4.7 Article

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

Journal

PLANT AND CELL PHYSIOLOGY
Volume 62, Issue 8, Pages 1311-1320

Publisher

OXFORD UNIV PRESS
DOI: 10.1093/pcp/pcab095

Keywords

Chloroplast; Gene; Intron; In vitro; Pre-mRNA; Splicing

Funding

  1. Japan Society for the Promotion of Science (JSPS) KAKENHI [17K0441, 04J06115]
  2. Grants-in-Aid for Scientific Research [04J06115] Funding Source: KAKEN

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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.
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.

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