Manabu Matsuura

Mechanism of maturase-promoted group II intron splicing.

Mobile group II introns encode reverse transcriptases that also function as intron‐specific splicing factors (maturases). We showed previously that the reverse transcriptase/maturase encoded by the Lactococcus lactis Ll.LtrB intron has a high affinity binding site at the beginning of its own coding region in an idiosyncratic structure, DIVa. Here, we identify potential secondary binding sites in conserved regions of the catalytic core and show via chemical modification experiments that binding of the maturase induces the formation of key tertiary interactions required for RNA splicing. The interaction with conserved as well as idiosyncratic regions explains how maturases in some organisms could evolve into general group II intron splicing factors, potentially mirroring a key step in the evolution of spliceosomal introns.

Retrotransposition of a yeast group II intron occurs by reverse splicing directly into ectopic DNA sites.

Group II introns, the presumed ancestors of nuclear pre-mRNA introns, are site-specific retroelements. In addition to “homing” to unoccupied sites in intronless alleles, group II introns transpose at low frequency to ectopic sites that resemble the normal homing site. Two general mechanisms have been proposed for group II intron transposition, one involving reverse splicing of the intron RNA directly into an ectopic DNA site, and the other involving reverse splicing into a site in RNA followed by reverse transcription and integration of the resulting cDNA by homologous recombination. Here, by using an “inverted-site” strategy, we show that the yeast mtDNA group II intron aI1 retrotransposes by reverse splicing directly into an ectopic DNA site. This same mechanism could account for other previously described ectopic transposition events in fungi and bacteria and may have contributed to the dispersal of group II introns into different genes.

Group II Intron-Based Gene Targeting Reactions in Eukaryotes

Background Mobile group II introns insert site-specifically into DNA target sites by a mechanism termed retrohoming in which the excised intron RNA reverse splices into a DNA strand and is reverse transcribed by the intron-encoded protein. Retrohoming is mediated by a ribonucleoprotein particle that contains the intron-encoded protein and excised intron RNA, with target specificity determined largely by base pairing of the intron RNA to the DNA target sequence. This feature enabled the development of mobile group II introns into bacterial gene targeting vectors (“targetrons”) with programmable target specificity. Thus far, however, efficient group II intron-based gene targeting reactions have not been demonstrated in eukaryotes. Methodology/Principal Findings By using a plasmid-based Xenopus laevis oocyte microinjection assay, we show that group II intron RNPs can integrate efficiently into target DNAs in a eukaryotic nucleus, but the reaction is limited by low Mg2+ concentrations. By supplying additional Mg2+, site-specific integration occurs in up to 38% of plasmid target sites. The integration products isolated from X. laevis nuclei are sensitive to restriction enzymes specific for double-stranded DNA, indicating second-strand synthesis via host enzymes. We also show that group II intron RNPs containing either lariat or linear intron RNA can introduce a double-strand break into a plasmid target site, thereby stimulating homologous recombination with a co-transformed DNA fragment at frequencies up to 4.8% of target sites. Chromatinization of the target DNA inhibits both types of targeting reactions, presumably by impeding RNP access. However, by using similar RNP microinjection methods, we show efficient Mg2+-dependent group II intron integration into plasmid target sites in zebrafish (Danio rerio) embryos and into plasmid and chromosomal target sites in Drosophila melanogster embryos, indicating that DNA replication can mitigate effects of chromatinization. Conclusions/Significance Our results provide an experimental foundation for the development of group II intron-based gene targeting methods for higher organisms.