Mol Genet Genomics

DOI 10.1007/s00438-009-0424-z

ORIGINAL PAPER

RNA editing: only eleven sites are present in the Physcomitrella patens mitochondrial transcriptome and a universal nomenclature proposal

Mareike Rüdinger · Helena T. Funk · Stefan A. Rensing · Uwe G. Maier · Volker Knoop

Received: 18 June 2008 / Accepted: 5 January 2009© Springer-Verlag 2009

Abstract RNA editing in mitochondria and chloroplastsof land plants alters the coding content of transcriptsthrough site-speciWc exchanges of cytidines into uridinesand vice versa. The abundance of RNA editing in modelplant species such as rice or Arabidopsis with some 500aVected sites in their organelle transcripts hinders straight-forward approaches to elucidate its mechanisms. The mossPhyscomitrella patens is increasingly being appreciated asan alternative plant model system, enhanced by the recentavailability of its complete chloroplast, mitochondrial, andnuclear genome sequences. We here report the transcrip-tomic analysis of Physcomitrella mitochondrial mRNAs asa prerequisite for future studies of mitochondrial RNA edit-ing in this moss. We Wnd a strikingly low frequency ofRNA editing aVecting only eleven, albeit highly important,sites of C-to-U nucleotide modiWcation in only nine mito-chondrial genes. Partial editing was seen for two of thesesites but no evidence for any silent editing sites (leavingthe identity of the encoded amino acid unchanged) as

commonly observed in vascular plants was found in Physc-omitrella, indicating a compact and eYcient organizationof the editing machinery. Furthermore, we here wish topropose a unifying nomenclature to clearly identify anddesignate RNA editing positions and to facilitate futurecommunication and database annotation.

Keywords Physcomitrella · RNA editing · Nomenclature · Mitochondrial DNA

Introduction

A number of gain-of-functions is apparently associatedwith the transition of what came to be land plants (embryo-phytes) from an aquatic to a terrestrial life style. One ofthese is the peculiar phenomenon of RNA editing of chloro-plast and mitochondrial transcripts commonly found in landplant, but not in green algal organelles (Gott and Emeson2000; Knoop 2004; Maier et al. 1996; Smith et al. 1997;Tillich et al. 2006). The targeted conversion of cytidinesto uridines, mostly in mRNAs and rarely also in tRNAor intron sequences, seems to have emerged concomitantwith the water-to-land transition (Covello and Gray 1993;Jobson and Qiu 2008; Maier et al. 2008; Steinhauser et al.1999). Originally reported in 1989 for mitochondrial mRNAsof angiosperms (Covello and Gray 1989; Gualbertoet al. 1989; Hiesel et al. 1989; Lamattina et al. 1989),C-to-U type of RNA editing was discovered in chloroplastsshortly thereafter (Hoch et al. 1991; Maier et al. 1992).Subsequent work has shown that RNA editing occurs inchloroplasts and mitochondria of all land plant clades withthe unique exception of the subclass of complex thalloidmarchantiid liverworts (Freyer et al. 1997; Groth-Maloneket al. 2007; Hiesel et al. 1994; Malek et al. 1996; Sper-Whitis

Communicated by B. Franz Lang.

M. Rüdinger · V. Knoop (&)Abteilung Molekulare Evolution, IZMB–Institut für Zelluläre und Molekulare Botanik, Universität Bonn, Kirschallee 1, 53115 Bonn, Germanye-mail: volker.knoop@uni-bonn.de

H. T. Funk · U. G. MaierFachbereich Biologie, Zellbiologie, Philipps-Universität Marburg, Karl-von-Frisch-Str., 35032 Marburg, Germany

S. A. RensingFRISYS, Faculty of Biology, University of Freiburg, Hauptstr. 1, 79104 Freiburg, Germany

123

Mol Genet Genomics

et al. 1994, 1996). Some basal land plant clades, most nota-bly the hornworts, lycophytes and ferns, also show a sig-niWcant amount of “reverse” (termed so for historicalreasons alone) U-to-C RNA editing (Groth-Malonek et al.2005; Kugita et al. 2003; Malek and Knoop 1998; Vange-row et al. 1999; Yoshinaga et al. 1996, 1997), a phenome-non that is only very rarely observed in angiosperms(Gualberto et al. 1990; Schuster et al. 1990a).

The overall frequency of RNA editing appears to be farmore lineage-dependent than gene-dependent. Among landplants, the general absence of RNA editing in marchantiidliverworts diVers from the high rates of RNA editing in thebasal liverwort Haplomitrium mnioides or in the quillwortIsoetes engelmannii (Lycopodiophyta), where the numberof RNA editing events in a single given mRNA may altermore than 100 nucleotides (F. Grewe and V. Knoop,unpublished observations). In total, some 300–500 RNAediting sites can be expected for the average angiospermmitochondrial transcriptome (Giegé and Brennicke 1999;Handa 2003; Mower and Palmer 2006; Notsu et al. 2002),signiWcantly more than the ca. 20–40 in the respective chlo-roplast transcriptomes (Inada et al. 2004; Sasaki et al. 2003;Tillich et al. 2001, 2005; Tsudzuki et al. 2001). Hence,plant RNA editing frequencies vary widely, which impli-cates the existence of diVerent rates of gains and losses ofediting sites and an array of speciWcity determinants forediting site recognition. The overwhelming majority ofediting sites serves to reconstruct evolutionarily conservedcodons in protein-encoding mRNAs and consequently,RNA editing can be reasonably well predicted from novelgenomic plant organelle sequences based on sequence com-parisons.

Like other popular model species in biology, the mossPhyscomitrella patens has assumed the role of a biologicalmodel organism due to its advantageous experimental qual-ities (Cove 2005; Frank et al. 2005; Quatrano et al. 2007;Rensing et al. 2002; Reski 1998; Reski and Cove 2004;Schaefer and Zryd 2001). A signiWcant further milestone inits ongoing establishment as a model plant species has beenthe recent completion of the Physcomitrella nucleargenome sequence (Rensing et al. 2008), shortly after theearlier completion of chloroplast (Sugiura et al. 2003) andmitochondrial (Terasawa et al. 2006) DNA sequencingprojects.

As a bryophyte, the life-cycle of P. patens is dominatedby the enduring haploid gametophyte stage. It occupies aninformative phylogenetic position halfway between greenalgae and Xowering plants such as the Brassicaceae Arabid-opsis thaliana, the model plant of molecular plant sciencespar excellence. Despite obvious and signiWcant diVerencesin angiosperm and bryophyte developmental processes dur-ing ontogenesis the molecular genetic data suggest thatmany genetic functions and the gene family diversity were

already present in the common ancestor of mosses and seedplants.

