Linear Group II Intron Retrohoming in Drosophila Figure 5. Characteristics of 59-integration junctions resulting from linear intron RNA retrohoming in wild-type and mutant strains. The bar graphs show the percentage of 59-integration junctions with (A) exon 1 deletions, (B) 59-intron truncations, (C) extra nucleotide additions, and (D) microhomologies in the indicated strains. For the polQ2 embryos, where the number of unique junction sequences recovered was smaller than for the other strains, the percentage of junctions having the indicated characteristics was calculated both as a percentage of total junctions (left bar) and a percentage of unique junctions (right bar, asterisk). doi:10.1371/journal.pgen.1002534.g005 investigate the function of PolQ in linear intron RNA retro- homing, we compared the sequences of 59-intregration junctions from parallel assays of this process in wild-type Or-R and polQ2 embryos (Figure 4E and 4F, Figure 5). Because the number of unique junction sequences recovered from the polQ mutant was lower than those for the other strains and some of these junctions were represented multiple times, we calculated the proportion of junctions with different characteristics in Figure 5 relative to both the total number of junctions (left bars) and the total number of unique junctions sequences (right bars, asterisks). Both compari- sons show that that the polQ mutation decreases the proportion of junctions containing extra nucleotide residues (4% of total junctions and 20% of unique junctions compared to .64% of junctions in wild-type Or-R and .48% in all other strains analyzed), as expected from the known function of PolQ. Further, the 59 junctions from the polQ2 embryos have a higher frequency of long ($15 bp) 59-exon deletions (72% of total junctions and 80% of unique junctions compared to 29% in wild-type Or-R) and a dramatically increased frequency of long ($5 nt) microhomol- ogies between exon and intron sequences (56% of total junctions and 40% of unique junctions compared to none among 170 total junctions from all the other strains analyzed). This increased frequency of long microhomologies may reflect that they are more stringently required for annealing of the 39 end of the cDNA to the upstream exon in the absence of PolQ. We note that among the unique junction sequences from the polQ mutant, two with large deletions were recovered $10 times each. Although we cannot exclude that the repeated recovery of these junctions reflects differential amplification by PCR, both have $5 nt microhomol- ogies that could have been used preferentially for annealing in multiple events, and indeed one of these junctions (Figure 4F bottom sequence) comprised 6 of 12 recovered junctions in an additional, separate experiment (data not included in Figure 4F). Considered together, the junction sequences indicate that PolQ functions in extra nucleotide addition to the 39 end of the cDNA during linear intron RNA retrohoming and that this extra PLoS Genetics | www.plosgenetics.org 7 February 2012 | Volume 8 | Issue 2 | e1002534