Plant organelle C‐to‐U <scp>RNA</scp> editing factors can operate successfully in yeast ( <i>Saccharomyces cerevisiae</i> ) as an easily amenable eukaryotic system for their functional analysis

Site-specific cytidine-to-uridine (C-to-U) RNA editing is essential for gene expression in plant mitochondria and chloroplasts. Specific RNA-binding pentatricopeptide repeat (PPR) proteins equipped with a DYW-type cytidine deaminase have been identified as the core RNA editing factors. Multiple circumstances impede their analysis in planta and call for experimental approaches in easily amenable heterologous setups for functional analyses of these intriguing proteins. Here, we report the establishment of the yeast Saccharomyces cerevisiae as such a system. Three plant RNA editing factors from the model moss Physcomitrium patens, namely PPR56 (PpPPR_56), PPR65 (PpPPR_65), and PPR78 (PpPPR_78), were successfully expressed in the yeast cytosol and faithfully edit not only their respective co-delivered targets but also variable ranges of off-targets with similar recognition sequences in the yeast background transcriptome. Convincingly, we found that the different editing factors behaved in the new system very much like previously found in bacteria or human cells, not only with respect to off-target conservation profiles but also with surprising differences in off-target numbers, championed by more than 125 off-targets for PPR56 with editing efficiencies of up to 41% in the S. cerevisiae transcriptome. We conclude that S. cerevisiae will be a helpful heterologous system for studying the intricacies of plant RNA editing.