Double methylation of tRNA-U54 to 2′-O-methylthymidine (Tm) synergistically decreases immune response by Toll-like receptor 7

Sensing of nucleic acids for molecular discrimination between self and non-self is a challenging task for the innate immune system. RNA acts as a potent stimulus for pattern recognition receptors including in particular human Toll-like receptor 7 (TLR7). Certain RNA modifications limit potentially harmful self-recognition of endogenous RNA. Previous studies had identified the 2′-O-methylation of guanosine 18 (Gm18) within tRNAs as an antagonist of TLR7 leading to an impaired immune response. However, human tRNA(Lys)(3) was non-stimulatory despite lacking Gm18. To identify the underlying molecular principle, interferon responses of human peripheral blood mononuclear cells to differentially modified tRNA(Lys)(3) were determined. The investigation of synthetic modivariants allowed attributing a significant part of the immunosilencing effect to the 2′-O-methylthymidine (m(5)Um) modification at position 54. The effect was contingent upon the synergistic presence of both methyl groups at positions C5 and 2’O, as shown by the fact that neither Um54 nor m(5)U54 produced any effect alone. Testing permutations of the nucleobase at ribose-methylated position 54 suggested that the extent of silencing and antagonism of the TLR7 response was governed by hydrogen patterns and lipophilic interactions of the nucleobase. The results identify a new immune-modulatory endogenous RNA modification that limits TLR7 activation by RNA.