3. How can animals defend their genomes without ping-pong piRNA amplification?

Animals have evolved sophisticated defence mechanisms to protect their genomes against transposons. One of these is the piRNA pathway, which produces small RNAs that recognise and silence transposon transcripts. In many animals, a mechanism known as ping-pong amplification generates large quantities of piRNAs while reinforcing the recognition and destruction of transposon RNAs. This mechanism has been extensively studied in the fruit fly Drosophila melanogaster, where it plays a major role in maintaining transposon silencing in the germline.

Unexpectedly, we discovered that a related fruit fly species, Drosophila eugracilis, has completely lost detectable ping-pong amplification yet continues to produce abundant piRNAs that target transposons (Chary and Hayashi, 2023, PLoS Biology; PMID: 37279192). Instead, this species relies on an alternative mechanism called phased piRNA biogenesis, in which a long precursor RNA is sequentially processed into multiple piRNAs.

This finding presents an evolutionary puzzle. How can phased biogenesis alone generate a sufficiently abundant and selectively antisense piRNA population to maintain transposon defence? Equally intriguingly, many genes associated with ping-pong amplification remain conserved in D. eugracilis, despite the apparent loss of the pathway itself. Why was ping-pong amplification lost, and have its molecular components acquired new functions?

In this project, we aim to uncover how D. eugracilis has reorganised its piRNA pathway to maintain transposon silencing without ping-pong amplification. Using CRISPR-based genome engineering, we will disrupt key piRNA biogenesis factors and investigate their contributions to piRNA production, transposon regulation and ovarian function. By combining genetic experiments with small RNA sequencing and comparative analyses, we will examine how phased biogenesis generates transposon-targeting piRNAs and whether conserved components of the ancestral pathway have been repurposed.

Ultimately, this research seeks to understand how an essential genome defence system can evolve radically different molecular strategies while retaining its biological function. More broadly, it may reveal how evolutionary changes in small RNA pathways allow animals to adapt to the changing threats posed by mobile genetic elements.

Techniques: Drosophila genetics, CRISPR-based genome engineering, molecular biology, small RNA sequencing, bioinformatics, and comparative genomics.