The Gating Domain of MEF28 is Essential for Editing Two Contiguous Cytidines in nad2 mRNA in Arabidopsis thaliana.

In plant organelles, each C-to-U RNA editing site is specifically recognized by PLS class pentatricopeptide repeat (PPR) proteins with E1-E2, E1-E2-E+, or E1-E2-DYW domain extensions at the C-terminus. The distance between the PPR domain binding site and the RNA editing site is usually fixed at four bases, increasing the specificity of target site recognition in this system. We here report, in contrast to the general case, on MEF28, which edits two adjacent mitochondrial sites, nad2-89 and nad2-90. When the sDYW domain of MEF28 was replaced with one derived from MEF11 or CRR22, the ability to edit downstream sites was lost, suggesting that the DYW domain of MEF28 provides unique target flexibility for two continuous cytidines. By contrast, substitutions of the entire E1-E2-DYW domains by MEF19E1-E2, SLO2E1-E2-E+, or the CRR22E1-E2-E+ target both nad2 sites. In these cases, access to the contiguous sites in the chimeric PPR proteins is likely to be provided by the trans-associated DYW1-like proteins via the replaced E1-E2 or E1-E2-E+ domains. Furthermore, we demonstrated that the gating domain of MEF28 plays an important role in specific target site recognition of the DYW domain. This finding suggests that the DYW domain and its internal gating domain fine-tune the specificity of the target site, which is valuable information for designing specific synthetic RNA editing tools based on plant RNA editing factors.

The Analysis of the Editing Defects in the dyw2 Mutant Provides New Clues for the Prediction of RNA Targets of Arabidopsis E+-Class PPR Proteins.

C to U editing is one of the post-transcriptional steps which are required for the proper expression of chloroplast and mitochondrial genes in plants. It depends on several proteins acting together which include the PLS-class pentatricopeptide repeat proteins (PPR). DYW2 was recently shown to be required for the editing of many sites in both organelles. In particular almost all the sites associated with the E+ subfamily of PPR proteins are depending on DYW2, suggesting that DYW2 is required for the function of E+-type PPR proteins. Here we strengthened this link by identifying 16 major editing sites controlled by 3 PPR proteins: OTP90, a DYW-type PPR and PGN and MEF37, 2 E+-type PPR proteins. A re-analysis of the DYW2 editotype showed that the 49 sites known to be associated with the 18 characterized E+-type PPR proteins all depend on DYW2. Considering only the 288 DYW2-dependent editing sites as potential E+-type PPR sites, instead of the 795 known editing sites, improves the performances of binding predictions systems based on the PPR code for E+-type PPR proteins. However, it does not compensate for poor binding predictions.

The conserved domain in MORF proteins has distinct affinities to the PPR and E elements in PPR RNA editing factors.

In plant organelles specific nucleotide motifs at C to U RNA editing sites are recognized by the PLS-class of pentatricopeptide repeat (PPR) proteins, which are additionally characterized by a C-terminal E domain. The PPR elements bind the nucleotides in the target RNA, while the function of the E domain has remained unknown. At most sites RNA editing also requires multiple organellar RNA editing factor (MORF) proteins. To understand how these two types of proteins are involved in RNA editing complexes, we systematically analyzed their protein-protein interactions. In vivo pull-down and yeast two-hybrid assays show that MORF proteins connect with selected PPR proteins. In a loss of function mutant of MORF1, a single amino acid alteration in the conserved MORF domain abrogates interactions with many PLS-class PPR proteins, implying the requirement of direct interaction to PPR proteins for the RNA editing function of MORF1. Subfragment analyses show that predominantly the N-terminal/central regions of the MORF domain in MORF1 and MORF3 bind the PPR proteins. Within the PPR proteins, the E domains in addition to PPR elements contact MORF proteins. In chimeric PPR proteins, different E domains alter the specificity of the interaction with MORF proteins. The selective interactions between E domain containing PPR and MORF proteins suggest that the E domains and MORF proteins play a key role for specific protein complexes to assemble at different RNA editing sites.

