To edit or not to edit RNAs: Exploring plant-type pentatricopeptide repeat RNA editing factors in various genetic contexts

Abstract

Up to thousands of cytidines are specifically converted into uridines in plant mitochondrial and chloroplast transcripts, mostly restoring conserved amino acid codons. Key players are the pentatricopeptide repeat (PPR) proteins of the PLS subgroup. They encompass N-terminal organellar targeting peptides, PLS-type PPR arrays that specifically bind to the target RNAs, and C-terminal DYW-type cytidine deaminase domains. Whereas RNA binding and nucleotide deamination functionalities may be split into two proteins that have to reassemble in higher land plants, we find a simple and likely ancestral editing apparatus with individual PPR proteins sufficing for the editing of one or two specific editing sites in the model moss Physcomitrium patens – the first plant with a complete mutual assignment of all (9) editing factors and (13) editing sites. The simplicity of its editing machinery makes it a favorable model organism for RNA editing research. <br /> Unraveling the co-evolution of mitochondrial RNA editing factor PPR78 and its known target sites cox1eU755SL and rps14eU137SL revealed highly variable editing rates at the latter in different mosses. Knockout complementation studies in Physcomitrium uncovered that rps14 editing rates are dictated by the PPR arrays of PPR78 orthologs. The functional assignment of PPR78 to a yet unknown additional editing target using a bacterial heterologous expression assay finally explained its conservation in mosses that do not require editing of the two previously known targets. <br /> The mitochondrial RNA editing factors PPR56 and PPR65 of P. patens were shown to edit co-transcribed native targets and additional off-targets in the cytosols of cultured human cells. Moreover, the modification of single amino acids crucial for protein-RNA interaction allowed a targeted shift in the binding specificity of PPR56. We conclude that plant-type RNA editing factors can operate in the entirely different genetic environment of a human cell and are not per se incompatible with eukaryotic cytosol. <br /> Nevertheless, the attempt to constitutively overexpress cytosolic editing factors in Physcomitrium failed. The switch to a hormone-inducible expression system, however, finally enabled editing activities of PPR56, PPR65 and PPR78 in the plant cytosol, too, even if their organellar targeting peptides are retained. Owing to a large nucleo-cytosolic transcriptome, huge off-target datasets allowed sophisticated profiling and comparison of nucleotide specificities. Obviously, plants must limit the expression of editing factors to restrict their activities to the organelles because they lack specificity in larger transcriptomes. In future, such assays can facilitate studying nucleotides specificities of native and synthetic PPR protein variants. <br /> With the help of our heterologous E. coli editing assay, the design of a custom, partially synthetic editing factor was optimized. This factor finally facilitated the efficient editing of a targeted artificial site in the Nicotiana benthamiana chloroplast upon transient expression in the hands of our collaborator. <br /> Altogether, this significantly contributes to our knowledge about the co-evolution and interplay of plant-type RNA editing factors and their targets. Moreover, it clearly confirms the value of the different heterologous expression setups for plant RNA editing research and their considerable potential to foster the successful generation of PPR-based site-specific RNA modification tools.