MITOCH-00911; No of Pages 7 Mitochondrion xxx (2014) xxx–xxx

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RNA editing in plant mitochondria —Connecting RNA target sequences and acting proteins Mizuki Takenaka ⁎, Daniil Verbitskiy, Anja Zehrmann, Barbara Härtel, Eszter Bayer-Császár, Franziska Glass, Axel Brennicke Molekulare Botanik, Universität Ulm, 89069 Ulm, Germany

a r t i c l e

i n f o

Article history: Received 28 January 2014 received in revised form 27 March 2014 accepted 4 April 2014 Available online xxxx Keywords: Plant mitochondria RNA editing PPR proteins Protein-RNA code MORF proteins

a b s t r a c t RNA editing changes several hundred cytidines to uridines in the mRNAs of mitochondria in flowering plants. The target cytidines are identified by a subtype of PPR proteins characterized by tandem modules which each binds with a specific upstream nucleotide. Recent progress in correlating repeat structures with nucleotide identities allows to predict and identify target sites in mitochondrial RNAs. Additional proteins have been found to play a role in RNA editing; their precise function still needs to be elucidated. The enzymatic activity performing the C to U reaction may reside in the C-terminal DYW extensions of the PPR proteins; however, this still needs to be proven. Here we update recent progress in understanding RNA editing in flowering plant mitochondria. © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

1. Introduction: RNA editing – the process A comparison of genomic and the thereof transcribed RNA sequences in the two plant extranuclear organelles with resident genomes, chloroplasts and mitochondria, reveals numerous sequence discrepancies in almost all land plants. These differences are caused by RNA editing, which changes specific nucleotide identities (Bock and Knoop, 2012; Chateigner-Boutin and Small, 2010; Finster et al., 2012; Shikanai, 2012). This type of RNA editing, which converts cytidines to uridines, evolved in land plants (Castandet and Araya, 2012; Gray, 2012; Knoop and Rüdinger, 2010) and was most likely subsequently lost in some marchantiid liverworts (Groth-Malonek et al., 2007; Knoop, 2013). The numbers of nucleotides altered vary between the plant lineages; only eight events occur in mitochondria of the moss Funaria hygrometrica and eleven in Physcomitrella patens (Rüdinger et al., 2009, 2011; Sugita et al., 2013; Ichinose et al., 2013). At the other end, the lycophytes Isoetes engelmanii and Selaginella moellendorfii modify more than 1.700 and 2.100 nucleotides, respectively (Grewe et al., 2011; Hecht et al., 2011). In these lycophytes as well as in ferns and hornworts reverse reactions converting U to C are observed in addition to the C to U alterations. In flowering plants, several hundred exclusively C to U alterations are seen in the two organelles, the majority occurring in mitochondria. Precise numbers are difficult to determine

⁎ Corresponding author. Tel.: +49 731 5022610; fax: +49 731 5022610. E-mail address: [email protected] (M. Takenaka).

(Giegé and Brennicke, 1999; Schuster and Brennicke, 1991), if not impossible or rather inappropriate, since all degrees of percentages of altered nucleotides are found as elegantly demonstrated in a recent analysis of deep cDNA sequencing in the model plant Arabidopsis thaliana (Bentolila et al., 2013). In most recent mitochondrial genome analyses, RNA editing sites have been estimated by prediction programs such as PREP-mt (Mower, 2009) or the improved PREPACT (Lenz and Knoop, 2013), but have been probed only sporadically by cDNA analysis for selected genes. Nevertheless, the numbers of mitochondrial editing sites given for Cycas taitungensis with over 1000 (Chaw et al., 2008; Salmans et al., 2010), Phoenix dactylifera with almost 600 (Fang et al., 2012), Spirodela polyrhiza with 540 (Wang et al., 2012), and Butomus umbellatus with about 560 (Cuenca et al., 2013) reflect the relative frequencies of editing. For the recently analysed mitochondrial and plastid genomes in Boea hygrometrica and the very large mitochondrial genome in Picea abies, the number of edits was unfortunately not given (Nystedt et al., 2013; Zhang et al., 2012). Interestingly, extensive editing has been observed in a non-eudicot or -monocot angiosperm species, Liriodendron tulipifera, where more than 700 editing sites have been documented by cDNA analysis in protein coding sequences (Richardson et al., 2013). For Amborella trichopoda, the sister to all other angiosperms, experimental cDNA analyses identified many more editing sites in bona fide Amborella genes than in any other eudicot or monocot species, but a total number would be difficult to assign in this plant due to the extensive integration of sequences from other plant species which are also partly transcribed and edited (Rice et al., 2013). Unfortunately, the predictions as well as cDNA probings are often limited to protein coding

http://dx.doi.org/10.1016/j.mito.2014.04.005 1567-7249/© 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Please cite this article as: Takenaka, M., et al., RNA editing in plant mitochondria —Connecting RNA target sequences and acting proteins, Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.04.005

