2’-O-methylation of the second transcribed nucleotide within mRNA 5’ cap impacts protein production level in a cell specific manner and contributes to RNA immune evasion

In higher eukaryotes, m7G-adjacent nucleotides undergo extensive modifications. Ribose of the first or first and second transcribed nucleotides can be subjected to 2’- O -methylation to form cap1 or cap2, respectively. Additionally, when the first transcribed nucleotide is adenosine, it can not only undergo 2’- O -methylation but can also be methylated at position N6 forming N6,2’- O -dimethyladenosine (m6Am). Recent studies have shed some light on the functions of cap1, showing that cap1 in mammalian cells plays a crucial role in distinguishing between ‘self’ and ‘non-self’ RNA during viral infection. Here, we attempted to understand the impact of other cap methylations on RNA-related processes. Therefore, we synthesized tetranucleotide cap analogs and used them for efficient co-transcriptional RNA capping during in vitro transcription. Using this tool, we found that 2’- O -methylation of the second transcribed nucleotide within the mRNA 5’ cap influences protein production levels in a cell-specific manner. The presence of this modification can strongly hamper protein biosynthesis or do not influence protein production levels. Interestingly, 2’- O -methylation of the second transcribed nucleotide as well as the presence of N6,2’- O -dimethyladenosine as the first transcribed nucleotide serve as determinants that define transcripts as ‘self’ and contribute to transcript escape from the host innate immune response. Additionally, cap methylation status does not influence transcript affinity towards translation initiation factor 4E or in vitro susceptibility to decapping by DCP2; however what we observe is resistance of RNA capped with cap2 to DXO-mediated decapping and degradation. Significance Statement Methylation of mRNA cap structure regulates protein biosynthesis in a cell-dependent manner. Among the three known m7G cap modifications, the 2’- O -methylation is dominant. 2’- O -methylation of the first transcribed nucleotide can boost protein production, whereas the same modification of the second transcribed nucleotide can strongly decrease translation. Interestingly, we show that in the JAWS II cell line, 2’- O -methylation of mRNA cap had a prominent impact on the composition of the protein interactome associated with the RNA bearing mentioned modifications. Further analysis revealed that 2’- O -methylation of the second transcribed nucleotide and N6-methylation of adenosine as the first transcribed nucleotide serve as determinants defining transcripts as ‘self’ and contribute to transcript escape from the host innate immune response. ### Competing Interest Statement The authors have declared no competing interest.