The Influence of cap methylations on recognition by immune receptors and components of the translation machinery
Final Report Abstract
Receptors of the innate immune system recognizes conserved structures of pathogens, so called pathogen associated molecular patterns (PAMPS). Detection of PAMPS leads to induction of defense mechanisms against invading microorganisms. The innate immune response against viruses consists of receptors that recognize viral nucleic acids and produce immune activating alarm signals and effector proteins that interfere with the integrity or function of viral RNA or DNA. While endogenous rRNA and tRNA have 5’-monophosphate (5’p) termini mRNA from higher eukaryotes bear a 5’-cap structure. RNA of viruses with RNA genome differs from endogenous RNA by untypical structure, (e.g. long base paired regions), unprocessed ends (5'tri/diphosphates) or missing modifications (methylations and mRNA cap structures) usually present at endogenous RNAs. Cap0-mRNA is characterized by a 5’-5’triphosphate-linked N7-methylated guanosine(m7G). In higher eukaryotes, the methyltransferase CMTR1 additionally methylates the 2'O-position of the penultimate mRNA nucleotide(N1) ribose (cap1-mRNA). While the m7G cap is essential for mRNA export and translation, the N1-2'O-methylation prevents recognition of cap1-mRNA by the antiviral RNA receptors RIG-I and IFIT1, but a function beyond immunotolerance has remained elusive until recently. Here, we generated CMTR1-knockout(CMTR1-/-) cells and found that type-I-interferon(IFN-I) treatment resulted in IFIT1-mediated reduction of cell viability and broad mRNA translation. Consequently, stimulation of the antiviral receptor RIG-I in CMTR1-/- cells revealed an IFIT1 dependent dramatic reduction of IFN-I and chemokine protein induction, demonstrating the importance of N1-2'O-methylation for antiviral responses. Additionally, IFN-I- and IFIT1-independent effects were observed: CMTR1-/- cells were smaller, divided slower, and exhibited a reduced transcription of mRNAs coding ribosomal proteins (RP), 5`TOP-RNA and snoRNA host genes(SNHG). Additionally, proteome and transcriptome analysis revealed that expression of NVL2, an essential factor in ribosome biogenesis, is strongly suppressed by an alternative-splicing event of NVL2 mRNA in CMTR1-/- cells. This reduction could only be rescued by catalytically active CMTR1. Altogether, besides antiviral immunity N1-2'O-methylation by CMTR1 has broad effects on cellular physiology and controls splicing of NVL2. While screening for the effect of 5'RNA modifications on the recognition of several RNA receptors we discovered that 5’p-termini prevented RIG-I activation while 5’unmodified hydroxyl(OH)-dsRNA demonstrated residual activation. Determination of RIG-I/dsRNA co-crystal structures and mutant activation studies revealed that the evolutionarily conserved I875 within the C-terminal binding cleft of RIG-I sterically inhibits 5’p-dsRNA binding. Mutant RIG-I(I875A) was activated by both synthetic 5’p-dsRNA and endogenous long dsRNA within the polyA-rich fraction of total cellular RNA. RIG-I(I875A) specifically interacted with long, polyA-bearing, mitochondrial(mt) RNA, and depletion of mtRNA from total RNA abolished its activation. These studies demonstrate that avoidance of 5’p-RNA recognition is crucial to prevent mtRNA-triggered RIG-I-mediated autoinflammation.
Publications
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A conserved isoleucine in the binding pocket of RIG-I controls immune tolerance to mitochondrial RNA. Nucleic Acids Research, 51(21), 11893-11910.
de Regt, Ann Kristin; Anand, Kanchan; Ciupka, Katrin; Bender, Felix; Gatterdam, Karl; Putschli, Bastian; Fusshöller, David; Hilbig, Daniel; Kirchhoff, Alexander; Hunkler, Charlotte; Wolter, Steven; Grünewald, Agathe; Wallerath, Christina; Schuberth-Wagner, Christine; Ludwig, Janos; Paeschke, Katrin; Bartok, Eva; Hagelueken, Gregor; Hartmann, Gunther; ... & Schlee, Martin
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mRNA N1-2’O-methylation by CMTR1 affects NVL2 mRNA splicing. (2023, 9, 29). Wallstein Verlag.
Wolter, Steven; Hennig, Thomas; Wallerath, Christina; Schlee-Guimaraes, Thais M.; Kirchhoff, Alexander; Urban, Christian; Piras, Antonio; Stukalov, Alexey; Juranek, Stefan; Engelke, Michael; Boehm, Volker; Gehring, Niels H.; Hartmann, Gunther; Pichlmair, Andreas; Friedel, Caroline C.; Dölken, Lars & Schlee, Martin
