The TRNA Half: A Strong Endogenous Toll-like Receptor 7 Ligand with a 5'-terminal Universal Sequence Signature

Toll-like receptors (TLRs) are crucial components of the innate immune system. Endosomal TLR7 recognizes single-stranded RNAs, yet its endogenous ssRNA ligands are not fully understood. We previously showed that extracellular (ex-) 5'-half molecules of tRNA (the 5'-tRNA half) in extracellular vesicles (EVs) of human macrophages activate TLR7 when delivered into endosomes of recipient macrophages. Here, we fully explored immunostimulatory ex-5'-tRNA half molecules and identified the 5'-tRNA half, the most abundant tRNA-derived RNA in macrophage EVs, as another 5'-tRNA half molecule with strong TLR7 activation capacity. Levels of the ex-5'-tRNA half were highly up-regulated in macrophage EVs upon exposure to lipopolysaccharide and in the plasma of patients infected with . The 5'-tRNA half-mediated activation of TLR7 effectively eradicated bacteria infected in macrophages. Mutation analyses of the 5'-tRNA half identified the terminal GUUU sequence as a determinant for TLR7 activation. We confirmed that GUUU is the optimal ratio of guanosine and uridine for TLR7 activation; microRNAs or other RNAs with the terminal GUUU motif can indeed stimulate TLR7, establishing the motif as a universal signature for TLR7 activation. These results advance our understanding of endogenous ssRNA ligands of TLR7 and offer insights into diverse TLR7-involved pathologies and their therapeutic strategies.