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 a selectable marker for mRNA editing. Editing of apo B 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 comprises all that is required for editing of apo B mRNA in vivo. Expression of APOBEC-1 and Gal4 fused to its inhibitor Gal80 by an intervening unedited apo B sequence (Gal4-apoB C -Gal80) did not result in the Gal4 dependent expression of HIS3 and ß-galactosidase in the yeast strain CG1945. Co-expression of APOBEC-1 and ACF/ASP induced editing of the apo B site in up to 13% of the Gal4-apoB C -Gal80 transcripts and enabled selection of yeast cells for robust expression of HIS3 and ß-galactosidase. Additional expression of the alternative splicing regulatory protein KSRP increased the editing of the apo B site by APOBEC-1 and ACF/ASP to 21%. Thus, APOBEC-1 and ACF/ASP represent the core apo B 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 application for the study of other mRNA processing reactions.


Introduction
Gal4-apoB-Gal80 as selectable marker for mRNA editing 5 complex is reconstituted in yeast by expression of APOBEC-1 and ASP/ACF. mRNA editing introduces a stop codon in the Gal4-Gal80 fusion transcript and allows to select for expression of active Gal4 protein. This is the first example that mRNA editing is reconstituted in yeast, and the selection system presented may have broader application for the study of other mRNA editing or processing reactions. The resulting construct GAL4-ApoB-GAL80 was amplified by PCR using oligonucleotides Gal4-s (CAAGCTTATGAAGCTACTGTCTTCTATCGAAC) and Gal80-rev (GGATCCAGCAATCTCGATCGAATTAATGTCGC), cloned into pGEM T Easy and entirely

Experimental Procedures
Gal4-apoB-Gal80 as selectable marker for mRNA editing 7 pBridge-Gal4-ApoB C -Gal80-KSRP: The full length cDNA of KSRP was excised from pGEM-T Easy by restriction enzyme digest with NotI and inserted into the unique NotI site of the multiple cloning site II of pBridge-Gal4-ApoB C -Gal80 to generate pBridge-Gal4-ApoB C -Gal80-KSRP.

Analysis of apo B mRNA editing in yeast:
Total RNA was prepared from yeast by acid phenol extraction. The yeast were grown to late log phase on synthetic drop-out media either with or without histidine in the presence of 5 mM 3-AT. The apo B cassette of the Gal4-ApoB-Gal80 transcript was amplified by RT-PCR with oligonucleotides GAL4-1 sense (CTTTCACAACCAATTGCCTCCTCTAAC) and apoB2 (CACGGATATGATA GTGCTCATCAAGAC) and analyzed for editing by primer extension assay (7,32). Primer extension products were quantitated as described (7,32).

Results
Gal4-apoB-Gal80 as a selectable marker for mRNA editing in yeast: The yeast transcription factor Gal4 is inhibited by complex formation with its specific inhibitor Gal80. A fusion protein consisting of Gal4 and Gal80 is inactive and does not promote Gal4 dependent transcription. To establish a functional assay system for apo B mRNA editing in vivo, we reasoned that this Gal4-Gal80 fusion transcript might be a useful selectable marker for mRNA editing in yeast. Therefore, we inserted 92 amino acids of the apo B sequence that encompass the editing site at C 6666 in frame between Gal4(1-841) and Gal80 to generate Gal4-apoB C -Gal80 (Gal4-C). As a positive control we constructed Gal4-apoB U -Gal80 (Gal4-U) that contains the edited version of the apo B sequence with the premature stop codon. Gal4-C should not be able to promote GAL4 dependent transcription because of complex formation with its specific inhibitor GAL80, while the edited version Gal4-U that contains only a tail of 25 amino acids of apo B sequence should be active similar to wild-type GAL4 (Fig. 1).
Gal4-C and Gal4-U were expressed in yeast CG1945 cells using the expression plasmid pAS in the absence or presence of APOBEC-1 (Fig. 2 A). The yeast strain CG1945 is commonly   Gal4-apoB C -Gal80 (Gal4-C) containing the unedited apo B sequence leads to a fusion protein in which gal4 is inhibited by complex formation with Gal80. In contrast, Gal4-apoB U -Gal80 (Gal4-U) gives rise to active Gal4 protein due to a premature stop translation codon in the edited apo B sequence.