The DYW Domains of Pentatricopeptide Repeat RNA Editing Factors Contribute to Discriminate Target and Non-Target Editing Sites

The DYW Domains of Pentatricopeptide Repeat RNA Editing Factors Contribute to Discriminate Target... Abstract In land plant organelles, many transcripts are modified by cytidine to uridine RNA editing. Target cytidines are specifically recognized by nuclear-encoded pentatricopeptide repeat (PPR) proteins via their sequence-specific RNA-binding motifs. In the moss Physcomitrella patens, all PPR editing factors have C-terminal E and DYW domains. To examine the contribution of E and DYW domains in RNA editing, we performed a complementation assay using mutated PpPPR_56 and PpPPR_71, which are responsible for mitochondrial editing sites. This assay showed that both E and DYW domains are required for RNA editing at the target sites, and that the conserved zinc-binding signature and the terminal triplet of the DYW domain are essential for editing. In addition, DYW domain-swapping experiments demonstrated that DYW domains are functionally different between PpPPR_56 and other mitochondrial PPR editing factors, and that residues 37–42 of the DYW domain are involved in site-specific editing. Our results suggest that PPR-DYW proteins specifically recognize their target editing sites via PPR motifs and the DYW domain. Introduction RNA editing, converting specific cytidines (C) to uridines (U), is a major post-transcriptional regulation to correct genetic information at the RNA level in mitochondria and chloroplasts of land plants (Takenaka et al. 2013b, Ichinose and Sugita 2017). The frequency of RNA editing in organelles varies greatly in vascular plants, as there are several hundred editing sites in flowering plants (Bentolila et al. 2013) and thousands of editing sites in lycophytes (Hecht et al. 2011, Oldenkott et al. 2014). On the other hand, RNA editing rarely occurs in the non-vascular moss Physcomitrella patens, as only two editing sites have been reported in chloroplasts (Miyata and Sugita 2004) and 11 sites in mitochondria (Rüdinger et al. 2009, Tasaki et al. 2010). Many pentatricopeptide repeat (PPR) proteins have been identified as site recognition factors for RNA editing at target sites in mitochondria and chloroplasts (Takenaka et al. 2013b, Barkan and Small 2014). In addition, recent studies revealed that non-PPR proteins, such as multiple organellar RNA editing factor/RNA editing factor-interacting proteins (MORF/RIPs), broadly affect RNA editing at multiple sites (Bentolila et al. 2012, Takenaka et al. 2012, Bentolila et al. 2013), although these are not likely to be site recognition factors for RNA editing. PPR proteins are characterized by tandem arrays of the degenerate 31–36 amino acid PPR motif that folds into a pair of antiparallel α-helices, which have been proposed to bind to specific RNA sequences (Barkan et al. 2012, Takenaka et al. 2013a, Yagi et al. 2013, Yin et al. 2013, Sun et al. 2016). PPR proteins are divided into P and PLS classes depending on their PPR motifs (Lurin et al. 2004, Cheng et al. 2016). Known PPR editing factors are members of the PLS class, which have characteristic C-terminal E or E-DYW domains (PPR-E or PPR-DYW). The PPR tract binds in a sequence-specific manner to approximately 20 nucleotides immediately upstream of target editing sites (Takenaka et al. 2013b, Barkan and Small 2014). While the function of the E domain (consisting of two PPR-like motifs) remains unclear, the DYW domain has been proposed to be a catalytic domain for C to U RNA editing, as DYW domains contain a highly conserved canonical zinc-binding motif, HxExnCxxC, and its putative secondary structure resembles cytidine deaminase (Salone et al. 2007, Iyer et al. 2011). A recent study demonstrated that the HxExnCxxC signature of Arabidopsis thaliana DYW1 is required for both zinc binding and RNA editing (Boussardon et al. 2014). However, RNA deamination activity of the DYW domain has not been demonstrated (Nakamura and Sugita 2008, Okuda et al. 2009), and the DYW domains of CRR22 and CRR28 editing factors were dispensable for RNA editing in vivo (Okuda et al. 2009). These observations further accentuate the current ambiguity of the role of the DYW domain. The moss P. patens has 10 PPR-DYW proteins, nine of which have been assigned as required for all 13 editing events (Ohtani et al. 2010, Tasaki et al. 2010, Rüdinger et al. 2011, Uchida et al. 2011, Ichinose et al. 2013, Schallenberg-Rüdinger et al. 2013, Ichinose et al. 2014). Thus, P. patens is the first organism with a complete set of PPR editing factors for all organellar editing sites. Among the nine PPR editing factors, PpPPR_56 is involved in RNA editing of two sites (nad3-C230 and nad4-C272) and PpPPR_71 is responsible for RNA editing of one site (ccmFC-C122) in mitochondrial transcripts (Ohtani et al. 2010, Tasaki et al. 2010). Although a PPR tract might be essential for editing site recognition, it is not known whether and how E and DYW domains recognize or specify target editing sites. To address this question, we performed an in vivo complementation assay using deleted or mutated PpPPR_56 and PpPPR_71. By using chimeric PPR-DYW protein versions in which E, DYW or both domains were replaced by other P. patens PPR editing factors, we demonstrated that DYW domains are essential for RNA editing and discriminate target and non-target editing sites. Results Both E and DYW domains of PpPPR_56 and 71 are required for RNA editing All P. patens PPR editing factors have E and DYW domains. To investigate whether both domains are required for RNA editing, we performed a complementation assay using previously generated knockout (KO) lines of PpPPR_56 and PpPPR_71 (Ohtani et al. 2010, Tasaki et al. 2010). PpPPR_56 KO (Δ56-22) showed impaired RNA editing of two sites, nad3-C230 and nad4-C272, and PpPPR_71 KO (Δ71 6-11) completely lost editing at the ccmFC-C122 site (Fig. 1). In this study, we adopted the definition of C-terminal E and DYW domains of Lurin et al. (2004). All transgenes in KO mosses were overexpressed under the control of the rice actin promoter. We analyzed at least two independent transgenic lines per construct, and the expression of transgenes was verified by reverse transcription–PCR (RT–PCR) (Supplementary Table S1). Fig. 1 View largeDownload slide Effects of deleting the E and/or DYW domains of PpPPR_56 and PpPPR_71 on RNA editing. (A) Schematic structure of truncated PpPPR_56. Gray boxes indicate transit peptide targeting mitochondria. (B) Direct sequencing was performed on the cDNA derived from wild-type, KO (Δ56-22 and Δ71 6-11) and complemented lines (FL, ΔE/DYW, ΔDYW and ΔE). Nucleotide sequences including the RNA editing sites of nad3-C230 and nad4-C272 for PpPPR_56 and ccmFC-C122 for PpPPR_71 are shown as sequencing chromatograms. Arrowheads indicate the editing sites. Fig. 1 View largeDownload slide Effects of deleting the E and/or DYW domains of PpPPR_56 and PpPPR_71 on RNA editing. (A) Schematic structure of truncated PpPPR_56. Gray boxes indicate transit peptide targeting mitochondria. (B) Direct sequencing was performed on the cDNA derived from wild-type, KO (Δ56-22 and Δ71 6-11) and complemented lines (FL, ΔE/DYW, ΔDYW and ΔE). Nucleotide sequences including the RNA editing sites of nad3-C230 and nad4-C272 for PpPPR_56 and ccmFC-C122 for PpPPR_71 are shown as sequencing chromatograms. Arrowheads indicate the editing sites. We transformed full-length (FL) or truncated versions (ΔE/DYW, ΔDYW and ΔE) of PpPPR_56 or PpPPR_71 into the respective KO moss backgrounds (Fig. 1A). RNA editing was fully rescued to the wild-type levels in the lines complemented with the FL version (56FL and 71FL), whereas C to U conversion was not detected at any of the target sites in transformants with E and/or DYW domain-truncated versions (Fig. 1B). These results indicated that both E and DYW domains of PpPPR_56 and PpPPR_71 are indispensable for RNA editing at their target sites. The E and DYW domains of PpPPR_56 and 71 are not mutually exchangeable for RNA editing To investigate further the role of E and DYW domains in RNA editing at specific sites, we designed a chimeric PpPPR_56 gene in which the E and/or DYW domains were replaced by those of PpPPR_71: 56PPR + 71E/71DYW, 56PPR + 71E/56DYW and 56PPR-E + 71DYW (Supplementary Fig. S1A). These chimeric constructs were transformed into the PpPPR_56 KO line (Δ56-22). Again, the complemented mosses transformed with the 56FL construct restored RNA editing at nad3-C230 and nad4-C272 sites, whereas transformants with the respective chimeric PpPPR_56 gene did not restore editing at these sites (Table 1; Supplementary Fig. S1B). This suggested that the E and DYW domains of PpPPR_56 and 71 are not compatible with each other for RNA editing, and their functions might differ between PpPPR_56 and PpPPR_71. Table 1 Effects of swapping the P. patens E and/or DYW domains on RNA editing Construct   Editing   PPR  E  DYW  Site  Efficiency  Site  Efficiency  PpPPR_56  71  71  nad3-C230  –  nad4-C272  –  (this work)  71  56  nad3-C230  –  nad4-C272  –    56  71  nad3-C230  –  nad4-C272  –  PpPPR_56  56  56  nad3-C230  95%  nad4-C272  100%  (this work)  56  45  nad3-C230  35%  nad4-C272  95%    56  65  nad3-C230  −  nad4-C272  −    56  77  nad3-C230  −  nad4-C272  −    56  78  nad3-C230  −  nad4-C272  −    56  79  nad3-C230  −  nad4-C272  −    56  91  nad3-C230  −  nad4-C272  −    56  98  nad3-C230  −  nad4-C272  −  PpPPR_71  71  71  ccmFc-C122  80%      (this work)  71  45  ccmFc-C122  −        71  56  ccmFc-C122  −        71  77  ccmFc-C122  −      PpPPR_78  78  79  cox1-C755  100%  rps14-C137  80%  (Schallenberg-Rüdinger et al. 2017)  Construct   Editing   PPR  E  DYW  Site  Efficiency  Site  Efficiency  PpPPR_56  71  71  nad3-C230  –  nad4-C272  –  (this work)  71  56  nad3-C230  –  nad4-C272  –    56  71  nad3-C230  –  nad4-C272  –  PpPPR_56  56  56  nad3-C230  95%  nad4-C272  100%  (this work)  56  45  nad3-C230  35%  nad4-C272  95%    56  65  nad3-C230  −  nad4-C272  −    56  77  nad3-C230  −  nad4-C272  −    56  78  nad3-C230  −  nad4-C272  −    56  79  nad3-C230  −  nad4-C272  −    56  91  nad3-C230  −  nad4-C272  −    56  98  nad3-C230  −  nad4-C272  −  PpPPR_71  71  71  ccmFc-C122  80%      (this work)  71  45  ccmFc-C122  −        71  56  ccmFc-C122  −        71  77  ccmFc-C122  −      PpPPR_78  78  79  cox1-C755  100%  rps14-C137  80%  (Schallenberg-Rüdinger et al. 2017)  Two editing mutants were transformed by various constructs obtained by swapping from the E and/or DYW domains of PpPPR_56 or PpPPR_71 to the cognate region