RNA Recognition Motif-Containing ... - Plant Physiology · RNA Recognition Motif-Containing Protein ORRM4 Broadly Affects Mitochondrial RNA Editing and Impacts Plant Development and

RNA Recognition Motif-Containing Protein ORRM4Broadly Affects Mitochondrial RNA Editing and ImpactsPlant Development and Flowering1[OPEN]

Xiaowen Shi, Arnaud Germain, Maureen R. Hanson*, and Stéphane Bentolila

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853 (X.S., A.G., M.R.H.,S.B.)

ORCID IDs: 0000-0003-0281-2953 (X.S.); 0000-0001-8141-3058 (M.R.H.); 0000-0003-2805-7298 (S.B.).

Plant RNA editosomes modify cytidines (C) to uridines (U) at specific sites in plastid and mitochondrial transcripts. Members ofthe RNA-editing factor interacting protein (RIP) family and Organelle RNA Recognition Motif-containing (ORRM) family areessential components of the Arabidopsis (Arabidopsis thaliana) editosome. ORRM2 and ORRM3 have been recently identified asminor mitochondrial editing factors whose silencing reduces editing efficiency at ;6% of the mitochondrial C targets. Here wereport the identification of ORRM4 (for organelle RRM protein 4) as a novel, major mitochondrial editing factor that controls;44% of the mitochondrial editing sites. C-to-U conversion is reduced, but not eliminated completely, at the affected sites. Theorrm4 mutant exhibits slower growth and delayed flowering time. ORRM4 affects editing in a site-specific way, though orrm4mutation affects editing of the entire transcript of certain genes. ORRM4 contains an RRM domain at the N terminus and aGly-rich domain at the C terminus. The RRM domain provides the editing activity of ORRM4, whereas the Gly-rich domain isrequired for its interaction with ORRM3 and with itself. The presence of ORRM4 in the editosome is further supported by itsinteraction with RIP1 in a bimolecular fluorescence complementation assay. The identification of ORRM4 as a majormitochondrial editing factor further expands our knowledge of the composition of the RNA editosome and reveals thatadequate mitochondrial editing is necessary for normal plant development.

C-to-U RNA editing in plants is confined to plastidand mitochondrial transcripts (Covello and Gray, 1989;Hiesel et al., 1989), with the exception of C-to-U editingevents that have been observed at two sites withinnuclear-encoded tRNAs of Arabidopsis (Arabidopsisthaliana; Zhou et al., 2014). A total of 43 Cs in plastidsand more than 600 Cs in mitochondria have beenreported to be editing sites in Arabidopsis (Chateigner-Boutin and Small, 2007; Bentolila et al., 2013; Ruweet al., 2013). A large proportion of RNA editing eventsresults in encoding of a different amino acid than theone predicted by the genome sequence, while in othercases, editing creates a start or stop codon (Wintz andHanson, 1991; Kotera et al., 2005). The editing eventusually generates a more conserved amino acid relative

to the residue in homologous proteins from other or-ganisms (Gualberto et al., 1989). RNA editing is regar-ded as a correction mechanism for T-to-C mutations inplastids andmitochondria that would otherwise impairthe proper function of gene products, even leading toseedling lethality (Smith et al., 1997; Chateigner-Boutinet al., 2008; Gray, 2012).

RNA is processed by editosomes that are found be-tween the 200 and 400 kD markers on size exclusioncolumns (Bentolila et al., 2012). Although the compo-sition of the RNA editosome is not yet fully understood,cis- and trans-factors involved in the editing machineryhave been identified. Cis-elements are sequences pre-sent in close proximity to the C targets (Chaudhuri andMaliga, 1996; Staudinger and Kempken, 2003; Hayesand Hanson, 2007). Members of the PLS subfamily ofpentatricopeptide repeat (PPR) motif-containing pro-teins are trans-factors that specifically recognize cis-elements (Kotera et al., 2005; Zehrmann et al., 2009).The recognition code that specifies how PPR proteinsinteract with cis-elements was proposed recently basedon in silico analysis and interactions were confirmedby RNA-protein binding assays (Barkan et al., 2012;Takenaka et al., 2013; Yin et al., 2013; Okuda et al.,2014). Many editing PPR proteins contain a C-terminalDYW domain (Lurin et al., 2004). The DYW domainexhibits sequence similarity to known cytidine deami-nase motifs (Salone et al., 2007). Mutagenesis of con-served deaminase residues in DYW1, QED1, RARE1,OTP84, and CREF7 leads to loss of editing activity

1 This work was supported by the National Science FoundationDivision of Molecular and Cellular Biosciences, Gene and GenomeSystems (grant no. MCB–1330294 to M.R.H. and S.B.).

* Address correspondence to [email protected] authors responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) are:Maureen R. Hanson ([email protected]) and Stéphane Bentolila([email protected]).

X.S., S.B., and A.G. performed all experiments; S.B. and M.R.H.supervised the experiments and conceived the project; X.S., S.B., andM.R.H. analyzed the data and wrote the article.

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(Boussardon et al., 2014; Wagoner et al., 2015; Hayeset al., 2015), indicating the enzyme activity for catalyzingthe C-to-U conversion may reside in the DYW domain.In addition to the PPR proteins, members of the RIP/

MORF familywere also identified as RNA editing factorsin plastids and mitochondria (Bentolila et al., 2012, 2013;Takenaka et al., 2012). RIP1 (for RNA-editing factorInteracting Protein 1) is a major editing factor that con-trols the editing extent of hundreds of plastid and mito-chondrial C targets (Bentolila et al., 2012). Another familyinvolved in RNA editing is called the ORRM (forOrganelle RNA Recognition Motif-containing protein)family. The ORRM family is composed of a number ofRRM (for RNA Recognition Motif)-containing proteinsthat are mostly predicted or proven to be organelle-targeted (Sun et al., 2013). The founder of this family,ORRM1, is a plastid editing factor discovered througha database search with the RIP1 protein sequence(Sun et al., 2013). Another plastid editing factor, OZ1 (forOrganelle Zinc finger 1), was recently discovered bycoimmunoprecipitation with ORRM1 (Sun et al., 2015).By analyzing the ORRM family, we identified two

proteins, ORRM2 and ORRM3, as minor mitochondrialediting factors (Shi et al., 2015). Unlike ORRM1, whichcontains a truncated RIP-RIP domain at its N terminusand an RRM domain at its C terminus, ORRM2 carriesan RRM domain, while ORRM3 contains an N-terminalRRM domain and a C-terminal GR (for Gly-rich) do-main. The fact that the RRM domain in ORRM1 andORRM3 by itself is able to provide the editing activity ofthe corresponding protein in complementation tests tes-tifies to the critical role of the RRM domain in plastid andmitochondrial editing (Sun et al., 2013; Shi et al., 2015).Here we report the identification of another member

of the ORRM family, encoded byAt1g74230, as amajor,novel mitochondrial editing factor. The protein, whichwe named ORRM4 (for Organelle RRM protein 4),contains an RRM domain at its N terminus and a GRdomain at its C terminus. The absence of ORRM4 ex-pression caused defective mitochondrial editing at;44% of all the mitochondrial sites assayed in thisstudy. ORRM4 is therefore second only to RIP1, which

controls the editing extent of 77% of all the mitochon-drial sites (Bentolila et al., 2013), in its impact on mito-chondrial RNA editing in Arabidopsis. ORRM4expression is also required for normal plant develop-ment, as the orrm4mutant exhibited slower growth anddelayed flowering time. We were able to restore theediting defects in the orrm4 mutant by generating astable transgenic line expressing the coding region ofORRM4. Our identification of ORRM4 further estab-lishes the ORRM family as an important group inplastid and mitochondrial editing.

RESULTS

Transient Silencing of ORRM4 Causes MitochondrialEditing Defects

To characterize the function of ORRM4 in RNA edit-ing, we performed virus-induced gene silencing (VIGS)to transiently silence ORRM4 in Arabidopsis seedlingsexpressing GFP under a 35S promoter. GFP is used as aselectivemarker to track the silencing effectiveness. Two-week-old seedlings were inoculated with Agrobacteriumtransformed with a GFP-silencing construct, or withAgrobacterium carrying an ORRM4/GFP cosilencing con-struct. Nomorphological defectswere observedupon thetransient silencing of ORRM4. Editing extent in uninoc-ulated plants, GFP-silenced plants, and ORRM4/GFPcosilenced plants was measured by a strand- andtranscript-specific RNA-seq method (STS-PCRseq;Bentolila et al., 2013; Shi et al., 2015; Supplemental DatasetS1). The two biological replicates assayed for each samplewere obtained from different plants grown and treatedunder the same conditions (Supplemental Fig. S1).

We performed quantitative reverse transcriptionPCR (qRT-PCR) to assay the reduction of ORRM4 ex-pression level. The RNA expression level of ORRM4 isreduced to ;16% compared to the uninoculated andthe GFP-silenced controls (Fig. 1A). The specificity ofthe silencing experiment is validated by the fact that theexpression level of ORRM4 is not significantly reducedin the ORRM2- and ORRM3-silenced plants (Fig. 1A).

Figure 1. Transient silencing of ORRM4 causes mi-tochondrial editing defects. A, Relative RNA expres-sion level of ORRM4 measured by qRT-PCR. B,Twenty-two editing sites that experienced a significantdecrease of editing extent $ 10% upon the silencingof ORRM4. Editing extents were measured by STS-PCRseq. Two biological replicates were used in eachsilencing experiment. Not inoculated, Plants not in-oculated with Agrobacterium; GFPsil, plants inocu-lated with Agrobacterium carrying a GFP-silencingconstruct; ORRM2sil, plants inoculated with Agro-bacteria harboring an ORRM2/GFP cosilencing con-struct; ORRM3sil, plants inoculated with Agrobacteriaharboring an ORRM3/GFP cosilencing construct;ORRM4sil, plants inoculated with Agrobacteriumcarrying an ORRM4/GFP cosilencing construct.

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ORRM4 Is a Major Mitochondrial RNA Editing Factor


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Out of the 564 mitochondrial editing sites analyzed, wefound that the transient silencing of ORRM4 leads to asignificant reduction of editing extents in both biologi-cal replicates at 51 mitochondrial sites (D $ 10%, P ,1.6e-6), which constitutes 9% of the total of the mito-chondrial sites assayed (Fig. 1B; Supplemental TableS1). No significant plastid editing defects were ob-served in both silenced biological replicates. A previousstudy demonstrated that ORRM4 is localized to mito-chondria in a GFP assay (Vermel et al., 2002).