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sequences and the extent of editing in rRNA and tRNA molecules as well as in introns remains open in most plant species. Deep transcriptome sequencing as was first done for Vitis vinifera (Picardi et al., 2010) detected 44 editing events in structural RNA sequences of tRNAs and introns. The recent deep sequence analysis of Nicotiana tabacum mitochondrial transcripts identified five editing events in tRNAs and 73 in non-coding regions in addition to 557 editing events in open reading frames (Grimes et al., 2014). The biochemical results of RNA editing in plants are a C to U deamination and in lycophytes also the U to C amination, but the underlying reactions and their catalysts are as yet unclear. Recent reviews summarize general and specific aspects of RNA editing and the reader is referred to these for general overviews (Several chapters in: Bock and Knoop, 2012; Fujii and Small, 2011; Hammani and Giegé, 2014; Knoop, 2004, 2011, 2013; Shikanai, 2006; Takenaka et al., 2013b; Takenaka, in press). We here focus on advances in understanding the proteins involved in editing and in deciphering the contacts between these proteins and the RNA. Major progress has been made in understanding how the C nucleotides to be edited to U are identified in the RNA population of the organelles in angiosperms. The proteins recognizing and binding to a specific RNA sequence are a subgroup of the PPR proteins (pentatricopeptide repeat proteins), which are involved in targeting the RNA editing reactions to specific nucleotides as detailed below (Barkan and Small, 2014; Lurin et al., 2004; O’Toole et al., 2008; Schmitz-Linneweber and Small, 2008; Small and Peeters, 2000; Takenaka et al., 2008). Additional proteins, unrelated to PPRs, are also involved in organellar RNA editing, suggesting that the process is mediated by complex editosomes consisting of several different proteins in chloroplasts as well as in mitochondria. A number of excellent reviews have been written on the functions, actions, structures and specializations of PPR proteins (Barkan and Small, 2014; Schmitz-Linneweber and Small, 2008; Yagi et al., 2013b). We therefore focus here on selected aspects to summarize recent developments in our understanding of the RNA editing process and point out several present lines of investigations. 2. Specific nucleotides are edited—the RNA-protein code In the organellar RNAs, nucleotides to be edited must be distinguished from those that have to be left unaltered. In flowering plants, no editing appears to alter the rRNAs (Schuster et al., 1991), but some tRNAs are edited (Binder et al., 1994; Grewe et al., 2009), suggesting that in these instances secondary structure folding of the RNA can be opened for editing and/or tRNA precursors are not as tightly folded (Grimes et al., 2014; Marchfelder et al., 1996). In vitro and in organello assays had shown that the sequence pattern 5–20 nucleotides upstream of the edited nucleotide contain the determining address (Bock et al., 1996; Farré et al., 2001; Hegeman et al., 2005; Neuwirt et al., 2005; Takenaka et al., 2004; van der Merwe et al., 2006). These unique RNA sequences are recognized and bound by the complementary unique arrangement of variant tandem elements of specific PPR proteins (Barkan and Small, 2014). PPR proteins are found in all eukaryotes, but their numbers have greatly increased in land plants in comparison to fungi and animals and also to alga. A recent survey of sequenced genomes identified 8– 17 PPR proteins in Rhodophyta and 14–25 in species of the Chlorophyta (Tourasse et al., 2013). In flowering plant genomes, several hundred PPR proteins are encoded in the nucleus, e.g. 450 in A. thaliana, 477 in Oryza sativa, 365 in Medicago trunculata, 629 in Glycine max, more than 1.000 in S. moellendorfii and 106 in P. patens, but only 10 in Schizosaccharomyces pombe and 15 in Saccharomyces cerevisiae (Fujii and Small, 2011; Lurin et al., 2004). Their PPR name-giving substructures are the variant elements of about 35 amino acids arranged in tandem in the protein. Elements in PPR proteins involved in processes other than editing are usually 35 amino acids long (P-type) and PPR proteins for RNA editing contain elements of variable length (PLStype). Each of these modules can bind to one nucleotide and the number

