Two DYW Subclass PPR Proteins are Involved in RNA Editing of ...

Two DYW Subclass PPR Proteins are Involved in RNA Editingof ccmFc and atp9 Transcripts in the Moss Physcomitrellapatens: First Complete Set of PPR Editing Factors inPlant MitochondriaMizuho Ichinose1, Chieko Sugita1, Yusuke Yagi2, Takahiro Nakamura2 and Mamoru Sugita1,*1Center for Gene Research, Nagoya University, Chikusa-ku, Nagoya, 464-8602 Japan2Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan*Corresponding author: E-mail, [email protected]; Fax, +81-52-789-3081.(Received July 5, 2013; Accepted September 10, 2013)

The moss Physcomitrella patens has 11 RNA editing sitesin mitochondrial transcripts. We previously identified sixDYW subclass pentatricopeptide repeat (PPR) proteins asRNA editing factors for nine out of 11 sites. In thisstudy, we identified two novel DYW subclass PPR proteins,PpPPR_65 and PpPPR_98, as RNA editing factors. Disruptionof the PpPPR_65 gene resulted in a complete loss of RNAediting at two neighboring sites, ccmFc-C103 and ccmFc-C122, in the mitochondrial ccmFc transcript. To confirmthis result, we further generated PpPPR_65 knockdown(KD) mutants by an inducible RNA interference (RNAi)system. The generated RNAi lines displayed reduced levelsof RNA editing at both ccmFc-C103 and ccmFc-C122 sites.Next, we characterized the function of PpPPR_98 by con-structing a KD mutant of PpPPR_98 expression. The KDmutant showed a 30% reduction in the level of atp9-C92editing. When PpPPR_98 cDNA was introduced into the KDmutant, RNA editing levels were restored to the wild-typelevel. This indicates that PpPPR_98 is an editing factor forthe atp9-C92 site. The recombinant PpPPR_98 proteinbound to the upstream sequence of the editing site thatwas created by splicing of atp9 transcript. This suggeststhat atp9 RNA editing occurs after splicing of atp9 tran-script. Our present and previous data provide the first evi-dence that all 11 known editing events require at least eightDYW subclass PPR proteins in the moss mitochondria.

Keywords: Mitochondria � Moss � Physcomitrella patens �

PPR protein � RNA editing.

Abbreviations: EMSA, electrophoresis mobility shift assay;GFP, green fluorescent protein; gfp, gene encoding greenfluorescent protein; hpt, gene encoding hygromycin phos-photransferase; KD, knockdown; KO, knockout; MORF/RIP,multiple organellar RNA editing factor/RNA editing factor-interacting protein; PPR, pentatricopeptide repeat; PPR-DYW,DYW-subclass PPR; RFP, red fluorescent protein: RNAi, RNAinterference; qRT–PCR, quantitative real-time reverse

transcription–PCR; RRM, RNA recognition motif; RT–PCR,reverse transcription–PCR; UTR, untranslated region.

The nucleotide sequences reported in this paper have beensubmitted to the DDBJ database under accession numbersAB830083 (PpPPR_65 mRNA) and AB830784 (PpPPR_98mRNA).

Introduction

RNA editing frequently occurs in many transcripts of plantorganelles. More than 400 RNA editing sites, mostly involvingcytidine (C) to uridine (U) transitions, have been identified inthe mitochondria and �40 sites in the plastids of floweringplants (Shikanai 2006, Takenaka et al. 2008, Knoop 2011).However, the molecular mechanism of RNA editing is notfully understood. Recent genetic studies show robust involve-ment of pentatricopeptide repeat (PPR) proteins in RNA edit-ing in plant organelles (Fujii and Small 2011). PPR proteinsusually comprise a tandem array of degenerate 35 amino acidmotifs (Small and Peeters 2000, Lurin et al. 2004). In particular,plant PPR proteins are encoded by a large gene family com-posed of 100 to >800 genes in land plants (Lurin et al. 2004,O’Toole et al. 2008, Banks et al. 2011). Most PPR proteins arelocalized in plastids and mitochondria (Lurin et al. 2004)and play a key role in various RNA-related processes such asstability, splicing, editing or translational regulation (Schmitz-Linneweber and Small 2008, Gutmann et al. 2012).

