Current Genetics

, Volume 48, Issue 2, pp 77–87 | Cite as

Identification and characterization of upstream open reading frames (uORF) in the 5′ untranslated regions (UTR) of genes in Saccharomyces cerevisiae

Research Article

Abstract

We have taken advantage of recently sequenced hemiascomycete fungal genomes to computationally identify additional genes potentially regulated by upstream open reading frames (uORFs). Our approach is based on the observation that the structure, including the uORFs, of the post-transcriptionally uORF regulated Saccharomyces cerevisiae genes GCN4 and CPA1 is conserved in related species. Thirty-eight candidate genes for which uORFs were found in multiple species were identified and tested. We determined by 5′ RACE that 15 of these 38 genes are transcribed. Most of these 15 genes have only a single uORF in their 5′ UTR, and the length of these uORFs range from 3 to 24 codons. We cloned seven full-length UTR sequences into a luciferase (LUC) reporter system. Luciferase activity and mRNA level were compared between the wild-type UTR construct and a construct where the uORF start codon was mutated. The translational efficiency index (TEI) of each construct was calculated to test the possible regulatory function on translational level. We hypothesize that uORFs in the UTR of RPC11, TPK1, FOL1, WSC3, and MKK1 may have translational regulatory roles while uORFs in the 5′ UTR of ECM7 and IMD4 have little effect on translation under the conditions tested.

Keywords

Yeast Translation Expression Post-transcription mRNA 

Notes

Acknowledgements

We thank John E.G. McCarthy at UMIST for providing YCp22FL series plasmids. We thank Joe Heitman for providing yeast strains and Miguel Arevalo-Rodriguez, Shihua Lu, and Carol Gallione for valuable technical help and advice. We are grateful to Joe Heitman, John McCusker, Douglas Marchuk, and Bryan Cullen and for generously providing laboratory facilities and comments on the whole project. The authors also thank Mark DeLong for his careful reading and revising of the manuscript.

