Dual Reporter Systems for the Analysis of Translational Readthrough in Mammals

Abstract

Translational readthrough, the decoding of stop codons as sense codons, leads to C-terminal extension of proteins which may lead to the formation of protein isoforms with distinct properties from the original protein. Two proteins have recently been identified that are targeted to the peroxisome via hidden peroxisomal targeting signals in their readthrough extensions. This noninduced basal translational readthrough can be distinguished from pharmacological induction of readthrough by aminoglycosides or other small molecules, which can be used for the treatment of diseases caused by premature stop (termination) codons (PTCs). Readthrough of both, natural stop codons and PTCs, can be quantified in cell culture using reporter systems. In the present article, we describe two dual reporter systems, based on combined fluorescence/luminescence measurement and flow cytometric fluorescence measurement, respectively. Further, we provide a protocol for a fast and efficient cloning procedure of reporter constructs. The dual reporter systems described here help to analyze the peroxisome-specific isoforms of readthrough enzymes as well as potential readthrough therapeutics.