Zucchini-dependent piRNA processing is triggered by recruitment to the cytoplasmic processing machinery

We show that heterologous RNA that lacks complementary piRNAs is processed into piRNAs upon recruitment of several piRNA pathway factors. Our approach utilizes tethering of components of the piRNA pathway to the transcript of a reporter and then small RNA cloning from total RNA from Drosophila ovaries. Our approach allows discrimination of proteins involved in transcription and export of piRNA precursors from components required for the cytoplasmic processing steps. piRNA processing correlates with localization of the substrate RNA to nuage, a distinct membraneless cytoplasmic compartment, which surrounds the nucleus of germ cells, suggesting that sequestration of RNA to this subcellular compartment is both necessary and sufficient for selecting piRNA biogenesis substrates. Examination of small RNAs mapping to the mKate2 reporter in D. melanogaster ovaries upon tethering and concomitant knockdown of piRNA pathway factors