Engelke, M. | bioRxiv Science

bioRxiv2023

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

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.

molecular biology↗

bioRxiv2023

Autoinhibition in the Signal Transducer CIN85 Modulates B Cell Activation

Signal transduction by the ligated B cell antigen receptor (BCR) depends on the pre-organization of its intracellular components, such as the effector proteins SLP65 and CIN85 within phase-separated condensates. These liquid-like condensates are based on the interaction between three Src homology 3 (SH3) domains and corresponding proline-rich recognition motifs (PRM) in CIN85 and SLP65, respectively. However, detailed information on the protein conformation and how it impacts on the capability of SLP65/CIN85 condensates to orchestrate BCR signal transduction is still lacking. This study identifies a hitherto unknown intramolecular SH3:PRM interaction between the C-terminal SH3 domain (SH3C) of CIN85 and an adjacent PRM. We used high-resolution nuclear magnetic resonance (NMR) experiments to study the flexible linker region containing the PRM and determined the extent of the interaction in multidomain constructs of the protein. Moreover, we observed that the phosphorylation of a serine residue located in the immediate vicinity of the PRM regulates this intramolecular interaction. This allows for a dynamic modulation of CIN85s valency towards SLP65, regulating the extent of liquid-liquid phase separation. B cell culture experiments further revealed that the PRM/SH3C interaction is crucial for maintaining the physiological level of SLP65/CIN85 condensate formation, activation-induced membrane recruitment of CIN85, and subsequent mobilization of Ca2+. Our findings therefore suggest that the intramolecular interaction to the adjacent disordered linker is effective in modulating CIN85s valency both in vitro and in vivo. This therefore constitutes a powerful way for the modulation of SLP65/CIN85 condensate formation and subsequent B cell signaling processes within the cell.

biophysics↗