DYW domain structures imply an unusual regulation principle in plant organellar RNA editing catalysis.

RNA editosomes selectively deaminate cytidines to uridines in plant organellar transcripts-mostly to restore protein functionality and consequently facilitate mitochondrial and chloroplast function. The RNA editosomal pentatricopeptide repeat proteins serve target RNA recognition, whereas the intensively studied DYW domain elicits catalysis. Here we present structures and functional data of a DYW domain in an inactive ground state and activated. DYW domains harbour a cytidine deaminase fold and a C-terminal DYW motif, with catalytic and structural zinc atoms, respectively. A conserved gating domain within the deaminase fold regulates the active site sterically and mechanistically in a process that we termed gated zinc shutter. Based on the structures, an autoinhibited ground state and its activation are cross-validated by RNA editing assays and differential scanning fluorimetry. We anticipate that, in vivo, the framework of an active plant RNA editosome triggers the release of DYW autoinhibition to ensure a controlled and coordinated cytidine deamination playing a key role in mitochondrial and chloroplast homeostasis.

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.

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.

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.

REDIdb: an upgraded bioinformatics resource for organellar RNA editing sites.

RNA editing is a post-transcriptional molecular process whereby the information in a genetic message is modified from that in the corresponding DNA template by means of nucleotide substitutions, insertions and/or deletions. It occurs mostly in organelles by clade-specific diverse and unrelated biochemical mechanisms. RNA editing events have been annotated in primary databases as GenBank and at more sophisticated level in the specialized databases REDIdb, dbRES and EdRNA. At present, REDIdb is the only freely available database that focuses on the organellar RNA editing process and annotates each editing modification in its biological context. Here we present an updated and upgraded release of REDIdb with a web-interface refurbished with graphical and computational facilities that improve RNA editing investigations. Details of the REDIdb features and novelties are illustrated and compared to other RNA editing databases. REDIdb is freely queried at http://biologia.unical.it/py_script/REDIdb/.

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.

Reverse genetic screening identifies five E-class PPR proteins involved in RNA editing in mitochondria of Arabidopsis thaliana.

RNA editing in flowering plant mitochondria post-transcriptionally alters several hundred nucleotides from C to U, mostly in mRNAs. Several factors required for specific RNA-editing events in plant mitochondria and plastids have been identified, all of them PPR proteins of the PLS subclass with a C-terminal E-domain and about half also with an additional DYW domain. Based on this information, we here probe the connection between E-PPR proteins and RNA editing in plant mitochondria. We initiated a reverse genetics screen of T-DNA insertion lines in Arabidopsis thaliana and investigated 58 of the 150 E-PPR-coding genes for a function in RNA editing. Six genes were identified to be involved in mitochondrial RNA editing at specific sites. Homozygous mutants of the five genes MEF18-MEF22 display no gross disturbance in their growth or development patterns, suggesting that the editing sites affected are not crucial at least in the greenhouse. These results show that a considerable percentage of the E-PPR proteins are involved in the functional processing of site-specific RNA editing in plant mitochondria.

A truncated MEF11 protein shows site-specific effects on mitochondrial RNA editing.

RNA editing in flowering plant mitochondria post-transcriptionally alters several hundred nucleotides from C to U, mostly in mRNAs. We recently identified the nuclear encoded gene MEF11 which is involved in RNA editing of three sites in mRNAs coding for subunits of respiratory chain complexes in Arabidopsis thaliana. In the mef11-2 mutant a T-DNA insert alters the C-terminal part of the DYW domain. This insertion blocks RNA editing at sites cox 3-422 and site nad4-124 completely, but only partially at ccb203-344. Transcription analysis shows that RNA editing is not defective due to disturbance of transcripts from the mef11-2 locus. These observations suggest that the enzymatic activity or its recruitment is not compromised by the alteration of the C-terminus of MEF11.

The PPR protein encoded by the LOVASTATIN INSENSITIVE 1 gene is involved in RNA editing at three sites in mitochondria of Arabidopsis thaliana.

Post-transcriptional RNA editing in flowering plant mitochondria alters several hundred nucleotides from cytidine to uridine, mostly in mRNAs. To characterize the factors involved in RNA editing in plant mitochondria, we initiated a screen for nuclear mutants defective in RNA editing at specific sites. Here we identify the nuclear-encoded gene MEF11, which is involved in RNA editing of the three sites cox3-422, nad4-124 and ccb203-344 in Arabidopsis thaliana. A T-DNA insertion line of this gene was previously characterized as showing enhanced tolerance to the compound lovastatin, an inhibitor of the mevalonate pathway of isoprenoid biosynthesis. The mef11-1 mutant described here shows similar tolerance to lovastatin. Identification of the function of the MEF11 protein in site-specific mitochondrial RNA editing suggests indirect effects of retrograde signalling from mitochondria to the cytoplasm to evoke alteration of the mevalonate pathway. The editing sites cox3-422 and ccb203-344 each alter amino acids that are conserved in the respective proteins, while the nad4-124 site is silent. The single amino acid change in the mef11-1 mutant occurs in the second pentatricopeptide repeat, suggesting that this motif is required for site-specific RNA editing.

A DYW domain-containing pentatricopeptide repeat protein is required for RNA editing at multiple sites in mitochondria of Arabidopsis thaliana.

RNA editing in flowering plant mitochondria alters 400 to 500 nucleotides from C to U, changing the information content of most mRNAs and some tRNAs. So far, none of the specific or general factors responsible for RNA editing in plant mitochondria have been identified. Here, we characterize a nuclear-encoded gene that is involved in RNA editing of three specific sites in different mitochondrial mRNAs in Arabidopsis thaliana, editing sites rps4-956, nad7-963, and nad2-1160. The encoded protein MITOCHONDRIAL RNA EDITING FACTOR1 (MEF1) belongs to the DYW subfamily of pentatricopeptide repeat proteins. Amino acid identities altered in MEF1 from ecotype C24, in comparison to Columbia, lower the activity at these editing sites; single amino acid changes in mutant plants inactivate RNA editing. These variations most likely modify the affinity of the editing factor to the affected editing sites in C24 and in the mutant plants. Since lowered and even absent RNA editing is tolerated at these sites, the amino acid changes may be silent for the respective protein functions. Possibly more than these three identified editing sites are addressed by this first factor identified for RNA editing in plant mitochondria.