Effects of mRNA ۵′ cap in protein expression in mammalian cells

Background and Aim: In mammals, the predominant cap structure is ۷-methylguanosine linked via a ۵′ to ۵′ triphosphate bridge to the first transcribed nucleotide, which is methylated on the ribose O-۲ position (denoted m۷G(۵′)ppp(۵′)Xm, X is the first transcribed nucleotide). ۷-Methylguanosine ۵′cap on mRNA is necessary for efficient protein expression in vitro and in vivo. ۲ -O-Methylation is the most common modification of the first transcribed nucleotide(s) in higher eukaryotes and corresponding viruses. In mammalian cells, mRNAs are usually ۵ -terminated with m۷GpppNm or m۷GpppN۱ mN۲ m structures called cap ۱ and cap ۲, respectively, wherein Nm is a ۲ -O-methylated nucleotide. The mRNA cap protects RNA from degradation, recruits complexes involved in RNA processing, export and translation initiation, and marks cellular mRNA as “self” to avoid recognition by the innate immune system.Methods: In order to Synthesis of trinucleotide cap analogs and purification and Protein expression studies were used from NMR, FQT, RT - qPCR , HPLC and flow cytometry.Results: The highest expression was observed for mRNAs carrying ۵ -terminal A/Am and m۶Am, whereas the lowest was observed for G and Gm. The mRNAs carrying ۲ -O-methyl at the first transcribed nucleotide (cap ۱) had significantly higher expression than unmethylated counterparts (cap ۰).The capping efficiencies for the obtained RNAs ranged from ۵۴ to ۹۰%. The highest capping efficiency was observed for trinucleotides featuring a purine nucleotides at the position of the first transcribed nucleotide (‘purine trinucleotides’): m۷GpppAmpG (۹۰%), m۷GpppApG (۸۹%), m۷GpppGmpG (۸۶%), m۷GpppGpG (۸۰%), while the lowest capping efficiencies were observed for pyrimidine trinucleotides. cells with transcripts capped with trinucleotide cap analogues containing G or Gm, that were found to be poorly translated, did not lead to immune response activation compared to trinucleotides with A and Am.Conclusion: Identical mRNAs that differing only in the first transcribed nucleotide (A, m۶A, C, U, G) may be expressed at significantly different levels under certain conditions., and minor changes in the ۵’ mRNA end alter the identity or methylation status of the first translated nucleotide, which may significantly change the expression of mRNA in living cells. Moreover, the biological effects of these changes may significantly vary depending on the purity of mRNA and the production line. However, in all cell lines studied, mRNAs carrying m۶Am and Am as the first translated nucleotide showed high protein expression .

Keywords:

Capping , mammalian , mRNA translation , protein expression.