The completed organelle genome sequences of P. patenssuggested a surprisingly low requirement for RNA editingto correct coding sequences on the RNA level. If this is thecase, Physcomitrella qualiWes as a suitable model systemfor the investigation of RNA editing in that it has a manage-able number of RNA editing sites (and probably co-factors)to determine the speciWcities of editing site recognition. Tothis end we have investigated mitochondrial cDNAs andindeed could verify a very low amount of RNA editing inthe Physcomitrella mitochondrial transcriptome. Further-more, we suggest a general nomenclature for designation ofRNA editing sites to facilitate and standardize scientiWccommunication and database annotation in the future.

Materials and methods

RNA was prepared from plant material using the Nucleo-Spin® RNA Plant kit (Macherey-Nagel, Düren, Germany)or TRIZOL reagent (Invitrogen, Karlsruhe, Germany).RNA was additionally treated with DNase I (FermentasLife Sciences, St. Leon-Rot, Germany; Roche AppliedScience, Mannheim, Germany) to remove potential ves-tiges of DNA. First strand cDNA was synthesized using theRevertAid™ M-MulV® Reverse Transcriptase kit (Fermen-tas) or Omniscript (Qiagen, Hilden, Germany) and thehexanucleotide random primer mix (10 �M per assay; CarlRoth, Karlsruhe, Germany). DiVerent primer pairs border-ing complete mitochondrial genes or regions with potentialediting sites were used for cDNA assays (the completeprimer list is available from the authors upon request).PCR ampliWcation assays contained 1 �l template cDNA(10 ng–1 �g), 1£ recommended PCR buVer, 1.5–2.0 mMMgCl2, 2 mM dNTPs, 0.2–0.4 �M of each primer, 1 UDNA polymerase and double distilled water up to 25 �l.DiVerent commercially available thermostable DNA poly-merase kits were used: GoTaq® DNA Pol (Promega,Mannheim, Germany) to amplify PCR products for directsequencing and the Triplemaster® PCR System (Eppendorf,Hamburg, Germany), which was mainly used to amplifyPCR products for cloning. AmpliWcation assays included5 min initial denaturation at 94°C followed by 30 cycleseach with 30 s denaturation at 94°C, 30 s annealing at 50–52°C, 1–3 min synthesis at 72°C and a Wnal step of synthe-sis for 5 min at 72°C. PCR products were cloned into thepGEM-T Easy vector (Promega) before sequencing or weresequenced directly (Macrogen Inc., Seoul, South Korea).Direct sequencing of PCR products was also done on anABI PRISM® 377 DNA sequencer (Applied Biosystems,Darmstadt, Germany) using the DYEnamic ET TerminatorCycle Sequencing Kit (GE-Healthcare, Munich, Germany).

123

Mol Genet Genomics

cDNA sequences were aligned to the correspondinggene sequences of the annotated mitochondrial genomeof P. patens (http://www.ncbi.nlm.nih.gov, accession:NC_007945) with Sequencher 4.7 (Gene Codes Corporation,Ann Arbor, MI, USA) or MEGA 4 (Tamura et al. 2007)using the ClustalW algorithm (Thompson et al. 1994) andsubsequent manual reWnement. Folding of tRNAs was doneusing the Lowe lab tRNAscan-SE search server (http://lowelab.ucsc.edu/tRNAscan-SE).

Results

A nomenclature for RNA editing site annotation

Here, we suggest a nomenclature for RNA editing follow-ing a similar proposal for the annotation of organellarintrons, based on their location in the respective readingframes with numbering beginning at the Wrst nucleotide ofthe start codon (Dombrovska and Qiu 2004). The nameof the gene is to be followed by “e” for editing, “U or C”for the respective nucleotide created by RNA editing, thenucleotide position in the reading frame and the inducedcodon change (if any) using the single-letter amino acidcode. As an example “cox1eU755SL” denotes a C-to-URNA editing of the cox1 mRNA at position 755 (position 1corresponding to the A of the AUG start codon) with theconsequential change of a genomically encoded serinecodon into a leucine codon on mRNA level. Stop codonswould be designated with an asterisk (*), silent editingpositions would be identiWed through the same one-letteramino acid designation occurring twice. Designations suchas “atp1eU1501R*”, “nad3eC298*Q” or “nad7eU36PP”would be examples. The degree of partial RNA editingcould be reXected through a “p” followed by the percent-age of edited nucleotides identiWed in a cDNA pool, e.g.,“nad1eU39SSp40” for an RNA editing site reXected inonly 40% of cDNAs, as frequently observed for silentsites.

RNA editing in Physcomitrella patens mitochondria

To identify potential sites of RNA editing in the P. patensmitochondrial transcriptome, we Wrst inspected all deducedtRNA cloverleaf structures to identify potential sites whereC-to-U transitions could correct A-C mismatches to restorebase-pairings in the helical stem regions or restore antico-don identities or other uridine consensus positions con-served in functional tRNA molecules. However, no suchsites could be identiWed in any one of the 24 tRNAsequences encoded in the Physcomitrella mtDNA, whichhence appear to encode functional tRNAs without anynecessity of RNA editing (not shown).

Next, we aligned all protein translations deduced fromPhyscomitrella mitochondrial genes with those of the algaChara vulgaris and the liverwort Marchantia polymorpha(two species with known lack of organellar RNA editing).This should allow for the identiWcation of sequence posi-tions with amino acid codons potentially subject to changeby pyrimidine exchange RNA editing (P-S, P-L, S-L, S-F,L-F, H-Y, R-W, R-C, T-M, T-I, A-V, Q-*, R-*) that wouldintroduce codons for the identical amino acid or one chemi-cally more similar to homologous sequences in Chara and/or Marchantia. Several well conserved mitochondrial genesrevealed no such sites, thus suggesting no codon correc-tions at the RNA level. That highly conserved sequencescontaining no predicted editing sites correspond to little orno actual RNA editing is seen in the typical editing exam-ple of the nad6 sequence, in that its transcript is rampantlyedited in the Xowering plant Arabidopsis in comparison toPhyscomitrella (Fig. 1a). On the opposite end of the scale,among generally less conserved protein sequences, such asthe ccm genes encoding components of the cytochrome cmaturation pathway, several potential RNA editing siteswere identiWed but unable to be substantiated in Physcomit-rella. ccmB is an exemplary case in which none of the fourpotential RNA editing positions were conWrmed uponcDNA analysis, again in contrast to Arabidopsis, whereseveral sites of RNA editing had been conWrmed in theccmB reading frame (Fig. 1b). A diVerent picture emergedfrom cDNA analysis of ccmFC or nad5, respectively(Fig. 1c). In both cases, two predicted RNA editing posi-tions were indeed conWrmed through Physcomitrella cDNAanalyses.

