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 (E8S1 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 premRNA, we analyzed short sequences corresponding to the above EBSs and IBSs. Mutation analyses revealed that EBS1-IBS1 pairing is essential, while ERS2-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.

Automated summary

Chloroplast genomes in land plants contain approximately 20 intron-containing genes, similar to group II introns found in fungi, algae, and some bacteria, but lacking self-splicing capability. Researchers developed an in vitro system using tobacco chloroplasts to study splicing reactions, revealing differences in splicing mechanisms between chloroplast pre-mRNA and self-splicing group II introns.