RNase H1介导的R-loop清除在卵子成熟和母源-合子过渡过程中的生理功能及病理影响

R-loop是细胞内一种三链核酸结构，包括互补杂交的DNA:RNA杂合链和一条被RNA替代而游离出来的单链DNA。R-loop伴随着转录活动产生，对基因组稳定性和基因表达都有广泛影响。但R-loop在卵泡发育和母源合子过渡中的功能、与胚胎发育潜能的关系还缺乏研究。本项目拟利用特异性识别R-loop的S9.6单克隆抗体结合微量测序技术，示踪R-loop在卵母细胞和受精卵基因组上的分布规律。通过Rnaseh1的卵母细胞特异性敲除调控R-loop含量，检测R-loop对卵母细胞减数分裂和早期胚胎发育过程中基因组稳定性和ZGA的影响。利用母源性不育小鼠模型和辅助生殖中发育阻滞的受精卵，研究R-loop的累积是否是造成早期胚胎发育阻滞的重要原因；通过实验手段清除阻滞胚胎中的R-loop能否挽救其发育命运。预期结果不仅能揭示R-loop在雌性生殖调控中的功能，也为提高辅助生殖成功率提供潜在理论支持。

R-loops are transient genomic structures that form during transcription when the nascent RNA anneals with the template DNA strand, thus displacing the non-template DNA strand. R-loops play multiple physiological roles in cellular processes, but often, they represent nonscheduled co-transcriptional structures with a negative impact on transcription, replication, and DNA repair. Accumulating evidence suggests that they constitute a source of replication stress, DNA breaks and genome instability. Cells avoid R-loop accumulation by either preventing R-loop formation or directly removing the DNA:RNA hybrids through RNase H1-coupled mechanisms. Given the R-loop impact on chromatin and genome organization and its potential relation with genetic diseases, we hypothesized that the RNase H1-involved R-loop homeostasis plays essential physiological and pathological roles during oocyte development and maternal-to-zygotic transition (MZT). However, the potential role of R-loops in oocyte homeostasis, follicle development, and MZT have not been reported. Furthermore, the impact of R-loop accumulation on the developmental potential of human preimplantation embryos in assisted reproduction was unclear..In this investigation, we aim to label and detect R-loops in developing oocytes and zygotes using S9.6, a monoclonal antibody that specifically recognizes DNA:RNA hybrids, and an exogenously expressed catalytic sites-mutated RNase H1, which binds but does not remove endogenous R-loops. Subsequently, the genome-wide localization/distribution of S9.6- or RNase H1-labeled R-loops in oocytes and zygotes will be determined using ultra-low input nucleic acid sequencing approaches. The levels of R-loop in oocytes can be upregulated or downregulated by oocyte-specific knockout of Rnaseh1 gene or overexpression of Rnaseh1 in oocytes, respectively. Using these approaches, the impacts of R-loop on genome stability, maternal mRNA accumulation, and zygotic genome activation will be evaluated. We will also determine that if the abnormal R-loop accumulation contributes to early embryo developmental arrest, and if the phenotypes of developmental arrest can be rescued by experimentally removing R-loops in the blastomeres. The expected results will not only reveal functions of R-loop in the regulation of female reproduction, but also provide potential guidance to improve the rate of success in assisted reproduction.