of elements defines the maximal length of RNA sequence that can be recognized. The number of elements in plant PPR proteins is usually larger than the minimal RNA sequence of about 6–8 nucleotides required in the RNA population to define a unique site. However, some PPR proteins have fewer such elements and would thus not be able to target a specific site without further additional guiding. The largest PPR protein in A. thaliana in terms of number of PPR motifs is MEF12 which contains 25 such elements (Härtel et al., 2013), much larger than the average number of 13–14 such PPR motifs in A. thaliana (13.7 average) and in Oryza sativa (13.1 average) (Fig. 1). In the moss P. patens, average numbers of PPR elements are higher than in flowering plants with about 20 such repeats (Fig. 1). The reason for these differences is not clear; the mitochondrial and plastid genomes in the moss are not more complex than in the flowering plants and thus do not require an extended specificity through a larger recognition motif. The coding system in the PPR elements basically relies on the amino acid identities at two specific positions in the PPR elements. These were first identified by in silico analyses comparing target RNA patterns and coinciding amino acid identities in non-editing P-type and editing PLS-type PPR proteins (Barkan et al., 2012; Takenaka et al., 2013a; Yagi et al., 2013a). These coincidences have been confirmed by crystal structural analyses of the PPR repeats with and without the target RNA sequence (Ban et al., 2013; Ke et al., 2013; Yin et al., 2013). Although these were obtained from a strongly, and possibly irreversibly, RNA binding P-type PPR protein (PPR10), the similarity between the code parameters suggests that each element in the RNA editing PPR proteins will analogously fold into two helical repeats and expose the same amino acid positions to the respective ribonucleotide. Still, the different lengths of the variant PLS elements in the RNA editing PPR proteins between 32 and 38 amino acids may exert an influence in the molecular interactions and contribute to the difference in affinity distinct from the group of tightly binding PPR proteins such as PPR10. PPR10 and other such P-type PPR proteins involved in stopping exonucleolytic DNases must attach strongly if not irreversibly to their target to resist dissociation by the progressive DNA degrading enzyme and to resolve secondary structures (reviewed in Barkan and Small, 2014). While the target sequence bound by e.g. PPR10 is detected as a small RNA molecule, none of the editing site motifs have been seen in such assays or by primer extension probings (Ruwe and Schmitz-Linneweber, 2012). It will be important in the near future to identify the parameters allowing an RNA editing PPR protein to attach to specific target sites and yet to be able to dissociate again rapidly from the RNA so as not to inhibit translation or further editing by other factors. The protein–protein connection in the PPR10 dimers suggests that the repeats not only bind to RNA but can also present a protein binding surface and undergo connections to other proteins in the editosome. The surprising observation of the dimer formation of PPR10 in the crystal structure analysis raises the question about its relevance in vivo. Does such a homodimer form in vivo and does it bind to two RNA molecules? How should we imagine this close vicinity of two mRNA molecules? Alternatively, is the dimer only formed in vitro in the absence of other proteins which would in vivo dissociate the PPR protein homodimers? Is the PPR homodimer then the nonphysiological product of sticky protein surfaces of the PPR elements? In any case, while one face of the PPR elements clearly attaches to the RNA, the other is accessible to protein–protein interactions which may be involved in the assembly of the editosome in vivo (Fig. 2). 3. Additional domains in the RNA editing PPR proteins—signatures of a deaminase In addition to the variable length of the PPR elements, the RNA editing PPR proteins are characterized by C-terminal extensions. Adjacent to the PPR elements is the so-called E-domain which is present in all of the RNA editing PPR proteins. Structural and some sequence similarities suggest that this region evolved from two PPR repeat modules.

