Divergence of piRNA pathway proteins affects piRNA biogenesis but not TE transcript level

Abstract

In metazoan germlines, the piRNA pathway acts as a genomic immune system, employing small RNA-mediated silencing to defend host DNA from the harmful effects of transposable elements (TEs). In response to dynamic changes in TE content, host genomes are proposed to alter the piRNAs that they produce in order to silence the most active TE families. Surprisingly, however, piRNA pathway proteins, which execute piRNA biogenesis and enforce silencing of targeted sequences, also evolve rapidly and adaptively in animals. If TE silencing evolves through changes in piRNAs, what necessitates changes in piRNA pathway proteins? Here we used interspecific complementation to test for functional differences between Drosophila melanogaster and D. simulans alleles of three adaptively evolving piRNA pathway proteins: Armitage, Aubergine and Spindle-E. Surprisingly, we observed interspecific divergence in the regulation of only a handful of TE families, which were more robustly silenced by the heterospecific piRNA pathway protein. This suggests that positive selection does not act on piRNA effector proteins to enhance their function in TE repression, but rather that TEs may evolve to "escape" silencing by homospecific alleles. We also discovered that D. simulans alleles of aub and armi exhibit enhanced off-target effects on host transcripts in a D. melanogaster background, suggesting the avoidance of genomic auto-immunity as a critical target of selection. Our observations suggest that piRNA effector proteins are subject to an evolutionary trade-off between defending the host genome from the harmful effect of TEs while also minimizing friendly fire against host genes. Author SummaryTransposable elements are mobile fragments of selfish DNA that burden host genomes with deleterious mutations and incite genome instability. Host cells employ a specialized small-RNA mediated silencing pathway, the piRNA pathway, to act as a genomic immune system suppressing the mobilization of TEs. Changes in genomic TE content are met with rapid changes in the piRNA pool, thereby maintain host control over transposition. However, piRNA pathway proteins--which enact piRNA biogenesis and silence target TEs--also evolve adaptively. To isolate forces that underlie this adaptive evolution, we examined functional divergence between two Drosophila species for three adaptively evolving piRNA pathway proteins. To our surprise, we found very few differences in TE regulation, suggesting that evolution has not generally acted to enhance control of TE parasites. Rather, we discovered interspecific differences in the regulation of host mRNAs for two proteins, which suggested that proteins evolve to avoid off-target silencing of host transcripts. We propose that the avoidance of such "genomic autoimmunity" is an important and underappreciated force driving the adaptive evolution of piRNA proteins.