of another P. patens editing factor. Restoration of editing was verified by direct sequencing (Supplementary Figs. S1–S3). Average editing efficiencies in independent transformants are shown (n ≤ 2) and non-restoration of editing is indicated by −. Table 1 Effects of swapping the P. patens E and/or DYW domains on RNA editing Construct   Editing   PPR  E  DYW  Site  Efficiency  Site  Efficiency  PpPPR_56  71  71  nad3-C230  –  nad4-C272  –  (this work)  71  56  nad3-C230  –  nad4-C272  –    56  71  nad3-C230  –  nad4-C272  –  PpPPR_56  56  56  nad3-C230  95%  nad4-C272  100%  (this work)  56  45  nad3-C230  35%  nad4-C272  95%    56  65  nad3-C230  −  nad4-C272  −    56  77  nad3-C230  −  nad4-C272  −    56  78  nad3-C230  −  nad4-C272  −    56  79  nad3-C230  −  nad4-C272  −    56  91  nad3-C230  −  nad4-C272  −    56  98  nad3-C230  −  nad4-C272  −  PpPPR_71  71  71  ccmFc-C122  80%      (this work)  71  45  ccmFc-C122  −        71  56  ccmFc-C122  −        71  77  ccmFc-C122  −      PpPPR_78  78  79  cox1-C755  100%  rps14-C137  80%  (Schallenberg-Rüdinger et al. 2017)  Construct   Editing   PPR  E  DYW  Site  Efficiency  Site  Efficiency  PpPPR_56  71  71  nad3-C230  –  nad4-C272  –  (this work)  71  56  nad3-C230  –  nad4-C272  –    56  71  nad3-C230  –  nad4-C272  –  PpPPR_56  56  56  nad3-C230  95%  nad4-C272  100%  (this work)  56  45  nad3-C230  35%  nad4-C272  95%    56  65  nad3-C230  −  nad4-C272  −    56  77  nad3-C230  −  nad4-C272  −    56  78  nad3-C230  −  nad4-C272  −    56  79  nad3-C230  −  nad4-C272  −    56  91  nad3-C230  −  nad4-C272  −    56  98  nad3-C230  −  nad4-C272  −  PpPPR_71  71  71  ccmFc-C122  80%      (this work)  71  45  ccmFc-C122  −        71  56  ccmFc-C122  −        71  77  ccmFc-C122  −      PpPPR_78  78  79  cox1-C755  100%  rps14-C137  80%  (Schallenberg-Rüdinger et al. 2017)  Two editing mutants were transformed by various constructs obtained by swapping from the E and/or DYW domains of PpPPR_56 or PpPPR_71 to the cognate region of another P. patens editing factor. Restoration of editing was verified by direct sequencing (Supplementary Figs. S1–S3). Average editing efficiencies in independent transformants are shown (n ≤ 2) and non-restoration of editing is indicated by −. The HxExnCxxC motif and C-terminal DYW triplet are essential for RNA editing The DYW domains of PpPPR_56 and PpPPR_71 contain the zinc-binding signature HxExnCxxC and the C-terminal aspartate (D)–tyrosine (Y)–tryptophan (W) triplet. To investigate their roles in RNA editing, we generated transgenic lines with mutated DYW domains in which the HxExnCxxC signature was changed to AxAxnCxxC (56M1 and 71M1) and HxExnAxxA (56M2 and 71M2), and the C-terminal DYW triplet was deleted in 56M3 and 71M3 lines (Fig. 2A). The RNA editing of the cognate sites was not restored in all three mutant lines (Fig. 2B). This indicated that the zinc-binding signature and the DYW triplet of PpPPR_56 and PpPPR_71 are essential for RNA editing. Fig. 2 View largeDownload slide RNA editing in complemented lines with mutated PPR-DYW variants. (A) PpPPR_56 variants with mutations in the zinc -binding motif (56M1 and 56M2) or with a deleted C-terminal DYW triplet (56M3). (B) Direct sequence chromatograms of RT–PCR products amplified from the cDNA of complemented lines with a transgene (M1, AxAxnCxxC; M2, HxExnAxxA; or M3, ΔDYW triplet) in PpPPR_56 or PpPPR_71 KO lines. Arrowheads indicate the editing sites. Fig. 2 View largeDownload slide RNA editing in complemented lines with mutated PPR-DYW variants. (A) PpPPR_56 variants with mutations in the zinc -binding motif (56M1 and 56M2) or with a deleted C-terminal DYW triplet (56M3). (B) Direct sequence chromatograms of RT–PCR products amplified from the cDNA of complemented lines with a transgene (M1, AxAxnCxxC; M2, HxExnAxxA; or M3, ΔDYW triplet) in PpPPR_56 or PpPPR_71 KO lines. Arrowheads indicate the editing sites. DYW domains discriminate target and non-target sites in RNA editing To investigate whether the DYW domain of other PPR-DYW editing factors is compatible with that of PpPPR_56, we further generated chimeric PpPPR_56 genes in which the DYW domain was replaced by that of other P. patens PPR-DYW editing factors (Supplementary Fig. S2). Fusion genes were inserted by three nucleotides (TCG coding for serine) between the E and DYW domains, due to technical reasons for the construction of chimeric genes. This insertion did not affect RNA editing as verified by using a self-fusion construct (56PPR-E + 56DYW; Supplementary Fig. S2). The PpPPR_56 fused to the DYW domain of PpPPR_45 (56PPR-E + 45DYW) showed complete editing of nad4-C272 and partial editing of nad3-C230 (Table 1; Supplementary Fig. S2). In contrast, none of the other chimeric PPRs complemented editing defects at both sites. To verify whether the DYW domain of PpPPR_45 was functionally interchangeable with any other DYW domains, fusion constructs of PpPPR_71 with the DYW domain swapped with that of PpPPR_45, 56 and 77 were introduced into the PpPPR_71 KO line. However, RNA editing at ccmFC-C122 was not restored in any of the transgenic lines (Table 1; Supplementary Fig. S3). The central portion of the DYW domain is involved in target site specificity As shown in the swapping experiments (Supplementary Figs. S1–S3), the DYW domain of each PPR editing factor can be considered to act toward its target editing sites only. To investigate this hypothesis, and which region of the DYW domain is involved in such editing site specificity, the DYW domain (95 amino acids) of PpPPR_56 was divided into three parts (dywA, residues 1–22; dywB, 23–61; and dywC, 62–95) and each part was swapped with the cognate region of the DYW domain in PpPPR_71 (Fig. 3A). These constructs were transformed into the PpPPR_56 KO moss. Six independent 56PPR-E-r71dywA lines exhibited approximately 70% and 90% RNA editing at nad3-C230 and nad4-C272, respectively (Fig. 3B, D). Similarly, RNA editing was partially restored to 35% at nad3-C230 and to 50% at nad4-C272 in the six independent 56PPR-E-r71dywB lines (Fig. 3B). In the 56PPR-E-r71dywC lines, RNA editing at the two sites was fully restored to the wild-type level (Fig. 3B). These results suggest that the dywB part of PpPPR_56 containing the putative zinc-binding signature includes the most critical part for specific editing at the two target sites. Fig. 3 View largeDownload slide Effects of partial swapping of DYW domains between PpPPR_56 and PpPPR_71 on RNA editing. (A) Alignment of PpPPR_56 and PpPPR_71 DYW domains. Regions with partial domain swapping are shown above. Identical amino acids are shaded in black, and the conserved zinc-binding signature is underlined in red. (B) RNA editing of nad3-C230 and nad4-C272 in various transgenic lines harboring chimeric PpPPR_56 constructs (56PPE-E-r71dywA, -dywB or -dywC) in a PpPPR_56 KO background. Arrowheads indicate the editing sites. The swapped parts of DYW domains are represented above. Blue and gray boxes indicate parts of the DYW domains of PpPPR_56 and PpPPR_71, respectively. Average editing efficiencies in independent transformants are shown below (n ≤ 3). (C) RNA editing in Δ56 complemented lines with dywB-1 and dywB-2, respectively (n = 3). (D) RT–PCR results for the PpPPR_56 transcript in the wild type (WT) and Δ56-22 and Δ56 complemented lines. The asterisk indicates an alternative splicing variant of PpPPR_56. PpActin1 was used as a control. Fig. 3 View largeDownload slide Effects of partial swapping of DYW domains between PpPPR_56 and PpPPR_71 on RNA editing. (A) Alignment of PpPPR_56 and PpPPR_71 DYW domains. Regions with partial domain swapping are shown above. Identical amino acids are shaded in black, and the conserved zinc-binding signature is underlined in red. (B) RNA editing of nad3-C230 and nad4-C272 in various transgenic lines harboring chimeric PpPPR_56 constructs (56PPE-E-r71dywA, -dywB or -dywC) in a PpPPR_56 KO background. Arrowheads indicate the editing sites. The swapped parts of DYW domains are represented above. Blue and gray boxes indicate parts of the DYW domains of PpPPR_56 and PpPPR_71, respectively. Average editing efficiencies in independent transformants are shown below (n ≤ 3). (C) RNA editing in Δ56 complemented lines with dywB-1 and dywB-2, respectively (n = 3). (D) RT–PCR results for the PpPPR_56 transcript in the wild type (WT) and Δ56-22 and Δ56 complemented lines. The asterisk indicates an alternative splicing variant of PpPPR_56. PpActin1 was used as a control. To determine which part of dywB is critical for the editing role of the PpPPR_56 editing factor, we generated two constructs in which region dywB-1 (residues 23–43) or dywB-2 (residues 44–61) of the DYW domain of PpPPR_56 was replaced by that of PpPPR_71 (Fig. 3A). The 56PPR-E-r71dywB-1 construct partially complemented nad3-C230 and nad4-C272 editing at 25% and 45%, respectively, whereas the 56PPR-E-r71dywB-2 construct perfectly complemented both editing sites (Fig. 3C, D). This suggested that the dywB-1 region is involved in the discrimination of target sites. The dywB-1 regions of PpPPR_56 and PpPPR_71 are mismatched at nine sites (Fig. 3A). To investigate which amino acid residues are involved in editing site discrimination, we introduced point mutations into the chimeric PpPPR_56 gene in which the DYW domain was replaced by that of PpPPR_71 (56PPR-E + 71DYW; Fig. 4A). First, one or two amino acids of the 71DYW domain were modified to those of PpPPR_56. The 56PPR-E + 71DYW-A23D construct (alanine to aspartic acid substitution at residue 23 of 71DYW) did not complement the editing defect in the PpPPR_56 KO line (Supplementary Fig. S4), and none of the transformants expressing 56PPR-E + 71DYW variants with single or double amino acid replacement showed detectable RNA editing of nad3-C230 and nad4-C272 (Supplementary Fig. S4). Next, 56PPR-E + 71DYW-m1 and -m2 constructs were introduced into the PpPPR_56 KO background (Fig. 4A). In 56PPR-E + 71DYW-m1 lines in which three residues (S25Y26A34) of 71DYW were modified to C25S26V34 of 56DYW (see Fig. 3A), there was no RNA editing of the cognate sites (Fig. 4B). On the other hand, in 56PPR-E + 71DYW-m2 lines in which five residues (V37L38S39L40S42) of 71DYW were modified to L37M38H39T40P42 of 56DYW, RNA editing was partially rescued to 15% at nad3-C230 and to 50% at nad4-C272 (Fig. 4B). Based on these complementation assays, it is suggested that the region from residues 37 to 42 in the DYW domain is involved in the discrimination between target and non-target editing sites. Fig. 4 View largeDownload slide The six amino acid region (residues 37–42) of