PLANT SIGNALING & BEHAVIOR 2016, VOL. 11, NO. 5, e1167299 (4 pages) http://dx.doi.org/10.1080/15592324.2016.1167299
ARTICLE ADDENDUM
Organelle RNA recognition motif-containing (ORRM) proteins are plastid and mitochondrial editing factors in Arabidopsis Xiaowen Shi, Stephane Bentolila, and Maureen R. Hanson Department of Molecular Biology and Genetics, Cornell University, Biotechnology Building, Ithaca, NY, USA
ABSTRACT
ARTICLE HISTORY
Post-transcriptional C-to-U RNA editing occurs at specific sites in plastid and plant mitochondrial transcripts. Members of the Arabidopsis pentatricopeptide repeat (PPR) motif-containing protein family and RNA-editing factor Interacting Protein (RIP, also known as MORF) family have been characterized as essential components of the RNA editing apparatus. Recent studies reveal that several organelle-targeted RNA recognition motif (RRM)-containing proteins are involved in either plastid or mitochondrial RNA editing. ORRM1 (Organelle RRM protein 1) is essential for plastid editing, whereas ORRM2, ORRM3 and ORRM4 are involved in mitochondrial RNA editing. The RRM domain of ORRM1, ORRM3 and ORRM4 is required for editing activity, whereas the auxiliary RIP and Glycine-Rich (GR) domains mediate the ORRM proteins’ interactions with other editing factors. The identification of the ORRM proteins as RNA editing factors further expands our knowledge of the composition of the editosome.
Received 18 February 2016 Revised 10 March 2016 Accepted 12 March 2016 Keywords
CP31; RNA editing; Glycine-rich domain; ORRM proteins; protein-protein interaction assays; RNA recognition motif
Abbreviations: RRM, RNA Recognition Motif; ORRM, Organelle RRM protein; GR, Glycine-Rich; PPR, pentatricopep-
tide repeat; RIP, RNA-editing factor Interacting Protein; MORF, Multiple Organelle RNA-editing Factor; OZ1, Organelle Zinc finger protein 1
In land plants, post-transcriptional C-to-U RNA editing modifies plastid and mitochondrial transcripts, often changing the amino acid sequence from what the genomic sequence predicts.1,2 It is regarded as a correction mechanism for defective genes affected by T-to-C mutations.3 Changes in the extent of RNA editing may impair the proper function of gene products, even leading to seedling lethality.4,5 In Arabidopsis, 43 Cs in plastids and over 600 Cs in mitochondria have been characterized as targets of RNA editing.6,7 Cis- and trans- factors essential for the editing process have been identified. Cis-elements, sequences present in close proximity to the editable C,8-10 are recognized by members of the pentatricopeptide repeat (PPR) motif-containing protein family.11,12 Members of the RNA-editing factor Interacting Protein (RIP, also known as MORF) family are trans-factors that function in both plastid and mitochondrial RNA editing.6,13,14,16 Several other proteins, such as OZ1, a zinc finger protein, and PPO1, a tetrapyrrole biosynthesis enzyme, have also been reported to be editing transfactors.5,15 The DYW domain present on some members of the PPR protein family may supply the cytidine deaminase activity required to convert C to U.16-18 In spite of these findings, the composition of the plant RNA editosome is not yet fully understood.
