Volume 8, Issue 10, Pages 1466-1477 (October 2015) MEF13 Requires MORF3 and MORF8 for RNA Editing at Eight Targets in Mitochondrial mRNAs in Arabidopsis thaliana  Franziska Glass, Barbara Härtel, Anja Zehrmann, Daniil Verbitskiy, Mizuki Takenaka  Molecular Plant  Volume 8, Issue 10, Pages 1466-1477 (October 2015) DOI: 10.1016/j.molp.2015.05.008 Copyright © 2015 The Author Terms and Conditions

Figure 1 RNA Editing at nad7-213 Is Reduced in Arabidopsis thaliana C24 and Abolished in the mef13-1 Mutant. (A) In Arabidopsis thaliana ecotype C24, RNA editing at nucleotide 213 in the mRNA of the nad7 gene is reduced to 70% and the EMS mutant line mef13-1 shows no editing at this nucleotide, whereas WT plants of Arabidopsis thaliana ecotype Columbia (Col) edit up to 100%. On the other hand, the T-DNA insertion line mef13-2 shows no RNA editing defect at the site, suggesting that expression is not disturbed and that the 67 C-terminal amino acids of the E domain beyond the T-DNA insertion site are not essential for function. Overexpression of At3g02330 restores the editing defects in C24 as well as in mef13-1 as shown in the right-hand panels. Color traces in the sequence analyses are as follows: C, blue; T, red; G, black; A, green. (B) Nucleotide changes in C24 and mef13-1 and the concomitant amino acid alterations against the reference sequence of Col are shown in the schematic structure of the MEF13 PPR protein deduced from the gene sequence. Only the Col/C24 difference at nucleotide 252 in the second PPR element and near the EMS mutation in the first PPR element correlates with the lower editing in C24 plants. The T-DNA insertion site of the mef13-2 line is indicated by a triangle. LB, left border of the T-DNA insertion; P, canonical PPR element; L, long PPR element; S, short PPR element; L2, a C-terminally located variation of the L element; S2, a C-terminally located variation of the S element; E, the extension domain. Molecular Plant 2015 8, 1466-1477DOI: (10.1016/j.molp.2015.05.008) Copyright © 2015 The Author Terms and Conditions

Figure 2 The EMS Mutation in Line mef13-1 Abolishes RNA Editing at Seven Sites in Addition to nad7-213. In the mef13-1 mutant line, no C to U RNA editing is observed at the nucleotides ccmFc-50, ccmFc-415, cox3-314, nad2-59, nad4-158, nad5-1665, nad5-1916, and nad7-213 (Figure 1A). Overexpression of At3g02330 restores the RNA editing at seven of the eight affected sites in transgenic mef13-1 plants to the levels found in WT Col plants. Editing at nad5-1665 in the overexpressing line is increased to 60%, whereas WT Col plants show only 20% U derived by RNA editing. Molecular Plant 2015 8, 1466-1477DOI: (10.1016/j.molp.2015.05.008) Copyright © 2015 The Author Terms and Conditions

Figure 3 Mutant Plants of MEF13 Develop Slower than WT Arabidopsis thaliana Plants. WT Arabidopsis thaliana plants, mutants mef13-1 and mef13-1 complemented with the intact MEF13 gene under control of the 35S promoter are compared after 2 weeks on agar medium and 4 weeks on soil. Plants were grown under a 16-h light/8-h dark regime. Bars represent 0.3 cm. Molecular Plant 2015 8, 1466-1477DOI: (10.1016/j.molp.2015.05.008) Copyright © 2015 The Author Terms and Conditions

Figure 4 Similarity between and Ranking of the RNA Editing Targets of MEF13 Suggest Unique Connections between the PPR Protein and the RNAs. (A) Alignment of the putative MEF13 RNA anchor region, the –25 to +5 nucleotide sequences around the eight target sites, shows only one U nucleotide conserved at identical positions in all sites (inverse shading). A further seven nucleotides are present in seven out of the eight nucleotide sequences at the RNA targets, all of which are also uridines (gray background). The few conserved nucleotides suggest that the different editing sites are targeted by MEF13 through distinct nucleotide connections. Sequences are shown 5′ to 3′ from left to right. U nucleotides derived by other editing events are bold and underlined. (B) Prediction of MEF13 target sites assigns the combination of the amino acids at positions 6 and 1′ of the individual 21 PPR elements to the nucleotide most often associated with this set in the previously identified PPR elements to which target RNA sequences have been characterized. Under the premise of one PPR element contacting one nucleotide, amino acid 6 of one element together with the first amino acid of the next element (1′) will by extrapolation thus give a probability of the nucleotide identity most likely recognized by this respective PPR. The top line counts the nucleotides from the edited C toward the 5′ end of the RNA (toward the left). The second line shows the type of PPR element, P having 35, the longer L 36–38, and the shorter S element having 32–34 amino acids. S2 is the C-terminal element just preceding the E domain in the MEF13 PPR protein (Figure 1B). Lines labeled 6 and 1′ give the amino acid present in the respective element. The probability with which each nucleotide is predicted for the amino acid combination in the respective PPR element is given in the bottom panel.The sum of these probabilities can be employed to determine the probability of editing sites being targeted by a given PPR protein. This analysis ranks six of the eight editing sites targeted by MEF13 among the top 20 editing sites as shown in the lower part. This result suggests that indeed not all PPR elements are equally well matched to their opposing nucleotide identity and that the overall match given by the prediction program identifies most of the connections, albeit not perfectly (Takenaka et al., 2013a). Molecular Plant 2015 8, 1466-1477DOI: (10.1016/j.molp.2015.05.008) Copyright © 2015 The Author Terms and Conditions

