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N6-methyladenosine in mRNA disrupts tRNA selection and translation-elongation dynamics

Abstract

N6-methylation of adenosine (forming m6A) is the most abundant post-transcriptional modification within the coding region of mRNA, but its role during translation remains unknown. Here, we used bulk kinetic and single-molecule methods to probe the effect of m6A in mRNA decoding. Although m6A base-pairs with uridine during decoding, as shown by X-ray crystallographic analyses of Thermus thermophilus ribosomal complexes, our measurements in an Escherichia coli translation system revealed that m6A modification of mRNA acts as a barrier to tRNA accommodation and translation elongation. The interaction between an m6A-modified codon and cognate tRNA echoes the interaction between a near-cognate codon and tRNA, because delay in tRNA accommodation depends on the position and context of m6A within codons and on the accuracy level of translation. Overall, our results demonstrate that chemical modification of mRNA can change translational dynamics.

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Figure 1: Single-molecule assay for observing translational dynamics on m6A-modified mRNA.
Figure 2: Single-base m6A modification of codons delays tRNA accommodation.
Figure 3: Single-base m6A modification slows down binding of ternary complexes to the A site of ribosomes during decoding and has a minor effect on the subsequent steps.
Figure 4: The effect of m6A in delaying tRNA-incorporation scales measured across methods.
Figure 5: Effect of m6A on different stages of decoding observed with tRNA-tRNA FRET.
Figure 6: Proposed model for protein-synthesis regulation via dynamic modification of mRNA.

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Acknowledgements

This work was supported by US National Institutes of Health (NIH) grants GM51266 and GM099687 to J.D.P.; by grants from the Knut and Alice Wallenberg Foundation (RiboCORE) and the Swedish Research Council and the Human Frontier Science Program to M.E.; by NIH grants GM111858 to S.E.O'L.; by grants from the Israel Science Foundation (ISF) grant no. 1667/12), the Israeli Centers of Excellence (I-CORE) Program (ISF grants no. 41/11 and no. 1796/12) and the Ernest and Bonnie Beutler Research Program to G.R.; by a Human Frontier Science Program long-term fellowship to D.D.; and by a Stanford Bio-X fellowship to J. Choi. 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. G.R. is supported as a member of the Sagol Neuroscience Network and by the Kahn Family Foundation. We thank P. Agris (University of Albany) for a human ASL reagent and members of Puglisi laboratory for discussion. J. Choi thanks J.B. Choi for support.

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Authors

Contributions

J. Choi, K.-W.I. and H.D. performed all the experiments and the data analysis; J. Choi performed single-molecule experiments; K.-W.I. performed bulk kinetic experiments; H.D. performed X-ray crystallography, with the help of S.M.S. in material preparations. D.D. and G.R. provided reagents and conceived the project with J. Choi, K.-W.I., H.D., J. Chen, M.E. and J.D.P. J. Chen, A. Petrov and A. Prabhakar assisted in reagent preparation. J. Choi, K.-W.I., H.D., S.E.O'L., M.E. and J.D.P. wrote manuscript.

Corresponding author

Correspondence to Joseph D Puglisi.

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Integrated supplementary information

Supplementary Figure 1 Comparing rotated- and nonrotated-state lifetimes among mRNA m6A modifications in different codon contexts.

a. mRNA sequences used for each experiments, as same as shown in Figure 2a.

b. The rotated state and non-rotated state lifetime measurements for all codons in the mRNAs used. First row shows the non-rotated state lifetimes for each codons within the mRNAs, and second row shows the rotated state lifetimes for each codons within the mRNAs. Last row shows the number of ribosomes observed to reach particular codon during translation prior to photobleaching of reporter fluorescence dye.

Supplementary Figure 2 Geometry of the m6A-uridine Watson-Crick base-pair interactions.

The final σA-weighted m2Fo-dFc electron density map contoured at 1σ level shows that a. m6A1•U36, b. m6A•U35 and c. m6A•mcm5s2U34 base pairs are in the Watson-Crick geometry. ASL carbons are colored wheat and mRNA carbons are gray. d. The overlay of all structures reveals a nearly identical orientation of the codon:anticodon interaction). The ASLLys3UUU –AAA complex (colored in gray), the ASLLys3UUU – (m6A)AA (colored in light pink), ASLLys3UUU–A(m6A)A (colored in cyan) and ASLLys3UUU–AA(m6A) (colored in green) are superposed and aligned with respect to the 16S rRNA.

