Chemical modifications of 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 acids. 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 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.
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This work was supported by the US National Institutes of Health (NIH) grants GM51266 and GM113078 to J.D.P. and by grants from the Knut and Alice Wallenberg Foundation (RiboCORE), the Swedish Research Council and the Human Frontier Science Program to M.E., the Kahn Family Foundation to D.D. and G.R., the Ernest and Bonnie Beutler Research Program, Flight Attendant Medical Research Institute (FAMRI) and the Israeli Centers of Excellence (I-CORE) Program (ISF grants no. 41/11 and no. 1796/12) to G.R., a Human Frontier Science Program long-term fellowship to D.D., by the NSF Science and Technology Centers grant NSF-1231306 (Biology with X-ray Lasers, BioXFEL) to H.D., by a Stanford Bio-X fellowship to J.C. and A. Prabhakar and by a Knut and Alice Wallenberg Foundation postdoc fellowship to J.W. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource (SSRL), a national user facility operated by Stanford University on behalf of the US Department of Energy, US Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the US Department of Energy, Office of Biological and Environmental Research, NIH, US National Center for Research Resources, Biomedical Technology Program, and the US National Institute of General Medical Sciences. C.H. is supported by the Howard Hughes Medical Institute (HHMI). We thank P. Agris (University of Albany) for a human ASL reagent and members of Puglisi laboratory for discussion.
The authors declare no competing financial interests.
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Integrated supplementary information
(a) A representative trace from tRNA-tRNA FRET experiments. On the modified codon (AAmA), we observe tRNA is bound to the A*/T site during sampling (prior to GTP-hydrolysis activation; FRET efficiency near 0.5), followed by the accommodation event (FRET efficiency near 0.7).(b) Contour plots of FRET-efficiency trajectories, generated by post-synchronizing traces to the successful tRNA accommodation events on AAmA codon at 15 mM Mg2+ concentration (n = 369). (c) Contour plots of FRET-efficiency trajectories, generated by post-synchronizing traces to the successful tRNA accommodation events on AAA codon at 15 mM Mg2+ concentration (n = 371). (d) Contour plots of FRET-efficiency trajectories, generated by post-synchronizing traces to the tRNA sampling events on AAmA codon at 15 mM Mg2+ concentration (n = 1244). (e) Contour plots of FRET-efficiency trajectories, generated by post-synchronizing traces to the tRNA sampling events on AAmA codon at 15 mM Mg2+ concentration in the presence of GDPNP (n = 565). (f)Contour plots of FRET-efficiency trajectories, generated by post-synchronizing traces to the tRNA sampling events on AAA codon at 15 mM Mg2+ concentration in the presence of GDPNP (n = 420). (g) tRNA-tRNA FRET efficiencies assigned to different stages of tRNA selection: Upon the initial binding of tRNA in the form of TC, the codon-anticodon interaction stabilizes tRNA to the A*/T-site (FRET-efficiency near 0.5), where monitoring bases has not made contact with codon-anticodon helices to activate GTP-hydrolysis. Activation of monitoring bases slightly moves tRNA to the A/T-site, which facilitates GTP-hydrolysis (FRET-efficiency near 0.6). GTP-hydrolysis followed by the peptide-bond formation accommodates tRNA to the A site (FRET-efficiency near 0.7).
Supplementary Figure 2 fMet consumption over time with Lys-tRNA only or with Lys-tRNA depleted bulk aa-tRNA.
0.2 µM ribosomes initiated with [3H]fMet-tRNAfMet and AAA or AAmA codon in the A site were reacted to 2 µM EF-Tu and 100 µM total E. coli tRNA charged with all 19 amino acids except lysine or only lysine, as indicated. The experiment was performed in polymix buffer containing 15 mM total Mg2+. The decrease of the fMet-tRNAfMet fraction over time reveals a corresponding increase in peptide bond formation. In the presence of Lys-tRNALys and unmodified AAA codon all available fMet was consumed by fMet-Lys formation (filled-circle data points), but in the absence of Lys-tRNALys and presence of charged bulk-tRNA the extent of peptide bond formation was negligible both for the modified AAmA (filled-triangle data points) and unmodified AAA (filled-diamond data points) codon.