the DYW domain involved in site-specific editing. (A) Schematic representation of 56PPR-E + 71DYW variants (m1 and m2). Blue boxes indicate the mutated region of PpPPR_56. Positions of amino acid substitutions correspond to those displayed in Fig. 3A. (B) RNA editing efficiency of nad3-C230 and nad4-C272 in Δ56 complemented lines with 56PPR-E + 71DYW-m1 and -m2, respectively. Average editing efficiencies in independent transformants are shown below. Fig. 4 View largeDownload slide The six amino acid region (residues 37–42) of the DYW domain involved in site-specific editing. (A) Schematic representation of 56PPR-E + 71DYW variants (m1 and m2). Blue boxes indicate the mutated region of PpPPR_56. Positions of amino acid substitutions correspond to those displayed in Fig. 3A. (B) RNA editing efficiency of nad3-C230 and nad4-C272 in Δ56 complemented lines with 56PPR-E + 71DYW-m1 and -m2, respectively. Average editing efficiencies in independent transformants are shown below. This region is well conserved across the DYW domains of P. patens editing factors, except for residues 38 and 39 (Fig. 5A). To check whether the above result could be generalized to other editing factors, we expressed the 56PPR-E + 78DYW I38M/S39H variant, in which two amino acids of the 78DYW domain were modified to those of PpPPR_56 in 56PPR-E + 78DYW, in the PpPPR_56 KO line (Fig. 5B). In this variant, RNA editing was partially restored as with the 56PPR-E + 71DYW-m2 variant (Fig. 5B), suggesting that six specific residues of DYW domains, especially those at positions 38 and 39, contribute to the discrimination of target sites. Fig. 5 View largeDownload slide The conserved six amino acid region of the DYW domain. (A) Alignment of the dywB-1 region in the DYW domains of P. patens editing factors. The six amino acid region (residues 37–42) is boxed. Conserved amino acids are shaded in black or gray. (B) RNA editing in the PpPPR_56 KO mosses complemented with 56PPR-E + 78DYW I38M/S39H. The cDNA chromatograms are shown. Black arrowheads indicate editing sites. RNA editing efficiency is shown as a percentage. Fig. 5 View largeDownload slide The conserved six amino acid region of the DYW domain. (A) Alignment of the dywB-1 region in the DYW domains of P. patens editing factors. The six amino acid region (residues 37–42) is boxed. Conserved amino acids are shaded in black or gray. (B) RNA editing in the PpPPR_56 KO mosses complemented with 56PPR-E + 78DYW I38M/S39H. The cDNA chromatograms are shown. Black arrowheads indicate editing sites. RNA editing efficiency is shown as a percentage. Discussion The present study revealed that target sites could be edited through the synergistic effect of site recognition by a PPR tract and site discrimination by specific amino acid residues in the central portion of the DYW domain. First, our complementation study demonstrated that the DYW domains of PpPPR_56 and PpPPR_71 are essential for editing at their target sites. Similarly, a recent study showed that the DYW domain of PpPPR_78 is required for efficient editing at sites rps14-C137 and cox1-C755 in P. patens (Schallenberg-Rüdinger et al. 2017). The DYW domain of PpPPR_56 could be replaced by that of the plastid editing factor PpPPR_45, but not by that of other moss PPR editing factors. However, the DYW domain of PpPPR_71 was not interchangeable with that of PpPPR_45, PpPPR_56 or PpPPR_77. These results indicate that the recognition of target editing sites is regulated not only by PPR motifs but also by the DYW domain. Thus, as shown in the experiment swapping the DYW of PpPPR_56 and 45, some DYW domains are functionally equivalent between mitochondria and chloroplasts in P. patens. The most interesting finding in the present study is that a six amino acid region (residues 37–42) of the DYW domain is involved in the discrimination of specific editing sites. This region is conserved between PpPPR_56 and PpPPR_45 (Fig. 5A). Similarly, a recent study showed that the DYW domain of PpPPR_78 could be functionally replaced by that of PpPPR_79 (Schallenberg-Rüdinger et al. 2017; Table 1), and that their six amino acid sequences are identical (Fig. 5A). Hence, how does the DYW domain discriminate its target editing sites? Previous studies showed that some DYW domains interact with its substrate RNAs (Tasaki et al. 2010, Okuda et al. 2014) and that the 5′-proximal region of the cis-element from –3 to 0 was bound by DYW domains (Okuda et al. 2014). In nad3-C230 and nad4-C272 sites, these regions are distinct from that of two plastid rps14 editing sites targeted by PpPPR_45, but similar to that of the ccmFC-C122 site targeted by PpPPR_71 (Supplementary Fig. S5). These observations suggest that the DYW domain can recognize editing sites without sequence preference. The region from residues 37 to 42 of the DYW domain might be involved in the interaction with its specific editing site or might contribute to structural integrity to access the catalytic site to editing sites, as this region is close to one of the zinc-binding sites HxE. However, the chimeric 56DYW domains, which replaced the dywB or dywB-1 parts in the 71 DYW domain, behaved similarly to 56PPR-E + 71DYW-m2, even without residues 37–42 of the 56DYW domain (Figs. 3B, C, 4B). Therefore, other regions might be sufficient for the specific recognition of PpPPR_56. A further study on the crystal structure of the DYW protein will help in understanding the mode of action of the DYW domain. We also showed that mutations in the zinc-binding signature of PpPPR_56 and PpPPR_71 resulted in the complete loss of their ability to edit cognate sites. Previous studies demonstrated that DYW domains bind to zinc ions (Hayes et al. 2013, Boussardon et al. 2014) and mutations in the zinc-binding signature of Arabidopsis DYW1 eliminate both zinc binding and RNA editing (Boussardon et al. 2014). Our results support the hypothesis that the DYW domain catalyzes the deaminase reaction. Additionally, the highly conserved C-terminal triplet in the DYW domains of PpPPR_56 and PpPPR_71 was shown to be required for editing, which is consistent with the function of DYW1 (Boussardon et al. 2014). The function of the DYW terminus remains unclear, although it might interact with protein(s) that have not been identified to date (Boussardon et al. 2014). In addition to the DYW domain, our swapping experiments showed that E domains are also essential for editing, and are not interchangeable between PpPPR_56 and PpPPR_71. Previous deletion studies demonstrated that the E domain is essential for RNA editing (Okuda et al. 2007, Okuda et al. 2009, Chateigner-Boutin et al. 2013). In contrast, the E domains of A. thaliana E-type OTP70 and moss DYW-type PpPPR_43, which were identified as organellar splicing factors, are not required for splicing (Chateigner-Boutin et al. 2011, Ichinose et al. 2012). Although the role of E domains is still unclear, recent studies revealed that those of CRR4, CRR21 and CLB19 PPR editing factors do not contribute to RNA binding (Okuda et al. 2014, Ramos-Vega et al. 2015), but some PPR proteins interact through their E domains with MORF/RIP proteins (Bayer-Császár et al. 2017). In A. thaliana, E-type OTP71 is involved in mitochondrial editing, and its E domain could be functionally replaced by that of the mitochondrial editing factor OTP72 but not by chloroplast editing factors CRR4 and CLB19 (Chateigner-Boutin et al. 2013). MORF8/RIP1 affects editing events at both ccmFN2-C176 and rpl16-C770 sites, which are targeted by OTP71 and OTP72, respectively (Bentolila et al. 2013). Indeed, the moss DYW-type PpPPR_65 was shown to have potential for protein–protein interaction with the A. thaliana MORF/RIP proteins by a yeast two-hybrid assay (Schallenberg-Rüdinger et al. 2013), supporting the idea that E domains recruit additional specific editing factors in P. patens. Materials and Methods Plant materials and culture conditions The moss P. patens was grown at 25ºC under continuous light, as previously described (Ichinose et al. 2012). BCDATG medium was used for regular culturing of protonemata. Procedures to obtain moss lines PpPPR_56 KO (Δ56-22) and PpPPR_71 KO (Δ71 6-11) have been previously described (Ohtani et al. 2010, Tasaki et al. 2010). Plasmids and moss transformation The PCR primers used in the present study are listed in Supplementary Table S2. For the in vivo complementation assay of PpPPR_56 and PpPPR_71 KO mosses, full-length PpPPR genes containing the native stop codon were amplified from genomic DNA. Resulting amplicons were then cloned into the SwaI site of the overexpression vector p9WmycH13 (Ichinose et al. 2012), and the resultant plasmids were named p56FL and p71FL, respectively. For constructing PpPPR proteins lacking their E/DYW domains, DYW domain or C-terminal DYW triplets, truncated genomic sequences of PpPPR proteins were amplified and cloned into p9WmycH13. Deletion of the E domain and the introduction of point mutations into the DYW domain were carried out by site-directed mutagenesis PCR using PrimeSTAR GXL polymerase (TaKaRa). For the chimeric PpPPR_56, in which the original DYW domain of PpPPR_56 was replaced by that of other PpPPR proteins, PpPPR_56 lacking a DYW domain was amplified by PCR using reverse primers containing the NruI site, and then cloned into p9WmycH13 to generate p56ΔDYW-NruI. The various DYW domains were obtained by PCR and cloned into the NruI site of p56ΔDYW-NruI. The swapping constructs of the E domain or partial DYW domain were generated using the In-Fusion cloning system (Clontech). The resulting plasmids were linearized by NotI and introduced into Δ56-22 or Δ71 6-11 KO lines by particle bombardment or polyethylene glycol-mediated DNA transformation (Nishiyama et al. 2000, Tasaki et al. 2010). Hygromycin-resistant mosses were selected and transformants were confirmed by PCR. The expression of transgenes was confirmed by RT–PCR. Analysis of RNA editing Total RNA was isolated from 4-day-old protonemata using ISOGEN II (NIPPON GENE), and treated with DNase I (TaKaRa). Reverse transcription was carried out using ReverTra Ace (TOYOBO) from 1 µg of DNA-free RNA primed with random hexamers. Amplified products, including the editing sites, were purified and sequenced directly as previously described (Ichinose et al. 2013). Editing efficiency was calculated by the ratio of T and C peak height. Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the Japan Society for the Promotion of Science (JSPS) [KAKENHI grant Nos. 2529105, 17K08195 (to M.S.) and 18K14435 (to M.I.), Research Fellow for Young Scientists (to M.I.)]; The Hori Science and Arts Foundation [to M.I.]; and the IGER program from Nagoya University [to M.I.]