Group II Intron-Based Gene Targeting Reactions in Eukaryotes

Abstract

BackgroundMobile 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 FindingsBy 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/SignificanceOur results provide an experimental foundation for the development of group II intron-based gene targeting methods for higher organisms.

Screening for differentially expressed genes in Anoectochilus roxburghii (Orchidaceae) during symbiosis with the mycorrhizal fungus Epulorhiza sp.

“…These nuclear maturase-like proteins may be imported into organelles to function in group II intron splicing and/or they may have other cellular functions. Nuclear-encoded maturases could regulate organellar gene expression and may reflect a step in the evolution of mobile group II introns into spliceosomal introns [40,41]. Because maturase-encoding group II introns are present in plant mitochondria and chloroplast genomes, it is likely that the open reading frames of nuclear maturases were transferred from an organelle to the nucleus, as has been documented for other organellar genes [42,43].…”

EcI5, a group IIB intron with high retrohoming frequency: DNA target site recognition and use in gene targeting

“…This feature made it possible to develop Ll.LtrB into a highly efficient bacterial gene targeting vector (''targetron''), which has programmable target specificity. The Ll.LtrB targetron has been used in diverse Gram-negative and Gram-positive bacteria (Karberg et al 2001;Frazier et al 2003;Perutka et al 2004;Chen et al 2005Chen et al , 2007Yao et al 2006;Heap et al 2007;Shao et al 2007;Yao and Lambowitz 2007;Malhotra and Srivastava 2008;Rodriguez et al 2008;Sayeed et al 2008), and has the potential to be used similarly for gene targeting in eukaryotes Mastroianni et al 2008). The ability to retarget the Ll.LtrB intron and use it as a gene targeting vector reflects a combination of beneficial properties, including that active intron RNA and IEP can be expressed readily from a donor plasmid, that IEP interactions with the DNA target site are relatively few and flexible, that the base-pairing interactions between the intron RNA and DNA target sequence can be modified, and that these base-pairing interactions are sufficiently long to confer high specificity.…”

The Role of Mg(II) in DNA Cleavage Site Recognition in Group II Intron Ribozymes

“…It has been shown both in bacterial and eukaryotic cells that the efficiency of retrohoming is strongly coupled to the Mg 2ϩ concentration in the cell (47,48). In fact, the lower Mg 2ϩ concentration of the eukaryotic cell limits the retrohoming efficiency of group II introns that are of bacterial origin.…”

“…Moreover, several metal ion binding sites have been located in the active site (38 -40), and a two-metal ion mechanism (41,42) has been suggested to underlie intron catalysis (43,44). In-cell studies establishing a correlation between the intracellular Mg 2ϩ concentration and the frequency of splicing and retrohoming buttress the relevance of Mg 2ϩ for group II intron catalysis (45)(46)(47)(48). The importance of the identity of the divalent metal ions bound to the intron is underscored by the finding that the presence of Mn 2ϩ can lead to a shift of the cleavage site (49) and that already low amounts of Ca 2ϩ decrease the turnover rate by 50% in the Sc.ai5␥ intron (50).…”