DYW cytidine deaminase domains have a long‐range impact on RNA recognition by the PPR array of chimeric plant C‐to‐U RNA editing factors and strongly affect target selection.

SUMMARY: The protein factors for the specific C‐to‐U RNA editing events in plant mitochondria and chloroplasts possess unique arrays of RNA‐binding pentatricopeptide repeats (PPRs) linked to carboxy‐terminal cytidine deaminase DYW domains via the extension motifs E1 and E2. The E1 and E2 motifs have distant similarities to tetratricopeptide repeats known to mediate protein–protein interactions but their precise function is unclear. Here, we investigate the tolerance of PPR56 and PPR65, two functionally characterized RNA editing factors of the moss Physcomitrium patens, for the creation of chimeras by variably replacing their C‐terminal protein regions. Making use of a heterologous RNA editing assay system in Escherichia coli we find that heterologous DYW domains can strongly restrict or widen the spectrum of off‐targets in the bacterial transcriptome for PPR56. Surprisingly, our data suggest that these changes are not only caused by the preference of a given heterologous DYW domain for the immediate sequence environment of the cytidine to be edited but also by a long‐range impact on the nucleotide selectivity of the upstream PPRs. Significance Statement: DYW domains affect upstream PPR arrays for RNA recognition. [ABSTRACT FROM AUTHOR]