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RNA modifications are a well-recognized way of gene expression regulation at the post-transcriptional level. Despite the importance of this level of regulation, current knowledge on modulation of tRNA modification status in response to stress conditions is far from being complete. While it is widely accepted that tRNA modifications are rather dynamic, such variations are mostly assessed in terms of total tRNA, with only a few instances where changes could be traced to single isoacceptor species. Using Escherichia coli as a model system, we explored stress-induced modulation of 2′-O-methylations in tRNAs by RiboMethSeq. This analysis and orthogonal analytical measurements by LC-MS show substantial, but not uniform, increase of the Gm18 level in selected tRNAs under mild bacteriostatic antibiotic stress, while other Nm modifications remain relatively constant. The absence of Gm18 modification in tRNAs leads to moderate alterations in E. coli mRNA transcriptome, but does not affect polysomal association of mRNAs. Interestingly, the subset of motility/chemiotaxis genes is significantly overexpressed in ΔTrmH mutant, this corroborates with increased swarming motility of the mutant strain. The stress-induced increase of tRNA Gm18 level, in turn, reduced immunostimulation properties of bacterial tRNAs, which is concordant with the previous observation that Gm18 is a suppressor of Toll-like receptor 7 (TLR7)-mediated interferon release. This documents an effect of stress induced modulation of tRNA modification that acts outside protein translation.

The abstract is available here: https://uscholar.univie.ac.at/o:1144837

Self/foreign discrimination by the innate immune system depends on receptors that identify molecular patterns as associated to pathogens. Among others, this group includes endosomal Toll-like receptors, among which Toll-like receptors (TLR) 3, 7, 8, and 13 recognize and discriminate mammalian from microbial, potentially pathogen-associated, RNA. One of the discriminatory principles is the recognition of endogenous RNA modifications. Previous work has identified a couple of RNA modifications that impede activation of TLR signaling when incorporated in synthetic RNA molecules. Of note, work that is more recent has now shown that RNA modifications in their naturally occurring context can have immune-modulatory functions: Gm, a naturally occurring ribose-methylation within tRNA resulted in a lack of TLR7 stimulation and within a defined sequence context acted as antagonist. Additional RNA modifications with immune-modulatory functions have now been identified and recent work also indicates that RNA modifications within the context of whole prokaryotic or eukaryotic cells are indeed used for immune-modulation. This review will discuss new findings and developments in the field of immune-modulatory RNA modifications.

Posttranscriptional RNA modifications are an important feature of self/non-self discrimination of nucleic acids by the innate immune system. Ribose 2’-O-methylation within RNA has been shown to limit recognition by Toll-like receptors (TLR) 7 and TLR8. In the natural RNA context, 2’-O-methylation of guanosine at position 18 (Gm18) within transfer RNAs (tRNAs) was identified to abolish RNA induced immune activation. However, the exact requirement of Gm18 within tRNA for affecting TLR responses remained unknown. Moreover, whether Gm18 plays a role as immune modulatory RNA modification in physiological settings or has different roles in pro- vs. eukaryotes was unexplored. Finally, preliminary data suggested the existence of further RNA modifications that affect immune recognition. This thesis therefore aimed to explore the function of Gm18 for immune regulation in more detail and to study further RNA modifications for immune regulatory properties. In a combined chemical-immunological approach on human tRNALys3 which showed no TLR7 activation but lacked Gm18, this work identified a new immune-silencing modification: Tm, a double-methylation of uridine, at position 54 of human tRNAs was sufficient to decrease immune stimulation of TLR7. However, in contrast to Gm18, the effects were not dominant negative in co-stimulation experiments. Thus, for the first time an immune silencing modification in a natural RNA context has been deciphered. To further define the inhibition of TLR7 and TLR8 activation by Gm18 modified RNA, a systematic variation of nucleotides within a conserved tRNA sequence was performed. The studies allowed identification of an optimized sequence motif antagonizing TLR7/8 responses. The minimal, naturally occurring GmGC tri-nucleotide motif within a 9-mer oligoribonucleotide was identified as most efficient to antagonize recognition of otherwise immune stimulatory RNA. The findings could be beneficial to design immune inhibitory oligoribonucleotides as therapeutic approach in autoimmune diseases associated with exaggerated TLR7/8 activation. In further series of experiments mutants lacking Gm18 modification in either bacterial or eukaryotic tRNA were used to study the functional impact on overall immune stimulation. tRNA from an Escherichia coli (E.coli) mutant lacking the enzyme trmH that incorporates Gm18 showed increased immune stimulation. However, stimulation of human peripheral blood mononuclear cells (PBMCs) and TLR8 deficient genetically engineered monocyte/macrophage–like BlaER1 cells, with whole E.coli wild-type and trmH deficient bacteria revealed RNA dependent recognition of E. coli but negligible effects of Gm18. Interestingly, results indicate that trmH mediated Gm18 modification might be subject to regulation under stress conditions. Similarly, CRISPR/Cas9 generated knockouts of TARBP1, the Gm18 methyltransferase in human cells, showed slightly increased immunostimulation for tRNA fractions but no change for whole RNA stimulation. In summary, RNA 2’-O-methyl modification within tRNA is capable to limit immune recognition by TLR7/8, yet for manipulation of the overall immune recognition of bacterial or eukaryotic RNA the identified modifications alone seem to be insufficient.