Enhanced group II intron retrohoming in magnesium-deficient Escherichia coli via selection of mutations in the ribozyme core David M. Truong1, David J. Sidote, Rick Russell, and Alan M. Lambowitz1 Institute for Cellular and Molecular Biology, Departments of Molecular Biosciences and Chemistry, University of Texas at Austin, Austin, TX 78712 Contributed by Alan M. Lambowitz, August 22, 2013 (sent for review July 5, 2013) Mobile group II introns are bacterial retrotransposons thought to group II introns retrohome and function efficiently for gene tar- be evolutionary ancestors of spliceosomal introns and retroele- geting in bacteria, their natural hosts, they do so inefficiently in ments in eukaryotes. They consist of a catalytically active intron eukaryotes, at least in part owing to lower free Mg2+ concentrations RNA (“ribozyme”) and an intron-encoded reverse transcriptase, (6), which decrease group II intron ribozyme activity (discussed 2+ which function together to promote RNA splicing and intron mo- below). These lower Mg concentrations constitute a natural bility via reverse splicing of the intron RNA into new DNA sites barrier that impedes group II introns from invading the nuclear (“retrohoming”). Although group II introns are active in bacteria, genomes of present-day eukaryotes and limits their utility as gene their natural hosts, they function inefficiently in eukaryotes, targeting vectors for higher organisms. where lower free Mg2+ concentrations decrease their ribozyme Recent X-ray crystal structures of a group II intron RNA activity and constitute a natural barrier to group II intron prolifer- provide a structural framework for investigating group II in- ation within nuclear genomes. Here, we show that retrohoming of tron splicing and retrohoming mechanisms and potentially for the Ll.LtrB group II intron is strongly inhibited in an Escherichia coli improving their function in gene targeting (7–9). Group II intron mutant lacking the Mg2+ transporter MgtA, and we use this sys- RNAs consist of six conserved domains (denoted DI–DVI) that tem to select mutations in catalytic core domain V (DV) that par- interact via tertiary contacts to fold the RNA into a catalytically tially rescue retrohoming at low Mg2+ concentrations. We thus active 3D structure (Fig. 1A) (1). DV is a small conserved domain identified mutations in the distal stem of DV that increase retro- that binds catalytic metal ions and interacts with DI and J2/3 to ’ homing efficiency in the MgtA mutant up to 22-fold. Biochemical form the intron RNA s active site. It is thought to be the cognate assays of splicing and reverse splicing indicate that the mutations of the U2/U6 snRNAs of the spliceosome, and consequently its increase the fraction of intron RNA that folds into an active con- architecture and function are central to understanding the mech- formation at low Mg2+ concentrations, and terbium-cleavage anism and evolution of RNA splicing in higher organisms (10, 11). assays suggest that this increase is due to enhanced Mg2+ binding DI, the largest domain, provides a structural scaffold for the as- to the distal stem of DV. Our findings indicate that DV is involved sembly of the other domains and contains exon-binding sites that ′ ′ in a critical Mg2+-dependent RNA folding step in group II introns position the 5 - and 3 -splice sites and ligated-exon junction at the and demonstrate the feasibility of selecting intron variants that ribozyme active site for RNA splicing and reverse splicing reac- function more efficiently at low Mg2+ concentrations, with impli- tions. DIII functions as a catalytic effector, DIV is the location of cations for evolution and potential applications in gene targeting. the ORF encoding the IEP, and DVI contains the branch-point adenosine used for lariat formation. directed evolution | Mg2+ transport | RNA structure Three major structural subclasses of group II intron RNAs, denoted IIA, IIB, and IIC, have been identified with differences in both peripheral and active-site elements (1). X-ray crystal obile group II introns are retrotransposons that are found structures of the Oceanobacillus iheyensis group IIC intron reveal Min prokaryotes and the mitochondrial and chloroplast the folded structure of DI-V, with and without bound exons (7–9). DNAs of some eukaryotes and are thought to be evolutionary ancestors of spliceosomal introns, the spliceosome, retro- Significance transposons, and retroviruses in higher organisms (1). They consist of two components—an autocatalytic intron RNA (“ribozyme”) and an intron-encoded protein (IEP) with re- Mobile group II introns are bacterial retrotransposons. They “ ” verse transcriptase activity—that function together in a ribo- consist of an autocatalytic intron RNA ( ribozyme ) and an in- nucleoprotein (RNP) complex to promote RNA splicing and tron-encoded reverse transcriptase and were likely ancestors of site-specific