Plant PPR proteins can be divided into four classes, P, PLS, Eand DYW (Lurin et al. 2004). PLS-type PPR proteins withC-terminal E or E + DYW domains (PPR-E or PPR-DYW)are required for RNA editing (Kotera et al. 2005, Okuda et al.2007, Hammani et al. 2009, Zehrmann et al. 2009). Mostly, asingle PPR protein is involved in RNA editing at either a single ormultiple sites. Many PPR-E and PPR-DYW proteins play a role inRNA editing as a site recognition factor that was demonstrated

Plant Cell Physiol. 54(11): 1907–1916 (2013) doi:10.1093/pcp/pct132, available online at www.pcp.oxfordjournals.org! The Author 2013. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.All rights reserved. For permissions, please email: [email protected]

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by an in vitro RNA binding assay for the target RNA molecules(Okuda et al. 2006, Tasaki et al. 2010, Hammani et al. 2011,Okuda and Shikanai 2012, Toda et al. 2012). In addition tothe PPR-E and PPR-DYW proteins, non-PPR proteins such asRNA recognition motif (RRM)-containing RNA-binding pro-teins and multiple organellar RNA editing factor/RNA editingfactor-interacting proteins (MORF/RIPs) were shown to takepart in RNA editing events at multiple sites (Tillich et al.2009, Bentolila et al. 2012, Takenaka et al. 2012, Sun et al.2013). Moreover, MORF/RIP proteins interacted with PPR pro-teins (Takenaka et al. 2012), and MORF8 (known as RIP1) wasdetected in a complex >200 kDa in Arabidopsis thaliana(Bentolila et al. 2012). It is intriguing that MORF/RIPs arefound in flowering plants but not in non-flowering plants,e.g. bryophytes. These observations led us to consider a contextin which PPR proteins work as a site recognition factor and inwhich MORF/RIPs and RRM-type RNA-binding proteins serveas a general regulator in RNA editing in flowering plants.However, the mechanism of RNA editing still remainsenigmatic.

Unlike the flowering plants, the genome of the mossPhyscomitrella patens encodes 10 DYW subclass PPR proteinsbut no E subclass PPR proteins (O’Toole et al. 2008, Sugita et al.2013). In P. patens, C to U RNA editing occurs at only two sitesin the plastid rps14 transcript (Miyata and Sugita 2004) and at11 sites in nine mitochondrial gene transcripts (Rudinger et al.2009, Tasaki et al. 2010). Single editing sites lie within mito-chondrial rps14, nad3, nad4, cox1, cox2, cox3 and atp9 tran-scripts and two sites in ccmFc and nad5 transcripts. To date,six out of 10 PPR-DYW proteins have been identified as editingfactors for nine sites (Ohtani et al. 2010, Tasaki et al. 2010,Rudinger et al. 2011, Uchida et al. 2011) and one PPR-DYWprotein PpPPR_43 is essential for splicing of cox1 pre-mRNAin the mitochondria (Ichinose et al. 2012). Plastid-localizedDYW-type PpPPR_45 is predicted to be a plastid rps14 RNAediting factor. This raises the question of whether PPR-DYWproteins are responsible for all RNA editing events in mosses. Tothis end, the editing factor(s) targeted to the remaining twoediting sites, ccmFc-C103 (formerly ccmF-1, Tasaki et al. 2010)and atp9-C92, need to be urgently identified. In this study,based on a reverse genetics approach, we identified a DYW-type PpPPR_65 as an editing factor required for two neighbor-ing sites, ccmFc-C103 and ccmFc-C122 (formerly ccmF-2), andPpPPR_98 for the atp9-C92 site.

Results

Disruption of PpPPR_65 resulted in severegrowth retardation of the moss protonemata

In this study, we first investigated whether PpPPR_65 isinvolved in RNA editing at either the ccmFc-C103 or theatp9-C92 site, or both. The PpPPR_65 gene corresponds tothe gene model Pp1s13_264V6.1 (http://www.cosmoss.org;Rensing et al. 2008). The gene and cDNA sequences were

predicted to encode a polypeptide composed of 15 PLS-typePPR motifs and C-terminal E/E+ and DYW domains (Fig. 1A).The TargetP program (Emanuelsson et al. 2000) predicted witha high score of 0.89 that PpPPR_65 is localized in the mitochon-dria. This was confirmed by microscopic observation of trans-genic moss plants expressing PpPPR_65–green fluorescentprotein (GFP) (Supplementary Fig. S1).

