Structural and functional study of human SAMD9, rabbit SAMD9, myxoma virus protein M62/M63, and SARS-CoV-2-N protein

As a defense strategy against viruses or competitors, some microbes use anticodon nucleases (ACNases) to deplete essential tRNAs, effectively halting global protein synthesis. However, this mechanism has not been observed in multicellular eukaryotes. Here, we report that human SAMD9 is an ACNase that specifically cleaves phenylalanine tRNA (tRNAᴾʰᵉ), resulting in codon-specific ribosomal pausing and stress signaling. While SAMD9 ACNase activity is normally latent in cells, it can be activated by poxvirus infection or rendered constitutively active by SAMD9 mutations associated with various human disorders, revealing tRNAᴾʰᵉ depletion as an antiviral mechanism and a pathogenic condition in SAMD9 disorders. We identified the N-terminal effector domain of SAMD9 as the ACNase, with substrate specificity primarily determined by a eukaryotic tRNAᴾʰᵉ–specific 2’-O-methylation at the wobble position, making virtually all eukaryotic tRNAᴾʰᵉ susceptible to SAMD9 cleavage. Notably, the structure and substrate specificity of SAMD9 ACNase differ from known microbial ACNases, suggesting convergent evolution of a common immune defense strategy targeting tRNAs.