ORRM4 Mutation Recapitulates Mitochondrial EditingDefects Observed in ORRM4-Silenced Plants

In order to verify the involvement of ORRM4 in mi-tochondrial RNA editing, we obtained a T-DNA in-sertional line SALK_023061C in Columbia backgroundfrom the ABRC stock center. The orrm4mutant containsa T-DNA insertion in an intron separating the 59UTRfrom the first exon ofORRM4 (Fig. 2A). It is a knockoutmutant since the ORRM4 expression is decreased to anundetectable level measured by qRT-PCR (Fig. 2B). Weperformed STS-PCRseq to examine the editing extent ofsites affected in the orrm4 mutant. The orrm4 mutationresults in a significant reduction (D$10%, P, 1.6e-6) ofediting extent at 270 mitochondrial sites (SupplementalTable S3), 44% of the total mitochondrial sites assayed.A total of 96% of the sites identified by transient si-lencing of ORRM4 also showed defects in the orrm4mutant (Supplemental Table S1). For example, at sitesrpl5 C58, rpl5 C47, rps4 C88, and rpl5 C92, decreasedediting extent was observed in both ORRM4-silencedplants and the orrm4 mutant compared to the controls(Fig. 2C). However, the effect on editing resulting fromthe orrm4 mutation is more drastic than that caused byORRM4 silencing, as more sites are affected and editing

extent is more intensely decreased in orrm4 mutantplants compared to the ORRM4-silenced plants (Fig.2C; Supplemental Tables S1 and S3). This result can beexplained by the absence of ORRM4 expression in theorrm4mutant, while someORRM4 expression occurs inthe silenced tissue (Figs. 1A and 2B).

Surprisingly, five plastid sites among the 38 sitesassayed show a significant reduction of editing extentin both biological replicates of the orrm4 mutant plant(Supplemental Table S4). Given the mitochondriallocalization of ORRM4 and the massive effect ofthe mutation on mitochondrial editing, it is likely thatthis effect is indirectly caused by altered mitochondrialfunction. Alternatively, the genetic background of thewild-type plant to which we are comparing the orrm4mutant plants might have resulted in detection of dif-ferent levels of plastid editing at the 5 sites. The inser-tional T-DNA orrm4 mutant plant is distributed as ahomozygous plant; its genetic background is Columbiaso that we used a generic Columbia wild-type as con-trol. However, very slight differences might exist be-tween the unavailable Columbia accession used togenerate the orrm4 mutant and the one we used ascontrol. Divergence between the mutant line’s wild-type sibling and our wild-type line could also explainthe occurrence of the two mitochondrial sites that weresignificantly reduced inORRM4-silenced plants but notin orrm4 mutant plants (Supplemental Table S1).

orrm4 Mutation or Silencing Affects Editing in aSite-Specific Way, Even When All the Sites on a TranscriptAre Reduced in Their Editing Extent

Many sites affected in the orrm4 mutant occur to-gether on a few transcripts (Fig. 3, A and B). An editingfactor can possibly affect editing extent in either a

Figure 2. ORRM4 gene model, RNA expression level, and editing extents in the orrm4 mutant. A, Gene structure of ORRM4.Triangle indicates the locus of the T-DNA insertion. Dashed box shows the gene-specific region selected for the VIGS experiment.B, Relative ORRM4 expression level measured by qRT-PCR. ORRM4 expression is reduced to an undetectable level in orrm4homozygousmutants relative towild-type plants (n = 3). ***, Significance of P, 0.001. C,ORRM4mutant plants recapitulate themitochondrial editing defects in theORRM4-silenced plants assayed by STS-PCRseq. Not inoculated, Plants not inoculated withAgrobacterium (n = 2); GFP_sil, plants inoculated with Agrobacterium carrying a GFP-silencing construct (n = 2); wt, wild-typeArabidopsis (n = 2); ORRM4sil_1, one plant inoculated with Agrobacterium carrying an ORRM4/GFP cosilencing construct;orrm4, orrm4 homozygous mutant plants (n = 2).

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site-specific or a transcript-specificmanner, or bothways.To examine how ORRM4 affects editing, we representedthe proportion of sites controlled byORRM4based on thetranscripts where they are located and on the complex towhich the transcripts belong (Fig. 3A).A total of 262 sites showed decreased editing in the

orrm4 mutant and are distributed on 31 mitochondrialgene transcripts or 43% of the sites assayed (orf114was notincluded in this analysis; Fig. 3A). Three transcriptsshoweda reductionof editing extent at all the sites assayed,whereas the proportion of sites affected at 10 transcripts fellinto a range from 60% to 90% (Fig. 3A). Eight transcriptsshowed a reduction of editing extent between 20% and60% of sites, while 10 transcripts exhibited a reduction ofediting extent on less than 20% of their sites (Fig. 3A).Among this latter group, three transcripts did not exhibitreduction of editing extent at any of their sites.The proportion of reduced edited sites/transcript

varies widely between and inside the mitochondrialcomplexes, with the transcripts encoding the ribosomalproteins being the most affected by the orrm4mutation,followed by the cytochrome c biogenesis transcripts(Fig. 3A). Among this latter group, the ccmFc transcriptis an outlier, as its proportion of reduced edited sitesfalls below 20% while the proportion of all theremaining ccm transcripts is above 60%. Likewise, twogroups can be easily observed in the complex I tran-scripts: one group that is highly affected by orrm4 mu-tation is comprised of nad3, nad4L, and nad9, and asecond group that is more weakly affected is comprised

of the remaining nad transcripts. In summary, eventhough all Cs targets on some transcripts are affected byorrm4mutation, other transcripts vary in the proportionof sites affected.

All the known editing sites on the rpl5 transcript wereaffected by mutation or silencing of ORRM4 (Fig. 3B,top; Supplemental Tables S1 and S3). However, thedecrease in editing extent in rpl5 transcripts showssome site specificity, as it is not constant along thetranscript and varies from 18% to 78% in the orrm4mutant and from 14% to 63% in the ORRM4-silencedtissue (Fig. 3B, bottom). The alteration in editing extentof sites on rpl5 caused by orrm4mutation correlates wellwith that caused by the silencing of ORRM4 (Fig. 3C).Likewise, all the known editing sites on the nad4Ltranscript are affected bymutation or silencing (Fig. 3A;Supplemental Fig. S2A; Supplemental Tables S1 andS3). As observed for rpl5, the reduction of editing extentalong the nad4L transcript varies from 13% to 35% andfrom 8% to 15% in the orrm4mutant and in theORRM4-silenced tissue, respectively (Supplemental Fig. S2B).The reduction of editing extent on nad4L sites caused bythe orrm4mutation or its silencing are highly correlated(R2 = 0.89, Supplemental Fig. S2C). The reduction ofediting extent at transcripts such as rpl5 and nad4L oc-curs on every editing site; however, the magnitude ofthe reduction is site specific as some sites experience amuch more pronounced decrease than the others.

The location of sites affected by the knockdown orknockout of ORRM4 is dispersed in location on certain

Figure 3. orrm4 mutation or silencingaffects editing extent in a site-specificway. A, Proportion of sites affected byorrm4mutation on each transcript. Eachbar represents a transcript color-codedaccording to the complex to which itbelongs. B, The decreased editing andthe alteration of editing extents of siteson the rpl5 transcript measured by STS-PCRseq. C, The decreases in editingcaused by ORRM4 silencing are highlycorrelated with the decreases caused bythe orrm4 mutation on the rpl5 tran-script. D, Editing at site nad4 C1101 issignificantly reduced while editing attwo sites next to it is not. E, Reduction ofediting is observed at site ccmFc C160,but not at ccmFc C155. Not inoc, Plantsnot inoculatedwithAgrobacterium (n=2);GFP_sil, plants inoculated with Agro-bacterium carrying a GFP-silencing con-struct (n = 2); wt, wild-type Arabidopsis(n = 2); ORRM4sil_1, one plant inocu-lated with Agrobacterium carrying anORRM4/GFP cosilencing construct; orrm4mutants (n = 2), Dorrm4 = (% editing ofwt2%editing of orrm4)/% editing of wt;DORRM4sil_1 = (% editing of Not inoc2% editing of ORRM4sil_1)/% editing ofNot inoc.











transcripts. For example, editing at site nad4 C1101 andsite ccmFc C160 are significantly reduced, but the sitesaround them are not (Fig. 3, D and E; SupplementalDataset S1; Supplemental Table S3).

orrm4 Mutation Affects the Abundance of CertainTranscripts Independently of Its Effect on Editing Extent

A possible reason for the strong effect of orrm4 mu-tation on all editing events located on the rpl5 transcriptcould be an alteration in the total abundance of rpl5transcripts. We therefore examined the transcriptabundance of two groups of transcripts showing eithera strong (rps4 and rpl5) or a slight effect (nad4 and nad7)toward orrm4 mutation on their editing extent. Likerpl5, rps4 shows a reduction of editing extent for themajority of its sites in the orrm4 mutant, whereas lessthan 10% of the sites on nad4 and nad7 transcripts areaffected by the orrm4 mutation (Fig. 3A). RNA blothybridization indicates that the orrm4 mutation resultsin significantly decreased transcript abundance onlyfor nad4 and rps4 when compared to the wild-type(P , 0.01), but not for nad7 or rpl5 transcripts (Fig. 4).The effect of orrm4 mutation on transcript abundancedoes not match with its effect on editing extent, as il-lustrated by the transcripts surveyed. Like rps4, nad4exhibits a clear and significant reduction of its transcriptabundance; but unlike rps4, nad4 shows very little effecton the editing extent of its sites (8% vs. 86%, respec-tively; Fig. 3A). Furthermore, both rps4 and rpl5 belongto the group with more sites controlled by ORRM4. Theabundance of rpl5 transcripts is similar in the orrm4mutants versus the wild-type, whereas rps4 shows amost pronounced reduction of transcript abundance inthe orrm4 mutant (Fig. 4). Thus, our data are consistentwith an effect of the orrm4 mutation on the abundanceof transcripts, as detected by RNA blots, but this effectdoes not correlate with the effect on editing extent.

