The efficiency of translation termination depends on the nature of the stop codon and the surrounding nucleotides. Some molecules, such as aminoglycoside antibiotics (gentamicin), decrease termination efficiency and are currently being evaluated for diseases caused by premature termination codons. However, the readthrough response to treatment is highly variable and little is known about the rules governing readthrough level and response to aminoglycosides. In this study, we carried out in-depth statistical analysis on a very large set of nonsense mutations to decipher the elements of nucleotide context responsible for modulating readthrough levels and gentamicin response. We quantified readthrough for 66 sequences containing a stop codon, in the presence and absence of gentamicin, in cultured mammalian cells. We demonstrated that the efficiency of readthrough after treatment is determined by the complex interplay between the stop codon and a larger sequence context. There was a strong positive correlation between basal and induced readthrough levels, and a weak negative correlation between basal readthrough level and gentamicin response (i.e. the factor of increase from basal to induced readthrough levels). The identity of the stop codon did not affect the response to gentamicin treatment. In agreement with a previous report, we confirm that the presence of a cytosine in +4 position promotes higher basal and gentamicin-induced readthrough than other nucleotides. We highlight for the first time that the presence of a uracil residue immediately upstream from the stop codon is a major determinant of the response to gentamicin. Moreover, this effect was mediated by the nucleotide itself, rather than by the amino-acid or tRNA corresponding to the −1 codon. Finally, we point out that a uracil at this position associated with a cytosine at +4 results in an optimal gentamicin-induced readthrough, which is the therapeutically relevant variable.
Vyšlo v časopise: Statistical Analysis of Readthrough Levels for Nonsense Mutations in Mammalian Cells Reveals a Major Determinant of Response to Gentamicin. PLoS Genet 8(3): e32767. doi:10.1371/journal.pgen.1002608 Kategorie: Research Article prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1002608
1. BertramGInnesSMinellaORichardsonJStansfieldI 2001 Endless possibilities: translation termination and stop codon recognition. Microbiology 147 255 269
2. KisselevLEhrenbergMFrolovaL 2003 Termination of translation: interplay of mRNA, rRNAs and release factors? EMBO J 22 175 182
3. BidouLHatinIPerezNAllamandVPanthierJJ 2004 Premature stop codons involved in muscular dystrophies show a broad spectrum of readthrough efficiencies in response to gentamicin treatment. Gene Ther 11 619 627
4. HowardMTShirtsBHPetrosLMFlaniganKMGestelandRF 2000 Sequence specificity of aminoglycoside-induced stop condon readthrough: potential implications for treatment of Duchenne muscular dystrophy. Ann Neurol 48 164 169
5. KeelingKMBedwellDM 2002 Clinically relevant aminoglycosides can suppress disease-associated premature stop mutations in the IDUA and P53 cDNAs in a mammalian translation system. J Mol Med 80 367 376
6. NamyOHatinIRoussetJP 2001 Impact of the six nucleotides downstream of the stop codon on translation termination. EMBO Rep 2 787 793
7. BonettiBFuLMoonJBedwellDM 1995 The efficiency of translation termination is determined by a synergistic interplay between upstream and downstream sequences in Saccharomyces cerevisiae. J Mol Biol 251 334 345
8. TorkSHatinIRoussetJPFabretC 2004 The major 5′ determinant in stop codon read-through involves two adjacent adenines. Nucleic Acids Res 32 415 421
9. BurkeJFMoggAE 1985 Suppression of a nonsense mutation in mammalian cells in vivo by the aminoglycoside antibiotics G-418 and paromomycin. Nucleic Acids Res 13 6265 6272
