Ataluren and aminoglycosides stimulate read-through of nonsense codons by orthogonal mechanisms

Abstract

During protein synthesis, nonsense mutations, resulting in premature stop codons (PSCs), produce truncated, inactive protein products. Such defective gene products give rise to many diseases, including cystic fibrosis, Duchenne muscular dystrophy (DMD), and some cancers. Small molecule nonsense suppressors, known as TRIDs (translational read-through-inducing drugs), stimulate stop codon read-through. The best characterized TRIDs are ataluren, which has been approved by the European Medicines Agency for the treatment of DMD, and G418, a structurally dissimilar aminoglycoside. Previously [1], we applied a highly purified in vitro eukaryotic translation system to demonstrate that both aminoglycosides like G418 and more hydrophobic molecules like ataluren stimulate read-through by direct interaction with the cell's protein synthesis machinery. Our results suggested that they might do so by different mechanisms. Here, we pursue this suggestion through a more-detailed investigation of ataluren and G418 effects on read-through. We find that ataluren stimulation of read-through derives exclusively from its ability to inhibit release factor activity. In contrast, G418 increases functional near-cognate tRNA mispairing with a PSC, resulting from binding to its tight site on the ribosome, with little if any effect on release factor activity. The low toxicity of ataluren suggests that development of new TRIDs exclusively directed toward inhibiting termination should be a priority in combatting PSC diseases. Our results also provide rate measurements of some of the elementary steps during the eukaryotic translation elongation cycle, allowing us to determine how these rates are modified when cognate tRNA is replaced by near-cognate tRNA ± TRIDs.

Keywords: G418; TRID; ataluren; nonsense suppression; read-through.

Fig. 1.

CrPV-IRES mRNAs and elongation, read-through, and termination schemes. (A) Trp-IRES encodes PheLysValArgGlnTrpLeuMet. (B) Elongation reactions of Trp-POST5 complex. (C) Stop-IRES encodes PheLysValArgGlnStopLeuMet. (D) Termination and elongation (read-through) reactions of Stop-POST5 complex. Both Trp-POST5 and Stop-POST5 complexes contain PheLysValArgGln-tRNA^Gln in the P-site. Rate constants are defined in the text.

Fig. 2.

Read-through and termination on mixing Stop-POST5 complex with other reactants. In these experiments, eRF3 concentration was always twice that of eRF1. (A–C) Octapeptide formation during stop codon read-through ± TRIDs, determined by [³⁵S]-FKVRQWLM-tRNA^Met cosedimenting with the ribosome as a function of (A) varying eRF1 concentration at fixed Trp-TC concentration, (B) varying Trp-TC concentration at fixed eRF1 concentration, and (C) varying ataluren concentration at fixed G418 concentration. Each of these experiments contained the following fixed concentrations: Stop-POST5 complex (0.02 μM), Leu-TC and [³⁵S]Met-TC (each 0.05 µM), GTP (1 mM), and, when present, G418 (10 µM). In addition, (A) contained 0.05 μM Trp-TC and varying eRF1; (B) contained 10 nM eRF1 and varying Trp-TC; and (C) contained 0.05 μM Trp-TC, 30 nM eRF1, and varying ataluren. (D) Direct comparison of ataluren (green ∆) and G418 (purple ♢) effects on octapeptide read-through, as demonstrated previously (1), and termination (eRF1/RF3-catalyzed pentapeptidyl-tRNA hydrolysis) (ataluren, red ●; G418, blue ■). Stop-POST5 (0.05 μM) was mixed with Trp-TC, Leu-TC, and [³⁵S]Met-TC (each 0.05 µM), GTP (1 mM), 0.1 μM eRF1, 0.2 μM eRF3, and either ataluren (500 µM) or G418 (10 µM). The concentration of G418 is plotted on the top axis and of ataluren on the bottom axis. The fraction of peptidyl-tRNA hydrolyzed is plotted on the left axis, and the read-through product expressed as octapeptide/POST5 is plotted on the right axis.

Fig. 3.