Characterization of four ORRM proteins as RNA editing factors We have recently discovered an additional group of trans-factors required for editing of particular Cs in chloroplasts and mitochondria. Due to the presence of an RNA recognition motif (RRM), we have named these trans-factors as Organelle RRM (ORRM) proteins. The founder of this family, ORRM1 (Organelle RNA recognition motif-containing protein 1), is a plastid editing factor that was discovered through a database search with the protein sequence of the major plastid and mitochondrial editing factor RIP1.13,19 Twenty-one plastid sites exhibit a reduction of editing in the orrm1 homozygous mutant relative to the wild-type (Table 1).19 Subsequently, mitochondrial RNA editing factors ORRM2, ORRM3 and ORRM4 were identified based on their sequence similarity to ORRM1 in the RRM domain (Fig. 1).20,21 Transient silencing of ORRM2 and ORRM3 resulted in reduced editing efficiency at over 30 of the mitochondrial sites (Table 1).21 ORRM4 was characterized as a major mitochondrial editing factor because ORRM4 mutation causes editing defects at 270 mitochondrial C targets (Table 1).20 The alteration of ORRM1, ORRM2 and ORRM3 expression did not cause any morphological defects in the corresponding mutant or silenced tissue, whereas a slow growth
CONTACT Maureen R. Hanson [email protected] Addendum to: Shi X, Germain A, Hanson MR, Bentolila S. RNA Recognition Motif-Containing Protein ORRM4 Broadly Affects Mitochondrial RNA Editing and Impacts Plant Development and Flowering. Plant Physiol. 2016; 170:294–309. doi: 10.1104/pp.15.01280. PMID: 26578708 Published with license by Taylor & Francis Group, LLC © Xiaowen Shi, Stephane Bentolila, and Maureen R. Hanson This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.
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Table 1. Description of the ORRM proteins, CP31A, CP31B, and CP29A. The table shows information relating to the proteins discussed in this paper, including their gene IDs, location of the proteins, names and phenotypes of T-DNA mutant or virus-induced gene silencing (VIGS) lines. Published data is from Sun et al., 2013; Shi et al., 2015; Shi et al., 2016; Tillich et al., 2009 and Kupsch et al., 2012.19-21,29,30 P, plastid. M, mitochondria. Gene ID
Location
ORRM1 ORRM2 ORRM3
At3g20930 At5g54580 At5g61030
P M M
ORRM4
At1g74230
M
CP31A
At4g24770
P
CP31B
At5g50250
P
CP29A
At3g53460
P
Name of T-DNA mutant or VIGS line
Growth phenotype
SALK_072648 ORRM2-VIGS ORRM3-VIGS SALK_038244 ORRM4-VIGS SALK_023061C SALK_109613 SAIL_258_h02 WiscDsLox383H9
No phenotypic defect No phenotypic defect No phenotypic defect No phenotypic defect No phenotypic defect Slow growth and late flowering Pale leaves under cold stress conditions No phenotypic defect
SALK_003066 001G06
Pale leaves under cold stress conditions
and late flowering phenotype was observed in the orrm4 mutant (Table 1).19-21 The impact of ORRM4 on plant growth and flowering indicates that adequate mitochondrial editing is necessary for normal plant development. Interaction between ORRM proteins and other editing factors RNA editing is performed by editosomes found between the 200 and 400 kD markers on size exclusion columns.13 Proteinprotein interaction assays have provided us with some information about the composition of RNA editosomes. ORRM1 interacts with the PPR proteins that are required for editing of the same C target as ORRM1(Fig. 2A) . For instance, both ORRM1 and OTP82 are required for the editing at ndhG C50 and ndhB C836, and as expected, ORRM1 interacts with OTP82.19,22 On the contrary, ORRM1 is dispensable for the editing at the sites controlled by RARE1, thus no interaction was observed (Fig. 2A).19,23 In addition to PPR proteins, ORRM1 interacts with RIP1, plastid editing factors RIP2 and OZ1 as shown in Fig. 2A.5,19 Unlike ORRM1, mitochondrial editing factors
Figure 1. Phylogenetic tree based on the amino acid sequences of the RRM motifs in RRM-containing proteins (80 amino acids considered). Protein alignments were achieved by using Clustal X version 2.1 (http://www.clustal.org/clustal2/) and adjusted manually. The construction of phylogenetic trees was performed with MEGA5 (www.megasoftware.net). For proteins with two RRM motifs, the difference caused by using either RRM motif for alignment was negligible. Thus, the second RRM motif was used for alignment. The tree was inferred using the Neighbor-Joining method, and evolutionary distances were computed using the P-distance method. The scale bar corresponds to 0.1 substitutions per site.