Please cite this article as: Takenaka, M., et al., RNA editing in plant mitochondria —Connecting RNA target sequences and acting proteins, Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.04.005

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Fig. 1. The average number of PPR elements in flowering plants such as Arabidopsis thaliana and Oryza sativa is smaller than in the moss Physcomitrella patens. The MEF12 protein with 25 PPR elements contains the largest number of these elements in the E and E + DYW classes of PPR proteins in Arabidopsis thaliana, which comprise all so far identified PPR proteins involved in RNA editing (Härtel et al., 2013). The next largest such protein in Arabidopsis thaliana contains only 23 PPR motif elements. The average number of PPR elements in these RNA editing PPR proteins is 13.7 in Arabidopsis thaliana and 13.1 in Oryza sativa. In the moss Physcomitrella patens, the E + DYW class of PPR proteins contains an average number of 20.2 PPR elements; the largest two proteins are predicted to have 26 PPR elements.

The function of this region is as yet unclear, but it must be important for the RNA editing process since several such proteins cannot complement respective mutants without these E domains. The similarity with the PPR protein elements suggest that they may also bind to the RNA sequence—although maybe not to specific nucleotide identities as suggested by the bias of nucleotides in the −1 position just upstream

of the edited nucleotide where most often pyrimidines are found (Fig. 2). Alternatively or in addition the E-domain may attract and attach to other proteins to build the editosome. About half of the RNA editing PPR proteins are further extended at their C-termini by the so-called DYW protein domain, named after the C-terminal amino acids D, Y and W. In flowering plants half, but in the

MORF/RIP proteins 5’ mRNA

Accessory proteins PPR PPR PPR PPR PPR

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C to U deaminase

DYW

PPR PPR PPR PPR PPR PPR PPR PPR PPR PPR PPR

-12 -11 -10 -9 -8 -7 -6 -5 -4 -3

E

-2 -1

DYW

U>C>>>A,G

C

? 3’ Fig. 2. Model of the organellar editosome in flowering plants. One E-class PPR protein binds to the specific RNA sequence 5′ from nucleotide –4 upstream of the edited C nucleotide. Each PPR element can contact one nucleotide, but not all of them may actually be bound. This PPR protein may or may not contain a DYW element. Through direct protein–protein interaction or with the support of the MORF/RIP proteins a second PPR protein is recruited which contributes the DYW function. The DYW domain possibly acts as the deaminase enzyme or another such activity is contributed by an as yet unknown enzyme. Various other proteins, such as the ORRM or PPO1 proteins, may bind to the core function proteins via the MORF/RIP proteins or directly at the PPR proteins to exert a regulatory influence at certain PPR + MORF/RIP combinations which assemble at specific editing sites. The E domain possibly exerts some steric influence on the nucleotide identity just upstream of the edited nucleotide at the –1 position where most often pyrimidines and far less frequently purines are present.

Please cite this article as: Takenaka, M., et al., RNA editing in plant mitochondria —Connecting RNA target sequences and acting proteins, Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.04.005

