Physiological role of 2’-O-methylation within prokaryotic and eukaryotic tRNA for innate immune activation via endosomal Toll-like receptors

Bacterial RNA has emerged as important activator of innate immune responses by stimulating the endosomal Toll-like receptors TLR7 and TLR8 in humans. Guanosine 2’-O-methylation at position 18 (Gm18) in bacterial tRNA was shown to antagonize tRNA induced TLR7/8 activation, giving rise to a potential role of this modification as immune escape mechanism. This modification also occurs in eukaryotic tRNA, yet a physiological immune function remains to be established. We therefore set out to investigate the physiological role of Gm18 in prokaryotic and eukaryotic microorganisms by using mutants deficient in the respective 2’-O-methyltransferase. In E. coli, lack of 2’-O-methyltransferase trmH enhanced immune stimulatory properties of both tRNA and whole cellular RNA. Yet, when using living microorganisms, trmH mutants did not differ from their wildtype counterparts in terms of immunostimulation although gene expression profiling demonstrated the induction of a TLR8/RNA dependent gene signature by E. coli in principle. In summary, the results demonstrate that Gm18 is a global immune inhibitory RNA modification across the kingdoms and contributes to RNA recognition by innate immune cells. Total RNA was isolated from four stimulation experiments (non-treated, LPS, WT E. coli and ΔtrmH E. coli) of transdifferntiated wild-type BlaER1 cells and four transdifferentiated ΔTLR8 BlaER1 cells. Each stimulation was performed three times (sample 1-3) in pooled triplicates.