10. MartinRMoggAEHeywoodLANitschkeLBurkeJF 1989 Aminoglycoside suppression at UAG, UAA and UGA codons in Escherichia coli and human tissue culture cells. Mol Gen Genet 217 411 418
11. CarterAPClemonsWMBrodersenDEMorgan-WarrenRJWimberlyBT 2000 Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature 407 340 348
12. OgleJMCarterAPRamakrishnanV 2003 Insights into the decoding mechanism from recent ribosome structures. Trends Biochem Sci 28 259 266
13. LindeLKeremB 2008 Introducing sense into nonsense in treatments of human genetic diseases. Trends Genet 24 552 563
14. ZingmanLVParkSOlsonTMAlekseevAETerzicA 2007 Aminoglycoside-induced translational read-through in disease: overcoming nonsense mutations by pharmacogenetic therapy. Clin Pharmacol Ther 81 99 103
15. HowardMTAndersonCBFassUKhatriSGestelandRF 2004 Readthrough of dystrophin stop codon mutations induced by aminoglycosides. Ann Neurol 55 422 426
16. AllamandVBidouLArakawaMFloquetCShiozukaM 2008 Drug-induced readthrough of premature stop codons leads to the stabilization of laminin alpha2 chain mRNA in CMD myotubes. J Gene Med 10 217 224
17. TateWPManneringSA 1996 Three, four or more: the translational stop signal at length. Mol Microbiol 21 213 219
18. BulyginKNRepkovaMNVen'yaminovaAGGraiferDMKarpovaGG 2002 Positioning of the mRNA stop signal with respect to polypeptide chain release factors and ribosomal proteins in 80S ribosomes. FEBS Lett 514 96 101
19. Sermet-GaudelusIRenouilMFajacABidouLParbailleB 2007 In vitro prediction of stop-codon suppression by intravenous gentamicin in patients with cystic fibrosis: a pilot study. BMC Med 5 5
20. FloquetCDeforgesJRoussetJPBidouL 2011 Rescue of non-sense mutated p53 tumor suppressor gene by aminoglycosides. Nucleic Acids Res 39 3350 3362
21. FloquetCRoussetJPBidouL 2011 Readthrough of premature termination codons in the adenomatous polyposis coli gene restores its biological activity in human cancer cells. PLoS ONE 6 e24125 doi:10.1371/journal.pone.0024125
22. BidouLStahlGHatinINamyORoussetJP 2000 Nonsense-mediated decay mutants do not affect programmed −1 frameshifting. RNA 6 952 961
23. DunantPWalterMCKarpatiGLochmullerH 2003 Gentamicin fails to increase dystrophin expression in dystrophin-deficient muscle. Muscle Nerve 27 624 627
24. ManuvakhovaMKeelingKBedwellDM 2000 Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system. RNA 6 1044 1055
25. NudelmanIRebibo-SabbahACherniavskyMBelakhovVHainrichsonM 2009 Development of novel aminoglycoside (NB54) with reduced toxicity and enhanced suppression of disease-causing premature stop mutations. J Med Chem 52 2836 2845
26. MattisVBEbertADFossoMYChangCWLorsonCL 2009 Delivery of a read-through inducing compound, TC007, lessens the severity of a spinal muscular atrophy animal model. Hum Mol Genet 18 3906 3913
27. NudelmanIGlikinDSmolkinBHainrichsonMBelakhovV 2010 Repairing faulty genes by aminoglycosides: Development of new derivatives of geneticin (G418) with enhanced suppression of diseases-causing nonsense mutations. Bioorg Med Chem
28. Mottagui-TabarSIsakssonLA 1998 The influence of the 5′ codon context on translation termination in Bacillus subtilis and Escherichia coli is similar but different from Salmonella typhimurium. Gene 212 189 196
29. Mottagui-TabarSTuiteMFIsakssonLA 1998 The influence of 5′ codon context on translation termination in Saccharomyces cerevisiae. Eur J Biochem 257 249 254
30. CassanMRoussetJP 2001 UAG readthrough in mammalian cells: effect of upstream and downstream stop codon contexts reveal different signals. BMC Mol Biol 2 3
31. SmithDYarusM 1989 tRNA-tRNA interactions within cellular ribosomes. Proc Natl Acad Sci U S A 86 4397 4401
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