. Acknowledgments We thank Dr. Bernard Gutmann (University of Western Australia) for valuable comments and discussion on an early draft of the manuscript. Disclosures The authors have no conflicts of interest to declare. References Barkan A., Small I. ( 2014) Pentatricopeptide repeat proteins in plants. Annu. Rev. Plant Biol.  65: 415– 442. Google Scholar CrossRef Search ADS PubMed  Barkan A., Rojas M., Fujii S., Yap A., Chong Y.S., Bond C.S. ( 2012) Combinatorial amino acid code for RNA recognition by pentatricopeptide repeat proteins. PLoS Genet.  8: e1002910. Google Scholar CrossRef Search ADS PubMed  Bayer-Császár E., Haag S., Jörg A., Glass F., Härtel B., Obata T., et al.   ( 2017) The conserved domain in MORF proteins has distinct affinities to the PPR and E elements in PPR RNA editing factors. Biochim. Biophys. Acta  1860: 813– 828. Google Scholar CrossRef Search ADS PubMed  Bentolila S., Heller W.P., Sun T., Babina A.M., Friso G., Van Wijk K.J., et al.   ( 2012) RIP1, a member of an Arabidopsis protein family, interacts with the protein RARE1 and broadly affects RNA editing. Proc. Natl. Acad. Sci. USA  109: E1453– E1461. Google Scholar CrossRef Search ADS   Bentolila S., Oh J., Hanson M.R., Bukowski R. ( 2013) Comprehensive high-resolution analysis of the role of an Arabidopsis gene family in RNA editing. PLoS Genet.  9: e1003584. Google Scholar CrossRef Search ADS PubMed  Boussardon C., Avon A., Kindgren P., Bond C.S., Challenor M., Lurin C., et al.   ( 2014) The cytidine deaminase signature HxE(x)nCxxC of DYW1 binds zinc and is necessary for RNA editing of ndhD-1. New Phytol.  203: 1090– 1095. Google Scholar CrossRef Search ADS PubMed  Chateigner-Boutin A.L., Colas Des Francs-Small C., Fujii S., Okuda K., Tanz S.K., Small I. ( 2013) The E domains of pentatricopeptide repeat proteins from different organelles are not functionally equivalent for RNA editing. Plant J.  74: 935– 945. Google Scholar CrossRef Search ADS PubMed  Chateigner-Boutin A.L., Des Francs-Small C.C., Delannoy E.E., Kahlau S., Tanz S.K., de Longevialle A.F., et al.   ( 2011) OTP70 is a pentatricopeptide repeat protein of the E subgroup involved in splicing of the plastid transcript rpoC1. Plant J . 65: 532– 542. Google Scholar CrossRef Search ADS PubMed  Cheng S., Gutmann B., Zhong X., Ye Y., Fisher M.F., Bai F., et al.   ( 2016) Redefining the structural motifs that determine RNA binding and RNA editing by pentatricopeptide repeat proteins in land plants. Plant J . 85: 532– 547. Google Scholar CrossRef Search ADS PubMed  Hayes M.L., Giang K., Berhane B., Mulligan R.M. ( 2013) Identification of two pentatricopeptide repeat genes required for RNA editing and zinc binding by C-terminal cytidine deaminase-like domains. J. Biol. Chem.  288: 36519– 36529. Google Scholar CrossRef Search ADS PubMed  Hecht J., Grewe F., Knoop V. ( 2011) Extreme RNA editing in coding islands and abundant microsatellites in repeat sequences of Selaginella moellendorffii mitochondria: the root of frequent plant mtDNA recombination in early tracheophytes. Genome Biol. Evol . 3: 344– 358. Google Scholar CrossRef Search ADS PubMed  Ichinose M., Sugita M. ( 2017) RNA editing and its molecular mechanism in plant organelles. Genes (Basel)  8: 5. Google Scholar CrossRef Search ADS   Ichinose M., Sugita C., Yagi Y., Nakamura T., Sugita M. ( 2013) Two DYW subclass PPR proteins are involved in RNA editing of ccmFc and atp9 transcripts in the moss Physcomitrella patens: first complete set of PPR editing factors in plant mitochondria. Plant Cell Physiol . 54: 1907– 1916. Google Scholar CrossRef Search ADS PubMed  Ichinose M., Tasaki E., Sugita C., Sugita M. ( 2012) A PPR-DYW protein is required for splicing of a group II intron of cox1 pre-mRNA in Physcomitrella patens. Plant J . 70: 271– 278. Google Scholar CrossRef Search ADS PubMed  Ichinose M., Uchida M., Sugita M. ( 2014) Identification of a pentatricopeptide repeat RNA editing factor in Physcomitrella patens chloroplasts. FEBS Lett . 588: 4060– 4064. Google Scholar CrossRef Search ADS PubMed  Iyer L.M., Zhang D., Rogozin I.B., Aravind L. ( 2011) Evolution of the deaminase fold and multiple origins of eukaryotic and mutagenic nucleic acid deaminase from bacterial toxin systems. Nucleic Acids Res . 39: 9473– 9497. Google Scholar CrossRef Search ADS PubMed  Lurin C., Andrés C., Aubourg S., Bellaoui M., Bitton F., Bruyère C., et al.   ( 2004) Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell  16: 2089– 2103. Google Scholar CrossRef Search ADS PubMed  Miyata Y., Sugita M. ( 2004) Tissue- and stage-specific RNA editing of rps14 transcripts in moss (Physcomitrella patens) chloroplasts. J. Plant Physiol . 161: 113– 115. Google Scholar CrossRef Search ADS PubMed  Nakamura T., Sugita M. ( 2008) A conserved DYW domain of pentatricopeptide repeat protein possesses a novel endoribonuclease activity. FEBS Lett . 582: 4163– 4168. Google Scholar CrossRef Search ADS PubMed  Nishiyama T., Hiwatashi Y., Sakakibara I., Kato M., Hasebe M. ( 2000) Tagged mutagenesis and gene-trap in the moss, Physcomitrella patens by shuttle mutagenesis. DNA Res.  7: 9– 17. Google Scholar CrossRef Search ADS PubMed  Ohtani S., Ichinose M., Tasaki E., Aoki Y., Komura Y., Sugita M. ( 2010) Targeted gene disruption identifies three PPR-DYW proteins involved in RNA editing for five editing sites of the moss mitochondrial transcripts. Plant Cell Physiol . 51: 1942– 1949. Google Scholar CrossRef Search ADS PubMed  Okuda K., Chateigner-Boutin A.L., Nakamura T., Delannoy E., Sugita M., Myouga F., et al.   ( 2009) Pentatricopeptide repeat proteins with the DYW motif have distinct molecular functions in RNA editing and RNA cleavage in Arabidopsis chloroplasts. Plant Cell  21: 146– 156. Google Scholar CrossRef Search ADS PubMed  Okuda K., Myouga F., Motohashi R., Shinozaki K., Shikanai T. ( 2007) Conserved domain structure of pentatricopeptide repeat proteins involved in chloroplast RNA editing. Proc. Natl. Acad. Sci. USA  104: 8178– 8183. Google Scholar CrossRef Search ADS   Okuda K., Shoki H., Arai M., Shikanai T., Small I., Nakamura T. ( 2014) Quantitative analysis of motifs contributing to the interaction between PLS-subfamily members and their target RNA sequences in plastid RNA editing. Plant J.  80: 870– 882. Google Scholar CrossRef Search ADS PubMed  Oldenkott B., Yamaguchi K., Tsuji-Tsukinoki S., Knie N., Knoop V. ( 2014) Chloroplast RNA editing going extreme: more than 3400 events of C-to-U editing in the chloroplast transcriptome of the lycophyte Selaginella uncinata. RNA  20: 1499– 1506. Google Scholar CrossRef Search ADS PubMed  Ramos-Vega M., Guevara-García A., Llamas E., Sánchez-León N., Olmedo-Monfil V., Cielle-Calzada J.P., et al.   ( 2015) Functional analysis of the Arabidopsis thaliana CHLOROPLAST BIOGENESIS 19 pentatricopeptide repeat editing protein. New Phytol . 208: 430– 441. Google Scholar CrossRef Search ADS PubMed  Rüdinger M., Funk H.T., Rensing S.A., Maier U.G., Knoop V. ( 2009) RNA editing: only eleven sites are present in the Physcomitrella patens mitochondria transcriptome and a universal nomenclature proposal. Mol. Genet. Genomics  281: 473– 481. Google Scholar CrossRef Search ADS PubMed  Rüdinger M., Szövényi P., Rensing S.A., Knoop V. ( 2011) Assigning DYW-type PPR proteins to RNA editing sites in the funariid mosses Physcomitrella patens and Funaria hygrometrica. Plant J . 67: 370– 380. Google Scholar CrossRef Search ADS PubMed  Salone V., Rüdinger M., Polsakiewicz M., Hoffmann B., Groth-Malonek M., Szurek B., et al.   ( 2007) A hypothesis on the identification of the editing enzyme in plant organelles. FEBS Lett . 581: 4132– 4138. Google Scholar CrossRef Search ADS PubMed  Schallenberg-Rüdinger M., Kindgren P., Zehrmann A., Small I., Knoop V. ( 2013) A DYW-protein knockout in Physcomitrella affects two closely spaced mitochondrial editing sites and causes a severe developmental phenotype. Plant J.  76: 420– 432. Google Scholar CrossRef Search ADS PubMed  Schallenberg-Rüdinger M., Oldenkott B., Hiss M., Trinh P.L., Knoop V., Rensing S.A. ( 2017) A single-target mitochondrial RNA editing factor of Funaria hygrometrica can fully reconstitute RNA editing at two sites in Physcomitrella patens. Plant Cell Physiol . 58: 496– 507. Google Scholar CrossRef Search ADS PubMed  Sun T., Bentolila S., Hanson M.R. ( 2016) The unexpected diversity of plant organelle RNA editosomes. Trends Plant Sci.  21: 962– 973. Google Scholar CrossRef Search ADS PubMed  Takenaka M., Zehrmann A., Brennicke A., Graichen K. ( 2013a) Improved computational target site prediction for pentatricopeptide repeat RNA editing factors. PLoS One  8: e65343. Google Scholar CrossRef Search ADS   Takenaka M., Zehrmann A., Verbitskiy D., Härtel B., Brennicke A. ( 2013b) RNA editing in plants and its evolution. Annu. Rev. Genet.  47: 335– 352. Google Scholar CrossRef Search ADS   Takenaka M., Zehrmann A., Verbitskiy D., Kugelmann M., Härtel B., Brennicke A. ( 2012) Multiple organellar RNA editing factor (MORF) family proteins are required for RNA editing in mitochondria and plastids of plants. Proc. Natl. Acad. Sci. USA  109: 5104– 5109. Google Scholar CrossRef Search ADS   Tasaki E., Hattori M., Sugita M. ( 2010) The moss pentatricopeptide repeat protein with a DYW domain is responsible for RNA editing of mitochondrial ccmFc transcript. Plant J . 62: 560– 570. Google Scholar CrossRef Search ADS PubMed  Uchida M., Ohtani S., Ichinose M., Sugita C., Sugita M. ( 2011) The PPR-DYW proteins are required for RNA editing of rps14, cox1 and nad5 transcripts in Physcomitrella patens mitochondria. FEBS Lett . 585: 2367– 2371. Google Scholar CrossRef Search ADS PubMed  Yagi Y., Hayashi S., Kobayashi K., Hirayama T., Nakamura T. ( 2013) Elucidation of the RNA recognition code for pentatricopeptide repeat proteins involved in organelle RNA editing in plants. PLoS One  8: e57286. Google Scholar CrossRef Search ADS PubMed  Yin P., Li Q., Yan C., Liu Y., Liu J., Yu F., et al.   ( 2013) Structural basis for the modular recognition of single-stranded RNA by PPR proteins. Nature  504: 168– 171. Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations FL full length KO knockout MORF multiple organellar RNA editing factor PPR pentatricopeptide repeat PPR-DYW DYW-subclass PPR RIP RNA editing factor-interacting protein RT–PCR reverse transcription–PCR © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

The DYW Domains of Pentatricopeptide Repeat RNA Editing Factors Contribute to Discriminate Target and Non-Target Editing Sites

Loading next page...
