A synthetic RNA editing factor edits its target site in chloroplasts and bacteria

Santana Royan,Ashley Soet,Catherine Colas Des Francs-Small,Ian Small,Yueming Kelly Sun,Lilian Vincis Pereira Sanglard,Charles S Bond,Jason Schmidberger,Bernard Gutmann,Suvi Honkanen

doi:10.1038/s42003-021-02062-9

Abstract

Members of the pentatricopeptide repeat (PPR) protein family act as specificity factors in C-to-U RNA editing. The expansion of the PPR superfamily in plants provides the sequence variation required for design of consensus-based RNA-binding proteins. We used this approach to design a synthetic RNA editing factor to target one of the sites in the Arabidopsis chloroplast transcriptome recognised by the natural editing factor CHLOROPLAST BIOGENESIS 19 (CLB19). We show that our synthetic editing factor specifically recognises the target sequence in in vitro binding assays. The designed factor is equally specific for the target rpoA site when expressed in chloroplasts and in the bacterium E. coli. This study serves as a successful pilot into the design and application of programmable RNA editing factors based on plant PPR proteins.

Highlights

Members of the pentatricopeptide repeat (PPR) protein family act as specificity factors in C-to-U RNA editing
A key difference is the residues at the 5th and last positions of each PPR motif, which in the case of dsn3PLS-DYW, were programmed to target the rpoA78691 site recognised by CHLOROPLAST BIOGENESIS 19 (CLB19), but not a second target of CLB19, clpP1-69942 (Fig. 1)
The synthetic editing factor dsn3PLS-DYW was designed to bind to the region upstream of the A. thaliana chloroplast rpoA-78691 editing site recognised by the natural editing factor CLB19

Summary

Introduction

Members of the pentatricopeptide repeat (PPR) protein family act as specificity factors in C-to-U RNA editing. 1234567890():,; RNA editing is a term denoting a type of post-transcriptional modification of a transcribed RNA such that it differs from the sequence predicted from the genomic DNA1 This process occurs in diverse forms across all kingdoms of life, including the deamination of adenosines to inosines (which are read as guanosines in the mRNA) by ADAR enzymes in animals[2], the deamination of cytidines to uridines by APOBEC enzymes in animals[3] and by PPR-DYW proteins in plants[4], and the unknown mechanism of uridine modification to cytidine in an as yet unelucidated pathway present in some groups of plants (hornworts, lycophytes, and some ferns)[5,6]. Of relevance to RNA editing are the PLS-class PPR proteins, which generally contain triplets of three different types of PPR motifs in a (P1-L1S1)n-P2-L2-S2 arrangement[11] These PPR arrays are followed by PPR-like E1 and E2 helix-turn-helix motifs, extending the characteristic repeating structure and a ~136 aa domain (named the DYW domain) with a conserved C-terminal D, Y, W tripeptide[11,12]. These specificity-determining interactions involve hydrogen bonding between the amino acids at the 5th and last positions in each motif and the aligned RNA base[28]

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Results

Conclusion