海洋线虫RNA选择性多聚腺苷酸化的检测及调控机制研究

Free-living marine nematodes, as one of the most abundant taxa among marine meiobenthos, play an important regulatory role in benthic ecosystems. However, there are no reports on the mechanism of regulation of gene expression in marine nematodes. Alternative Polyadenylation (APA) shows ubiquitous effect in regulation of gene expression in eukaryotes. Polyadenylation may take place at different sites on 3` end of mRNA, thereby creating a variety of mRNA isoforms, which influence mRNA localization, stability and translational efficiency. Studies have shown that APA not only plays an important regulatory role in gene expression, but also its abnormal expression is related to a variety of human diseases including cancer. Combined with Oxford Nanopore Technologies, this project intends to perform full-length transcriptome sequencing on the inbreeding line of marine nematodes of different stages, and construct a transcriptome database. APA will be identified in the transcriptome of marine nematodes, and the regulation of APA in gene expression will be analyzed to characterize the dynamic changes of APA during development. We will also perform gene-knockout experiments of APA cleavage factors using CRISPR/Cas9 technology to explore how they affect APA site selection. It is expected that this project will help to understand the regulation mechanisms of APA in gene expression, and pave the way for the study of genetic regulation network of marine nematodes and the detection and prevention of APA-related human diseases.

自由生活海洋线虫在底栖生态环境中有着重要的调节作用。然而，有关海洋线虫基因表达调控机制的研究目前尚无报道。RNA选择性多聚腺苷酸化（Alternative Polyadenylation，APA）是一种普遍存在于真核生物中的基因表达调控机制，通过调控单个基因编码多种不同的mRNA亚型，对mRNA定位、稳定性以及翻译效率等产生影响。研究表明APA不仅对基因表达谱有着重要的调控作用，而且其异常表达与包括癌症在内的多种人类疾病有关。本项目拟利用海洋线虫纯培养系，结合纳米孔测序技术，对不同生长阶段的海洋线虫纯系进行全长转录组测序分析，构建全长转录组数据库；分析APA在基因表达过程中的调控作用及动态变化规律；利用CRISPR/Cas9基因编辑技术探究不同剪切因子对APA位点选择的调控机制。预期研究成果将有助于深入了解海洋线虫的基因表达调控网络，为APA相关人类疾病的检测与防控提供参考。

自由生活海洋线虫在底栖生态环境中有着重要的调节作用。然而，有关海洋线虫基因表达调控机制的研究目前尚无报道。RNA选择性多聚腺苷酸化（Alternative Polyadenylation， APA）是一种普遍存在于真核生物中的基因表达调控机制，通过调控单个基因编码多种不同的mRNA亚型，对mRNA定位、稳定性以及翻译效率等产生影响。本项目利用海洋线虫纯培养系，结合纳米孔测序技术，对不同生长阶段的海洋线虫纯系进行全长转录组测序分析，构建了全长转录组数据库，并对转录组范围内的APA现象进行了全面筛查与统计分析。揭示了L. marina不同生长阶段的mRNA可变剪切模式以及转录本亚型变化情况；阐述了L. marina转录组中lncRNA的组成情况和动态变化规律；完成了L. marina不同发育阶段的RNA甲基化水平检测，差异甲基化位点分析；完成了对L. marina雌雄成虫样品的小RNA测序工作，通过多组学联合分析，探究了竞争性内源RNA在L. marina转录组中的调控机制。