Conservation of the moss RNA editing factor PPR78 despite the loss of its known cytidine-to-uridine editing sites is explained by a hidden extra target.

Cytidine (C)-to-uridine (U) RNA editing in plant organelles relies on specific RNA-binding pentatricopeptide repeat (PPR) proteins. In the moss Physcomitrium patens, all such RNA editing factors feature a C-terminal DYW domain that acts as the cytidine deaminase for C-to-U conversion. PPR78 of Physcomitrium targets 2 mitochondrial editing sites, cox1eU755SL and rps14eU137SL. Remarkably, the latter is edited to highly variable degrees in different mosses. Here, we aimed to unravel the coevolution of PPR78 and its 2 target sites in mosses. Heterologous complementation in a Physcomitrium knockout line revealed that the variable editing of rps14eU137SL depends on the PPR arrays of different PPR78 orthologues but not their C-terminal domains. Intriguingly, PPR78 has remained conserved despite the simultaneous loss of editing at both known targets among Hypnales (feather mosses), suggesting it serves an additional function. Using a recently established RNA editing assay in Escherichia coli, we confirmed site-specific RNA editing by PPR78 in the bacterium and identified 4 additional off-targets in the bacterial transcriptome. Based on conservation profiles, we predicted ccmFNeU1465RC as a candidate editing target of PPR78 in moss mitochondrial transcriptomes. We confirmed editing at this site in several mosses and verified that PPR78 targets ccmFNeU1465RC in the bacterial editing system, explaining the conservation and functional adaptation of PPR78 during moss evolution.

DYW cytidine deaminase domains have a long-range impact on RNA recognition by the PPR array of chimeric plant C-to-U RNA editing factors and strongly affect target selection.

The protein factors for the specific C-to-U RNA editing events in plant mitochondria and chloroplasts possess unique arrays of RNA-binding pentatricopeptide repeats (PPRs) linked to carboxy-terminal cytidine deaminase DYW domains via the extension motifs E1 and E2. The E1 and E2 motifs have distant similarities to tetratricopeptide repeats known to mediate protein-protein interactions but their precise function is unclear. Here, we investigate the tolerance of PPR56 and PPR65, two functionally characterized RNA editing factors of the moss Physcomitrium patens, for the creation of chimeras by variably replacing their C-terminal protein regions. Making use of a heterologous RNA editing assay system in Escherichia coli we find that heterologous DYW domains can strongly restrict or widen the spectrum of off-targets in the bacterial transcriptome for PPR56. Surprisingly, our data suggest that these changes are not only caused by the preference of a given heterologous DYW domain for the immediate sequence environment of the cytidine to be edited but also by a long-range impact on the nucleotide selectivity of the upstream PPRs.

One C-to-U RNA Editing Site and Two Independently Evolved Editing Factors: Testing Reciprocal Complementation with DYW-Type PPR Proteins from the Moss Physcomitrium (Physcomitrella) patens and the Flowering Plants Macadamia integrifolia and Arabidopsis.

Cytidine-to-uridine RNA editing is a posttranscriptional process in plant organelles, mediated by specific pentatricopeptide repeat (PPR) proteins. In angiosperms, hundreds of sites undergo RNA editing. By contrast, only 13 sites are edited in the moss Physcomitrium (Physcomitrella) patens Some are conserved between the two species, like the mitochondrial editing site nad5eU598RC. The PPR proteins assigned to this editing site are known in both species: the DYW-type PPR protein PPR79 in P. patens and the E+-type PPR protein CWM1 in Arabidopsis (Arabidopsis thaliana). CWM1 also edits sites ccmCeU463RC, ccmBeU428SL, and nad5eU609VV. Here, we reciprocally expressed the P. patens and Arabidopsis editing factors in the respective other genetic environment. Surprisingly, the P. patens editing factor edited all target sites when expressed in the Arabidopsis cwm1 mutant background, even when carboxy-terminally truncated. Conversely, neither Arabidopsis CWM1 nor CWM1-PPR79 chimeras restored editing in P. patens ppr79 knockout plants. A CWM1-like PPR protein from the early diverging angiosperm macadamia (Macadamia integrifolia) features a complete DYW domain and fully rescued editing of nad5eU598RC when expressed in P. patens. We conclude that (1) the independently evolved P. patens editing factor PPR79 faithfully operates in the more complex Arabidopsis editing system, (2) truncated PPR79 recruits catalytic DYW domains in trans when expressed in Arabidopsis, and (3) the macadamia CWM1-like protein retains the capacity to work in the less complex P. patens editing environment.

