Using evolution to guide the engineering of PPR proteins as customizable RNA processing tools

Bernath-Levin K, Colas Des Francs-Small C, Gutmann B, Honkanen S, McDowell R, Melonek J, Pereira Vincis Sanglard L, Royan S, Sun Y and Small ID

ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, University of Western Australia.

Pentatricopeptide repeat (PPR) proteins are modular nucleic acid binding proteins that are highly prevalent in plants, with some species producing thousands of different PPR proteins, each binding a different target sequence. PPR proteins function almost exclusively inside organelles, where they are involved in post-transcriptional steps in gene expression, including RNA cleavage, intron splicing, RNA stabilization, RNA editing and initiation of translation. Their sequence specificity resides in base-specific contacts made by 2-3 amino acids in each repeat that can be described by a simple code, making the target specificity of PPR proteins not only predictable, but programmable. Amongst the many different PPR proteins known, two groups show unusual evolutionary behavior that make them particularly interesting for protein engineering. Restorer-of-fertility factors are a small clade of PPR proteins showing a high degree of inter- and intra-specific variation and strong diversifying selection on the residues that determine RNA binding specificity. We have found that this natural variability makes them ideal subjects for re-engineering and we are currently using them to make targeted knock-downs in mitochondrial gene expression. In a similar vein, but on a much larger scale, PPR RNA editing factors have undergone massive independent expansions in early-diverging land plants such as hornworts, lycophytes and ferns. Inspired by their natural functions, we are attempting to engineer these factors to control gene expression by the targeted creation and removal of start and stop codons using RNA editing.