RNA m5C调控早期胚胎发育分子机制研究

RNA m5C methylation modification plays an important role in regulating mRNA processing, metabolism and nuclear export. Epigenetic regulatory mechanism in early embryonic development has always been the research highlight in the field of reproduction, while the function of RNA m5C and its regulatory proteins during embryonic development has not been reported yet. We found that ALYREF expressed after zygotic genome activation in mouse, the same as in human early embryo development, indicating its function during the process of RNA transport after genome activation. However, the expression patterns of RNA methyltransferase NSUN2 in human and mouse early embryos suggest NSUN2 working as a maternal factor. This result indicates that NSUN2 and RNA m5C may play important roles during zygotic genome activation, while the distribution and function of RNA m5C are still needed to be further investigated. Focusing on early embryonic development, we plan to combine the multidisciplinary techniques of embryology, biochemistry, transcriptome and low-input-cell RNA m5C single-base resolution high-throughput sequencing and bioinformatics analysis; and to reveal the distribution of RNA m5C modifications during early embryonic development, as well as the molecular mechanism by which m5C regulates early embryonic genome activation; and also to analyze the functions of NSUN2 (Writer) and ALYREF (Reader) during embryonic development. This project will broaden the research areas related to RNA modifications in embryonic development and provide a new theoretical basis for reproductive medicine.

RNA m5C在mRNA加工代谢及转运出核调控中发挥重要功能，早期胚胎发育表观遗传调控机制一直是生殖领域热点，而RNA m5C及其调控蛋白在胚胎发育过程中功能研究尚未见报道。申请人前期研究发现：人与鼠胚胎中ALYREF均在基因组激活后开始高表达，提示其在基因组激活后RNA转运过程中发挥功能；而RNA甲基转移酶NSUN2表达模式呈现其为母源因子，提示RNA m5C调控胚胎基因组激活，但其分布规律、功能和机制尚不清楚；项目拟针对胚胎发育调控，结合胚胎、转录组、少量细胞RNA m5C单碱基高通量测序和信息分析等多学科技术，揭示胚胎发育过程RNA m5C分布规律，阐明m5C调控早期胚胎基因组激活分子机制，解析NSUN2和ALYREF在胚胎发育调控中的功能，开拓胚胎发育RNA修饰研究领域，为生殖医学提供新的理论依据。

项目负责人主要针对人胚胎发育过程中RNA m5C 修饰、组蛋白修饰的调控机制以及单基因遗传病子代传递阻断问题进行研究。项目执行期间，项目负责人完成了项目既定目标，取得了原创性成果：项目组对13-28周人类胎儿组织四类器官的转录组、DNA 5hmC和RNA m5C表观基因组图谱进行绘制，首次阐明了人类胎儿器官发生不同阶段的表观遗传图谱，分别鉴定出70091个器官阶段特异性差异羟甲基化区域（DhMR）和503个mRNA m5C修饰，发现关键转录因子（TFs）、T-box转录因子20（TBX20）、配对盒蛋白pax-8（PAX-8）、krueppel样因子1（KLF1）、转录因子21（TCF21）和CCAAT增强子结合蛋白β（CEBPB）特别有助于在不同阶段形成不同的器官。此外，分析结果还发现5hmC富集的Alu元件可以参与TF靶向基因表达的调节，本研究揭示了DNA修饰和RNA甲基化之间的重要联系，并说明了人类胎儿器官发生过程中的表观遗传图谱，为理解早期发育和出生缺陷的深入表观遗传机制提供了基础。项目组创新性提出了人胚胎发育“表观基因组重启”理论，即精子、卵子受精后，人类早期胚胎清除亲本的表观遗传记忆（内存清理），重构初始化状态（加载基础架构），重建表观基因组调控细胞分化（加载高级功能模块），首次揭示人类早期发育经典组蛋白修饰重编程规律，奠定人早期发育组蛋白修饰研究基础，该研究入选2019年度中国医学重大进展；此外，项目组首次创新性构建了人孤雄胚胎，并发现可以发育至囊胚阶段这一奇特现象，进而结合孤雌胚胎解析了人亲本表观遗传记忆的转换；项目组创建了微量细胞单体型识别基因片段重复新技术，解决了“基因片段重复类遗传病”子代传递阻断难题；创建了单细胞遗传连锁技术识别真假基因技术，解决了此类遗传性疾病子代传递阻断难题。