Linear Group II Intron Retrohoming in Drosophila Figure 2. Retrohoming efficiencies of linear and lariat group II intron RNAs in wild-type and mutant Drosophila. (A) Microinjection assay for retrohoming of lariat and linear group II intron RNAs. D. melanogaster precellular blastoderm are microinjected with the AmpR-recipient plasmid pBRR3-ltrB, which contains an Ll.LtrB target site (ligated E1–E2 sequence) cloned upstream of a promoterless tetR gene, followed by separate microinjection of Ll.LtrB RNPs containing linear or lariat intron RNAs with a phage T7 promoter sequence inserted near their 39 end. The embryos are incubated at 30uC for 1 h, during which the intron integrates into the target site in the recipient plasmid, placing the T7 promoter upstream of the promoterless tetR gene. Nucleic acids are then extracted and transformed into E. coli HMS174(DE3) for plating assays, and retrohoming efficiencies are calculated as the ratio of (TetR+AmpR)/AmpR colonies. T1 and T2, E. coli rrnB transcription terminators; Tw, phage T7 transcription terminator. (B) Retrohoming efficiencies of lariat and linear Ll.LtrB RNPs in D. melanogaster wild-type (w1118 and Or-R) and mutant embryos were determined, as described in panel A and Materials and Methods. The bar graphs show retrohoming efficiency in the indicated mutant embryos relative to that of wild-type embryos assayed in parallel in ten independent experiments with different combinations of strains (Table S1). The values are the mean for at least three independent determinations for each mutant, with the error bars indicating the standard error. The retrohoming efficiency of #0.5% wild type for linear RNPs in the polQ mutant is an upper limit, as only a single TetR+AmpR colony was recovered in three separate experiments, although 59-intron integration junctions could be detected by using a more sensitive PCR assay in all experiments (see Figure 3). doi:10.1371/journal.pgen.1002534.g002 retrohoming efficiencies for both lariat and linear intron retro- homing (67% and 46% wild type, respectively). The latter result was surprising because Ku and Lig4 ordinarily function together in the same NHEJ pathway [41,42]. The strong differential inhibition of linear compared to lariat RNA retrohoming in the lig4 and polQ mutants supports models in which these enzymes function directly at unique steps in this process, presumably by providing the DNA ligase and repair DNA polymerase activities needed to link the intron cDNA to the upstream exon. The similar moderate decreases in lariat and linear intron retrohoming efficiency in the ku70 mutant could reflect that Ku functions at a common step in both pathways or could be an indirect effect (see Discussion). Comparison of 59-integration junctions from linear intron RNA retrohoming in wild-type and lig4 and ku70 mutant embryos D. melanogaster uses at least two NHEJ pathways to repair double-strand breaks: classical NHEJ (C-NHEJ), which is dependent upon Lig4 and Ku70, and alternate end-joining (alt- EJ), which operates without either factor and could be a mixture of different pathways [31,38,39,43]. In a genetic assay for repair of double-strand breaks induced in the germline by the meganuclease I-SceI, lig4 and ku70 mutants inhibited NHEJ activity by 76–78%, leaving 22–24% residual activity that was attributed to alt-EJ [43]. Our finding above that the lig4 mutant shows similar degrees of inhibition and residual activity for linear intron retrohoming (82% and 18%, respectively), most simply suggests that Lig4-indepen- dent retrohoming occurs by using the alt-EJ pathway or components thereof. Previous studies showed that the DNA repair junctions resulting from alt-EJ in Drosophila ku70 and lig4 mutants are generally similar to those for C-NHEJ [31], although in some assays, the lig4 mutant gave somewhat increased frequencies of junctions with extra nucleotide additions (55–63% compared to 30–36% for wild type [38,39]). To further investigate whether Lig4-independent retro- homing occurs via alt-EJ, we compared 59- and 39-intron integration junctions resulting from linear intron retrohoming in the mutant and two commonly used wild-type strains (w1118 and Or- PLoS Genetics | www.plosgenetics.org 4 February 2012 | Volume 8 | Issue 2 | e1002534