Mechanistic divergence of piRNA biogenesis in Drosophila [ncRNA-seq]

Organisms require mechanisms to distinguish self and non-self RNA. This distinction is crucial to initiate the biogenesis of piRNAs. In Drosophila ovaries, PIWI-guided slicing and the recognition of piRNA precursor transcripts by the DEAD-box RNA helicase Yb are the two known mechanisms to initiate biogenesis in the germline and the soma, respectively. Both, the PIWI proteins and Yb are highly conserved across most Drosophila species and are thought to be essential to the piRNA pathway and for silencing TEs. However, we find that species closely related to D. melanogaster have lost the yb gene, as well as the PIWI gene Ago3. We show that neither mechanism is required to efficiently produce TE antisense piRNAs in Drosophila. Thus, there are more possible routes through which the piRNA pathway can achieve specificity than previously suggested. Overall design: The study aims at understanding the mechanism of Piwi-interacting RNA (piRNA) in a variety of Drosophila species. We generated small RNA sequencing libraries from freshly collected ovaries and embryos as well as immuno-precipitated samples using antibodies against PIWI proteins. We also generated RNA sequencing libraries from poly-A plus RNA and ChIP sequencing libraries using antibodies against RNA polymerase II to determine the genomic origin of the piRNA precursor RNA.