Fig. 7: Transposon silencing.
a Piwi-mediated silencing of the mdg1 transposon. FLAG-tagged wild-type Piwi or slicer-Piwi was expressed in endogenous Piwi-depleted OSCs, and the expression levels of the mdg1 transposon were examined by quantitative RT-PCR (n = 3; error bars indicate SEM). Empty, empty vector control; Slicer, the slicer-Piwi K617H/A625S/V653E/K818H mutant. b Binding of Piwi to Arx. FLAG-tagged wild-type Piwi or slicer-Piwi was expressed in endogenous Piwi-depleted OSCs, and the proteins were then immunoprecipitated using anti-FLAG beads. The cell lysates and immunoprecipitates were analyzed by western blotting, using the indicated antibodies. H3 was used as a loading control. c Requirement of Arx for Piwi-mediated silencing. FLAG-tagged wild-type Piwi or slicer-Piwi was expressed in endogenous Piwi/Arx-depleted OSCs, and the expression levels of the mdg1 transposon were examined by quantitative RT-PCR (n = 3; error bars indicate SEM). d Binding of slicer-Piwi to the cleavage products. FLAG-tagged wild-type Piwi or slicer-Piwi was immunopurified from OSCs, and the proteins were then incubated with the internally 32P-labeled substrate RNA (flam target). The supernatant and beads fractions were analyzed by denaturing urea-PAGE. Signal intensities of the fragments are shown on the right. e Binding of Piwi to partially complementary targets. The immunopurified FLAG-tagged wild-type Piwi and slicer-Piwi were incubated with the 5′ 32P-labeled substrate RNA, and the supernatant and beads fractions were then analyzed by denaturing urea-PAGE. Sequences of the target RNAs (WT, mt1, and mt2) and the piRNA (mdg1-piRNA) are shown below. Source data are provided as a Source Data file.
