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• W2090953675 abstract "RNA editing in flowering plant mitochondria post-transcriptionally alters several hundred nucleotides from C to U, mostly in mRNAs. Several factors required for specific RNA-editing events in plant mitochondria and plastids have been identified, all of them PPR proteins of the PLS subclass with a C-terminal E-domain and about half also with an additional DYW domain. Based on this information, we here probe the connection between E-PPR proteins and RNA editing in plant mitochondria. We initiated a reverse genetics screen of T-DNA insertion lines in Arabidopsis thaliana and investigated 58 of the 150 E-PPR-coding genes for a function in RNA editing. Six genes were identified to be involved in mitochondrial RNA editing at specific sites. Homozygous mutants of the five genes MEF18-MEF22 display no gross disturbance in their growth or development patterns, suggesting that the editing sites affected are not crucial at least in the greenhouse. These results show that a considerable percentage of the E-PPR proteins are involved in the functional processing of site-specific RNA editing in plant mitochondria. RNA editing in flowering plant mitochondria post-transcriptionally alters several hundred nucleotides from C to U, mostly in mRNAs. Several factors required for specific RNA-editing events in plant mitochondria and plastids have been identified, all of them PPR proteins of the PLS subclass with a C-terminal E-domain and about half also with an additional DYW domain. Based on this information, we here probe the connection between E-PPR proteins and RNA editing in plant mitochondria. We initiated a reverse genetics screen of T-DNA insertion lines in Arabidopsis thaliana and investigated 58 of the 150 E-PPR-coding genes for a function in RNA editing. Six genes were identified to be involved in mitochondrial RNA editing at specific sites. Homozygous mutants of the five genes MEF18-MEF22 display no gross disturbance in their growth or development patterns, suggesting that the editing sites affected are not crucial at least in the greenhouse. These results show that a considerable percentage of the E-PPR proteins are involved in the functional processing of site-specific RNA editing in plant mitochondria. IntroductionRNA editing in mitochondria of flowering plants changes 400–500 selected cytosines to uridines mostly in coding regions of mRNAs and some tRNAs (1Giegé P. Brennicke A. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 15324-15329Crossref PubMed Scopus (311) Google Scholar, 2Handa H. Nucleic Acids Res. 2003; 31: 5907-5916Crossref PubMed Scopus (292) Google Scholar, 3Takenaka M. Verbitskiy D. van der Merwe J.A. Zehrmann A. Brennicke A. Mitochondrion. 2008; 8: 35-46Crossref PubMed Scopus (109) Google Scholar). In non-flowering plants such as Isoetes species, the number of edited nucleotides is estimated to be more than 1,500 (4Grewe F. Viehoever P. Weisshaar B. Knoop V. Nucleic Acids Res. 2009; 37: 5093-5104Crossref PubMed Scopus (105) Google Scholar), raising the question of how the to-be-edited nucleotides are distinguished from other unaltered moieties.Specific sequence contexts in the pre-mRNA yield the first part of an answer; such cis-elements are required to identify a bona fide editing site as in vivo, in organelle, and in vitro analyses of several mitochondrial RNA-editing sites have shown (5Bock R. Hermann M. Kössel H. EMBO J. 1996; 15: 5052-5059Crossref PubMed Scopus (133) Google Scholar, 6Bock R. Koop H.U. EMBO J. 1997; 16: 3282-3288Crossref PubMed Scopus (59) Google Scholar, 7Chateigner-Boutin A.L. Hanson M.R. Mol. Cell. Biol. 2002; 22: 8448-8456Crossref PubMed Scopus (66) Google Scholar, 8Farré J.C. Leon G. Jordana X. Araya A. Mol. Cell. Biol. 2001; 21: 6731-6737Crossref PubMed Scopus (90) Google Scholar, 9Kempken F. Bolle N. Bruhs A. Endocytobiosis Cell Res. 2009; 19: 1-10Crossref PubMed Scopus (8) Google Scholar, 10Neuwirt J. Takenaka M. van der Merwe J.A. Brennicke A. RNA. 2005; 11: 1563-1570Crossref PubMed Scopus (67) Google Scholar, 11van der Merwe J.A. Takenaka M. Neuwirt J. Verbitskiy D. Brennicke A. FEBS Lett. 2006; 580: 268-272Crossref PubMed Scopus (40) Google Scholar). The second part of the answer seems to be provided by the identification of nuclear encoded specificity factors in Arabidopsis thaliana such as MEF1, MEF9, and MEF11 (mitochondrial-editing factor (MEF) 2The abbreviations used are: MEFmitochondrial-editing factorPPRpentatricopeptide repeat proteinColColumbia.), which are required intact for correct editing of specific different sites (12Takenaka M. Plant Physiol. 