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Molecular Genetics and Genomics

, Volume 272, Issue 3, pp 297–307 | Cite as

Nonsense mutations in the essential gene SUP35 of Saccharomyces cerevisiae are non-lethal

  • S. Chabelskaya
  • D. Kiktev
  • S. Inge-Vechtomov
  • M. Philippe
  • G. ZhouravlevaEmail author
Original Paper

Abstract

In the present work we have characterized for the first time non-lethal nonsense mutations in the essential gene SUP35, which codes for the translation termination factor eRF3 in Saccharomyces cerevisiae. The screen used was based on selection for simultaneous suppression of two auxotrophic nonsense mutations. Among 48 mutants obtained, sixteen were distinguished by the production of a reduced amount of eRF3, suggesting the appearance of nonsense mutations. Fifteen of the total mutants were sequenced, and the presence of nonsense mutations was confirmed for nine of them. Thus a substantial fraction of the sup35 mutations recovered are nonsense mutations located in different regions of SUP35, and such mutants are easily identified by the fact that they express reduced amounts of eRF3. Nonsense mutations in the SUP35 gene do not lead to a decrease in levels of SUP35 mRNA and do not influence the steady-state level of eRF1. The ability of these mutations to complement SUP35 gene disruption mutations in different genetic backgrounds and in the absence of any tRNA suppressor mutation was demonstrated. The missense mutations studied, unlike nonsense mutations, do not decrease steady-state amounts of eRF3.

Keywords

Yeast Translation termination Release factor eRF3 Readthrough 

Notes

Acknowledgements

The work was supported by grants from CRDF ST-012, CRDF N RB1-2336-ST-02, RFBR 03-04-48886 and to S.C. from NATO, ESF 2002/EG02, the Ministry of Education of the Russian Federation A03-2.12-2. We are very grateful to K. Gull for anti-tubulin antibodies, to K. Volkov for plasmids and strains and to H.B. Osborne for critical reading of the manuscript.

