Single-molecule visualization of sequence-specific RNA binding by a designer PPR protein

Abstract

Pentatricopeptide repeat (PPR) proteins are a large family of modular RNA-binding proteins that recognize specific ssRNA target sequences. There is significant interest in developing designer PPRs for use in diagnostics or as tools to detect and localize target RNA sequences. However, it is unclear how PPRs search for target sequences within complex transcriptomes and current models to predict PPR binding sites struggle to reconcile the effects that RNA mismatches and secondary structure have on PPR binding. To address this, we determined the structure of a designer PPR (dPPR10) bound to its target sequence and used two- and three-colour single-molecule FRET to interrogate the mechanism of ssRNA binding to individual PPR proteins in real time. We demonstrate that longer RNA sequences were significantly slower to bind (or could not bind at all) and that this is, in part, due to their propensity to form stable secondary structures that sequester the target sequence from dPPR10. Importantly, dPPR10 does not associate with non-target flanking sequences, binding specifically to its target sequence within longer ssRNA species. This data provides evidence that PPRs have limited to no capacity to scan RNA transcripts for target sequences and instead rely on diffusion for cognate searching. The kinetic constraints imposed by random three-dimensional diffusion may explain the long-standing conundrum of why PPR proteins are abundant in organelles, but almost unknown outside them (i.e. in the cytosol and nucleus). These findings will inform improved prediction of PPR binding sites for the development of designer PPRs. SummaryPentatricopeptide repeat proteins (PPR) are a large family of modular RNA-binding proteins, whereby each module can be designed to bind to a specific ssRNA nucleobase and thus any RNA sequence of interest. As such, there is substantial interest in developing designer PPRs for a range of biotechnology applications, including diagnostics or in vivo localisation of RNA species; however, the mechanistic details regarding how PPRs search for and bind to target sequences is unclear. As such, we combined structure-based and single- molecule approaches and determined that PPRs bind only to their target sequences (i.e., they do not associate with non-target RNA sequences) and do not scan longer RNA oligonucleotides for the target sequence. Instead, target searching appears kinetically-constrained by random three-dimensional diffusion, providing an explanation as to why PPRs are found almost exclusively in organelle compartments that typically have smaller transcriptomes. Collectively, this work identifies several key considerations for future designer PPR developments.