Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

Abstract

A premature termination codon (PTC) in the ORF of an mRNA generally leads to production of a truncated polypeptide, accelerated degradation of the mRNA, and depression of overall mRNA expression. Accordingly, nonsense mutations cause some of the most severe forms of inherited disorders. The small-molecule drug ataluren promotes therapeutic nonsense suppression and has been thought to mediate the insertion of near-cognate tRNAs at PTCs. However, direct evidence for this activity has been lacking. Here, we expressed multiple nonsense mutation reporters in human cells and yeast and identified the amino acids inserted when a PTC occupies the ribosomal A site in control, ataluren-treated, and aminoglycoside-treated cells. We find that ataluren's likely target is the ribosome and that it produces full-length protein by promoting insertion of near-cognate tRNAs at the site of the nonsense codon without apparent effects on transcription, mRNA processing, mRNA stability, or protein stability. The resulting readthrough proteins retain function and contain amino acid replacements similar to those derived from endogenous readthrough, namely Gln, Lys, or Tyr at UAA or UAG PTCs and Trp, Arg, or Cys at UGA PTCs. These insertion biases arise primarily from mRNA:tRNA mispairing at codon positions 1 and 3 and reflect, in part, the preferred use of certain nonstandard base pairs, e.g., U-G. Ataluren's retention of similar specificity of near-cognate tRNA insertion as occurs endogenously has important implications for its general use in therapeutic nonsense suppression.

Keywords: Translarna; base mispairing; nonsense suppression; readthrough.

Fig. 1.

Ataluren treatment of mammalian or yeast cells increases PTC readthrough. Dose–response of ataluren treatment in 293H cells stably transfected with (A) PTC(UGA_W12X) or (B) wild-type (WT) NanoLuc reporters. The 293H cells stably expressing the NanoLuc reporters were treated with ataluren at the indicated concentrations. The data are expressed as the mean ± SD of the NanoLuc activity units normalized to total protein. (C) Western analyses of yeast cells showing readthrough products expressed from HA-HIS3_(UAA100)-SF reporters after ataluren or gentamicin treatment. Upper and Lower blots, respectively, were probed for the HA (N-terminal) and FLAG (C-terminal) epitopes. The predominant band at ∼17 kDa in the HA blot is an HA artifact detected only in cells expressing HIS3_(UAA100)-SF, but not HIS3_(WT)-SF.

Fig. 2.

Ataluren-mediated decoding of nonsense codons. Comparison of amino acid insertion at PTCs during termination readthrough of HA-LUC_(PTC20)-SF reporters after ataluren treatment in yeast and 293H cells. Epitope-tagged luciferase was purified from the respective cells and subjected to mass spectrometry analyses. Box and whisker graphs depict the amino acids inserted for each nonsense codon. Horizontal lines are the means and vertical lines are the SD. (A) Data from WT [PSI−] yeast cells treated with 30 µM ataluren (n = 3). (B) Data from 293H cells treated with 30 µM ataluren. Boxes represent the range of at least four determinations. Where there are no boxes, three determinations were done. Data used to generate graphs in B are in SI Appendix, Table S6.

Fig. 3.

Characterization of the amino acid insertions at human CFTR-G542X. (A) Western blot showing an increased amount of full-length protein (containing both TGFP and HA tag) (yellow-green bands) compared to the TGFP protein lacking the HA tag (red bands) after G418 treatment in HEK293 cells transiently transfected with the G542X construct. (B) Western blot showing the level of CFTR maturation observed in HEK293 cells transiently transfected with CFTR G542 variants. “Band B” is the ER form of CFTR, whereas “Band C” is the mature form. CFTR-F508del is a mutant form of CFTR that is retained in the ER and consequently only produces Band B. (C) Representative tracings (Left) and quantitation results (Right) of short-circuit current (Isc) measurements of CFTR chloride channel activity in FRT epithelial cell monolayers stably expressing the CFTR G542 variant constructs. (D) Representative tracings (Left) and quantitation (Right) of Conductance (Gt) measurements of CFTR chloride channel activity in FRT monolayers stably expressing the CFTR G542 variant constructs.

Fig. 4.

Tobramycin is a potent inhibitor of ataluren-mediated readthrough. The effect of tobramycin on NanoLuc activity in HEK293 cells expressing a UGA (W12X) NanoLuc reporter with either no prior treatment or following treatment with 10 µM ataluren for 48 hours. Initial stimulation of readthrough by ataluren in the absence of tobramycin was 15-fold above background.