title: Retrohoming of a Mobile Group II Intron in Human Cells Suggests How Eukaryotes Limit Group II Intron Proliferation.
abstract: Mobile bacterial group II introns are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a "ribozyme") and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites by a mechanism termed "retrohoming". Although mobile group II introns splice and retrohome efficiently in bacteria, all examined thus far function inefficiently in eukaryotes, where their ribozyme activity is limited by low Mg2+ concentrations, and intron-containing transcripts are subject to nonsense-mediated decay (NMD) and translational repression. Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1%, in viable HEK-293 cells. Surprisingly, under these conditions, the Ll.LtrB intron reverse transcriptase is required for retrohoming but not for RNA splicing as in bacteria. By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but <4-fold and not without added Mg2+. Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations. Our results reveal differences between group II intron retrohoming in human cells and bacteria and suggest constraints on critical nucleotide residues of the ribozyme core that limit how much group II intron retrohoming in eukaryotes can be enhanced. These findings have implications for group II intron use for gene targeting in eukaryotes and suggest how differences in intracellular Mg2+ concentrations between bacteria and eukarya may have impacted the evolution of introns and gene expression mechanisms.
authors.affiliation: Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, United States of America.
authors.forename: David M
authors.initials: DM
authors.lastname: Truong
authors.affiliation: Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, United States of America.
authors.forename: F Curtis
authors.initials: FC
authors.lastname: Hewitt
authors.affiliation: Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, United States of America.
authors.forename: Joseph H
authors.initials: JH
authors.lastname: Hanson
authors.affiliation: Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, United States of America.
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Cui
authors.affiliation: Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, United States of America.
authors.forename: Alan M
authors.initials: AM
authors.lastname: Lambowitz
mesh_terms: D021581:Active Transport, Cell Nucleus; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002470:Cell Survival; D019020:Directed Molecular Evolution; D057809:HEK293 Cells; D006801:Humans; D007438:Introns; D055029:Inverted Repeat Sequences; D008969:Molecular Sequence Data; D059365:Nonsense Mediated mRNA Decay; D010957:Plasmids / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D018626:Retroelements
chemical_list: D001426:Bacterial Proteins; D018626:Retroelements; C099785:LtrA protein, Lactococcus lactis; D012194:RNA-Directed DNA Polymerase