CRISPR-Cas9 genetic knockout screens to identify genetic modifiers of cellular response to azacytidine

Excessive RNA damage activates cellular stress responses, triggering cell death. However, pathways that negatively regulate RNA damage responses are largely uncharacterized. Using genetic screens, we find that the ubiquitin ligase RNF25 provides tolerance to RNA damage caused by the nucleoside analogue azacytidine (aza-C), a chemotherapeutic agent used to treat acute myeloid leukemia (AML). Modifier screens reveal that the aza-C-hypersensitivity of RNF25-deficient cells is suppressed by loss of the GCN2-dependent integrated stress response. Mechanistically, we show that aza-C is incorporated into mRNA, where its pyrimidine ring spontaneously decomposes, creating a lesion that stalls elongating ribosomes. The resulting ribosome collisions are sensed by GCN1, which recruits RNF25 to ubiquitylate the ribosome. RNF25 prevents GCN1 from activating GCN2 and thereby suppresses cell death. Our study reveals an mRNA damage tolerance mechanism that determines cellular survival in response to aza-C, highlighting RNA damage as a potentially critical component of chemosensitivity in AML.