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In plant organelles, each C-to-U RNA-editing site is specifically recognized by pentatricopeptide repeat (PPR) proteins with E1-E2, E1-E2-E+ or E1-E2-DYW domain extensions at the C-terminus. The distance between the PPR domain–binding site and the RNA-editing site is usually fixed at four bases, increasing the specificity of target-site recognition in this system. We here report, in contrast to the general case, on MEF28, which edits two adjacent mitochondrial sites, nad2 -89 and nad2 -90. When the sDYW domain of MEF28 was replaced with one derived from MEF11 or CRR22, the ability to edit downstream sites was lost, suggesting that the DYW domain of MEF28 provides unique target flexibility for two continuous cytidines. By contrast, substitutions of the entire E1-E2-DYW domains by MEF19E1-E2, SLO2E1-E2-E+ or CRR22E1-E2-E+ target both nad2 sites. In these cases, access to the contiguous sites in the chimeric PPR proteins is likely to be provided by the trans -associated DYW1-like proteins via the replaced E1-E2 or E1-E2-E+ domains. Furthermore, we demonstrated that the gating domain of MEF28 plays an important role in specific target-site recognition of the DYW domain. This finding suggests that the DYW domain and its internal gating domain fine-tune the specificity of the target site, which is valuable information for designing specific synthetic RNA-editing tools based on plant RNA-editing factors. [ABSTRACT FROM AUTHOR]