Position-dependent effects of regioisomeric methylated adenine and guanine ribonucleosides on translation

Abstract

Reversible methylation of the N6 or N1 position of adenine in RNA has recently been shown to play significant roles in regulating the functions of RNA. RNA can also be alkylated upon exposure to endogenous and exogenous alkylating agents. Here we examined how regio-specific methylation at the hydrogen bonding edge of adenine and guanine in mRNA affects translation. When situated at the third codon position, the methylated nucleosides did not compromise the speed or accuracy of translation under most circumstances. When located at the first or second codon position, N1-methyladenosine (m1A) and m1G constituted robust blocks to both Escherichia coli and wheat germ extract translation systems, whereas N2-methylguanosine (m2G) moderately impeded translation. While m1A, m2G and N6-methyladenosine (m6A) did not perturb translational fidelity, O6-methylguanosine (m6G) at the first and second codon positions was strongly and moderately miscoding, respectively, and it was decoded as an adenosine in both systems. The effects of methylated ribonucleosides on translation could be attributed to the methylation-elicited alterations in base pairing properties of the nucleobases, and the mechanisms of ribosomal decoding contributed to the position-dependent effects. Together, our study afforded important new knowledge about the modulation of translation by methylation of purine nucleobases in mRNA.

Figure 1.

Experimental outline. (A) Chemical structures of A, m¹A, m⁶A, G, m¹G, m²G and m⁶G. ‘Rib’ indicates ribose. (B) A schematic diagram illustrating the procedures employed for assessing the impact of the methylated ribonucleosides on translation. ‘X’ indicates a methylated adenosine or guanosine. ‘RBS’ and ‘STOP’ designate the ribosome binding site and the stop codon, respectively. Only the mRNA containing an m¹G, m²G, m⁶G or G at the first position of sixth codon of the mRNA, as well as the wild-type peptide product MVGAGGVGK and the competitor peptide product MVVGAGGVGK are shown.

Figure 2.

Representative LC-MS results for monitoring the influence of m⁶G on translation. The relative abundances of the wild-type peptide product MVGAGGVGK (i.e. 9AA-WT), the mutant product MVGAGSVGK (i.e. 9AA-Mu) and the competitor peptide product MVVGAGGVGK (i.e. 10AA-Comp) from the reconstituted Escherichia coli translation reactions, where GGC and m⁶GGC were used as the sixth codon of the unmodified (A) and the m⁶G-bearing (B) mRNA templates, respectively. ‘RT’, retention time; ‘MA’, peak area found in the selected-ion chromatogram for monitoring the formation of the [M+2H]²⁺ ions of the peptide products. (C) The MS/MS of the [M+2H]²⁺ ion of the mutant peptide product MVGAGSVGK from the reconstituted E. coli translation reaction, where m⁶GGC was the sixth codon of the m⁶G-bearing mRNA template.

Figure 3.

The effects of m⁶G and m¹G in mRNA on the translational fidelity. (A and B) Quantification of the peptides carrying an a glycine (Gly), serine (Ser) or aspartic acid (Asp) at the sixth codon of the m⁶G-bearing mRNA templates during the reactions mediated by Escherichia coli (A) or the wheat germ extract (B) translation systems. (C) Quantification of the peptides carrying a glycine (Gly), cysteine (Cys) or arginine (Arg) at the sixth codon of the m¹G-bearing mRNA templates produced from the reactions mediated by the wheat germ extract translation systems. ‘1^st’, ‘2^nd’ and ‘3^rd’ indicate the first, second and third positions of the codon, respectively. The data represent the mean and standard error of results from three independent experiments. (D) The codon-anticodon pair involved in the generation of translation products for the m⁶G- and m¹G-bearing mRNAs.

Figure 4.

Effects of methylated adenosine and guanosine derivatives in mRNA on translation efficiency. Shown are translation bypass efficiencies (TBEs) of m¹G, m²G, m⁶G, m¹A and m⁶A in the translation reactions mediated by the reconstituted Escherichia coli (A) and wheat germ extract (B) translation systems. A summary of TBE values are also displayed in heatmaps (C). ‘1^st’, ‘2^nd’ and ‘3^rd’ indicate the first, second and third positions of the codon, respectively. The data represent the mean and standard error of results from three independent experiments.