RIPseq of AtMKIP1-YFP and AtBE3-YFP in Arabidopsis thaliana

Chloroplast group IIA introns derive from bacterial ribozymes. Their splicing likely requires Maturase K (MatK), which has been largely inaccessible to functional analyses being itself a chloroplast intron-encoded protein. We show that MatK physically interacts with a conserved, essential plastid-localized homolog of starch branching enzymes (BEs), dubbed MATURASE K INTERACTING PROTEIN1 (MKIP1). We demonstrate that MKIP1 proteins have lost BE activity and acquired an insertion enabling direct interaction with the N-terminal region of MatK. Arabidopsis MKIP1 specifically co-precipitates all known intron targets of MatK. Induced MKIP1 silencing results in pale newly emerging leaves, in which the splicing of these intron targets is strongly reduced. Our data suggest that MKIP1 functionally diverged from canonical BEs to facilitate splicing in conjunction with MatK. We propose that the N-terminus of MatK, in turn, has evolved from an RNA-binding domain into a platform for protein interaction, helping its transition towards a general splicing factor. Overall design: To identify RNA regions specifically associated with AtMKIP1 or its complex in vivo, we performed RNA co-immunoprecipitation (co-IP) followed by RNA sequencing (RIP-seq) on 10-days old Arabidopsis thaliana seedlings (Col-0 ecotype) stably expressing PUBQ10::AtMKIP1-YFP in the mkip1 mutant background. An Arabidopsis thaliana line stably expressing PUBQ10::AtBE3-YFP (in the wild-type background) served as control. The input RNA and immunoprecipitated RNA associated with either bait (AtMKIP1-YFP or AtBE3-YFP) in Arabidopsis seedlings were subjected to RNA sequencing. Three replicates, each consisting of ~10 g (fresh weight) of pooled 10-day old seedlings, were analyzed per line.