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



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.,The dataset includes source data files for Figure 2, Supplementary Figure S2, Supplementary Figure S4 and Supplementary Figure S5 from the article in Communications Biology. For Figure 2, four .tif files are provided which contain the results of electrophoretic mobility shift analysis using a fluorophore-labelled RNA probe. Details are provided in the article. The fluorescence signal was imaged with an Amersham Typhoon system (GE Healthcare), with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. For Supplementary Figure S2, six .tif files are provided containing uncropped images corresponding to the protein gels shown in Supplementary Figure S2 panels c and d. For Supplementary Figure S4, tab-separated text files are provided containing count values generated with Salmon (the RNA-seq mapping methodology is explained in the article). Each row in each file is an extract from a Salmon quants file. Columns are as follows: sample_file:gene_id, length, effective length, transcripts per million (TPM), normalised counts. 'FigS4.ipynb' is a Jupyter notebook containing Python code used to produce Supplementary Figure S4 from this data. For Supplementary Figure S5, 'ycf3_splicing.xls' contains read counts for RNA-seq reads crossing the donor, acceptor and splice junctions of the ycf3 transcript (intron 2). 'FigS5.ipynb' is a Jupyter notebook containing Python code used to produce Supplementary Figure S5 from this data.,
Date made available27 Mar 2022

Cite this