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

Author:

Roy Bijoyita,Friesen Westley J.,Tomizawa Yuki,Leszyk John D.,Zhuo Jin,Johnson Briana,Dakka Jumana,Trotta Christopher R.,Xue Xiaojiao,Mutyam Venkateshwar,Keeling Kim M.,Mobley James A.,Rowe Steven M.,Bedwell David M.,Welch Ellen M.,Jacobson Allan

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.

Funder

HHS | NIH | National Institute of General Medical Sciences

HHS | NIH | National Institute of Neurological Disorders and Stroke

HHS | NIH | National Institute of Diabetes and Digestive and Kidney Diseases

Cystic Fibrosis Foundation

HHS | NIH | National Cancer Institute

Publisher

Proceedings of the National Academy of Sciences

Subject

Multidisciplinary

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