Identification of modifications in microbial, native tRNA that suppress immunostimulatory activity.
Naturally occurring nucleotide modifications within RNA have been proposed to be structural determinants for innate immune recognition. We tested this hypothesis in the context of native nonself-RNAs. Isolated, fully modified native bacterial transfer RNAs (tRNAs) induced significant secretion of IFN-α from human peripheral blood mononuclear cells in a manner dependent on TLR7 and plasmacytoid dendritic cells. As a notable exception, tRNA(Tyr) from Escherichia coli was not immunostimulatory, as were all tested eukaryotic tRNAs. However, the unmodified, 5'-unphosphorylated in vitro transcript of tRNA(Tyr) induced IFN-α, thus revealing posttranscriptional modifications as a factor suppressing immunostimulation. Using a molecular surgery approach based on catalytic DNA, a panel of tRNA(Tyr) variants featuring differential modification patterns was examined. Out of seven modifications present in this tRNA, 2'-O-methylated G(m)18 was identified as necessary and sufficient to suppress immunostimulation. Transplantation of this modification into the scaffold of yeast tRNA(Phe) also resulted in blocked immunostimulation. Moreover, an RNA preparation of an E. coli trmH mutant that lacks G(m)18 2'-O-methyltransferase activity was significantly more stimulatory than the wild-type sample. The experiments identify the single methyl group on the 2'-oxygen of G(m)18 as a natural modification in native tRNA that, beyond its primary structural role, has acquired a secondary function as an antagonist of TLR7.
Department of Chemistry, Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, 69117 Heidelberg, Germany.
Stefanie
S
Gehrig
Mariel-Esther
ME
Eberle
Flavia
F
Botschen
Katharina
K
Rimbach
Florian
F
Eberle
Tatjana
T
Eigenbrod
Steffen
S
Kaiser
Walter M
WM
Holmes
Volker A
VA
Erdmann
Mathias
M
Sprinzl
Guillaume
G
Bec
Gérard
G
Keith
Alexander H
AH
Dalpke
Mark
M
Helm
D017931:DNA Primers / Q000235:genetics; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007113:Immunity, Innate / Q000276:immunology*; D007114:Immunization; D016898:Interferon-alpha / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics / Q000276:immunology*; D012346:RNA, Transfer, Amino Acyl / Q000276:immunology*; D051199:Toll-Like Receptor 7 / Q000276:immunology / Q000378:metabolism; D012359:tRNA Methyltransferases / Q000378:metabolism*
D017931:DNA Primers; D016898:Interferon-alpha; D012346:RNA, Transfer, Amino Acyl; C495348:TLR7 protein, human; D051199:Toll-Like Receptor 7; D012359:tRNA Methyltransferases