Supplementary Figure 3 Proofreading of tRNALys ternary complexes reading (m6A)AA.

Dipeptide formation was measured at 1µM ribosomes and 0.5 µM ternary complexes. Experiments were done in parallel using the very same ternary complexes reacting with initiation complexes displaying AAA or (m6A)AA codon in the A site. The lower plateau of dipeptide fMet-Lys formation for (m6A)AA indicates the rejection of tRNALys after GTP hydrolysis.

a. Experiments were done in low Mg2+ buffer (see Methods). Proofreading factor, f = 1.5, was calculated as the ratio between the plateaus of dipeptide formation for AAA and (m6A)AA.

b. Same experiment was performed in high Mg2+ buffer (see Methods), where proofreading factor was measured to be f = 1.3.

Supplementary Figure 4 Reducing decoding accuracy reduces the effect of m6A on translational dynamics in high-Mg2+ conditions.

(a) Kinetics of GTP hydrolysis after binding of Lys-tRNALys ternary complexes (0.2 µM) to 70S initiation complexes (0.7µM) programmed with AAA or (m6A)AA in the A site. (b) Estimates of kcat/KM-values for GTP hydrolysis. (c) and (d) Kinetics of GTP hydrolysis and dipeptide fMet-Lys formation measured simultaneously in the very same experiment. The grey areas represent the total time for all subsequent steps after GTP hydrolysis up to and including peptidyl transfer. (e) Estimates of the compounded rate constant, kpep, for the steps after GTP hydrolysis on EF-Tu up to and including peptidyl transfer, from experiments shown in c and d. See Supplementary Data Table 1 for data in b and e. Kinetic data in a, c, and d are representative of three independent experiments. Error bars in b and e represent SD (n = 3, technical replicates) as calculated from the fitting procedure (Johansson, M. et al. Proc. Natl. Acad. Sci. U. S. A. 108, 79–84 (2011)).

Supplementary Figure 5 m6A shifts FRET value between P-site tRNA and A-site tRNA in the GTPase-activated state.

FRET histogram of the GDPNP-induced prolonged GTPase-activated state and codon-recognition states for unmodified (left) and first-base m6A modified (right) cases at 15mM magnesium concentration. Values inserted indicate fitted center of Gaussian distribution to histogram, and values in parenthesis indicate 95% confidence interval of fitting. While peaks near 0 FRET efficiency indicates no FRET state, peaks near 0.6 FRET efficiency corresponds to GTPase-activated state. FRET efficiency decreases slightly from 0.643 to 0.585 when cognate AAA codon is modified to (m6A)AA codon, mirroring previous comparison between cognate and near-cognate pairing on Phe (Lee, T., Blanchard, S. C., Kim, H. D., Puglisi, J. D. & Chu, S. The role of fluctuations in tRNA selection by the ribosome. Proc. Natl. Acad. Sci. 104, 13661–13665 (2007)).

Supplementary Figure 6 tRNA-tRNA FRET lifetime decreases severely with m6A at low magnesium.

Comparing representative synchronized FRET time evolution between different conditions. Low magnesium condition aggravates the effect of m6A in tRNA recognition by increasing accuracy of tRNA selection of ribosome (top left, top middle panel), which inverts ratio between successful accommodation event and transient sampling event compared to high magnesium condition. When decoding is further hindered by GDPNP at low magnesium condition, FRET lifetime at GTPase-activated state decreases and the effect of m6A cannot be detected accurate in our current time resolution of 100 millisecond; decreased FRET event lifetime due to m6A might be less than 100 millisecond, which our measurement would only sample long-lived FRET events rather than giving a correct lifetime. We also performed tRNA FRET between P site fMet-(Cy3)tRNAfMet and A site Phe-(Cy5)tRNAPhe binding to UUC codon at A site, similar to previously published result for comparison (rightmost two panels). Number of FRET events post-synchronized for each experiment is 316, 213, 497, 577, 221, and 781 for GTP-AAA, GTP-(m6A)AA, GDPNP-AAA, GDPNP-(m6A)AA, GTP-UUC and GDPNP-UUC, respectively.

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Choi, J., Ieong, KW., Demirci, H. et al. N6-methyladenosine in mRNA disrupts tRNA selection and translation-elongation dynamics. Nat Struct Mol Biol 23, 110–115 (2016). https://doi.org/10.1038/nsmb.3148

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