Beyond a PPR-RNA recognition code: Many aspects matter for the multi-targeting properties of RNA editing factor PPR56.

The mitochondrial C-to-U RNA editing factor PPR56 of the moss Physcomitrium patens is an RNA-binding pentatricopeptide repeat protein equipped with a terminal DYW-type cytidine deaminase domain. Transferred into Escherichia coli, PPR56 works faithfully on its two native RNA editing targets, nad3eU230SL and nad4eU272SL, and also converts cytidines into uridines at over 100 off-targets in the bacterial transcriptome. Accordingly, PPR56 is attractive for detailed mechanistic studies in the heterologous bacterial setup, allowing for scoring differential RNA editing activities of many target and protein variants in reasonable time. Here, we report (i) on the effects of numerous individual and combined PPR56 protein and target modifications, (ii) on the spectrum of off-target C-to-U editing in the bacterial background transcriptome for PPR56 and two variants engineered for target re-direction and (iii) on combinations of targets in tandem or separately at the 5'- and 3'-ends of large mRNAs. The latter experimentation finds enhancement of RNA editing at weak targets in many cases, including cox3eU290SF as a new candidate mitogenome target. We conclude that C-to-U RNA editing can be much enhanced by transcript features also outside the region ultimately targeted by PPRs of a plant editing factor, possibly facilitated by its enrichment or scanning along transcripts. Author summary: RNA-binding pentatricopeptide repeat (PPR) proteins convert specific cytidines to uridines in mitochondrial and plastid transcripts of land plants to correct genetic information. PPR protein PPR56 is assigned to two editing sites in nad3 and nad4 transcripts of the moss Physcomitrium patens. Transferred into Escherichia coli, PPR56 edits its co-delivered native targets, but also more than 100 specific cytidines in endogenous bacterial transcripts. We have used the E. coli editing system to intensively study the editing properties of PPR56 as a prime example of a plant-type C-to-U RNA editing factor. We confirm that the selection of editing sites depends on the PPR region of the editing factor, which recognizes the RNA target upstream of the C to be edited. Some single amino acid modifications within this region can re-direct the editing factor to new targets, but others reduce or prevent editing completely. Modifications on the target side even outside of the RNA region recognized by the editing factor also affect editing efficiencies significantly. The combination of targets, for example, enhances the editing of the weaker target in many cases. Thus, not only the direct target-protein interaction, but also other transcript features influence the final selection of editing sites. [ABSTRACT FROM AUTHOR]