template switches to exon 1 RNA showed similar characteristics, but with a higher proportion of junctions containing extra nucleotide residues (61% compared to 33% for exon 1 DNA; Figure 7B; bands j–l). The second set of larger bands (120–140 nts; bands f and g in lane 5 and m and n in lane 6) contains products resulting from two sequential template switches to exon 1 DNA or RNA (Figure 7A and 7B, respectively) and/or the Ll.LtrB RNA (Figure S1). These products of multiple template switches have characteristics similar to those resulting from a single template switch, including addition of extra nucleotide residues, mostly A residues, at some template- switching junctions and at the 39 ends of the cDNAs. The above results were obtained under reaction optimized for reverse transcription by the Ll.LtrB RT in vitro (450 mM NaCl, 5 mM Mg2+), the high salt concentration helping to stabilize free protein and minimize aggregation of this RT [9]. However, similar results were obtained for template-switching reactions under near- physiological salt conditions (100 or 200 mM KCl, 5 mM Mg2+). Although the RT activity of the protein was lower under these conditions, the gel profiles show roughly equal levels of template switching to exon 1 RNA and DNA (Figure S2), and sequencing of the products showed similar template-switching junctions and patterns of non-templated nucleotide addition (Figure S3). Finally, we tested whether the Ll.LtrB RT could template switch to double-stranded exon 1 DNA or RNA with an annealed bottom-strand DNA leaving either a blunt end or a 59 bottom- strand overhang identical to that generated in vivo by the staggered double-strand break accompanying group II intron insertion (Figure 1; complete annealing confirmed by native gel analysis; Figure S4). Neither of these configurations significantly decreased the formation of the 100-nt product resulting from template switching to exon 1 DNA or RNA (Figure 6, lanes 7–9). DNA sequencing confirmed the template switch to double-stranded E1 DNA with a 59-bottom-strand overhang and showed that this template switch was seamless in most cases (Figure 7C). The sequencing also showed several instances in which the template switch occurred to the penultimate rather than the 39 terminal residue of exon 1 (Figure 7C), as well as template switches to Ll.LtrB RNA and the bottom-strand overhang oligonucleotide (Figure S5). Template switching to the penultimate nucleotide residue was not seen for single-strand acceptor DNA templates and could be related to the presence of the complementary DNA strand. Together, the biochemical assays show that the Ll.LtrB RT can template switch from the 59 end of the intron RNA to exon 1 and surprisingly that template switching is similarly efficient regardless of whether the exon 1 template is RNA or DNA or single- or double-stranded. However, the junctions resulting from template switching differ from those generated during retrohoming of linear intron RNA in vivo in that extra nucleotide additions are uniformly short and mostly A-residues. Discussion Considered together, our results lead to the model shown in Figure 8 for the key steps in linear intron RNA retrohoming. The finding of strong differential inhibition of linear relative to lariat intron retrohoming in D. melanogaster mutants indicates that the NHEJ factor Lig4 is the predominant enzyme involved in ligating the intron cDNA to the upstream exon and that extra nucleotide addition by the DNA repair polymerase h (PolQ) also plays a crucial role. Although Lig4 and PolQ appear to be the major enzymes playing these roles in D. melanogaster, residual linear intron RNA retrohoming with extra nucleotide addition occurs in both Linear Group II Intron Retrohoming in Drosophila Figure 8. Model for ligation of the intron cDNA to exon 1 during linear group II intron retrohoming. Partial reverse splicing of the linear intron RNA into the DNA target site is followed by bottom- strand cleavage between E2 positions +9 and +10 and synthesis of a cDNA of the attached linear intron RNA. After cDNA synthesis reaches the 59 end of the intron RNA template, extra nucleotide residues are added to the 39 end of the cDNA generating microhomologies that enable annealing of the cDNA strand to the top strand of E1. In Drosophila, the repair DNA polymerase h is the major enzyme responsible for this extra nucleotide addition, but some extra nucleotide addition may also be done by other host DNA polymerases or by the Ll.LtrB RT. The annealing of the cDNA requires unwinding and/or resection of the bottom strand, leading to loss of the bottom- strand 59 overhang resulting from the initial double-strand break by the group II intron RNP. In the final step, the annealed cDNA is ligated to the bottom strand of E1 by DNA ligase 4 or an alternate ligase. The retrohoming of the linear intron RNA is completed by degradation or displacement of the intron RNA template strand, second-strand DNA synthesis, and sealing of nicks by host enzymes. E1 and E2, 59 and 39 exons, respectively. doi:10.1371/journal.pgen.1002534.g008 PLoS Genetics | www.plosgenetics.org 10 February 2012 | Volume 8 | Issue 2 | e1002534