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. 2019 Aug 1;38(17):e102870. doi: 10.15252/embj.2019102870

Figure EV3. The PPNP complex is required for the co‐transcriptional silencing in the Piwi‐piRNA‐targeted reporter system.

Figure EV3

  1. Schematic of piRNA reporter gene assay. Reporter plasmid carries flam element in its intron. Luc mRNA harboring flam element in the antisense direction is targeted by Piwi carrying piRNAs derived from the flam locus.
  2. Experimental scheme. OSCs or S2 cells were transfected with reporter plasmids transiently and harvested at 24 or 48 hpt.
  3. Left panel shows reporter gene constructs harboring flam elements (0.6 kbp or 1.3 kbp) inserted in the intron. Blue and red arrows indicate sense and antisense directions, respectively. Middle graph shows luciferase activity relative to that of reporter gene without flam elements in OSC. Right graph shows luciferase activity relative to that of reporter gene without flam elements in S2 cells. Error bar indicates SD (n = 4). The sense reporters were expressed similar to control reporter gene lacking the flam element, whereas the antisense (piRNA‐targeted) reporters were completely silenced, even though we introduced the reporter gene as a plasmid into OSCs, suggesting that the co‐transcriptional silencing occurs independent of the chromatin context.
  4. Time course of luciferase activity in OSCs. Blue and red arrows indicate reporter gene bearing a flam element (1.3 kbp) in the sense or antisense direction, respectively. Error bar indicates SD (n = 3).
  5. Silencing of reporter gene harboring flam element (1.3 kbp, antisense direction) occurs in a Piwi‐dependent manner. Graph shows fold change of luciferase activity in OSCs treated with siPiwi or siEGFP (control). Error bar indicates SD (n = 3).
  6. Effects of knockdown of the indicated genes on piRNA reporter activity (1.3 kbp, flam). Red bar shows the luciferase activities of the antisense reporter relative to that of the sense reporter (blue). Error bars indicate SD (n = 3). Transfection schedule of siRNA and reporter plasmids is shown at the top of the figure. Depletion of Nxf2, Panx, p15, and known cofactors Gtsf1 and Mael, de‐silenced the piRNA‐targeted reporter in OSCs. In contrast, Eggless‐KD, H1‐KD, and HP1‐KD had no impact on the silencing.