2010; 152: 939-947Crossref PubMed Scopus (80) Google Scholar, 13Tang J. Kobayashi K. Suzuki M. Matsumoto S. Muranaka T. Plant J. 2010; 61: 456-466Crossref PubMed Scopus (61) Google Scholar, 14Verbitskiy D. Zehrmann A. van der Merwe J.A. Brennicke A. Takenaka M. Plant J. 2010; 61: 446-455Crossref PubMed Scopus (72) Google Scholar, 15Zehrmann A. Verbitskiy D. van der Merwe J.A. Brennicke A. Takenaka M. Plant Cell. 2009; 21: 558-567Crossref PubMed Scopus (145) Google Scholar).The present theory is that the cis-elements in the mitochondrial RNA molecules are individually recognized by RNA-binding proteins, which may be these MEF proteins. This model makes several predictions, which need to be tested to correct or substantiate this connection. First, the nuclear-encoded MEF proteins must show an evolutionary diversity and a variation sufficient to recognize and target the more than 400 editing sites in plant mitochondria in, for example, A. thaliana. Secondly, the MEF proteins must be able to bind RNA and do so selectively to specific RNA sequences, the identified cis-elements. Specific RNA binding has been experimentally verified for one of the proteins required for a plastid RNA-editing event, the CRR4 protein (16Okuda K. Nakamura T. Sugita M. Shimizu T. Shikanai T. J. Biol. Chem. 2006; 281: 37661-37667Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) in addition to similar proteins involved in other RNA maturation steps (see Refs. 17Beick S. Schmitz-Linneweber C. Williams-Carrier R. Jensen B. Barkan A. Mol. Cell. Biol. 2008; 28: 5337-5347Crossref PubMed Scopus (142) Google Scholar, 18Williams-Carrier R. Kroeger T. Barkan A. RNA. 2008; 14: 1930-1941Crossref PubMed Scopus (92) Google Scholar; for reviews, see Refs. 19Delannoy E. Stanley W.A. Bond C.S. Small I.D. Biochem. Soc. Trans. 2007; 35: 1643-1647Crossref PubMed Scopus (184) Google Scholar, 20Schmitz-Linneweber C. Small I.D. Trends Plant Sci. 2008; 13: 663-670Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar).In this study, we addressed the first question and initiated an analysis to test if other proteins similar to MEF1, MEF9, and MEF11 are involved in RNA editing in plant mitochondria. The Arabidopsis mitochondrial-editing factors MEF1 and MEF11 as well as the OGR1 factor in rice (13Tang J. Kobayashi K. Suzuki M. Matsumoto S. Muranaka T. Plant J. 2010; 61: 456-466Crossref PubMed Scopus (61) Google Scholar, 14Verbitskiy D. Zehrmann A. van der Merwe J.A. Brennicke A. Takenaka M. Plant J. 2010; 61: 446-455Crossref PubMed Scopus (72) Google Scholar, 15Zehrmann A. Verbitskiy D. van der Merwe J.A. Brennicke A. Takenaka M. Plant Cell. 2009; 21: 558-567Crossref PubMed Scopus (145) Google Scholar, 21Kim S.R. Yang J.I. Moon S. Ryu C.H. An K. Kim K.M. Yim J. An G. Plant J. 2009; 59: 738-749Crossref PubMed Scopus (128) Google Scholar), are pentatricopeptide repeat proteins (PPR proteins) of the DYW subgroup as are several PPR proteins required for specific RNA-editing events in plastids. These DYW-PPR proteins are extended by the about 100-amino acid long DYW region at the C terminus beyond the preceding E-domain. The mitochondrial protein MEF9 as well as several plastid RNA-editing factors do not contain the DYW C terminus and terminate with the E-domain (12Takenaka M. Plant Physiol. 2010; 152: 939-947Crossref PubMed Scopus (80) Google Scholar, 20Schmitz-Linneweber C. Small I.D. Trends Plant Sci. 2008; 13: 663-670Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar, 22Cai W. Ji D. Peng L. Guo J. Ma J. Zou M. Lu C. Zhang L. Plant Physiol. 2009; 150: 1260-1271Crossref PubMed Scopus (94) Google Scholar, 23Chateigner-Boutin A.L. Ramos-Vega M. Guevara-García A. Andrés C. Gutierrez-Nava M. Cantero A. Delannoy E. Jiménez L.F. Lurin C. Small I.D. León P. Plant J. 2008; 56: 590-602Crossref PubMed Scopus (205) Google Scholar, 24Kotera E. Tasaka M. Shikanai T. Nature. 