Role of RNA modifications in immune regulation
Year: 2024
Type: dissertation
Abstract: RNA recognition plays an important role in the host defence against invading pathogens. Endosomal Toll-like receptors TLR7 and TLR8 recognize viral and bacterial single stranded RNA (ssRNA). To prevent autoinflammation, posttranscriptional RNA modifications represent a decisive feature in the discrimination of endogenous from foreign nucleic acids. The naturally occurring 2’-O-ribose methylation was identified to inhibit TLR activation even in the presence of otherwise stimulatory RNA, with the modified nucleobase and the sequence context being important. The present work aimed to further elucidate the impact of RNA modifi¬cations with particular consideration of 2’-O-ribose methylations on innate immune recognition. Initially, physiological relevance of RNA recognition was demonstrated identifying different bacteria that were dependent on TLR8 recognition in human monocytes using a TLR8 knockout model. Strikingly, with Haemophilus influenzae even a gram-negative bacterium showed strong TLR8 dependency. Further analysis revealed, that missing TLR8 recognition affects a broad portion of the innate immune response to bacteria. Since it is known that certain bacteria such as Escherichia coli (E. coli) express the tRNA 2’-O-methyltransferase TrmH that incorporates the immune-inhibitory ribose methylation of guanosine at position 18 (Gm18) into tRNA, it was further hypothesized that this enzyme could be upregulated upon exposure to stress in order to decrease immunostimulatory properties as a possible immune evasion mechanism. This could be confirmed for E. coli DH5α and clinical strains as different stress conditions significantly increased Gm18 levels correlating with decreased immunostimulatory potential of tRNA. Furthermore, two additional E. coli tRNA 2’ O-methyltransferases TrmJ and TrmL were analysed for their so far unknown impact on the immune modulatory properties of RNA using single, double and triple knockout strains in combination with the well-studied TrmH. RiboMethSeq analysis revealed methylation sites dependent on these enzymes but TrmJ- and TrmL-dependent sites were significantly less than TrmH-dependent sites and involved other modified nucleobases resulting in no significant phenotypic differences of trmJ-and trmL-deficient strains regarding the immunostimulatory potential of RNA. Of note, Am32 was newly identified to be dependent on TrmJ. To further evaluate the downstream effects of 2’-O-ribose methylations on TLR activation beyond cytokine secretion, antigen presentation ability of innate cells was addressed. It was found that 2’-O-methylation affects the recall response of antigen-specific memory T cells upon antigen presentation by innate immune cells. Additionally, long-term priming of bone marrow-derived macrophages was abolished by 2’-O-methyl modifications demonstrating far-reaching impacts of this RNA modification. Finally, the antagonistic interactions of RNA modifications with the TLR7 and TLR8 receptor were addressed in a structure-activity relationship study. It was found that the 2’-O-ribose substituent can be varied but only to a limited extent. More extensive alkylation abolished the immune inhibitory effect. Moreover, the size of the hydrophobic surface of the modified nucleobase was observed to especially affect the antagonistic activity on TLR8. Of note, new immunomodulatory derivatives were identified which elicit varying degrees of antagonistic activity such as the 2’-O-methyl-5-bromouridine modification which was identified to suppress TLR8 activation but not TLR7. In summary, this work investigated the relevance of 2’-O-ribose methylations for innate immune RNA recognition.
Author: Grober, Antonia
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7134026080