Number of editing sites affected 21 of 34 35 of 618 32 of 618 110 of 576 51 of 564 270 of 618 13 of 34; 3 of 34 under cold stress conditions 8 of 34, dependent on CP31A mutation 3 of 34, only under cold stress conditions
ORRM2, ORRM3 and ORRM4 do not interact with the tested PPR proteins in yeast two-hybrid assays. ORRM3 interacts with ORRM4, and both of them interact with RIP1 and with themselves (Fig. 2B). ORRM2 interacts with ORRM3, but does not interact with ORRM4, RIP1 or itself (Fig. 2B).20,21 Functional analysis of the RRM domain and the GR domain in ORRM proteins The RRM domain is a conserved ~80 amino-acid domain that binds RNA molecules with a variety of specificities and affinities.24,25 Each of the four ORRM proteins contains one RRM domain either at its N terminus or C terminus (Fig. 3). Complementation tests revealed that the RRM domain of ORRM1, ORRM3 and ORRM4 is necessary for the editing ability of the
Figure 2. Known interactions of ORRM proteins with themselves and other editing factors. (A) Interaction between ORRM1 and other editing factors. (B) Interaction between mitochondrial ORRM proteins and RIP1. Data is from yeast two-hybrid and bimolecular fluorescence complementation assays.5,19-21 Positive signals are represented by a C (shaded light red), while negative signals are shown by a - (shaded light green). n.d., not determined (shaded light yellow).
PLANT SIGNALING & BEHAVIOR
Figure 3. The motif diagram of ORRM1, ORRM2, ORRM3, ORRM4, CP31A, CP31B and CP29A. The scale bar represents 100 amino acids.
protein,19-21 thus the RRM domain plays a key role in both plastid and mitochondrial RNA editing. In addition to the RRM domain, ORRM1 carries a truncated RIP-RIP domain at its N terminus, while ORRM3 and ORRM4 contain a C-terminal Glycine-Rich (GR) domain (Fig. 3). The RIP domain or the GR domain is dispensable for the editing activity of the protein. However, the N-terminal RIP domain is required for ORRM1 to interact with PPR proteins (Fig. 2A), and the GR domain of ORRM4 is responsible for mediating its interaction with ORRM3 and with itself.19,20 Functional redundancy between the mitochondrial ORRM proteins In our study, the editing extent was reduced but not eliminated by the knockdown or knockout of ORRM2, ORRM3 and ORRM4 in mutants or silenced tissue,20,21 likely due to the redundant function of the ORRM proteins. We have shown that the editing efficiency of sites under the control of both ORRM2 and ORRM3 was further impaired by transiently silencing ORRM2 in the orrm3 mutant background.21 Thus, the redundancy hypothesis is plausible for ORRM2 and ORRM3. Additionally, overexpression of ORRM3 or the RRM domain of ORRM3 improves the editing defects in the orrm4 mutant,20 suggesting that ORRM3 is able to compensate (at least partially) for the function of ORRM4 in the editosome. Other plant RRM-containing proteins The RRM domain is one of the most abundant protein domains in eukaryotes, and is involved in multiple biological functions, such as pre-mRNA processing, splicing, mRNA stability, translation regulation, and RNA editing.25 Other RRM-containing proteins have been previously analyzed in terms of their function in RNA editing. CP31A, CP31B and CP29A belong to a small family of chloroplast ribonucleoproteins (cpRNPs) that are characterized by a twin RRM domain.26,27 The tobacco
