ИСТИНА

mRNA transfection is a promising approach for gene delivery that may be applied for substitutional therapy and cell reprogramming. It is also a powerful tool for studying protein biosynthesis and translational control. We developed the fleeting mRNA transfection technique (FLERT), a method for the measurement of a luciferase mRNA translation in cultured mammalian and insect cells. We applied FLERT to study the immediate effects of oxidative stress, unfolded protein response, and small molecule inhibitors on translation efficiency of several reporter mRNAs that differ in their translation initiation mechanism. We then further improved the method by establishing a technique of continuous luciferase imaging in mRNA transfected cultured cells (real-time FLERT). Using this approach, the dynamics of the reporter protein synthesis during cell stress response, adaptation and recovery were investigated with a high time resolution. We compared expression kinetics of the luciferase gene delivered by FLERT with that of a plasmid DNA transfection and a virus mediated delivery. Next, we unexpectedly found that in vitro synthesized transcripts containing an intron in the coding region are efficiently spliced when transfected into proliferating mammalian cells. Thus, not only protein synthesis but also splicing can be studied with this assay. We also showed that modified nucleosides m5C and N1mΨ within mRNA, that restrain the innate immune response in primary cells, negatively affect translation when introduced in a 5’ untranslated region, presumably due to interference with ribosomal scanning. Finally, we found that mRNA molecules, which invade the cytoplasm de novo, are preferentially translated in a cell recovering from a stress. In summary, we developed a highly sensitive method for the continuous measurement of the reporter mRNA activity in cultured mammalian cells and demonstrated its applicability for studying gene expression and cell adaptation to the changing environment. The work was supported by the grant of the Russian Federation government №14.W03.31.0012 and RSF grant 18-14-00291.