RNF25 restrains GCN2 hyperactivation to sustain protein synthesis and cell proliferation in response to RNA damage

Regulation of protein synthesis is essential for maintaining cellular homeostasis during stress. The integrated stress response (ISR) is a conserved signaling pathway that modulates global mRNA translation through four eIF2α kinases—GCN2, PKR, PERK, and HRI. However, how these kinases are selectively activated and tuned to distinct stress signals to direct appropriate cell fate decisions remains poorly understood. Here, we employ ultra-deep mutagenesis screens to systematically map regulators of protein synthesis across diverse stress perturbations in human cells. This comparative approach identifies stress-specific translational control factors, including a previously unrecognized role for the E3 ubiquitin ligase RNF25 in selectively sustaining translation following UV irradiation and other RNA-damaging treatments. In this context, we demonstrate that RNF25 operates independently of its partner RNF14, and that its ubiquitin ligase activity, as well as its RWD-domain, is required to restrain excessive activation of the eIF2α kinase GCN2. Accordingly, loss of RNF25 results in hyperactivation of GCN2, exacerbated translation shutdown, and impaired cell proliferation following RNA damage—phenotypes that can be fully reversed by genetic or pharmacological inhibition of GCN2. Together, these findings uncover a previously unappreciated RNF25–GCN2 signaling axis and identify ISR-driven toxicity as a potential vulnerability in combination with RNA-damaging chemotherapeutics.