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

DNA 损伤控制、小 RNA 和物种隔离的建立

• 批准号: RGPIN-2020-07024
• 负责人: Lasko, Paul
• 金额: $ 3.06万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=745776
• 关键词: DNA; damage; control; small; RNAs

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.

本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，我们将研究piwi相互作用RNA （piRNA）和ATM/ATR DNA损伤反应途径的扰动导致种间D. melanogaster/D不育的假设。simulans混合动力车。我们将重点调查瓦萨。我们还将探讨Chk2激酶活性是否调节原始生殖细胞数量。真核生物基因组包含许多转座因子（te）拷贝，在种系中必须抑制其迁移以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。TE转位的抑制涉及称为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piRNA过程的基因具有进化极快的区域，甚至在密切相关的物种（如D. melanogaster和D. simulans）之间也显示出较高的非同义替换率，它们大约在500万年前从共同祖先分化而来。te的进化速度也很快，在D. melanogaster和D. simulans中都有特定的家族。解析：D。在杂交雌性中，piRNA的生物发生和转座子沉默受到破坏，这表明piRNA途径基因的快速进化可能导致杂交不育。我们认为，piRNA通路中其他快速进化的蛋白质可能有助于种间生殖隔离。一个强有力的候选是Vasa (Vas)，这是一种DEAD-box型RNA解旋酶，其同源物遍布动物界。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沉默有关的Vasa的特定快速进化区域（Vas）。在这里，我们建议确定这些Vas区域是否导致了黑腹龙眼和拟马眼龙眼之间的生殖隔离。我们还将通过确定在嵌合D. melanogaster/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种情况发生的机制。模拟不支持生育的输精管蛋白。最后，我们将使用基于遗传和化学的方法在果蝇中操纵Chk2活性，以探索对生殖细胞发育以及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（以及PI）资助的学员将在国家和国际会议上展示他们的成果。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学和成像技术，这将使他们能够在公共或私营部门从事研究工作。