Reconstitution of mRNA Editing in Yeast Using a Gal4-ApoB-Gal80 Fusion Transcript as the Selectable Marker

Heinrich Lellek,Sybille Welker,Ines Diehl,Romy Kirsten,Jobst Greeve

doi:10.1074/jbc.m203517200

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-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.

Highlights

MRNA editing is a genetic regulation that alters gene expression by posttranscriptional nucleotide changes within the coding regions of transcripts. mRNA editing of nuclear-encoded genes consists of site-specific deamination reactions that convert A to I and C to U
Several studies demonstrated that APOBEC-1 and APOBEC-1-stimulating protein (ASP)/APOBEC-1 complementing factor (ACF) exert strong apoB mRNA editing activity in vitro, it is unknown whether APOBEC-1 and ASP/ACF are all that is required for editing of apoB mRNA in vivo
To establish a functional assay system for apoB mRNA editing in vivo, we reasoned that this Gal4-Gal80 fusion transcript might be a useful selectable marker for mRNA editing in yeast

Summary

Introduction

MRNA editing is a genetic regulation that alters gene expression by posttranscriptional nucleotide changes within the coding regions of transcripts. mRNA editing of nuclear-encoded genes consists of site-specific deamination reactions that convert A to I and C to U. The A to I editing described for glutamate receptors, serotonin 5-HT2C receptors, and the hepatitis delta virus RNA is mediated by a family of adenosine deaminases known as adenosine deaminases acting on RNAs [3, 4] These enzymes function as single peptides and contain both RNA binding and deaminase activity [3]. In 2000, the cloning of the second essential component of the apoB mRNA editing enzyme complex was reported by Driscoll and co-workers [23] and simultaneously by our group [24] This protein, termed APOBEC-1 complementing factor (ACF) by Driscoll and co-workers [23] and APOBEC-1-stimulating protein (ASP) by us, represents a novel type of RNA-binding protein with three non-identical binding domains for singlestranded RNA at the amino terminus and a putative binding domain for double-stranded RNA at the carboxyl terminus. Several studies demonstrated that APOBEC-1 and ASP/ACF exert strong apoB mRNA editing activity in vitro, it is unknown whether APOBEC-1 and ASP/ACF are all that is required for editing of apoB mRNA in vivo

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