To analyze the function of PpPPR_65, we deleted the cog-nate gene by replacing the coding region with the gfp and hpt(gene cassette encoding GFP and hygromycin phosphotransfer-ase, respectively; Fig. 1B). A single hygromycin-resistant moss�65-1 was generated (Fig. 1C; Supplementary Fig. S2), andits absence of PpPPR_65 transcripts was verified by reversetranscription–PCR (RT–PCR) (Fig. 1D). This indicated that�65-1 is a bona fide genetic knockout (KO) mutant. The pro-tonemata of the PpPPR_65 disruptant displayed severe growthretardation (Fig. 1C). This phenotype was stronger than that ofthe PpPPR_71 disruptants, which displayed an RNA editingdefect at ccmFc-C122 (Tasaki et al. 2010).

The PpPPR_65 disruptant completely lostRNA editing at two neighboring sites inccmFc transcripts

We then investigated RNA editing for all 11 known sites inmitochondria and the rps14-C2 site in plastids in the disrup-tant. Direct sequencing of RT–PCR products showed a com-plete defect of RNA editing at both the ccmFc-C103 andccmFc-C122 sites in the disruptant (Fig. 2) while the othernine editing sites in mitochondria and the rps14 site in plastidswere not affected (Supplementary Fig. S3). Since PpPPR_71transcripts were detected in the PpPPR_65 disruptant (Fig. 1D),the defect of ccmFc-C122 editing was caused by the loss ofPpPPR_65 but not PpPPR_71. This suggests that PpPPR_65 isessential for RNA editing at the two ccmFc sites. These data arethe same as observed by Schallenberg-Rudinger et al. (2013).

To confirm our results, we further generated PpPPR_65 RNAinterference (RNAi) plants by introducing an inducible RNAiconstruct into the transgenic GH moss line expressing GFP–tubulin and histone H2B–red fluorescent protein (RFP)(Nakaoka et al. 2012). Several RNAi moss plants were selectedand their PpPPR_65 transcript levels were measured by quan-titative real-time RT–PCR (qRT–PCR) (Fig. 3A). Two independ-ent RNAi mosses with reduced PpPPR_65 transcript levels wereanalyzed for RNA editing. In the GH lines, RNA editing atccmFc-C103 and ccmFc-C122 sites occurred normally eitherwith or without treatment by b-estradiol. In contrast, thelevel of RNA editing at the two sites decreased by 45–60% inthe 65RNAi#5 and #16 mosses when treated with b-estradiol(Fig. 3B). To quantify the level of RNA editing at the two sites,we randomly isolated 100 independent ccmFc cDNA clones anddetermined their cDNA sequences. This analysis revealed thatediting efficiency at ccmFc-C103 was 92.5% (99 edited cDNAclones out of 107 sequenced cDNA clones) in the GH linetreated with b-estradiol and 67.6% (71 out of 105 cDNA

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clones) and 61.3% (65 out of 106 cDNA clones) in the inducedRNAi lines #5 (+) and #16 (+), respectively. Notably, we couldnot isolate cDNA edited at ccmFc-C122 only in the RNAimosses. This coincides with a previous result in the wild-typeprotonemata (Tasaki et al. 2010). We therefore concluded thatPpPPR_65 is an essential factor for RNA editing at both ccmFc-C103 and ccmFc-C122 sites.

PpPPR_65 presumably recognizes the upstreamsequence of the ccmFc-C103 site but not of theccmFc-C122 site

To investigate whether PpPPR_65 can recognize the target up-stream RNA sequences of both ccmFc sites, we performed com-putational prediction to infer the amino acid code for RNArecognition by PPR motifs (Yagi et al. 2013). The predictedtarget RNA sequence of PpPPR_65 fitted well the upstreamsequence of ccmFc-C103 but not that of ccmFc-C122 (Fig. 4).This result was also supported by analysis using a similar PLS-dependent two-letter RNA recognition code (Barkan et al.2012). This suggests that PpPPR_65 is directly associated withthe proximal sequence of ccmFc-C103 but not of ccmFc-C122.On the other hand, the amino acid codes of PpPPR_71 for RNArecognition were predicted to interact with the upstreamsequence of ccmFc-C122 (Yagi et al. 2013). Direct binding of

Fig. 1 Generation and characterization of the PpPPR_65 gene KO mutant. (A) Predicted PpPPR_65 protein motif structure. (B) Gene structure ofPpPPR_65. Most of the coding region of PpPPR_65 was replaced by the gfp and hpt gene cassette, and the gene disruptant �65-1 was selected.(C) Protonemata colony morphologies of the wild-type (WT) and disruptant �65-1. As a control, the disruptant �71-6-11 line (Tasaki et al.2010) is also shown. The mosses were grown for 3 weeks on BCD medium plates without hygromycin B. Scale bar = 10 mm. (D) RT–PCR fordetection of cognate transcripts was performed in WT, �65-1 and �71-6-11 lines. PpActin1 transcript was used as the control.

