Impact of eIF2a phosphorylation on the translational landscape of mouse embryonic stem cells [RNA-Seq]

The integrated stress response (ISR) is critical for cell survival under stress. In response to diverse environmental cues, eIF2abecomes phosphorylated, engendering a dramatic change in mRNA translation. The activation of ISR plays a pivotal role in the early embryogenesis but the eIF2-dependent translational landscape in pluripotent embryonic stem cells (ESC) is largely unexplored. We employ a multi-omics approachconsisting of ribosome profiling, proteomics, and metabolomics inwild-type(eIF2a+/+)andphosphorylation-deficient mutant eIF2a(eIF2aA/A)mouse ESCs (mESCs) to investigate phosphorylated (p)-eIF2a-dependent translational control of naïve pluripotency. We show a transient increase in p-eIF2a in the naïve epiblast layer of E4.5 embryos. Absence of eIF2a phosphorylation engenders an exit from naive pluripotency following 2i (two chemical inhibitors of MEK1/2 and GSK3a/ß) withdrawal. p-eIF2a controls translation of mRNAs encoding proteins which govern pluripotency, chromatin organization, and glutathione synthesis.Thus, p-eIF2aacts as a key regulator of the naïve pluripotency gene regulatory network. Overall design: To delineate the mechanisms by which phosphorylation of eIF2a controls the naïve state of pluripotency, we performed ribosome profiling (Ribo-Seq) and RNA-Seq on eIF2aA/A (KI) and eIF2a+/+ (WT) mouse embryonic stem cells (mESCs) grown in LIF/2i and LIF conditions