RNA结合蛋白hnRNPH1在精子发生过程的作用及其分子机制研究

RNA binding proteins (RBPs) participate in gene transcriptional and posttranscriptional regulation by controlling the biosynthesis, localization, stabilization, translation and degradation of RNA. Mammalian spermatogenesis follows a distinct gene transcriptional and posttranscriptional regulation program due to the existence of two phases for transcriptional arrest. Therefore, RNA binding proteins are thought to play a significant role in spermatogenesis. However, their physiological functions and molecular mechanism are still unclear. We previously found that RNA binding protein hnRNPH1 was highly expressed in mouse testis, and germ cell conditioned Hnrnph1 gene knockout male mice were sterile. Therefore, we speculated that hnRNPH1 is involved in the regulation of mouse spermatogenesis. In this project, we plan to utilize the conditioned gene knockout mouse model to analyze the male sterile phenotype, screen the interacting proteins of hnRNPH1, identify its binding RNA sequence, and detect the changes of gene transcriptome upon gene knockout by sequencing. Finally, this research will reveal the biological functions and molecular mechanism of hnRNPH1 during spermatogenesis, providing a new theoretical basis for the diagnosis and treatment of spermatogenesis disorders caused by genetic factors.

RNA结合蛋白通过调控RNA的生物合成、定位、稳定、翻译和降解等过程，进而参与基因转录和转录后调控。哺乳动物精子发生过程因出现两次转录抑制现象，其基因转录和转录后调控模式存在特殊性，因此RNA结合蛋白被认为在精子发生过程中具有重要调控作用，但其生理功能和分子机理目前尚不清楚。申请人在前期工作中发现RNA结合蛋白hnRNPH1在小鼠睾丸组织中高表达，生殖细胞条件性敲除Hnrnph1基因导致雄性小鼠不育，因此推测该蛋白参与调控小鼠精子发生过程。本课题拟利用生殖细胞条件性基因敲除Hnrnph1小鼠模型，通过分析敲除小鼠不育表型、筛选与hnRNPH1相互作用的蛋白、鉴定其结合的RNA序列，再利用高通量测序技术检测hnrnph1基因敲除后对转录组的影响，最终揭示RNA结合蛋白hnRNPH1在精子发生过程中的生物学功能和分子调控机制，为遗传学因素所致精子发生障碍的诊疗提供新的理论依据。

mRNA前体的选择性剪接对生殖细胞的发育至关重要，然而，调控选择性剪切的分子机制在生殖细胞中尚知之甚少。此研究中，我们发现RNA结合蛋白hnRNPH1在生殖系统优势表达，定位在减数分裂生精细胞的染色体上（粗线期精母细胞的性染色体除外），并可招募剪接蛋白PTBP2和SRSF3进而协同调控精子发生关键基因的选择性剪切。在生精细胞中条件性敲除Hnrnph1导致诸多减数分裂和生殖细胞-支持细胞之间通信相关基因的异常剪接事件，染色体不联会且生精细胞-支持细胞间通讯受损，最终导致雄性不育。我们进一步发现，hnRNPH1可以直接与SPO11蛋白结合并能招募剪接调控因子PTBP2和SRSF3，协同调控靶基因的选择性剪接。引人注目的是，生殖细胞中条件性敲除Hnrnph1基因的雌性小鼠也完全不育，Hnrnph1敲除的卵母细胞也表现出染色体不联会和细胞-细胞连接缺陷，这与Hnrnph1敲除的雄性生殖细胞相似。总之，该研究揭示了hnRNPH1在精子发生和卵子发生过程中调节mRNA前体选择性剪接的重要作用，证明了hnRNPH1通过招募PTBP2和SRSF3调控生殖细胞中选择性剪接事件的分子网络模型。