Abstract
A subfamily of pentatricopeptide repeat (PPR) proteins, known as PPR-DYW:PG, catalyzes the cytidine to uridine (C-to-U) RNA editing in plant organelles. A related PPR subfamily, PPR-DYW:KP, catalyzes the uridine to cytidine (U-to-C) reaction, via a crosslinking mechanism involving a lysine residue. We demonstrate that Lys88 in the DYW:KP domain is essential for the U-to-C editing activity of PPR-DYW:KP proteins. Substituting Lys88 with other amino acids in designer proteins switches the protein activity to C-to-U and prevents crosslinking with the edited RNA. However, this mutation leads to C-to-U off-target editing downstream the targeted site. Finally, other modifications can modulate the catalytic activity and alter the type of reaction catalyzed by the DYW domain. Altogether, our results suggest that subtle modifications in the DYW domain can influence the position of the edited nucleotide and the type of RNA editing reaction.