References

  1. Beier H, Grimm M (2001) Misreading of termination codons in eukaryotes by natural nonsense suppressor tRNAs. Nucleic Acids Res 29:4767–4782CrossRefPubMedGoogle Scholar
  2. Bertram G, Innes S, Minella O, Richardson J, Stansfield I (2001) Endless possibilities: translation termination and stop codon recognition. Microbiology 147:255–269PubMedGoogle Scholar
  3. Boeck R, Lapeyre B, Brown CE, Sachs AB (1998) Capped mRNA degradation intermediates accumulate in the yeast spb8-2 mutant. Mol Cell Biol 18:5062–5072PubMedGoogle Scholar
  4. Boeke JD, Lacroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197:345–346PubMedGoogle Scholar
  5. Bonetti B, Fu L, Moon J, Bedwell DM (1995) The efficiency of translation termination is determined by a synergistic interplay between upstream and downstream sequences in Saccharomyces cerevisiae. J Mol Biol 251:334–345CrossRefPubMedGoogle Scholar
  6. Bradley ME, Bagriantsev S, Vishveshwara N, Liebman SW (2003) Guanidine reduces stop codon read-through caused by missense mutations in SUP35 or SUP45. Yeast 20:625–632CrossRefPubMedGoogle Scholar
  7. Cai T, Reilly TR, Cerio M, Schmitt ME (1999) Mutagenesis of SNM1, which encodes a protein component of the yeast RNase MRP, reveals a role for this ribonucleoprotein endoribonuclease in plasmid segregation. Mol Cell Biol 19:7857–7869PubMedGoogle Scholar
  8. Carr-Schmid A, Durko N, Cavallius J, Merrick WC, Kinzy TG (1999) Mutations in a GTP-binding motif of eukaryotic elongation factor 1A reduce both translational fidelity and the requirement for nucleotide exchange. J Biol Chem 274:30297–30302CrossRefPubMedGoogle Scholar
  9. Chernoff YO (2001) Mutation processes at the protein level: is Lamarck back? Mutat Res 488:39–64CrossRefPubMedGoogle Scholar
  10. Cosson B, Couturier A, Chabelskaya S, Kiktev D, Inge-Vechtomov S, Philippe M, Zhouravleva G (2002) Poly(A)-binding protein acts in translation termination via eukaryotic release factor 3 interaction and does not influence [PSI(+)] propagation. Mol Cell Biol 22:3301–3315CrossRefPubMedGoogle Scholar
  11. Eurwilaichitr L, Graves FM, Stansfield I, Tuite MF (1999) The C-terminus of eRF1 defines a functionally important domain for translation termination in Saccharomyces cerevisiae. Mol Microbiol 32:485–496CrossRefPubMedGoogle Scholar
  12. Fearon K, McClendon V, Bonetti B, Bedwell DM (1994) Premature translation termination mutations are efficiently suppressed in a highly conserved region of yeast Ste6p, a member of the ATP-binding cassette (ABC) transporter family. J Biol Chem 269:17802–17808PubMedGoogle Scholar
  13. Gietz D, St Jean A, Woods RA, Schiestl RH (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425PubMedGoogle Scholar
  14. Harrell L, Melcher U, Atkins JF (2002) Predominance of six different hexanucleotide recoding signals 3′ of read-through stop codons. Nucleic Acids Res 30:2011–2017CrossRefPubMedGoogle Scholar
  15. Inge-Vechtomov S, Zhouravleva G, Philippe M (2003) Eukaryotic release factors (eRFs) history. Biol Cell 95:195–209CrossRefPubMedGoogle Scholar
  16. Ito-Harashima S, Hartzog PE, Sinha H, McCusker JH (2002) The tRNA-Tyr gene family of Saccharomyces cerevisiae: agents of phenotypic variation and position effects on mutation frequency. Genetics 161:1395–1410PubMedGoogle Scholar
  17. Kisselev L, Ehrenberg M, Frolova L (2003) Termination of translation: interplay of mRNA, rRNAs and release factors? EMBO J 22:175–182CrossRefPubMedGoogle Scholar
  18. Kokoska RJ, Stefanovic L, Buermeyer AB, Liskay RM, Petes TD (1999) A mutation of the yeast gene encoding PCNA destabilizes both microsatellite and minisatellite DNA sequences. Genetics 151:511–519PubMedGoogle Scholar
  19. Kong C, Ito K, Walsh MA, Wada M, Liu Y, Kumar S, Barford D, Nakamura Y, Song H (2004) Crystal structure and functional analysis of the eukaryotic Class II release factor eRF3 from S. pombe. Mol Cell 14:233–245Google Scholar
  20. Kopczynski JB, Raff AC, Bonner JJ (1992) Translational readthrough at nonsense mutations in the HSF1 gene of Saccharomyces cerevisiae. Mol Gen Genet 234:369–378PubMedGoogle Scholar
  21. Liu JJ, Sondheimer N, Lindquist SL (2002) Changes in the middle region of Sup35 profoundly alter the nature of epigenetic inheritance for the yeast prion [PSI+]. Proc Natl Acad Sci USA 99 Suppl 4:16446–16453CrossRefGoogle Scholar
