Mitochondrial RNA editing PPR proteins can tolerate protein tags at E as well as at DYW domain termini

INTRODUCTION In plastids and mitochondria of plants, RNA editing changes numerous cytidines to uridines. The nucleotides to be edited are selected by trans-acting proteins which are structurally characterized as pentatricopeptide repeat (PPR) proteins (Kotera et al., 2005; Schmitz-Linneweber and Small, 2008; Takenaka et al., 2013b). The approximately 35 amino acids long elements each attach to a specific nucleotide moiety and several tandemly arranged elements establish selective contact to a unique RNA sequence pattern. The PPR parameters defining the nucleotide specificity were identified by computational analysis and confirmed by experimental retargeting and by crystal structures (Barkan et al., 2012; Ke et al., 2013; Takenaka et al., 2013a; Yagi et al., 2013; Yin et al., 2013). Half of the PPR family of about 450 proteins in flowering plants consists only of these repeats and few further domains if any. This group of PPR proteins is involved in RNA processing events other than RNA editing (SchmitzLinneweber and Small, 2008). The about 200 PPR proteins associated with RNA editing are C-terminally extended by the so-called E domains, the function of which is so far unknown. About half of these proteins contain an additional C-terminal domain with key features of deaminases in the form of characteristic amino acids which may bind an essential zinc atom (Hayes et al., 2013). However, so far no deaminase activity has been found, one of these domains rather shows an RNase activity (Nakamura and Sugita, 2008). This domain terminates in most instances with the name giving amino acid triplet DYW. In several such PPR RNA editing factors in plastids and mitochondria, the DYW domain can be deleted without compromising the function of the protein in editing (Okuda et al., 2009), while in others the DYW domain is required (Zehrmann et al., 2010). In some of the E class PPR RNA editing factors, addition of a DYW domain does not affect their competence in editing (Verbitskiy et al., 2012a). So far the functional parameters are unclear which distinguish those protein factors that require the C-terminal DYW extension beyond the E domain from those that do not. The E domain is in all of these RNA editing PPR proteins essential and cannot be removed. To learn more about the function and structural features of these extension domains, we probed the importance of native C-termini of several PPR proteins in mitochondria by adding an additional protein domain. These chimeric proteins were assayed for their in vivo RNA editing competence to complement respective Arabidopsis thaliana mutants.