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protein CP31 was reported to be a plastid editing factor in an in vitro RNA editing system.28 However, its close relatives in Arabidopsis, CP31A, CP31B and CP29A, only have a minor impact on editing (Table 1).29,30 Compared to drastic alterations of RNA accumulation that occurred, cp31a and cp29a mutants showed minor editing defects under cold stress conditions.29 The effect of CP31B on editing is mostly dependent on CP31A mutation.30 Although the RRM domains of the ORRM proteins show high sequence similarity with the one in these cpRNPs, they are in separate phylogenetic groups (Fig. 1), have a distinct domain arrangement (Fig. 3), and different expression pattern under stress conditions. For instance, cold stress does not affect ORRM4 expression but it induces the accumulation of CP29A and CP31A.29,31 These facts may point to different functions of ORRM proteins and the RRM-containing cpRNPs. The identification of the ORRM proteins as RNA editing factors further expands our knowledge of the composition of the editosome. There are now three major protein families known to be required for plant organelle RNA editing: the PPR protein family, the RIP/MORF family and the ORRM family. Further experiments investigating the interaction between mRNA and the ORRM proteins, analyzing the redundancy between the ORRM proteins, and identifying other components of the RNA editosome should give us more insights into the RNA editing apparatus.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
Funding Work on the ORRM family was funded by the National Science Foundation Division of Molecular and Cellular Biosciences, Gene and Genome Systems (grant no. MCB–1330294 to M.R.H. and S.B., grant MCB1020636 to S.B. and grant MCB-0929423 to M.R.H.)
References 1. Hanson MR, Sutton CA, Lu B. Plant organelle gene expression: altered by RNA editing. Trends Plant Sci 1996; 1:57–64; http://dx.doi.org/ 10.1016/S1360-1385(96)80030-6 2. Stern DB, Goldschmidt-Clermont M, Hanson MR. Chloroplast RNA metabolism. Annu Rev Plant Biol 2010; 61:125–55; PMID:20192740; http://dx.doi.org/10.1146/annurev-arplant-042809-112242 3. Gray MW, Covello PS. RNA editing in plant mitochondria and chloroplasts. FASEB J 1993; 7:64–71; PMID:8422976; http://dx.doi.org/ 10.1007/BF00039390 4. Chateigner-Boutin AL, Ramos-Vega M, Guevara-Garcıa A, Andres C, De La Luz Gutierrez-Nava M, Cantero A, Delannoy E, Jimenez LF, Lurin C, Small I, et al. CLB19, a pentatricopeptide repeat protein required for editing of rpoA and clpP chloroplast transcripts. Plant J 2008; 56:590–602; PMID:18657233; http://dx.doi.org/10.1111/j.1365313X.2008.03634.x 5. Sun T, Shi X, Friso G, Van Wijk K, Bentolila S, Hanson MR. A zinc finger motif-containing protein is essential for chloroplast RNA editing. PLoS Genet 2015; 11:e1005028; PMID:25768119; http://dx. doi.org/10.1371/journal.pgen.1005028 6. Bentolila S, Oh J, Hanson MR, Bukowski R. Comprehensive high-resolution analysis of the role of an Arabidopsis gene family in RNA
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2013; 110:E1169–78; PMID:23487777; http://dx.doi.org/10.1073/ pnas.1220162110 Shi X, Germain A, Hanson MR, Bentolila S. RNA recognition motif-containing protein ORRM4 broadly affects mitochondrial RNA editing and impacts plant development and flowering. Plant Physiol 2016; 170:294–309; PMID:26578708; http://dx.doi.org/10.1104/ pp.15.01280 Shi X, Hanson MR, Bentolila S. Two RNA recognition motifcontaining proteins are plant mitochondrial editing factors. Nucleic Acids Res 2015; 43:3814–25; PMID:25800738; http://dx.doi.org/ 10.1093/nar/gkv245 Okuda K, Hammani K, Tanz SK, Peng L, Fukao Y, Myouga F, Motohashi R, Shinozaki K, Small I, Shikanai T. The pentatricopeptide repeat protein OTP82 is required for RNA editing of plastid ndhB and ndhG transcripts. Plant J 2010; 61:339–49; PMID:19845878; http://dx. doi.org/10.1111/j.1365-313X.2009.04059.x Robbins JC, Heller WP, Hanson