The m5C reader Ybx1 regulates embryonic cortical neurogenesis by promoting the progenitor cell cycle progression [E18.5-cKO_RNAseq]

The reversible epitranscriptomic mark, 5-methylcytosine (m5C) modification, is implicated in numerous cellular processes, but its role in neuronal development remains largely unexplored. In this study, we discovered high expression of the m5C reader Ybx1 in the developing mouse cortex. To elucidate its role in cortical development, Ybx1 was deleted in mouse embryonic cortical neural stem cells (NSCs). Interestingly, the deletion of Ybx1 led to perinatal mortality in mice, along with abnormal cortical development. Cortical progenitor cells lacking Ybx1 exhibited impaired proliferation and differentiation. Multi-omics analysis identified the target mRNAs of Ybx1, which encode the key cell cycle regulatory proteins, such as cyclin D2 (Ccnd2). Ybx1 was found to regulate the stability of its target transcripts. Both knockdown and overexpression of Ybx1 targets via in utero electroporation confirmed that they mediated the Ybx1 regulation of proliferation and differentiation of neural precursor cells. Further mechanistic analysis showed that deletion of Ybx1 resulted in the blocking of G1 to S phase transition in the cortical progenitor cells. This study highlights the crucial function of the m5C reader protein Ybx1 in promoting cell cycle progression of the embryonic cortical progenitors, essential for proper cortical development. We perform high-throughput sequencing of mouse cortex with or without deletion of Ybx1 to profile RNA expression. For the RNA-seq experiments, we performed 3 batches of sequencing experiments in different biological replicates to account for the technical variation可逆表观转录组修饰标记5-甲基胞嘧啶（m5C）参与诸多细胞生命进程，但其在神经元发育中的作用仍未得到充分探索。本研究发现，m5C阅读蛋白（m5C reader）Ybx1在发育中的小鼠大脑皮层中高表达。为阐明其在皮层发育中的功能，我们在小鼠胚胎皮层神经干细胞（NSCs）中特异性敲除了Ybx1基因。有趣的是，Ybx1缺失会导致小鼠出现围产期致死，并伴随皮层发育异常。缺失Ybx1的皮层祖细胞表现出增殖与分化能力受损。多组学分析鉴定出Ybx1的靶mRNA，其编码包括细胞周期蛋白D2（Ccnd2）在内的关键细胞周期调控蛋白。研究证实，Ybx1可调控其靶转录本的稳定性。通过子宫内电转染技术对Ybx1靶基因进行敲低与过表达实验，结果证实这些靶基因介导了Ybx1对神经前体细胞增殖与分化的调控作用。进一步的机制分析显示，Ybx1缺失会阻断皮层祖细胞的G1到S期细胞周期转换。本研究揭示了m5C阅读蛋白Ybx1在促进胚胎皮层祖细胞细胞周期进程中的关键功能，该进程对于正常皮层发育至关重要。我们对Ybx1缺失与未缺失的小鼠皮层样本开展高通量测序以解析RNA表达谱。在RNA测序（RNA-seq）实验中，我们设置了3批独立的生物学重复测序实验，以控制技术变异带来的影响。