Impact of RNA modifications on TLR7 recognition

Steffen Kaiser¹, Stefanie Gehrig², Mariel-Esther Kaschel³, Flavia Botschen⁴, Katharina Schmidt⁵, Florian Eberle⁶, Tatjana Eigenbrod⁷, Walter M. Holmes⁸, Volker A. Erdmann⁹, Mathias Sprinzl¹⁰, Guillaume Bec¹¹, Gérard Keith¹², Alexander H. Dalpke¹³, Mark Helm¹⁴

1 Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University Mainz, Mainz, Germany; steffen.kaiser@uni-mainz.de

2 Institute of Pharmacy and Molecular Biotechnology, Department of Chemistry, Heidelberg University, Heidelberg, Germany

3 Department of Infectious Diseases - Medical Microbiology and Hygiene, Heidelberg University, Heidelberg, Germany

4 Institute of Pharmacy and Molecular Biotechnology, Department of Chemistry, Heidelberg University, Heidelberg, Germany

5 Department of Infectious Diseases - Medical Microbiology and Hygiene, Heidelberg University, Heidelberg, Germany

6 Department of Infectious Diseases - Medical Microbiology and Hygiene, Heidelberg University, Heidelberg, Germany

7 Department of Infectious Diseases - Medical Microbiology and Hygiene, Heidelberg University, Heidelberg, Germany

8 Departments of Microbiology/Immunology and Biochemistry, Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, Virginia, USA

9 Institute for Chemistry and Biochemistry, Free University Berlin, Berlin, Germany

10 Universitat Bayreuth, Fakultat fur Biologie, Chemie und Geowissenschaften, Lehrstuhl für Biochemie, D-95440 Bayreuth

11 Architecture et Réactivité de l'ARN, Universite de Strasbourg, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes F-67084 Strasbourg, France

12 Architecture et Réactivité de l'ARN, Universite de Strasbourg, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes F-67084 Strasbourg, France

13 Department of Infectious Diseases - Medical Microbiology and Hygiene, Heidelberg University, Heidelberg, Germany

14 Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University Mainz, Mainz, Germany; Institute of Pharmacy and Molecular Biotechnology, Department of Chemistry, Heidelberg University, Heidelberg, Germany

Abstract

The human innate immune system can distinguish between nucleic acids of the host and nucleic acids of invading microbial pathogens. In case of an infection, pathogen-associated molecular patterns (PAMPs) are recognized by Toll-like receptors (TLRs) of immune cells which trigger a signaling cascade leading to the expression of cytokine and interferon encoding genes.

For TLR7, which recognizes single-stranded RNA of microbial pathogens, the exact structural motif recognized remains unknown. However, it has been shown that RNA modifications play an important role in TLR recognition.

As tRNAs represent the highest modified subtype of RNA, and as they are freely accessible in non-protein-bound forms in the cytoplasm, they are promising candidates for investigations of the impact of modified nucleotides on recognition by TLR7. To quantify the TLR7-mediated immunostimulation, IFN-α produced by human peripheral blood mononuclear cells (PBMCs) after transfection with tRNA was measured by ELISA.

In general bacterial tRNAs were shown to be more immunostimulative than eukaryotic tRNAs, in line with the trend that eukaryotic tRNAs contain more and chemically more complex modifications. However, we also found a bacterial tRNA that showed only a very low immunostimulation and could trace the reason for this low stimulation to a single modified nucleotide. We further investigated the effect of this single modification at various alternative positions in the tRNA structure.

DOI^®: 10.3288/contoo.paper.1570