2005; 433: 326-330Crossref PubMed Scopus (431) Google Scholar, 25Okuda K. Myouga F. Motohashi R. Shinozaki K. Shikanai T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 8178-8183Crossref PubMed Scopus (225) Google Scholar, 26Robbins J.C. Heller W.P. Hanson M.R. RNA. 2009; 15: 1142-1153Crossref PubMed Scopus (100) Google Scholar, 27Shikanai T. Cell. Mol. Life Sci. 2006; 63: 689-708Crossref Scopus (204) Google Scholar, 28Yu Q.B. Jiang Y. Chong K. Yang Z.N. Plant J. 2009; 59: 1011-1023Crossref PubMed Scopus (128) Google Scholar, 29Zhou W. Cheng Y. Yap A. Chateigner-Boutin A.L. Delannoy E. Hammani K. Small I.D. Huang J. Plant J. 2009; 58: 82-96Crossref PubMed Scopus (160) Google Scholar). Thus, all of the RNA-editing factors identified today belong to the E-class, suggesting that this E-domain is important for their function in the editing process.In this reported analysis, we, therefore, focus on this E-subgroup of the 450 PPR proteins encoded in the nuclear genome of Arabidopsis (30Andrés C. Lurin C. Small I.D. Physiol. Plant. 2007; 129: 14-22Crossref Scopus (87) Google Scholar, 31Lurin C. Andrés C. Aubourg S. Bellaoui M. Bitton F. Bruyère C. Caboche M. Debast C. Gualberto J.M. Hoffmann B. Lecharny A. Le Ret M. Martin-Magniette M.L. Mireau H. Peeters N. Renou J.P. Szurek B. Taconnat L. Small I. Plant Cell. 2004; 16: 2089-2103Crossref PubMed Scopus (987) Google Scholar, 32Small I.D. Peeters N. Trends Biochem. Sci. 2000; 25: 46-47Abstract Full Text Full Text PDF PubMed Google Scholar). About 150 of the PPR proteins contain the E region and 87 of these also the DYW domain (19Delannoy E. Stanley W.A. Bond C.S. Small I.D. Biochem. Soc. Trans. 2007; 35: 1643-1647Crossref PubMed Scopus (184) Google Scholar, 30Andrés C. Lurin C. Small I.D. Physiol. Plant. 2007; 129: 14-22Crossref Scopus (87) Google Scholar, 33Nakamura T. Sugita M. FEBS Lett. 2008; 582: 4163-4168Crossref PubMed Scopus (69) Google Scholar). Consequently we consider the DYW-containing PPR proteins a subgroup of the E-domain-containing PPR proteins. The DYW C-terminal extension is particularly intriguing in proteins involved in organellar RNA editing in plants because it displays a signature characteristic of Zn-containing cytidine deaminases. The enzymatic activity involved in the C to U RNA editing has not yet been identified, and the DYW domain has been proposed to potentially mediate this reaction (34Rüdinger M. Polsakiewicz M. Knoop V. Mol. Biol. Evol. 2008; 25: 1405-1414Crossref PubMed Scopus (73) Google Scholar, 35Salone V. Rüdinger M. Polsakiewicz M. Hoffmann B. Groth-Malonek M. Szurek B. Small I. Knoop V. Lurin C. FEBS Lett. 2007; 581: 4132-4138Crossref PubMed Scopus (183) Google Scholar). On the other hand, truncated proteins without the DYW domain can still be competent in editing in vivo (36Okuda K. Hammani K. Tanz S.K. Peng L. Fukao Y. Myouga F. Motohashi R. Shinozaki K. Small I. Shikanai T. Plant J. 2010; 61: 339-349Crossref PubMed Scopus (80) Google Scholar, 37Okuda K. Chateigner-Boutin A.L. Nakamura T. Delannoy E. Sugita M. Myouga F. Motohashi R. Shinozaki K. Small I. Shikanai T. Plant Cell. 2009; 21: 146-156Crossref PubMed Scopus (185) Google Scholar).To investigate whether more of the about 150 E-group proteins with 65 E-only and 87 DYW-E-PPR proteins are involved in RNA editing at specific sites in plant mitochondria, we here screened 58 T-DNA insertion lines of genes coding for DYW-E-PPR proteins and E-domain PPR proteins in Arabidopsis by a direct analysis for impaired RNA editing with the recently developed multiplexed SNaPshot procedure (38Takenaka M. Brennicke A. Nucleic Acids Res. 2009; 37: e13Crossref PubMed Scopus (31) Google Scholar).DISCUSSIONHere we functionally assign the five PPR proteins MEF18-MEF22 to be involved in RNA editing in plant mitochondria and identify target sites for each of these proteins. The five T-DNA insertions disrupt the respective reading frames and have induced the deletion of fragments of the coding sequences (Fig. 5). The insertions in MEF18 and MEF21 are within the PPR repeats, the insert in MEF20 is just upstream of the ATG codon. These three insertions and sequence deletions appear to disable gene functions because editing is lost at their target