  22. Moskalenko SE, Chabelskaya SV, Inge-Vechtomov SG, Philippe M, Zhouravleva GA (2003) Viable nonsense mutants for the essential gene SUP45 of Saccharomyces cerevisiae. BMC Mol Biol 4:2CrossRefPubMedGoogle Scholar
  23. Percudani R, Pavesi A, Ottonello S (1997) Transfer RNA gene redundancy and translational selection in Saccharomyces cerevisiae. J Mol Biol 268:322–330CrossRefPubMedGoogle Scholar
  24. Pure GA, Robinson GW, Naumovski L, Friedberg EC (1985) Partial suppression of an ochre mutation in Saccharomyces cerevisiae by multicopy plasmids containing a normal yeast tRNAGln gene. J Mol Biol 183:31–42PubMedGoogle Scholar
  25. Riles L, Olson MV (1988) Nonsense mutations in essential genes of Saccharomyces cerevisiae. Genetics 118:601–607PubMedGoogle Scholar
  26. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Google Scholar
  27. Serio TR, Lindquist SL (1999) [PSI+]: an epigenetic modulator of translation termination efficiency. Annu Rev Cell Dev Biol 15:661–703Google Scholar
  28. Sherman F, Fink GR, Hicks JB (1986) Laboratory course manual for methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
  29. Song JM, Liebman SW (1985) Interaction of UAG suppressors and omnipotent suppressors in Saccharomyces cerevisiae. J Bacteriol 161:778–780PubMedGoogle Scholar
  30. Stansfield I, Jones KM, Kushnirov VV, Dagkesamanskaya AR, Poznyakovski AI, Paushkin SV, Nierras CR, Cox BS, Ter-Avanesyan MD, Tuite MF (1995) The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae. EMBO J 14:4365–4373PubMedGoogle Scholar
  31. Stansfield I, Eurwilaichitr L, Akhmaloka, Tuite MF (1996) Depletion in the levels of the release factor eRF1 causes a reduction in the efficiency of translation termination in yeast. Mol Microbiol 20:1135–1143PubMedGoogle Scholar
  32. Ter-Avanesyan MD, Kushnirov VV, Dagkesamanskaya AR, Didichenko SA, Chernoff YO, Inge-Vechtomov SG, Smirnov VN (1993) Deletion analysis of the SUP35 gene of the yeast Saccharomyces cerevisiae reveals two non-overlapping functional regions in the encoded protein. Mol Microbiol 7:683–692PubMedGoogle Scholar
  33. Tork S, Hatin I, Rousset JP, Fabret C (2004) The major 5′ determinant in stop codon read-through involves two adjacent adenines. Nucleic Acids Res 32:415–421Google Scholar
  34. Valouev IA, Kushnirov VV, Ter-Avanesyan MD (2002) Yeast polypeptide chain release factors eRF1 and eRF3 are involved in cytoskeleton organization and cell cycle regulation. Cell Motil Cytoskeleton 52:161–173CrossRefPubMedGoogle Scholar
  35. Volkov KV, Aksenova AY, Soom MJ, Osipov KV, Svitin AV, Kurischko C, Shkundina IS, Ter-Avanesyan MD, Inge-Vechtomov SG, Mironova LN (2002) Novel non-Mendelian determinant involved in the control of translation accuracy in Saccharomyces cerevisiae. Genetics 160:25–36PubMedGoogle Scholar
  36. Wang W, Czaplinski K, Rao Y, Peltz SW (2001) The role of Upf proteins in modulating the translation read-through of nonsense-containing transcripts. EMBO J 20:880–890CrossRefPubMedGoogle Scholar
  37. Weiss WA, Friedberg EC (1986) Normal yeast tRNA(CAGGln) can suppress amber codons and is encoded by an essential gene. J Mol Biol 192:725–735PubMedGoogle Scholar
  38. Weiss WA, Edelman I, Culbertson MR, Friedberg EC (1987) Physiological levels of normal tRNA(CAGGln) can effect partial suppression of amber mutations in the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci USA 84:8031–8034PubMedGoogle Scholar
  39. Woods A, Sherwin T, Sasse R, MacRae TH, Baines AJ, Gull K (1989) Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies. J Cell Sci 93:491–500PubMedGoogle Scholar
  40. Zhou P, Derkatch IL, Uptain SM, Patino MM, Lindquist S, Liebman SW (1999) The yeast non-Mendelian factor [ETA+] is a variant of [PSI+], a prion-like form of release factor eRF3. EMBO J 18:1182–1191CrossRefPubMedGoogle Scholar
  41. Zhouravleva G, Frolova L, Le Goff X, Le Guellec R, Inge-Vechtomov S, Kisselev L, Philippe M (1995) Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3. EMBO J 14:4065–4072PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • S. Chabelskaya
    • 1
    • 2
  • D. Kiktev
    • 1
  • S. Inge-Vechtomov
    • 1
  • M. Philippe
    • 2
  • G. Zhouravleva
    • 1
    • 2
    Email author
  1. 1.Department of Genetics and BreedingSt Petersburg State UniversitySt PetersburgRussia
  2. 2.CNRS UMR 6061, IFR 97Université de Rennes 1Rennes CedexFrance

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