 
/lp/ou_press/the-dyw-domains-of-pentatricopeptide-repeat-rna-editing-factors-UjBAvS7c1v
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com
ISSN
0032-0781
eISSN
1471-9053
D.O.I.
10.1093/pcp/pcy086
Publisher site
See Article on Publisher Site

Abstract

Abstract In land plant organelles, many transcripts are modified by cytidine to uridine RNA editing. Target cytidines are specifically recognized by nuclear-encoded pentatricopeptide repeat (PPR) proteins via their sequence-specific RNA-binding motifs. In the moss Physcomitrella patens, all PPR editing factors have C-terminal E and DYW domains. To examine the contribution of E and DYW domains in RNA editing, we performed a complementation assay using mutated PpPPR_56 and PpPPR_71, which are responsible for mitochondrial editing sites. This assay showed that both E and DYW domains are required for RNA editing at the target sites, and that the conserved zinc-binding signature and the terminal triplet of the DYW domain are essential for editing. In addition, DYW domain-swapping experiments demonstrated that DYW domains are functionally different between PpPPR_56 and other mitochondrial PPR editing factors, and that residues 37–42 of the DYW domain are involved in site-specific editing. Our results suggest that PPR-DYW proteins specifically recognize their target editing sites via PPR motifs and the DYW domain. Introduction RNA editing, converting specific cytidines (C) to uridines (U), is a major post-transcriptional regulation to correct genetic information at the RNA level in mitochondria and chloroplasts of land plants (Takenaka et al. 2013b, Ichinose and Sugita 2017). The frequency of RNA editing in organelles varies greatly in vascular plants, as there are several hundred editing sites in flowering plants (Bentolila et al. 2013) and thousands of editing sites in lycophytes (Hecht et al. 2011, Oldenkott et al. 2014). On the other hand, RNA editing rarely occurs in the non-vascular moss Physcomitrella patens, as only two editing sites have been reported in chloroplasts (Miyata and Sugita 2004) and 11 sites in mitochondria (Rüdinger et al. 2009, Tasaki et al. 2010). Many pentatricopeptide repeat (PPR) proteins have been identified as site recognition factors for RNA editing at target sites in mitochondria and chloroplasts (Takenaka et al. 2013b, Barkan and Small 2014). In addition, recent studies revealed that non-PPR proteins, such as multiple organellar RNA editing factor/RNA editing factor-interacting proteins (MORF/RIPs), broadly affect RNA editing at multiple sites (Bentolila et al. 2012, Takenaka et al. 2012, Bentolila et al. 2013), although these are not likely to be site recognition factors for RNA editing. PPR proteins are characterized by tandem arrays of the degenerate 31–36 amino acid PPR motif that folds into a pair of antiparallel α-helices, which have been proposed to bind to specific RNA sequences (Barkan et al. 2012, Takenaka et al. 2013a, Yagi et al. 2013, Yin et al. 2013, Sun et al. 2016). PPR proteins are divided into P and PLS classes depending on their PPR motifs (Lurin et al. 2004, Cheng et al. 2016). Known PPR editing factors are members of the PLS class, which have characteristic C-terminal E or E-DYW domains (PPR-E or PPR-DYW). The PPR tract binds in a sequence-specific manner to approximately 20 nucleotides immediately upstream of target editing sites (Takenaka et al. 2013b, Barkan and Small 2014). While the function of the E domain (consisting of two PPR-like motifs) remains unclear, the DYW domain has been proposed to be a catalytic domain for C to U RNA editing, as DYW domains contain a highly conserved canonical zinc-binding motif, HxExnCxxC, and its putative secondary structure resembles cytidine deaminase (Salone et al. 2007, Iyer et al. 2011). A recent study demonstrated that the HxExnCxxC signature of Arabidopsis thaliana DYW1 is required for both zinc binding and RNA editing (Boussardon et al. 2014). However, RNA deamination activity of the DYW domain has not been demonstrated (Nakamura and Sugita 2008, Okuda et al. 2009), and the DYW domains of CRR22 and CRR28 editing factors were dispensable for RNA editing in vivo (Okuda et al. 2009). These observations further accentuate the current ambiguity of the role of the DYW domain. The moss P. patens has 10 PPR-DYW proteins, nine of which have been assigned as required for all 13 editing events (Ohtani et al. 2010, Tasaki et al. 2010, Rüdinger et al. 2011, Uchida et al. 2011, Ichinose et al. 2013, Schallenberg-Rüdinger et al. 2013, Ichinose et al. 2014). Thus, P. patens is the first organism with a complete set of PPR editing factors for all organellar editing sites. Among the nine PPR editing factors, PpPPR_56 is involved in RNA editing of two sites (nad3-C230 and nad4-C272) and PpPPR_71 is responsible for RNA editing of one site (ccmFC-C122) in mitochondrial transcripts (Ohtani et al. 2010, Tasaki et al. 2010). Although a PPR tract might be essential for editing site recognition, it is not known whether and how E and DYW domains recognize or specify target editing sites. To address this question, we performed an in vivo complementation assay using deleted or mutated PpPPR_56 and PpPPR_71. By using chimeric PPR-DYW protein versions in which E, DYW or both domains were replaced by other P. patens PPR editing factors, we demonstrated that DYW domains are essential for RNA editing and discriminate target and non-target editing sites. Results Both E and DYW domains of PpPPR_56 and 71 are required for RNA editing All P. patens PPR editing factors have E and DYW domains. To investigate whether both domains are required for RNA editing, we performed a complementation assay using previously generated knockout (KO) lines of PpPPR_56 and PpPPR_71 (Ohtani et al. 2010, Tasaki et al. 2010). PpPPR_56 KO (Δ56-22) showed impaired RNA editing of two sites, nad3-C230 and nad4-C272, and PpPPR_71 KO (Δ71 6-11) completely lost editing at the ccmFC-C122 site (Fig. 1). In this study, we adopted the definition of C-terminal E and DYW domains of Lurin et al. (2004). All transgenes in KO mosses were overexpressed under the control of the rice actin promoter. We analyzed at least two independent transgenic lines per construct, and the expression of transgenes was verified by reverse transcription–PCR (RT–PCR) (Supplementary Table S1). Fig. 1 View largeDownload slide Effects of deleting the E and/or DYW domains of PpPPR_56 and PpPPR_71 on RNA editing. (A) Schematic structure of truncated PpPPR_56. Gray boxes indicate transit peptide targeting mitochondria. (B) Direct sequencing was performed on the cDNA derived from wild-type, KO (Δ56-22 and Δ71 6-11) and complemented lines (FL, ΔE/DYW, ΔDYW and ΔE). Nucleotide sequences including the RNA editing sites of nad3-C230 and nad4-C272 for PpPPR_56 and ccmFC-C122 for PpPPR_71 are shown as sequencing chromatograms. Arrowheads indicate the editing sites. Fig. 1 View largeDownload slide Effects of deleting the E and/or DYW domains of PpPPR_56 and PpPPR_71 on RNA editing. (A) Schematic structure of truncated PpPPR_56. Gray boxes indicate transit peptide targeting mitochondria. (B) Direct sequencing was performed on the cDNA derived from wild-type, KO (Δ56-22 and Δ71 6-11) and complemented lines (FL, ΔE/DYW, ΔDYW and ΔE). Nucleotide sequences including the RNA editing sites of nad3-C230 and nad4-C272 for PpPPR_56 and ccmFC-C122 for PpPPR_71 are shown as sequencing chromatograms. Arrowheads indicate the editing sites. We transformed full-length (FL) or truncated versions (ΔE/DYW, ΔDYW and ΔE) of PpPPR_56 or PpPPR_71 into the respective KO moss backgrounds (Fig. 1A). RNA editing was fully rescued to the wild-type levels in the lines complemented with the FL version (56FL and 71FL), whereas C to U conversion was not detected at any of the target sites in transformants with E and/or DYW domain-truncated versions (Fig. 1B). These results indicated that both E and DYW domains of PpPPR_56 and PpPPR_71 are indispensable for RNA editing at their target sites. The E and DYW domains of PpPPR_56 and 71 are not mutually exchangeable for RNA editing To investigate further the role of E and DYW domains in RNA editing at specific sites, we designed a chimeric PpPPR_56 gene in which the E and/or DYW domains were replaced by those of PpPPR_71: 56PPR + 71E/71DYW, 56PPR + 71E/56DYW and 56PPR-E + 71DYW (Supplementary Fig. S1A). These chimeric constructs were transformed into the PpPPR_56 KO line (Δ56-22). Again, the complemented mosses transformed with the 56FL construct restored RNA editing at nad3-C230 and nad4-C272 sites, whereas transformants with the respective chimeric PpPPR_56 gene did not restore editing at these sites (Table 1; Supplementary Fig. S1B). This suggested that the E and DYW domains of PpPPR_56 and 71 are not compatible with each other for RNA editing, and their functions might differ between PpPPR_56 and PpPPR_71. Table 1 Effects of swapping the P. patens E and/or DYW domains on RNA editing Construct   Editing   PPR  E  DYW  Site  Efficiency  Site  Efficiency  PpPPR_56  71  71  nad3-C230  –  nad4-C272  –  (this work)  71  56  nad3-C230  –  nad4-C272  –    56  71  nad3-C230  –  nad4-C272  –  PpPPR_56  56  56  nad3-C230  95%  nad4-C272  100%  (this work)  56  45  nad3-C230  35%  nad4-C272  95%    56  65  nad3-C230  −  nad4-C272  −    56  77  nad3-C230  −  nad4-C272  −    56  78  nad3-C230  −  nad4-C272  −    56  79  nad3-C230  −  nad4-C272  −    56  91  nad3-C230  −  nad4-C272  −    56  98  nad3-C230  −  nad4-C272  −  PpPPR_71  71  71  ccmFc-C122  80%      (this work)  71  45  ccmFc-C122  −        71  56  ccmFc-C122  −        71  77  ccmFc-C122  −      PpPPR_78  78  79  cox1-C755  100%  rps14-C137  80%  (Schallenberg-Rüdinger et al. 2017)  Construct   Editing   PPR  E  DYW  Site  Efficiency  Site  Efficiency  PpPPR_56  71  71  nad3-C230  –  nad4-C272  –  (this work)  71  56  nad3-C230  –  nad4-C272  –    56  71  nad3-C230  –  nad4-C272  –  PpPPR_56  56  56  nad3-C230  95%  nad4-C272  100%  (this work)  56  45  nad3-C230  35%  nad4-C272  95%    56  65  nad3-C230  −  nad4-C272  −    56  77  nad3-C230  −  nad4-C272  −    56  78  nad3-C230  −  nad4-C272  −    56  79  nad3-C230  −  nad4-C272  −    56  91  nad3-C230  −  nad4-C272  −    56  98  nad3-C230  −  nad4-C272  −  PpPPR_71  71  71  ccmFc-C122  80%      (this work)  71  45  ccmFc-C122  −        71  56  ccmFc-C122  −        71  77  ccmFc-C122  −      PpPPR_78  78  79  cox1-C755  100%  rps14-C137  80%  (Schallenberg-Rüdinger et al. 2017)  Two editing mutants were transformed by various constructs obtained by swapping from the E and/or DYW domains of PpPPR_56 or