In Arabidopsis, the PPR proteins EMB1006, EMB1270, and EMB976 are all essential for the splicing of plastid clpP1 intron 2 (clpP1.2), although each also targets other distinct RNAs. The precise mechanism underlying their coordinated action in clpP1.2 splicing remains unclear. In this study, RNA electrophoretic mobility shift assays (REMSA), guided by PPR code prediction, confirm that EMB1006 specifically binds to a sequence near the 3′ end of clpP1 exon 2. Additionally, immunoprecipitation coupled with mass spectrometry (IP-MS) reveals that EMB1006 forms a complex with EMB1270, EMB976, and CFM2. Direct interactions between EMB1006 and EMB1270 or CFM2 are further supported by yeast two-hybrid (Y2H) and semi-in vivo pull-down assays. However, no direct interactions between EMB976 and EMB1006, CFM2 or EMB1270 are detected by Y2H. Based on these findings and previous evidence that EMB1270 binds to clpP1 intron 2 and interacts with CFM2, we propose a model in which EMB1006 and EMB1270 bind to distinct sites on clpP1 pre-mRNA. Together with CFM2 and possible indirect association with EMB976, they assemble into a protein-RNA complex that facilitates the splicing of clpP1.2 in chloroplasts.
Zhang et al. (Wed,) studied this question.