Several proteins form variable RNA editing complexes in plant organelles

In RNA editing in plant organelles, PPR (Pentatricopeptide Repeat) proteins with an E or E and DYW domains at the C-terminus are important participants for recognizing the specific cis-elements. These proteins are able to connect to their target RNA sequence via the PPR domains. Recognition of the specific target RNA sequence by PPR proteins is based on the specific binding of one PPR motif to one nucleotide. There are several possibilities for the function of the E-domains in the PPR RNA editing factors: they may bind to the RNA as well as the PPR motifs, or provide the interacting surface for other proteins involved also in the RNA editing process. It is also not excluded that E domains perform these two possible tasks simultaneously. The DYW domain contains a conserved motif (HxExnCxxC) of cytidine deaminases and shows zinc-binding capability, suggesting the domain works as the so far missing deaminase enzyme. But its cytidine deaminase activity has not yet been proofed. The MORFs (Multiple Organellar RNA Editing Factors) also termed as RIPs (RNA editing factor Interacting Proteins) are also involved in several C-to-U substitution events in flowering plants mitochondria and/or in chloroplasts. Nine members of this family contain the conserved 100 amino acids long MORF box and one protein contains only the C-terminally half of that. The exact function of these proteins is unknown yet, but it is suggested that they work together with PPR proteins to perform certain RNA editing events maybe through their direct connections to PPR proteins at each MORF box. To understand further detailed functions of the three domains PPR, E and DYW in RNA editing factors, I employed three different approaches. The first one is functional complementation of the mef28-1 RNA editing mutants with chimeric PPR type RNA editing factors. The MEF28 is involved in the editing of two neighboring cytidines at the nad2-89 and 90 sites. We are interested in how this rarely event is performed by a single PPR protein. The DYW domain of MEF28 is not able to substitute to other DYW domains to edit the downstream cytidine in the two nad2 sites, suggesting its requirement for the flexible targeting function. In the second approach I used Y2H analyses for mapping the binding sites between MEFs (Mitochondrial RNA Editing Factors) and MORFs. In the most cases the bait wild type and partial MEF proteins could connect to the MORF1, MORF2, MORF8 and MORF9, rarely with MORF3 and very rarely with MORF4, MORF5, MORF6 and MORF7. In contrast, the wild type and the partial MEF8 bait constructs (except the MEF8_ECE+) could bind to almost all MORF preys, indicates that MEF8 is a very unique PPR protein. After Y2H analyses of several different partial E domain constructs, I found that the MEF21 is able to bind with the MORF1 through the N-terminal part of the E domain. Finally, I established the Pichia pastoris expression system to express recombinant PPR type RNA editing proteins. The system is now available for further molecular functional analysis of PPR proteins that have been difficult to be expressed in E.coli system

The DYW Subgroup PPR Protein MEF35 Targets RNA Editing Sites in the Mitochondrial rpl16, nad4 and cob mRNAs in Arabidopsis thaliana.

RNA editing in plant mitochondria and plastids alters specific nucleotides from cytidine (C) to uridine (U) mostly in mRNAs. A number of PLS-class PPR proteins have been characterized as RNA recognition factors for specific RNA editing sites, all containing a C-terminal extension, the E domain, and some an additional DYW domain, named after the characteristic C-terminal amino acid triplet of this domain. Presently the recognition factors for more than 300 mitochondrial editing sites are still unidentified. In order to characterize these missing factors, the recently proposed computational prediction tool could be of use to assign target RNA editing sites to PPR proteins of yet unknown function. Using this target prediction approach we identified the nuclear gene MEF35 (Mitochondrial Editing Factor 35) to be required for RNA editing at three sites in mitochondria of Arabidopsis thaliana. The MEF35 protein contains eleven PPR repeats and E and DYW extensions at the C-terminus. Two T-DNA insertion mutants, one inserted just upstream and the other inside the reading frame encoding the DYW domain, show loss of editing at a site in each of the mRNAs for protein 16 in the large ribosomal subunit (site rpl16-209), for cytochrome b (cob-286) and for subunit 4 of complex I (nad4-1373), respectively. Editing is restored upon introduction of the wild type MEF35 gene in the reading frame mutant. The MEF35 protein interacts in Y2H assays with the mitochondrial MORF1 and MORF8 proteins, mutation of the latter also influences editing at two of the three MEF35 target sites. Homozygous mutant plants develop indistinguishably from wild type plants, although the RPL16 and COB/CYTB proteins are essential and the amino acids encoded after the editing events are conserved in most plant species. These results demonstrate the feasibility of the computational target prediction to screen for target RNA editing sites of E domain containing PLS-class PPR proteins

The <i>rpl16</i>-209 editing site is located in a highly conserved environment.

