addressing the protein to the plasma membrane where it is not efficient. Internal ribosome entry upstream of the Firefly cistron is therefore under the dependence of the mevalonate pathway inhibitors Indeed products able to inhibit protein farnesylation, rescue the cytoplasmic location of the R17-eIF4G-CAAX protein which once more becomes a translation factor for the expression of the second cistron. To exemplify the system the present work checks the ability of various antiestrogens to interfere with the mevalonate pathway. It appears that pure antiestrogen, able to selectively bind the estrogen receptor, is unable to switch on the second Firefly cistron although selective antiestrogen-binding-site ligands are able to.

Key words: Ras, Regulation - post-transcriptional

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