A Conserved Lysine in an Ion-Pair with a Catalytic Glutamate Is Critical for U-to-C RNA Editing but Restricts C-to-U RNA Editing.

Abstract

Plants make pyrimidine base substitutions in organellar mRNAs through the action of sequence-specific nuclear-encoded enzymes. Pentatricopeptide repeat (PPR) proteins are essential for ensuring specificity, while the enzymatic DYW domain is often present at the C-terminus of a PPR protein and dependent on the variant possessing C-to-U and/or U-to-C RNA editing activities. Expression of exogenous DYW-KP variant enzymes in bacteria leads to the modification of RNAs suggestive of U-to-C base changes. The modified RNAs could only be purified from the interphase of an acidic guanidinium thiocyanate-phenol-chloroform experiment. It was projected that in bacteria stable RNA-enzyme cross-links form from a lysyl attack. In this study, RNA editing was examined for dual U-to-C/C-to-U editing enzyme KP6 with conserved lysine residues substituted by alanine. A single lysine was found to be essential for U-to-C editing and, based on the crystal structures of DYW domains, would likely be present in the active site. Crystal structures also suggest that the lysine can potentially form an ion pair with the catalytic glutamate critical for C-to-U RNA editing. Mutation of lysine to alanine greatly stimulated the C-to-U RNA editing by KP6. A ∼319 Da adduct observed on DYW-KP proteins could not be detected on the U-to-C-deficient lysine to alanine point mutant enzymes. This work establishes the critical role for a single lysine in the DYW-KP domain specifically for U-to-C editing activity but also highlights a secondary role for the lysine in modulating C-to-U editing through the formation of an inhibitory ion pair with the catalytic glutamate.