Plant mitochondrial RNA editing factors can perform targeted C-to-U editing of nuclear transcripts in human cells.

RNA editing processes are strikingly different in animals and plants. Up to thousands of specific cytidines are converted into uridines in plant chloroplasts and mitochondria whereas up to millions of adenosines are converted into inosines in animal nucleo-cytosolic RNAs. It is unknown whether these two different RNA editing machineries are mutually incompatible. RNA-binding pentatricopeptide repeat (PPR) proteins are the key factors of plant organelle cytidine-to-uridine RNA editing. The complete absence of PPR mediated editing of cytosolic RNAs might be due to a yet unknown barrier that prevents its activity in the cytosol. Here, we transferred two plant mitochondrial PPR-type editing factors into human cell lines to explore whether they could operate in the nucleo-cytosolic environment. PPR56 and PPR65 not only faithfully edited their native, co-transcribed targets but also different sets of off-targets in the human background transcriptome. More than 900 of such off-targets with editing efficiencies up to 91%, largely explained by known PPR-RNA binding properties, were identified for PPR56. Engineering two crucial amino acid positions in its PPR array led to predictable shifts in target recognition. We conclude that plant PPR editing factors can operate in the entirely different genetic environment of the human nucleo-cytosol and can be intentionally re-engineered towards new targets.

The Expansion and Diversification of Pentatricopeptide Repeat RNA-Editing Factors in Plants.

The RNA-binding pentatricopeptide repeat (PPR) family comprises hundreds to thousands of genes in most plants, but only a few dozen in algae, indicating massive gene expansions during land plant evolution. The nature and timing of these expansions has not been well defined due to the sparse sequence data available from early-diverging land plant lineages. In this study, we exploit the comprehensive OneKP datasets of over 1000 transcriptomes from diverse plants and algae toward establishing a clear picture of the evolution of this massive gene family, focusing on the proteins typically associated with RNA editing, which show the most spectacular variation in numbers and domain composition across the plant kingdom. We characterize over 2 250 000 PPR motifs in over 400 000 proteins. In lycophytes, polypod ferns, and hornworts, nearly 10% of expressed protein-coding genes encode putative PPR editing factors, whereas they are absent from algae and complex-thalloid liverworts. We show that rather than a single expansion, most land plant lineages with high numbers of editing factors have continued to generate novel sequence diversity. We identify sequence variations that imply functional differences between PPR proteins in seed plants versus non-seed plants and variations we propose to be linked to seed-plant-specific editing co-factors. Finally, using the sequence variations across the datasets, we develop a structural model of the catalytic DYW domain associated with C-to-U editing and identify a clade of unique DYW variants that are strong candidates as U-to-C RNA-editing factors, given their phylogenetic distribution and sequence characteristics.

Frequent chloroplast RNA editing in early-branching flowering plants: pilot studies on angiosperm-wide coexistence of editing sites and their nuclear specificity factors.

BACKGROUND: RNA editing by cytidine-to-uridine conversions is an essential step of RNA maturation in plant organelles. Some 30-50 sites of C-to-U RNA editing exist in chloroplasts of flowering plant models like Arabidopsis, rice or tobacco. We now predicted significantly more RNA editing in chloroplasts of early-branching angiosperm genera like Amborella, Calycanthus, Ceratophyllum, Chloranthus, Illicium, Liriodendron, Magnolia, Nuphar and Zingiber. Nuclear-encoded RNA-binding pentatricopeptide repeat (PPR) proteins are key editing factors expected to coevolve with their cognate RNA editing sites in the organelles. RESULTS: With an extensive chloroplast transcriptome study we identified 138 sites of RNA editing in Amborella trichopoda, approximately the 3- to 4-fold of cp editing in Arabidopsis thaliana or Oryza sativa. Selected cDNA studies in the other early-branching flowering plant taxa furthermore reveal a high diversity of early angiosperm RNA editomes. Many of the now identified editing sites in Amborella have orthologues in ferns, lycophytes or hornworts. We investigated the evolution of CRR28 and RARE1, two known Arabidopsis RNA editing factors responsible for cp editing events ndhBeU467PL, ndhDeU878SL and accDeU794SL, respectively, all of which we now found conserved in Amborella. In a phylogenetically wide sampling of 65 angiosperm genomes we find evidence for only one single loss of CRR28 in chickpea but several independent losses of RARE1, perfectly congruent with the presence of their cognate editing sites in the respective cpDNAs. CONCLUSION: Chloroplast RNA editing is much more abundant in early-branching than in widely investigated model flowering plants. RNA editing specificity factors can be traced back for more than 120 million years of angiosperm evolution and show highly divergent patterns of evolutionary losses, matching the presence of their target editing events.

