Linear Group II Intron Retrohoming in Drosophila Figure 1. Models for retrohoming of Ll.LtrB group II intron lariat and linear RNAs. (A) Retrohoming of lariat RNA. RNPs containing lariat RNA recognize the DNA target site (ligated E1–E2 sequence) and carry out both steps of reverse splicing, resulting in insertion of the intron RNA between E1 and E2. The IEP uses its En domain to cleave the bottom strand between positions +9 and +10 of E2, and then uses the 39 end of the cleaved DNA strand as a primer for reverse transcription of the inserted intron RNA. The resulting full-length intron cDNA is extended directly into E1 by continued DNA synthesis. Retrohoming is completed by a process that includes removal of the 59 overhang on the bottom strand, degradation or displacement of the intron RNA template strand, top-strand DNA synthesis by a host DNA polymerase, and sealing of nicks by a host DNA ligase [21]. (B) Retrohoming of linear RNA. RNPs containing linear intron RNA recognize the DNA target site and carry out the first step of reverse splicing, resulting in ligation of the 39 end of the intron RNA to the 59 end of E2. The IEP then uses its En domain to cleave the bottom strand between positions +9 and +10, generating a primer for reverse transcription of the intron RNA, as in lariat RNA retrohoming. However, because the 59 end of the linear intron RNA is unattached, the resulting cDNA cannot be extended directly into E1 and is instead linked to the 59 exon DNA by an error- prone process that sometimes leads to precise insertion of the intron RNA, but often gives imprecise 59 junctions due to deletion of E1 sequences, 59- intron truncations, and/or insertion of extra nucleotide residues at the ligation junction. As for lariat RNA, retrohoming of the linear intron RNA is completed by degradation or displacement of the intron RNA template strand, top-strand DNA synthesis, and sealing of nicks by host enzymes. E1 and E2, 59 and 39 exon, respectively; CS, bottom-strand cleavage site; IS, intron-insertion site. doi:10.1371/journal.pgen.1002534.g001 reconstituted in vitro from the purified IEP and intron RNA (see Materials and Methods). After incubating the embryos for 1 h at 30uC, nucleic acids were extracted and transformed into an E. coli strain (HMS174(DE3)), which expresses T7 RNA polymerase. The transformed bacteria were then plated on medium containing ampicillin or ampicillin and tetracycline, and mobility efficiencies were quantified as the ratio of (TetR+AmpR)/AmpR colonies. Figure 2B compares the retrohoming efficiencies of lariat and linear intron RNAs in wild-type and mutant embryos, based on parallel assays in ten separate experiments (summarized in Table S1). For each strain in each experiment, 80 injected embryos were pooled prior to extracting nucleic acids and transforming them into E. coli. The results for the lig4 mutant show the retrohoming efficiency of the lariat intron was unchanged, whereas the retrohoming efficiency of the linear intron was decreased strongly (18% wild type), but could be restored to wild-type levels by ectopic expression of Lig4 from an integrated P-element (lig42; P{lig4+} embryos). In the polQ mutant, the retrohoming efficiency of the linear intron was decreased to #0.5% of wild type, compared to 27% wild type for lariat RNPs. Finally, the ku70 mutant, a trans-heterozygote of two putative alleles (see Materials and Methods), showed only moderately decreased PLoS Genetics | www.plosgenetics.org 3 February 2012 | Volume 8 | Issue 2 | e1002534