PpPPR_71 to the cognate region of another P. patens editing factor. Restoration of editing was verified by direct sequencing (Supplementary Figs. S1–S3). Average editing efficiencies in independent transformants are shown (n ≤ 2) and non-restoration of editing is indicated by −. Table 1 Effects of swapping the P. patens E and/or DYW domains on RNA editing Construct   Editing   PPR  E  DYW  Site  Efficiency  Site  Efficiency  PpPPR_56  71  71  nad3-C230  –  nad4-C272  –  (this work)  71  56  nad3-C230  –  nad4-C272  –    56  71  nad3-C230  –  nad4-C272  –  PpPPR_56  56  56  nad3-C230  95%  nad4-C272  100%  (this work)  56  45  nad3-C230  35%  nad4-C272  95%    56  65  nad3-C230  −  nad4-C272  −    56  77  nad3-C230  −  nad4-C272  −    56  78  nad3-C230  −  nad4-C272  −    56  79  nad3-C230  −  nad4-C272  −    56  91  nad3-C230  −  nad4-C272  −    56  98  nad3-C230  −  nad4-C272  −  PpPPR_71  71  71  ccmFc-C122  80%      (this work)  71  45  ccmFc-C122  −        71  56  ccmFc-C122  −        71  77  ccmFc-C122  −      PpPPR_78  78  79  cox1-C755  100%  rps14-C137  80%  (Schallenberg-Rüdinger et al. 2017)  Construct   Editing   PPR  E  DYW  Site  Efficiency  Site  Efficiency  PpPPR_56  71  71  nad3-C230  –  nad4-C272  –  (this work)  71  56  nad3-C230  –  nad4-C272  –    56  71  nad3-C230  –  nad4-C272  –  PpPPR_56  56  56  nad3-C230  95%  nad4-C272  100%  (this work)  56  45  nad3-C230  35%  nad4-C272  95%    56  65  nad3-C230  −  nad4-C272  −    56  77  nad3-C230  −  nad4-C272  −    56  78  nad3-C230  −  nad4-C272  −    56  79  nad3-C230  −  nad4-C272  −    56  91  nad3-C230  −  nad4-C272  −    56  98  nad3-C230  −  nad4-C272  −  PpPPR_71  71  71  ccmFc-C122  80%      (this work)  71  45  ccmFc-C122  −        71  56  ccmFc-C122  −        71  77  ccmFc-C122  −      PpPPR_78  78  79  cox1-C755  100%  rps14-C137  80%  (Schallenberg-Rüdinger et al. 2017)  Two editing mutants were transformed by various constructs obtained by swapping from the E and/or DYW domains of PpPPR_56 or PpPPR_71 to the cognate region of another P. patens editing factor. Restoration of editing was verified by direct sequencing (Supplementary Figs. S1–S3). Average editing efficiencies in independent transformants are shown (n ≤ 2) and non-restoration of editing is indicated by −. The HxExnCxxC motif and C-terminal DYW triplet are essential for RNA editing The DYW domains of PpPPR_56 and PpPPR_71 contain the zinc-binding signature HxExnCxxC and the C-terminal aspartate (D)–tyrosine (Y)–tryptophan (W) triplet. To investigate their roles in RNA editing, we generated transgenic lines with mutated DYW domains in which the HxExnCxxC signature was changed to AxAxnCxxC (56M1 and 71M1) and HxExnAxxA (56M2 and 71M2), and the C-terminal DYW triplet was deleted in 56M3 and 71M3 lines (Fig. 2A). The RNA editing of the cognate sites was not restored in all three mutant lines (Fig. 2B). This indicated that the zinc-binding signature and the DYW triplet of PpPPR_56 and PpPPR_71 are essential for RNA editing. Fig. 2 View largeDownload slide RNA editing in complemented lines with mutated PPR-DYW variants. (A) PpPPR_56 variants with mutations in the zinc -binding motif (56M1 and 56M2) or with a deleted C-terminal DYW triplet (56M3). (B) Direct sequence chromatograms of RT–PCR products amplified from the cDNA of complemented lines with a transgene (M1, AxAxnCxxC; M2, HxExnAxxA; or M3, ΔDYW triplet) in PpPPR_56 or PpPPR_71 KO lines. Arrowheads indicate the editing sites. Fig. 2 View largeDownload slide RNA editing in complemented lines with mutated PPR-DYW variants. (A) PpPPR_56 variants with mutations in the zinc -binding motif (56M1 and 56M2) or with a deleted C-terminal DYW triplet (56M3). (B) Direct sequence chromatograms of RT–PCR products amplified from the cDNA of complemented lines with a transgene (M1, AxAxnCxxC; M2, HxExnAxxA; or M3, ΔDYW triplet) in PpPPR_56 or PpPPR_71 KO lines. Arrowheads indicate the editing sites. DYW domains discriminate target and non-target sites in RNA editing To investigate whether the DYW domain of other PPR-DYW editing factors is compatible with that of PpPPR_56, we further generated chimeric PpPPR_56 genes in which the DYW domain was replaced by that of other P. patens PPR-DYW editing factors (Supplementary Fig. S2). Fusion genes were inserted by three nucleotides (TCG coding for serine) between the E and DYW domains, due to technical reasons for the construction of chimeric genes. This insertion did not affect RNA editing as verified by using a self-fusion construct (56PPR-E + 56DYW; Supplementary Fig. S2). The PpPPR_56 fused to the DYW domain of PpPPR_45 (56PPR-E + 45DYW) showed complete editing of nad4-C272 and partial editing of nad3-C230 (Table 1; Supplementary Fig. S2). In contrast, none of the other chimeric PPRs complemented editing defects at both sites. To verify whether the DYW domain of PpPPR_45 was functionally interchangeable with any other DYW domains, fusion constructs of PpPPR_71 with the DYW domain swapped with that of PpPPR_45, 56 and 77 were introduced into the PpPPR_71 KO line. However, RNA editing at ccmFC-C122 was not restored in any of the transgenic lines (Table 1; Supplementary Fig. S3). The central portion of the DYW domain is involved in target site specificity As shown in the swapping experiments (Supplementary Figs. S1–S3), the DYW domain of each PPR editing factor can be considered to act toward its target editing sites only. To investigate this hypothesis, and which region of the DYW domain is involved in such editing site specificity, the DYW domain (95 amino acids) of PpPPR_56 was divided into three parts (dywA, residues 1–22; dywB, 23–61; and dywC, 62–95) and each part was swapped with the cognate region of the DYW domain in PpPPR_71 (Fig. 3A). These constructs were transformed into the PpPPR_56 KO moss. Six independent 56PPR-E-r71dywA lines exhibited approximately 70% and 90% RNA editing at nad3-C230 and nad4-C272, respectively (Fig. 3B, D). Similarly, RNA editing was partially restored to 35% at nad3-C230 and to 50% at nad4-C272 in the six independent 56PPR-E-r71dywB lines (Fig. 3B). In the 56PPR-E-r71dywC lines, RNA editing at the two sites was fully restored to the wild-type level (Fig. 3B). These results suggest that the dywB part of PpPPR_56 containing the putative zinc-binding signature includes the most critical part for specific editing at the two target sites. Fig. 3 View largeDownload slide Effects of partial swapping of DYW domains between PpPPR_56 and PpPPR_71 on RNA editing. (A) Alignment of PpPPR_56 and PpPPR_71 DYW domains. Regions with partial domain swapping are shown above. Identical amino acids are shaded in black, and the conserved zinc-binding signature is underlined in red. (B) RNA editing of nad3-C230 and nad4-C272 in various transgenic lines harboring chimeric PpPPR_56 constructs (56PPE-E-r71dywA, -dywB or -dywC) in a PpPPR_56 KO background. Arrowheads indicate the editing sites. The swapped parts of DYW domains are represented above. Blue and gray boxes indicate parts of the DYW domains of PpPPR_56 and PpPPR_71, respectively. Average editing efficiencies in independent transformants are shown below (n ≤ 3). (C) RNA editing in Δ56 complemented lines with dywB-1 and dywB-2, respectively (n = 3). (D) RT–PCR results for the PpPPR_56 transcript in the wild type (WT) and Δ56-22 and Δ56 complemented lines. The asterisk indicates an alternative splicing variant of PpPPR_56. PpActin1 was used as a control. Fig. 3 View largeDownload slide Effects of partial swapping of DYW domains between PpPPR_56 and PpPPR_71 on RNA editing. (A) Alignment of PpPPR_56 and PpPPR_71 DYW domains. Regions with partial domain swapping are shown above. Identical amino acids are shaded in black, and the conserved zinc-binding signature is underlined in red. (B) RNA editing of nad3-C230 and nad4-C272 in various transgenic lines harboring chimeric PpPPR_56 constructs (56PPE-E-r71dywA, -dywB or -dywC) in a PpPPR_56 KO background. Arrowheads indicate the editing sites. The swapped parts of DYW domains are represented above. Blue and gray boxes indicate parts of the DYW domains of PpPPR_56 and PpPPR_71, respectively. Average editing efficiencies in independent transformants are shown below (n ≤ 3). (C) RNA editing in Δ56 complemented lines with dywB-1 and dywB-2, respectively (n = 3). (D) RT–PCR results for the PpPPR_56 transcript in the wild type (WT) and Δ56-22 and Δ56 complemented lines. The asterisk indicates an alternative splicing variant of PpPPR_56. PpActin1 was used as a control. To determine which part of dywB is critical for the editing role of the PpPPR_56 editing factor, we generated two constructs in which region dywB-1 (residues 23–43) or dywB-2 (residues 44–61) of the DYW domain of PpPPR_56 was replaced by that of PpPPR_71 (Fig. 3A). The 56PPR-E-r71dywB-1 construct partially complemented nad3-C230 and nad4-C272 editing at 25% and 45%, respectively, whereas the 56PPR-E-r71dywB-2 construct perfectly complemented both editing sites (Fig. 3C, D). This suggested that the dywB-1 region is involved in the discrimination of target sites. The dywB-1 regions of PpPPR_56 and PpPPR_71 are mismatched at nine sites (Fig. 3A). To investigate which amino acid residues are involved in editing site discrimination, we introduced point mutations into the chimeric PpPPR_56 gene in which the DYW domain was replaced by that of PpPPR_71 (56PPR-E + 71DYW; Fig. 4A). First, one or two amino acids of the 71DYW domain were modified to those of PpPPR_56. The 56PPR-E + 71DYW-A23D construct (alanine to aspartic acid substitution at residue 23 of 71DYW) did not complement the editing defect in the PpPPR_56 KO line (Supplementary Fig. S4), and none of the transformants expressing 56PPR-E + 71DYW variants with single or double amino acid replacement showed detectable RNA editing of nad3-C230 and nad4-C272 (Supplementary Fig. S4). Next, 56PPR-E + 71DYW-m1 and -m2 constructs were introduced into the PpPPR_56 KO background (Fig. 4A). In 56PPR-E + 71DYW-m1 lines in which three residues (S25Y26A34) of 71DYW were modified to C25S26V34 of 56DYW (see Fig. 3A), there was no RNA editing of the cognate sites (Fig. 4B). On the other hand, in 56PPR-E + 71DYW-m2 lines in which five residues (V37L38S39L40S42) of 71DYW were modified to L37M38H39T40P42 of 56DYW, RNA editing was partially rescued to 15% at nad3-C230 and to 50% at nad4-C272 (Fig. 4B). Based on these complementation assays, it is suggested that the region from residues 37 to 42 in the DYW domain is involved in the discrimination between target and non-target editing sites. Fig. 4 View largeDownload slide The six amino acid