Fig. 2 RNA editing defects in the PpPPR_65 disruptant. Directsequence chromatograms of PCR-amplified cDNA from the wildtype (WT) and the disruptants �65-1 and �71-6-11 are shown.Gray shaded areas indicate the two ccmFc editing sites. RNA editingefficiencies are shown as a percentage.


PPR-DYW editing factors in moss mitochondria


PpPPR_71 to the target RNA covering ccmFc-C122 was previ-ously demonstrated (Tasaki et al. 2010).

PpPPR_98 is an RNA editing factor for theatp9 site

Since the atp9-C92 was normally edited in the PpPPR_65 dis-ruptant (Supplementary Fig. S3), PpPPR_98 is a candidate foran RNA editing factor for this site. The transgenic moss plantsexpressing PpPPR_98–GFP clearly showed its mitochondriallocalization (Supplementary Fig. S1). The gene (gene model;Pp1s276_42V6.1) and cDNA sequences were predicted toencode a polypeptide composed of 21 PLS-type PPR motifsand C-terminal E/E+ and DYW domains (Fig. 5A). To investi-gate the function of PpPPR_98, we intensively tried to generatePpPPR_98 KO mosses by insertion of an hpt gene cassette into

the third exon or the first intron of the gene. However, we wereunable to generate any KO mutant mosses. Instead, we insertedthe hpt cassette into the 50-untranslated region (UTR) ofthe gene (Fig. 5B), and subsequently an hpt cassette-taggedmoss plant was raised as confirmed by genomic PCR(Supplementary Fig. S4). Although the mutant grew as wellas the wild-type mosses (Fig. 5C), the level of PpPPR_98 tran-script was 35% less than that of the wild type (Fig. 5D). Thus,this moss could be defined as a KD mutant and was designatedas the 98KD line. Then, we tested RNA editing events in the98KD line. Direct sequencing of RT–PCR products showed a30% reduction of atp9-C92 RNA editing in this line (Fig. 5E).The other 10 editing sites in mitochondria were not affected inthe 98KD line (Supplementary Fig. S5). To confirm this result,we generated transformants of the 98KD mutant comple-mented with a PpPPR_98 full-length cDNA (Fig. 5D). In thecomplemented 98KD line thus obtained, RNA editing at atp9-C92 was restored to 100% (Fig. 5E). This strongly suggests thatPpPPR_98 is an RNA editing factor for the atp9-C92 site.

The moss mitochondrial atp9 gene is interrupted by threeintrons, and the atp9-C92 site lies within the third exon(Rudinger et al. 2009). The predicted target RNA sequence ofPpPPR_98 fits well into the upstream sequence of the atp9-C92site in spliced mRNA and also shares, to a lower extent, hom-ology with that of unspliced pre-mRNA (Fig. 4), predicted by aP-value of 2.6� 10�3 and 4.2� 10�1, respectively. However, thereduction in the atp9 RNA editing level might be caused byindirect effects, e.g. disturbed splicing of the atp9 transcripts,rather than a direct interference of PpPPR_98 at the editing site.To eliminate this possibility, we performed RT–PCR to comparethe splicing efficiencies of atp9 transcripts between wild-typeand KD mutant mosses. There were similar amounts of splicedand unspliced atp9 transcripts between the wild type andmutant (data not shown).

