NXF2在piRNA介导的反转座子沉默中的机制研究

PIWI-interacting RNAs (piRNAs) are endogenous small non-coding RNAs which associate with PIWI proteins in various organisms. piRNAs are mainly expressed in germline cells and act as guardians of the genome, protecting it from invasive transposable elements (TE). Animals lacking piRNA show severe defects in gametogenesis and exhibit sterility. In Drosophila melanogaster, a growing number of studies on piRNAs have unveiled the mechanisms of piRNA-mediated TE silencing post-transcriptionally and/or transcriptionally. However, the mechanisms of piRNA biogenesis and function remain mysterious in mammals because of their complexity and lack of a convenient research system. We have established the in vitro culture system of mouse spermatogonial stem cell (SSC) and screened the candidate genes which might involved in piRNA biogenesis or function using CRISPR-Cas9. Depletion of Nuclear RNA Export Factor 2 (NXF2) shows no effect on piRNA biogenesis, but causes an up-regulation of retrotransposons, indicating a role of NXF2 in the piRNA-mediated retrotransposon silencing. Further studies on the interactions between NXF2 and piRNA/PIWI complex might reveal the mechanisms of piRNAs in maintaining genome stability during mouse spermatogenesis.

piRNA（PIWI-interacting RNA）是一类主要在生殖细胞中特异性表达的小分子非编码RNA，通过与PIWI家族蛋白质相互作用沉默基因组中的移动遗传元件，从而维持生殖细胞基因组的稳定和完整，为精子发生所必需。但目前对于哺乳动物中piRNA的作用机制知之甚少。我们建立并优化了小鼠精原干细胞的体外培养和基因组编辑技术，对可能参与piRNA功能的潜在蛋白质因子进行了筛选，结果发现敲除NXF2基因后不影响piRNA的表达，但会引起反转座子表达上调，提示其可能参与piRNA对反转座子的沉默。本项目将利用NXF2敲除的精原干细胞和小鼠模型，通过Tethering、免疫沉淀、GRO-seq和CHIP-seq等方法，结合生物信息学分析，研究NXF2与piRNA分子机器间的相互作用，揭示其在转录和转录后水平沉默反转座子的分子机制，为精子生成异常和男性不育的诊治提供理论基础。