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

阐明小RNA途径在精子发生过程中热应激诱导的DNA损伤中的作用

• 批准号: 10222443
• 负责人: Nicole A Kurhanewicz
• 金额: $ 3.47万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-28 至 2021-03-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10222443
• 关键词: Affect; Biological Assay; Biological Models; Biological Process; Body Temperature; Caenorhabditis elegans; Cell Death; Complement; Complex; DNA Damage; DNA Repair; Data; Development; Developmental Process; Disease; Excision; Exposure to; Female; Fertility; Gametogenesis; Generations; Genetic Models; Genome; Genomics; Germ Cells; Germ Lines; Heat Stress Disorders; Heat-Shock Response; High temperature of physical object; Human; Infertility; Light; Link; Location; Maintenance; Male Infertility; Malignant Neoplasms; Mammals; Maps; Mediating; Mediator of activation protein; Meiosis; Monitor; Nature; Nematoda; Oocytes; PPBP gene; Pathway interactions; Phenotype; Play; Population; Production; Proteins; RNA; RNA Interference; Regulation; Role; Small RNA; Southern Blotting; Spermatocytes; Spermatogenesis; Spontaneous abortion; System; Temperature; Testing; Testis; Thermogenesis; Tissues; Untranslated RNA; Work; deep sequencing; differential expression; egg; experimental study; extreme temperature; follow-up; genetic approach; genome integrity; genome-wide; improved; insight; kinetosome; male; mutant; piRNA; prevent; programs; sex; sperm cell; transcriptome sequencing

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.

项目概要 在减数分裂期间，基因组的忠实遗传对于配子的成功形成是必要的。尽管 许多组织都会受到极端温度变化的影响，睾丸中发育的精子尤其受到影响 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显示出生育能力下降。研究于 哺乳动物已将高温与精母细胞 DNA 损伤的增加联系起来，然而 根本机制仍不清楚。 Libuda 实验室之前的工作发现，与哺乳动物类似， 暴露于热应激会导致 DNA 损伤，特别是在秀丽隐杆线虫精母细胞中，而不是 卵母细胞。利用线虫作为模型系统，转座子动员被认为是一种可能的方法 热诱导 DNA 损伤产生的机制。小非编码 RNA，与 相关蛋白是种系发育和维持的重要调节因子，包括调节 RNAi 和转座子活性。某些小 RNA 途径也被认为是精母细胞特异性的，并且 在温度引起的不孕症中发挥作用。因此，种系中的小 RNA 通路代表了 作为热应激诱导的精母细胞 DNA 损伤调节剂的有希望的目标。因此，我 假设热应激诱导的 DNA 损伤（特别是在精母细胞中）是由转座子引起的 动员是由种系中的小RNA途径调节的。为了测试这一点，我将采取 多管齐下的方法，将候选突变方法与无偏 RNA 测序相结合来识别 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，监测小RNA途径突变体中温度诱导的DNA损伤。我也会使用RNA 测序以公正的方式表征所有温度敏感的小 RNA 群体。在目标 2 中，我 将跟进我的发现 PRG-1，它与种系中的 piRNA 相互作用以抑制转座子， 是热应激诱导的 DNA 损伤所必需的。我将研究我的假设：PRG-1 调节特定的 piRNA 亚类介导精母细胞中温度诱导的 DNA 损伤的产生 针对 piRNA 分析优化的小 RNA 测序实验。为了进一步探索这个结果，我将 表征 PRG-1、相关 piRNA 和其他物质的热休克依赖性定位和相互作用 已知作用于 PRG-1 下游的小 RNA 通路成分。在目标 3 中，我将评估转座子 热激后的动员并表征参与热应激诱导 DNA 的转座子类别 损害。我建议结合深度测序和遗传方法来探索温度如何诱导 DNA 损伤特别发生在使用线虫秀丽隐杆线虫的精母细胞中。总体而言，这些数据将 为提高我们对这些重要生物过程的理解做出重大贡献 与人类不育和疾病有关。