Proposing a mechanism of action for ataluren.

Protein synthesis follows the advice given in Alice in Wonderland: “‘Begin at the beginning,’ the King said gravely, ‘and go on till you come to the end: then stop.’” (1). For most protein synthesis, “the beginning” is the first methionine codon (AUG) encountered by the ribosome downstream of the cap, which is situated at the 5′ end of the mRNA (Fig. 1). “The end” is a signal that consists of one of three termination or stop codons: UAG, UAA, or UGA. The path between the initiating AUG and the proper stop codon delineates the open reading frame of the encoded protein. Nonsense mutations occur when a point mutation in a sense codon, which codes for an amino acid, is altered to one of three stop codons. This results in a truncated protein product due to premature termination of mRNA translation, hence the term premature termination codon (PTC) (Fig. 1). When PTCs are encountered by the elongating ribosome, mRNAs are subjected to an accelerated degradation process through a mechanism referred to as nonsense-mediated decay (NMD) (2). NMD is a conserved surveillance mechanism that involves the recruitment of several proteins to degrade and reduce the expression of nonsense-containing mRNAs (3). This process engenders minimal expression of the defective gene, that is, reduced amounts of truncated, and usually inactive, protein are synthesized. Indeed, it is thought that NMD has evolved to reduce the generation of truncated proteins that could function in a dominantnegative manner and compromise the activity of the complexes in which they serve as subunits. Nonsense suppression is a process by which PTCs can be functionally overridden by alterations in protein synthesis that affect the efficiency of termination. For instance, whenever a ribosome positioned with a nonsense codon in its A site incorporates a nearcognate tRNA, rather than engaging the termination machinery, it bypasses translation termination and results in stop codon readthrough. PTCs encountered during protein synthesis are the underlying cause of a large number of genetic disorders caused by nonsense mutations. Approximately 10–15% of all human genetic diseases are thought to result from PTC mutations (4, 5); diseases associated with PTC mutations include cystic fibrosis, β-thalassemia, Duchenne muscular dystrophy (DMD), hemophilia, and some forms of cancer (6, 7). Ataluren, previously known as PTC124, is a bioactive molecule that is thought to modulate the translation machinery (8, 9). The compound allows for the readthrough of PTCs during mRNA translation and thereby facilitates the production of full-length functional proteins (8). This drug (under the name Translarna) has been approved in the European Union for the treatment of nonsense mutation DMD and is currently being evaluated in the clinic for treatment of other diseases, including cystic fibrosis, aniridia, and mucopolysaccharidosis I (10). In PNAS, Roy et al. (11) address the efficacy and mechanism of action of ataluren. This study shows that ataluren-mediated readthrough of different PTCs (UAG, UAA, and UGA) can be observed with multiple reporter systems in human cells as well as yeast, and identifies the specific amino acids inserted during nonsense suppression when premature termination is bypassed. Enhancement of near-cognate tRNA insertion by ataluren favors a subset of tRNAs, which generally leads to the incorporation of specific amino acids at the PTC (Gln, Lys, and Tyr at UAA and UAG codons, and Trp, Arg, and Cys at the UGA codon). These results show that tRNA selection is attributable to base mispairing at codon positions 1 and 3, that is, not just at the classic wobble position, and to the preferred use of certain nonstandard base pairs, such as U–G. 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