<p><b>(A)</b> Comparison of nucleotide identities in the <i>cis</i>-recognition sequence around the <i>rpl16</i>-209 editing site with the homologous editing sites in other plant species reveals the high degree of conservation. This is presumably imposed by the functional constraints on the conservation of the amino acids surrounding the <i>rpl16</i>-209 editing site. Some of the plants compared here encode a genomic T at this position to maintain the amino acid identity. <b>(B)</b> The absence of RNA editing event <i>rpl16</i>-209 results in <i>Arabidopsis</i> in the incorporation of the genomically encoded threonine rather than the isoleucine specified by the edited codon number 70. The amino acid isoleucine is conserved in even distant plant species. Nucleotides and amino acids derived by RNA editing are given in bold letters and are underlined. Nucleotides and amino acids differing from the consensus are shown in inverse shading.</p

Prediction of the target sites of the mitochondrial RNA editing factor MEF35.

<p>The prediction tool derived from nucleotide–amino acid coincidences between a given PPR element and the corresponding nucleotide was used to predict and rank editing targets for MEF35 by probability [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140680#pone.0140680.ref008" target="_blank">8</a>]. In this listing, the observed target editing sites <i>rpl16</i>-209 and <i>nad4</i>-1373 are highlighted. The third site <i>cob</i>-286 was not predicted by this approach. The putative binding sequences are aligned and the PPR scores are given. These indicate less intensive binding in the N-terminal elements. As generally assumed, alignment begins at nucleotide -4 from the edited C (marked E; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140680#pone.0140680.ref005" target="_blank">5</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140680#pone.0140680.ref009" target="_blank">9</a>]. The nucleotide sequences of the three bona fide target sequences are shown in the bottom part.</p

MEF35 interacts with the MORF1, MORF2 and MORF8 proteins.

<p>Yeast 2-hybrid (Y2H) assays reveal interactions with the mitochondrially located MORF1, the plastid MORF2 and the dual targeted MORF8. In a mutant of MORF1, the MEF35 target sites are not affected [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140680#pone.0140680.ref022" target="_blank">22</a>], some slight effects are seen upon knock-down in other assays [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140680#pone.0140680.ref028" target="_blank">28</a>]. In a respective MORF8 (also termed RIP1) mutant, editing at two of the three MEF35 target sites is reduced, suggesting that the interactions with MORF8 maybe functionally relevant. Controls include cotransfection of the pGBKT7-MEF35 construct with the pGADT7 vector without any MORF insert (empty) as well as the positive interaction control of murine p53 (pGBKT7-53) with the SV40 large T-antigen in pGADT7 (T) and no connection of human lamin C (pGBKT7-Lam) with the SV40 large T-antigen (T). The fusion protein pGBKT7-53 does not interact with the protein product of the empty pGADT7 vector (empty).</p

The MEF35 protein is required for RNA editing at the <i>rpl16</i>-209, <i>nad4</i>-1373 and <i>cob</i>-286 editing sites in mitochondria of <i>Arabidopsis thaliana</i>.

<p><b>(A)</b> Schematic structure of the MEF35 PPR-protein encoded by locus At4g14050. Locations of the T-DNA insertions in the two mutants and their respective left borders (LB) are indicated. The predicted types of PPR elements, P, L or S, as well as the E and DYW motifs are labelled. The region marked E+ is also considered part of the E domain. <b>(B)</b> Analysis of editing in <i>mef35-1</i> and <i>mef35-2</i> mutant plants. Comparison of the cDNA sequence analysis of three RNA editing sites (boxed) in the mitochondrial <i>rpl16</i>, <i>nad4</i> and <i>cob</i> mRNAs between wild type <i>Arabidopsis thaliana</i> (wt) and the <i>mef35-1</i> and <i>mef35-2</i> mutant plants shows that both mutants have lost the ability of C to U editing at these sites. Three other editing sites in the respective same mRNAs are shown as controls, these sites are correctly edited in wild type and both mutant plants. In the cDNA strands analysed, the detected T nucleotide (red trace) corresponds to the edited U, the observed C (blue trace) is derived from an unedited C. The right hand analyses (<i>mef35-2+MEF35</i>) show that the Col <i>MEF35</i> gene sequence restores the ability for RNA editing in transgenic plants of mutant line <i>mef35-2</i>. (C) The plants of mutant line <i>mef35-1</i> show developmental and adult phenotypes indistinguishable from the wild type. Seedlings of mutant line <i>mef35-2</i> initially develop a little slower in comparison to the wild type Col plantlets, but look similar at the flowering stage (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140680#pone.0140680.s001" target="_blank">S1 Fig</a>). Transgenic complemented plants of <i>mef35-2</i> also display the retarded growth indicating that this phenotype is not related to the <i>mef35-2</i> T-DNA insertion. Four weeks old plants grown side by side are shown.</p