Reduced Mitochondrial Editing Results in Slow Growthand Late Flowering

As shown in Figure 5A, the orrm4 homozygousmutant grows slower than the wild-type Arabidopsisecotype Columbia. orrm4 mutants have lower freshweight than the wild-type plants grown under long-day conditions (14 h of light/day) at the three timepoints that were measured (Fig. 5B). In order to exam-ine the flowering phenotype of the mutant, we exam-ined three flowering time-related traits. The resultsindicate that it took the mutants three more days onaverage for their first flower bud to become visible inthe center of the rosette, for theirs inflorescence stems toreach 1 cm in height, and for their first flower to openin contrast to the wild-type (Fig. 5C). To determinewhether the slow-growth phenotype is the cause of thelate-flowering phenotype, we recorded the freshweightand the number of total leaves of the mutant versus thewild type when the first flower opened. As shown in

Figure 5, D and E, orrm4 mutant plants have higherfresh weight and number of total leaves than the con-trols when their first flower opened. The fact that themutant requires a greater mass than the wild-type toflower indicates that the late-flowering phenotype isnot solely a consequence of its slow-growth phenotype.

Stable Overexpression of ORRM4 in the orrm4 MutantBackground Complements the MitochondrialEditing Defects

To verify that the mitochondrial editing defects inthe orrm4 mutant are truly caused by the absence ofORRM4 expression, we transformed orrm4 homozy-gous mutants with a construct expressing ORRM4driven by a 35S promoter.We performed genotyping ofplants that survived in the Basta selection to verify theirhomozygosity for the orrm4 T-DNA insertion allele andthe presence of the ORRM4 transgene. Afterward, wecollected the seeds from one transgenic line carrying thetransgene in the orrm4 mutant background and cul-tured them under long-day conditions for 4 weeks.Eleven segregating T1 plants were genotyped, and theirediting extents were measured by poisoned primer

Figure 4. orrm4mutation can affect the transcript abundance. A, RNAblots hybridized with nad4, nad7, rps4, or rpl5 probes; wt, three wild-type plants; orrm42/2, three homozygousmutant plants. The negative ofthe ethidium bromide staining gel in the bottom panel serves as aloading control. B, Quantification of the transcript abundance visual-ized in the RNA blots and normalized to the amount of RNA loadeddemonstrates a significant reduction for nad4 and rps4 (**P, 0.01), butnot for nad7 or rpl5.


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extension (PPE) assays. Our genotyping data indicatesthat three plants did not carry any ORRM4 transgene,while eight plants carried one or two copies of thetransgene. Two wild-type plants were grown under thesame conditions, and their editing extents were alsoassayed. Compared to the homozygous mutants with-out the transgene, all plants expressing ORRM4showed restoration of editing (Fig. 6). At site rpl5 C59,the editing extent increased in some transgenic lines to;50% or even to ;65% relative to ;40% in the mutantplants without the transgene (Fig. 6A). Likewise,

editing extent at site rps4 C235 increases from ;30% to;55% or;80% (Fig. 6B). Mitochondrial editing defectsin the orrm4mutant can be restored to a wild-type levelor even higher.

In order to test whether expression of ORRM4 in-creased editing not only in sites affected in the mutant,but also had any effect on other sites, we measured theediting extent of a site that was not affected in the orrm4mutant by a PPE assay. As shown in Figure 6C, theediting extent of cox2 C138 in orrm4 mutants were un-changed from the wild-type level. However, three outof the eight transgenic plants carrying the ORRM4transgene exhibited an unexpected decrease of editingfrom ;85% to ;70% (Fig. 6C). These three plants cor-responded to the ones showing a higher extent ofcomplementation at sites rpl5 C59 and rps4 C235 thanthe wild-type level (Fig. 6, A and B). The measurementof ORRM4 expression by qRT-PCR indicates that thesethree plants possess the highest level of ORRM4 ex-pression (Fig. 6D), probably because they contain twocopies of the transgene. Observation of the ORRM4expression level clearly divides the transgenic lines intotwo groups: three plants presumably homozygous inORRM4 transgene and five plants hemizygous for theORRM4 transgene have an averageORRM4 expressionthat is ;14 times and 5 times the wild-type level, re-spectively (Fig. 6). The ORRM4 expression level corre-lates well with the increase or decrease of editingextents as shown in Figure 6.

Because we found an effect of orrm4 mutation on theabundance of nad4 and rps4 transcripts, we measuredthe abundance of these transcripts in the complementedlines. As seen in Supplemental Figure S3, the presenceof the transgene is able to significantly increase theabundance of nad4 and rps4 transcripts to close to or atwild-type levels (P , 0.01 and P , 0.001, respectively).The abundance of the nad7 transcript is increased, butnot significantly, in the complemented plants, while theamount of rpl5 is invariant (Supplemental Fig. S3). Thislatter result further supports an absence of clear rela-tionship between the effect of ORRM4 on transcriptabundance and editing extent, as the restoration of rpl5editing extent in complemented lines is not accompa-nied by a change in transcript abundance.

ORRM4 Interacts with RIP1

Both RIP1 and ORRM4 have a major impact on mi-tochondrial editing, as decreased expression of eitherresult in editing defects at more than 250 mitochondrialsites. We consolidated the sites affected in either rip1mutants or orrm4 mutants by subdividing them basedon the transcripts where they are located. After rulingout the outliers, which are cox2, nad6, and genes en-coding complex V subunits, we found that the pro-portion of sites per gene affected by rip1 mutationcorrelates well with those affected by the orrm4 muta-tion (Fig. 7A). Additionally, we compiled the data fromthis study with previous data (Bentolila et al., 2013) todetermine which sites are affected by rip1, rip3, or orrm4

Figure 5. orrm4 mutant plants show a slow-growth and late-floweringphenotype. A, Plant growth phenotype of orrm4 homozygous mutants(bottom) and wild-type plants (top) grown at 14 h of light per day for29 d. B, Fresh weight of orrm4 homozygous mutants and wild-typeplants grown at 14 h of light per day for 24 d, 31 d, and 37 d, respec-tively (n = 6). C, orrm4 homozygous mutants flower late compared tothe wild-type plants. First bud, Days until visible flower buds in thecenter of the rosette; 1 cm, days until inflorescence stem reached 1 cmin height; first flower, days until first open flower (n = 30). D and E, Thefresh weight and the number of total leaves of the orrm4mutants versusthe wild-type plants at the opening of the first flower (n = 30). ** P ,0.01, ***P , 0.001.







mutations jointly or independently of each other(Supplemental Dataset S2). A total of 253 sites, or 95%of the sites affected in the orrm4 mutant, are also af-fected in the rip1 mutant (Fig. 7B). As a comparisonpoint, 88% of the sites affected in the rip3 mutant arealso affected in the rip1 mutant (Supplemental DatasetS2; Fig. 7B). The large overlap of mitochondrial sitescontrolled by RIP1 and RIP3 is supported by a physicalinteraction between these two proteins, as demon-strated by yeast two-hybrid (Y2H), pulldown, and bi-molecular fluorescence complementation (BiFC) assays(Zehrmann et al., 2015).

To test whether the genetic interaction between RIP1and ORRM4 is dependent on a physical interactionbetween their encoded products, we performed BiFCassays by transiently coexpressing a protein fused tothe N-terminal half of GFP (GFPN) with another proteinfused to the C-terminal half of GFP (GFPC) in Nicotianabenthamiana. No interactionwould be expected betweenORRM4 and MEF9, as editing at site nad7 C200 wasimpaired only in the mef9mutant (Takenaka, 2010) andnot in the orrm4 mutant (Supplemental Table S3). Aspredicted, no signal was observed when ORRM4-GFPN

was coexpressed with MEF9-GFPC, or in the reciprocalpair MEF9-GFPN with ORRM4-GFPC (Fig. 8, A and B).In contrast, positive protein-protein interaction signals

Figure 6. Stable expression of ORRM4 driven by a 35S promoter inorrm4 homozygous mutants restores the editing defects caused by theorrm4mutation. A, Editing of rpl5C59 assayed by PPE. B, Editing of rps4C235 assayed by PPE. C, Editing of cox2C138 assayed by PPE. orrm42/2,Three plants from the T1 segregating population that do not contain theORRM4 transgene; orrm42/2 w/ 35S::ORRM4, plants from the T1 seg-regating population that carry one or two copies of ORRM4 transgenedriven by a 35S promoter in the orrm4 mutant background; wt, wild-type plants. Editing extents are shown as dark orange bars in com-plemented lines with excess editing of rpl5 C59 and rps4 C235 anddecreased editing of cox2 C138. D, ORRM4 expression measured byqRT-PCR. The level of ORRM4 expression was normalized to the av-erage detected in wild-type plants. The average expression in wild-typeplants was arbitrarily chosen to be 1 (n = 3, log10 scale).

Figure 7. The genetic interaction between ORRM4 and other mito-chondrial editing factors. A, The correlation between percent of re-duced sites per gene affected in the orrm4mutant versus that in the rip1mutant. B, Number of mitochondrial sites affected in rip1, rip3, ororrm4 mutants. C, Number of mitochondrial sites affected in ORRM2-silenced, ORRM3-silenced, or orrm4 mutant plants. Numbers in over-lap region indicates the sites under the control of both/all. Numberoutside the circles shows the sites that are not affected by any of thethree mutations.


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were observed when ORRM4-GFPN and RIP1-GFPC

were coinoculated (Fig. 8C). Also, RIP1-GFPN interactswith RIP1-GFPC (Fig. 8D). In our Y2H assays, the pre-dicted mature coding sequence (with the predictedtransient peptides removed) was fused to the AD or BDdomain. However, the interaction between ORRM4and RIP1 is not informative in yeast, due to the auto-activation of both when fused to the BD domain.

ORRM4 Interacts with ORRM3 and Itself

When we compared the editing sites affected by thesilencing of ORRM3 (Shi et al., 2015) and those affectedby the orrm4 mutation, we found that 15 sites are af-fected in both cases (Fig. 7C). These 15 sites constitute47% of all the sites affected by the silencing of ORRM3.Likewise, about 54% of sites affected in ORRM2-si-lenced plants are also affected in the orrm4 mutant

(Fig. 7C). Therefore, it is possible that ORRM4 interactswith ORRM2 and ORRM3. We performed Y2H andBiFC assays to test this hypothesis. Interaction betweenORRM4 and ORRM3 was observed in both BiFC andY2H assays (Figs. 8E and 9A). Our result also demon-strates that ORRM4 forms homo-dimers (Figs. 8F and9B) but does not interact with ORRM2 in yeast (Fig. 9C).To examine the interaction between ORRM4 and PPRproteins, we selected MEF1 and MEF20, since de-creased editing at site rps4 C956 was observed by eithermef1 or orrm4mutation while editing at site rps4 C226 isimpaired in both mef20 and orrm4 mutants (Takenakaet al., 2010; Zehrmann et al., 2010). The results indicatethat ORRM4 does not interact with MEF1 or MEF20 inY2H (Fig. 9D).