MR. A comparative genomics approach identifies a PPR-DYW protein that is essential for C-to-U editing of the Arabidopsis chloroplast accD transcript. RNA 2009; 15:1142–53; PMID:19395655; http://dx.doi.org/10.1261/rna.1533909 Kenan DJ, Query CC, Keene JD. RNA recognition: towards identifying determinants of specificity. Trends Biochem Sci 1991; 16:214–20; PMID:1716386; http://dx.doi.org/10.1016/0968-0004(91)90088-D Maris C, Dominguez C, Allain FH-T. The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression. FEBS J 2005; 272:2118–31; PMID:15853797; http://dx.doi. org/10.1111/j.1742-4658.2005.04653.x Lorkovic ZJ, Barta A. Genome analysis: RNA recognition motif (RRM) and K homology (KH) domain RNA-binding proteins from the flowering plant Arabidopsis thaliana. Nucleic Acids Res 2002; 30:623–35; PMID:11809873; http://dx.doi.org/10.1093/nar/30.3.623 Ohta M, Sugita M, Sugiura M. Three types of nuclear genes encoding chloroplast RNA-binding proteins (cp29, cp31 and cp33) are present in Arabidopsis thaliana: presence of CP31 in chloroplasts and its homologue in nuclei/cytoplasms. Plant Mol Biol 1995; 27:529–39; PMID:7894017; http://dx.doi.org/10.1007/BF00019319 Hirose T, Sugiura M. Involvement of a site-specific trans-acting factor and a common RNA-binding protein in the editing of chloroplast mRNAs: development of a chloroplast in vitro RNA editing system. EMBO J 2001; 20:1144–52; PMID:11230137; http://dx.doi.org/ 10.1093/emboj/20.5.1144 Kupsch C, Ruwe H, Gusewski S, Tillich M, Small I, Schmitz-Linneweber C. Arabidopsis chloroplast RNA binding proteins CP31A and CP29A associate with large transcript pools and confer cold stress tolerance by influencing multiple chloroplast RNA processing steps. Plant Cell 2012; 24:4266–80; PMID:23110894; http://dx.doi.org/ 10.1105/tpc.112.103002 Tillich M, Hardel SL, Kupsch C, Armbruster U, Delannoy E, Gualberto JM, Lehwark P, Leister D, Small ID, Schmitz-Linneweber C. Chloroplast ribonucleoprotein CP31A is required for editing and stability of specific chloroplast mRNAs. Proc Natl Acad Sci 2009; 106:6002–7; PMID: 19297624; http://dx.doi.org/10.1073/pnas.0808529106 Kwak KJ, Kim YO, Kang H. Characterization of transgenic Arabidopsis plants overexpressing GR-RBP4 under high salinity, dehydration, or cold stress. J Exp Bot 2005; 56:3007–16; PMID: 16207746; http://dx.doi.org/10.1093/jxb/eri298
ARTICLE ADDENDUM
Organelle RNA recognition motif-containing (ORRM) proteins are plastid and mitochondrial editing factors in Arabidopsis Xiaowen Shi, Stephane Bentolila, and Maureen R. Hanson Department of Molecular Biology and Genetics, Cornell University, Biotechnology Building, Ithaca, NY, USA
ABSTRACT
ARTICLE HISTORY
Post-transcriptional C-to-U RNA editing occurs at specific sites in plastid and plant mitochondrial transcripts. Members of the Arabidopsis pentatricopeptide repeat (PPR) motif-containing protein family and RNA-editing factor Interacting Protein (RIP, also known as MORF) family have been characterized as essential components of the RNA editing apparatus. Recent studies reveal that several organelle-targeted RNA recognition motif (RRM)-containing proteins are involved in either plastid or mitochondrial RNA editing. ORRM1 (Organelle RRM protein 1) is essential for plastid editing, whereas ORRM2, ORRM3 and ORRM4 are involved in mitochondrial RNA editing. The RRM domain of ORRM1, ORRM3 and ORRM4 is required for editing activity, whereas the auxiliary RIP and Glycine-Rich (GR) domains mediate the ORRM proteins’ interactions with other editing factors. The identification of the ORRM proteins as RNA editing factors further expands our knowledge of the composition of the editosome.
