Reconstitution of mRNA editing in yeast using a Gal4-apoB-Gal80 fusion transcript as the selectable marker.
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-apoB(C)-Gal80) did not result in the Gal4-dependent expression of HIS3 and beta-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-apoB(C)-Gal80 transcripts and enabled selection of yeast cells for robust expression of HIS3 and beta-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.
Lellek
Heinrich
H
Klinik und Poliklinik für Innere Medizin, Kernklinik und Poliklinik, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
Welker
Sybille
S
Diehl
Ines
I
Kirsten
Romy
R
Greeve
Jobst
J
D000071479:APOBEC-1 Deaminase; D001055:Apolipoproteins B / Q000235:genetics*; D020134:Catalytic Domain; D003564:Cytidine Deaminase / Q000502:physiology; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000502:physiology*; D017393:RNA Editing; D016601:RNA-Binding Proteins / Q000502:physiology; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D015003:Yeasts / Q000235:genetics*
C411647:A1CF protein, human; D001055:Apolipoproteins B; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; C055002:GAL80 protein, S cerevisiae; C105789:KHSRP protein, human; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000071479:APOBEC-1 Deaminase; C000605089:APOBEC1 protein, human; D003564:Cytidine Deaminase