Selective targeting of type II tRNAs underlies SLFN14-mediated translational repression and its dysregulation by thrombocytopenia-linked mutations

Schlafens (SLFNs) are interferon-inducible proteins with emerging roles in RNA metabolism and antiviral defense. SLFN14 is a ribosome-associated endoribonuclease whose pathogenic variants cause an autosomal dominant form of inherited thrombocytopenia (IT), yet the molecular basis of this disorder has remained unclear. Here, we show that SLFN14 represses global protein synthesis and viral replication primarily through selective cleavage of type II tRNAs. Using cell lines engineered to express SLFN14 variants, we demonstrate that thrombocytopenia-linked mutations alter RNA substrate specificity, enhancing degradation of type II tRNAs while diminishing rRNA cleavage. This shift in substrate preference increases ribosome stalling at codons decoded by type II tRNAs, leading to global translational arrest, stress signalling, and ultimately cell death. The heightened degradation of type II tRNAs by SLFN14 mutants provides a mechanistic explanation for the translational defects and impaired megakaryocyte maturation observed in SLFN14-associated IT. Our findings suggest that type II tRNA targeting is a conserved regulatory strategy within the SLFN family and identify enhanced tRNA cleavage and the resulting impaired translation as the central pathogenic processes driven by thrombocytopenia-linked SLFN14 mutations. Overall design: Ribo-seq from HEK293T cells expressing SLFN14 variants treated with Dox at different timepoints (0 and 24 h).