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: 9794650
• 负责人: Nicole A Kurhanewicz
• 金额: $ 6.37万
• 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/9794650
• 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 许多组织会受到极端温度变化的影响，睾丸中发育中的精子尤其会受到影响。 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 低于核心体温睾丸暴露在高温下会降低生育能力。研究 哺乳动物已经将温度升高与精母细胞DNA损伤的增加联系起来，然而， 潜在的机制仍然未知。之前的研究发现，与哺乳动物类似， 暴露于热应激产生DNA损伤，特别是在秀丽隐杆线虫精母细胞中， 卵母细胞利用C.作为一个模式系统，转座子动员被确定为一个可能的 热诱导DNA损伤的产生机制。小的非编码RNA，与 相关蛋白，是生殖系发育和维持的关键调节因子，包括调节 RNA干扰和转座子活性。还已知某些小RNA途径是精母细胞特异性的， 在温度引起的不孕症中发挥作用。因此，生殖细胞系中的小RNA途径代表了一种 作为调节热应激诱导的精母细胞DNA损伤的有前途的目标。所以我 推测热应激引起精母细胞DNA损伤是转座子引起的 在生殖细胞系中由小RNA途径调节的动员。为了验证这一点，我将采取一个 多管齐下的方法，将候选突变体方法与无偏RNA测序相结合， 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。我也会用RNA 测序以无偏的方式表征所有温度敏感的小RNA群体。在目标2中，我 将继续我的发现，PRG-1，它与生殖系中的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 在精母细胞中介导温度诱导的DNA损伤产生的皮尔纳亚类， 针对皮尔纳分析优化的小RNA测序实验。为了进一步研究这个结果，我将 表征PRG-1、相关的piRNA和另外的piRNA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途径组分。在目标3中，我将评估转座子 热休克后的动员和表征热应激诱导的DNA中涉及的转座子类别 损害我建议将联合收割机和遗传学方法结合起来，探索温度诱导的 利用线虫C.优美的总的来说，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重大贡献 与人类不育和疾病有关的基因。