Supplementary material

294_2005_1_MOESM1_ESM.pdf (274 kb)
Supplementary material

References

  1. Albrecht G, Mosch HU, Hoffmann B, Reusser U, Braus GH (1998) Monitoring the Gcn4 protein-mediated response in the yeast Saccharomyces cerevisiae. J Biol Chem 273:12696–12702CrossRefPubMedGoogle Scholar
  2. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410CrossRefPubMedGoogle Scholar
  3. Brachat S, Dietrich FS, Voegeli S, Zhang Z, Stuart L, Lerch A, Gates K, Gaffney T, Philippsen P (2003) Reinvestigation of the Saccharomyces cerevisiae genome annotation by comparison to the genome of a related fungus: Ashbya gossypii. Genome Biol 4:R45CrossRefPubMedGoogle Scholar
  4. Brown CY, Mize GJ, Pineda M, George DL, Morris DR (1999) Role of two upstream open reading frames in the translational control of oncogene mdm2. Oncogene 18:5631–5637CrossRefPubMedGoogle Scholar
  5. Chedin S, Riva M, Schultz P, Sentenac A, Carles C (1998) The RNA cleavage activity of RNA polymerase III is mediated by an essential TFIIS-like subunit and is important for transcription termination. Genes Dev 12:3857–3871PubMedGoogle Scholar
  6. Cherry JM, Ball C, Weng S, Juvik G, Schmidt R, Adler C, Dunn B, Dwight S, Riles L, Mortimer RK, Botstein D (1997) Genetic and physical maps of Saccharomyces cerevisiae. Nature 387:67–73CrossRefPubMedGoogle Scholar
  7. Cliften P, Sudarsanam P, Desikan A, Fulton L, Fulton B, Majors J, Waterston R, Cohen BA, Johnston M (2003) Finding functional features in Saccharomyces genomes by phylogenetic footprinting. Science 301:71–76CrossRefPubMedGoogle Scholar
  8. Dietrich FS, Voegeli S, Brachat S, Lerch A, Gates K, Steiner S, Mohr C, Pohlmann R, Luedi P, Choi S, Wing RA, Flavier A, Gaffney TD, Philippsen P (2004) The Ashbya gossypii genome as a tool for mapping the ancient Saccharomyces cerevisiae genome. Science 304:304–307CrossRefPubMedGoogle Scholar
  9. Dujon B, Sherman D, Fischer G, Durrens P, Casaregola S, Lafontaine I, De Montigny J, Marck C, Neuveglise C, Talla E, Goffard N, Frangeul L, Aigle M, Anthouard V, Babour A, Barbe V, Barnay S, Blanchin S, Beckerich JM, Beyne E, Bleykasten C, Boisrame A, Boyer J, Cattolico L, Confanioleri F, De Daruvar A, Despons L, Fabre E, Fairhead C, Ferry-Dumazet H, Groppi A, Hantraye F, Hennequin C, Jauniaux N, Joyet P, Kachouri R, Kerrest A, Koszul R, Lemaire M, Lesur I, Ma L, Muller H, Nicaud JM, Nikolski M, Oztas S, Ozier-Kalogeropoulos O, Pellenz S, Potier S, Richard GF, Straub ML, Suleau A, Swennen D, Tekaia F, Wesolowski-Louvel M, Westhof E, Wirth B, Zeniou-Meyer M, Zivanovic I, Bolotin-Fukuhara M, Thierry A, Bouchier C, Caudron B, Scarpelli C, Gaillardin C, Weissenbach J, Wincker P, Souciet JL (2004) Genome evolution in yeasts. Nature 430:35–44CrossRefPubMedGoogle Scholar
  10. Gaba A, Wang Z, Krishnamoorthy T, Hinnebusch AG, Sachs MS (2001) Physical evidence for distinct mechanisms of translational control by upstream open reading frames. EMBO J 20:6453–6463CrossRefPubMedGoogle Scholar
  11. Gerstel B, McCarthy JE (1989) Independent and coupled translational initiation of atp genes in Escherichia coli: experiments using chromosomal and plasmid-borne lacZ fusions. Mol Microbiol 3:851–859PubMedCrossRefGoogle Scholar
  12. Goyer C, Altmann M, Lee HS, Blanc A, Deshmukh M, Woolford JL Jr, Trachsel H, Sonenberg N (1993) TIF4631 and TIF4632: two yeast genes encoding the high-molecular-weight subunits of the cap-binding protein complex (eukaryotic initiation factor 4F) contain an RNA recognition motif-like sequence and carry out an essential function. Mol Cell Biol 13:4860–4874PubMedGoogle Scholar
  13. Hampsey M (1998) Molecular genetics of the RNA polymerase II general transcriptional machinery. Microbiol Mol Biol Rev 62:465–503PubMedGoogle Scholar
  14. Harigai M, Miyashita T, Hanada M, Reed JC (1996) A cis-acting element in the BCL-2 gene controls expression through translational mechanisms. Oncogene 12:1369–1374PubMedGoogle Scholar
  15. Hashimoto S, Suzuki Y, Kasai Y, Morohoshi K, Yamada T, Sese J, Morishita S, Sugano S, Matsushima K (2004) 5′-end SAGE for the analysis of transcriptional start sites. Nat Biotechnol 22:1146–1149CrossRefPubMedGoogle Scholar
