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

这项提案中的研究将利用果蝇来调查确保种间杂交不能繁殖的分子机制。更具体地说，我们将调查的假设，在piwi相互作用RNA（皮尔纳）和ATM/ATR DNA损伤反应途径的扰动有助于种间D。黑腹果蝇D. simulans hybrids我们将集中在瓦萨调查这一点。我们还将探索Chk 2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大量的转座因子（transposable elements，TEs），为了保持染色体的完整性和确保遗传信息传递到下一代的准确性，必须抑制TEs在生殖细胞中的迁移。TE转座的抑制涉及称为piRNA的小RNA，其靶向TE衍生的mRNA，使其不稳定并抑制其翻译和转录。参与皮尔纳过程的基因具有进化非常迅速的区域，并且即使在密切相关的物种之间也显示出非同义取代的速率升高，例如D。melanogaster和D. simulans，大约500万年前从一个共同的祖先分化出来。TE也迅速进化，在两个D. melanogaster和D. simulans。In D.黑腹果蝇D.在simulans杂种雌性中，皮尔纳生物发生和转座子沉默受到损害，表明皮尔纳途径基因的快速进化可能有助于杂种不育。我们认为皮尔纳途径中的其他快速进化的蛋白质可能有助于种间生殖隔离。一个强有力的候选者是Vasa（Vas），这是一种死亡盒型RNA解旋酶，在整个动物界具有直向同源物。 在之前NSERC资助的研究中，我们对Vas进行了广泛的结构-功能分析，并发现了与TE沉默相关的Vasa（Vas）的特定快速进化区域。在这里，我们建议，以确定这些区域的Vas有助于生殖隔离之间的D。melanogaster和D. simulans。我们还将通过确定在嵌合D中哪些涉及Vas的蛋白质-蛋白质相互作用被破坏来研究这是如何发生的机制。黑腹果蝇D. simulans Vas不支持生育的蛋白质。最后，我们将使用基于遗传和化学的方法操纵果蝇中的Chk 2活性，以探索对生殖细胞发育以及皮尔纳和TE群体的影响。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PI）将在国家和国际会议上介绍他们的成果。学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.