sites. The insertion in MEF22 in the E-domain and the thus disabled partial E-domain potentially does not completely abolish the function of the mutant protein. If stably transcribed and translated, the mutant protein may still be partially functional and thus may cause the only partial reduction of editing at the target site. The insertion in the MEF19 mutant is also in the E-domain, but most likely abolishes the competence in RNA editing because no nucleotide conversion is observed at the target site of MEF19.We detected no gross detrimental phenotypic effect on plant growth and development in the homozygous T-DNA insertion lines in which no editing is seen at the target sites for MEF18-MEF21. The second finding in common for these five factors is that as yet only single targets have been identified for each of them. Both common features may be connected to the selection of homozygous and basically healthy T-DNA plants, because loss of a single editing event may be less likely to be detrimental to plant health than the elimination of several editing events. However, fully functional mitochondrial proteins specified by fully edited mRNAs may be crucial under more stressful growth conditions in the wild.Analogous observations have been reported for plastid-editing sites, several of which, although highly conserved, show no phenotypical consequences when their cognate-editing factors are disabled by T-DNA mutations (42Hammani K. Okuda K. Tanz S.K. Chateigner-Boutin A.L. Shikanai T. Small I. Plant Cell. 2009; 21: 3686-3699Crossref PubMed Scopus (148) Google Scholar). These congruent findings in both organelles impact considerations underlying analyses of evolutionarily conserved editing sites and their loss in individual plant lineages (47Tillich M. Le Sy V.L. Schulerowitz K. von Haeseler A. Maier U.G. Schmitz-Linneweber C. BMC Evol. Biol. 2009; 9: 201Crossref PubMed Scopus (14) Google Scholar).The Target Sites of MEF18-MEF22 Alter Conserved Amino AcidsAll target-editing sites alter the encoded amino acid sequence; none of the events is silent. The nad4-1355 site of MEF18 alters a serine to a conserved leucine codon in the NAD4 protein, the ccb206-566-editing event of MEF19 results in encoding a conserved phenylalanine rather than a serine in CCB206, the rps4-226 site requires MEF20 to change a proline amino acid to a serine residue codon at amino acid residue 76 in RPS4. The cox3-257 site targeted by MEF21 changes a serine amino acid to a conserved phenylalanine residue at amino acid 86 in COX3 and MEF22 enhances editing at nad3-149 to code for a conserved phenylalanine rather than a serine.At most of these positions, all land plants including the moss and the liverwort incorporate the amino acid into the protein specified after the respective editing event in Arabidopsis (supplemental Fig. S3), suggesting that these conserved residues are important for proper function. From this high degree of conservation, one would expect impaired proteins synthesized from mRNA intermediates unedited at these positions and thus detectable physiological consequences on the growth pattern and phenotypes of the plants. The apparently normal growth and absence of any gross malformation in the greenhouse is similar to the phenotypes seen for MEF1 and MEF9 (12Takenaka M. Plant Physiol. 2010; 152: 939-947Crossref PubMed Scopus (80) Google Scholar, 15Zehrmann A. Verbitskiy D. van der Merwe J.A. Brennicke A. Takenaka M. Plant Cell. 2009; 21: 558-567Crossref PubMed Scopus (145) Google Scholar). These observations suggest that the criterion of conservation of an amino acid (and an underlying editing event) between different plant species is not a straightforward indicator of functional importance.The Five Identified MEF Proteins Have Different CompositionsThe predicted gene structures of the five PPR proteins and their annotated T-DNA insertion sites show that only one, MEF22, belongs to the DYW subclass of the E-PPR proteins. The other four predicted PPR proteins, MEF18-MEF21, are also members of the E-group, but do not contain the C-terminal DYW extension (Fig. 5). The five proteins MEF18-MEF22 show very little structural similarity in the order and sizes of the various PPRs. MEF20 is, with about 8 repeats, the shortest, MEF18 with 17 predicted repeats the longest. These two proteins display a rather degenerated E-domain, in which only a C-terminal part is fairly well conserved, whereas the region between the C-terminal discernible PPR and this conserved E-internal region displays little similarity. Furthermore, MEF18 and MEF20 terminate within the actual E-domain just beyond the conserved E-internal region. MEF19 is only few amino acids longer than MEF18 and MEF20 and of the same size as MEF9 (12Takenaka M. Plant Physiol. 