The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4– Dependent and –Independent Mechanisms Travis B. White, Alan M. Lambowitz* Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry and Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas, United States of America Abstract Mobile group II introns are bacterial retrotransposons that are thought to have invaded early eukaryotes and evolved into introns and retroelements in higher organisms. In bacteria, group II introns typically retrohome via full reverse splicing of an excised intron lariat RNA into a DNA site, where it is reverse transcribed by the intron-encoded protein. Recently, we showed that linear group II intron RNAs, which can result from hydrolytic splicing or debranching of lariat RNAs, can retrohome in eukaryotes by performing only the first step of reverse splicing, ligating their 39 end to the downstream DNA exon. Reverse transcription then yields an intron cDNA, whose free end is linked to the upstream DNA exon by an error-prone process that yields junctions similar to those formed by non-homologous end joining (NHEJ). Here, by using Drosophila melanogaster NHEJ mutants, we show that linear intron RNA retrohoming occurs by major Lig4-dependent and minor Lig4-independent mechanisms, which appear to be related to classical and alternate NHEJ, respectively. The DNA repair polymerase h plays a crucial role in both pathways. Surprisingly, however, mutations in Ku70, which functions in capping chromosome ends during NHEJ, have only moderate, possibly indirect effects, suggesting that both Lig4 and the alternate end-joining ligase act in some retrohoming events independently of Ku. Another potential Lig4-independent mechanism, reverse transcriptase template switching from the intron RNA to the upstream exon DNA, occurs in vitro, but gives junctions differing from the majority in vivo. Our results show that group II introns can utilize cellular NHEJ enzymes for retromobility in higher organisms, possibly exploiting mechanisms that contribute to retrotransposition and mitigate DNA damage by resident retrotransposons. Additionally, our results reveal novel activities of group II intron reverse transcriptases, with implications for retrohoming mechanisms and potential biotechnological applications. Citation: White TB, Lambowitz AM (2012) The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4–Dependent and –Independent Mechanisms. PLoS Genet 8(2): e1002534. doi:10.1371/journal.pgen.1002534 Editor: Harmit S. Malik, Fred Hutchinson Cancer Research Center, United States of America Received September 29, 2011; Accepted December 24, 2011; Published February 16, 2012 Copyright: 2012 White, Lambowitz. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NIH grant GM37949 and Welch Foundation grant F-1607. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Lambowitz@mail.utexas.edu Introduction Mobile group II introns are site-specific retrotransposons that consist of a catalytically active intron RNA (ribozyme) and an intron-encoded protein (IEP), with reverse transcriptase (RT) activity [1]. Although they are found mainly in bacterial and organellar genomes, group II introns are thought to have played a major role in eukaryotic genome evolution as evolutionary ancestors of nuclear spliceosomal introns and retrotransposons in higher organisms [2–4]. Group II intron RNAs catalyze their own splicing via two sequential transesterification reactions that are the same as those for spliceosomal introns and yield an excised intron lariat with a branched 29-59 phosphodiester linkage [5,6]. For mobile group II introns, the splicing reactions are assisted by the IEP, which binds specifically to the intron RNA and stabilizes the catalytically active RNA structure [7,8]. The IEP then remains bound to the excised intron lariat RNA in a ribonucleoprotein particle (RNP) that promotes intron integration into new DNA sites [9,10]. Intron integration is targeted to the ligated-exon junction in an intronless alleles in a process called ‘‘retrohoming’’, but can also occur at lower frequency into ectopic sites that resemble the homing site in a process called ‘‘retrotransposition’’ or ‘‘ectopic retrohoming’’. In both cases, the intron inserts into the new DNA site by a novel mechanism in which the excised intron lariat RNA fully reverse splices into a DNA strand and is reverse transcribed by the IEP, yielding an intron cDNA that is integrated into the genome by host enzymes [1,10–14]. Retrohoming leads to the expansion of intron-containing alleles in a population, while ectopic retrohoming provides a means of intron dispersal to new sites. Group II intron RNAs can also splice without branching by an alternate pathway, termed ‘‘hydrolytic splicing’’ [1]. In this pathway, the first transesterification, 59-splice site cleavage, occurs by hydrolysis rather than branching, and the second transester- ification yields ligated exons and an excised linear intron RNA. Hydrolytic splicing was first observed as a side reaction of group II intron self-splicing under non-physiological conditions [15,16] and was demonstrated to occur in vivo by using a mutant yeast mitochondrial intron that was deleted for the branch-point A residue [17]. Some subclasses of group II introns lack the branch- point A residue and rely entirely on the hydrolytic mechanism for splicing in vivo [18,19]. Linear group II intron RNAs can also be PLoS Genetics | www.plosgenetics.org 1 February 2012 | Volume 8 | Issue 2 | e1002534