Cofactor-independent RNA editing by a synthetic S-type PPR protein

Abstract

Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that are attractive tools for RNA processing in synthetic biology applications given their modular structure and ease of design. Several distinct types of motifs have been described from natural PPR proteins, but almost all work so far with synthetic PPR proteins has focused on the most widespread P-type motifs. We have investigated synthetic PPR proteins based on tandem repeats of the more compact S-type PPR motif found in plant organellar RNA editing factors, and particularly prevalent in the lycophyte Selaginella. With the aid of a novel plate-based screening method we show that synthetic S-type PPR proteins are easy to design, bind with high affinity and specificity, and are functional in a wide range of pH, salt and temperature conditions. We find that they outperform a synthetic P-type PPR scaffold in many situations. We designed an S-type editing factor to edit an RNA target in E. coli and demonstrate that it edits effectively without requiring any additional cofactors to be added to the system. These qualities make S-type PPR scaffolds ideal for developing new RNA processing tools.

Plant mitochondrial RNA editing factors can perform targeted C-to-U editing of nuclear transcripts in human cells

“…These studies have been based on variations of natural PPR proteins ( 71 , 72 ) or alternatively used synthetic PPR proteins based on consensus profiles of the widely distributed P-type PPRs ( 64 , 68 , 73–77 ). A very recent new approach has focused on particular S-type PPRs ( 78 ) modeling the binding properties of the widely studied natural CLB19 editing factor ( 50 , 78–85 ). This work extended and complemented an earlier attempt using a synthetic PLS-type PPR array as a surrogate for the PPR array of CLB19, which, however, turned out to require MORF2, a ‘Multiple Organelle RNA editing Factor’, as a necessary co-factor for obtaining moderate RNA editing efficiency ( 86 ).…”

“…This work extended and complemented an earlier attempt using a synthetic PLS-type PPR array as a surrogate for the PPR array of CLB19, which, however, turned out to require MORF2, a ‘Multiple Organelle RNA editing Factor’, as a necessary co-factor for obtaining moderate RNA editing efficiency ( 86 ). Using the E. coli assay setup, the synthetic PLS-type PPR protein did not cause an off-target editing, while the artificial S-type approach resulted in 50% of RNA editing at its natural CLB19 rpoA target and only weakly affected one single off-target ( tufB ) in E. coli ( 78 ).…”

Emergence of Novel RNA-Editing Sites by Changes in the Binding Affinity of a Conserved PPR Protein

“…It shows that the introduction of a foreign PPR editing factor can result in novel editing events that cause deleterious phenotypes. Recent reports have uncovered extensive off-target effects of native and synthetic PPR editing factors expressed in planta ( Royan et al 2021 ), in E. coli ( Oldenkott et al 2019 ; Bernath-Levin et al 2021 ) and in human cells ( Ichinose et al 2022 ; Lesch et al 2022 ). Our analyses of the QED1 protein demonstrate that a PPR editing factor can also edit numerous off-target sites in planta .…”

Knockdown of mitochondrialatp1mRNA by a custom-designed pentatricopeptide repeat protein alters F1FoATP synthase

“…The PPR code (Barkan et al, 2012; Miranda et al, 2018; Yan et al, 2019; Bernath-Levin et al, 2021) describing sequence specific binding ability between PPR proteins and their organelle target RNAs makes custom design usable for organelle biotechnology (Colas des Francs-Small et al, 2018). We show here that an engineered PPR RFL protein can be used to cleave a new target transcript within the coding sequence of atp1 .…”

“…That research established a new method using chloroplasts as biofactories to synthesize and store valuable biological molecules. With more natural PPR proteins being functionally validated, the artificially designed PPR proteins have potential to expand their application in organelles (Bernath-Levin et al, 2021; Royan et al, 2021).…”