2010; 152: 939-947Crossref PubMed Scopus (80) Google Scholar). The P, L2, and S repeats (nomenclature according to Ref. 48Rivals E. Bruyère C. Toffano-Nioche C. Lecharny A. Plant Physiol. 2006; 141: 825-839Crossref PubMed Scopus (73) Google Scholar) just upstream of the E-domain are much better conserved between these and the other MEF proteins than any of the further N-terminal PPR repeats. Assignments of further PPR proteins to the functional group of RNA-editing factors will show which of these similar features is a characteristic feature of MEF-PPR proteins distinguishing these from non-MEF-PPRs. Recently, a PPR protein of the P subfamily without E- or DYW domain has been found to influence RNA-editing levels, in fact to lower the percentage of edited steady state mRNAs for a ribosomal protein (49Doniwa Y. Ueda M. Ueta M. Wada A. Kadowaki K. Tsutsumi N. Gene. 2010; 454: 39-46Crossref PubMed Scopus (44) Google Scholar).When searching the genomic databases of other plant species for orthologs of the five proteins MEF18-MEF22, an interesting distribution emerges for MEF20: the three fully sequenced dicot plants Arabidopsis, Brassica oleracea, and Vitis vinifera encode clearly assigned orthologs and show this editing event at the mitochondrial rps4-226 site. In rice, where the mitochondrial genome already encodes a T residue, no ortholog of MEF20 is detected. This finding may suggest that since this editing event is not required in rice, the dedicated factor MEF20 has been lost from the genome. This scenario would support the assumption that MEF20 is required only for this single editing event in the mitochondrial transcriptome of Arabidopsis.On the other hand, orthologs of MEF22 are detected in all four flowering plants, although Vitis does not require this editing event since a U corresponding to the editing site can be directly transcribed from the mitochondrial genomic sequence. In Arabidopsis, we observe that disruption of MEF22 only lowers editing at this site, and there may be another target site not yet identified that requires this factor in Arabidopsis and also in the grape. Alternatively, if stably transcribed and translated, the mutant protein of mef22 may still be partially functional and thus may cause only partial reduction of the editing at the target site. Orthologs can be found for MEF18, 19, and 21 in the four vascular plants, with the most similar grapevine PPR protein varying more from Arabidopsis MEF18 than the orthologs in the other plants.Further E-class PPR Proteins May Be Required for Functionally Important Editing EventsThe selection criterion of having yielded viable homozygous T-DNA insertion mutants will have preselected for editing events with minor biochemical and physiological impact. The other about 90 E-class PPR proteins may be required for editing reactions resulting in amino acid changes with major structural and functional consequences. This may be the reason why no homozygous lines had been annotated for these other genes. A number of the E-class proteins will not be involved in RNA editing at all but in other functionally important RNA-processing steps (20Schmitz-Linneweber C. Small I.D. Trends Plant Sci. 2008; 13: 663-670Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar, 27Shikanai T. Cell. Mol. Life Sci. 2006; 63: 689-708Crossref Scopus (204) Google Scholar, 50de Longevialle A.F. Meyer E.H. Andrés C. Taylor N.L. Lurin C. Millar A.H. Small I.D. Plant Cell. 2007; 19: 3256-3265Crossref PubMed Scopus (224) Google Scholar, 51Schmitz-Linneweber C. Williams-Carrier R. Barkan A. Plant Cell. 2005; 17: 2791-2804Crossref PubMed Scopus (221) Google Scholar, 52Schmitz-Linneweber C. Williams-Carrier R.E. Williams-Voelker P.M. Kroeger