Finally, to confirm PpPPR_98 as a site recognition factor, weperformed an electrophoresis mobility shift assay (EMSA) usingthe recombinant PpPPR_98 and RNA probes. Prior to this ana-lysis, we checked the extent of atp9-C92 editing because theatp9 gene is interrupted by three introns and the atp9-C92 sitelies within the third exon, being only 8 nucleotides (nt) long(Supplementary Fig. S6). In the wild-type protonemata,the atp9-C92 site was completely edited in fully splicedmRNA while this site was unedited in unspliced transcripts(Supplementary Fig. S6). Accordingly, we used two differentoligo RNA probes, spliced or unspliced (34 nt) for EMSA experi-ments (Fig. 6A). The RNA was labeled with 32P and incubatedwith the recombinant protein. The protein–RNA complex wasdetected as shifted bands that migrated more slowly than freeRNA probe in the gel. As shown in Fig. 6B, recombinantPpPPR_98 (r-98) strongly bound to the spliced atp9 RNA butvery weakly bound to the unspliced atp9 RNA. In contrast, therecombinant PpPPR_71 (r-71) did not bind to the atp9-splicedRNA. The binding of r-98 to the labeled atp9-spliced RNA wasslightly more strongly inhibited by addition of cold atp9-splicedRNA than cold atp9-unspliced RNA (Supplementary Fig. S7).

Fig. 3 RNA editing in the PpPPR_65 RNAi lines. (A) qRT–PCR analysisto quantify PpPPR_65 mRNA levels in mosses with or withoutb-estradiol. Error bars indicate the SD of 2–3 independent experi-ments. (B) Direct sequence chromatograms of RT–PCR productsamplified from cDNA of RNAi lines treated with or without b-estra-diol. Gray shaded areas indicate the editing sites.


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This result indicated that PpPPR_98 is a bona fide site recog-nition factor for the spliced atp9 transcript.

Discussion

In this study, a defect of DYW type PpPPR_65 completely abol-ished editing of not only ccmFc-C103 but also the ccmFc-C122site. This indicates that PpPPR_65 is an essential editing factorfor the two neighboring sites. Although PpPPR_56, 77 and 78are known to target two editing sites (Ohtani et al. 2010, Uchidaet al. 2011), the result of the PpPPR_65 disruptant is a bitsurprising because PpPPR_71 was previously identified as accmFc-C122-specific editing factor (Tasaki et al. 2010). Loss ofPpPPR_71 abolishes editing at ccmFc-C122 but not at ccmFc-C103 (Tasaki et al. 2010). Even though PpPPR_71 transcriptsaccumulated in the PpPPR_65 disruptant, the C122 editingevent was completely defective in the disruptant. These

observations indicate that ccmFc-C122 editing requires bothPpPPR_71 and 65, whereas ccmFc-C103 editing requiresPpPPR_65 but not PpPPR_71.

In silico prediction of the target RNA sequence supportsthat PpPPR_65 can recognize the upstream sequence ofccmFc-C103 but not that of ccmFc-C122 (Fig. 4). This providesnew insights into the RNA editing mechanism in plant organ-elles. Involvement of PpPPR_65 in ccmFc-C122 editing may bedue to some secondary effect rather than direct recognition ofccmFc-C122. The question is how PpPPR_65 is involved inccmFc-C122 editing. One option can be considered thatccmFc-C122 editing requires some cooperative interaction be-tween the two PPR-DYW proteins. A recent work done bySchallenberg-Rudinger et al. (2013) showed a weak interactionbetween PpPPR_65 and 71 by yeast two-hybrid assay. However,it is unclear whether such a weak interaction is essential forediting at both sites. Our previous cDNA analysis strongly sug-gested that RNA editing of ccmFc transcripts occurs

Fig. 4 PPR motif contexts and in silico target assignments for the PpPPR proteins. The nucleotide-specifying residues (NSRs; residues 1, 4 and ‘ii’)were extracted from the PPR motifs of PpPPR_65 and PpPPR_98. The NSRs were converted into a probability matrix that indicated the decodingnucleotide frequency according to the PPR code using table S4 in Yagi et al. (2013). The probability matrix was also shown by logo (http://weblogo.threeplusone.com/create.cgi). The NSRs that did not match known PPR codes (shown as ‘–’) were converted to the background frequency. Theasterisks indicate any amino acid. The target RNA sequences are shown: ccmFc-C103 for PpPPR65 and atp9-C92 for PpPPR_98. The intron region ofunspliced RNA is underlined. The right upper panel displays the result of in silico target assignment. The target assignment was conducted asdescribed in Yagi et al. (2013), using PpPPR_65 and _98 against all 13 RNA editing sites in P. patens. The diamond represents the P-value for thematching score against the editing site. The red diamond indicates the editing site for which a deficiency was observed in the mutant.




