Nonsense mutations promote premature translational termination and cause anywhere from 5-70% of the individual cases of most inherited diseases. Studies on nonsense-mediated cystic fibrosis have indicated that boosting specific protein synthesis from <1% to as little as 5% of normal levels may greatly reduce the severity or eliminate the principal manifestations of disease. To address the need for a drug capable of suppressing premature termination, we identified PTC124-a new chemical entity that selectively induces ribosomal readthrough of premature but not normal termination codons. PTC124 activity, optimized using nonsense-containing reporters, promoted dystrophin production in primary muscle cells from humans and mdx mice expressing dystrophin nonsense alleles, and rescued striated muscle function in mdx mice within 2-8 weeks of drug exposure. PTC124 was well tolerated in animals at plasma exposures substantially in excess of those required for nonsense suppression. The selectivity of PTC124 for premature termination codons, its well characterized activity profile, oral bioavailability and pharmacological properties indicate that this drug may have broad clinical potential for the treatment of a large group of genetic disorders with limited or no therapeutic options.

Transcripts regulated by the yeast nonsense-mediated and 5' to 3' mRNA decay pathways were identified by expression profiling of wild-type, upf1Delta, nmd2Delta, upf3Delta, dcp1Delta, and xrn1Delta cells. This analysis revealed that inactivation of Upf1p, Nmd2p, or Upf3p has identical effects on global RNA accumulation; inactivation of Dcp1p or Xrn1p exhibits both common and unique effects on global RNA accumulation but causes upregulation of only a small fraction of transcripts; and the majority of transcripts upregulated in upf/nmd strains are also upregulated to similar extents in dcp1Delta and xrn1Delta strains. Our results define the core transcripts regulated by NMD, identify several novel structural classes of NMD substrates, demonstrate that nonsense-containing mRNAs are primarily degraded by the 5' to 3' decay pathway even in the absence of functional NMD, and indicate that 3' to 5' decay, not 5' to 3' decay, may be the major mRNA decay activity in yeast cells.

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Mutations gef1, stp22, STP26, and STP27 in Saccharomyces cerevisiae were identified as suppressors of the temperature-sensitive ␣-factor receptor (mutation ste2-3) and arginine permease (mutation can1 ts ). These suppressors inhibited the elimination of misfolded receptors (synthesized at 34°C) as well as damaged surface receptors (shifted from 22 to 34°C). The stp22 mutation (allelic to vps23 [M. Babst and S. Emr, personal communication] and the STP26 mutation also caused missorting of carboxypeptidase Y, and ste2-3 was suppressed by mutations vps1, vps8, vps10, and vps28 but not by mutation vps3. In the stp22 mutant, both the mutant and the wild-type receptors (tagged with green fluorescent protein [GFP]) accumulated within an endosome-like compartment and were excluded from the vacuole. GFP-tagged Stp22p also accumulated in this compartment. Upon reaching the vacuole, cytoplasmic domains of both mutant and wild-type receptors appeared within the vacuolar lumen. Stp22p and Gef1p are similar to tumor susceptibility protein TSG101 and voltage-gated chloride channel, respectively. These results identify potential elements of plasma membrane quality control and indicate that cytoplasmic domains of membrane proteins are translocated into the vacuolar lumen.Plasma membrane proteins link the interior of the cell with the extracellular environment. Removal of defective membrane proteins prevents the accumulation of damage that might otherwise compromise the ability of the cell to maintain electrochemical gradients, transport nutrients, and respond to sensory information. However, degradation of integral membrane proteins presents special problems for the cell because the internal and external sides of the protein are exposed to different biochemical environments. In eucaryotic cells, membrane proteins which have not folded or assembled properly are normally eliminated by the endoplasmic reticulum (ER) quality control process (25); however, examples of post-ER quality control are known (16,31). Degradation of defective membrane proteins in the yeast vacuole has been recognized; however, the molecular details of this process are unknown. This report describes a genetic approach toward elucidating the steps that comprise the quality control of integral plasma membrane proteins.Recent work with Saccharomyces cerevisiae has shown that temperature-sensitive forms of plasma membrane ATPase (Pma1p) (5) and ␣-factor receptors (19) are delivered directly to the vacuole, where they are degraded. In a previous report (19), we described a temperature-sensitive form of the yeast ␣-factor receptor (Ste2-3p) as a model for investigating the consequences of structural defects of integral plasma membrane proteins. Operationally, we define "misfolded receptors" as mutant receptors that are synthesized at the nonpermissive temperature, whereas "damaged cell surface receptors" are receptors that are exposed to the nonpermissive temperature only after they have been expressed at the cell surface. Misfolded receptors are delivered to the va...

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades transcripts containing premature translation termination codons. Gene expression profiling experiments have shown that inactivation of the NMD pathway leads to the accumulation of both aberrant, nonsense-containing mRNAs, and many apparently wild-type transcripts. Such increases in transcript steady-state levels could arise from direct changes in the respective mRNA half-lives, or indirectly, as a consequence of the stabilization of transcripts encoding specific regulatory proteins. Here, we distinguished direct from indirect substrates by virtue of their association with the Saccharomyces cerevisiae Upf1 protein.Analyses of this dataset, and its comparison to the sets of transcripts that respectively increase or decrease in abundance when NMD is either inactivated or reactivated, indicate that the number of direct NMD substrates is larger than previously thought and that low abundance, alternatively transcribed mRNAs, i.e., mRNAs whose 5 ends are derived from previously unannotated 5 flanking sequences, comprise a significant class of direct substrates. Using thiamine metabolism as an example, we also show that apparent NMD-regulated cellular pathways may actually reflect the detection of low-abundance alternative transcripts under conditions where a pathway is repressed.microarray analysis ͉ mRNA half-lives ͉ nonsense-mediated mRNA decay ͉ translation termination ͉ Upf proteins