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. 2021 Feb 8;22(4):e51313. doi: 10.15252/embr.202051313

Figure EV3. In vivo reporter assay and in vitro unwinding assay.

Figure EV3

  1. Experimental procedure for the in vivo reporter assay (see Materials and Methods for further details) used to detect artificial Siwi‐piRNA produced by a reporter plasmid encoding the PiggyBac‐piRNA target sequence.
  2. Analysis of the Yamato‐based and Pao‐based artificial piRNA biogenesis in DDX43‐depleted BmN4 cells related to Fig 3C.
  3. The experimental procedure for the Ago3‐cleaved target RNA‐unwinding assay (see Materials and Methods for further details).
  4. In vitro target RNA cleavage by Ago3 and fractionation of the cleaved RNAs. n.i.: non‐immune mouse IgG. Sup: supernatant. The cleaved target RNA remained bound to Ago3‐piRISC upon cleavage.
  5. Model of step‐by‐step pre‐Siwi‐piRISC assembly: Upon cleavage, the 5′‐end of 3′‐fragment is first bound to Siwi while the 3′‐end of the piRNA precursor is still attached to Ago3‐piRISC. The 3′‐end of the fragment is then released from Ago3‐piRISC, producing pre‐Siwi‐piRISC for Zuc‐dependent maturation.