The Influence of cap methylations on recognition by immune receptors and components of the translation machinery

• Applicant: Professor Dr. Martin Schlee
• Subject Area: Immunology
• Term: from 2012 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 204346300

Since viruses use the cellular biosynthesis machinery to replicate, they produce few distinctly recognizable molecules, such as sugars, lipids or proteins, which mark them as foreign. The innate immune response against viruses consists of receptors, which recognize viral nucleic acids and produce immune activating alarm signals, and effector proteins, which interfere with the integrity or function of viral RNA or DNA. Viral genomic RNA differs from endogenous RNA in its untypical structure, e.g. long base-paired regions and lack of modifications, such as methylation and cap structures usually present in endogenous RNAs. These common patterns would suggest that both RNA receptors and effector proteins recognize and attack similar RNA structures and modifications. Higher eukaryotes have a cap1 structure at the 5´-terminal end of all their mRNAs. It consists of the N7-methylated guanosine which is 5´-5´ linked via a triphosphate group (ppp) to the 5´terminal nucleotide (N1) and a 2´O-methyl group at N1. 50% of the mRNAs also have a 2´O-methylation on the adjacent nucleotide (N2). In previous studies, we were able to show that the activity of the type I-IFN-inducing cytosolic helicase RIG-I, which is essential for the immune recognition of RNA genome viruses, can be completely inhibited by the presence of 2´O-methylation of the 5´-terminal nucleotide (N1). N1-2´O methylation, a highly conserved modification of mRNA of higher eukaryotes, is used by flaviviruses to mask its own RNA from detection by RIG-I. In cells without N1-2´O-methyltransferase (MTr1-/-), we were able to detect IFN-dependent and IFN-independent effects of missing N1-2´O methylation. In MTr1 deficient cells, we observed a reduction of 5´TOP mRNA, which encode proteins involved in mRNA translation. In the continuation of the project, we will investigate the effect of cap-methylation on translational control, RNA transcription and processing, and activation of further cytosolic antiviral effector proteins. In particular, we are focusing on the translation-inhibiting effector proteins IFIT1 and PKR and the RNA degradation-inducing protein OAS1 as well as on IFN-independent mechanisms of the reduction of 5´TOP mRNAs. The central techniques that will be employed are chemical pppRNA synthesis for the preparation of stimulatory and reporter RNA, the CRISPR / Cas9 nuclease method for the deletion of genes, 4Thio-uridine (4sU) labeling and ribosome profiling techniques for the analysis of genome-wide mRNA transcription, processing and translation.

• DFG Programme: Research Grants