Taken together, including the more remote hypotheticalpossibilities of RNA editing such as those assumed for themore variable ccm genes (Fig. 1b), we could identify a totalof 65 potential editing sites in 27 of the 42 annotated Physc-omitrella mitochondrial genes (including orf187, orf533and orf622). Due to the obviously limited predictability ofRNA editing as judged from analysis of the ccm genes, weperformed cDNA analyses of all those 42 Physcomitrellamitochondrial protein coding regions through RT-PCRbased approaches, in total targeting more than 31,000 kb ofcoding sequences. DiVerent sources of PhyscomitrellaRNA were used for independent approaches in the partici-pating laboratories with direct RT-PCR sequencing (Mar-burg) and cloning (Bonn) pursued in parallel. Upon cloningof RT-PCR products, at least three independent cDNAclones were sequenced to avoid overlooking only partiallyand/or silent RNA editing sites.

We generally found that only those sites with clear andunequivocal predictions for codon exchanges re-establish-ing conserved sequence positions in Chara and Marchantia(as well as in other taxa) were indeed altered on RNA level.In total, these amounted to only 11 positions of C-to-U

123

Mol Genet Genomics

Fig. 1 Exemplary alignment of mitochondrial DNA coding regions of nad6 (a), ccmB (b) and nad5 (c) in the liverwort Marchantia polymorpha, the alga Chara vulgaris, the moss Physcomitrella patens and the angiosperm Arabidopsis thaliana. Dots indicate amino acid identities with the top sequence. Highlighted are those amino acid positions where altered codons would reconsti-tute identities conserved in Chara or Marchantia through C to U RNA editing. RNA editing events conWrmed or identiWed through corresponding cDNA sequencing are indicated in bold and underlined. Only the core region of the ccmB coding region is shown in b which is aminoterminally extended with less conserved reading frames in Arabidopsis, Chara and Marchantia

a – nad6Marchantia MILFYVFVVL ALVSGAMVIR AKNPVHSVLF LILVFCNTSG LLVLLGLDFF AMIFLVVYVG AIAVLFLFVV Chara ....SI.SSI .....V.... .R........ .......... .......... .......... .......... Physcomitrella ....S..SSI ....SV.... .......... F....F.... .......... .......... .......... Arabidopsis ...-S.LSSP .....L..A. .......... P.P..RD... ..L....... ....P..HI. ....S.....

Marchantia MMLHIRIEEI HENVLRYLPV GGIIGLIFLL EIFLMVDNDY IPILPTKLSA TYLTYTVYAG KIHSWTNLET Chara ...N.K.A.. .......... .......... ....V..... ......E..T .VFQ.....S ..Q....... Physcomitrella ...N.K.A.. .......... .....V.... ...FI..... .........T .........E ..Q....... Arabidopsis ..F..Q.A.. ..E....... S.......WW .M.FIL..ES ..L...QRNT .S.R...... .VR.......

Marchantia LGNLLYTTYF FLFLVSSLIL LVALIGAIVL TMHKTTKVKR QDVFIQNAID FQNTIKKVR* Chara .......... Y......... ...M...... ......Q... ....Q..M.. ..K....I.Q * Physcomitrella .......... V......... ...M...... ......Q... ....R..... .K.....I.D I* Arabidopsis .......Y.S VW...P.... ...M...... ...R...... ....RR.... .RR..MRRTT DPLTIY*

b – ccmBMarchantia MIGFSKDFLC HFHLGLIWIC LLFSFLP--E RFFQNDFEDG TLELYYLSG- -YCLQKILLS KLYGHWVLQI Chara L....TNL.F ..N.....M. I......NMD .L........ .....C...P STR.....I. Q.LS..L.K. Physcomitrella .......... .......... .......--. ..L....... .....CS..- -......... .VV....... Arabidopsis .L..E...S. .S...P.R.P P..P.P.--A P.PR.EK... ........T- -...P....L Q.V..R.I..

Marchantia SGVFCSFPVL QLLYQFDQSK MNWFTIIIGS QIFTLMCGIH SCLALGITSN G--WNSLQNL TTLPTLLPLI Chara MAILL..... ....H.QY.T V.CL..VV.. LV...I.... ...T..LR.T DSRKT.FFFF .......... Physcomitrella .....T...V ........L. .....L.... L......... .......I.H S--....... .......... Arabidopsis .R...G..M. ..S...GR.G .DRLN.PL.. LVL..L.... .RS......S SG-...S..P ..S......T

Marchantia VFCTSIETEW FHVILLMGYL LLFLFFYPIL VSITLQTLLA K* Chara L....YQ..D EAMT.F.... ...F.C.... .....RN.IS Q. Physcomitrella I..A.T.... ...V...... .....L.A.. ......K.IS Q. Arabidopsis .SR....... ...LSSI..S S..VSLF..S ...S..D*-- --

c – nad5Marchantia MYLLIVILPL IGSFAAGFFG RFLGSRGVAV VTTTCVSLSS IFSCIAFYEV ALCASACYIK IAPWIFSELF Chara ......V... L..LV..V.. .......A.L .......I.. GLCF...... ..G....... F....L..M. Physcomitrella ......T... L..CV..A.. ....L..T.I .........F .L.L...... ..G....... ........M. Arabidopsis ......F... L..SV..... .....E.S.I M......F.. .L.L...... .PG.....LR .....S..M.

Marchantia DAAWGFLFDS LTVILLLVVT IVSSLVHIYS ISYMSEDPHS PRFFCYLSIF TFFMLMLVTG DNFIQLFLGW Chara ..S......T ...VM.I... F......... ........FL ...M...... .......... ..L..M.... Physcomitrella ..S...F... ...VM.I... F......L.. .......... ...M...... .......... .......... Arabidopsis ..S....... P..VM.I... SI.....L.. .......... ...M.....L ....P..... ..SL......

