Protein of Interest Synthesis Using Non-formylated Methionine−Escherichia coli initiator tRNA in HeLa cells

Abstract In eukaryotic cells and Escherichia coli, protein synthesis is initiated through non-formylated and formylated methionine-conjugated initiator tRNA (Met-tRNAi and fMet-tRNAi, respectively). Studies have shown that protein of interest (POI) reduced EGFP mRNA expression in human Met-tRNAi/human methionyl-tRNA synthetase (HMRS) pairs under methionine-deficient conditions. Intracellular L-Met depletion can result in the inhibition of endogenously non-formylated human Met-tRNAi/HMRS in HeLa cells. We investigated the effect of non-formylated Met-conjugated E. coli initiator tRNA (E. coli Met-tRNAi) on POI synthesis in HeLa cells under intracellular L-Met levels. The expression of GFP—a model protein—in L-methionine-deficient HeLa cells, in which endogenous tRNAi was replaced with two kinds of E. coli tRNAi, E. coli Met-tRNAi/E. coli methionyl-tRNA synthetase (EcMRS) and E. coli Met-tRNAi/HMRS, was analyzed. The results indicated that E. coli Met-tRNAi can offset human Met-tRNAi, and E. coli methionyl tRNA synthetase (EcMRS) can drive the expression of EGFP mRNA. The non-formylated E. coli Met-tRNAi/EcMRS pairs reduced the expression of EGFP mRNA, resulting in reduced HeLa cell survival. Our results provide new insights into the potential use of E. coli Met-tRNAi as novel therapeutics that can control POI synthesis initiation. The E. coli Met-tRNAi/MRS pair has the potential to fine-tune protein synthesis in mammalian cells.