ORRM4 contains an RRM domain at the N terminusand a GR domain at the C terminus (Fig. 10A). To fur-ther probe the role of different domains of ORRM4 inprotein-protein interactions, we also performed Y2Hassays between truncated proteins carrying either theN-terminal or C-terminal regions of the ORRMs. Ourdata indicate that the C-terminal GR domain of ORRM4is indispensable for it to form a homo-dimer, while theN-terminal RRM domain is not (Fig. 9E). Likewise, theC-terminal GR of ORRM4, but not the N-terminal RRMdomain, is required for its interaction with ORRM3 (Fig.9F). The C-terminal GR domain of ORRM4 interacts withthe N-terminal RRM domain of ORRM3 (Fig. 9F).

Stable Overexpression of the N-Terminal RRM Domain ofORRM4 Restores the Editing Defects in the orrm4 Mutant,While the C-Terminal GR Domain Only Has a MinorEffect on Editing

Given that the RRM domain of ORRM3 is requiredfor its editing activity in mitochondria (Shi et al., 2015),we hypothesized that the RRM domain in ORRM4might also rescue the editing defects caused by theorrm4 mutation. Thus, we transformed homozygousorrm4 mutants with two different transgenes. Oneconstruct expresses the N-terminal RRM domain ofORRM4 under the control of a 35S promoter (Fig. 10B).The other transgene expresses a mitochondrial transitpeptide fused to the C-terminal GR domain of ORRM4driven by a 35S promoter (Fig. 10B).

All the transgenic lines were morphologically nor-mal. We selected three independent transgenic plantsfrom the T0 generation of plants transformed with theN-terminal region of ORRM4, and six plants fromthe T0 generation of plants transformed with themitochondrial transit peptide fused to the C-terminalORRM4. Mutants expressing the N-terminal part ofORRM4 showed increased editing extent at the sites wemonitored by PPE assays compared to the mutantswithout the transgene, whereas mutants expressing theC terminus of ORRM4 only exhibited a minor changeof editing extent (Fig. 10C). For instance, editing extentat site rps4 C77 varies from 62% to 87% when theN-terminal portion of ORRM4 is expressed, indicatingthat editing can be recovered to a wild-type level.

Figure 8. ORRM4 interacts with RIP1, ORRM3, and ORRM4 whentransiently coexpressed in N. benthamiana leaves by BiFC assays. Eachconfocal image shows the merge of GFP signal (green) and chlorophyllautofluorescence (red). Scale bar (white line) represents 20 mm. Whitearrowheads point to the GFP signals indicating the interaction. A and B,ORRM4 does not interact with MEF9 serving as a negative control. C,ORRM4 interacts with RIP1. D, RIP1 forms homo-dimers. E, ORRM4forms hetero-dimers with ORRM3. F, ORRM4 forms homo-dimers.





However, in mutants expressing the C terminus ofORRM4, the editing extent is altered only from 44% to56% (Fig. 10C). The restoration of editing upon the ex-pression of the N-terminal portion of ORRM4 was alsoobserved at site rps4 C235 (Fig. 10C). In order to de-termine whether the increase of editing extent in thecomplemented lines is site-specific, we analyzed theediting extent of a site that was not affected in the orrm4mutant. We found that editing at site cox2C138 was notaffected in any of the transgenic lines assayed; it wasnot decreased as has been observed previously for thethree T1 plants transformed with the full lengthORRM4 (Figs. 6C and 10D).

Examination of ORRM4 expression by qRT-PCR inthe complemented lines shows a correlation betweenthe editing restoration and the level of ORRM4 ex-pression in the mutants expressing the N-terminal partof ORRM4 (nORRM4; Fig. 10E). The highest nORRM4-expressing transgenic plant shows also the highest levelof editing restoration, while the lowest nORRM4-expressing transgenic plant shows the lowest level ofediting restoration (Fig. 10, C and E). Given that theexpression of nORRM4 in two of the transgenic plantsreaches a comparable level as the one observed intransgenic T1 plants expressing the full length ORRM4without a decrease in editing extent at site cox2 C138

Figure 9. In the Y2H assay, ORRM4 interacts with ORRM3 and itself,and its C-terminal GR domain is required for the interaction. A, ORRM4forms hetero-dimers with ORRM3. B, ORRM4 forms homo-dimers. C,ORRM4 does not interact with ORRM2 in yeast. D, ORRM4 does notinteract with MEF1 or MEF20 in yeast. E, The C-terminal GR domain ofORRM4 is required for its interaction with itself, whereas its N-terminalRRM domain is not. F, The N-terminal RRMdomain of ORRM3 interactswith the C-terminalGR domain ofORRM4. EM, Yeasts transformedwitha vector carrying an empty GW cassette as a negative control.

Figure 10. The N-terminal RRM domain of ORRM4 is able to rescueRNA editing activity in the orrm4 mutant, whereas the C-terminal GRdomain does not. A, The motif diagram of ORRM1, ORRM2, ORRM3,and ORRM4. B, Constructs used for transformation. nORRM4, Aminoacids 1 to 112; cORRM4, amino acids 113 to 290;mtp, amino acids 1 to29 of the nuclear-encoded coxIV from yeast. C, Editing of rps4 C77 andrpl5 C59 assayed by PPE. D, Editing of rps4 C77, rps4 C235, andrpl5 C59 assayed by PPE. wt, Wild-type plants (n = 2); orrm4 homo-zygous mutants (n = 2); orrm4 w/ 35S::nORRM4, orrm4 homozygousmutants transformed with the N-terminal RRM domain of ORRM4 un-der a 35S promoter; orrm4 w/ 35S::mtp-cORRM4, orrm4 homozygousmutants transformed with the C-terminal GR domain of ORRM4 underthe control of a 35S promoter and an mtp. E, ORRM4 expressionmeasured by qRT-PCR. The level ofORRM4 expressionwas normalizedto the average detected in wild-type plants. The average expression inwild-type plants was arbitrarily chosen to be one (two plants assayed forwild type and orrm4; no. of technical replicates, three). The expressionof the two constructs was measured with two different set of primers.


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(Figs. 6, C andD, and 10, D and E), we can conclude thatthe maintenance of wild-type levels of cox2 C138 edit-ingmight result from the absence of the C-terminal partof ORRM4.The level of expression of the C-terminal GR domain

of ORRM4 in transgenic lines with a range of 303 to903 of the wild type demonstrates that the inability ofthis portion of ORRM4 to restore editing is not due to afaulty expression of the transgene (Fig. 10E). A level ofnORRM4 expression that is 10 times higher than wildtype is sufficient to restore editing extent in two of thethree sites surveyed whereas a level of cORRM4 that is30 times higher than wild type does not restore editingextent in the same two sites (Fig. 10, C and E). Thus, ourdata suggest that the editing ability of ORRM4 ismostlyconferred by its RRM motif.

Stable Overexpression of ORRM3 Restores the EditingDefects in the orrm4 Mutant

Because ORRM3 andORRM4 are bothmitochondrialediting factors sharing high sequence similarity, par-ticularly in the RRMdomain (48% identical at thewholeprotein level, 70% identical in the RRM domain;Supplemental Fig. S4), we wondered whether we could

compensate the editing default in the orrm4 mutantby overexpressing ORRM3. We transformed orrm4homozygous mutants with a construct expressingORRM3 driven by a 35S promoter (Fig. 11A). After se-lection and genotyping by PCR, transgenic plants alongwith two wild-type plants and two orrm4 homozygousmutant plants were grown and assayed for editing ex-tent at three sites by PPE. Editing at site rps4 C77 andrpl5 C59 is controlled by both ORRM3 and ORRM4,whereas editing at site cox2 C138 is controlled by nei-ther (Fig. 1A; Supplemental Tables S1 and S3; Shi et al.,2015). As seen in Figure 11B (left) at site rps4 C77,ORRM3 is able to restore editing in the orrm4mutant upto the wild-type level in one T0 plant and even higherfor another T0 plant. This latter plant also shows atransgressive editing extent at site rpl5 C59. Survey ofORRM3 expression by qRT-PCR indicates that thetransgenic plant showing the highest editing extent alsopossesses the highest level of ORRM3 expressionamong the transgenic plants (110 times the wild-typelevel; Fig. 11C). As observed previously with certain T1plants transformed with ORRM4 under the control ofthe 35S promoter (Fig. 6C), the level of editing extent isdecreased at site cox2C138 for the two plants exhibitingthe highest level of ORRM3 expression (Fig. 11, B andC). This phenomenon occurs here at a much higher

Figure 11. Stable expression of ORRM3 or theN-terminal RRM domain of ORRM3 driven by a 35Spromoter is able to rescue RNA editing activity in theorrm4 mutant. A, Constructs used for transformation.nORRM3, Amino acids 1 to 120; ORRM3, the codingregion of ORRM3. B, Editing of rps4 C77, rpl5 C59,and cox2C138 assayed by PPE. Left, Editing in mutantplants transformed with the full-length ORRM3;right, editing in mutant plants transformed with theN-terminal RRM domain of ORRM3; wt, wild-typeplants (n = 2); orrm4 homozygous mutants (n = 2);orrm4 w/ 35S::ORRM3, orrm4 homozygous mutantstransformed with the coding region of ORRM3 undera 35S promoter; orrm4 w/ 35S::nORRM3, orrm4 ho-mozygous mutants transformed with the N-terminalRRM domain of ORRM3 under a 35S promoter. C,ORRM3 expression measured by qRT-PCR. The levelof ORRM3 expression was normalized to the averagedetected in wild-type plants. The average expressionin wild-type plants was arbitrarily chosen to be one(n = 3).







level of expression of the transgene ;100 times thewild-type level forORRM3 vs.;10 times the wild-typelevel for ORRM4 (Figs. 6D and 11C).

