Prokaryotic Schlafen proteins function as widespread modular antiviral defense systems in bacteria, where diverse sensor domains detect phage infection triggers such as tail assembly chaperones and activate a conserved ribonuclease effector domain that cleaves tRNA in the anticodon arm to mediate abortive infection, revealing that tRNA-targeting antiviral immunity represents an ancient mechanistically conserved strategy across all domains of life.

Human Schlafen proteins restrict viral replication by cleaving tRNA, thereby suppressing protein synthesis. Although the ribonuclease domain of Schlafen proteins is conserved across all domains of life, its function in prokaryotes has remained unclear. Here, we show that prokaryotic Schlafen nucleases (pSlfns) are widespread antiviral effectors that protect bacteria from phages. These nucleases are fused to diverse protein domains that sense phage infection. We focus on a system where Schlafen nuclease is fused to a previously unknown immunoglobulin-like sensor domain and demonstrate that it recognizes T5-like phage tail assembly chaperones and cleaves both bacterial and viral tRNA, triggering abortive infection. Our findings redefine Schlafens as an ancient, mechanistically conserved family of immune effectors, revealing the deep evolutionary origin of tRNA-targeting antiviral immunity in humans.