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moss P. patens as well as in several protists, all RNA editing PPR proteins contain a DYW domain (Knoop and Rüdinger, 2010; SchallenbergRüdinger et al., 2013). All these DYW domains show signature amino acids of nucleotide deaminases with histidines and cysteines at positions characteristic for a zinc binding motif (Salone et al., 2007). Recently, a zinc ion has indeed been identified to copurify with the DYW domain, which further supports a deaminase activity for the DYW domain (Hayes et al., 2013). Now experimental evidence is required to prove that the DYW domain can indeed catalyse the deamination reaction. The enzymatic activity performing the C to U deamination may however require additional protein factors and need an intact editosome complex to catalyse the reaction on the RNA. The amination reactions found in RNA editing in non-flowering plant mitochondria and plastids may require different enzymes and it will be interesting to see why these activities are not present, possibly by having been lost in flowering plants. 4. The DYW domain is required for editing—in cis or in trans The observation that only half of the PPR proteins involved in RNA editing at specific sites contain a DYW protein domain could be interpreted to suggest that this additional region may not be required for editing (Barkan and Small, 2014; Fujii and Small, 2011; Schmitz-Linneweber and Small, 2008; Shikanai and Fujii, 2013; Yagi et al., 2013a, 2013b). Furthermore, in some DYW–PPR proteins the DYW domain can be deleted without great effect on their function in RNA editing (Okuda et al., 2007, 2009; Verbitskiy et al., 2010; Zehrmann et al., 2010, 2011). In other PPR proteins, however, the DYW domain cannot be removed and such truncated proteins are not competent in complementation assays (Chateigner-Boutin et al., 2013; Zehrmann et al., 2010, 2011). The explanation that is presently most likely postulates that the function of the DYW domain can be supplied in trans by another PPR protein. The observation of homodimer formation between PPR proteins in the recent crystallization studies suggests that heterodimer assemblies may be possible in which one of the two PPR proteins supplies the DYW domain (Ban et al., 2013; Ke et al., 2013; Yin et al., 2013). Direct support for such complex formations comes from the study of the short DYW1 protein and its function in plastid RNA editing (Boussardon et al., 2012). The DYW1 protein consists of a DYW domain but does not contain any clear PPR repeat elements in Arabidopsis. DYW1 is necessary for editing the plastid site ndhD-1 which also requires the PPR protein CRR4 which does not contain a DYW domain (Boussardon et al., 2012). Physical interaction between the DYW1 and CRR4 proteins was shown in vivo. This interaction was manipulated from a trans to a cis configuration with the DYW1 protein fused to the C-terminus of CRR4. This fusion protein can now complement a double mutant in which both DYW1 and CRR4 genes are disrupted. The DYW1 protein seems to be involved in editing only this site; other DYW domains may be required for the other PPR proteins lacking a DYW domain. In A. thaliana mitochondria, the MEF8 and MEF8S and three further PPR proteins contain only few PPR repeats which appear to be too few to allow sequence specific binding (Fig. 1). The MEF8 and MEF8S PPR proteins are very similar in sequence and structure and target the same sites, but their diverged transcription patterns result in differential effects in their respective mutants (Verbitskiy et al., 2012). By analogy to DYW1 they could provide the DYW domain in trans to mitochondrial PPR editing proteins which do not have this domain. Specific editing sites are affected in mutants of MEF8 or MEF8S for which so far no other PPR protein has been assigned. However, double mutants of MEF8 and MEF8S are not viable, which may be caused by other editing sites being affected which are essential in mitochondria and thus in the plant (Verbitskiy et al., 2012). In this scenario, the DYW domain supplied in trans by MEF8 and MEF8S can not be substituted from other RNA editing factors. Such heterodimer-formations between different PPR proteins need to be shown and their specificities will have to be

determined in the near future to extend the homodimer formation seen in the structural analyses (Ban et al., 2013; Ke et al., 2013; Yin et al., 2013).

5. More proteins make up the editosome—the MORF/RIP family Several further proteins are directly or indirectly involved in RNA editing in both organelles (Fig. 2). Most prominent is the family of the MORF proteins (Takenaka et al., 2012), which have also been designated as RIP proteins (Bentolila et al., 2012). The first name of these proteins, multiple site organellar RNA editing factors (MORFs), is derived from their participation at many RNA editing events; the second, RNAediting factor interacting proteins (RIP), was coined from the identification procedure of one such factor. The small family of MORF/RIP proteins is very different from the PPR proteins and is unrelated to any other known protein. The MORF/RIP proteins are characterized by a common, rather well conserved domain which, however, is not similar to any known functional protein domain and its function remains unclear. If it is indeed involved in protein contacts, it may represent a novel type of protein–protein interaction surface. Similarities outside of this region are seen for example between the MORF5/RIP5 and MORF6/RIP6 proteins, which are present as a pair only in Arabidopsis and closely related species but not in other flowering plants and probably arose by a rather recent gene duplication event (Takenaka, in press; Takenaka et al., 2013b). Although several MORF/RIP proteins or their genes, respectively, are found in all flowering plant species, their numbers differ and they are not visible in some other plant species (Table 1). Nine full-length MORF/RIP proteins are encoded in A. thaliana, but their number already differs in such closely related species as Arabidopsis lyrata and Thellungiella halophila (Table 1; Takenaka, in press). Genes for MORF/RIP proteins are not detected in Selaginella which boasts more than twice as many editing sites as flowering plants (Banks et al., 2011). In this plant, E-PPR proteins and DYW containing E-PPR proteins are specified in the nuclear genome and, if they interact, they do not require the MORF/RIP proteins. In the moss P. patens, all PPR proteins involved in RNA editing contain a DYW domain and there are no genes for MORF/RIP proteins. These comparisons raise the question of the actual role of the MORF/RIP proteins in the editosome and why they evolved in the flowering plants but are apparently not required in other plant species. Experimental investigation needs to identify the functions and functional interactions of the MORF/RIP, the PPR and other proteins. The MORF/RIP proteins interact with specific PPR proteins and it may be possible that they support the interactions between PPR proteins with and those without a DYW domain and/or other proteins in the editosome (Bentolila et al., 2012; Takenaka et al., 2012). Two such specific interactions between the dual (i.e. to plastids and mitochondria) targeted MORF8/RIP1 and PPR proteins involve RARE1 in plastids (Bentolila et al., 2012) and MEF10 proteins in mitochondria, respectively, (Härtel et al., 2013), both of which are DYW containing PPR proteins yet require the function of MORF8/RIP1. These DYW proteins could be providing the deaminase function of the DYW domains and other

