The interaction between nucleotides 32 and 38 of the tRNA anticodon loop is known to impact codon recognition and tRNA binding to the ribosome in Escherichia coli15. Thus, we investigated the possible effects of m3C32 modification on tRNA-Arg function in protein synthesis using codon-dependent reporter constructs. We generated lentiviral reporters expressing nanoluciferase protein fused downstream of ten consecutive AGA or AGG codons, which are decoded by m3C-containing tRNA-Arg-UCU or CCU (Supplementary Fig. 5a). We also generated reporter plasmids containing codon runs of CGA or CGC, which are decoded by tRNA-Arg-UCG or ACG lacking m3C. Based upon this system, we found no significant difference in protein expression between the WT or D3-KO cell lines for any of the arginine codon reporters (Supplementary Fig. 5b and c). We also detected no major change in expression of a control GFP construct between WT and D3-KO cell lines (Supplementary Fig. 5b and c). These results suggest that the m3C modification has minimal effect on translation efficiency in these codon reporters and/or plays a role in arginine tRNAs that cannot be detected using this experimental system.
