High conservation of translation-enabling RNA editing sites in hyper-editing ferns implies they are not selectively neutral.

RNA editing is essential for processing transcripts in plant chloroplasts and mitochondria. Levels of RNA editing vary between lineages but some hornworts, lycophytes and ferns have an extraordinary abundance of RNA editing. A feature of 'hyper-editing' species is the prevalence of RNA editing events that promote translation by creating start codons or removing stop codons via C-to-U or U-to-C editing respectively. These 'translation-enabling' RNA editing events may play roles in regulating organelle gene expression. To investigate the importance of translation-enabling RNA editing events, we generated DNA and RNA sequence libraries for four Salviniales water ferns: Azolla rubra, Azolla pinnata, Marsilea mutica and Salvinia molesta. We assembled chloroplast genomes, mitochondrial genes and transcriptomes, and identified RNA editing sites and candidate RNA editing factors. We re-analysed sequence data of Azolla filiculoides and conducted a comparative analysis of RNA editing in chloroplasts and mitochondrial transcripts. Estimates of pyrimidine transition rates show that translation-enabling RNA editing sites are more conserved than other nonsynonymous editing sites, suggesting an emergent role in organelle gene expression that is not selectively neutral. This makes these events rare examples of RNA editing conferring adaptive advantage, in contrast to the prevailing view that RNA editing arises via constructive neutral evolutionary processes. Shotgun proteomics of Marsilea mutica chloroplast thylakoid fractions verified the expected consequences of RNA editing on translation of chloroplast transcripts and implies that mechanisms exist to avoid translation of partially edited transcripts. Start codon editing may be one of those mechanisms.