Moss DYW-type pentatricopeptide repeat (PPR) proteins as modifiable C-to-U RNA editing factors in diverse genetic systems.
Plant Physiology
Final Report Abstract
We come to the following conclusions: 1. Organelle RNA editing factors of the moss Physcomitrium are functional not only upon heterologous expression in a bacterial setup but also in the cytosol of human and plant cells. Accordingly, they are very unlikely to rely on specific biochemic machineries present only in plant mitochondria or chloroplasts. 2. The different RNA editing factors show strikingly different capacities to address different amounts of off-targets with PPR56 exceeding PPR65 and PPR78 by far in the number of detectable offtargets independent of expression in bacteria or the human or plant cytosol. Surprisingly, we find that single amino acid exchanges in individual PPRs or the exchange of the terminal DYW domain in PPR56 also result in vastly different numbers of off-targets. We conclude that despite their apparently clearly defined modular makeup, the different components of RNA editing factors combine in a still enigmatic way for target recognition and C-to-U conversion. 3. We assume that the large number of off-targets and accordingly a restricted specificity is the key reason why plant RNA editing factors are confined in evolution to operate only in the much smaller transcriptomes of chloroplasts and mitochondria. Nevertheless, the now available possibility to score off-targets in the much larger nuclear transcriptomes is a unique opportunity to study targeting properties of natural, recombinantly engineered or synthetic “designer” plant-type RNA editing factors, likely in the future also those from flowering plants. 4. Recombinant engineering of plant RNA editing factors and their targets has clearly shown that their successful operation may rely on transcript features beyond the RNA region ultimately targeted by the respective PPR array. The changes to the initial cloning strategy adapted for 6 successful expression of PPR78 suggest that native N-terminal sequences upstream of a PPR array may be more beneficial than the upstream fusion to MBP. The increased RNA editing efficiencies observed for tandem target arrangements hints at an enrichment of editing factors in the vicinity of their ultimate target on a transcript. 5. The large numbers of constructs tested for the effects of mutations on the protein and target side largely confirm the PPR-RNA code but also pose multiple caveats for predictability: individual PPRs may behave differently, conceptual improvements of PPR-RNA matches do not necessarily improve RNA editing efficiency and changes to the carboxyterminal domains behind the PPR array contribute significantly to target recognition. The prediction of ccmFNeU1465RC as a third target for PPR78 is a bona fide example for strongly improved binding prognoses based on off-target profiling extending the concept of 1:1 matches for PPRs and juxtaposed ribonucleotides. Vice versa, attempts to redirect PPR65 to new targets in Physcomitrium patens mitochondria, for example, remained unsuccessful despite testing several different PPR versions with modified individual PPR repeats. Synthetic designer PPRs may ultimately prove superior to modifying natural PPR arrays for engineering towards new targets. In any case, we strongly recommend the testing of modified natural or synthetic designer PPRs in large transcriptome environments as now demonstrated and established.
Publications
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Plant mitochondrial RNA editing factors can perform targeted C-to-U editing of nuclear transcripts in human cells. Nucleic Acids Research, 50(17), 9966-9983.
Lesch, Elena; Schilling, Maximilian T.; Brenner, Sarah; Yang, Yingying; Gruss, Oliver J.; Knoop, Volker & Schallenberg-Rüdinger, Mareike
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Conservation of the moss RNA editing factor PPR78 despite the loss of its known cytidine-to-uridine editing sites is explained by a hidden extra target. The Plant Cell, 36(3), 727-745.
Lesch, Elena; Stempel, Maike Simone; Dressnandt, Vanessa; Oldenkott, Bastian; Knoop, Volker & Schallenberg-Rüdinger, Mareike
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DYW cytidine deaminase domains have a long‐range impact on RNA recognition by the PPR array of chimeric plant C‐to‐U RNA editing factors and strongly affect target selection. The Plant Journal, 116(3), 840-854.
Yang, Yingying; Oldenkott, Bastian; Ramanathan, Shyam; Lesch, Elena; Takenaka, Mizuki; Schallenberg‐Rüdinger, Mareike & Knoop, Volker
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Conquering new grounds: plant organellar C‐to‐U RNA editing factors can be functional in the plant cytosol. The Plant Journal, 119(2), 895-915.
Thielen, Mirjam; Gärtner, Béla; Knoop, Volker; Schallenberg‐Rüdinger, Mareike & Lesch, Elena
