Germline silencing of UASt depends on the piRNA pathway

One of the most extensively used techniques in Drosophila is the Gal4/UAS binary system, which allows tissue-specific misexpression or knockdown of specific genes of interest. The original UAS vector, UASt, can only be activated for transgene expression in somatic tissues and not in the germline cells. Rorth (1998) generated UASp, a modified UAS vector that is responsive to Gal4 in both somatic and germline tissues, by replacing both the hsp70 promoter and the SV40 3UTR with the P transposase promoter and the K10 3UTR respectively. At present, the mechanisms by which UASt is silenced in germline cells are not fully understood. Here, we report that the piRNA pathway is involved in suppressing UASt expression in ovarian germline cells. Individually knocking down or mutating components of the piRNA biogenesis pathway (e.g., Piwi, AGO3, Aub, Spn-E, and Vasa) resulted in the expression of the UASt-reporter (GFP or RFP) in the germline. An RNA-seq analysis of small RNAs revealed that the hsp70 promoter of UASt is targeted by piRNAs, and in the aub mutant ovary, the amount of piRNAs targeting the hsp70 promoter is reduced by around 40 folds. In contrast, the SV40 3UTR of the UASt, which happens to be targeted by the Nonsense-mediated RNA decay (NMD) pathway, is not responsible for germline UASt suppression, as UASt-reporters with NMD-insensitive 3UTRs fail to show germline expression. Taken together, our studies reveal a crucial role of the piRNA pathway, potentially via the suppression of the hsp70 promoter, in germline UASt silencing in Drosophila ovaries.