We also tested the ability of the N-terminal RRMdomain of ORRM3 driven by a 35S promoter to com-plement the editing defects of the orrm4 mutant. orrm4homozygous mutants were transformed with a con-struct expressing the N-terminal RRM domain ofORRM3 driven by a 35S promoter (Fig. 11A). As seen inthe right panel of Figure 11B, one of the transgenic T0plants is restored to wild-type level for the editing ex-tent at site rpl5 C59. The same plant exhibits a pro-nounced increase in editing extent at the other siteanalyzed, rps4 C77. This result is somewhat similar tothe experiment performed previously with N-terminalRRM domain ofORRM4 and further supports the RRMdomain as being necessary for the editing ability of theprotein.

DISCUSSION

In this study, we demonstrate that ORRM4 is a majormitochondrial editing factor that controls about 44%of mitochondrial sites in Arabidopsis. Impaired RNAediting was observed in both the orrm4mutant and theORRM4-silenced tissue. Stable overexpression of theORRM4 coding region complements the editing defectsin orrm4 homozygous mutants, further confirmingORRM4 as a mitochondrial editing factor.

ORRM4 resembles ORRM3 in many aspects. Theyhave high similarity in gene structure and domain ar-rangement. As mitochondrial editing factors, both ofthem interact with RIP1 and form homo-dimers. Nev-ertheless, ORRM4 is a very different editing factor thanORRM3, as it has a more general impact on mitochon-drial editing. The transient silencing ofORRM3 resultedin decreased editing extent at 32 mitochondrial sites,with one to five sites affected on each transcript (Shiet al., 2015). Even though the realm of influence ofORRM3 is underestimated, as ORRM3 expression isonly reduced but not eliminated in the silenced tissuewe analyzed, it is clear that the effect of ORRM3 onmitochondrial editing is site-specific and more limitedthan the effect of ORRM4 (Shi et al., 2015). orrm4 mu-tation has a more significant impact on mitochondrialediting; it affects the editing of 270 mitochondrial sites.Among all the mitochondrial editing factors knowntoday, only RIP1 has been reported to affect more sites(Bentolila et al., 2012). However, unlike RIP1, whosedefective expression resulted in an absence of detect-able editing extent for ;100 mitochondrial sites, resid-ual editing extent was observed for all the sites under thecontrol of ORRM4 in the mutant plant. Functional re-dundancy betweenmitochondrial ORRMs could preventthe elimination of editing in sites under the control ofORRM4 in the mutant plant. We have shown previouslythat this hypothesis was plausible for ORRM2 andORRM3 (Shi et al., 2015). Silencing of ORRM2 in orrm3mutant plants further impaired the editing extent of sitesunder the control of both ORRM2 and ORRM3.

The sites under the impact of ORRM4 are sometimesclustered together on particular transcripts, which wasnot observed when ORRM3 was silenced. However,evidence still supports ORRM4 as a site-specific, notmerely transcript-specific, editing factor. First, eventhough sites affected by orrm4 mutation sometimesencompass the entire transcript, the reduction in editingextent at different sites is variable. Second, orrm4 mu-tation does not always affect all the editing sites on onetranscript. In 90% of cases (28 out of 31 gene tran-scripts), reduced editing extent was only observed in afraction of the editing sites, and the distribution of thesesites is not contiguous. It is therefore unlikely that suchspecificity could be explained by general effects such astranscript stability, even though changes in transcriptstability are expected to affect editing extent (Lu andHanson, 1992). While orrm4 mutation affects theabundance of certain transcripts, our data clearlydemonstrate an absence of relationship between theeffect of orrm4mutation on editing extent and transcriptabundance. rpl5 transcript abundance is not reduced inthe orrm4mutant while its editing extent is decreased inall the editing sites. Restoration of rpl5 editing extentin orrm4mutants expressingORRM4 is not accompaniedby a change in its abundance. Finally, we observed adirect interaction between ORRM4 and the mitochon-drial editing factors RIP1and ORRM3, which furtherimplicates ORRM4 as a component of the mitochondrialeditosome. The interaction between ORRM4 andORRM3 was shown to be mediated by the C-terminalGR domain of ORRM4. NMR studies of NtGR-RBP1,which contains an N-terminal RRM domain and aC-terminal GR domain like ORRM4, show that the GRdomain is responsible for mediating self-association bytransient interactions with its RRM domain (NtRRM;Khan et al., 2014).

In the complementation assay, we observed an un-expected decrease of editing at cox2 C138, a site unaf-fected in the orrm4 mutant, upon the stable expressionofORRM4. It only occurred in the transgenic plants thatdisplayed a higher extent of complementation at sitescontrolled by ORRM4. The decreased editing in theseplants is likely due to the overexpression of ORRM4.The overexpression of ORRM4 in these transgenicplants (;10 times the wild-type level) might sequesterother editing factors and thus result in a decrease ofediting at sites for which the sequestered editing factorsare needed. For example, sufficient RIP1 is obviouslyrequired for cox2 C138 editing, given that editing at sitethis site decreased from ;86% to ;8% in the rip1 mu-tant (Bentolila et al., 2012). Because ORRM4 interactswith RIP1, an excessive amount of ORRM4 proteinsmay withhold a large number of RIP1 proteins. Con-sequently, the amount of RIP1 proteins at the RIP1 Ctargets would be deficient, leading to editing defects atsites not controlled by ORRM4. We noticed that theunexpected drop of editing extent did not occur whenwe transformed orrm4 mutants with a construct carry-ing the N-terminal RRM domain driven by a 35S pro-moter. Given that we have shown that the interaction of


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ORRM4 with ORRM3 is mediated through theC-terminal GR domain of ORRM4, the absence of areduction in editing in transgenic lines overexpressingthe N-terminal RRM domain is expected, because otherediting factors cannot be sequestered due to the absenceof the C-terminal domain. This theory is also supportedby the fact that a decrease of editing at cox2 C138 isobserved in some orrm4mutants expressing full-lengthORRM3, but not in the plants expressing theN-terminalRRM domain of ORRM3.Like ORRM3, ORRM4 carries an RRM domain at its

N terminus and a GR domain at its C terminus. In thisstudy, we demonstrated that the expression of the RRMdomain of ORRM4, rather than the expression of its GRdomain, is able to restore the editing defects in theorrm4 mutant. However, in the transgenic line com-plemented for editing extent at sites rps4 C77 and rps4C235, editing extent at site rpl5 C59 did not reach thewild-type level. The level of expression of the transgenein this plant (;10 times the wild-type level) is compa-rable to the one measured in transgenic line expressingthe full length ORRM4 and complemented for rpl5 C59editing extent. Thus, we cannot exclude a supportiverole for the C-terminal GR domain of ORRM4 in con-tributing to the editing ability of the protein for certainsites. We showed that the N-terminal RRM domainof ORRM4 and ORRM3 were overexpressed in bothcomplementation experiments. The excess amount ofnORRM4 or nORRM3 may impair the specificity con-tributed by GR domain through interacting with otherediting factors. In addition to our previous resultsshowing that the RRM domain by itself is able to pro-vide the editing activity of ORRM1 and ORRM3, wehave now further demonstrated that the RRM domainplays a key role in both plastid and mitochondrialediting, whereas the GR domain may play a supportiverole in editing.Expression of ORRM3 in the orrm4 background

supports the hypothesis that some specificity exists inthe RRM domains of different editing factors. Ap-proximately 100 times the expression of the wild-typelevel of ORRM3 was necessary to complement theediting defects in a transgenic plant expressing the full-length ORRM3, whereas ;10 times the expression ofthe full-length ORRM4 was sufficient to restore editingin all the sites analyzed.The RRM domain is a conserved ;80-amino acid

domain that binds to a wide variety of RNA moleculeswith a range of specificities and affinities (Kenan et al.,1991). It is one of the most abundant protein domains ineukaryotes and is involved in various processes of RNAmetabolism, such as premRNA processing, mRNAsplicing, mRNA stability, and RNA editing (Maris et al.,2005). The numerous biological functions of the RRM-containing proteins are likely due to the structuralversatility of the RRM domain, as well as the presenceof variable auxiliary domains. The extreme structuralversatility of the RRM domain makes it capable ofinteracting with diverse partners, not only RNA mole-cules but also proteins, whereas the auxiliary domains,

such as the zinc fingers, are also involved in differentaspects of RNA metabolism (Maris et al., 2005). Takentogether, the data presented in this paper and previousliterature suggest that ORRM4 may specifically recog-nize editing targets; nevertheless, the specificity ofORRM4 for its RNA targets might be enhanced by itsC-terminal GR domain, which binds to cognate editingfactors.

In addition to the ORRMs, other RRM-containingproteins have also been identified as RNA-editing fac-tors. For instance, the mammalian ACF (for apobec-1complementation factor) contains three RRM domains(Mehta et al., 2000). It binds to the apo-B mRNA anddocks APOBEC1 to deaminate its C target (Mehta et al.,2000). Another Arabidopsis RRM-containing protein,CP31A, belongs to a family of chloroplast ribonucleo-proteins with an acidic residue-rich domain and twoRRM domains (Hirose and Sugiura, 2001; Tillich et al.,2009; Kupsch et al., 2012). CP31A was first character-ized as a plastid editing factor as the cp31a mutantshowed editing defects at several plastid sites com-pared to the wild type under normal growth conditions(Tillich et al., 2009). Even though decreased abundanceof some transcripts was observed, the RNA alterationsdid not correlate with the editing defects that weredetected (Tillich et al., 2009). A subsequent studyshowed that CP31A is likely to play a more importantrole inmRNA stability than in editing, as the effect of itsmutation on RNA editing is minor compared to theeffect on rapid change of mRNA accumulation andRNA processing in response to cold stress (Kupschet al., 2012). Whether CP31A interacts with knowncomponents of the RNA editosome is unknown, whileinteractions between ORRM4 and known editing factorswere observed in this study. Additionally, the responseof ORRM4 toward stress conditions is different from thatof CP31A, as ORRM4 expression was not affected bycold, hydration, or salt stress (Kwak et al., 2005).

The GR domain is a feature that is present in the plantGly-rich proteins (GRPs), consisting of sequence re-peats that can be summarized by the formula (Gly)n-X

Figure 12. Model of the mitochondrial editosome that operates onccmC C618 based on protein-protein interaction data. Data are fromY2H and BiFC assays (this study; Bentolila et al., 2012; Shi et al., 2015).The stoichiometry of the components is not indicated, as it is unknown.The editable C at position 618 on the ccmC transcript is represented byastar. The cis-element upstream of the target C is shown as a red bar.