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sequentially from the upstream C103 to the downstream C122site (Tasaki et al. 2010). In addition, PpPPR_71 bound preferablyto the edited RNA rather than to the unedited RNA at ccmFc-C103 (Tasaki et al. 2010). From these observations, it is sug-gested that binding of PpPPR_65 to the target C103 site couldprovide a scaffold for RNA editing at the second site C122recognized by PpPPR_71. This might be related to alterationof a secondary or tertiary structure of the RNA region encom-passing the two ccmFc editing sites. Because, Prikryl et al. (2011)proposed a model that binding of the maize PPR10 to the50-UTR of atpH transcript refolds its stem–loop structure anda ribosome-binding site is released from an RNA duplex.A similar PPR protein-induced RNA rearrangement is suggestedfor the moss clpP pre-mRNA (Hattori and Sugita 2009).

In Arabidopsis plastids, ndhD-C878 and ndhD-C887 sites ofndhD mRNA are separated by only 8 nt, and are edited by a PPR-DYW protein CRR28 and CRR22, respectively. Mutations of the

CRR28 gene caused a loss of RNA editing for ndhD-C878 but notfor ndhD-C887 (Okuda et al. 2009). Thus, RNA editing of thedownstream site occurs independently of whether the upstreamsite is edited or not. Likewise, Arabidopsis OTP82 is required forndhB-C836 editing but not for ndhB-C830 in plastids (Okudaet al. 2010). In Arabidopsis mitochondria, there are many closelyspaced editing sites. MEF25 targets to nad1-C308 but not tonad1-C307 (Arenas-M. et al. 2013), MEF11 recognizes cob203-C344 but not cob203-C356 (Verbitskiy et al. 2010), and SLO1 isrequired for nad4-C449 editing but not for nad4-C436 and C437(Sung et al. 2010). Rice mitochondrial OGR1 is essential for nad4-C433 editing but not for nad4-C437 (Kim et al. 2009). Thus, singlePPR editing factors do not recognize neighboring editing sites ofthe target site. It is interesting to note that the dependence ofccmFc-C122 on C103 editing might be unique to mosses.

RNA editing at the accD-C794 site requires two distinct PPR-DYW proteins, RARE1 and VAC1 (also known as AtECB2) in

Fig. 5 Generation and characterization of the PpPPR_98 KD mutant. (A) Predicted PpPPR_98 protein motif structure. (B) Gene structure ofPpPPR_98. Filled boxes indicate the translated region of the gene model. The hpt gene cassette was inserted into the 50-UTR at the XbaI site 11 bpupstream from the putative translation initiation codon. (C) Protonemata colony morphologies of the wild type (WT) and 98KD line. The mosseswere grown for 2 weeks on a BCD medium plate without hygromycin B. Scale bar = 10 mm. (D) RT–PCR for detection of cognate transcript wasperformed. RT–PCR for PpActin1 gene transcript was used as the control. (E) RNA editing in the WT, 98KD and complementation (comp.) lines.Direct sequence chromatograms of PCR-amplified cDNA are shown. Gray shaded areas indicate the editing sites.


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Arabidopsis plastids (Robbins et al. 2009, Tseng et al. 2010). Lossof RARE1 function resulted in the complete loss of RNA editingat this site. In contrast, loss of VAC1 reduced the level of RNAediting to 62% at accD-C794 (Tseng et al. 2010). Thus, VAC1protein might act as an auxiliary factor for site-specific editingevents. In addition, two interacting editing factors, CRR4 andDYW1, perform RNA editing at a single site, the ndhD-1 site, inArabidopsis plastids (Boussardon et al. 2012). E-subclass PPRprotein CRR4 is a site recognition factor demonstrated to bedirectly associated with the target ndhD-1 site (Okuda et al.2006). In addition to PPR editing factors, RRM-type RNA-bind-ing proteins and MORF/RIPs have recently been identified as aprotein family broadly affecting RNA editing of mitochondriaand plastids (Tillich et al. 2009, Bentolila et al. 2012, Takenakaet al. 2012, Sun et al. 2013). Some MORF/RIPs were demon-strated to interact with PPR-E or PPR-DYW editing factors.These observations suggest that multiple protein componentsare involved in RNA editing and may constitute a large protein–RNA complex, the editosome, in plant organelles (Bentolilaet al. 2012). The P. patens genome does not encode MORF/RIPs but does code various RRM-type RNA-binding proteins.