Marchantia EGVGLASYLL INFWFTRIQA NKAAIKAMLI NRVGDFGLAL GIMGCFTIFQ TVDFSTIFAC ASAFSEPHHY Chara .......... .......L.. .........V .......... ......A... .......... ....-VWEPS Physcomitrella .......... .......L.. .........V .......... .......... .........R .......... Arabidopsis .......... .H.....L.. D...T....V .......... ..S.R..L.. .........R ...---.RNS

Marchantia FLFCNMGFHA ITVICILVFI GAVGKSAQIG LHTWLPDAME GPTPVSALIH AATMVTAGVF MIARCSPLFE Chara .I.L..KI.. L.A..V.L.. .......... .......... .......... .......... .M........ Physcomitrella .I....R... .......L.. .......... ...R...... E......... .......... .......... Arabidopsis WIS...RLN. .SL....LL. .......... S...S..... .......S.. .......... ..........

Marchantia YSPNALIVIT FVGAMTSFFA ATTGILQNDL KRVIAYSTCS QLGYMIFACG ISNYSVSVFH LMNHACFKAL Chara .A.K...... .......... .......... .......... .....V.... .......... .....L.... Physcomitrella ...T...... .......... .......... .......... .......... .......... .....F.... Arabidopsis .P.T...... SA..T...L. .......... .......... .......... .......... .....F....

Marchantia LFLSAGSVIH AMSDEQDMRK MGGLASLLPF TYAMMLIGSL SLIGFPFLTG FYSKDVILEL AYTKYTISGN Chara .......... .......... .......... .........M .......... .......... .......... Physcomitrella .......... .......... .......... .......... .......C.. .......... .......... Arabidopsis .......... .......... ......SF.L .......... .......... .......... ..........

Marchantia FAFWLGSVSV FFTSYYSFRL LFLTFLAPTN SFKRDLSRCH DAPILMAIPL ILLAFGSIFV GYLAKDMMIG Chara L......L.. .L........ .......... A....IE... ...T...... .......L.. .......... Physcomitrella .......I.. .......... .......... .....IL... .......... .F........ ..V....... Arabidopsis .......... L......... ......V... ..G..I.... ....P..... ....L..L.. ..........

Marchantia LGTNFWANSL FILPKNEILA ESEFATPTII KLIPILFSTL GSFVAYSVNF VVNPLIFALK TSTFGNRLYC Chara ...H.....I ........MF S......RMM ........AI .A.L..R... CA.KF.YV.. ...L.TKC.. Physcomitrella .......... .......... .......... ....L..... .A.M..NI.. .A........ .......... Arabidopsis .........P LV........ .....A...T .........S .A....N..P .ADQFQR.FQ ....C....S

Marchantia FFNKRWFFDK VFNDFLARSF LRFGYEVSFK ALDKGAIEIL GPYGISYTIR KMAQQISKIQ SGFVYHYAFV Chara .L....L... .....I.K.. ........L. T......S.. ......T.F. .L.K...TL. .......... Physcomitrella .L........ I....IL.F. .......... V......... ........F. .L.K....L. .......... Arabidopsis .........Q .L....V... .........E .......... ........F. RL.ER..QL. .........A

Marchantia MLLGLTIFIS VIGLWDFISF WVDNRLYFIY IVSFLFINI* ------- Chara ..I......T I......... .......... LL.....HLE RDVRTN* Physcomitrella ..I......T I......... .......... .......HFE KDISTN* Arabidopsis ....S.L.VT FSRM..SL.S .....SS..L ...SFYTKSS QE*----

123

Mol Genet Genomics

exchange in the Physcomitrella mitochondrial transcripts(Fig. 2). Single positions of RNA editing were identiWed inseven genes (atp9, cox1, cox2, cox3, nad3, nad4 and rps14)and two genes were each found to carry two RNA editingsites (nad5, ccmFC). No evidence for any accompanyingsilent RNA editing in third codon positions (or Wrst codonposition of YUR leucine codons) or in the transcribed inter-genic regions were identiWed in the cDNA sequences,which is fully understandable in the context of the overallvery low degree of RNA editing in Physcomitrella.

To assess the general observation of approximately ten-fold lower RNA editing in chloroplasts than in mitochon-dria of land plants, we investigated possible RNA editingsites in P. patens chloroplasts in a computational approach.However, we identiWed no further candidate editing sitesother than the two already determined (Miyata et al. 2002;Miyata and Sugita 2004), to be labelled rps14eU2TM andrps14eU-1 according to our nomenclature proposal.

Partial RNA editing and co-transcription in Physcomitrella mitochondria

Sequences of independent cDNA clones as well as directsequencing of RT-PCR products generally reXected completeRNA editing in the Physcomitrella mitochondrial steady-statemRNA pool with the exception of two editing events:rps14eU137SL and nad3eU230SL. Direct sequencing of therespective RT-PCR products showed partial editing of therespective sites in rps14 and nad3 (Fig. 3). Independent rps14cDNA clones reXected the editing event in eight out of twelveclones and, according to our proposal, this editing wouldhence be designated as rps14eU137SLp66.

In some cases our RT-PCR-based cDNA studies led theidentiWcation of the co-transcription of adjacent genes: rpl2with rps19 and rps3 (separated by spacers of only 3 or 6 bp,respectively), rpl5 with rps14 (separated by a 4-bp spacer),but also of ccmB with ccmC (separated by a 463-bpspacer).

Discussion

Considering the total of only eleven sites, of which two arepartially edited, RNA editing is a comparatively rare phe-nomenon in mitochondrial transcripts of P. patens. Wefound that only the codon positions reconstituting highlyconserved amino acids in the encoded proteins were subjectto sense-correction through C-to-U exchanges in mRNAs

Fig. 2 Locations of all eleven RNA editing sites identiWed in the transcripts of nine mitochondrial genes of Physcomitrella patens. The newnomenclature proposed in this manuscript is used to designate RNA editing positions

atp9

atp9eU92SL

cox2

cox2eU370RW

cox3

cox3eU733RW

nad3

nad3eU230SLp

nad4

nad4eU272SL

ccmFC

ccmFCeU103PS ccmFCeU122SF

rps14

rps14eU137SLp66

cox1eU755SL

cox1

nad5

nad5eU598RC nad5eU730RW

100 bp

Fig. 3 Pherograms of the rps14 (left) and nad3 (right) RT-PCR prod-uct direct sequencing. The simultaneous occurrence of a T and C peakin the pherograms at the positions labelled N reXect the partial RNAediting events rps14eU137SL and nad3eU230SL

123

Mol Genet Genomics

(Fig. 2). Other than its general scarcity in Physcomitrella,RNA editing follows the usual pattern known from otherland plants. In this species it restores, on RNA level,leucine, serine, phenylalanine, tryptophane and cysteinecodons from genomically encoded serine, proline or argi-nine codons (Fig. 2). The transformations of codons forthreonine to isoleucine or for alanine to valine are muchrarer in general and no examples at all were identiWed inPhyscomitrella (although rps3eU1397TI seemed to be areasonable candidate site). This most likely reXects thesame underlying biochemical mechanisms as in vascularplants, for example, a likely sterical hindrance by the largerpurine nucleotides preceding such second codon positionediting events. No silent editing, no editing in non-codingregions and no examples for reverse U-to-C exchangescould be identiWed. The low degree of RNA editing inPhyscomitrella is, however, in no way indicative of a gen-erally low RNA editing activity in mosses, which varieswidely in this clade (Beckert et al. 2001).

