Predicting and Verifying Binding Sites for Pentatricopeptide Repeat (PPR) Proteins

Hundreds of pentatricopeptide repeat proteins participate in the complex post-transcriptional processing that prepare plant organellar RNA for translation. They act in determining 5' and 3' RNA termini, splicing, editing, endonucleolytic cleavage and promoting or blocking initiation of translation. Most show a high degree of sequence specificity in their binding preferences and the physiological role of the protein is entirely determined by which target RNA it recognises and where on that target it binds. Thus determining the exact binding site is the often the most sought after goal of researchers studying these proteins. Recently it has been shown that PPR proteins recognise RNA through a simple code involving 2-3 amino acids in each repeat. This code has proven extremely useful for predicting binding sites for editing and splicing factors in both chloroplasts and mitochondria. Verifying editing site predictions is technically straightforward and comparing libraries of small RNAs between wild-type and mutants lacking a specific PPR protein gives a rapid and effective way of identifying probable binding sites through characterization of putative PPR 'footprints'. Coupling these two independent bioinformatic and experimental methods is proving a fast and powerful way of getting at the function of PPR proteins. Knowledge of how PPR proteins recognise RNA sequences also allows the design of custom RNA-binding proteins and progress towards this goal is looking extremely encouraging.