Received 18 February 2016 Revised 10 March 2016 Accepted 12 March 2016 Keywords
CP31; RNA editing; Glycine-rich domain; ORRM proteins; protein-protein interaction assays; RNA recognition motif
Abbreviations: RRM, RNA Recognition Motif; ORRM, Organelle RRM protein; GR, Glycine-Rich; PPR, pentatricopep-
tide repeat; RIP, RNA-editing factor Interacting Protein; MORF, Multiple Organelle RNA-editing Factor; OZ1, Organelle Zinc finger protein 1
In land plants, post-transcriptional C-to-U RNA editing modifies plastid and mitochondrial transcripts, often changing the amino acid sequence from what the genomic sequence predicts.1,2 It is regarded as a correction mechanism for defective genes affected by T-to-C mutations.3 Changes in the extent of RNA editing may impair the proper function of gene products, even leading to seedling lethality.4,5 In Arabidopsis, 43 Cs in plastids and over 600 Cs in mitochondria have been characterized as targets of RNA editing.6,7 Cis- and trans- factors essential for the editing process have been identified. Cis-elements, sequences present in close proximity to the editable C,8-10 are recognized by members of the pentatricopeptide repeat (PPR) motif-containing protein family.11,12 Members of the RNA-editing factor Interacting Protein (RIP, also known as MORF) family are trans-factors that function in both plastid and mitochondrial RNA editing.6,13,14,16 Several other proteins, such as OZ1, a zinc finger protein, and PPO1, a tetrapyrrole biosynthesis enzyme, have also been reported to be editing transfactors.5,15 The DYW domain present on some members of the PPR protein family may supply the cytidine deaminase activity required to convert C to U.16-18 In spite of these findings, the composition of the plant RNA editosome is not yet fully understood.
Characterization of four ORRM proteins as RNA editing factors We have recently discovered an additional group of trans-factors required for editing of particular Cs in chloroplasts and mitochondria. Due to the presence of an RNA recognition motif (RRM), we have named these trans-factors as Organelle RRM (ORRM) proteins. The founder of this family, ORRM1 (Organelle RNA recognition motif-containing protein 1), is a plastid editing factor that was discovered through a database search with the protein sequence of the major plastid and mitochondrial editing factor RIP1.13,19 Twenty-one plastid sites exhibit a reduction of editing in the orrm1 homozygous mutant relative to the wild-type (Table 1).19 Subsequently, mitochondrial RNA editing factors ORRM2, ORRM3 and ORRM4 were identified based on their sequence similarity to ORRM1 in the RRM domain (Fig. 1).20,21 Transient silencing of ORRM2 and ORRM3 resulted in reduced editing efficiency at over 30 of the mitochondrial sites (Table 1).21 ORRM4 was characterized as a major mitochondrial editing factor because ORRM4 mutation causes editing defects at 270 mitochondrial C targets (Table 1).20 The alteration of ORRM1, ORRM2 and ORRM3 expression did not cause any morphological defects in the corresponding mutant or silenced tissue, whereas a slow growth
CONTACT Maureen R. Hanson [email protected] Addendum to: Shi X, Germain A, Hanson MR, Bentolila S. RNA Recognition Motif-Containing Protein ORRM4 Broadly Affects Mitochondrial RNA Editing and Impacts Plant Development and Flowering. Plant Physiol. 2016; 170:294–309. doi: 10.1104/pp.15.01280. PMID: 26578708 Published with license by Taylor & Francis Group, LLC © Xiaowen Shi, Stephane Bentolila, and Maureen R. Hanson This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.
