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.

五肽重复序列（pentatricopeptide repeat, PPR）蛋白家族成员可作为C到U（C-to-U）RNA编辑的特异性因子。植物PPR超家族的扩张，为基于共识序列的RNA结合蛋白设计提供了必要的序列变异基础。我们依托该策略设计了一款合成RNA编辑因子，靶向拟南芥叶绿体转录组中天然编辑因子叶绿体生物发生19（CHLOROPLAST BIOGENESIS 19, CLB19）所识别的其中一个编辑位点。体外结合实验证实，我们设计的合成编辑因子可特异性识别该靶序列；当该因子在叶绿体与大肠杆菌（E. coli）中表达时，对靶标rpoA位点同样表现出高度专一性。本研究成功为基于植物PPR蛋白的可编程RNA编辑因子的设计与应用提供了先导性验证范例。