propiR1 has gene regulatory potential in trans. (A) Schematic representation of the firefly luciferase (FLuc) reporters bearing a propiR1 target site or a control sequence in the 3′ UTR. Numbers correspond to the position of residues in the target RNA (t1–30) expected to base-pair with propiR1 nucleotides 1–30 (from 5′ to 3′). Sequences of propiR1, a fully complementary target site, and the scrambled control (used in C) are shown. (B) Luciferase assay using reporters bearing a fully complementary target site (full) or no target site. FLuc activity was normalized to a cotransfected Renilla luciferase (RLuc) reporter (same in C and D). Bars represent mean ± SD of a representative of five independent experiments, each performed with three biological replicates. (***) P < 0.0005 (unpaired t-test). (C) Luciferase assay of reporters with propiR1 target sites harboring three consecutive mismatches (dark blue) or single nt mismatches (light blue), or an increasing number of mismatches corresponding to the 3′ end of the piRNA (yellow). Numbers indicate positions in the target site that were mutated (t1–30, shown in A). In all cases, guanosine residues were changed to cytidine, adenosine to uridine, and vice versa. Bars depict mean ± SD of a representative of three independent experiments, each performed with three biological replicates. (D) Luciferase assay using reporters with either a fully complementary (full) or scrambled target site upon PIWI gene knockdown in Aag2 cells. dsRNA targeting GFP (dsGFP) was used as a control. Bars represent mean ± SD of a representative of three independent experiments, each performed with three biological replicates. Asterisks denote statistically significant differences in fold silencing between dsGFP control and PIWI gene knockdowns (unpaired two-tailed t-tests with Holm–Sidak correction; [*] P < 0.05, [**] P < 0.005).
