Fertility Gene Introns Harbor Transposable Elements that Shape Y-Loop Architecture

Transposable elements (TEs) are powerful drivers of genome evolution, yet how they persist under selection and become incorporated into host regulatory networks remains poorly understood. In the Drosophila male germline, TEs are highly expressed during the spermatocyte stage, coinciding with activation of giant fertility genes on the Y chromosome. These genes contain megabase-scale introns enriched for repetitive DNA, and three of these genes form prominent nuclear structures known as Y-loops, providing a unique system to investigate gene regulation. Here, we show that multiple TEs expressed in spermatocytes are transcribed from the introns of Y-linked fertility genes. RNA fluorescence in situ hybridization (FISH) targeting several TEs, including accord2, Juan, and HMS Beagle, illuminates distinct nuclear regions corresponding to kl-2, kl-3, and kl-5, respectively. Genetic perturbation of these fertility gene loci or disruption of RNA-processing factors eliminates these TE transcripts, demonstrating that these TE sequences are embedded within Y chromosome-associated nascent transcripts rather than being independently transcribed. The identity and expression patterns of Y-loop-associated TEs vary extensively among closely related Drosophila species, consistent with the previously documented rapid evolution of Y-linked loci and suggesting that TEs may contribute to the genetic diversification of these giant fertility genes. We propose that continual turnover of repetitive elements within Y-linked introns provides a mechanism by which rapidly evolving repetitive DNA influences germline gene regulation, male fertility, and speciation.