Figure 5 RNA Editing Target Sites of MEF13 Are Affected in Mutants of Different MORF Editing Proteins in Arabidopsis thaliana. The extent of RNA editing of the nucleotides affected in the MEF13-1 mutant is compared with the respective editing levels in mutants of MORF1, MORF3, and MORF8. For comparison, MEF21 target cox3-257 and MEF19 target ccmB-566 are shown, which are most strongly affected in MORF1 and MORF8 mutants, respectively. All information is extracted from the supplemental data of Bentolila et al. (2013). Molecular Plant 2015 8, 1466-1477DOI: (10.1016/j.molp.2015.05.008) Copyright © 2015 The Author Terms and Conditions

Figure 6 Y2H Assays and BiFC Experiments Reveal Distinct Interactions between Arabidopsis thaliana MEF13 and MORF RNA Editing Factors. (A) In Y2H analyses, MEF13 interacts with mitochondrial MORF1 and less prominently with the dual-targeted MORF8, but not with MORF3, the mutation of which most strongly affects MEF13 target sites. Interactions with the chloroplast located MORF2 and MORF9 are most likely physiologically not relevant since these proteins will not be in contact with the mitochondrial MEF13 in planta. Colony images were taken after 9 days of incubation at 28°C. SD-TL and SD-TLHA indicate SD/-Trp-Leu and SD/-Trp-Leu-His-Ade dropout plates, respectively. To inhibit the interactions, 3AT was added. (B) Fluorescence complementation assays detect interactions between MEF13 and MORF1 as well as with MORF3. Both interactions are located in mitochondria. Individual panels show, from left to right, YFP fluorescence, the mtRFP signal, chlorophyll autofluorescence, and the overlay. Arrows point out congruent locations of mitochondria. Bars represent 10 μm. Molecular Plant 2015 8, 1466-1477DOI: (10.1016/j.molp.2015.05.008) Copyright © 2015 The Author Terms and Conditions

Figure 7 Y3H Assays of MEF13 and MORF Proteins Reveal Distinct Interactions between these Arabidopsis thaliana RNA Editing Factors. Analogous Y2H assays as in Figure 6, but in the inverse orientation with MEF13 connected to the activation domain (AD) and with the MORF3 protein connected to the binding domain (BD) in pBridge detect an interaction between MORF3 and MEF13 (pBridge-MORF3(MORF8), upper left panels). The MORF3–MEF13 connection is, however, too weak to support full growth on 1 mM 3AT. 3AT suppresses the activity of the HIS3 enzyme and thus requires a higher expression level of HIS3, which depends on a strongly transcription activating interaction. In Y3H assays of these same constructs, expression of free MORF8 (induced in the absence of methionine) stabilizes the MORF3–MEF13 interaction, which sustains growth on 2.5 and 10 mM 3AT (upper right panels). MORF3 from the pBridge vector additionally coding for MORF1 does not interact with MEF13 alone (pBridge-MORF3(MORF1), lower left panels) and additional expression of free MORF1 in the Y3H assay conditions does not support or stabilize the potential MORF3–MEF13 interaction (lower right panels). Images of yeast colonies were taken after 6 days of incubation at 28°C. SD-TL, SD-TLHA, SD-TLM, and SD-TLHAM indicate SD/-Trp-Leu, SD/-Trp-Leu-His-Ade, SD/-Trp-Leu-Met, and SD/-Trp-Leu-His-Ade-Met dropout plates, respectively. 3AT was added to probe for the strength of the interactions and the induced activation of the HIS3 transcription. Molecular Plant 2015 8, 1466-1477DOI: (10.1016/j.molp.2015.05.008) Copyright © 2015 The Author Terms and Conditions