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Table 1. Description of the ORRM proteins, CP31A, CP31B, and CP29A. The table shows information relating to the proteins discussed in this paper, including their gene IDs, location of the proteins, names and phenotypes of T-DNA mutant or virus-induced gene silencing (VIGS) lines. Published data is from Sun et al., 2013; Shi et al., 2015; Shi et al., 2016; Tillich et al., 2009 and Kupsch et al., 2012.19-21,29,30 P, plastid. M, mitochondria. Gene ID
Location
ORRM1 ORRM2 ORRM3
At3g20930 At5g54580 At5g61030
P M M
ORRM4
At1g74230
M
CP31A
At4g24770
P
CP31B
At5g50250
P
CP29A
At3g53460
P
Name of T-DNA mutant or VIGS line
Growth phenotype
SALK_072648 ORRM2-VIGS ORRM3-VIGS SALK_038244 ORRM4-VIGS SALK_023061C SALK_109613 SAIL_258_h02 WiscDsLox383H9
No phenotypic defect No phenotypic defect No phenotypic defect No phenotypic defect No phenotypic defect Slow growth and late flowering Pale leaves under cold stress conditions No phenotypic defect
SALK_003066 001G06
Pale leaves under cold stress conditions
and late flowering phenotype was observed in the orrm4 mutant (Table 1).19-21 The impact of ORRM4 on plant growth and flowering indicates that adequate mitochondrial editing is necessary for normal plant development. Interaction between ORRM proteins and other editing factors RNA editing is performed by editosomes found between the 200 and 400 kD markers on size exclusion columns.13 Proteinprotein interaction assays have provided us with some information about the composition of RNA editosomes. ORRM1 interacts with the PPR proteins that are required for editing of the same C target as ORRM1(Fig. 2A) . For instance, both ORRM1 and OTP82 are required for the editing at ndhG C50 and ndhB C836, and as expected, ORRM1 interacts with OTP82.19,22 On the contrary, ORRM1 is dispensable for the editing at the sites controlled by RARE1, thus no interaction was observed (Fig. 2A).19,23 In addition to PPR proteins, ORRM1 interacts with RIP1, plastid editing factors RIP2 and OZ1 as shown in Fig. 2A.5,19 Unlike ORRM1, mitochondrial editing factors
Figure 1. Phylogenetic tree based on the amino acid sequences of the RRM motifs in RRM-containing proteins (80 amino acids considered). Protein alignments were achieved by using Clustal X version 2.1 (http://www.clustal.org/clustal2/) and adjusted manually. The construction of phylogenetic trees was performed with MEGA5 (www.megasoftware.net). For proteins with two RRM motifs, the difference caused by using either RRM motif for alignment was negligible. Thus, the second RRM motif was used for alignment. The tree was inferred using the Neighbor-Joining method, and evolutionary distances were computed using the P-distance method. The scale bar corresponds to 0.1 substitutions per site.
Number of editing sites affected 21 of 34 35 of 618 32 of 618 110 of 576 51 of 564 270 of 618 13 of 34; 3 of 34 under cold stress conditions 8 of 34, dependent on CP31A mutation 3 of 34, only under cold stress conditions
ORRM2, ORRM3 and ORRM4 do not interact with the tested PPR proteins in yeast two-hybrid assays. ORRM3 interacts with ORRM4, and both of them interact with RIP1 and with themselves (Fig. 2B). ORRM2 interacts with ORRM3, but does not interact with ORRM4, RIP1 or itself (Fig. 2B).20,21 Functional analysis of the RRM domain and the GR domain in ORRM proteins The RRM domain is a conserved ~80 amino-acid domain that binds RNA molecules with a variety of specificities and affinities.24,25 Each of the four ORRM proteins contains one RRM domain either at its N terminus or C terminus (Fig. 3). Complementation tests revealed that the RRM domain of ORRM1, ORRM3 and ORRM4 is necessary for the editing ability of the
Figure 2. Known interactions of ORRM proteins with themselves and other editing factors. (A) Interaction between ORRM1 and other editing factors. (B) Interaction between mitochondrial ORRM proteins and RIP1. Data is from yeast two-hybrid and bimolecular fluorescence complementation assays.5,19-21 Positive signals are represented by a C (shaded light red), while negative signals are shown by a - (shaded light green). n.d., not determined (shaded light yellow).
