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

这项计划中的研究将使用果蝇来研究确保种间杂交不能繁殖的分子机制。更具体地说，我们将调查这一假说，即piwi相互作用RNA(PiRNA)和ATM/ATR DNA损伤反应通路中的扰动导致种间黑腹果蝇/拟果蝇杂交种的不育性。我们将重点关注Vasa来调查这一点。我们还将探索Chk2激酶活性是否调节原始生殖细胞数量。真核生物基因组中含有许多转座元件(TES)的拷贝，为了保持染色体的完整性并确保遗传信息传递给后代的保真度，必须抑制TES在生殖系中的迁移率。抑制TE转座涉及到称为piRNAs的小RNA，它以TE衍生的mRNA为靶点，破坏它们的稳定，并抑制它们的翻译和转录。参与piRNA过程的基因具有进化极其迅速的区域，甚至在密切相关的物种之间也显示出高的非同义替换率，例如大约500万年前从共同祖先分化而来的黑腹夜蛾和拟态夜蛾。TES也进化得很快，在黑腹毛虫和拟黑腹毛虫中都存在物种特有的家族。在黑腹粉虱/拟黑粉蝶杂交雌性中，piRNA的生物发生和转座子沉默都受到了影响，这表明piRNA途径基因的快速进化可能是造成杂交不育的原因之一。我们认为，piRNA途径中其他快速进化的蛋白质可能有助于物种间的生殖隔离。一个强有力的候选者是Vasa(Vas)，它是一种死盒型RNA解旋酶，在整个动物界都有同源基因。在之前由NSERC资助的研究中，我们对Vas进行了广泛的结构-功能分析，发现了Vasa(Vas)的特定快速进化区域，我们将其与TE沉默联系在一起。在这里，我们建议确定这些Vas区域是否有助于黑腹夜蛾和拟黑腹夜蛾之间的生殖隔离。我们还将通过确定哪些涉及Vas的蛋白质-蛋白质相互作用在不支持生育的嵌合黑腹果蝇/D·模拟菌Vas蛋白中被破坏，来研究这种情况发生的机制。最后，我们将使用基于遗传和化学的方法来操纵果蝇中Chk2的活性，以探索由此对生殖细胞发育以及对piRNA和TE种群的影响。这项工作的预期影响将包括在高质量的国际科学期刊上发表文章，由赠款资助的受训人员(以及国际和平研究所)将在国内和国际会议上介绍他们的成果。受训人员还将完成博士论文项目，这些项目将涉及获得最先进的分子遗传和成像技术，这将使他们能够在公共或私营部门从事研究事业。

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.