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 chloroplast-based in vitro system. We optimized the splicing reaction using atpF precursor mRNA (pre-mRNA). Our system requires a high ATP concentration, whereas ATP is unnecessary for self-splicing group II introns. Self-splicing group II introns possess two exon-binding sites (EBS1 & 2) complementary to two intron-binding sites (IBS1 & 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.