Targeted introduction of premature stop codon in plant mitochondrial mRNA by a designer pentatricopeptide repeat protein with C-to-U editing function

Abstract

RNA editing is a crucial post-transcriptional modification in endosymbiotic plant organelles, predominantly involving C-to-U conversions. Pentatricopeptide repeat (PPR) proteins play a key role in this process. To establish a system for gene expression manipulation in genetically inaccessible mitochondria, we engineered a synthetic PPR protein, dPPR-nad7-DYW, to induce de novo C-to-U editing in the NADH dehydrogenase subunit 7 (nad7) mRNA of Arabidopsis thaliana, thereby creating a premature stop codon. This designer protein, composed of 13 P-type PPR domains, was fused with the DYW-type cytidine deaminase domain from Physcomitrium patens PpPPR_56 and programmed to bind a specific nad7 mRNA segment. In vitro binding assays confirmed the specificity of dPPR-nad7-DYW for its target sequence. When expressed in Arabidopsis plants, dPPR-nad7-DYW achieved up to 85% editing efficiency at the target site, successfully introducing a premature stop codon in nad7 mRNA. This resulted in reduced polysome loading of nad7 transcripts and a phenotype characteristic of mitochondrial complex I dysfunction. RNA-sequencing revealed potential off-target editing events, albeit at lower frequencies. Our study demonstrates the successful application of an editing factor with a synthetic P-type PPR tract targeting a de novo editing site in plant mitochondria, achieving high editing efficiency. This approach opens new avenues for manipulating organellar gene expression and studying mitochondrial gene function in plants and other eukaryotes.