(Sachetto-Martins et al., 2000). The presence of the Gly-rich repeats in GRPs is usually accompanied by someother conserved domains, such as RRM domains, zincfingers, and cold-shock domains (Mangeon et al., 2010).Plant GRPs generally exhibit a developmentally regu-lated and tissue-specific expression pattern, and theirexpression is also modulated by several biotic andabiotic factors (Sachetto-Martins et al., 2000; Mangeonet al., 2010). Plant GRPs have diverse functions in var-iable aspects of cell metabolism. They were proposed tofunction in cell elongation, plant defense, plant stressresponses, flowering, and development, etc. (Fusaroet al., 2007; Fu et al., 2007; Kim et al., 2007; Streitneret al., 2008; Mangeon et al., 2009), likely due to thevariation of their domain arrangements. Despite of allthe efforts in characterizing the functions of plant GRPs,little is known about the function of the GR domain inthese GRPs. With regard to ORRM4, we found that theRRM domain of ORRM4 provides its editing activity,whereas the GR domain of ORRM4 is required for itsinteractionwith other editing factors. This result revealsa possible supportive function of the GR domain inplant GRPs. In other words, when the GRP is part of aprotein complex, the additional domains (such as theRRMdomain) in plant GRPs play amore important rolein the protein function while the GR domain is re-sponsible for its interaction with other members of theprotein complex.

This study provides new insights into the molecularfunction of GR-RBPs. ORRM4 was first identified asa mitochondrial RNA-binding protein named At-mRBP2a (for mitochondrial RNA-binding protein 2a),isolated by its affinity to ssDNA in purified potatomitochondria (Vermel et al., 2002). ORRM4 was alsodescribed as GR-RBP5 (for Gly-rich RNA-binding Pro-tein 5), one of the eight GR-RBP family members inArabidopsis (Lorkovi�c and Barta, 2002). All these GR-RBPs are classified as Class IVa GRPs (Mangeon et al.,2010) based on a N-terminal RRM domain and aC-terminal GR domain of variable length. Plant GR-RBPs have been implicated in the responses to vari-ous stress conditions, particularly to cold stress (Vermelet al., 2002; Kwak et al., 2005; Kim et al., 2007, 2008;Mangeon et al., 2010). Except for ORRM4 (GR-RBP5)and GR-RBP6, cold treatment greatly increased theexpression of all the other GR-RBPs (GR-RBP-1 to -4, -7,and -8; Kwak et al., 2005). Though the molecular ap-paratus relevant to the function of these other GR-RBPsis still at large, several studies have revealed their in-volvement in RNA metabolism (Kwak et al., 2005;Schöning et al., 2008; Mangeon et al., 2010).

We observed a late-flowering phenotype in the orrm4mutant compared to the wild type. Mutation or si-lencing of two other GR-RBPs, GR-RBP7 and GR-RBP8(also named AtGRP7 and AtGRP8), also showed a late-flowering phenotype (Streitner et al., 2008). Thelate-flowering phenotype in the atgrp7 mutant isassociated with an elevated transcript level of a MADS-box protein FLC (for Flowering Locus C), a key re-pressor of flowering (Streitner et al., 2008). Another

study revealed that the silencing of CSDP2 (for ColdShockDomain Protein 2, also namedAtGRP2) resulted inan early flowering phenotype (Fusaro et al., 2007). GR-RBP7, GR-RBP8, and CSDP2 are all nucleo-cytoplasmicproteins (Fusaro et al., 2007; Fu et al., 2007); as a result ofwhich, the mechanism underlying how these proteinsregulate the flowering time is likely to be very differentfrom the influence of ORRM4.

This study further supports the involvement of theORRM family in organelle editosomes. Based on all ofthe interaction data we obtained in this study as wellas previous studies, we propose an updated modelfor mitochondrial editosome. The mitochondrialeditosome is a complex that contains various numbersof RIP/MORFs, ORRMs, and at least one PPR protein.It is heterogenous in its composition and has an un-known stoichiometry of subunits. Figure 12 is an ex-ample of a model for the editosome acting upon siteccmC C618 based on the interaction data obtained fromY2H and BiFC assays reported here and previously(Bentolila et al., 2012; Shi et al., 2015). In this model,RIP1 is a key component in the editosome as it acts likea bridge holding the PPR proteins and the ORRMs to-gether, whereas ORRM proteins exert their functionsthrough interacting with RIP1 and with each other. Inthis model, we have not represented the binding ofORRMs with the RNA even though it is likely to occur,given there is ample support from literature, includingour own data from the study of the chloroplast editingORRM1 (Sun et al., 2013). In addition, we representthis editosome model as a static complex where allthe proteins are present all together, but the interac-tion between the different components might occurconsecutively.

MATERIALS AND METHODS

Plant Material and Morphological Analysis

The Arabidopsis (Arabidopsis thaliana) T-DNA insertion line SALK_023061Cin the ORRM4 gene was ordered from the Arabidopsis Biological ResourceCenter (https://abrc.osu.edu/). After 3 d of stratification, seeds from the mu-tant line were planted in soil and grown in a growth room (14 h of light/10 h ofdark) at 26°C. Plants were genotyped by PCR with BioMix Red (Bioline) usingprimer pair SALK023061C_LP and SALK023061C_RP listed in SupplementalTable S5. The PCR product was sequenced at Cornell University Life SciencesCore Laboratories Center. Leaves were collected from 5-week-old plants forfurther analysis. The ORRM4 expression level was measured by qRT-PCR.

The freshweightofSALK_023061Cplantswasmeasured24, 31, and37dafterplanted.We recorded the number of days it took for visible flower buds to showin the center of the rosette, for inflorescence stem of the plant to reach 1 cm inheight, and for its first flower to open. Information regarding to the freshweightand the number of total leaves of the mutant versus the wild-type was recordedwhen the first flower bloomed.

VIGS

To performVIGS, primers flanking a gene-specific regionwere selected fromthe CATMA database (Crowe et al., 2003). Fragments were amplified usingArabidopsis Columbia ecotype genomic DNA as a template with primer pairsORRM4_VIGS_F and ORRM4_VIGS_R. The PCR product was first integratedinto a PCR8/GW/TOPO vector (Invitrogen) and then transferred to a silencingvector PTRV2/GW/GFP upon a LR Clonase II (Invitrogen) recombination reac-tion. The silencing construct was subsequently transformed into Agrobacterium


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https://abrc.osu.edu/




tumefaciensGV3101 by electroporation. Agrobacterium expressing theORRM4/GFPsilencing construct was used to infiltrate 2-week-old Arabidopsis seedlingsexpressing 35S-GFP as previously described (Bentolila et al., 2012). The green flu-orescence of the inoculated plants was examined under UV light to track the si-lencing efficiency 2 weeks after infiltration. Leaves from 5-week-old GFP-silencedplants that exhibited dark red chlorophyll fluorescence, but no green fluorescence,were collected for further analysis. The silencing efficiency of ORRM4 was mea-sured by qRT-PCR.

Use of STS-PCRseq Method to Assay Editing Extent

The STS-PCRseq technique was presented in detail in a previous paper(Bentolila et al., 2013). In brief, we amplified all transcripts encoding eitherplastid or mitochondrial genes with organelle-specific primers from the RNAextracted from the silenced plants, the mutant plants, and the controls. The RT-PCR products were mixed in equimolar ratio, sheared by sonication, and thenused as a template to produce an Illumina TruSeqDNALibrary. All the samplesin this study were pooled in one sequencing lane of an Illumina HiSEquation2500 instrument. The data we obtained was processed according to theguideline in Bentolila et al. (2013). Subsequent statistical analysis was previ-ously described as in Bentolila et al. (2013) and in Shi et al. (2015). Briefly, wedetected 618 mitochondrial editing sites and 38 plastid editing sites. Because ofrepetitive testing we adjusted the nominal significance test by a Bonferronicorrection to P, 1.6e-6 or P, 2.6e-5 for themitochondrial or plastid editing set,respectively. In the silencing experiments, we discarded 54 mitochondrial sitesand 3 plastid sites from our analyses because they showed a significant re-duction of editing extent in the GFP-silenced control when compared to theuninoculated plants. Finally, the only sites that were declared to be significantlyreduced in their editing extent were those sites that exhibited a significant re-duction in ORRM4-silenced plants when compared with both control plants,uninoculated and GFP-silenced.

Use of PPE Assay to Measure Editing Extents

RNA was extracted using PureLink RNA Mini Kit (Life Technologies),cleared from DNA contamination by using a turbo DNA-free kit (Ambion),followedby reverse transcriptionusing theSuperScript IIIReverseTranscriptase(Life Technologies). Primers used are listed in Supplemental Table S5 or pro-vided in previous studies (Bentolila et al., 2008, 2012, 2013; Shi et al., 2015).Editing extent was examined by PPE assays as described previously (Peetersand Hanson, 2002; Robbins et al., 2009).

Generation of Transgenic Plants

The coding sequence ofORRM4was reverse-transcribedwith SuperScript IIIReverse Transcriptase (Life Technologies) from RNA extracted from wild-typeArabidopsis Columbia using PureLink RNA Mini Kit (Life Technologies) andthen cloned into a PCR8/GW/TOPO vector. Primers used were listed inSupplemental Table S5. The fragment was subsequently shuffled into a modi-fied pBI121 vector using LR Clonase II. The sequence encoding the N-terminusof ORRM4 was amplified from the reverse-transcribed ORRM4 coding se-quence with primer pair ORRM4_F and ORRM4_336R_TAG. The C-terminalORRM4 with the mitochondrial transient peptide (amino acids 1–29 of thenuclear-encoded coxIV from yeast as described by Köhler et al. [1997]) wascloned using an overlap extension PCR method. The fragment amplified usingprimer pair ScCOX4_F and ScCOX4_R and the fragment amplified usingScCOX4R_ORRM4_337F and ORRM4_R were mixed and used as a templatefor a third PCR reactionwith primer pair ScCOX4_F andORRM4_R. The clonedN-terminalORRM4 and the C-terminalORRM4with the transient peptidewereeach integrated into a PCR8/GW/TOPO vector and then transferred into thepBI121 vector by LR reaction. 35S-ORRM4, 35S-nORRM4, and 35S-ScCOX4-cORRM4 in the pBI121 vector were transformed into A. tumefaciens GV3101.Agrobacterium transformed with 35S-nORRM3 or 35S-ORRM3 was from aprevious study (Shi et al., 2015). Floral dip transformation of homozygousorrm4mutant plants was performed as described by Zhang et al. (2006). Plantswere sprayed with Basta twice on soil for selection. The presence of the trans-gene and the homozygosity of the orrm4 mutant allele were verified by PCRreactions using primers listed in Supplemental Table S5. Leaves from 4- to6-week-old transgenic plants were collected for further analysis. T1 generationof a 35S-ORRM4 transgenic line was planted on soil, and leaves from 4-weeksegregating T1 plants were collected.