Across the plant kingdom, the frequency of organellarRNA editing varies from zero to several hundred sites. Ifpresent, RNA editing always co-exists in both organelleswith an approximately tenfold lower frequency in chloro-plasts as compared to mitochondria. The two RNA editingsites in chloroplasts versus the eleven now identiWed inmitochondria of P. patens nicely mirror the editing ratio ofplant organelles hitherto observed.

There is reason to assume that RNA editing in plantorganelles is monophyletic. In this scenario, the earliestland plants would have evolved the organellar editing sys-tem as a mechanism to correct mutations after exposure tothe new terrestrial environment. Extant taxa accordinglywould reXect the secondary gains and losses of individualRNA editing sites in their editotypes during »450 MY ofevolution. In the light of signiWcantly higher rates of RNAediting in other bryophytes, most notably in the basal mossgenus Takakia; (Miyata et al. 2008; Pruchner et al. 2001;Sugita et al. 2006), Physcomitrella would be an example ofan impressive rate of back mutations, leaving behind onlyeleven mitochondrial and two chloroplastic editing sites.

Soon after the discovery of organelle RNA editing inplants it became clear that the steady-state pool of mRNAsfor a given gene contained either diVerentially or partiallyedited molecules (Gualberto et al. 1991; Schuster et al.1990c; Yang and Mulligan 1991). In our study we couldidentify two of the eleven editing sites in P. patens mito-chondria to be partially edited. A resulting protein hetero-geneity from diVerentially edited RNAs was reported inone case (Lu et al. 1996; Phreaner et al. 1996) but not inothers (Lu and Hanson 1994; Williams et al. 1998). Thereis as yet no reasonable evidence for an additional level ofmodulating protein heterogeneity with physiological conse-quences through partially edited mRNAs. We consider it

likely that partial editing of essential positions is detectedfrom transcripts that are still being processed (Verbitskiyet al. 2006). RNA editing of silent positions, which are fre-quently found only partially edited, (Hirose et al. 1996;Kempken et al. 1991) may simply reXect non-speciWc bind-ing of editing speciWcity factors as a side-eVect of targetingmore relevant sites.

Apparently, certain nuclear-encoded proteins are speciW-cally associated with individual RNA editing sites. Amongthese, the RNA-binding pentatricopeptide repeat (PPR)proteins seem to play prominent roles (Kotera et al. 2005;Okuda et al. 2006, 2007). The PPR gene family is not onlyvastly extended in the land plant lineage (to more than 400members in angiosperms), but also displays a surprisingevolutionary dynamic in the nuclear genomes (Andrés et al.2007; Delannoy et al. 2007; Geddy and Brown 2007; Lurinet al. 2004; Rivals et al. 2006; Saha et al. 2007; Small andPeeters 2000). Although the overall expansion of PPR pro-tein family members in the course of land plant evolution(O’Toole et al. 2008) is striking in itself, even more com-pelling is the correlation of the relative level of editingamong land plants and the abundance of members of onesubclade of PPR, the DYW proteins, containing a speciWccarboxyterminal extension (Rüdinger et al. 2008; Saloneet al. 2007). The Physcomitrella nuclear genome containsonly 103 PPR genes of which only 10 are DYW-typegenes, the rarity of which correlates well with the rarity ofRNA editing in its mitochondrial and chloroplast tran-scripts, making the model moss an attractive object forfunctional investigation of plant organelle RNA editing.

The suggested nomenclature allows Xexibility throughextension; an obvious example is the designation of partialRNA editing as described here. For non-silent editing oftwo adjacent sites in YYN codons aVecting the proline-serine-leucine-phenylalanine group, we suggest that suchtandem editing should be designated with a nucleotideduplet labelling, e.g., as nad5eUU13PF.

RNA editing of tRNAs or introns could be annotatedaccordingly, obviously without resulting amino acid changes.As an example, the well characterized RNA editing eventin the acceptor arm of mitochondrial tRNA-Phe(GAA) inangiosperms (Binder et al. 1994), reconstituting a canonicalU-A basepair as a prerequisite for subsequent processing fromthe precursor (Marchfelder et al. 1996), would be designatedtrnF(GAA)eU4. The three RNA editing sites identiWed indomains V and VI of wheat mitochondrial group II intronsin nad7 (Carrillo and Bonen 1997) would be labelledaccordingly as nad7i676eU887, nad7i917eU1589, andnad7i917eU1657, respectively.

For the annotation of the rare editing events upstream ordownstream of coding regions or in transcribed spacers,we suggest a nomenclature referring to the distance to therespective closest coding region with “¡” (upstream of the

123

Mol Genet Genomics

start codon) or “+” (downstream of the stop codon). TheRNA editing event reported in the 5�-UTR (untranslatedregion) four nucleotides upstream of the mitochondrialrps14 reading frame in Oenothera berteriana, suggested toimprove a ribosome binding site (Schuster et al. 1990b),would accordingly be labelled rps14eU-4.

Acknowledgments Research in the authors’ laboratories is sup-ported by grants from the Deutsche Forschungsgemeinschaft DFG, inparticular SFB-TR1 (Marburg), and BMBF FRISYS (Freiburg). Wethank Andrew Bozarth for comments on the manuscript. H.T.F. thanksChristopher Grosche for skilful lab assistance. M.R. is grateful to JuliaDreistein for concentrated and helpful work during a laboratory prac-tical training and to Judith Schleppenbäumer and Thomas Münster,MPI Cologne, who have kindly provided the P. patens Gransden strain.We wish to dedicate this publication to the memory of Rudolf Hiesel,colleague, friend and co-discoverer of RNA editing in plants, whopassed away much too early.