region (residues 37–42) of the DYW domain involved in site-specific editing. (A) Schematic representation of 56PPR-E + 71DYW variants (m1 and m2). Blue boxes indicate the mutated region of PpPPR_56. Positions of amino acid substitutions correspond to those displayed in Fig. 3A. (B) RNA editing efficiency of nad3-C230 and nad4-C272 in Δ56 complemented lines with 56PPR-E + 71DYW-m1 and -m2, respectively. Average editing efficiencies in independent transformants are shown below. Fig. 4 View largeDownload slide The six amino acid region (residues 37–42) of the DYW domain involved in site-specific editing. (A) Schematic representation of 56PPR-E + 71DYW variants (m1 and m2). Blue boxes indicate the mutated region of PpPPR_56. Positions of amino acid substitutions correspond to those displayed in Fig. 3A. (B) RNA editing efficiency of nad3-C230 and nad4-C272 in Δ56 complemented lines with 56PPR-E + 71DYW-m1 and -m2, respectively. Average editing efficiencies in independent transformants are shown below. This region is well conserved across the DYW domains of P. patens editing factors, except for residues 38 and 39 (Fig. 5A). To check whether the above result could be generalized to other editing factors, we expressed the 56PPR-E + 78DYW I38M/S39H variant, in which two amino acids of the 78DYW domain were modified to those of PpPPR_56 in 56PPR-E + 78DYW, in the PpPPR_56 KO line (Fig. 5B). In this variant, RNA editing was partially restored as with the 56PPR-E + 71DYW-m2 variant (Fig. 5B), suggesting that six specific residues of DYW domains, especially those at positions 38 and 39, contribute to the discrimination of target sites. Fig. 5 View largeDownload slide The conserved six amino acid region of the DYW domain. (A) Alignment of the dywB-1 region in the DYW domains of P. patens editing factors. The six amino acid region (residues 37–42) is boxed. Conserved amino acids are shaded in black or gray. (B) RNA editing in the PpPPR_56 KO mosses complemented with 56PPR-E + 78DYW I38M/S39H. The cDNA chromatograms are shown. Black arrowheads indicate editing sites. RNA editing efficiency is shown as a percentage. Fig. 5 View largeDownload slide The conserved six amino acid region of the DYW domain. (A) Alignment of the dywB-1 region in the DYW domains of P. patens editing factors. The six amino acid region (residues 37–42) is boxed. Conserved amino acids are shaded in black or gray. (B) RNA editing in the PpPPR_56 KO mosses complemented with 56PPR-E + 78DYW I38M/S39H. The cDNA chromatograms are shown. Black arrowheads indicate editing sites. RNA editing efficiency is shown as a percentage. Discussion The present study revealed that target sites could be edited through the synergistic effect of site recognition by a PPR tract and site discrimination by specific amino acid residues in the central portion of the DYW domain. First, our complementation study demonstrated that the DYW domains of PpPPR_56 and PpPPR_71 are essential for editing at their target sites. Similarly, a recent study showed that the DYW domain of PpPPR_78 is required for efficient editing at sites rps14-C137 and cox1-C755 in P. patens (Schallenberg-Rüdinger et al. 2017). The DYW domain of PpPPR_56 could be replaced by that of the plastid editing factor PpPPR_45, but not by that of other moss PPR editing factors. However, the DYW domain of PpPPR_71 was not interchangeable with that of PpPPR_45, PpPPR_56 or PpPPR_77. These results indicate that the recognition of target editing sites is regulated not only by PPR motifs but also by the DYW domain. Thus, as shown in the experiment swapping the DYW of PpPPR_56 and 45, some DYW domains are functionally equivalent between mitochondria and chloroplasts in P. patens. The most interesting finding in the present study is that a six amino acid region (residues 37–42) of the DYW domain is involved in the discrimination of specific editing sites. This region is conserved between PpPPR_56 and PpPPR_45 (Fig. 5A). Similarly, a recent study showed that the DYW domain of PpPPR_78 could be functionally replaced by that of PpPPR_79 (Schallenberg-Rüdinger et al. 2017; Table 1), and that their six amino acid sequences are identical (Fig. 5A). Hence, how does the DYW domain discriminate its target editing sites? Previous studies showed that some DYW domains interact with its substrate RNAs (Tasaki et al. 2010, Okuda et al. 2014) and that the 5′-proximal region of the cis-element from –3 to 0 was bound by DYW domains (Okuda et al. 2014). In nad3-C230 and nad4-C272 sites, these regions are distinct from that of two plastid rps14 editing sites targeted by PpPPR_45, but similar to that of the ccmFC-C122 site targeted by PpPPR_71 (Supplementary Fig. S5). These observations suggest that the DYW domain can recognize editing sites without sequence preference. The region from residues 37 to 42 of the DYW domain might be involved in the interaction with its specific editing site or might contribute to structural integrity to access the catalytic site to editing sites, as this region is close to one of the zinc-binding sites HxE. However, the chimeric 56DYW domains, which replaced the dywB or dywB-1 parts in the 71 DYW domain, behaved similarly to 56PPR-E + 71DYW-m2, even without residues 37–42 of the 56DYW domain (Figs. 3B, C, 4B). Therefore, other regions might be sufficient for the specific recognition of PpPPR_56. A further study on the crystal structure of the DYW protein will help in understanding the mode of action of the DYW domain. We also showed that mutations in the zinc-binding signature of PpPPR_56 and PpPPR_71 resulted in the complete loss of their ability to edit cognate sites. Previous studies demonstrated that DYW domains bind to zinc ions (Hayes et al. 2013, Boussardon et al. 2014) and mutations in the zinc-binding signature of Arabidopsis DYW1 eliminate both zinc binding and RNA editing (Boussardon et al. 2014). Our results support the hypothesis that the DYW domain catalyzes the deaminase reaction. Additionally, the highly conserved C-terminal triplet in the DYW domains of PpPPR_56 and PpPPR_71 was shown to be required for editing, which is consistent with the function of DYW1 (Boussardon et al. 2014). The function of the DYW terminus remains unclear, although it might interact with protein(s) that have not been identified to date (Boussardon et al. 2014). In addition to the DYW domain, our swapping experiments showed that E domains are also essential for editing, and are not interchangeable between PpPPR_56 and PpPPR_71. Previous deletion studies demonstrated that the E domain is essential for RNA editing (Okuda et al. 2007, Okuda et al. 2009, Chateigner-Boutin et al. 2013). In contrast, the E domains of A. thaliana E-type OTP70 and moss DYW-type PpPPR_43, which were identified as organellar splicing factors, are not required for splicing (Chateigner-Boutin et al. 2011, Ichinose et al. 2012). Although the role of E domains is still unclear, recent studies revealed that those of CRR4, CRR21 and CLB19 PPR editing factors do not contribute to RNA binding (Okuda et al. 2014, Ramos-Vega et al. 2015), but some PPR proteins interact through their E domains with MORF/RIP proteins (Bayer-Császár et al. 2017). In A. thaliana, E-type OTP71 is involved in mitochondrial editing, and its E domain could be functionally replaced by that of the mitochondrial editing factor OTP72 but not by chloroplast editing factors CRR4 and CLB19 (Chateigner-Boutin et al. 2013). MORF8/RIP1 affects editing events at both ccmFN2-C176 and rpl16-C770 sites, which are targeted by OTP71 and OTP72, respectively (Bentolila et al. 2013). Indeed, the moss DYW-type PpPPR_65 was shown to have potential for protein–protein interaction with the A. thaliana MORF/RIP proteins by a yeast two-hybrid assay (Schallenberg-Rüdinger et al. 2013), supporting the idea that E domains recruit additional specific editing factors in P. patens. Materials and Methods Plant materials and culture conditions The moss P. patens was grown at 25ºC under continuous light, as previously described (Ichinose et al. 2012). BCDATG medium was used for regular culturing of protonemata. Procedures to obtain moss lines PpPPR_56 KO (Δ56-22) and PpPPR_71 KO (Δ71 6-11) have been previously described (Ohtani et al. 2010, Tasaki et al. 2010). Plasmids and moss transformation The PCR primers used in the present study are listed in Supplementary Table S2. For the in vivo complementation assay of PpPPR_56 and PpPPR_71 KO mosses, full-length PpPPR genes containing the native stop codon were amplified from genomic DNA. Resulting amplicons were then cloned into the SwaI site of the overexpression vector p9WmycH13 (Ichinose et al. 2012), and the resultant plasmids were named p56FL and p71FL, respectively. For constructing PpPPR proteins lacking their E/DYW domains, DYW domain or C-terminal DYW triplets, truncated genomic sequences of PpPPR proteins were amplified and cloned into p9WmycH13. Deletion of the E domain and the introduction of point mutations into the DYW domain were carried out by site-directed mutagenesis PCR using PrimeSTAR GXL polymerase (TaKaRa). For the chimeric PpPPR_56, in which the original DYW domain of PpPPR_56 was replaced by that of other PpPPR proteins, PpPPR_56 lacking a DYW domain was amplified by PCR using reverse primers containing the NruI site, and then cloned into p9WmycH13 to generate p56ΔDYW-NruI. The various DYW domains were obtained by PCR and cloned into the NruI site of p56ΔDYW-NruI. The swapping constructs of the E domain or partial DYW domain were generated using the In-Fusion cloning system (Clontech). The resulting plasmids were linearized by NotI and introduced into Δ56-22 or Δ71 6-11 KO lines by particle bombardment or polyethylene glycol-mediated DNA transformation (Nishiyama et al. 2000, Tasaki et al. 2010). Hygromycin-resistant mosses were selected and transformants were confirmed by PCR. The expression of transgenes was confirmed by RT–PCR. Analysis of RNA editing Total RNA was isolated from 4-day-old protonemata using ISOGEN II (NIPPON GENE), and treated with DNase I (TaKaRa). Reverse transcription was carried out using ReverTra Ace (TOYOBO) from 1 µg of DNA-free RNA primed with random hexamers. Amplified products, including the editing sites, were purified and sequenced directly as previously described (Ichinose et al. 2013). Editing efficiency was calculated by the ratio of T and C peak height. Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the Japan Society for the Promotion of Science (JSPS) [KAKENHI grant Nos. 2529105, 17K08195 (to M.S.) and 18K14435 (to M.I.), Research Fellow for Young Scientists (to M.I.)]; The Hori Science and Arts Foundation [to M.I.]; and the IGER program from Nagoya University [to M.I.]