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.

RETRACTED: Gentamicin B1 is a minor gentamicin component with major nonsense mutation suppression activity

“…The application of PTC readthrough as a therapeutic approach is premised on the inserted amino acid not negatively impacting protein function. The nature of the amino acid introduced at PTCs during readthrough stimulated by the aminoglycoside G418 was recently studied in human HEK293 cells using a proteomic approach (16). Readthrough introduced Arg, Trp, and Cys in a 3/1/1 ratio at a UGA PTC, whereas Gln and Tyr were incorporated in a 8.5/1.5 ratio at UAG, and Gln and Tyr were inserted in equal proportions at UAA (16).…”

Proposing a mechanism of action for ataluren

“…1). This conclusion is supported by results in Roy et al (11) where they showed that the aminoglycoside, Tobramycin, which binds to the ribosomal A site (15), engendered a dose-dependent reduction of ataluren's readthrough activity. Further research investigating the physical interaction between ataluren and the ribosome, including 3D structural studies, will be important for the development of more potent ataluren derivatives in the future.…”

“…Previous studies reported that ataluren's nonsense suppression activity might be attributable to stabilization of the luciferase enzyme used as a reporter (13,14). The confirmation of ataluren's readthrough activity in numerous nonluciferase systems (including those in Roy et al) (11) and the demonstration that the hypothetical inhibitory molecule, PTC124-AMP, is rapidly converted to ataluren under conditions of in vitro translation, effectively alleviate this concern.…”

“…Combined with a previous study (12), and with the observation that similar readthrough specificities appear to be operating endogenously (albeit at much lower efficiency), the data demonstrate that the rules of near-cognate tRNA insertion at PTCs are conserved in eukaryotes. Strikingly, Roy et al (11) show that the proteins resulting from such nonsense suppression retain substantial function, notwithstanding the different amino acids inserted at the PTCs during ataluren-mediated readthrough. These proteins included CFTR (cystic fibrosis transmembrane conductance regulator), the protein defective in nonsense mutation CF.…”

“…This appears to be a unique asset of ataluren, because Roy et al (11) demonstrate that other readthrough-promoting drugs (G418 and gentamicin) generate products that deviate significantly from endogenous readthrough proteins and are therefore more likely to have dominant-negative functions or elicit an immune response.…”