Moss DYW-type pentatricopeptide repeat (PPR) proteins as modifiable C-to-U RNA editing factors in diverse genetic systems.

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Specific pentatricopeptide repeat (PPR) proteins mediate cytidine-to-uridine (C-to-U) RNA editing in mitochondrial and chloroplast transcripts of land plants. DYW-type PPR proteins are the key factors of plant-type C-to-U RNA editing. They recognize specific RNA targets to define the C to be edited and perform cytidine deamination to create uridines. How exactly the conversion of specific cytidines is accomplished, is still elusive, although the crucial PPR-RNA binding code is elucidated and the C-terminal DYW domain is identified as the enzymatic domain by now. The RNA editing system of the moss Physcomitrella patens with only nine DYW-type PPR proteins assigned to 13 editing sites in its organellar transcriptomes is one of the simplest in the land plant kingdom. In other plants like the model flowering plant Arabidopsis thaliana the editing system has evolved towards much more complexity with more than 450 sites to be edited and various combinations of recognition- and deamination factors plus additional helper editing factors important for efficient conversion of many sites. We therefore focused our research on the moss editing system. By insertion of single moss DYW-type PPR genes and their targets, we were able to transfer C-to-U RNA editing into the bacterium Escherichia coli. This setup offers a unique possibility to quickly investigate modifications both on the protein factor and the RNA target side. We here aim to further elucidate the functionality of altogether five different DYW-type PPR proteins. Re-direction of these five editing factors by modification of binding positions or protein motifs can now easily be tested in the bacterial system prior transfer into the plant system of choice (here Physcomitrella patens and Arabidopsis thaliana). While genetic modification of the mitochondrial genome is not feasible in plants up to date, DYW-type PPR proteins as engineerable mitochondrial RNA editors would open new ways for basic and applied organellar research. Within this project, we aim to analyze the mutants generated via insertion of engineered PPR proteins to get deeper insights into the mitochondrial energy metabolism. In parallel, we will investigate whether DYW-type PPR proteins are also capable to edit transcripts in the plant cytosol. Ultimately, we might be able to introduce specific C-to-U changes in organellar and nuclear transcripts and thereby engineer functions of diverse plant systems of choice.