Reconstitution of mRNA editing in yeast using a Gal4-ApoB-Gal80 fusion transcript as the selectable marker

Abstract

We describe a fusion transcript of Gal4 linked to its specific inhibitor protein Gal80 by 276 nucleotides of apolipoprotein (apo) B sequence as a selectable marker for mRNA editing. Editing of apoB mRNA is catalyzed by an editing enzyme complex that introduces a stop codon by deamination of C to U. The catalytic subunit APOBEC-1 is a cytidine deaminase and requires a second essential component recently cloned and termed APOBEC-1 complementing factor (ACF) or APOBEC-1-stimulating protein (ASP). The aim of this study was to demonstrate that APOBEC-1 plus ACF/ASP comprise all that is required for editing of apoB mRNA in vivo. Expression of APOBEC-1 and Gal4 fused to its inhibitor Gal80 by an intervening unedited apoB sequence (Gal4-apoBc-Gal80) did not result in the Gal4-dependent expression of HIS3 and β-galactosidase in the yeast strain CG1945. Co-expression of APOBEC-1 and ACF/ASP induced editing of the apoB site in up to 13% of the Gal4-apoBc-Gal80 transcripts and enabled selection of yeast cells for robust expression of HIS3 and β-galactosidase. Additional expression of the alternative splicing regulatory protein KSRP increased the editing of the apoB site by APOBEC-1 and ACF/ASP to 21%. Thus, APOBEC-1 and ACF/ ASP represent the core apoB mRNA editing enzyme in vivo. This study demonstrates for the first time the successful use of a selectable marker for mRNA editing. The Gal4-Gal80 system is analogous to the two-hybrid assay and may have broader applications for the study of other mRNA processing reactions.