MEF13 Requires MORF3 and MORF8 for RNA Editing at Eight Targets in Mitochondrial mRNAs in Arabidopsis thaliana.

RNA editing sites in plant mitochondria and plastids are addressed by pentatricopeptide repeat (PPR) proteins with E or E and DYW domains, which recognize a specific nucleotide motif upstream of the edited nucleotide. In addition, some sites require MORF proteins for efficient RNA editing. Here, we assign the novel E domain-containing PPR protein, MEF13, as being required for editing at eight sites in Arabidopsis thaliana. A SNP in ecotype C24 altering the editing level at only one of the eight target sites was located by genomic mapping. An EMS mutant allele of the gene for MEF13 was identified in a SNaPshot screen of a mutated plant population. At all eight target sites of MEF13, editing levels are reduced in both morf3 and morf8 mutants, but at only one site in morf1 mutants, suggesting that specific MEF13-MORF interactions are required. Yeast two-hybrid analyses detect solid connections of MEF13 with MORF1 and weak contact with MORF3 proteins. Yeast three-hybrid (Y3H) analysis shows that the presence of MORF8 enhances the connection between MEF13 and MORF3, suggesting that a MORF3-MORF8 heteromer may form stably or transiently to establish interaction with MEF13.

Selective homo- and heteromer interactions between the multiple organellar RNA editing factor (MORF) proteins in Arabidopsis thaliana.

RNA editing in plastids and mitochondria of flowering plants requires pentatricopeptide repeat proteins (PPR proteins) for site recognition and proteins of the multiple organellar RNA editing factor (MORF) family as cofactors. Two MORF proteins, MORF5 and MORF8, are dual-targeted to plastids and mitochondria; two are targeted to plastids, and five are targeted to mitochondria. Pulldown assays from Arabidopsis thaliana tissue culture extracts with the mitochondrial MORF1 and the plastid MORF2 proteins, respectively, both identify the dual-targeted MORF8 protein, showing that these complexes can assemble in the organelles. We have now determined the scope of potential interactions between the various MORF proteins by yeast two-hybrid, in vitro pulldown, and bimolecular fluorescence complementation assays. The resulting MORF-MORF interactome identifies specific heteromeric MORF protein interactions in plastids and in mitochondria. Heteromers are observed for MORF protein combinations affecting a common site, suggesting their functional relevance. Most MORF proteins also undergo homomeric interactions. Submolecular analysis of the MORF1 protein reveals that the MORF-MORF protein connections require the C-terminal region of the central conserved MORF box. This domain has no similarity to known protein modules and may form a novel surface for protein-protein interactions.

RNA editing in plant mitochondria­connecting RNA target sequences and acting proteins.

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

RNA editing in plants and its evolution.

RNA editing alters the identity of nucleotides in RNA molecules such that the information for a protein in the mRNA differs from the prediction of the genomic DNA. In chloroplasts and mitochondria of flowering plants, RNA editing changes C nucleotides to U nucleotides; in ferns and mosses, it also changes U to C. The approximately 500 editing sites in mitochondria and 40 editing sites in plastids of flowering plants are individually addressed by specific proteins, genes for which are amplified in plant species with organellar RNA editing. These proteins contain repeat elements that bind to cognate RNA sequence motifs just 5' to the edited nucleotide. In flowering plants, the site-specific proteins interact selectively with individual members of a different, smaller family of proteins. These latter proteins may be connectors between the site-specific proteins and the as yet unknown deaminating enzymatic activity.

The longest mitochondrial RNA editing PPR protein MEF12 in Arabidopsis thaliana requires the full-length E domain.

