mRNA N1-2'O-methylation by CMTR1 affects NVL2 mRNA splicing

Wolter, S.; Henning, T.; Wallerath, C.; Schlee-Guimaraes, T. M.; Kirchhoff, A.; Urban, C.; Piras, A.; Stukalov, A.; Juranek, S.; Engelke, M.; Boehm, V.; Gehring, N. H.; Hartmann, G.; Pichlmair, A.; Friedel, C. C.; Doelken, L.; Schlee, M.

doi:10.1101/2023.09.28.560047

bioRxiv · 10.1101/2023.09.28.560047

mRNA N1-2'O-methylation by CMTR1 affects NVL2 mRNA splicing

Wolter, S.·Henning, T.·Wallerath, C.·Schlee-Guimaraes, T. M.·Kirchhoff, A.·Urban, C.·Piras, A.·Stukalov, A.·Juranek, S.·Engelke, M.·Boehm, V.·Gehring, N. H.·Hartmann, G.·Pichlmair, A.·Friedel, C. C.·Doelken, L.·Schlee, M.

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Abstract

Cap0-mRNA is characterized by a 5-5triphosphate-linked N7-methylated guanosine(m7G). In higher eukaryotes, the methyltransferase CMTR1 additionally methylates the 2O-position of the penultimate mRNA nucleotide(N1) ribose (cap1-mRNA). While the m7G cap is essential for mRNA export and translation initiation by the eIF4F complex, the N1-2O-methylation prevents recognition of cap1-mRNA by the antiviral RNA receptors RIG-I and IFIT1, but a function beyond immunotolerance remained elusive. 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 N1-2O-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), 5TOP-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 N1-2O-methylation by CMTR1 has broad effects on cellular physiology and controls splicing of NVL2.

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Wolter, S., Henning, T., Wallerath, C., Schlee-Guimaraes, T. M., Kirchhoff, A., Urban, C., Piras, A., Stukalov, A., Juranek, S., Engelke, M., Boehm, V., Gehring, N. H., Hartmann, G., Pichlmair, A., Friedel, C. C., Doelken, L., Schlee, M.. 2023-09-29. mRNA N1-2'O-methylation by CMTR1 affects NVL2 mRNA splicing. https://doi.org/10.1101/2023.09.28.560047

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