Kinetics of (prf)Trp-TC binding to POST5 complexes. Experiments were carried out with fixed concentrations of (prf)Trp-TC (0.05 µM), 1 mM GTP, and, when added, 500 µM ataluren or 10 µM G418. (A) The increase in fluorescence intensity, measured as the voltage detected by the photomultiplier tube on the stopped-flow spectrofluorometer, following rapid mixing of (prf)Trp-TC with Trp-POST5 (0.075 μM, red line) or Stop-POST5 (0.075 μM) in the presence of either G418 (blue line) or ataluren (green line) or in the absence of TRID (black line). These increases are much larger than the small increase observed (gray line) on rapid mixing of 0.075 μM Trp-POST5 with 0.05 μM Trp-tRNA^Trp(prf) in the absence of eEF1A. (B) Normalized fluorescence changes of (prf)Trp-TC mixed with 0.075 to 0.30 μM added Stop-POST5 in the presence of G418. Changes for (prf)Trp-TC mixing with either Stop-POST5 ± ataluren or Trp-POST5 are shown in SI Appendix, Fig. S2. Dependence of (C) k_app,1 and (D) k_app,2 on free POST5 complex concentration, calculated as presented in Materials and Methods.

Fig. 4.

Kinetics of PRE6 formation and translocation. (A) Rates of FKVRQW-tRNA^Trp formation were measured by TLE analysis following strong base quenching. POST5 complexes (0.02 μM) were rapidly mixed with [³H]Trp-tRNA^Trp (0.15 μM), eEF1A (0.3 μM), eRF1 (0.02 μM), eRF3 (0.04 μM), and GTP (1 mM) in the presence of either G418 (10 μM) or ataluren (500 μM) or in the absence of TRIDs. Aliquots were quenched at various times with 0.4 M NaOH. (B) The dependence of apparent rate constants of FKVRQW-tRNA^Trp formation on free Trp-TC concentration, calculated as presented in Materials and Methods. (C and D) Rates of PRE6 translocation to POST6 were determined from the eEF2-dependent increase in prf fluorescence on rapid mixing of POST5 (0.15 μM) with (Prf)Trp-tRNA^Trp (0.05 μM), eEF1A (0.2 µM), GTP (1 mM), and eEF2. (C) Trp-PRE6 with varying eEF2 concentrations. (D) Stop-PRE6 ± TRID. Time-dependent changes in prf fluorescence on rapid mixing of Stop-POST5 (0.15 μM) with 1 μM eEF2 and either 10 μM G418 or 500 μM ataluren or in the absence of TRID. Raising the eEF2 concentration to 3 µM had negligible effects on the traces shown. For Stop-POST5 ± ataluren, extending the observations to 600 s gave no evidence of a significant eEF2-dependent change. (E) Rate of Trp-PRE6 translocation to Trp-POST6 as determined by FKVRQW-puromycin formation. Trp-PRE6 or Trp-POST6 (0.025 μM) was incubated with ±1 μM eEF2 and ±5 μM puromycin for various times and quenched with ice-cold pH 6 buffer. Hexapeptidyl-tRNA remaining on the ribosome was determined by cosedimentation.

Scheme 1.

Fig. 5.

Kinetics of octapeptide formation. Octapeptide formation was determined as described in Fig. 2. k₅₈ and k₆₈ were measured for (A) Trp-POST5, (B) Stop-POST5 + 10 μM G418, (C) Stop-POST5 + 600 μM ataluren, and (D) Stop-POST5, no added TRID. Measurements used to determine k₅₈ are indicated as 5 to 8 in the figures and were carried out following mixing 0.02 μM POST5 complex with 0.5 μM each of Trp-TC, Leu-TC, and [³⁵S]-Met-TC, 1 μM or 4 μM eEF2 as indicated, and 1 mM GTP. Measurements used to determine k₆₈ are indicated as 6 to 8 in the figures and were carried out similarly, except that 0.02 μM POST5 complex was premixed with 0.5 μM Trp-TC and 1 μM eEF2 concentration for either 20 s (A and B) or 2 min (C and D), followed by further mixing with 0.5 μM each of Leu-TC and [³⁵S]Met-TC while maintaining eEF2 concentration. Octapeptide yields were normalized to 0.79 (k₅₈) and 0.61 (k₆₈) for B, 0.42 (k₅₈) and 0.33 (k₆₈) for C, and 0.42 (k₅₈) and 0.34 (k₆₈) for D. Increasing eEF2 concentration from 1 to 4 μM had negligible effects on octapeptide yield.

Scheme 2.