Y2H Assay

The ORRM4 coding sequence with its predicted transit peptide removed(amino acids 1–30), was amplified from the reverse-transcribed ORRM4 cDNAclone with primer pair ORRM4_91F and ORRM4_R as listed in SupplementalTable S5. The coding sequence of N-terminal ORRM4was cloned with primersORRM4_91F and ORRM4_336R_TAG, whereas the coding sequence ofC-terminal ORRM4 was cloned with primers ORRM4_337F and ORRM4_Rusing the ORRM4 cDNA clone as a template. The N-terminal ORRM3 (aminoacids 1–36 removed) was amplified with primer pair ORRM3_109F andORRM3_360R_TAG from the reverse-transcribedORRM3 cDNA clone as in Shiet al. (2015). All these PCR products were integrated into PCR8/GW/TOPOvectors and then shuffled to pGADT7GW and pGBKT7GW vectors (Horáket al., 2008), respectively. The ORRM3 construct was from Shi et al. (2015).

Twomating types of yeast strain PJ69-4, a and a, were transformed with theconstructs above followed by a protocol in Gietz et al. (1995). Double trans-formants were produced by mating single transformants of mating type a tomating type a, cultured in Leu- and Trp-dropout media (Clontech), and sub-sequently diluted with sterile water to 1 3 106, 1 3 105 cells /mL. A total of10mL of each dilutionwas spotted on Leu-, Trp-, adenine-, andHis-dropoutmediaplates. Yeasts transformed with empty vectors were used as negative controls todetect auto-activation. Data were collected from 2 d to 5 d after spotting.

Bimolecular Fluorescence Complementation Assay

The coding sequences ofORRM3,ORRM4, andRIP1without the stop codonwere amplified from the full-length cDNAs as described above and in previousstudies (Bentolila et al., 2012; Shi et al., 2015), and cloned into pENTR/D-TOPOvectors (Invitrogen). These fragments were then shuffled into XNGW andXCGW vectors (Ohashi-Ito and Bergmann, 2006) by LR reactions. The codingsequence of MEF9 without the stop codon was reverse-transcribed from RNAextracted from wild-type Arabidopsis and subsequently cloned into a PCR8/GW/TOPO vector. The DNA was incubated with restriction endonucleasesXhoI and XbaI for 4 h under 37°C and then transferred into the destinationvectors XNGW and XCGW by LR reactions. Final vectors were validated bysequencing and transformed intoA. tumefaciensGV3101. A range of 5 to 7mL ofeach agrobacterial culture were incubated for 2 d at 28°C, and resuspended in2-(N-morpholine)-ethanesulphonic acid buffer, pH 5.6, with 10 mM MgCl2 and150 mM acetosyringone to an optical density of 1.0. Then we mixed the cultureexpressing GFPN to another culture expressing GFPC in equal volume to a finaloptical density of 0.5 each and incubated the mixtures in the dark at 28°C forabout three hours. Afterward, these mixtures were used to infiltrate leaves from4- to 6-week-old Nicotiana benthamiana plants grown at 10 h of light per day asdescribed in Lin et al. (2014). Within 2 to 4 d after agroinfiltration, the leaf tis-sues were examined with a confocal microscope (Zeiss LSM 710) through a LDLCI Plan-Apochromat 253/0.8 Imm Korr DIC M27 objective. The 488 nm lineof an argon laser was used to excite GFP and chlorophyll. The images werecollected and processed with ZEN 2012 microscope software (Carl ZeissMicroscopy).

Real-Time qRT-PCR Conditions and Analysis

The real-time qRT-PCRwas performed as described in a previous study (Shiet al., 2015). Primers used in the reaction are listed in Supplemental Table S5.The expression levels of ORRM4 and the sequence encoding the N-terminalRRM domain of ORRM4 were measured with qRT-ORRM4-F and qRT-ORRM4-R. C-terminal GR domain of ORRM4 expression was measured withqRT-cORRM4-F and qRT-cORRM4-R.ORRM3 andN-terminal RRMdomain ofORRM3 expression were measured with primer pairs qRT-ORRM3-F + qRT-ORRM3-R and qRT-nORRM3-F + qRT-nORRM3-R.

RNA Blots

RNA gel bot analysis was performed as described by Germain et al. (2011).Primers used to make the probes are listed in Bentolila et al. (2013).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession numbers BT002037.











Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. High reproducibility of editing extent measuredby STS-PCRseq between biological replicates.

Supplemental Figure S2. Variable effect of orrm4 mutation on nad4Lediting extent.

Supplemental Figure S3. Effect of orrm4mutation on transcript abundancecan be reverted in complemented lines.

Supplemental Figure S4. Alignment of ORRM3 and ORRM4.

Supplemental Table S1. Sites showing a significant reduction of editingextent in ORRM4-silenced plants.

Supplemental Table S2. Plastid sites editing extent in ORRM4-silencedplants.

Supplemental Table S3. Mitochondrial sites showing a significant reduc-tion of editing extent in orrm4 mutant plants.

Supplemental Table S4. Plastid sites showing a significant reduction ofediting extent in orrm4 mutant plants.

Supplemental Table S5. Primers used in this study.

Supplementary Dataset S1. Number of reads at each editing site (gene-position) for each library (genotype).

Supplementary Dataset S2. Analysis of the influence of rip1, rip3, andorrm4 mutations on the editing extent of mitochondrial sites.

ACKNOWLEDGMENT

We thank Robert Bukowski for performing the primary analysis of theIllumina sequencing reads.

Received August 13, 2015; accepted November 13, 2015; published November17, 2015.

LITERATURE CITED

Barkan A, Rojas M, Fujii S, Yap A, Chong YS, Bond CS, Small I (2012) Acombinatorial amino acid code for RNA recognition by pentatricopep-tide repeat proteins. PLoS Genet 8: e1002910

Bentolila S, Elliott LE, Hanson MR (2008) Genetic architecture of mito-chondrial editing in Arabidopsis thaliana. Genetics 178: 1693–1708

Bentolila S, Heller WP, Sun T, Babina AM, Friso G, van Wijk KJ, HansonMR (2012) RIP1, a member of an Arabidopsis protein family, interactswith the protein RARE1 and broadly affects RNA editing. Proc NatlAcad Sci USA 109: E1453–E1461

Bentolila S, Oh J, Hanson MR, Bukowski R (2013) Comprehensive high-resolution analysis of the role of an Arabidopsis gene family in RNAediting. PLoS Genet 9: e1003584

Boussardon C, Avon A, Kindgren P, Bond CS, Challenor M, Lurin C,Small I (2014) The cytidine deaminase signature HxE(x)n CxxC ofDYW1 binds zinc and is necessary for RNA editing of ndhD-1. NewPhytol 203: 1090–1095 10.1111/nph.12928

Chateigner-Boutin A-L, Ramos-Vega M, Guevara-García A, Andrés C, de laLuz Gutiérrez-Nava M, Cantero A, Delannoy E, Jiménez LF, Lurin C, SmallI, et al (2008) CLB19, a pentatricopeptide repeat protein required for editing ofrpoA and clpP chloroplast transcripts. Plant J 56: 590–602

Chateigner-Boutin A-L, Small I (2007) A rapid high-throughput methodfor the detection and quantification of RNA editing based on high-resolution melting of amplicons. Nucleic Acids Res 35: e114

Chaudhuri S, Maliga P (1996) Sequences directing C to U editing of theplastid psbL mRNA are located within a 22 nucleotide segment span-ning the editing site. EMBO J 15: 5958–5964

Covello PS, Gray MW (1989) RNA editing in plant mitochondria. Nature341: 662–666

Crowe ML, Serizet C, Thareau V, Aubourg S, Rouzé P, Hilson P, BeynonJ, Weisbeek P, van Hummelen P, Reymond P, et al (2003) CATMA: acomplete Arabidopsis GST database. Nucleic Acids Res 31: 156–158

Fusaro AF, Bocca SN, Ramos RLB, Barrôco RM, Magioli C, Jorge VC,Coutinho TC, Rangel-Lima CM, De Rycke R, Inzé D, et al (2007)AtGRP2, a cold-induced nucleo-cytoplasmic RNA-binding protein, has arole in flower and seed development. Planta 225: 1339–1351

Fu ZQ, Guo M, Jeong BR, Tian F, Elthon TE, Cerny RL, Staiger D, AlfanoJR (2007) A type III effector ADP-ribosylates RNA-binding proteins andquells plant immunity. Nature 447: 284–288

Germain A, Herlich S, Larom S, Kim SH, Schuster G, Stern DB (2011)Mutational analysis of Arabidopsis chloroplast polynucleotide phos-phorylase reveals roles for both RNase PH core domains in polyade-nylation, RNA 39-end maturation and intron degradation. Plant J 67:381–394

Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on thetransformation of intact yeast cells by the LiAc/SS-DNA/PEG proce-dure. Yeast 11: 355–360

Gray MW (2012) Evolutionary origin of RNA editing. Biochemistry 51:5235–5242

Gualberto JM, Lamattina L, Bonnard G, Weil J-H, Grienenberger J-M(1989) RNA editing in wheat mitochondria results in the conservation ofprotein sequences. Nature 341: 660–662

Hayes ML, Dang KN, Diaz MF, Mulligan RM (2015) A conserved gluta-mate residue in the C-terminal deaminase domain of pentatricopeptiderepeat proteins is required for RNA editing activity. J Biol Chem 290:10136–10142

Hayes ML, Hanson MR (2007) Identification of a sequence motif critical forediting of a tobacco chloroplast transcript. RNA 13: 281–288

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

Hirose T, Sugiura M (2001) Involvement of a site-specific trans-actingfactor and a common RNA-binding protein in the editing of chloro-plast mRNAs: development of a chloroplast in vitro RNA editing sys-tem. EMBO J 20: 1144–1152

Horák J, Grefen C, Berendzen KW, Hahn A, Stierhof Y-D, Stadelhofer B,Stahl M, Koncz C, Harter K (2008) The Arabidopsis thaliana responseregulator ARR22 is a putative AHP phospho-histidine phosphatase ex-pressed in the chalaza of developing seeds. BMC Plant Biol 8: 77

