PLD6-dependent small RNAs

Recently it was shown that a functional somatic piRNA pathway is not only a special trait of lower metazoans but is also widespread in protostomian species like arthropods and mollusks. While it seems that a somatic piRNA pathway got lost in the branch of vertebrates, several studies indicate that it may still be active in specific niches like stem cells of mammalian brains. To identify piRNAs, co-immunoprecipitation with PIWI proteins is the gold standard. As appropriate antibodies against PIWI proteins are not available, crawling small RNA sequencing data for sequence-homology and certain characteristics is the general approach to identify putative piRNAs in the soma. This method is only predictive and does not allow the identification of novel characteristics and functions of this pathway. Here, we developed an RNAi-based approach to verify the expression of small RNAs whose expression depend on Phospholipase D family member 6 (PLD6). PLD6 is an endonuclease that is critical for production of primary piRNAs in the animal germline. By siRNA-mediated knockdown of PLD6 in cultured cells, followed by small RNA sequencing, we identify somatic PLD6-dependent small RNAs (putative piRNAs) in vitro. We demonstrate that in somatic cells PLD6 acts as a functional endonuclease generating small RNAs with the typical size range of piRNAs. Overall design: HEK293T cells were transfected with anti-PLD6 siRNA or a non-target siRNA, total RNA was isolated 24 h after transfection, small RNA libraries were generated and 50 bp single-end deep sequenced on a BGISEQ-500 platform.