Membrane protein interactions between different Arabidopsis thaliana MRS2-type magnesium transporters are highly permissive.

Membrane proteins of the Arabidopsis thaliana MRS2 (MGT) family have been characterised as magnesium transporters. Like their bacterial CorA homologues, the plant MRS2 proteins are characterised by an invariable GMN tripeptide motif terminating the first of two closely spaced transmembrane domains at the carboxy-termini. The functional Mg(2+) transport channel is assembled as a pentamer in the case of CorA. However, in contrast to the single CorA genes of bacteria, plant genomes encode up to 10 highly divergent MRS2 proteins. To elucidate structure-function relationships and the possibility of plant MRS2 hetero-pentamer formation, we performed protein-protein interaction studies in the yeast mating-based split-ubiquitin system (mbSUS) and concomitant protein modelling using I-TASSER. Despite very restricted sequence similarities and variable polypeptide insertions all AtMRS2 proteins feature the key structural elements determined for the CorA crystal structure. The mbSUS setup conclusively demonstrates protein-protein interactions of any given AtMRS2 protein not only with itself but also highly permissive interactions to varying degrees among all AtMRS2 proteins. AtMRS2-3 seems particularly prone to non-selective, strong interactions with the other homologues. Deletion constructs show that six amino acids may be deleted from the carboxy-terminus and 27 (but not 41) from the amino-terminus of AtMRS2-7 without impairment of homologous or heterologous protein interactions. Despite significant diversification, the plant MRS2 proteins have obviously retained an ancient CorA/MRS2 core structure and the capacity for protein-protein interactions. Plant magnesium homeostasis may be influenced by hetero-oligomer channel formation where different plant MRS2 proteins meet in the same membrane naturally or in transgenic approaches.

A root-expressed magnesium transporter of the MRS2/MGT gene family in Arabidopsis thaliana allows for growth in low-Mg2+ environments.

The MRS2/MGT gene family in Arabidopsis thaliana belongs to the superfamily of CorA-MRS2-ALR-type membrane proteins. Proteins of this type are characterized by a GMN tripeptide motif (Gly-Met-Asn) at the end of the first of two C-terminal transmembrane domains and have been characterized as magnesium transporters. Using the recently established mag-fura-2 system allowing direct measurement of Mg(2+) uptake into mitochondria of Saccharomyces cerevisiae, we find that all members of the Arabidopsis family complement the corresponding yeast mrs2 mutant. Highly different patterns of tissue-specific expression were observed for the MRS2/MGT family members in planta. Six of them are expressed in root tissues, indicating a possible involvement in plant magnesium supply and distribution after uptake from the soil substrate. Homozygous T-DNA insertion knockout lines were obtained for four members of the MRS2/MGT gene family. A strong, magnesium-dependent phenotype of growth retardation was found for mrs2-7 when Mg(2+) concentrations were lowered to 50 microM in hydroponic cultures. Ectopic overexpression of MRS2-7 from the cauliflower mosaic virus 35S promoter results in complementation and increased biomass accumulation. Green fluorescent protein reporter gene fusions indicate a location of MRS2-7 in the endomembrane system. Hence, contrary to what is frequently found in analyses of plant gene families, a single gene family member knockout results in a strong, environmentally dependent phenotype.