The homeodomain transcription factors (TFs) Pax6 (OMIM: 607108) and Prox1 (OMIM: 601546) critically regulate gene expression in lens development. While PAX6 mutations in humans can cause cataract, aniridia, microphthalmia, and anophthalmia, among other defects, Prox1 deletion in mice causes severe lens abnormalities, in addition to other organ defects. Furthermore, the optimal dosage/spatiotemporal expression of these key TFs is essential for development. In lens development, Pax6 expression is elevated in cells of the anterior epithelium compared to fiber cells, while Prox1 exhibits the opposite pattern. Whether post-transcriptional regulatory mechanisms control these precise TF expression patterns is unknown. Here, we report the unprecedented finding that the cataract-linked RNA-binding protein (RBP), Celf1 (OMIM: 601074), post-transcriptionally regulates Pax6 and Prox1 protein expression in lens development. Immunostaining shows that Celf1 lens-specific conditional knockout (Celf1^cKO) mice exhibit abnormal elevation of Pax6 protein in fiber cells and abnormal Prox1 protein levels in epithelial cells—directly opposite to their normal expression patterns in development. Furthermore, RT-qPCR shows no change in Pax6 and Prox1 transcript levels in Celf1^cKO lenses, suggesting that Celf1 regulates these TFs on the translational level. Indeed, RNA-immunoprecipitation assays using Celf1 antibody indicate that Celf1 protein binds to Pax6 and Prox1 transcripts. Furthermore, reporter assays in Celf1 knockdown and Celf1-overexpression cells demonstrate that Celf1 negatively controls Pax6 and Prox1 translation via their 3′ UTRs. These data define a new mechanism of RBP-based post-transcriptional regulation that enables precise control over spatiotemporal expression of Pax6 and Prox1 in lens development, thereby uncovering a new etiological mechanism for Celf1 deficiency-based cataract.

同源域转录因子 (TF) Pax6 (OMIM: 607108) 和 Prox1 (OMIM: 601546) 在晶状体发育过程中严格调控基因表达。虽然人类的PAX6突变会导致白内障、无虹膜、小眼症和无眼症等缺陷，但Prox1除了其他器官缺陷外，小鼠中的缺失还会导致严重的晶状体异常。此外，这些关键 TF 的最佳剂量/时空表达对于开发至关重要。在晶状体发育中，与纤维细胞相比，前上皮细胞中 Pax6 的表达升高，而 Prox1 表现出相反的模式。转录后调控机制是否控制这些精确的 TF 表达模式尚不清楚。在这里，我们报告了前所未有的发现，即白内障相关的 RNA 结合蛋白 (RBP) Celf1 (OMIM: 601074) 在晶状体发育过程中转录后调节 Pax6 和 Prox1 蛋白表达。免疫染色显示 Celf1 晶状体特异性条件敲除 ( Celf1 ^cKO) 小鼠在纤维细胞中表现出异常升高的 Pax6 蛋白和在上皮细胞中异常的 Prox1 蛋白水平——与它们在发育中的正常表达模式直接相反。此外，RT-qPCR 显示Celf1 ^cKO晶状体中Pax6和Prox1转录水平没有变化，表明 Celf1 在翻译水平上调节这些 TF。事实上，使用 Celf1 抗体的 RNA 免疫沉淀分析表明 Celf1 蛋白与Pax6和Prox1结合成绩单。此外，Celf1 敲低和 Celf1 过表达细胞中的报告基因分析表明，Celf1 通过其 3' UTR 负控制 Pax6 和 Prox1 的翻译。这些数据定义了一种新的基于 RBP 的转录后调控机制，可以精确控制晶状体发育过程中 Pax6 和 Prox1 的时空表达，从而揭示 Celf1 缺陷型白内障的新病因机制。