Laboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.
Graduate School of Frontier Sciences, The University of Tokyo, Tokyo 108-8639, Japan.
J Reprod Dev. 2020 Oct 13;66(5):459-467. doi: 10.1262/jrd.2020-060. Epub 2020 Jul 6.
PTBP1, a well-conserved RNA-binding protein, regulates cellular development by tuning posttranscriptional mRNA modification such as alternative splicing (AS) or mRNA stabilization. We previously revealed that the loss of Ptbp1 in spermatogonia causes the dysregulation of spermatogenesis, but the molecular mechanisms by which PTBP1 regulates spermatogonium homeostasis are unclear. In this study, changes of AS or transcriptome in Ptbp1-knockout (KO) germline stem cells (GSC), an in vitro model of proliferating spermatogonia, was determined by next generation sequencing. We identified more than 200 differentially expressed genes, as well as 85 genes with altered AS due to the loss of PTBP1. Surprisingly, no differentially expressed genes overlapped with different AS genes in Ptbp1-KO GSC. In addition, we observed that the mRNA expression of Nanos3, an essential gene for normal spermatogenesis, was significantly decreased in Ptbp1-KO spermatogonia. We also revealed that PTBP1 protein binds to Nanos3 mRNA in spermatogonia. Furthermore, Nanos3;Ptbp1 mice exhibited abnormal spermatogenesis, which resembled the effects of germ cell-specific Ptbp1 KO, whereas no significant abnormality was observed in mice heterozygous for either gene alone. These data implied that PTBP1 regulates alternative splicing and transcriptome in spermatogonia under different molecular pathways, and contributes spermatogenesis, at least in part, in concert with NANOS3.
PTBP1 是一种高度保守的 RNA 结合蛋白，通过调节转录后 mRNA 修饰，如选择性剪接 (AS) 或 mRNA 稳定化，来调控细胞发育。我们之前的研究表明，生精细胞中 Ptbp1 的缺失会导致精子发生失调，但 PTBP1 调节精原细胞稳态的分子机制尚不清楚。在这项研究中，通过下一代测序确定了 Ptbp1 敲除 (KO) 生殖干细胞 (GSC)，即增殖性精原细胞的体外模型中的 AS 或转录组的变化。我们鉴定出了 200 多个差异表达基因，以及 85 个由于 PTBP1 缺失而改变 AS 的基因。令人惊讶的是，Ptbp1-KO GSC 中没有差异表达基因与不同 AS 基因重叠。此外，我们观察到 Nanos3 的 mRNA 表达，一种正常精子发生所必需的基因，在 Ptbp1-KO 精原细胞中显著降低。我们还发现 PTBP1 蛋白在精原细胞中与 Nanos3 mRNA 结合。此外，Nanos3;Ptbp1 小鼠表现出异常的精子发生，这类似于生殖细胞特异性 Ptbp1 KO 的影响，而在杂合子小鼠中单独敲除任一基因都没有观察到明显的异常。这些数据表明，PTBP1 通过不同的分子途径调节精原细胞中的选择性剪接和转录组，并与 NANOS3 一起至少部分贡献于精子发生。
