Elucidating the role of small RNA pathways in heat-stress induced DNA damage during spermatogenesis

项目摘要 在减数分裂过程中，基因组的忠实遗传是成功形成配子所必需的。而当 许多组织都会受到极端温度变化的影响，尤其是睾丸中发育的精子 对温度的微小波动敏感，精子发生需要2-7°C的狭窄等温线 低于核心体温。暴露在高温下的睾丸表现出生育力下降。研究项目： 哺乳动物已经将温度升高与精母细胞DNA损伤增加联系在一起，然而 潜在的机制仍不清楚。利布达实验室之前的研究发现，与哺乳动物类似， 暴露于热应激可引起秀丽线虫精母细胞DNA的特异性损伤 卵母细胞。利用线虫作为模型系统，确定了转座子动员的可能性。 热诱导DNA损伤产生的潜在机制。小的非编码RNA，在复杂的 相关蛋白是生殖系发育和维持的关键调节因子，包括调节 RNAi和转座子活性。已知的某些小RNA通路也是精母细胞所特有的 在温度诱导的不孕症中起到一定作用。因此，胚系中的小RNA途径代表 有望作为热应激诱导精母细胞DNA损伤的调节靶点。因此，我 热应激导致精母细胞DNA损伤是由转座子引起的假设 由生殖系中的小RNA途径调节的动员。为了测试这一点，我将使用一个 多管齐下的方法，结合候选突变方法和无偏RNA测序来识别 参与温度诱导的DNA损伤的成分。在目标1中，我将完成我的候选突变体 筛选、监测温度诱导的小RNA途径突变体的DNA损伤。我也会使用RNA 测序，以无偏见的方式表征所有温度敏感的小RNA群体。在目标2中，我 我将跟进我的发现，与生殖系中的piRNAs相互作用以抑制转座子的PRG-1， 是热应激引起的DNA损伤所必需的。我将调查我的假设，即PRG-1调节特定的 介导温度诱导的精母细胞DNA损伤的piRNA亚类 针对piRNA分析优化的小RNA测序实验。为了进一步探索这一结果，我将 表征依赖于热休克的PRG-1的定位和相互作用、相关的piRNAs和其他 已知作用于PRG-1下游的小RNA途径组件。在目标3中，我将评估转座子 热应激诱导的DNA转座子种类及其在热应激中的动员 损坏。我建议将深度测序和遗传方法结合起来，探索温度如何导致 DNA损伤专门发生在使用线虫的精母细胞中。总的来说，这些数据将 为提高我们对这些重要生物过程的理解做出了实质性的贡献 与人类不孕不育和疾病有关。

Project Summary During meiosis the faithful inheritance of the genome is necessary for successful gamete formation. While many tissues are affected by extreme temperature changes, developing sperm in the testes are particularly sensitive to small fluctuations in temperature, with spermatogenesis requiring a narrow isotherm of 2-7°C below core body temperature. Testes exposed to high temperature display reduced fertility. Studies in mammals have linked elevated temperatures with an increase in DNA damage in spermatocytes, however the underlying mechanisms remain unknown. Previous work from the Libuda lab found that, similar to mammals, exposure to heat-stress produces DNA damage specifically in Caenorhabditis elegans spermatocytes and not oocytes. Utilizing C. elegans as a model system, transposon mobilization was identified as a possible mechanism underlying the production of heat-induced DNA damage. Small non-coding RNAs, in complex with associated proteins, are crucial regulators of germ line development and maintenance, including the regulation of RNAi and transposon activity. Certain small RNA pathways are also known to be spermatocyte-specific and play a role in temperature-induced infertility. As such, small RNA pathways in the germ line represent a promising target as regulators of heat-stress induced DNA damage in spermatocytes. Therefore, I hypothesize that heat-stress induced DNA damage specifically in spermatocytes is due to transposon mobilization which is regulated by small RNA pathways in the germ line. To test this, I will take a multipronged approach, combining a candidate mutant approach with unbiased RNA sequencing to identify components involved in temperature-induced DNA damage. In Aim 1, I will complete my candidate mutant screen, monitoring temperature-induced DNA damage in small RNA pathway mutants. I will also use RNA sequencing to characterize all temperature-sensitive small RNA populations in an unbiased manner. In Aim 2, I will follow up on my finding that PRG-1, which interacts with piRNAs in the germ line to suppress transposons, is required for heat-stress induced DNA damage. I will investigate my hypothesis that PRG-1 regulates specific piRNA subclasses that mediate the production of temperature-induced DNA damage in spermatocytes with a small RNA sequencing experiment optimized for piRNA analysis. To further explore this result, I will characterize heat shock-dependent localization and interactions of PRG-1, associated piRNAs, and additional small RNA pathway components known to act downstream of PRG-1. In Aim 3, I will assess transposon mobilization upon heat-shock and characterize transposon classes involved in heat-stress induced DNA damage. I propose to combine deep sequencing and genetic approaches to explore how temperature-induced DNA damage occurs specifically in spermatocytes using the nematode C. elegans. Overall, these data will make a substantial contribution toward improving our understanding of these important biological processes that are relevant to human infertility and disease.