T.S. Vichas A. Barkan A. Plant Cell. 2006; 18: 2650-2663Crossref PubMed Scopus (231) Google Scholar).Nevertheless many more E-class PPR proteins, also within the group analyzed, may be involved in mitochondrial-editing sites. Several lines of reasoning contribute to this assumption: Foremost, one-third of the T-DNA lines investigated here are annotated to have a T-DNA insertion outside of the actual reading frames and may not be affected in the function of the respective PPR protein. Likewise not or little affected may be proteins from genes with insertions near their C termini (14Verbitskiy D. Zehrmann A. van der Merwe J.A. Brennicke A. Takenaka M. Plant J. 2010; 61: 446-455Crossref PubMed Scopus (72) Google Scholar). Further factors may have been missed if homozygous seeds were not present, and the original seed batches contained only heterozygous seeds. Other, small effects of specific PPR proteins like that observed for MEF22 at nad3-149 may have escaped our screen. Furthermore, we screened only 2/3 of the editing sites in the Arabidopsis mitochondrial transcriptome and thus will have missed the E-class PPR proteins involved in the editing events that are not included.Outside of the T-DNA insertion lines of genes for E-class PPR proteins annotated as yielding homozygous plants, there will be more of such proteins involved in RNA editing in plant mitochondria. These other factors may affect more than one editing site as observed for some of the previously identified plant mitochondrial-editing factors MEF1, MEF11, and OGR1 (13Tang J. Kobayashi K. Suzuki M. Matsumoto S. Muranaka T. Plant J. 2010; 61: 456-466Crossref PubMed Scopus (61) Google Scholar, 14Verbitskiy D. Zehrmann A. van der Merwe J.A. Brennicke A. Takenaka M. Plant J. 2010; 61: 446-455Crossref PubMed Scopus (72) Google Scholar, 15Zehrmann A. Verbitskiy D. van der Merwe J.A. Brennicke A. Takenaka M. Plant Cell. 2009; 21: 558-567Crossref PubMed Scopus (145) Google Scholar, 21Kim S.R. Yang J.I. Moon S. Ryu C.H. An K. Kim K.M. Yim J. An G. Plant J. 2009; 59: 738-749Crossref PubMed Scopus (128) Google Scholar). Chances are that knock-out mutants of such factors will accumulate additive effects from disturbances of several unedited mitochondrially encoded proteins and therefore will show defects in their physiology even in the greenhouse. These genes will then not be amenable to identification by T-DNA insertions, which abolish gene function, but will require the analysis of more mild mutations such as the EMS mutants we are investigating in parallel (38Takenaka M. Brennicke A. Nucleic Acids Res. 2009; 37: e13Crossref PubMed Scopus (31) Google Scholar). These may allow access to such further trans-factors dedicated to editing sites, which need to be processed for proper mitochondrial function. IntroductionRNA editing in mitochondria of flowering plants changes 400–500 selected cytosines to uridines mostly in coding regions of mRNAs and some tRNAs (1Giegé P. Brennicke A. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 15324-15329Crossref PubMed Scopus (311) Google Scholar, 2Handa H. Nucleic Acids Res. 2003; 31: 5907-5916Crossref PubMed Scopus (292) Google Scholar, 3Takenaka M. Verbitskiy D. van der Merwe J.A. Zehrmann A. Brennicke A. Mitochondrion. 2008; 8: 35-46Crossref PubMed Scopus (109) Google Scholar). In non-flowering plants such as Isoetes species, the number of edited nucleotides is estimated to be more than 1,500 (4Grewe F. Viehoever P. Weisshaar B. Knoop V. Nucleic Acids Res. 2009; 37: 5093-5104Crossref PubMed Scopus (105) Google Scholar), raising the question of how the to-be-edited nucleotides are distinguished from other unaltered moieties.Specific sequence contexts in the pre-mRNA yield the first part of an answer; such cis-elements are required to identify a bona fide editing site as in vivo, in organelle, and in vitro analyses of several mitochondrial RNA-editing sites have shown (5Bock R. Hermann M. Kössel H. EMBO J. 1996; 15: 5052-5059Crossref PubMed Scopus (133) Google Scholar, 6Bock R. Koop H.U. EMBO J. 1997; 16: 3282-3288Crossref PubMed Scopus (59) Google Scholar, 7Chateigner-Boutin A.L. Hanson M.R. Mol. Cell. Biol. 2002; 22: 8448-8456Crossref PubMed Scopus (66) Google Scholar, 8Farré J.C. Leon G. Jordana