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.

中文翻译：

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RNA结合蛋白Ptbp1调节精原细胞中的选择性剪接和转录组，并与Nanos3协同维持精子发生

PTBP1 是一种高度保守的 RNA 结合蛋白，通过调节转录后 mRNA 修饰（例如选择性剪接 (AS) 或 mRNA 稳定化）来调节细胞发育。我们之前揭示了精原细胞中 Ptbp1 的缺失导致精子发生失调，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 基因敲除 (KO) 生殖系干细胞 (GSC)（一种增殖精原细胞的体外模型）中 AS 或转录组的变化。我们鉴定了 200 多个差异表达基因，以及 85 个因 PTBP1 缺失而改变 AS 的基因。令人惊讶的是，在 Ptbp1-KO GSC 中没有差异表达的基因与不同的 AS 基因重叠。此外，我们观察到 Nanos3 的 mRNA 表达，正常精子发生的必需基因，在 Ptbp1-KO 精原细胞中显着降低。我们还发现 PTBP1 蛋白与精原细胞中的 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，而在单独对任一基因杂合的小鼠中未观察到显着异常。这些数据暗示 PTBP1 在不同分子途径下调节精原细胞中的选择性剪接和转录组，并至少部分与 NANOS3 协同促进精子发生。这类似于生殖细胞特异性 Ptbp1 KO 的影响，而在单独对任一基因杂合的小鼠中未观察到显着异常。这些数据暗示 PTBP1 在不同分子途径下调节精原细胞中的选择性剪接和转录组，并至少部分与 NANOS3 协同促进精子发生。这类似于生殖细胞特异性 Ptbp1 KO 的影响，而在单独对任一基因杂合的小鼠中未观察到显着异常。这些数据暗示 PTBP1 在不同分子途径下调节精原细胞中的选择性剪接和转录组，并至少部分与 NANOS3 协同促进精子发生。