2′-O-methylation within prokaryotic and eukaryotic tRNA inhibits innate immune activation by endosomal Toll-like receptors but does not affect recognition of whole organisms

Abstract

Bacterial RNA has emerged as an important activator of innate immune responses by stimulating Toll-like receptors TLR7 and TLR8 in humans. Guanosine 2 ′ ′ ′ ′ ′-O-methylation at position 18 (Gm18) in bacterial tRNA was shown to antagonize tRNAinduced TLR7/8 activation, suggesting a potential role of Gm18 as an immune escape mechanism. This modification also occurs in eukaryotic tRNA, yet a physiological immune function remained to be tested. We therefore set out to investigate the immune modulatory role of Gm18 in both prokaryotic and eukaryotic microorganisms, Escherichia coli and Saccharomyces cerevisiae, and in human cells. Using RiboMethSeq analysis we show that mutation of trmH in E. coli, trm3 in S. cereviase, and CRISPR/Cas9-induced knockout of TARBP1 in H. sapiens results in loss of Gm18 within tRNA. Lack of Gm18 across the kingdoms resulted in increased immunostimulation of peripheral blood mononuclear cells when activated by tRNA preparations. In E. coli, lack of 2 ′ ′ ′ ′ ′-O-methyltransferase trmH also enhanced immune stimulatory properties by whole cellular RNA. In contrast, lack of Gm18 in yeasts and human cells did not affect immunostimulation by whole RNA preparations. When using live E. coli bacteria, lack of trmH did not affect overall immune stimulation although we detected a defined TLR8/RNA-dependent gene expression signature upon E. coli infection. Together, these results demonstrate that Gm18 is a global immune inhibitory tRNA modification across the kingdoms and contributes to tRNA recognition by innate immune cells, but as an individual modification has insufficient potency to modulate recognition of the investigated microorganisms.

Holistic Optimization of Bioinformatic Analysis Pipeline for Detection and Quantification of 2′-O-Methylations in RNA by RiboMethSeq

“…Recently, we published a high-throughput deep sequencingbased approach, named RiboMethSeq, for mapping of 2′-Omethylations in highly abundant RNAs, mostly in rRNA (Marchand et al, 2016;Erales et al, 2017), with possible extension to tRNA (Marchand et al, 2017a;Freund et al, 2019). This protocol is also suitable for low abundance RNAs (Krogh et al, 2017).…”

Bacterial tRNA 2′-O-methylation is dynamically regulated under stress conditions and modulates innate immune response

“…A logical next step, namely to address these findings in vivo , currently suffers from the lack of a suitable in vivo infection model. Given that the RNA/TLR7 axis is the only one in a large arsenal of PAMP/PRR systems ( 43 , 81 ) in the innate immune system, it stands to reason, that the contribution of tRNA to immunostimulation by living bacteria ( 46 , 82–84 ) might constitute an effect that is difficult to tease out in vivo . Clearly, while an assessment of the overall impact of bacterial Gm18 modulation under stress on infection remains to be characterized, we have here provide a causal chain along with a molecular mechanism that links exposure to antibiotics to a reduction of interferon emission by cells of the human innate immune system.…”

“…Next to spatial restrictions and sequence positions in nucleic acids, ribose-methylated guanosine in tRNA (Gm18) was among the first RNA modifications to be identified as crucial in the distinction between ‘self’ and ‘non-self’ RNA molecules by the innate immune system ( 44 ). In vitro studies showed that incorporation of a 2′-O-methylation in synthetic RNA transcripts, siRNA ( 45 ) or 18S rRNA prevents TLR activation ( 40 , 46 ). Given that mammalian tRNA and rRNA, which together amount to over 90% of cellular RNA mass, contain high amounts of ribose-methylated nucleosides, the current hypothesis views ribose methylation, in particular Gm, as a positive identifier of ‘self’ i.e.…”

The tRNA‐associated dysregulation in immune responses and immune diseases

“…[30][31][32] Of these, pathogen tRNAs perform a vital function 33,34 (Figure 2). In recent years, a variety of ncR-NAs have been reported to be involved in this physiological process.…”

“…In recent years, a variety of ncR-NAs have been reported to be involved in this physiological process. [30][31][32] Of these, pathogen tRNAs perform a vital function 33,34 (Figure 2).…”