RNA editosomes selectively deaminate cytidines to uridines in plant organellar transcripts—mostly to restore protein functionality and consequently facilitate mitochondrial and chloroplast function. The RNA editosomal pentatricopeptide repeat proteins serve target RNA recognition, whereas the intensively studied DYW domain elicits catalysis. Here we present structures and functional data of a DYW domain in an inactive ground state and activated. DYW domains harbour a cytidine deaminase fold and a C-terminal DYW motif, with catalytic and structural zinc atoms, respectively. A conserved gating domain within the deaminase fold regulates the active site sterically and mechanistically in a process that we termed gated zinc shutter. Based on the structures, an autoinhibited ground state and its activation are cross-validated by RNA editing assays and differential scanning fluorimetry. We anticipate that, in vivo, the framework of an active plant RNA editosome triggers the release of DYW autoinhibition to ensure a controlled and coordinated cytidine deamination playing a key role in mitochondrial and chloroplast homeostasis.

Analysis of RNA editing in plant mitochondria has at least in vitro been hampered by very low activity. Consequently, none of the trans-acting factors involved has yet been identified. We here report that in vitro RNA editing increases dramatically when additional cognate recognition motifs are introduced into the template RNA molecule. Substrate RNAs with tandemly repeated recognition elements enhance in vitro RNA editing from 2-3% to 50-80%. The stimulation is not influenced by the editing status of a respective RNA editing site, suggesting that specific recognition of a site can be independent of the edited nucleotide itself. In vivo, attachment of the editing complex may thus be analogously initiated at sequence similarities in the vicinity of bona fide editing sites. This cis-acting enhancement decreases with increasing distance between the duplicated specificity signals; a cooperative effect is detectable up to approximately 200 nucleotides. Such repeated template constructs promise to be powerful tools for the RNA affinity identification of the as yet unknown trans-factors of plant mitochondrial RNA editing.

C to U editing is one of the post-transcriptional steps which are required for the proper expression of chloroplast and mitochondrial genes in plants. It depends on several proteins acting together which include the PLS-class pentatricopeptide repeat proteins (PPR). DYW2 was recently shown to be required for the editing of many sites in both organelles. In particular almost all the sites associated with the E+ subfamily of PPR proteins are depending on DYW2, suggesting that DYW2 is required for the function of E+-type PPR proteins. Here we strengthened this link by identifying 16 major editing sites controlled by 3 PPR proteins: OTP90, a DYW-type PPR and PGN and MEF37, 2 E+-type PPR proteins. A re-analysis of the DYW2 editotype showed that the 49 sites known to be associated with the 18 characterized E+-type PPR proteins all depend on DYW2. Considering only the 288 DYW2-dependent editing sites as potential E+-type PPR sites, instead of the 795 known editing sites, improves the performances of binding predictions systems based on the PPR code for E+-type PPR proteins. However, it does not compensate for poor binding predictions.

DYW-type PPR proteins independently evolved in mosses and flowering plants restore RNA editing defects across wide phylogenetic distances.