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

Abstract

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.

M7G-related molecular subtypes can predict the prognosis and correlate with immunotherapy and chemotherapy responses in bladder cancer patients

“…Furthermore, these pathways could also influence the regulation of immune cells. Recently, a study reported that the cap structure of m7G can induce immune evasion by enhancing the identity of RNA as a ‘self-molecule’ for cellular recognition factors [ 32 ]. This finding might explain why the cluster was mainly involved in immune-related pathways.…”

The cap epitranscriptome: Early directions to a complex life as mRNA

“…However, in vitro transcribed mRNAs transfected into cells were differentially processed and translated in two different cell types depending on sequence and methylation status. [ 61 ] Hence, 5′UTR heterogeneity resulting from RNA Pol II promoters with multiple initiation sites can impact on translation efficiency (Figure 2) for differential regulation during development or in response to changing conditions. Here, addition of cOMe to some transcripts but not others could further increase the potential for differentially regulating gene expression, particularly in the context of local translation in neurons.…”

“…[ 60 ] Although this analysis has some bias from interference with transcription for some sequences, in vitro transcribed mRNAs transfected into cells were differentially processed and translated in two different cell types depending on sequence and methylation status. [ 61 ]…”

“…[67,68] Later it was shown that cOMe can also stimulate protein synthesis in in vitro translations and from mRNA transfected into human cells. [18,61] In Drosophila, cOMe was found to increase CBC binding and a knock-out of both CMTrs reduces protein synthesis at synapses and the ability to learn upon reward conditioning. [14] Intriguingly, the CBC prominently localises to synapses together with another nuclear complex, the exon junction complex (EJC), which has roles in synaptic protein expression.…”

Metabolomics reveals nucleoside analogs for regulating mucosal-associated invariant T cell responses

“…The copyright holder for this preprint this version posted February 2, 2023. ; https://doi.org/10.1101/2023.01.30.526332 doi: bioRxiv preprint Page11 methylation, which commonly locates at the 2'-OH position of ribose (Ayadi et al, 2019;Drazkowska et al, 2022).…”