Differentiating stem cells must coordinate their metabolism and fate trajectories. Here, we report that the catalytic activity of the glycolytic enzyme Enolase 1 (ENO1) is directly regulated by RNAs leading to metabolic rewiring in mouse embryonic stem cells (mESCs). We identify RNA ligands that specifically inhibit ENO1’s enzymatic activity in vitro and diminish glycolysis in cultured human cells and mESCs. Pharmacological inhibition or RNAi-mediated depletion of the protein deacetylase SIRT2 increases ENO1’s acetylation and enhances its RNA binding. Similarly, induction of mESC differentiation leads to increased ENO1 acetylation, enhanced RNA binding, and inhibition of glycolysis. Stem cells expressing mutant forms of ENO1 that escape or hyper-activate this regulation display impaired germ layer differentiation. Our findings uncover acetylation-driven riboregulation of ENO1 as a physiological mechanism of glycolytic control and of the regulation of stem cell differentiation. Riboregulation may represent a more widespread principle of biological control.

分化的干细胞必须协调它们的新陈代谢和命运轨迹。在这里，我们报告糖酵解酶烯醇化酶 1 (ENO1) 的催化活性受 RNA 直接调节，导致小鼠胚胎干细胞 (mESCs) 中的代谢重新布线。我们鉴定了在体外特异性抑制 ENO1 酶活性的 RNA 配体并减少培养的人类细胞和 mESCs 中的糖酵解。蛋白质去乙酰化酶 SIRT2 的药理抑制或 RNAi 介导的消耗增加了 ENO1 的乙酰化并增强了其 RNA 结合。同样，诱导 mESC 分化导致 ENO1 乙酰化增加、RNA 结合增强和糖酵解抑制。表达 ENO1 突变形式的干细胞逃脱或过度激活该调节显示受损的胚层分化。我们的研究结果揭示了 ENO1 的乙酰化驱动核糖调节作为糖酵解控制和干细胞分化调节的生理机制。核糖调节可能代表更广泛的生物控制原理。