N(6)-methyladenosine in mRNA disrupts tRNA selection and translation-elongation dynamics

Abstract

N(6)-methylation of adenosine (forming m(6)A) 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 m(6)A in mRNA decoding. Although m(6)A 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 m(6)A modification of mRNA acts as a barrier to tRNA accommodation and translation elongation. The interaction between an m(6)A-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 m(6)A within codons and on the accuracy level of translation. Overall, our results demonstrate that chemical modification of mRNA can change translational dynamics.

Figure 1

Single-molecule assay for observing translational dynamics on m6A-modified mRNA. (a) Experimental setup for single-molecule assay^,,. Pre-Initiation Complex (PIC) containing Cy3B labeled 30S ribosomal subunit, Initiation Factor 2 (IF2), fMet-tRNA^fMet, and 5′-biotinylated mRNA of interest is immobilized to partially neutravidin-biotinylated polyethylene glycol covered zero-mode waveguides (ZMWs). Experiment is started by illuminating ZMWs with green and red lasers and delivering BHQ-2 labeled 50S, Elongation Factor G (EF-G), Cy5-labeled and unlabeled tRNA ternary complexes to immobilized PICs. (b) Expected sequences of fluorescence signals using Cy3B-BHQ-2 FRET and Cy5 pulses. Cy3B signal reports the rotational state of ribosomal subunits for translating each codon, while long Cy5 pulses indicating stable binding of Cy5-tRNA to translational complex. (c) Sample fluorescent signal observed during experiment using m6A-modified mRNA sequence shown. Each Cy5 pulses are correlated with two Cy3B low-high-low cycle, corresponding to two rounds of elongation during tRNA is bound to translational complex (A-to-P and P-to-E movement in the perspective of transiting tRNA).

Figure 2

Single-base m6A-modification of codon delays tRNA accommodation. (a) mRNA constructs used in single-molecule assay. All mRNA constructs have six codons in the coding region with m6A-modified codon in the fourth codon, except Lys3, where twelve-codon long construct was used to test the effect of m6A entering and leaving ribosome completely. (b) The non-rotated state lifetimes at Lys, Gln or Pro codons present in the mRNA used. The error bars represent s.e.m. (95% confidence interval) from fitting a single-exponential distribution to number of molecule specified. (c) Fold-increases of m6A-modified codon rotated state lifetime are compared across experimental conditions and mRNAs used. Error bars are calculated using the propagation of error method from data shown in (b).

Figure 3

Single-base m6A-modification slows down binding of ternary complexes to the A site of ribosomes during decoding and has minor effect on the subsequent steps. (a) Kinetics of GTP hydrolysis after binding of Lys-tRNA^Lys ternary complexes (0.3 μM) to 70S (1 μM) initiation complexes programmed with AAA or (m6A)AA in the A site. (b) Dependence of the rate of GTP-hydrolysis, k_GTP, on ribosome concentration. (c) Estimates of k_cat/K_M-values for GTP-hydrolysis from b. (d) and (e) Kinetics of GTP-hydrolysis and dipeptide fMet-Lys formation measured simultaneously in the same experiment. The grey areas represent the total time for all subsequent steps after GTP-hydrolysis up to and including peptidyl transfer. (f) Estimates of the compounded rate constant, k_pep, for the steps after GTP-hydrolysis on EF-Tu up to and including peptidyl transfer, from experiments shown in d and e. See Supplementary Data Table 1 for data in c and f. Kinetic data in a, d and e are representative of three independent experiments. Error bars in b, c, and f represent s.d. (n = 3, technical replicates) as calculated from the fitting procedure.

Figure 4

The effect of m6A in delaying tRNA incorporation scales measured across methods. Comparing fold difference in m6A-induced delay in tRNA shows agreement across different methods. Inter-subunit FRET at 1.7mM Mg data point corresponds to data from Figure 2, while Quench-flow at 1.3 mM and 7.5 mM Mg data points corresponds to data from Figure 3 and Figure 4, respectively. Inter-subunit FRET at 11 mM magnesium concentration was measured taking ratio between two fittings of single-exponential distributions to time between 50S joining event and Lys-(Cy5)tRNA^Lys arrival event happening near simultaneously with increase in inter-subunit FRET efficiency, for both AAA and (m6A)AA (number of molecule analyzed = 113 and 80, for AAA and (m6A)AA, respectively). The error for this measurement is calculated by the propagation of error method from s.e.m calculated in these two experiments. tRNA-tRNA FRET at 11mM Mg data point has been measured as indicated in the text.

Figure 5

Effect of m6A on different stages of decoding observed using tRNA-tRNA FRET. (a) A model of tRNA selection steps and corresponding approximate FRET values. Monitoring FRET between fMet-(Cy3)tRNA^fMet and Lys-(Cy5)tRNA^Lys can be used to track the tRNA selection process. (b) Contour plots of the time evolution of population FRET at various conditions, generated by superimposing all the observed FRET events (in the case of the GTP experiment (left panels), first observed event per immobilized molecule) synchronized to the start of the event defined by the FRET efficiency larger than 0.35. Contours are plotted from tan (lowest population) to red (highest population). The number of FRET events post-synchronized for each experiment is 350, 350, 948, 1573, 520 and 396 for experimental conditions with GTP–and–AAA, GTP–and–(m6A)AA, GDPNP–and–AAA, GDPNP–and–(m6A)AA, GTP–and–tetracycline–and–AAA and GTP–and–tetracycline–and–(m6A)AA, respectively.

Figure 6

Proposed model for protein synthesis regulation via dynamic modification of mRNA. Regulation of mRNA modification could act as a localized toggle switch between two translation elongation dynamics for a single gene. Each set of elongation dynamic could influence folding of proteins or interaction between nascent protein and other factors.