X. Araya A. Mol. Cell. Biol. 2001; 21: 6731-6737Crossref PubMed Scopus (90) Google Scholar, 9Kempken F. Bolle N. Bruhs A. Endocytobiosis Cell Res. 2009; 19: 1-10Crossref PubMed Scopus (8) Google Scholar, 10Neuwirt J. Takenaka M. van der Merwe J.A. Brennicke A. RNA. 2005; 11: 1563-1570Crossref PubMed Scopus (67) Google Scholar, 11van der Merwe J.A. Takenaka M. Neuwirt J. Verbitskiy D. Brennicke A. FEBS Lett. 2006; 580: 268-272Crossref PubMed Scopus (40) Google Scholar). The second part of the answer seems to be provided by the identification of nuclear encoded specificity factors in Arabidopsis thaliana such as MEF1, MEF9, and MEF11 (mitochondrial-editing factor (MEF) 2The abbreviations used are: MEFmitochondrial-editing factorPPRpentatricopeptide repeat proteinColColumbia.), which are required intact for correct editing of specific different sites (12Takenaka M. Plant Physiol. 2010; 152: 939-947Crossref PubMed Scopus (80) Google Scholar, 13Tang J. Kobayashi K. Suzuki M. Matsumoto S. Muranaka T. Plant J. 2010; 61: 456-466Crossref PubMed Scopus (61) Google Scholar, 14Verbitskiy D. Zehrmann A. van der Merwe J.A. Brennicke A. Takenaka M. Plant J. 2010; 61: 446-455Crossref PubMed Scopus (72) Google Scholar, 15Zehrmann A. Verbitskiy D. van der Merwe J.A. Brennicke A. Takenaka M. Plant Cell. 2009; 21: 558-567Crossref PubMed Scopus (145) Google Scholar).The present theory is that the cis-elements in the mitochondrial RNA molecules are individually recognized by RNA-binding proteins, which may be these MEF proteins. This model makes several predictions, which need to be tested to correct or substantiate this connection. First, the nuclear-encoded MEF proteins must show an evolutionary diversity and a variation sufficient to recognize and target the more than 400 editing sites in plant mitochondria in, for example, A. thaliana. Secondly, the MEF proteins must be able to bind RNA and do so selectively to specific RNA sequences, the identified cis-elements. Specific RNA binding has been experimentally verified for one of the proteins required for a plastid RNA-editing event, the CRR4 protein (16Okuda K. Nakamura T. Sugita M. Shimizu T. Shikanai T. J. Biol. Chem. 2006; 281: 37661-37667Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) in addition to similar proteins involved in other RNA maturation steps (see Refs. 17Beick S. Schmitz-Linneweber C. Williams-Carrier R. Jensen B. Barkan A. Mol. Cell. Biol. 2008; 28: 5337-5347Crossref PubMed Scopus (142) Google Scholar, 18Williams-Carrier R. Kroeger T. Barkan A. RNA. 2008; 14: 1930-1941Crossref PubMed Scopus (92) Google Scholar; for reviews, see Refs. 19Delannoy E. Stanley W.A. Bond C.S. Small I.D. Biochem. Soc. Trans. 2007; 35: 1643-1647Crossref PubMed Scopus (184) Google Scholar, 20Schmitz-Linneweber C. Small I.D. Trends Plant Sci. 2008; 13: 663-670Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar).In this study, we addressed the first question and initiated an analysis to test if other proteins similar to MEF1, MEF9, and MEF11 are involved in RNA editing in plant mitochondria. The Arabidopsis mitochondrial-editing factors MEF1 and MEF11 as well as the OGR1 factor in rice (13Tang J. Kobayashi K. Suzuki M. Matsumoto S. Muranaka T. Plant J. 2010; 61: 456-466Crossref PubMed Scopus (61) Google Scholar, 14Verbitskiy D. Zehrmann A. van der Merwe J.A. Brennicke A. Takenaka M. Plant J. 2010; 61: 446-455Crossref PubMed Scopus (72) Google Scholar, 15Zehrmann A. Verbitskiy D. van der Merwe J.A. Brennicke A. Takenaka M. Plant Cell. 2009; 21: 558-567Crossref PubMed Scopus (145) Google Scholar, 21Kim S.R. Yang J.I. Moon S. Ryu C.H. An K. Kim K.M. Yim J. An G. Plant J. 2009; 59: 738-749Crossref PubMed Scopus (128) Google Scholar), are pentatricopeptide repeat proteins (PPR proteins) of the DYW subgroup as are several PPR proteins required for specific RNA-editing events in plastids. These DYW-PPR proteins are extended by the about 100-amino acid long DYW region at the C terminus beyond the preceding E-domain. The mitochondrial protein MEF9 as well as several plastid RNA-editing factors do not contain the DYW C terminus and terminate with the E-domain (12Takenaka M. Plant Physiol. 