Development of an In Vitro Chloroplast Splicing System: Sequences Required for Correct pre-mRNA Splicing

Chloroplast genomes in land plants include approximately 20 intron-containing genes. Most of the introns are similar to the group II introns found in fungi, algae and some bacteria, but no self-splicing has been reported. To analyze splicing reactions in chloroplasts, we developed a tobacco (Nicotiana tabacum) chloroplast-based in vitro system. We optimized the splicing reaction using atpF precursor messenger RNA (pre-mRNA). Our system requires a high ATP concentration, whereas ATP is not necessary for self-splicing group II introns. Self-splicing group II introns possess two exon-binding sites (EBS1 and 2) complementary to two intron-binding sites (IBS1 and 2) in the 3′ end of 5′ exons, which are involved in 5′ splice-site selection. Using our in vitro system and atpF pre-mRNA, we analyzed short sequences corresponding to the above EBSs and IBSs. Mutation analyses revealed that EBS1–IBS1 pairing is essential, while EBS2–IBS2 pairing is important but not crucial for splicing. The first 3′ exon nucleotide determines the 3′ splice sites of self-splicing introns. However, mutations to this nucleotide in atpF pre-mRNA did not affect splicing. This result suggests that the mechanism underlying chloroplast pre-mRNA splicing differs partly from that mediating the self-splicing of group II introns.