title: 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.
authors.affiliation: Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA 01655-0122.|PTC Therapeutics Inc., South Plainfield, NJ 07080.
authors.forename: Bijoyita
authors.initials: B
authors.lastname: Roy
authors.affiliation: PTC Therapeutics Inc., South Plainfield, NJ 07080.
authors.forename: Westley J
authors.initials: WJ
authors.lastname: Friesen
authors.affiliation: PTC Therapeutics Inc., South Plainfield, NJ 07080.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Tomizawa
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655-0122.
authors.forename: John D
authors.initials: JD
authors.lastname: Leszyk
authors.affiliation: PTC Therapeutics Inc., South Plainfield, NJ 07080.
authors.forename: Jin
authors.initials: J
authors.lastname: Zhuo
authors.affiliation: PTC Therapeutics Inc., South Plainfield, NJ 07080.
authors.forename: Briana
authors.initials: B
authors.lastname: Johnson
authors.affiliation: PTC Therapeutics Inc., South Plainfield, NJ 07080.
authors.forename: Jumana
authors.initials: J
authors.lastname: Dakka
authors.affiliation: PTC Therapeutics Inc., South Plainfield, NJ 07080.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Trotta
authors.affiliation: PTC Therapeutics Inc., South Plainfield, NJ 07080.|Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294.|Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL 35294.
authors.forename: Xiaojiao
authors.initials: X
authors.lastname: Xue
authors.affiliation: Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL 35294.|Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294.
authors.forename: Venkateshwar
authors.initials: V
authors.lastname: Mutyam
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294.|Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL 35294.
authors.forename: Kim M
authors.initials: KM
authors.lastname: Keeling
authors.affiliation: Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294.
authors.forename: James A
authors.initials: JA
authors.lastname: Mobley
authors.affiliation: Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL 35294.|Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Rowe
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294.|Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL 35294.
authors.forename: David M
authors.initials: DM
authors.lastname: Bedwell
authors.affiliation: PTC Therapeutics Inc., South Plainfield, NJ 07080.
authors.forename: Ellen M
authors.initials: EM
authors.lastname: Welch
authors.affiliation: Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA 01655-0122; allan.jacobson@umassmed.edu.
authors.forename: Allan
authors.initials: A
authors.lastname: Jacobson
mesh_terms: D018389:Codon, Nonsense / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D010069:Oxadiazoles / Q000494:pharmacology*; D014176:Protein Biosynthesis / Q000187:drug effects; D020871:RNA Stability / Q000187:drug effects; D012343:RNA, Transfer / Q000235:genetics* / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D018389:Codon, Nonsense; D010069:Oxadiazoles; D012343:RNA, Transfer; C515878:ataluren
keywords: Translarna; base mispairing; nonsense suppression; readthrough