. Acknowledgments We thank Dr. Bernard Gutmann (University of Western Australia) for valuable comments and discussion on an early draft of the manuscript. Disclosures The authors have no conflicts of interest to declare. References Barkan A., Small I. ( 2014) Pentatricopeptide repeat proteins in plants. Annu. Rev. Plant Biol.  65: 415– 442. Google Scholar CrossRef Search ADS PubMed  Barkan A., Rojas M., Fujii S., Yap A., Chong Y.S., Bond C.S. ( 2012) Combinatorial amino acid code for RNA recognition by pentatricopeptide repeat proteins. PLoS Genet.  8: e1002910. Google Scholar CrossRef Search ADS PubMed  Bayer-Császár E., Haag S., Jörg A., Glass F., Härtel B., Obata T., et al.   ( 2017) The conserved domain in MORF proteins has distinct affinities to the PPR and E elements in PPR RNA editing factors. Biochim. Biophys. Acta  1860: 813– 828. Google Scholar CrossRef Search ADS PubMed  Bentolila S., Heller W.P., Sun T., Babina A.M., Friso G., Van Wijk K.J., et al.   ( 2012) RIP1, a member of an Arabidopsis protein family, interacts with the protein RARE1 and broadly affects RNA editing. Proc. Natl. Acad. Sci. USA  109: E1453– E1461. Google Scholar CrossRef Search ADS   Bentolila S., Oh J., Hanson M.R., Bukowski R. ( 2013) Comprehensive high-resolution analysis of the role of an Arabidopsis gene family in RNA editing. PLoS Genet.  9: e1003584. Google Scholar CrossRef Search ADS PubMed  Boussardon C., Avon A., Kindgren P., Bond C.S., Challenor M., Lurin C., et al.   ( 2014) The cytidine deaminase signature HxE(x)nCxxC of DYW1 binds zinc and is necessary for RNA editing of ndhD-1. New Phytol.  203: 1090– 1095. Google Scholar CrossRef Search ADS PubMed  Chateigner-Boutin A.L., Colas Des Francs-Small C., Fujii S., Okuda K., Tanz S.K., Small I. ( 2013) The E domains of pentatricopeptide repeat proteins from different organelles are not functionally equivalent for RNA editing. Plant J.  74: 935– 945. Google Scholar CrossRef Search ADS PubMed  Chateigner-Boutin A.L., Des Francs-Small C.C., Delannoy E.E., Kahlau S., Tanz S.K., de Longevialle A.F., et al.   ( 2011) OTP70 is a pentatricopeptide repeat protein of the E subgroup involved in splicing of the plastid transcript rpoC1. Plant J . 65: 532– 542. Google Scholar CrossRef Search ADS PubMed  Cheng S., Gutmann B., Zhong X., Ye Y., Fisher M.F., Bai F., et al.   ( 2016) Redefining the structural motifs that determine RNA binding and RNA editing by pentatricopeptide repeat proteins in land plants. Plant J . 85: 532– 547. Google Scholar CrossRef Search ADS PubMed  Hayes M.L., Giang K., Berhane B., Mulligan R.M. ( 2013) Identification of two pentatricopeptide repeat genes required for RNA editing and zinc binding by C-terminal cytidine deaminase-like domains. J. Biol. Chem.  288: 36519– 36529. Google Scholar CrossRef Search ADS PubMed  Hecht J., Grewe F., Knoop V. ( 2011) Extreme RNA editing in coding islands and abundant microsatellites in repeat sequences of Selaginella moellendorffii mitochondria: the root of frequent plant mtDNA recombination in early tracheophytes. Genome Biol. Evol . 3: 344– 358. Google Scholar CrossRef Search ADS PubMed  Ichinose M., Sugita M. ( 2017) RNA editing and its molecular mechanism in plant organelles. Genes (Basel)  8: 5. Google Scholar CrossRef Search ADS   Ichinose M., Sugita C., Yagi Y., Nakamura T., Sugita M. ( 2013) Two DYW subclass PPR proteins are involved in RNA editing of ccmFc and atp9 transcripts in the moss Physcomitrella patens: first complete set of PPR editing factors in plant mitochondria. Plant Cell Physiol . 54: 1907– 1916. Google Scholar CrossRef Search ADS PubMed  Ichinose M., Tasaki E., Sugita C., Sugita M. ( 2012) A PPR-DYW protein is required for splicing of a group II intron of cox1 pre-mRNA in Physcomitrella patens. Plant J . 70: 271– 278. Google Scholar CrossRef Search ADS PubMed  Ichinose M., Uchida M., Sugita M. ( 2014) Identification of a pentatricopeptide repeat RNA editing factor in Physcomitrella patens chloroplasts. FEBS Lett . 588: 4060– 4064. Google Scholar CrossRef Search ADS PubMed  Iyer L.M., Zhang D., Rogozin I.B., Aravind L. ( 2011) Evolution of the deaminase fold and multiple origins of eukaryotic and mutagenic nucleic acid deaminase from bacterial toxin systems. Nucleic Acids Res . 39: 9473– 9497. Google Scholar CrossRef Search ADS PubMed  Lurin C., Andrés C., Aubourg S., Bellaoui M., Bitton F., Bruyère C., et al.   ( 2004) Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell  16: 2089– 2103. Google Scholar CrossRef Search ADS PubMed  Miyata Y., Sugita M. ( 2004) Tissue- and stage-specific RNA editing of rps14 transcripts in moss (Physcomitrella patens) chloroplasts. J. Plant Physiol . 161: 113– 115. Google Scholar CrossRef Search ADS PubMed  Nakamura T., Sugita M. ( 2008) A conserved DYW domain of pentatricopeptide repeat protein possesses a novel endoribonuclease activity. FEBS Lett . 582: 4163– 4168. Google Scholar CrossRef Search ADS PubMed  Nishiyama T., Hiwatashi Y., Sakakibara I., Kato M., Hasebe M. ( 2000) Tagged mutagenesis and gene-trap in the moss, Physcomitrella patens by shuttle mutagenesis. DNA Res.  7: 9– 17. Google Scholar CrossRef Search ADS PubMed  Ohtani S., Ichinose M., Tasaki E., Aoki Y., Komura Y., Sugita M. ( 2010) Targeted gene disruption identifies three PPR-DYW proteins involved in RNA editing for five editing sites of the moss mitochondrial transcripts. Plant Cell Physiol . 51: 1942– 1949. Google Scholar CrossRef Search ADS PubMed  Okuda K., Chateigner-Boutin A.L., Nakamura T., Delannoy E., Sugita M., Myouga F., et al.   ( 2009) Pentatricopeptide repeat proteins with the DYW motif have distinct molecular functions in RNA editing and RNA cleavage in Arabidopsis chloroplasts. Plant Cell  21: 146– 156. Google Scholar CrossRef Search ADS PubMed  Okuda K., Myouga F., Motohashi R., Shinozaki K., Shikanai T. ( 2007) Conserved domain structure of pentatricopeptide repeat proteins involved in chloroplast RNA editing. Proc. Natl. Acad. Sci. USA  104: 8178– 8183. Google Scholar CrossRef Search ADS   Okuda K., Shoki H., Arai M., Shikanai T., Small I., Nakamura T. ( 2014) Quantitative analysis of motifs contributing to the interaction between PLS-subfamily members and their target RNA sequences in plastid RNA editing. Plant J.  80: 870– 882. Google Scholar CrossRef Search ADS PubMed  Oldenkott B., Yamaguchi K., Tsuji-Tsukinoki S., Knie N., Knoop V. ( 2014) Chloroplast RNA editing going extreme: more than 3400 events of C-to-U editing in the chloroplast transcriptome of the lycophyte Selaginella uncinata. RNA  20: 1499– 1506. Google Scholar CrossRef Search ADS PubMed  Ramos-Vega M., Guevara-García A., Llamas E., Sánchez-León N., Olmedo-Monfil V., Cielle-Calzada J.P., et al.   ( 2015) Functional analysis of the Arabidopsis thaliana CHLOROPLAST BIOGENESIS 19 pentatricopeptide repeat editing protein. New Phytol . 208: 430– 441. Google Scholar CrossRef Search ADS PubMed  Rüdinger M., Funk H.T., Rensing S.A., Maier U.G., Knoop V. ( 2009) RNA editing: only eleven sites are present in the Physcomitrella patens mitochondria transcriptome and a universal nomenclature proposal. Mol. Genet. Genomics  281: 473– 481. Google Scholar CrossRef Search ADS PubMed  Rüdinger M., Szövényi P., Rensing S.A., Knoop V. ( 2011) Assigning DYW-type PPR proteins to RNA editing sites in the funariid mosses Physcomitrella patens and Funaria hygrometrica. Plant J . 67: 370– 380. Google Scholar CrossRef Search ADS PubMed  Salone V., Rüdinger M., Polsakiewicz M., Hoffmann B., Groth-Malonek M., Szurek B., et al.   ( 2007) A hypothesis on the identification of the editing enzyme in plant organelles. FEBS Lett . 581: 4132– 4138. Google Scholar CrossRef Search ADS PubMed  Schallenberg-Rüdinger M., Kindgren P., Zehrmann A., Small I., Knoop V. ( 2013) A DYW-protein knockout in Physcomitrella affects two closely spaced mitochondrial editing sites and causes a severe developmental phenotype. Plant J.  76: 420– 432. Google Scholar CrossRef Search ADS PubMed  Schallenberg-Rüdinger M., Oldenkott B., Hiss M., Trinh P.L., Knoop V., Rensing S.A. ( 2017) A single-target mitochondrial RNA editing factor of Funaria hygrometrica can fully reconstitute RNA editing at two sites in Physcomitrella patens. Plant Cell Physiol . 58: 496– 507. Google Scholar CrossRef Search ADS PubMed  Sun T., Bentolila S., Hanson M.R. ( 2016) The unexpected diversity of plant organelle RNA editosomes. Trends Plant Sci.  21: 962– 973. Google Scholar CrossRef Search ADS PubMed  Takenaka M., Zehrmann A., Brennicke A., Graichen K. ( 2013a) Improved computational target site prediction for pentatricopeptide repeat RNA editing factors. PLoS One  8: e65343. Google Scholar CrossRef Search ADS   Takenaka M., Zehrmann A., Verbitskiy D., Härtel B., Brennicke A. ( 2013b) RNA editing in plants and its evolution. Annu. Rev. Genet.  47: 335– 352. Google Scholar CrossRef Search ADS   Takenaka M., Zehrmann A., Verbitskiy D., Kugelmann M., Härtel B., Brennicke A. ( 2012) Multiple organellar RNA editing factor (MORF) family proteins are required for RNA editing in mitochondria and plastids of plants. Proc. Natl. Acad. Sci. USA  109: 5104– 5109. Google Scholar CrossRef Search ADS   Tasaki E., Hattori M., Sugita M. ( 2010) The moss pentatricopeptide repeat protein with a DYW domain is responsible for RNA editing of mitochondrial ccmFc transcript. Plant J . 62: 560– 570. Google Scholar CrossRef Search ADS PubMed  Uchida M., Ohtani S., Ichinose M., Sugita C., Sugita M. ( 2011) The PPR-DYW proteins are required for RNA editing of rps14, cox1 and nad5 transcripts in Physcomitrella patens mitochondria. FEBS Lett . 585: 2367– 2371. Google Scholar CrossRef Search ADS PubMed  Yagi Y., Hayashi S., Kobayashi K., Hirayama T., Nakamura T. ( 2013) Elucidation of the RNA recognition code for pentatricopeptide repeat proteins involved in organelle RNA editing in plants. PLoS One  8: e57286. Google Scholar CrossRef Search ADS PubMed  Yin P., Li Q., Yan C., Liu Y., Liu J., Yu F., et al.   ( 2013) Structural basis for the modular recognition of single-stranded RNA by PPR proteins. Nature  504: 168– 171. Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations FL full length KO knockout MORF multiple organellar RNA editing factor PPR pentatricopeptide repeat PPR-DYW DYW-subclass PPR RIP RNA editing factor-interacting protein RT–PCR reverse transcription–PCR © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Plant and Cell PhysiologyOxford University Press

Published: Apr 27, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off