The differentiation of embryonic stem cells (ESCs) into a lineage-committed state is a dynamic process involving changes in cellular metabolism, epigenetic modifications, post-translational modifications, gene expression, and RNA processing. Here we integrated data from metabolomic, proteomic, and transcriptomic assays to characterize how alterations in NAD⁺ metabolism during the differentiation of mouse ESCs lead to alteration of the PARP1-mediated ADP-ribosylated (ADPRylated) proteome and mRNA isoform specialization. Our metabolomic analyses indicate that mESCs use distinct NAD⁺ biosynthetic pathways in different cell states: the de novo pathway in the pluripotent state, and the salvage and Preiss–Handler pathways as differentiation progresses. We observed a dramatic induction of PARP1 catalytic activity driven by enhanced nuclear NAD⁺ biosynthesis during the early stages of mESC differentiation (e.g., within 12 h of LIF removal). PARP1-modified proteins in mESCs are enriched for biological processes related to stem cell maintenance, transcriptional regulation, and RNA processing. The PARP1 substrates include core spliceosome components, such as U2AF35 and U2AF65, whose splicing functions are modulated by PARP1-mediated site-specific ADP-ribosylation. Finally, we observed that splicing is dysregulated genome-wide in Parp1 knockout mESCs. Together, these results demonstrate a role for the NAD⁺–PARP1 axis in the maintenance of mESC state, specifically in the splicing program during differentiation.

中文翻译：

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NAD+–PARP1 轴的多组学分析揭示了位点特异性 ADP-核糖基化在胚胎干细胞剪接中的作用

胚胎干细胞 (ESC) 分化为谱系定型状态是一个动态过程，涉及细胞代谢、表观遗传修饰、翻译后修饰、基因表达和 RNA 加工的变化。在这里，我们整合了来自代谢组学、蛋白质组学和转录组学分析的数据，以描述小鼠胚胎干细胞分化过程中NAD ^{+代谢的改变如何导致 PARP1 介导的 ADP 核糖基化 (ADPRylated) 蛋白质组和 mRNA 亚型特化的改变。}我们的代谢组学分析表明，mESCs 使用不同的 NAD ⁺不同细胞状态下的生物合成途径：多能状态下的从头途径，以及随着分化进展的补救和Preiss-Handler途径。我们观察到在 mESC 分化的早期阶段（例如，在去除 LIF 的 12 小时内），由增强的核 NAD ⁺生物合成驱动的 PARP1 催化活性的显着诱导。mESC 中的 PARP1 修饰蛋白丰富了与干细胞维持、转录调控和 RNA 加工相关的生物过程。PARP1 底物包括核心剪接体成分，例如 U2AF35 和 U2AF65，其剪接功能由 PARP1 介导的位点特异性 ADP-核糖基化调节。最后，我们观察到Parp1中的剪接在全基因组范围内失调敲除 mESC。^{总之，这些结果证明了 NAD +} –PARP1 轴在维持 mESC 状态中的作用，特别是在分化过程中的剪接程序中。