piRNA-mediated gene regulation and adaptation to sex-specific transposon expression in D. melanogaster male germline

Abstract

Small noncoding piRNAs act as sequence-specific guides to repress complementary targets in Metazoa. Prior studies in Drosophila ovaries have demonstrated the function of the piRNA pathway in transposon silencing and therefore genome defense. However, the ability of the piRNA program to respond to different transposon landscapes and the role of piRNAs in regulating host gene expression remain poorly understood. Here, we comprehensively analyzed piRNA expression and defined the repertoire of their targets in Drosophila melanogaster testes. Comparison of piRNA programs between sexes revealed sexual dimorphism in piRNA programs that parallel sex-specific transposon expression. Using a novel bioinformatic pipeline, we identified new piRNA clusters and established complex satellites as dual-strand piRNA clusters. While sharing most piRNA clusters, the two sexes employ them differentially to combat the sex-specific transposon landscape. We found two piRNA clusters that produce piRNAs antisense to four host genes in testis, including CG12717/pirate, a SUMO protease gene. piRNAs encoded on the Y chromosome silence pirate, but not its paralog, to exert sex- and paralog-specific gene regulation. Interestingly, pirate is targeted by endogenous siRNAs in a sibling species, Drosophila mauritiana, suggesting distinct but related silencing strategies invented in recent evolution to regulate a conserved protein-coding gene.

RDC complex executes a dynamic piRNA program during Drosophila spermatogenesis to safeguard male fertility

“…Interestingly, dependence of dual-strand cluster expression on Rhi varies between sexes: 38C is more affected by loss of rhi than 42AB in testis, while the opposite is found in ovary. Furthermore, piRNA production from both strands of complex satellites, which we recently found to behave as dual-strand piRNA clusters [ 20 ], also drastically declined in rhi mutant testes and ovaries ( Fig 4B ). These results show that Rhi is essential for piRNA production from a large fraction of piRNA clusters in the male germline.…”

“…To verify TE de-repression, we employed a copia-lacZ reporter, where the long terminal repeat (LTR) of copia containing copia promoter is fused upstream to the lacZ gene and its expression can be directly examined by X-gal staining [ 18 ]. copia is known to be active in the male germline [ 19 ] and has the highest expression level among all TEs in testes [ 20 ]. Whereas no detectable X-gal staining was seen in control testes, robust staining was observed in rhi mutants ( Fig 3F ), confirming strong de-repression of copia at the absence of rhi .…”

“…To understand the role of RDC complex in piRNA cluster expression in testis, we analyzed effects of rhi mutation on piRNA production from major piRNA clusters. Genomic loci that generate piRNAs in testis were recently de novo defined leading to identification of several novel piRNA clusters [ 20 ]. Rhi was dispensable for expression of uni-strand piRNA clusters, flam and 20A ( Fig 4A ), similar to results from ovary studies [ 2 , 4 , 14 ].…”

“…Rhi was dispensable for expression of uni-strand piRNA clusters, flam and 20A ( Fig 4A ), similar to results from ovary studies [ 2 , 4 , 14 ]. Surprisingly, we found that Rhi was also dispensable for piRNA production from the Y-linked Su(Ste) locus, which is the most active piRNA cluster in testis [ 20 ]. Unlike flam and 20A , Su(Ste) is a dual-strand cluster that generates piRNAs from both genomic strands.…”

“…piRNA profiles are distinct in male and female gonads, and expression of dual-strand piRNA clusters are sexually dimorphic [ 20 ]. To explore if RDC complex might be involved in orchestrating sex-specific piRNA programs, we profiled Rhi binding on the genome in ovaries under identical ChIP-seq conditions as in testes.…”