Mitochondrial RNA editing factor 12 (MEF12) was identified in a screen for editing defects of a chemically mutated plant population in Arabidopsis thaliana. The MEF12 editing protein is required for the C to U change of nucleotide nad5-374. The MEF12 polypeptide is characterized by an exceptionally long stretch of 25 pentatricopeptide repeats (PPR) and a C-terminal extension domain. Editing is lost in mutant plants with a stop codon in the extending element. A T-DNA insertion substituting the 10 C-terminal amino acids of the extension domain reduces RNA editing to about 20% at the target site in a mutant plant. These results support the importance of the full-length extension module for functional RNA editing in plant mitochondria.

MEF10 is required for RNA editing at nad2-842 in mitochondria of Arabidopsis thaliana and interacts with MORF8.

A forwards genetic screen of a chemically mutated plant population identified mitochondrial RNA editing factor 10 (MEF10) in Arabidopsis thaliana. MEF10 is a trans-factor required specifically for the C to U editing of site nad2-842. The MEF10 protein is characterized by a stretch of pentatricopeptide repeats (PPR) and a C-terminal extension domain ending with the amino acids DYW. Editing is lost in mutant plants but is recovered by transgenic introduction of an intact MEF10 gene. The MEF10 protein interacts with multiple organellar RNA editing factor 8 (MORF8) but not with other mitochondrial MORF proteins in yeast two hybrid assays. These results support the model that specific combinations of MORF and MEF proteins are involved in RNA editing in plant mitochondria.

Two related RNA-editing proteins target the same sites in mitochondria of Arabidopsis thaliana.

The facilitators for specific cytosine-to-uridine RNA-editing events in plant mitochondria and plastids are pentatricopeptide repeat (PPR)-containing proteins with specific additional C-terminal domains. Here we report the related PPR proteins mitochondrial editing factor 8 (MEF8) and MEF8S with only five such repeats each to be both involved in RNA editing at the same two sites in mitochondria of Arabidopsis thaliana. Mutants of MEF8 show diminished editing in leaves but not in pollen, whereas mutants of the related protein MEF8S show reduced RNA editing in pollen but not in leaves. Overexpressed MEF8 or MEF8S both increase editing at the two target sites in a mef8 mutant. Double mutants of MEF8 and MEF8S are not viable although both identified target sites are in mRNAs for nonessential proteins. This suggests that MEF8 and MEF8S may have other essential functions beyond these two editing sites in complex I mRNAs.

Multiple organellar RNA editing factor (MORF) family proteins are required for RNA editing in mitochondria and plastids of plants.

RNA editing in plastids and mitochondria of flowering plants changes hundreds of selected cytidines to uridines, mostly in coding regions of mRNAs. Specific sequences around the editing sites are presumably recognized by up to 200 pentatricopeptide repeat (PPR) proteins. The here identified family of multiple organellar RNA editing factor (MORF) proteins provides additional components of the RNA editing machinery in both plant organelles. Two MORF proteins are required for editing in plastids; at least two are essential for editing in mitochondria. The loss of a MORF protein abolishes or lowers editing at multiple sites, many of which are addressed individually by PPR proteins. In plastids, both MORF proteins are required for complete editing at almost all sites, suggesting a heterodimeric complex. In yeast two-hybrid and pull-down assays, MORF proteins can connect to form hetero- and homodimers. Furthermore, MORF proteins interact selectively with PPR proteins, establishing a more complex editosome in plant organelles than previously thought.

The DYW-class PPR protein MEF7 is required for RNA editing at four sites in mitochondria of Arabidopsis thaliana.

In plant mitochondria and plastids, RNA editing alters about 400 and about 35 C nucleotides into Us, respectively. Four of these RNA editing events in plant mitochondria specifically require the PPR protein MEF7, characterized by E and DYW extension domains. The gene for MEF7 was identified by genomic mapping of the locus mutated in plants from EMS treated seeds. The SNaPshot screen of the mutant plant population identified two independent EMS mutants with the same editing defects as a corresponding T-DNA insertion line of the MEF7 gene. Although the amino acid codons introduced by the editing events are conserved throughout flowering plants, even the combined failure of four editing events does not impair the growth efficiency of the mutant plants. Five nucleotides are conserved between the four affected editing sites, but are not sufficient for specific recognition by MEF7 since they are also present at three other sites which are unaffected in the mutants.

