Group III intron structure and evolutionary analysis in euglenoid chloroplast genomes

Abstract

The Euglena gracilis chloroplast genome contains over 150 introns, including the only known examples of group III introns. Group III introns may belong to the intron super-family consisting of group II and nuclear pre-mRNA introns. It has been suggested that group III intron evolution may have paralleled that of nuclear pre-mRNA introns through loss of domains from a group II-like ancestor. Homologs of psbC14, a group III twintron containing the coding locus for the mat1 polypeptide, have been identified in 7 Euglenoids. All of the species examined contain both the group III twintron and the mat1 locus. As determined by comparative phylogenetic analysis, group III introns contain a structural homolog of group II intron domain VI. The mat1 loci encode peptide motifs characteristic of group II intron maturases. A group III intron-encoded protein whose predicted sequence is similar to group II intron-encoded maturases, and a bona fide domain VI within group III introns, are compelling evidence for a common ancestor of group II and group III introns. A novel mixed operon, psbK-ycf12-psaM-trnR, has been identified in the photosynthetic protist, E. gracilis. The tetracistronic transcripts are processed through endonucleolytic cleavage of the intergenic spacers and splicing of two group III introns and a group III twintron to produce three monocistronic mRNAs and a tRNA. The psbK operon was cloned by PCR amplification from 9 related Euglenoid species. In each species, gene order and content is conserved. The psbK operons contain phylogenetically conserved eubacterial promoter, translational, and 3' processing elements. Based on comparison of homologous intron content to phylogenetic analysis, group III intron evolution within the Euglenoid lineage is more complex than previously believed. Surprisingly, based on psbCi4 and psbK intron distribution data, group III introns may pre-date group II introns in the Euglenoid lineage. An aadA transformation cassette was adapted for expression in Euglena gracilis chloroplasts. Transgenic DNA was introduced into E. gracilis chloroplast by biolisitc transformation and stably maintained as an episomal element during continuous antibiotic selection. When a truncated E. stellata psbK operon was introduced into E. gracilis chloroplasts, transgenic DNA was transcribed and the resultant pre-mRNA accurately spliced.