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

Abstract

In mammals, 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. When the first transcribed nucleotide is 2′-O-methylated adenosine, it can be additionally modified to N6,2′-O-dimethyladenosine (m6Am). Recently, the crucial role of cap1 in distinguishing between ‘self’ and ‘non-self’ in mammalian cells during viral infection was revealed. Here, we attempted to understand the impact of cap methylations on RNA-related processes. Therefore, we synthesized tetranucleotide cap analogues and used them for 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. This modification can strongly hamper protein biosynthesis or have no influence on protein production levels, depending on the cell line. Interestingly, 2′-O-methylation of the second transcribed nucleotide and the presence of m6Am 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 eIF4E or in vitro susceptibility to decapping by DCP2; however, we observe the resistance of cap2-RNA to DXO (decapping exoribonuclease)-mediated decapping and degradation.

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

“…6D). Thus, the precursor ion m/z 305.086 can be predicted as a methylinosine analog with a ribosyl methylation, which commonly locates at the 2’-OH position of ribose (Ayadi et al, 2019; Drazkowska et al, 2022). The synthetic reference 1-methylinosine compound with methylation at the N1 position of the purine base was detected at a similar retention time to the predicted methylinosine analog (Fig.…”

“…6D). Thus, the precursor ion m/z 305.086 can be predicted as a methylinosine analog with a ribosyl Page11 methylation, which commonly locates at the 2'-OH position of ribose (Ayadi et al, 2019;Drazkowska et al, 2022).…”

Chemical Modifications of mRNA Ends for Therapeutic Applications

“…32 Interestingly, the trinucleotide-based approach did not require methylation of the m 7 G ribose to prevent reverse incorporation thanks to an additional base pair between adenosine of the trinucleotide primer and thymidine at the −1 position of the template. 31 A few years ago, several trinucleotide cap analogs became commercially available as CleanCap reagents, 33 and a modified variant of a trinucleotide cap, m 2 7,3 ′ -O GpppA m pG (Figure 3B), has been used for the production of the Comirnaty vaccine. 5 Recently, we reevaluated the trinucleotide analogs of m 7 GpppA*pG and expanded this set with m 7 GpppNpG trinucleotides containing nucleobases other than adenosine (C, G, and U).…”

“…In a follow-up study, we showed that tetranucleotide cap analogs m 7 GpppA m pG m pG are also efficient IVT primers (ca. 90% capping using ϕ6.5 promoter) and provide direct access to mRNAs with cap-2 structures . We then applied a similar approach to incorporate noncanonical caps including NAD, FAD, and UDP-sugars …”

“…Although some GTP analogs are tolerated by the vaccinia enzyme as substrates for GMP transfer, the co-transcriptional capping with trinucleotides offers a more general platform for incorporating modifications. Those include natural methylations of 5′-terminal nucleotides (cap-1, cap-2, m 6 A m ), ,, noncanonical caps, synthetic modulators of translational properties, , and functional groups for molecular labeling. − …”