The E-class PPR protein MEF3 of Arabidopsis thaliana can also function in mitochondrial RNA editing with an additional DYW domain.

In plants, RNA editing is observed in mitochondria and plastids, changing selected C nucleotides into Us in both organelles. We here identify the PPR (pentatricopeptide repeat) protein MEF3 (mitochondrial editing factor 3) of the E domain PPR subclass by genetic mapping of a variation between ecotypes Columbia (Col) and Landsberg erecta (Ler) in Arabidopsis thaliana to be required for a specific RNA editing event in mitochondria. The Ler variant of MEF3 differs from Col in two amino acids in repeats 9 and 10, which reduce RNA editing levels at site atp4-89 to about 50% in Ler. In a T-DNA insertion line, editing at this site is completely lost. In Vitis vinifera the gene most similar to MEF3 continues into a DYW extension beyond the common E domain. Complementation assays with various combinations of PPR and E domains from the vine and A. thaliana proteins show that the vine E region can substitute for the A. thaliana E region with or without the DYW domain. These findings suggest that the additional DYW domain does not disturb the MEF3 protein function in mitochondrial RNA editing in A. thaliana.

PPR proteins network as site-specific RNA editing factors in plant organelles.

RNA editing in flowering plant mitochondria targets several hundred C nucleotides mostly in mRNAs to be altered to U. Several nuclear encoded genes have been recently identified predominantly in Arabidopsis thaliana which code for proteins involved in specific RNA editing events in plastids or mitochondria. These nuclear genes code for proteins characterized by a stretch of 4-20 repeats of 34-36 amino acids each, accordingly classified as pentatricopeptide repeat (PPR) proteins. These repeats most likely participate in recognizing and binding the specific nucleotide motifs around editing sites which have been defined as essential cis-elements. All of the RNA editing PPR proteins contain at their C-termini an extension of as yet unclear function, the E domain, and some of these are further extended by another domain which terminates with the triplet DYW. While the E domain seems to be always required for their function in RNA editing, the DYW domain can sometimes be removed. At some editing sites a given PPR protein seems to be required, while at others their function can at least partially be compensated by presumably other PPR proteins. These observations suggest that the PPR proteins may act in a complex network to define and to target RNA editing sites.

The DYW-E-PPR protein MEF14 is required for RNA editing at site matR-1895 in mitochondria of Arabidopsis thaliana.

We here identify the PPR protein MEF14 of the DYW subclass as a specific trans-factor required for C to U editing of site matR-1895 by genetic mapping of an EMS induced editing mutant in Arabidopsis thaliana. The wild type Col MEF14 gene complements mutant protoplasts. A T-DNA insertion in the MEF14 gene abolishes detectable editing at the matR-1895 site. Lack of RNA editing at the matR-1895 site does not alter the level of mature and precursor nad1 mRNA molecules. Such RNA editing mutants can be used to analyse the function of genes like this maturase related reading frame in plant mitochondria.

RNA editing competence of trans-factor MEF1 is modulated by ecotype-specific differences but requires the DYW domain.

RNA editing in plant mitochondria posttranscriptionally changes multiple cytidines to uridines. The RNA editing trans-factor MEF1 was identified via ecotype-specific editing polymorphisms in Arabidopsis thaliana. Complementation assays reveal that none of the three amino acid changes between Columbia (Col) and C24 individually alters RNA editing. Only one combination of these polymorphisms lowers editing at two of the three target sites, suggesting additive effects of the involved SNPs. Functional importance of the C-terminal DYW domain was analysed with DYW-truncated and extended constructs. These do not recover RNA editing in protoplasts and regain only low levels in stable transformants. In MEF1, the DYW domain is thus required for full competence in RNA editing and its C-terminus has to be accessible.