DNA damage control, small RNAs, and the establishment of species isolation

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The research in this proposal will use Drosophila to investigate the molecular mechanisms that ensure that interspecific hybrids cannot reproduce. More specifically, we will investigate the hypothesis that perturbations in the piwi-interacting RNA (piRNA) and ATM/ATR DNA damage response pathways contribute to sterility of interspecific D. melanogaster/D. simulans hybrids. We will focus on Vasa to investigate this. We will also explore whether Chk2 kinase activity regulates primordial germ cell number. Eukaryotic genomes contain many copies of transposable elements (TEs), whose mobility has to be suppressed in the germline to maintain chromosome integrity and ensure the fidelity of transmission of genetic information to the subsequent generation. Suppression of TE transposition involves small RNAs called piRNAs that target TE-derived mRNAs, destabilizing them and repressing both their translation and transcription. Genes involved in piRNA processes have regions that evolve extremely rapidly and show elevated rates of non-synonymous substitution even between closely related species, such as D. melanogaster and D. simulans, which diverged from a common ancestor approximately 5 million years ago. TEs also evolve rapidly, with species-specific families present in both D. melanogaster and D. simulans. In D. melanogaster/D. simulans hybrid females, piRNA biogenesis and transposon silencing are compromised, suggesting that rapid evolution of piRNA pathway genes may contribute to hybrid sterility. We believe it is likely that other rapidly evolving proteins in the piRNA pathway contribute to interspecific reproductive isolation. One strong candidate is Vasa (Vas), a DEAD-box type RNA helicase with orthologues throughout the animal kingdom.  In previous NSERC-funded research, we carried out an extensive structure-function analysis of Vas and discovered specific rapidly evolving regions of Vasa (Vas) that we linked to TE silencing. Here, we propose to determine whether these regions of Vas contribute to reproductive isolation between D. melanogaster and D. simulans. We also will investigate the mechanism of how this occurs by determining which protein-protein interactions involving Vas are disrupted in chimeric D. melanogaster/D. simulans Vas proteins that do not support fertility. Finally, we will manipulate Chk2 activity in flies using genetic- and chemical-based approaches, to explore resulting effects on germ cell development and on piRNA and TE populations. The expected impact of this work will include publications in high-quality international scientific journals, and the trainees funded by the grant (as well as the PI) will present their results at national and international conferences. The trainees will also complete PhD thesis projects that will have involved gaining facility in state-of-the-art molecular genetic and imaging techniques, which will enable them to pursue research careers in either the public or private sectors.