References

Andrés C, Lurin C, Small ID (2007) The multifarious roles of PPR pro-teins in plant mitochondrial gene expression. Physiol Plantarum129(1):14–22

Beckert S, Muhle H, Pruchner D, Knoop V (2001) The mitochondrialnad2 gene as a novel marker locus for phylogenetic analysis ofearly land plants: a comparative analysis in mosses. Mol Phyloge-net Evol 18(1):117–126

Binder S, Marchfelder A, Brennicke A (1994) RNA editing of tRNAPhe

and tRNACys in mitochondria of Oenothera berteriana is initiatedin precursor molecules. Mol Gen Genet 244(1):67–74

Carrillo C, Bonen L (1997) RNA editing status of nad7 intron domainsin wheat mitochondria. Nucleic Acids Res 25(2):403–409

Cove D (2005) The moss Physcomitrella patens. Annu Rev Genet39:339–358

Covello PS, Gray MW (1989) RNA editing in plant mitochondria.Nature 341(6243):662–666

Covello PS, Gray MW (1993) On the evolution of RNA editing.Trends Genet 9(8):265–268

Delannoy E, Stanley WA, Bond CS, Small ID (2007) Pentatricopep-tide repeat (PPR) proteins as sequence-speciWcity factors in post-transcriptional processes in organelles. Biochem Soc Trans 35(Pt6):1643–1647

Dombrovska E, Qiu YL (2004) Distribution of introns in the mitochon-drial gene nad1 in land plants: phylogenetic and molecular evolu-tionary implications. Mol Phylogenet Evol 32(1):246–263

Frank W, Decker EL, Reski R (2005) Molecular tools to study Physc-omitrella patens. Plant Biol (Stuttg) 7(3):220–227

Freyer R, Kiefer-Meyer M-C, Kössel H (1997) Occurrence of plastidRNA editing in all major lineages of land plants. Proc Natl AcadSci USA 94(12):6285–6290

Geddy R, Brown GG (2007) Genes encoding pentatricopeptiderepeat (PPR) proteins are not conserved in location in plantgenomes and may be subject to diversifying selection. BMCGenomics 8:130

Giegé P, Brennicke A (1999) RNA editing in Arabidopsis mitochon-dria eVects 441 C to U changes in ORFs. Proc Natl Acad Sci USA96(26):15324–15329

Gott JM, Emeson RB (2000) Functions and mechanisms of RNAediting. Annu Rev Genet 34:499–531

Groth-Malonek M, Pruchner D, Grewe F, Knoop V (2005) Ancestorsof trans-splicing mitochondrial introns support serial sister group

relationships of hornworts and mosses with vascular plants. MolBiol Evol 22(1):117–125

Groth-Malonek M, Wahrmund U, Polsakiewicz M, Knoop V (2007)Evolution of a pseudogene: exclusive survival of a functionalmitochondrial nad7 gene supports Haplomitrium as the earliestliverwort lineage and proposes a secondary loss of RNA editingin Marchantiidae. Mol Biol Evol 24(4):1068–1074

Gualberto JM, Lamattina L, Bonnard G, Weil JH, Grienenberger JM(1989) RNA editing in wheat mitochondria results in the conser-vation of protein sequences. Nature 341(6243):660–662

Gualberto JM, Weil JH, Grienenberger JM (1990) Editing of the wheatcoxIII transcript—evidence for 12 C to U and one U to C conver-sions and for sequence similarities around editing sites. NucleicAcids Res 18(13):3771–3776

Gualberto JM, Bonnard G, Lamattina L, Grienenberger JM (1991)Expression of the wheat mitochondrial nad3-rps12 transcriptionunit: correlation between editing and mRNA maturation. PlantCell 3(10):1109–1120

Handa H (2003) The complete nucleotide sequence and RNA editing con-tent of the mitochondrial genome of rapeseed (Brassica napus L.):comparative analysis of the mitochondrial genomes of rapeseedand Arabidopsis thaliana. Nucleic Acids Res 31(20):5907–5916

Hiesel R, Wissinger B, Schuster W, Brennicke A (1989) RNA editingin plant mitochondria. Science 246(4937):1632–1634

Hiesel R, Combettes B, Brennicke A (1994) Evidence for RNA editingin mitochondria of all major groups of land plants except theBryophyta. Proc Natl Acad Sci USA 91(2):629–633

Hirose T, Fan H, Suzuki JY, Wakasugi T, Tsudzuki T, Kössel H,Sugiura M (1996) Occurrence of silent RNA editing in chloro-plasts: its species speciWcity and the inXuence of environmentaland developmental conditions. Plant Mol Biol 30(3):667–672

Hoch B, Maier RM, Appel K, Igloi GL, Kössel H (1991) Editing of achloroplast mRNA by creation of an initiation codon. Nature353(6340):178–180

Inada M, Sasaki T, Yukawa M, Tsudzuki T, Sugiura M (2004) Asystematic search for RNA editing sites in pea chloroplasts: anediting event causes diversiWcation from the evolutionarily con-served amino acid sequence. Plant Cell Physiol 45(11):1615–1622

Jobson RW, Qiu YL (2008) Did RNA editing in plant organellargenomes originate under natural selection or through geneticdrift? Biol Direct 3(1):43

Kempken F, Mullen JA, Pring DR, Tang HV (1991) RNA editing ofsorghum mitochondrial atp6 transcripts changes 15 amino acidsand generates a carboxy-terminus identical to yeast. Curr Genet20(5):417–422

Knoop V (2004) The mitochondrial DNA of land plants: peculiaritiesin phylogenetic perspective. Curr Genet 46(3):123–139

Kotera E, Tasaka M, Shikanai T (2005) A pentatricopeptide repeatprotein is essential for RNA editing in chloroplasts. Nature433(7023):326–330

Kugita M, Yamamoto Y, Fujikawa T, Matsumoto T, Yoshinaga K(2003) RNA editing in hornwort chloroplasts makes more thanhalf the genes functional. Nucleic Acids Res 31(9):2417–2423

Lamattina L, Weil JH, Grienenberger JM (1989) RNA editing at asplicing site of NADH dehydrogenase subunit IV gene transcriptin wheat mitochondria. FEBS Lett 258(1):79–83

Lu B, Hanson MR (1994) A single homogeneous form of ATP6protein accumulates in petunia mitochondria despite thepresence of diVerentially edited atp6 transcripts. Plant Cell6(12):1955–1968

