Deciphering the role of accessory proteins in Arabidopsis chloroplast editosomes via interaction with a synthetic PPR-PLS factor in <i>E. coli</i>

RNA editing modifies cytidines to uridines in plant organelle transcripts so that their sequences differ from the ones predicted from the genomic DNA. This process involves a family of RNA-binding proteins that has significantly expanded, the pentatricopeptide repeat (PPR)-containing proteins. In angiosperms, PPR proteins are found in editosomes associated with accessory proteins. The exact function of these accessory proteins has been unclear. Bacterial co-expression of an angiosperm synthetic factor and different accessory proteins, RIP2, RIP9, and ORRM1, demonstrates their essential role in editing of an RNA target. The presence of ORRM1 and RIP2 or ORRM1 and RIP9 in bacteria with the PPR factor results in a target editing extent of 80%, which is similar to what is observed in planta. Accessory proteins increase the affinity of the PPR factor for the target RNA, likely the explanation of their role in improving editing efficiency. RNA-seq analysis of bacterial transcriptome in samples expressing various combinations of accessory proteins along with the synthetic factor identified a total of 34 off-target editing events. Investigation of their upstream sequences that are recognized and bound by the synthetic factor will facilitate the optimization of future designs to improve the specificity of this programmable RNA-editing factor.