2010; 152: 939-947Crossref PubMed Scopus (80) Google Scholar, 20Schmitz-Linneweber C. Small I.D. Trends Plant Sci. 2008; 13: 663-670Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar, 22Cai W. Ji D. Peng L. Guo J. Ma J. Zou M. Lu C. Zhang L. Plant Physiol. 2009; 150: 1260-1271Crossref PubMed Scopus (94) Google Scholar, 23Chateigner-Boutin A.L. Ramos-Vega M. Guevara-García A. Andrés C. Gutierrez-Nava M. Cantero A. Delannoy E. Jiménez L.F. Lurin C. Small I.D. León P. Plant J. 2008; 56: 590-602Crossref PubMed Scopus (205) Google Scholar, 24Kotera E. Tasaka M. Shikanai T. Nature. 2005; 433: 326-330Crossref PubMed Scopus (431) Google Scholar, 25Okuda K. Myouga F. Motohashi R. Shinozaki K. Shikanai T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 8178-8183Crossref PubMed Scopus (225) Google Scholar, 26Robbins J.C. Heller W.P. Hanson M.R. RNA. 2009; 15: 1142-1153Crossref PubMed Scopus (100) Google Scholar, 27Shikanai T. Cell. Mol. Life Sci. 2006; 63: 689-708Crossref Scopus (204) Google Scholar, 28Yu Q.B. Jiang Y. Chong K. Yang Z.N. Plant J. 2009; 59: 1011-1023Crossref PubMed Scopus (128) Google Scholar, 29Zhou W. Cheng Y. Yap A. Chateigner-Boutin A.L. Delannoy E. Hammani K. Small I.D. Huang J. Plant J. 2009; 58: 82-96Crossref PubMed Scopus (160) Google Scholar). Thus, all of the RNA-editing factors identified today belong to the E-class, suggesting that this E-domain is important for their function in the editing process.In this reported analysis, we, therefore, focus on this E-subgroup of the 450 PPR proteins encoded in the nuclear genome of Arabidopsis (30Andrés C. Lurin C. Small I.D. Physiol. Plant. 2007; 129: 14-22Crossref Scopus (87) Google Scholar, 31Lurin C. Andrés C. Aubourg S. Bellaoui M. Bitton F. Bruyère C. Caboche M. Debast C. Gualberto J.M. Hoffmann B. Lecharny A. Le Ret M. Martin-Magniette M.L. Mireau H. Peeters N. Renou J.P. Szurek B. Taconnat L. Small I. Plant Cell. 2004; 16: 2089-2103Crossref PubMed Scopus (987) Google Scholar, 32Small I.D. Peeters N. Trends Biochem. Sci. 2000; 25: 46-47Abstract Full Text Full Text PDF PubMed Google Scholar). About 150 of the PPR proteins contain the E region and 87 of these also the DYW domain (19Delannoy E. Stanley W.A. Bond C.S. Small I.D. Biochem. Soc. Trans. 2007; 35: 1643-1647Crossref PubMed Scopus (184) Google Scholar, 30Andrés C. Lurin C. Small I.D. Physiol. Plant. 2007; 129: 14-22Crossref Scopus (87) Google Scholar, 33Nakamura T. Sugita M. FEBS Lett. 2008; 582: 4163-4168Crossref PubMed Scopus (69) Google Scholar). Consequently we consider the DYW-containing PPR proteins a subgroup of the E-domain-containing PPR proteins. The DYW C-terminal extension is particularly intriguing in proteins involved in organellar RNA editing in plants because it displays a signature characteristic of Zn-containing cytidine deaminases. The enzymatic activity involved in the C to U RNA editing has not yet been identified, and the DYW domain has been proposed to potentially mediate this reaction (34Rüdinger M. Polsakiewicz M. Knoop V. Mol. Biol. Evol. 2008; 25: 1405-1414Crossref PubMed Scopus (73) Google Scholar, 35Salone V. Rüdinger M. Polsakiewicz M. Hoffmann B. Groth-Malonek M. Szurek B. Small I. Knoop V. Lurin C. FEBS Lett. 2007; 581: 4132-4138Crossref PubMed Scopus (183) Google Scholar). On the other hand, truncated proteins without the DYW domain can still be competent in editing in vivo (36Okuda K. Hammani K. Tanz S.K. Peng L. Fukao Y. Myouga F. Motohashi R. Shinozaki K. Small I. Shikanai T. Plant J. 2010; 61: 339-349Crossref PubMed Scopus (80) Google Scholar, 37Okuda K. Chateigner-Boutin A.L. Nakamura T. Delannoy E. Sugita M. Myouga F. Motohashi R. Shinozaki K. Small I. Shikanai T. Plant Cell. 2009; 21: 146-156Crossref PubMed Scopus (185) Google Scholar).To investigate whether more of the about 150 E-group proteins with 65 E-only and 87 DYW-E-PPR proteins are involved in RNA editing at specific sites in plant mitochondria, we here screened 58 T-DNA insertion lines of genes coding for DYW-E-PPR proteins and E-domain PPR proteins in Arabidopsis by a direct analysis for impaired RNA editing with the recently developed multiplexed SNaPshot procedure (38Takenaka M. Brennicke A. Nucleic Acids Res. 2009; 37: e13Crossref PubMed Scopus (31) Google Scholar)." @default.
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