ИСТИНА

mRNA transfection has emerged as a paradigm-shifting strategy for gene delivery, offering advantages such as minimal risk of genomic integration and independence from cell proliferation status. Synthetic mRNAs generated through in vitro transcription have applications in vaccination, replacement therapy, and cell reprogramming. However, these transcripts might activate innate immune response, which complicates transfection of primary cells and limits the application of this technology to living animals. Incorporation of modified nucleotides into the transcript can mitigate immune response, enhance mRNA stability and expression, thus optimizing translational outcomes. However, the literature presents conflicting evidence regarding the impact of nucleotide modifications on translation efficiency of different mRNAs. We investigated the effects of N1-methyl-pseudouridine (N1mΨ), a commonly used modified nucleoside in mRNA therapeutics, on the expression of reporter mRNAs that employ different translation initiation mechanisms in cultured cells and a number of cell-free systems. The efficiency and kinetics of luciferase accumulation were analyzed by time-resolved mRNA transfection and in vitro translation assays [1]. The impacts of the modification varied significantly depending on the mechanism of mRNA translation initiation. While the modification generally increased both translation efficiency and duration of expression of cap-dependent mRNAs with regular 5’ untranslated regions, it almost completely inhibited translation directed by internal ribosome entry sites (IRESs), specific viral regulatory elements widely used to facilitate mRNA translation under stress conditions. This inhibition appears to be a consequence of disrupted secondary and tertiary structure essential for IRES activity. We showed that these effects were consistent across viral IRES elements of all four types [2], exemplified by IRESs from RNA viruses such as HRV, EMCV, CSFV, BQCV, and others. Next, we explored the impact of U to N1mΨ substitution on translation directed by the 5'-end cap-independent translation enhancers (5' CITE), such as EMCV JK domain. We found that, in contrast to IRESs, the CITE-containing transcripts with 100% N1mΨ were efficiently translated and provided a resistance to cell stress. These findings may have implications for the design of new effective therapeutic mRNA platforms. 1. Akulich et al. (2016) Sci Rep 6, 37905. 2. Sorokin et al. (2021) Biochemistry (Moscow) 86, 1060–1094. The study was supported by the RSF grant no. 23-14-00218.