integration of the intron into new DNA sites in a spliceosomal introns and retroelements in eukaryotes. Al- though active in bacteria, group II introns function inefficiently process called retrohoming (1). Like spliceosomal introns, 2+ group II introns splice via two sequential transesterification in eukaryotes, where lower Mg concentrations decrease their reactions that yield an excised intron lariat RNA (2). For ribozyme activity and constitute a natural barrier to group II in- Escher- group II introns, the splicing reactions are catalyzed by the tron proliferation within nuclear genomes. By using an ichia coli 2+ intron RNA with the assistance of the IEP, which binds spe- Mg -transport mutant, we selected mutations near the fi intron RNA’s active site that enhance group II intron function ci cally to the intron RNA and stabilizes the catalytically 2+ active RNA structure. The IEP then remains bound to the at low Mg concentrations. Our results have implications for excised intron lariat RNA in an RNP that promotes retro- ribozyme mechanisms, evolution, and biotechnology. homing via reverse splicing of the intron RNA directly into Author contributions: D.M.T. and A.M.L. designed research; D.M.T. and D.J.S. performed DNA sites followed by reverse transcription by the IEP. The research; D.M.T., D.J.S., R.R., and A.M.L. analyzed data; and D.M.T., D.J.S., R.R., and A.M.L. resulting intron cDNA is integrated into the genome by host wrote the paper. fi enzymes (3, 4). The ribozyme-based, site-speci cDNAin- The authors declare no conflict of interest. tegration mechanism used by group II introns enabled their 1To whom correspondence may be addressed. E-mail: [email protected] development into gene targeting vectors (“targetrons”), which or [email protected]. fi combine high integration ef ciency with high and readily This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. programmable DNA target specificity (5). However, although 1073/pnas.1315742110/-/DCSupplemental. E3800–E3809 | PNAS | Published online September 16, 2013 www.pnas.org/cgi/doi/10.1073/pnas.1315742110 Downloaded by guest on October 1, 2021 many of the same metal ion-binding sites but without the bend PNAS PLUS A Ll.LtrB-ΔORF (12, 13). Besides the catalytic metal ions, several other Mg2+ ions are seen at different sites in DV in the O. iheyensis RNA crystal structures. Three putative Mg2+ ions lie within the major and minor grooves near the κ′ motif in the proximal stem, and three others (denoted here as M3–M5) are bound to the distal stem, one (M3) within a kink adjacent to the G of the GNRA tetra- loop, and another (M4) at the R of the GNRA tetraloop. The third Mg2+ bound to the distal stem (M5) forms a bridge be- tween the base pair distal to λ′ and the third nucleotide of the catalytic triad, potentially stabilizing the sharp bend in DV. These additional Mg2+-binding sites in the O. iheyensis intron are Tetraloop generally consistent with the locations of terbium-cleavage sites B CT Bulge in DV of the aI5γ intron (14). 5 4 2+ 1 2 Like other ribozymes, group II introns use Mg for both RNA 3 2+ 2 3 folding and catalysis (15, 16). However, the Mg concentrations 1 4 required for group II intron function are higher than those for 1 2 5 other ribozymes. Thus, mutations in the yeast mitochondrial Mg2+ Bulge CT Tetraloop transporter Mrs2 specifically inhibit the splicing of all four yeast mt C O. iheyensis DV Ll.LtrB DV group II introns, while having minimal effects on the transcription or splicing of group I introns (17). Moreover, efficient retrohoming in Xenopus laevis oocytes, Drosophila melanogaster embryos, and zebrafish (Danio rerio) embryos requires the coinjection of addi- tional Mg2+ to achieve an intracellular concentration of 5–10 mM (6). Bacteria, the natural hosts of group II introns, typically have free intracellular Mg2+ concentrations of 1–4 mM (18), whereas X. Fig. 1. Group II intron RNA and DV structures. (A) Secondary structure 2+ Δ laevis oocyte nuclei contain 0.3 mM Mg (19), and mammalian model of the Ll.LtrB- ORF group II intron, with DV highlighted in red. Greek – 2+ letters indicate sequence elements involved in long-range tertiary inter- cells contain 0.2 1mMMg during the majority of the cell cycle 2+ actions (dashed red lines). The locations of the ApaLI, KpnI, and MluI sites (20). The latter values are well below the optimal Mg concen- used in library construction and the inserted phage T7 promoter sequence trations for protein-assisted group II intron RNA splicing and (PT7) used for genetic selections are indicated. (B) Metal ions bound to DV in reverse splicing in vitro (5–10 mM) (21). For some ribozymes, it an ensemble of 15 superposed X-ray crystal structures of the O. iheyensis has been possible to select new variants that function at lower group IIC intron (3BWP, 3EOG, 3EOH, 3IGI, 4E8M, 4E8P, 4E8Q, 4E8R, 4E8V, Mg2+ concentrations or use different metal ions (22–24).