Recurrent expansion and rapid evolution of the Drosophilid RNAi pathway in testis

Elicker, K.; Setia, G.; Quraishi, M.; Jiang, X. Y. Z.; Kan, L.; Flynt, A. S.; Wen, J.; Wahba, L.; Lai, E. C.

doi:10.1101/2025.11.28.691189

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bioRxiv · 10.1101/2025.11.28.691189

Recurrent expansion and rapid evolution of the Drosophilid RNAi pathway in testis

Elicker, K.·Setia, G.·Quraishi, M.·Jiang, X. Y. Z.·Kan, L.·Flynt, A. S.·Wen, J.·Wahba, L.·Lai, E. C.

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Abstract

Multiple classes of selfish genetic elements, including transposable elements, meiotic drivers, and viruses, are suppressed by small interfering RNAs (siRNAs) that guide RNA interference (RNAi). These are interlocked within continually evolving genetic conflicts that are characterized by extremely rapid dynamics of change in both sequence and copy number. Indeed, it was shown that across Drosophila, the core RNAi machinery evolves under positive selection, and that the RNAi effector AGO2 has additional copies in some species. This contrasts with the core microRNA (miRNA) machinery in Drosophila, which evolves under negative selection and maintains one-to-one orthologs not only amongst flies, but even to mammals. Here, we analyze >300 long read genome assemblies of Drosophila species to generalize these attributes. Not only do we find recurrent expansion of AGO2 across [~]100 species, including lineages with ongoing amplification of AGO2, we also find dozens of species with extra copies of the other core RNAi factors, r2d2 and dicer-2. In many cases, these additional RNAi factor copies co-exist, or are nested, within a lineage bearing an ancestral expansion of AGO2. Transcriptome data provide evidence that core RNAi factors, including certain lineage-specific copies, are biased for testis expression. Finally, we use small RNA data to annotate hairpin RNAs (hpRNAs) in D. pseudoobscura, one of the species with prominent amplification of RNAi factors. We find rampant de novo hpRNA loci in this species, whose siRNAs are predominantly expressed in testis. All together, these findings highlight evolutionary plasticity of the fly RNAi pathway and affirm that it is preferentially deployed in the germline of the heterogametic sex. When considered alongside abundant genetic data for recurrent sex ratio meiotic drive against the Y chromosome, these genomic data strongly imply that a fundamental role of endogenous RNAi is to control sex chromosome conflicts.

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Elicker, K., Setia, G., Quraishi, M., Jiang, X. Y. Z., Kan, L., Flynt, A. S., Wen, J., Wahba, L., Lai, E. C.. 2025-11-29. Recurrent expansion and rapid evolution of the Drosophilid RNAi pathway in testis. https://doi.org/10.1101/2025.11.28.691189

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