By contrast, treatment of HeLa cells with sodium butyrate, an inhibitor of protein deacetylases, profoundly induced ENO1’s acetylation and RNA binding ( Figure 4A). Multiple experimental interrogations yielded no evidence for relevant ubiquitination (data not shown). What may explain the difference between the enhanced RNA binding of the ENO1up mutant in cellulo ( Figures 3E and 3F) and the normal RNA binding of the recombinant protein in vitro ( Figure S3D)? Considering that the ENO1up mutant represents a change of three lysine residues to alanine, we hypothesized that a post-translational lysine modification such as ubiquitination or acetylation in cellulo could activate ENO1’s RNA binding. Taken together, ENO1 binds RNA at numerous transcriptomic sites in human cells with two orders of magnitude difference between specific and non-specific interactions. Using NMR, we observed RNA-induced chemical shift perturbations and line broadening of ENO1 resonances in 1H, 15N-HSQC spectra, confirming direct RNA binding in vitro (shortened FTH1 ligand RNA-18-mer, Figures 1H and S1L). Highly consistent results were obtained for two additional ligand and control pairs derived from the FTH1 and PTP4A1 mRNAs, respectively ( Figures 1G, S1J, and S1K). Representative ligand and control RNAs, derived from the PABPC1 5′ UTR, were analyzed in a competition electromobility shift assay (EMSA) using recombinant human ENO1 ( Figures 1F and 1G K i(target): 27 ± 19 nM K i(control): 2,587 ± 9 nM, Figure S1I). We used these in an orthogonal assay to validate the eCLIP results and further assess the specificity of binding. Next, we synthesized RNAs of 35 nucleotides in length that either correspond to ENO1-binding regions or GC content-matched controls, derived from the same mRNAs (schematic in Figure 1E sequences are given in Table S1). Results Human Enolase 1 is a bona fide RNA-binding protein RNA-mediated inhibition of enzymatic activity-riboregulation-constitutes a physiologically relevant form of metabolic control, which plays a relevant role during stem cell differentiation. ENO1’s RNA binding is activated by acetylation, as shown by pharmacological, RNAi and cell differentiation experiments. However, when mutating ENO1 to be hyper-inhibited by RNA, stem cells are dramatically compromised in their differentiation toward the definitive endoderm, whereas RNA-binding-deficient ENO1 promotes endodermal differentiation. Under physiological conditions, the metabolic shift from glycolysis to OXPHOS during stem cell differentiation occurs concomitantly with an increase in ENO1’s RNA binding. Synthetic RNA ligands corresponding to these regions inhibit ENO1’s enzymatic activity in vitro, diminish glycolysis in HeLa cells, and specifically alter glycolytic metabolite levels and serine synthesis in pluripotent mouse embryonic stem cells (mESCs). Here, we find that human ENO1 binds hundreds of mRNAs of the cellular transcriptome via specific binding regions. Riboregulation may represent a more widespread principle of biological control. Our findings uncover acetylation-driven riboregulation of ENO1 as a physiological mechanism of glycolytic control and of the regulation of stem cell differentiation. Stem cells expressing mutant forms of ENO1 that escape or hyper-activate this regulation display impaired germ layer differentiation. Similarly, induction of mESC differentiation leads to increased ENO1 acetylation, enhanced RNA binding, and inhibition of glycolysis. Pharmacological inhibition or RNAi-mediated depletion of the protein deacetylase SIRT2 increases ENO1’s acetylation and enhances its RNA binding. We identify RNA ligands that specifically inhibit ENO1’s enzymatic activity in vitro and diminish glycolysis in cultured human cells and mESCs. 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). Differentiating stem cells must coordinate their metabolism and fate trajectories.
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