DYW deaminase domain has a distinct preference for neighboring nucleotides of the target RNA editing sites.

C-to-U RNA editing sites in plant organelles show a strong bias for neighboring nucleotides. The nucleotide upstream of the target cytidine is typically C or U, whereas A and G are less common and rare, respectively. In pentatricopeptide repeat (PPR)-type RNA editing factors, the PPR domain specifically binds to the 5' sequence of target cytidines, whereas the DYW domain catalyzes the C-to-U deamination. We comprehensively analyzed the effects of neighboring nucleotides of the target cytidines using an Escherichia coli orthogonal system. Physcomitrium PPR56 efficiently edited target cytidines when the nucleotide upstream was U or C, whereas it barely edited when the position was G or the nucleotide downstream was C. This preference pattern, which corresponds well with the observed nucleotide bias for neighboring nucleotides in plant organelles, was altered when the DYW domain of OTP86 or DYW1 was adopted. The PPR56 chimeric proteins edited the target sites even when the -1 position was G. Our results suggest that the DYW domain possesses a distinct preference for the neighboring nucleotides of the target sites, thus contributing to target selection in addition to the existing selection determined by the PPR domain.