2′-O-methylation in mRNA disrupts tRNA decoding during translation elongation

Chemical modifications of messenger RNA (mRNA) may regulate many aspects of mRNA processing and protein synthesis. Recently, 2′-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acid. Here, using single-molecule, bulk kinetics and structural methods, we show that 2′-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate transfer RNA (tRNA) selection. Our results suggest that 2′-O-methylation sterically perturbs interactions of ribosomal monitoring bases (G530, A1492 and A1493) with cognate codon-anticodon helices, thereby inhibiting downstream GTP-hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated-tRNAs in initial selection and proofreading. Our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.