Supplementary Figure 3 Aminoglycosides decrease the time lag between tRNA binding and accommodation.
Contour plots of the normalized Cy3B and Cy5 intensity trajectories, generated by post-synchronizing to the ribosomal intersubunit rotation event upon the peptidyl transfer reaction. Without any drug (lower-left panel), the Cy5 signal (upper plots; binding of (Cy5)-tRNALys to the ribosome) happens about 2 seconds earlier than the unquenching of Cy3B signal (lower plots; intersubunit rotation to the rotated state after peptidyl-transfer reaction) on the 2′-O-methylated codon. This time delay (indicated as Rotation Lag with a red bar) is minimized in the presence of paromomycin or neomycin (middle and right panels).
Structures of the decoding center (ASL and A1492, A1493 and G530 monitoring bases) while decoding (a) the unmodified, (b) the first base modified, (c) the second base modified, and (d) the third base modified Lys codon, soaked in the 30S crystals in the absence of paromomycin. Without paromomycin, crystal samples prepared in parallel with the modified Lys codon did not exhibit successful binding of ASL and mRNA nucleotides to the ribosome, marked by the lack of corresponding 2Fo-Fc electron densities contoured at 1.5 sigma level.
Composite omit 2mFo − DFc electron density maps of decoding center region (ASL and A1492, A1493 and G530 monitoring bases) while decoding (a) the unmodified, (b) the first base modified, (c) the second base modified, and (d) the third base modified Lys codon, soaked in the 30S crystals in the absence of paromomycin. Without paromomycin, crystal samples prepared in parallel with the modified Lys codon did not exhibit successful binding of ASL and mRNA nucleotides to the ribosome, marked by the lack of corresponding 2Fo-DFc composite omit map electron densities.
X-ray crystallography structures of the decoding center (UUU anticodon and A1492, A1493 and G530 monitoring bases) while decoding unmodified (AAA) (a) the first base unmodified (b) the second base unmodified (c) the third base unmodified (d) the first base modified (AmAA), (e) the second base modified (AAmA), and (f) the third base modified Lys codon (AAAm), soaked in the 30S subunit crystals in the presence of paromomycin.
Structures of the decoding center (ASL and A1492, A1493 and G530 monitoring bases) while decoding (a) the unmodified, (b) the first base modified, (c) the second base modified, and (d) the third base modified Lys codon, soaked in the 30S crystals in the presence of paromomycin. 2Fo-Fc electron density maps are contoured at 1.5 sigma level.
(a) The 2′-O-methylation within the A-site codon sterically disrupts the activation of monitoring bases (A1492 shown as it interacts with the second base within the A site codon). (b) Schematics of tRNA selection to the 2′-O-methylated codon: While the 2′-O-methylation disrupts the activation of monitoring bases, aminoglycosides such as paromomycin and neomycin counter its effect by forcing the activation of monitoring bases. The disruption of interactions among monitoring bases and mRNA-tRNA duplex may also delay EF-Tu•GDP dissociation, favoring tRNA rejection by aa-tRNA•EF-Tu•GDP dissociation. The increase of Mg2+ concentration may slow down both aa-tRNA•EF-Tu•GTP and aa-tRNA•EF-Tu•GDP dissociation rate, favoring tRNA accommodation on the 2′-O-methylated codon during the initial selection process and proofreading steps.
Supplementary Figures 1–8 and Supplementary Notes 1–3
Lifetimes and ribosome survival plots within the coding region for Figure 1.
Lifetimes and ribosome survival plots within the coding region for Figure 2.
Source data and statistical measures underlying figures.
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Choi, J., Indrisiunaite, G., DeMirci, H. et al. 2′-O-methylation in mRNA disrupts tRNA decoding during translation elongation. Nat Struct Mol Biol 25, 208–216 (2018). https://doi.org/10.1038/s41594-018-0030-z
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