In this study, we also identified PpPPR_98 as an atp9-specificediting factor. RNA editing at this site is accomplished afteratp9 pre-mRNA is spliced at the second intron. RNA editingat atp9-C92 alters a serine codon (UCG) to a leucine codon(UUG), the latter being an evolutionarily conserved amino acidamong various land plants. The encoded ATP9 is a c subunit ofFo-ATP synthase that forms the proton-translocating sector of

the ATP synthase rotor (Kehrein and Ott 2012). Loss of atp9-C92 editing probably causes severe defects in respiration andmay result in lethal phenotypes. This may be the reason why wewere unable to isolate any KO mutants in this study.

Taken together with all results of the present and previousstudies, eight PPR-DYW proteins required for all 11 known editingsites have been assigned in P. patens. This is the first evidence ofidentification of a complete set of PPR editing factors involved inall RNA editing events in plant mitochondria. PPR-DYW proteinsinteract with an unidentified editing factor(s) and control the C toU RNA editing reaction. We cannot exclude the possibility thatnon-PPR editing factors are involved in RNA editing. Cytidinedeaminase for RNA editing still remains to be identified in P.patens as well as in flowering plants. To address these questions,further genetic and biochemical experiments need to progressusing the model plant, P. patens.

Materials and Methods

Plant material and culture conditions

Protonemata of the moss P. patens subsp. patens were grown at25�C under constant white light as described previously(Sugiura et al. 2003).

Subcellular localization

The amplified cDNA fragment encoding the N-terminal 145and 104 amino acids of PpPPR_65 and 98, respectively, wascloned in-frame into the SmaI site in pKSPGFP9 (Tasaki et al.2010) to generate plasmids p65-GFP and p98-GFP. The PpPPR–GFP-coding region was amplified from the respective plasmidsand cloned into the SwaI site of p9WmycH13 (Ichinose et al.2012) to generate p65GFP-OX and p98GFP-OX. The generatedplasmid was linearized by NotI and introduced into the trans-genic Mt-RFP OX moss line expressing the mitochondria-loca-lized RFP protein (originally encoded by pMt-RFP, Uchida et al.2011). Finally, the transgenic PpPPR_65–GFP or PpPPR_98–GFP moss lines were selected on BCDAT plates containing30mg ml�1 hygromycin B and 50mg ml�1 zeocin (Invitrogen).GFP fluorescence was monitored using a confocal fluorescentmicroscope FLUOVIEW FV10i (Olympus).

Construction of plasmid and moss transformation

The 50 region (1,439 bp) and 30 region (1,374 bp) of thePpPPR_65 gene were amplified from the moss genomic DNAwith appropriate primers (Supplementary Table S1), andcloned into SwaI and SmaI sites, respectively, of the modifiedpKI-GFP (a derivative of pKSPGFP9, Tasaki et al. 2010). Theresulting plasmid was named pMI65-KO. The 35S promoterwas removed and the htp cassette was inserted into pKI-GFP.The 3�Myc tag, enterokinase cleavage site was generated bymutagenesis using PrimeSTAR GXL polymerase (TAKARA) andprimers containing 3�Myc and enterokinase cleavage sitesequences in-frame with the gfp gene.

Fig. 6 Detection of binding of the recombinant PpPPR_98 to thetarget RNA. (A) RNA sequence of RNA probes used for EMSA. Theintron region of the atp9-unspliced oligo RNA is underlined. (B) EMSAwas performed with the recombinant PPR proteins (r-98 and r-71) andlabeled oligo RNAs (atp9-spliced and atp9-unspliced). The concentra-tion of recombinant proteins is indicated above each lane. The pos-itions of the protein–RNA complex and free RNA are indicated bywhite and black arrowheads, respectively. An asterisk indicates a traceamount of unassigned RNA molecule.





The 50 region (3,188 bp) of the PpPPR_98 gene was amplifiedusing primers (Supplementary Table S1) and cloned intopGEM-T Easy (Promega) to generate plasmid pMI98. The hptgene expression cassette was amplified by PCR from p35-Hyg1(Uchida et al. 2011). The PCR product was inserted into theXbaI site in pMI98 and plasmid pMI98-hpt was generated.These plasmids were digested by NaeI or NotI, respectively,and introduced into P. patens protonemata by particle bom-bardment, and gene disruptants were selected as describedpreviously (Tasaki et al. 2010).