Table 1 Number of MORF/RIP genes in various plant species. Plant species

No. of MORF/RIP

Arabidopsis thaliana Arabidopsis lyrata Thellungiella halophila Populus trichocarpa Glycine max Cucumis sativa Oryza sativa Selaginella moellendorffii Physcomitrella patens

9 11 10 8 12 10 8 0 0

Please cite this article as: Takenaka, M., et al., RNA editing in plant mitochondria —Connecting RNA target sequences and acting proteins, Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.04.005

M. Takenaka et al. / Mitochondrion xxx (2014) xxx–xxx

E-class PPRs could be the primary RNA binding proteins. However, the refined target site prediction programme suggests that both RARE1 and MEF10 proteins do bind to the RNA sequence motifs at these target sites, respectively (Takenaka et al., 2013a). In addition, one would expect that the DYW donators would be more promiscuous and activate many sites, since the number of potential E- and E-DYW-PPR proteins available for individual editing sites would be halved if actually two cooperating PPR proteins were generally involved in editing one or few sites. Thus the role of MORF/RIP proteins particularly in conjunction with the DYW domains is still unclear; some have been surmised to be not involved in RNA editing at all and lower editing at only few if any sites when knocked out or knocked down (Bentolila et al., 2013). It is possible that the MORF/RIP proteins connect the PPR proteins to other nonPPR proteins, which could be feasible if an enzyme other than the DYW domain has to be recruited and would fit with the (partial) redundance of the MORFs/RIPs observed at some RNA editing events. 6. Further proteins may be involved in RNA editing—directly or indirectly Often effects of mutants are seen in many different aspects of the cellular metabolism which are physiologically actually very distant from the direct function of the respective mutated protein. At present, several instances have been reported for mutants of various proteins in which RNA editing in the organelles is altered. Some of these will be indirect effects and the mutated protein has no connection to RNA editing. Among these mutants, the best candidates for more direct functions or actions at least close to RNA editing would be RNA binding proteins. Also among these direct connections and indirect influences have to be distinguished. For example, individual cpRNP proteins influence RNA editing at many individual sites in chloroplasts, but in respective mutants the relative amounts of many RNAs are reduced and various RNA processing steps are influenced, so that their influence on editing is likely to be indirect (Kupsch et al., 2012; Tillich et al., 2009). These proteins with two RNA recognition motifs (RRM) are found only in plastids and not in mitochondria. In mitochondria, another group of RRM-containing proteins has been described, some of which may have a more direct function in RNA editing (Vermel et al., 2002). An RRM and two partial MORF/RIP motifs are present in the ORRM protein, which may be more closely involved in RNA editing in the chloroplast since the levels of mRNAs are not altered in a mutant, but individual editing events are affected, usually reduced (Sun et al., 2013). The RRM domain appears to be the important functional element in this protein and can fully complement the respective mutant. Similar proteins form a small family with the ORRM protein; some of which are predicted to be imported into plastids and/or mitochondria and thus may provide similar functions for RNA editing in both organelles. The recently identified connection of the protoporphyrinogen IX oxidase 1 (PPO1) to RNA editing in plastids via the MORF proteins suggests that the RNA editing process may be used as a new level of regulating gene expression (Zhang et al., 2014). The three plastid located MORF/ RIP proteins 2, 8 and 9 interact with PPO1 through a short protein domain in the enzyme. This interaction is required for editing, showing that the MORF/RIP proteins mediate protein–protein interactions and may act as connectors between the site-specific PPR proteins and regulatory accessories such as PPO1. 7. RNA editing in the organelles—one of many coordinated RNA processing steps In both organelles with genomes in plant cells, plastids and mitochondria, RNA molecules of all types, rRNAs, tRNAs and mRNAs are synthesized as larger precursor molecules. These require trimming of their termini as well as internal cuts in some polycistronic precursors (Fujii and Small, 2011; Schmitz-Linneweber and Small, 2008). Introns

