The molecular basis and evolution of the organellar RNA editosome by complementary DYW deaminases in seed plants

The DYW deaminase domain catalyzes the conversion of cytidines (C) to uridines (U) in RNA editing of plant organelles. While the DYW subgroup contains a complete DYW deaminase domain at the C-terminus, the E2 and E+ subgroups rely on complementary deaminases, in which catalytic activity depends on interactions with short DYW proteins, such as DYW1, DYW2, and MITOCHONDRIAL EDITING FACTOR 8 (MEF8)/MITOCHONDRIAL EDITING FACTOR 8 SIMILAR (MEF8S). Although orthogonal RNA editing in bacteria by a DYW subgroup pentatricopeptide repeat (PPR) has been reported, attempts to activate the DYW deaminase through molecular complementation in bacteria have been unsuccessful, leaving its molecular basis unresolved. In this study, we reconstituted the simplest editosome in Escherichia coli, composed of PPR56PPRE1E2-CRR4PG and DYW1 alone. Systematical mutational analysis of the PG-box of CHLORORESPIRATORY REDUCTION 4 (CRR4) in bacteria and in planta revealed the critical role of serine, isoleucine, and phenylalanine residues in DYW deaminase complementation and catalysis. CRR4-like PPR proteins, termed the "PG-type" characterized by the PG-box with these 3 key amino acid residues at the C-terminus, are minor in angiosperms but constitute one of the major subgroups in gymnosperms. Putative orthologs of Arabidopsis thaliana DYW1 are present in limited angiosperm species, suggesting that in other species, other short DYW proteins serve as the interaction partners for PG-type PPR proteins. Our findings reveal a minimal functional editosome module, shedding light on the conserved and diverse mechanisms of RNA editing in plant organelles.