Lu B, Wilson RK, Phreaner CG, Mulligan RM, Hanson MR (1996)Protein polymorphism generated by diVerential RNA editing of aplant mitochondrial rps12 gene. Mol Cell Biol 16(4):1543–1549

Lurin C, Andrés C, Aubourg S, Bellaoui M, Bitton F, Bruyère C,Caboche M, Debast C, Gualberto J, HoVmann B, Lecharny A, LeRet M, Martin-Magniette ML, Mireau H, Peeters N, Renou JP,

123

Mol Genet Genomics

Szurek B, Taconnat L, Small I (2004) Genome-wide analysis ofArabidopsis pentatricopeptide repeat proteins reveals their essen-tial role in organelle biogenesis. Plant Cell 16(8):2089–2103

Maier RM, Hoch B, Zeltz P, Kössel H (1992) Internal editing of themaize chloroplast ndhA transcript restores codons for conservedamino acids. Plant Cell 4(5):609–616

Maier RM, Zeltz P, Kössel H, Bonnard G, Gualberto JM, Grienenber-ger JM (1996) RNA editing in plant mitochondria and chloro-plasts. Plant Mol Biol 32(1–2):343–365

Maier UG, Bozarth A, Funk HT, Zauner S, Rensing SA, Schmitz-Linneweber C, Börner T, Tillich M (2008) Complex chloroplastRNA metabolism: just debugging the genetic programme? BMCBiol 6:36

Malek O, Knoop V (1998) Trans-splicing group II introns in plantmitochondria: the complete set of cis-arranged homologs in ferns,fern allies, and a hornwort. RNA 4(12):1599–1609

Malek O, Lättig K, Hiesel R, Brennicke A, Knoop V (1996) RNA edit-ing in bryophytes and a molecular phylogeny of land plants.EMBO J 15(6):1403–1411

Marchfelder A, Brennicke A, Binder S (1996) RNA editing is requiredfor eYcient excision of tRNA(Phe) from precursors in plant mito-chondria. J Biol Chem 271(4):1898–1903

Miyata Y, Sugita M (2004) Tissue- and stage-speciWc RNA editing ofrps14 transcripts in moss (Physcomitrella patens) chloroplasts.J Plant Physiol 161(1):113–115

Miyata Y, Sugiura C, Kobayashi Y, Hagiwara M, Sugita M (2002)Chloroplast ribosomal S14 protein transcript is edited to create atranslation initiation codon in the moss Physcomitrella patens.Biochim Biophys Acta 1576(3):346–349

Miyata Y, Sugita C, Maruyama K, Sugita M (2008) RNA editing in theanticodon of tRNA(Leu) (CAA) occurs before group I intronsplicing in plastids of a moss Takakia lepidozioides S. Hatt. &Inoue. Plant Biol (Stuttg) 10(2):250–255

Mower JP, Palmer JD (2006) Patterns of partial RNA editing in mito-chondrial genes of Beta vulgaris. Mol Genet Genomics276(3):285–293

Notsu Y, Masood S, Nishikawa T, Kubo N, Akiduki G, Nakazono M,Hirai A, Kadowaki K (2002) The complete sequence of the rice(Oryza sativa L.) mitochondrial genome: frequent DNA sequenceacquisition and loss during the evolution of Xowering plants. MolGenet Genomics 268(4):434–445

O’Toole N, Hattori M, Andres C, Iida K, Lurin C, Schmitz-Linne-weber C, Sugita M, Small I (2008) On the expansion of thepentatricopeptide repeat gene family in plants. Mol Biol Evol25(6):1120–1128

Okuda K, Nakamura T, Sugita M, Shimizu T, Shikanai T (2006) Apentatricopeptide repeat protein is a site-recognition factor inchloroplast RNA editing. J Biol Chem 281(49):37661–37667

Okuda K, Myouga F, Motohashi R, Shinozaki K, Shikanai T (2007)Conserved domain structure of pentatricopeptide repeat proteinsinvolved in chloroplast RNA editing. Proc Natl Acad Sci USA104(19):8178–8183

Phreaner CG, Williams MA, Mulligan RM (1996) Incomplete editingof rps12 transcripts results in the synthesis of polymorphic poly-peptides in plant mitochondria. Plant Cell 8(1):107–117

Pruchner D, Nassal B, Schindler M, Knoop V (2001) Mosses sharemitochondrial group II introns with Xowering plants, not withliverworts. Mol Genet Genomics 266(4):608–613

Quatrano RS, McDaniel SF, Khandelwal A, Perroud PF, Cove DJ(2007) Physcomitrella patens: mosses enter the genomic age.Curr Opin Plant Biol 10(2):182–189

Rensing SA, Rombauts S, Van de Peer Y, Reski R (2002) Moss tran-scriptome and beyond. Trends Plant Sci 7(12):535–538

Rensing SA, Lang D, Zimmer AD, Terry A, Salamov A, Shapiro H,Nishiyama T, Perroud PF, Lindquist EA, Kamisugi Y, TanahashiT, Sakakibara K, Fujita T, Oishi K, Shin I, Kuroki Y, Toyoda A,

Suzuki Y, Hashimoto S, Yamaguchi K, Sugano S, Kohara Y,Fujiyama A, Anterola A, Aoki S, Ashton N, Barbazuk WB,Barker E, Bennetzen JL, Blankenship R, Cho SH, Dutcher SK,Estelle M, Fawcett JA, Gundlach H, Hanada K, Heyl A, HicksKA, Hughes J, Lohr M, Mayer K, Melkozernov A, Murata T,Nelson DR, Pils B, Prigge M, Reiss B, Renner T, Rombauts S,Rushton PJ, Sanderfoot A, Schween G, Shiu SH, Stueber K,Theodoulou FL, Tu H, Van de Peer Y, Verrier PJ, Waters E,Wood A, Yang L, Cove D, Cuming AC, Hasebe M, Lucas S,Mishler BD, Reski R, Grigoriev IV, Quatrano RS, Boore JL(2008) The Physcomitrella genome reveals evolutionary insightsinto the conquest of land by plants. Science 319(5859):64–69

Reski R (1998) Physcomitrella and Arabidopsis: the David and Goli-ath of reverse genetics. Trends Plant Sci 3(6):209–210

