RNA editing converting cytidines into uridines is a hallmark of gene expression in land plant chloroplasts and mitochondria. Pentatricopeptide repeat (PPR) proteins have a key role in target recognition, but the functional editosome in the plant organelles has remained elusive. Here we show that individual Physcomitrella patens DYW-type PPR proteins alone can perform efficient C-to-U editing in Escherichia coli reproducing the moss mitochondrial editing. Single amino acid exchanges in the DYW domain abolish RNA editing, confirming it as the functional cytidine deaminase. The modification of RNA targets and the identification of numerous off-targets in the E. coli transcriptome reveal nucleotide identities critical for RNA recognition and cytidine conversion. The straightforward amenability of the new E. coli setup will accelerate future studies on RNA target recognition through PPRs, on the C-to-U editing deamination machinery and towards future establishment of transcript editing in other genetic systems.

RNA editing processes are strikingly different in animals and plants. Up to thousands of specific cytidines are converted into uridines in plant chloroplasts and mitochondria whereas up to millions of adenosines are converted into inosines in animal nucleo-cytosolic RNAs. It is unknown whether these two different RNA editing machineries are mutually incompatible. RNA-binding pentatricopeptide repeat (PPR) proteins are the key factors of plant organelle cytidine-to-uridine RNA editing. The complete absence of PPR mediated editing of cytosolic RNAs might be due to a yet unknown barrier that prevents its activity in the cytosol. Here, we transferred two plant mitochondrial PPR-type editing factors into human cell lines to explore whether they could operate in the nucleo-cytosolic environment. PPR56 and PPR65 not only faithfully edited their native, co-transcribed targets but also different sets of off-targets in the human background transcriptome. More than 900 of such off-targets with editing efficiencies up to 91%, largely explained by known PPR-RNA binding properties, were identified for PPR56. Engineering two crucial amino acid positions in its PPR array led to predictable shifts in target recognition. We conclude that plant PPR editing factors can operate in the entirely different genetic environment of the human nucleo-cytosol and can be intentionally re-engineered towards new targets.