A Single-Target Mitochondrial RNA Editing Factor of Funaria hygrometrica Can Fully Reconstitute RNA Editing at Two Sites in Physcomitrella patens.

Nuclear-encoded pentatricopeptide repeat (PPR) proteins are key factors for site-specific RNA editing, converting cytidines into uridines in plant mitochondria and chloroplasts. All editing factors in the model moss Physcomitrella patens have a C-terminal DYW domain with similarity to cytidine deaminase. However, numerous editing factors in flowering plants lack such a terminal DYW domain, questioning its immediate role in the pyrimidine base conversion process. Here we further investigate the Physcomitrella DYW-type PPR protein PPR_78, responsible for mitochondrial editing sites cox1eU755SL and rps14eU137SL. Complementation assays with truncated proteins demonstrate that the DYW domain is essential for full PPR_78 editing functionality. The DYW domain can be replaced, however, with its counterpart from another editing factor, PPR_79. The PPR_78 ortholog of the related moss Funaria hygrometrica fully complements the Physcomitrella mutant for editing at both sites, although the editing site in rps14 is lacking in Funaria. Editing factor orthologs in different taxa may thus retain editing capacity for multiple sites despite the absence of editing requirement.

Chloroplast RNA editing going extreme: more than 3400 events of C-to-U editing in the chloroplast transcriptome of the lycophyte Selaginella uncinata.

RNA editing in chloroplasts and mitochondria of land plants differs significantly in abundance. For example, some 200-500 sites of cytidine-to-uridine RNA editing exist in flowering plant mitochondria as opposed to only 30-50 such C-to-U editing events in their chloroplasts. In contrast, we predicted significantly more chloroplast RNA editing for the protein-coding genes in the available complete plastome sequences of two species of the spike moss genus Selaginella (Lycopodiophyta). To evaluate these predictions we investigated the Selaginella uncinata chloroplast transcriptome. Our exhaustive cDNA studies identified the extraordinary number of 3415 RNA-editing events, exclusively of the C-to-U type, in the 74 mRNAs encoding intact reading frames in the S. uncinata chloroplast. We find the overwhelming majority (61%) of the 428 silent editing events leaving codon meanings unaltered directly neighboring other editing events, possibly suggesting a sterically more flexible RNA-editing deaminase activity in Selaginella. No evidence of RNA editing was found for tRNAs or rRNAs but we identified a total of 74 editing sites in cDNA sequences of four group II introns (petBi6g2, petDi8g2, ycf3i124g2, and ycf3i354g2) retained in partially matured transcripts, which strongly contribute to improved base-pairing in the intron secondary structures as a likely prerequisite for their splicing.

DYW domain structures imply an unusual regulation principle in plant organellar RNA editing catalysis.

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.

Plant-type pentatricopeptide repeat proteins with a DYW domain drive C-to-U RNA editing in Escherichia coli.

RNA editing converting cytidines into uridines is a hallmark of gene expression in land plant chloroplasts and mitochondria. Pentatricopeptide repeat (PPR) proteins have a key role in target recognition, but the functional editosome in the plant organelles has remained elusive. Here we show that individual Physcomitrella patens DYW-type PPR proteins alone can perform efficient C-to-U editing in Escherichia coli reproducing the moss mitochondrial editing. Single amino acid exchanges in the DYW domain abolish RNA editing, confirming it as the functional cytidine deaminase. The modification of RNA targets and the identification of numerous off-targets in the E. coli transcriptome reveal nucleotide identities critical for RNA recognition and cytidine conversion. The straightforward amenability of the new E. coli setup will accelerate future studies on RNA target recognition through PPRs, on the C-to-U editing deamination machinery and towards future establishment of transcript editing in other genetic systems.