Author Summary Group II introns are bacterial mobile elements thought to be ancestors of introns and retrotransposons in higher organisms. They consist of a catalytically active intron RNA and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites in a process called ‘‘retrohoming.’’ In bacteria, retrohoming occurs by the excised intron lariat RNA fully reverse splicing into a DNA site, where it is reverse transcribed, yielding an intron cDNA that is copied directly into the host genome. However, little is known about how group II introns behave in higher organisms. Here, we find that linear group II intron RNAs, which cannot fully reverse splice, retrohome in Drosophila melanogaster by attaching themselves to only one end of a DNA site. Reverse transcription then yields an intron cDNA, which is integrated into the recipient DNA by host enzymes that function in non-homologous end joining, a critical cellular DNA–repair pathway. Biochemical experi- ments exploring alternate mechanisms show that group II intron reverse transcriptases can also template switch efficiently from one RNA template to a second RNA or DNA template, thereby directly linking the two template sequences. Our findings have implications for retotran- sposition and DNA repair mechanisms and potential biotechnological applications. generated from excised intron lariat RNAs by debranching, which is believed to accelerate RNA turnover [20]. However, the physiological and evolutionary significance of hydrolytic splicing and linear group II intron RNAs have remained largely unclear. The Lactococcus lactis Ll.LtrB intron, which has been used extensively as a model system for studying group II intron mobility mechanisms, has a broad host range and is actively mobile in Escherichia coli, making it possible to use E. coli genetic approaches to dissect mobility pathways [11,21]. The major retrohoming pathway used by the Ll.LtrB intron in E. coli is shown in Figure 1A. After promoting splicing, the Ll.LtrB IEP, denoted LtrA protein, remains bound to the excised intron lariat RNA in RNPs that recognize a DNA target site at the ligated-exon junction (denoted E1–E2) of an intronless allele. The intron lariat RNA initiates retrohoming by fully reverse splicing into the top DNA strand, leading to insertion of the intron RNA between the two DNA exons. The IEP then uses a DNA endonuclease activity to cleave the bottom strand and uses the 39 DNA end at the cleavage site as a primer for reverse transcription of the inserted intron RNA. In E. coli, the resulting intron cDNA is integrated into the host genome by a mechanism that involves degradation of the intron RNA template strand by a host RNase H and second-strand DNA synthesis by a host DNA polymerase [11,21]. In variations of this mechanism, Ll.LtrB and other group II introns can also retrohome without bottom-strand DNA cleavage by using a nascent strand at a DNA replication fork to prime reverse transcription of the intron RNA [14,22,23], and yeast mitochondrial group II introns retrohome by using recombination rather than DNA repair for cDNA integration [24]. Recently, while carrying out experiments to test whether microinjected group II intron RNPs could be used for gene targeting in Xenopus laevis oocyte nuclei and Drosophila melanogaster embryos, we found that linear as well as lariat group II intron RNAs can retrohome in vivo [25]. This finding was surprising because, unlike lariat RNAs, linear group II intron RNAs can carry out only the first reverse-splicing step, ligation of the 39 end of the intron RNA to the 59 end of the 39- exon DNA [26,27]. Linear Group II Intron Retrohoming in Drosophila While reverse transcription of fully reverse-spliced intron RNA yields an intron cDNA that can be extended directly by continued DNA synthesis into the upstream exon (Figure 1A), reverse transcription of a partially reverse-spliced intron RNA yields an intron cDNA with an unattached 39 end that must be linked to the upstream exon DNA in a separate step (Figure 1B). Sequencing of 59-integration junctions showed that this step occurs by an error- prone process. Although some events result in the precise insertion of the intron between the two DNA exons, most give 59- integration junctions with 59-exon deletions, intron 59-end truncations, insertion of extra nucleotides at the intron-exon junction, or indications of DNA repair via base pairing of microhomologies on opposite sides of the break [25], similar to ligation junctions resulting from double-strand break repair by non-homologous end joining (NHEJ) [28–31]. NHEJ activities have been found to contribute to the retrotransposition of LINE elements and other retrotransposons in eukaryotes [32,33] and may be exploited preferentially by retrotransposons to gain advantage in genetic conflict with their hosts, which rely on these enzymes for survival [34]. Thus, although group II introns are alien to Xenopus and Drosophila, they could be utilizing mechanisms that contribute to the retrotransposition of resident retroelements in eukaryotes and could be subject to host defenses that evolved to counter or mitigate such retrotransposition. Although NHEJ seemed the most likely mechanism for attachment of the free cDNA to the 59 exon in linear intron RNA retrohoming, an alternate possibility was that the RT template switches to the 59-exon DNA, either directly or following incorporation of extra nucleotide residues at the end of the cDNA, resulting in synthesis of a continuous DNA bottom strand containing intron and 59-exon sequences. Both template switching and incorporation of extra nucleotide residues at the ends of cDNA have been found for other non-LTR-retroelement RTs [35–37]. Although we thought this possibility unlikely because group II intron RTs appeared to have low DNA-dependent DNA polymerase activity in vitro [21], template switching and non- templated nucleotide addition by group II intron RTs have not been investigated previously. Here, we used Drosophila melanogaster mutants to investigate the contribution of NHEJ activities to linear intron RNA retrohoming and assessed the involvement of template switching by comparing junctions formed by this mechanism in vitro with those formed during linear intron RNA retrohoming in vivo. Our results indicate that linear intron RNA retrohoming occurs primarily by a novel variation of NHEJ that uses host enzymes, including DNA ligase 4 (Lig4) and DNA repair polymerase h (PolQ), but is minimally dependent upon Ku. Results Retrohoming of linear and lariat RNA in D. melanogaster mutant embryos To investigate the involvement of NHEJ factors, we compared lariat and linear group II intron retrohoming in D. melanogaster embryos with mutations in the genes encoding DNA ligase 4 (Lig4), Ku70, and the DNA repair polymerase h (PolQ) [38,39]. For these experiments, we used a plasmid-based retrohoming assay in which an Ll.LtrB-DORF intron with a phage T7 promoter sequence inserted near its 39 end integrates into a target site cloned in an AmpR-recipient plasmid upstream of a promoterless tetR reporter gene, thereby activating that gene (Figure 2A) [25,26,40]. The recipient plasmid was injected into the posterior of precellular blastoderm stage embryos, followed within 5 min by injection of lariat or linear RNPs, which were PLoS Genetics | www.plosgenetics.org 2 February 2012 | Volume 8 | Issue 2 | e1002534