A DYW ‐protein knockout in P hyscomitrella affects two closely spaced mitochondrial editing sites and causes a severe developmental phenotype

Summary RNA ‐binding pentatricopeptide repeat ( PPR ) proteins carrying a carboxy‐terminal DYW domain similar to cytidine deaminases have been characterized as site‐specific factors for C‐to‐U RNA editing in plant organelles. Here we report that knockout of DYW ‐ PPR _65 in P hyscomitrella patens causes a severe developmental phenotype in the moss and specifically affects two editing sites located 18 nucleotides apart on the mitochondrial ccmFC mRNA . Intriguingly, PPR _71, another DYW ‐type PPR , had been identified previously as an editing factor specifically affecting only the downstream editing site, ccmFCeU122SF. The now characterized PPR _65 binds specifically only to the upstream target site, ccmFCeU103PS, in full agreement with a recent RNA ‐recognition code for PPR arrays. The functional interference between the two editing events may be caused by a combination of three factors: (i) the destabilization of an RNA secondary structure interfering with PPR _71 binding by prior binding of PPR _65; (ii) the resulting upstream C–U conversion; or (iii) a direct interaction between the two DYW proteins. Indeed, we find the Physcomitrella DYW ‐ PPR s to interact in yeast‐two‐hybrid assays. The moss DYW ‐ PPR s also interact yet more strongly with MORF ( M ultiple O rganellar RNA editing F actor)/ RIP ( RNA editing factor interacting proteins) proteins of A rabidopsis known to be general editing factors in flowering plants, although MORF homologues are entirely absent in the moss. Finally, we demonstrate binding of P hyscomitrella DYW ‐ PPR _98, for which no KO lines could be raised, to its predicted target sequence upstream of editing site atp9eU92SL. Together with the functional characterization of DYW ‐ PPR _65, this completes the assignment of RNA editing factors to all editing sites in the P hyscomitrella mitochondrial transcriptome.