RNAi line selection

RNAi moss line selection was essentially performed accordingto Nakaoka et al. (2012). A target 0.8 kb DNA region encodingthe sixth to 14th PPR motifs of PpPPR_65 was introduced intothe pGG626 RNAi vector by the Gateway LR reaction (Nakaokaet al. 2012) and pMI-65RNAi was generated. The linearizedplasmid was introduced into 4-day-old protonemata of theGFP–tubulin/histone H2B–RFP expression line (GH line) byparticle bombardment, and hygromycin-resistant moss colo-nies were selected. To analyze RNAi mosses, the protonematawere cultured for 4 d in the presence of 1 mM b-estradiol.

Complementation line selection

The PpPPR_98 coding region was amplified from cDNAusing the primers listed in Supplementary Table S1. ThePCR product was cloned into the SwaI site of the overexpres-sion vector pOX9WZ1 (Sugita et al. 2012) and plasmid p98OXwas generated. This plasmid was digested by SacII and intro-duced into the KD line 98KD by particle bombardment. Zeocin-resistant transformants were selected on BCDAT platescontaining 30mg ml�1 hygromycin B and 50mg ml�1 zeocin.Transformants were confirmed by PCR using genomic DNAand appropriate primers (Supplementary Table S1).

RNA analysis

Isolation of RNA from 4-day-old protonemata, preparation ofRNA-free cDNA and RT–PCR, and detection of RNA editing bydirect sequencing were carried out as described previously(Ichinose et al. 2012). RNA editing efficiency was calculatedfrom the number of edited cDNA clones in sequenced cDNAclones. qRT–PCR was performed using Power SYBR Green PCRMaster Mix and the StepOnePlus Real-Time PCR system (modelStepOnePlus; Applied Biosystems). The data were normalizedwith the results for TUA1 (a-tubulin). Primers for qRT–PCR arelisted in Supplementary Table S1.

Production of the recombinant protein

The DNA sequence (2,748 bp) encoding PpPPR_98 proteinexcluding a putative transit peptide was amplified by PCRfrom cDNA using appropriate primers (SupplementaryTable S1). The amplified fragment was inserted in-frame intothe pBAD/Thio-TOPO vector (Invitrogen), allowing the proteinto be expressed as an N-terminal thioredoxin fusion protein

with six histidine residues at the C-terminus. The recombinantprotein was expressed in Escherichia coli BL21 (Novagen) andpurified as described previously (Tasaki et al. 2010).

EMSA

Synthetic 34-mer oligo RNAs, atp9-spliced (50- CUAUAGGUAUUGGAAACGUAUUUAGUUCUUCGAU-30) and atp9-unspliced(50-GGGGGGGUUUUGCACCCUAUCGGAAUUCUUCGAU-30,an intron region is underlined), were used for EMSA. Each syn-thetic oligo RNA was 50-end labeled with T4 polynucleotidekinase (TAKARA) and [g-32P]ATP at 37�C for 1 h, and thenextracted by ethanol precipitation. EMSA was performed bymixing various amounts of recombinant PPR protein with the32P-labeled synthetic oligo RNA probe (0.05 nM) as previouslydescribed (Tasaki et al. 2010). A synthetic 27-mer oligo RNA,unrelated RNA (50-AAAAAAAAAUAUAUAUAUAUUUUUUUU-30), was also used as non-labeled competitor RNA.

Supplementary data

Supplementary data are available at PCP online.

Funding

This work was supported by Japan Society for the Promotion ofScience (JSPS) KAKENHI [grant Nos. 25291059 and 25660292(to M.S.), 25660296 and 2592219 (to T.N.)]; JSPS [Grant-in-Aidfor JSPS Fellows (to M.I. and Y.Y.)]; the Novartis Foundation(Japan) for the Promotion of Science [to M.S.]; DAIKOFOUNDATION [research grant (to M.S.)]; the Adaptable andSeamless Technology Transfer Program through Target-drivenR&D [JST (to T.N.)].

Acknowledgments

We thank Professor Gota Goshima (Nagoya University) for thepGG626 RNAi vector and GH moss line, and Professor VolkerKnoop (University of Bonn, Germany) for exchanging unpub-lished results of PpPPR_65 function.

Disclosures

The authors have no conflicts of interest to declare.

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