PLANT SIGNALING & BEHAVIOR
Figure 3. The motif diagram of ORRM1, ORRM2, ORRM3, ORRM4, CP31A, CP31B and CP29A. The scale bar represents 100 amino acids.
protein,19-21 thus the RRM domain plays a key role in both plastid and mitochondrial RNA editing. In addition to the RRM domain, ORRM1 carries a truncated RIP-RIP domain at its N terminus, while ORRM3 and ORRM4 contain a C-terminal Glycine-Rich (GR) domain (Fig. 3). The RIP domain or the GR domain is dispensable for the editing activity of the protein. However, the N-terminal RIP domain is required for ORRM1 to interact with PPR proteins (Fig. 2A), and the GR domain of ORRM4 is responsible for mediating its interaction with ORRM3 and with itself.19,20 Functional redundancy between the mitochondrial ORRM proteins In our study, the editing extent was reduced but not eliminated by the knockdown or knockout of ORRM2, ORRM3 and ORRM4 in mutants or silenced tissue,20,21 likely due to the redundant function of the ORRM proteins. We have shown that the editing efficiency of sites under the control of both ORRM2 and ORRM3 was further impaired by transiently silencing ORRM2 in the orrm3 mutant background.21 Thus, the redundancy hypothesis is plausible for ORRM2 and ORRM3. Additionally, overexpression of ORRM3 or the RRM domain of ORRM3 improves the editing defects in the orrm4 mutant,20 suggesting that ORRM3 is able to compensate (at least partially) for the function of ORRM4 in the editosome. Other plant RRM-containing proteins The RRM domain is one of the most abundant protein domains in eukaryotes, and is involved in multiple biological functions, such as pre-mRNA processing, splicing, mRNA stability, translation regulation, and RNA editing.25 Other RRM-containing proteins have been previously analyzed in terms of their function in RNA editing. CP31A, CP31B and CP29A belong to a small family of chloroplast ribonucleoproteins (cpRNPs) that are characterized by a twin RRM domain.26,27 The tobacco
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protein CP31 was reported to be a plastid editing factor in an in vitro RNA editing system.28 However, its close relatives in Arabidopsis, CP31A, CP31B and CP29A, only have a minor impact on editing (Table 1).29,30 Compared to drastic alterations of RNA accumulation that occurred, cp31a and cp29a mutants showed minor editing defects under cold stress conditions.29 The effect of CP31B on editing is mostly dependent on CP31A mutation.30 Although the RRM domains of the ORRM proteins show high sequence similarity with the one in these cpRNPs, they are in separate phylogenetic groups (Fig. 1), have a distinct domain arrangement (Fig. 3), and different expression pattern under stress conditions. For instance, cold stress does not affect ORRM4 expression but it induces the accumulation of CP29A and CP31A.29,31 These facts may point to different functions of ORRM proteins and the RRM-containing cpRNPs. The identification of the ORRM proteins as RNA editing factors further expands our knowledge of the composition of the editosome. There are now three major protein families known to be required for plant organelle RNA editing: the PPR protein family, the RIP/MORF family and the ORRM family. Further experiments investigating the interaction between mRNA and the ORRM proteins, analyzing the redundancy between the ORRM proteins, and identifying other components of the RNA editosome should give us more insights into the RNA editing apparatus.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
Funding Work on the ORRM family was funded by the National Science Foundation Division of Molecular and Cellular Biosciences, Gene and Genome Systems (grant no. MCB–1330294 to M.R.H. and S.B., grant MCB1020636 to S.B. and grant MCB-0929423 to M.R.H.)
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