Background

C to U RNA editing is one of the most important post-transcriptional modifications that occur in the plant mitochondrial or chloroplast genes, which usually changes the first or second positions of nucleic acid triplet codons leading to altered protein sequences and is essential for their normal functions (Takenaka et al., 2013; Yan et al., 2018). The RNA editing and processing in mitochondria or chloroplasts have been reported to function in plant male sterility, seed development, adaptations to the environment, and resistance to pathogens (Hammani et al., 2011; Dahan and Mireau, 2013; Garcia-Andrade et al., 2013; Barkan and Small, 2014; Ren et al., 2020; Yang et al., 2020). Several methods have been established for the detection of RNA editing sites or editing levels, such as poisoned primer extension (PPE) assays, high-resolution melting (HRM) analysis, and DNA sequencing (Roberson et al., 2006; Chateigner-Boutin et al., 2007; Hayes and Hanson, 2007). However, the PPE assays usually require radiolabeled oligonucleotides (Hayes and Hanson, 2007). It is hard to distinguish the editing levels at two very close editing sites using HRM assays (Chateigner-Boutin et al., 2007). Currently, DNA sequencing has been an accurate, economic, and widely used method for the RNA editing assays (Bentolila et al., 2012; Brehme et al., 2015; Yang et al., 2017; He et al., 2018). Here, we describe a method for the RNA editing detection by DNA sequencing of cDNA to further evaluate the effects of different treatments or plant mutants on the C to U RNA editing in mitochondrial and chloroplast transcripts (Yang et al., 2020).