Human SAMD9 is a Poxvirus-Activatable Anticodon Nuclease Inhibiting Codon-Specific Protein Synthesis

As a defense strategy against viruses or competitors, some microbes employ 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. Ribo-seq and RNA-seq of uninfected HeLa cells as well as HeLa cells infected with vK1-C7- and WT VACV. HeLa cells in 2x150 mM dishes were left uninfected or infected with vK1-C7- or WT VACV WR in the absence or presence of AraC (50 ug/ml final) for 8 hours. The cells were treated with cycloheximide (0.1 mg/ml) for 1 min at 37oC. The cells were lysed with 800 ul mammalian lysis buffer. 100 ul of lysate was used for extracting total RNA, while 200 ul of lysate was used for ribosome foot printing.