Kenan DJ, Query CC, Keene JD (1991) RNA recognition: towards identi-fying determinants of specificity. Trends Biochem Sci 16: 214–220

Khan F, Daniëls MA, Folkers GE, Boelens R, Saqlan Naqvi SM, vanIngen H (2014) Structural basis of nucleic acid binding by Nicotianatabacum glycine-rich RNA-binding protein: implications for its RNAchaperone function. Nucleic Acids Res 42: 8705–8718

Kim JS, Jung HJ, Lee HJ, Kim KA, Goh C-H, Woo Y, Oh SH, Han YS,Kang H (2008) Glycine-rich RNA-binding protein 7 affects abiotic stressresponses by regulating stomata opening and closing in Arabidopsisthaliana. Plant J 55: 455–466

Kim JY, Park SJ, Jang B, Jung C-H, Ahn SJ, Goh C-H, Cho K, Han O, Kang H(2007) Functional characterization of a glycine-rich RNA-binding protein 2 inArabidopsis thaliana under abiotic stress conditions. Plant J 50: 439–451

Köhler RH, Zipfel WR, Webb WW, Hanson MR (1997) The green fluo-rescent protein as a marker to visualize plant mitochondria in vivo.Plant J 11: 613–621

Kotera E, Tasaka M, Shikanai T (2005) A pentatricopeptide repeat proteinis essential for RNA editing in chloroplasts. Nature 433: 326–330

Kupsch C, Ruwe H, Gusewski S, Tillich M, Small I, Schmitz-LinneweberC (2012) Arabidopsis chloroplast RNA binding proteins CP31A andCP29A associate with large transcript pools and confer cold stress tol-erance by influencing multiple chloroplast RNA processing steps. PlantCell 24: 4266–4280

Kwak KJ, Kim YO, Kang H (2005) Characterization of transgenic Arabi-dopsis plants overexpressing GR-RBP4 under high salinity, dehydra-tion, or cold stress. J Exp Bot 56: 3007–3016

Lin MT, Occhialini A, Andralojc PJ, Devonshire J, Hines KM, Parry MAJ,Hanson MR (2014) b-Carboxysomal proteins assemble into highly or-ganized structures in Nicotiana chloroplasts. Plant J 79: 1–12

Lorkovi�c ZJ, Barta A (2002) Genome analysis: RNA recognition motif(RRM) and K homology (KH) domain RNA-binding proteins from theflowering plant Arabidopsis thaliana. Nucleic Acids Res 30: 623–635

Lu B, Hanson MR (1992) A single nuclear gene specifies the abundance andextent of RNA editing of a plant mitochondrial transcript. Nucleic AcidsRes 20: 5699–5703

Lurin C, Andrés C, Aubourg S, Bellaoui M, Bitton F, Bruyère C, CabocheM, Debast C, Gualberto J, Hoffmann B, et al (2004) Genome-wide


Shi et al.














analysis of Arabidopsis pentatricopeptide repeat proteins reveals theiressential role in organelle biogenesis. Plant Cell 16: 2089–2103

Mangeon A, Junqueira RM, Sachetto-Martins G (2010) Functional diversity ofthe plant glycine-rich proteins superfamily. Plant Signal Behav 5: 99–104

Mangeon A, Magioli C, Menezes-Salgueiro AD, Cardeal V, de OliveiraC, Galvão VC, Margis R, Engler G, Sachetto-Martins G (2009) AtGRP5,a vacuole-located glycine-rich protein involved in cell elongation. Planta230: 253–265

Maris C, Dominguez C, Allain FH-T (2005) The RNA recognition motif, aplastic RNA-binding platform to regulate post-transcriptional gene ex-pression. FEBS J 272: 2118–2131

Mehta A, Kinter MT, Sherman NE, Driscoll DM (2000) Molecular cloningof apobec-1 complementation factor, a novel RNA-binding protein involvedin the editing of apolipoprotein B mRNA. Mol Cell Biol 20: 1846–1854

Ohashi-Ito K, Bergmann DC (2006) Arabidopsis FAMA controls the finalproliferation/differentiation switch during stomatal development. PlantCell 18: 2493–2505

Okuda K, Shoki H, Arai M, Shikanai T, Small I, Nakamura T (2014)Quantitative analysis of motifs contributing to the interaction betweenPLS-subfamily members and their target RNA sequences in plastid RNAediting. Plant J 80: 870–882

Peeters NM, Hanson MR (2002) Transcript abundance supercedes editingefficiency as a factor in developmental variation of chloroplast geneexpression. RNA 8: 497–511

Robbins JC, Heller WP, Hanson MR (2009) A comparative genomics ap-proach identifies a PPR-DYW protein that is essential for C-to-U editingof the Arabidopsis chloroplast accD transcript. RNA 15: 1142–1153

Ruwe H, Castandet B, Schmitz-Linneweber C, Stern DB (2013) Arabi-dopsis chloroplast quantitative editotype. FEBS Lett 587: 1429–1433

Sachetto-Martins G, Franco LO, de Oliveira DE (2000) Plant glycine-richproteins: a family or just proteins with a common motif? Biochim Bio-phys Acta 1492: 1–14

Salone V, Rüdinger M, Polsakiewicz M, Hoffmann B, Groth-Malonek M,Szurek B, Small I, Knoop V, Lurin C (2007) A hypothesis on the identifica-tion of the editing enzyme in plant organelles. FEBS Lett 581: 4132–4138

Schöning JC, Streitner C, Meyer IM, Gao Y, Staiger D (2008) Reciprocalregulation of glycine-rich RNA-binding proteins via an interlockedfeedback loop coupling alternative splicing to nonsense-mediated decayin Arabidopsis. Nucleic Acids Res 36: 6977–6987

Shi X, Hanson MR, Bentolila S (2015) Two RNA recognition motif-containing proteins are plant mitochondrial editing factors. NucleicAcids Res 43: 3814–3825

Smith HC, Gott JM, Hanson MR (1997) A guide to RNA editing. RNA 3:1105–1123

Staudinger M, Kempken F (2003) Electroporation of isolated higher-plantmitochondria: transcripts of an introduced cox2 gene, but not an atp6gene, are edited in organello. Mol Genet Genomics 269: 553–561

Streitner C, Danisman S, Wehrle F, Schöning JC, Alfano JR, Staiger D(2008) The small glycine-rich RNA binding protein AtGRP7 promotesfloral transition in Arabidopsis thaliana. Plant J 56: 239–250

Sun T, Germain A, Giloteaux L, Hammani K, Barkan A, Hanson MR,Bentolila S (2013) An RNA recognition motif-containing protein is

required for plastid RNA editing in Arabidopsis and maize. Proc NatlAcad Sci USA 110: E1169–E1178

Sun T, Shi X, Friso G, Van Wijk K, Bentolila S, Hanson MR (2015) A zincfinger motif-containing protein is essential for chloroplast RNA editing.PLoS Genet 11: e1005028

Takenaka M (2010) MEF9, an E-subclass pentatricopeptide repeat protein,is required for an RNA editing event in the nad7 transcript in mito-chondria of Arabidopsis. Plant Physiol 152: 939–947

Takenaka M, Verbitskiy D, Zehrmann A, Brennicke A (2010) Reverse geneticscreening identifies five E-class PPR proteins involved in RNA editing inmitochondria of Arabidopsis thaliana. J Biol Chem 285: 27122–27129

Takenaka M, Zehrmann A, Brennicke A, Graichen K (2013) Improvedcomputational target site prediction for pentatricopeptide repeat RNAediting factors. PLoS One 8: e65343

Takenaka M, Zehrmann A, Verbitskiy D, Kugelmann M, Härtel B,Brennicke A (2012) Multiple organellar RNA editing factor (MORF)family proteins are required for RNA editing in mitochondria andplastids of plants. Proc Natl Acad Sci USA 109: 5104–5109

Tillich M, Hardel SL, Kupsch C, Armbruster U, Delannoy E, GualbertoJM, Lehwark P, Leister D, Small ID, Schmitz-Linneweber C (2009)Chloroplast ribonucleoprotein CP31A is required for editing and stability ofspecific chloroplast mRNAs. Proc Natl Acad Sci USA 106: 6002–6007

Vermel M, Guermann B, Delage L, Grienenberger J-M, Maréchal-Drouard L,Gualberto JM (2002) A family of RRM-type RNA-binding proteins specific toplant mitochondria. Proc Natl Acad Sci USA 99: 5866–5871

Wagoner JA, Sun T, Lin L, Hanson MR (2015) Cytidine deaminase motifswithin the DYW domain of two pentatricopeptide repeat-containingproteins are required for site-specific chloroplast RNA editing. J BiolChem 290: 2957–2968

Wintz H, Hanson MR (1991) A termination codon is created by RNA editing inthe petunia mitochondrial atp9 gene transcript. Curr Genet 19: 61–64

Yin P, Li Q, Yan C, Liu Y, Liu J, Yu F, Wang Z, Long J, He J, Wang H-W,et al (2013) Structural basis for the modular recognition of single-stranded RNA by PPR proteins. Nature 504: 168–171

Zehrmann A, Härtel B, Glass F, Bayer-Császár E, Obata T, Meyer E,Brennicke A, Takenaka M (2015) Selective homo- and heteromer in-teractions between the multiple organellar RNA editing factor (MORF)proteins in Arabidopsis thaliana. J Biol Chem 290: 6445–6456

Zehrmann A, Verbitskiy D, Härtel B, Brennicke A, Takenaka M (2010)RNA editing competence of trans-factor MEF1 is modulated by ecotype-specific differences but requires the DYW domain. FEBS Lett 584: 4181–4186

Zehrmann A, Verbitskiy D, van der Merwe JA, Brennicke A, Takenaka M(2009) A DYW domain-containing pentatricopeptide repeat protein isrequired for RNA editing at multiple sites in mitochondria of Arabidopsisthaliana. Plant Cell 21: 558–567

Zhang X, Henriques R, Lin S-S, Niu Q-W, Chua N-H (2006) Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method.Nat Protoc 1: 641–646

Zhou W, Karcher D, Bock R (2014) Identification of enzymes foradenosine-to-inosine editing and discovery of cytidine-to-uridine edit-ing in nucleus-encoded transfer RNAs of Arabidopsis. Plant Physiol 166:1985–1997





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