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have to be removed by splicing processes distinct from the nuclear spliceosome and mRNAs have to enter the ribosomes eventually. RNA editing takes place sometimes between transcription and translation, but when and how still need to be worked out (Barkan and Small, 2014; Fujii and Small, 2011; Schmitz-Linneweber and Small, 2008). The editosomes appear to attach to the mRNA before as well as after splicing of the introns. In some introns editing events seem to be required for proper activation of the intron and thus occur before the intron is excised (Bonen, 2008; Börner et al., 1995; Wissinger et al., 1991). In other instances, exons need to be ligated after intron excision to generate the PPR signature target site for editing (Bonen, 2008). It will also be interesting to determine how the editosome processes along the RNA molecule and which helper functions of e.g. a helicase or other RNA binding proteins are involved. In plastids, transcriptionally active chromosomes are attached to the membrane as large protein complexes and may include components for some of these RNA processing steps as well (Gualberto and Kuhn, 2014, in this issue; Krause and Krupinska, 2000; Krupinska et al., 2013). For example, the ribosome binding factor RbfA, which is also involved in processing the 16S rRNA, is associated with the thylakoid membranes (Fristedt et al., 2014). The individual steps have to be dissected and reassembled to determine the minimal composition of e.g. the editosome. The separation of RNA editing from other processing steps most likely complexed in vivo is experimentally feasible as the preparation of editing-active lysates from both organelles shows. In these lysates, some editing events can be processed, but most are not accessed by the components present in the mitochondrial or plastid extracts. This suggests that the active protein complexes are different for individual sites, definitely in composition as different PPR proteins are required, but possibly also in their association with other necessary proteins. The composition of the active editosomes has not yet been dissected in the lysates, but a size fractionation of the RARE1-RIP1/MORF8 associated proteins from plastids showed the RARE1 protein to be present in a complex of more than 200 MDa. Now the minimal editosome size needs to be determined of protein complexes functionally competent in editing added RNA molecules (Fig. 2). The in vitro activity suggests that these editosomes may form a robust complex of the editing protein components at least surviving the preparation for some editing sites (Takenaka and Brennicke, 2003). It will be important to determine which of the different proteins influencing RNA editing are actually assembled in an active editosome complex. This information will also be a prerequisite to designing PPR proteins and employing any of the other participants in editing for biotechnological applications (Colas des Francs-Small and Small, 2013; Yagi et al., in press)

Acknowledgements We thank Dagmar Pruchner, Angelika Müller and Bianca Wolf for excellent experimental help. We gratefully acknowledge all members of the lab for their dedicated contributions. This work was supported by grants from the Deutsche Forschungsgemeinschaft (TA624/4-1, TA624/6-1, TA624/2-2, BR726/9-5, and BR726/11-1). Mizuki Takenaka is a Heisenberg fellow.

References Ban, T., et al., 2013. Structure of a PLS-class pentatricopeptide repeat protein provides insights into mechanism of RNA recognition. J. Biol. Chem. 288, 31540–31548. Banks, J.A., et al., 2011. The Selaginella genome identifies genetic changes associated with the evolution of vascular plants. Science 332, 960–963. Barkan, A., Small, I., 2014. Pentatricopeptide repeat proteins in plants. Ann. Rev. Plant Biol. 65, 1. Barkan, A., et al., 2012. A combinatorial amino acid code for RNA recognition by pentatricopeptide repeat proteins. PLoS Genet. 8, e1002910. Bentolila, S., Heller, W.P., Sun, T., Babina, A.M., Friso, G., et al., 2012. RIP1, a member of an Arabidopsis protein family, interacts with the protein RARE1 and broadly affects RNA editing. Proc. Natl. Acad. Sci. U. S. A. 109, E1453–E1461.

Please cite this article as: Takenaka, M., et al., RNA editing in plant mitochondria —Connecting RNA target sequences and acting proteins, Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.04.005

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