Reski R, Cove DJ (2004) Physcomitrella patens. Curr Biol 14(7):R261–R262

Rivals E, Bruyere C, ToVano-Nioche C, Lecharny A (2006) Formationof the Arabidopsis pentatricopeptide repeat family. Plant Physiol141(3):825–839

Rüdinger M, Polsakiewicz M, Knoop V (2008) Organellar RNA edit-ing and plant-speciWc extensions of pentatricopeptide repeat(PPR) proteins in jungermanniid but not in marchantiid liver-worts. Mol Biol Evol 25(7):1405–1414

Saha D, Prasad AM, Srinivasan R (2007) Pentatricopeptide repeat pro-teins and their emerging roles in plants. Plant Physiol Biochem45(8):521–534

Salone V, Rüdinger M, Polsakiewicz M, HoVmann B, Groth-MalonekM, Szurek B, Small I, Knoop V, Lurin C (2007) A hypothesis onthe identiWcation of the editing enzyme in plant organelles. FEBSLett 581(22):4132–4138

Sasaki T, Yukawa Y, Miyamoto T, Obokata J, Sugiura M (2003) Iden-tiWcation of RNA editing sites in chloroplast transcripts from thematernal and paternal progenitors of tobacco (Nicotiana taba-cum): comparative analysis shows the involvement of distincttrans-factors for ndhB editing. Mol Biol Evol 20(7):1028–1035

Schaefer DG, Zryd JP (2001) The moss Physcomitrella patens, nowand then. Plant Physiol 127(4):1430–1438

Schuster W, Hiesel R, Wissinger B, Brennicke A (1990a) RNA editingin the cytochrome b locus of the higher plant Oenothera berteri-ana includes a U-to-C transition. Mol Cell Biol 10(5):2428–2431

Schuster W, Unseld M, Wissinger B, Brennicke A (1990b) Ribosomalprotein S14 transcripts are edited in Oenothera mitochondria.Nucleic Acids Res 18(2):229–233

Schuster W, Wissinger B, Unseld M, Brennicke A (1990c) Transcriptsof the NADH-dehydrogenase subunit 3 gene are diVerentiallyedited in Oenothera mitochondria. EMBO J 9(1):263–269

Small ID, Peeters N (2000) The PPR motif—a TPR-related motifprevalent in plant organellar proteins. Trends Biochem Sci25(2):46–47

Smith HC, Gott JM, Hanson MR (1997) A guide to RNA editing. RNA3(10):1105–1123

Sper-Whitis GL, Russell AL, Vaughn JC (1994) Mitochondrial RNAediting of cytochrome c oxidase subunit II (coxII) in the primitivevascular plant Psilotum nudum. Biochem Biophys Acta GeneStruct Expr 1218(2):218–220

Sper-Whitis GL, Moody JL, Vaughn JC (1996) Universality of mito-chondrial RNA editing in cytochrome-c oxidase subunit I (coxI)among the land plants. Biochem Biophys Acta Gene Struct Expr1307(3):301–308

Steinhauser S, Beckert S, Capesius I, Malek O, Knoop V (1999) Plantmitochondrial RNA editing: extreme in hornworts and dividingthe liverworts? J Mol Evol 48(3):303–312

Sugita M, Miyata Y, Maruyama K, Sugiura C, Arikawa T, Higuchi M(2006) Extensive RNA editing in transcripts from the psbB oper-on and rpoA gene of plastids from the enigmatic moss Takakialepidozioides. Biosci Biotechnol Biochem 70(9):2268–2274

123

Mol Genet Genomics

Sugiura C, Kobayashi Y, Aoki S, Sugita C, Sugita M (2003) Completechloroplast DNA sequence of the moss Physcomitrella patens:evidence for the loss and relocation of rpoA from the chloroplastto the nucleus. Nucleic Acids Res 31(18):5324–5331

Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evo-lutionary genetics analysis (MEGA) software version 4.0. MolBiol Evol 24(8):1596–1599

Terasawa K, Odahara M, Kabeya Y, Kikugawa T, Sekine Y, FujiwaraM, Sato N (2006) The mitochondrial genome of the moss Physc-omitrella patens sheds new light on mitochondrial evolution inland plants. Mol Biol Evol 24(3):699–709

Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improv-ing the sensitivity of progressive multiple sequence alignmentthrough sequence weighting, position-speciWc gap penalties andweight matrix choice. Nucleic Acids Res 22(22):4673–4680

Tillich M, Schmitz-Linneweber C, Herrmann RG, Maier RM (2001)The plastid chromosome of maize (Zea mays): update of the com-plete sequence and transcript editing sites. Maize Genet CoopNewslett 75:42–44

Tillich M, Funk HT, Schmitz-Linneweber C, Poltnigg P, Sabater B,Martin M, Maier RM (2005) Editing of plastid RNA in Arabidop-sis thaliana ecotypes. Plant J 43(5):708–715

Tillich M, Lehwark P, Morton BR, Maier UG (2006) The evolution ofchloroplast RNA editing. Mol Biol Evol 23(10):1912–1921

Tsudzuki T, Wakasugi T, Sugiura M (2001) Comparative analysis ofRNA editing sites in higher plant chloroplasts. J Mol Evol 53(4–5):327–332

Vangerow S, Teerkorn T, Knoop V (1999) Phylogenetic informationin the mitochondrial nad5 gene of pteridophytes: RNA editingand intron sequences. Plant Biol 1(2):235–243

Verbitskiy D, Takenaka M, Neuwirt J, van der Merwe JA, BrennickeA (2006) Partially edited RNAs are intermediates of RNA editingin plant mitochondria. Plant J 47(3):408–416

Williams MA, Tallakson WA, Phreaner CG, Mulligan RM (1998)Editing and translation of ribosomal protein S13 transcripts:unedited translation products are not detectable in maize mito-chondria. Curr Genet 34(3):221–226

Yang AJ, Mulligan RM (1991) RNA editing intermediates of cox2transcripts in maize mitochondria. Mol Cell Biol 11(8):4278–4281

Yoshinaga K, Iinuma H, Masuzawa T, Uedal K (1996) Extensive RNAediting of U to C in addition to C to U substitution in the rbcLtranscripts of hornwort chloroplasts and the origin of RNA editingin green plants. Nucleic Acids Res 24(6):1008–1014

Yoshinaga K, Kakehi T, Shima Y, Iinuma H, Masuzawa T, Ueno M(1997) Extensive RNA editing and possible double-strandedstructures determining editing sites in the atpB transcripts ofhornwort chloroplasts. Nucleic Acids Res 25(23):4830–4834

123