  16. van den Heuvel JJ, Bergkamp RJ, Planta RJ, Raue HA (1989) Effect of deletions in the 5′-noncoding region on the translational efficiency of phosphoglycerate kinase mRNA in yeast. Gene 79:83–95CrossRefPubMedGoogle Scholar
  17. Hinnebusch AG (1997) Translational regulation of yeast GCN4. A window on factors that control initiator-tRNA binding to the ribosome. J Biol Chem 272:21661–21664CrossRefPubMedGoogle Scholar
  18. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59CrossRefPubMedGoogle Scholar
  19. Hoffmann B, Valerius O, Andermann M, Braus GH (2001) Transcriptional autoregulation and inhibition of mRNA translation of amino acid regulator gene cpcA of filamentous fungus Aspergillus nidulans. Mol Biol Cell 12:2846–2857PubMedGoogle Scholar
  20. Holstege FC, Jennings EG, Wyrick JJ, Lee TI, Hengartner CJ, Green MR, Golub TR, Lander ES, Young RA (1998) Dissecting the regulatory circuitry of a eukaryotic genome. Cell 95:717–728CrossRefPubMedGoogle Scholar
  21. Iizuka N, Najita L, Franzusoff A, Sarnow P (1994) Cap-dependent and cap-independent translation by internal initiation of mRNAs in cell extracts prepared from Saccharomyces cerevisiae. Mol Cell Biol 14:7322–7330PubMedGoogle Scholar
  22. Kellis M, Patterson N, Endrizzi M, Birren B, Lander ES (2003) Sequencing and comparison of yeast species to identify genes and regulatory elements. Nature 423:241–254CrossRefPubMedGoogle Scholar
  23. Kellis M, Birren BW, Lander ES (2004) Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae. Nature 428:617–624CrossRefPubMedGoogle Scholar
  24. Komar AA, Lesnik T, Cullin C, Merrick WC, Trachsel H, Altmann M (2003) Internal initiation drives the synthesis of Ure2 protein lacking the prion domain and affects [URE3] propagation in yeast cells. EMBO J 22:1199–1209CrossRefPubMedGoogle Scholar
  25. Kozak M (2002) Pushing the limits of the scanning mechanism for initiation of translation. Gene 299:1–34CrossRefPubMedGoogle Scholar
  26. Lodder AL, Lee TK, Ballester R (1999) Characterization of the Wsc1 protein, a putative receptor in the stress response of Saccharomyces cerevisiae. Genetics 152:1487–1499PubMedGoogle Scholar
  27. McIntosh EM, Haynes RH (1986) Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae. Mol Cell Biol 6:1711–1721PubMedGoogle Scholar
  28. Messenguy F, Vierendeels F, Pierard A, Delbecq P (2002) Role of RNA surveillance proteins Upf1/CpaR, Upf2 and Upf3 in the translational regulation of yeast CPA1 gene. Curr Genet 41:224–231CrossRefPubMedGoogle Scholar
  29. Miyasaka H (1999) The positive relationship between codon usage bias and translation initiation AUG context in Saccharomyces cerevisiae. Yeast 15:633–637CrossRefPubMedGoogle Scholar
  30. Oliveira CC, van den Heuvel JJ, McCarthy JE (1993) Inhibition of translational initiation in Saccharomyces cerevisiae by secondary structure: the roles of the stability and position of stem-loops in the mRNA leader. Mol Microbiol 9:521–532PubMedCrossRefGoogle Scholar
  31. Pestova TV, Kolupaeva VG, Lomakin IB, Pilipenko EV, Shatsky IN, Agol VI, Hellen CU (2001) Molecular mechanisms of translation initiation in eukaryotes. Proc Natl Acad Sci USA 98:7029–7036CrossRefPubMedGoogle Scholar
  32. Polymenis M, Schmidt EV (1997) Coupling of cell division to cell growth by translational control of the G1 cyclin CLN3 in yeast. Genes Dev 11:2522–2531PubMedGoogle Scholar
  33. Ruiz-Echevarria MJ, Peltz SW (2000) The RNA binding protein Pub1 modulates the stability of transcripts containing upstream open reading frames. Cell 101:741–751CrossRefPubMedGoogle Scholar
  34. Schumperli D, McKenney K, Sobieski DA, Rosenberg M (1982) Translational coupling at an intercistronic boundary of the Escherichia coli galactose operon. Cell 30:865–871CrossRefPubMedGoogle Scholar
  35. Sherman D, Durrens P, Beyne E, Nikolski M, Souciet JL (2004) Genolevures: comparative genomics and molecular evolution of hemiascomycetous yeasts. Nucleic Acids Res 32(Database issue):D315–D318CrossRefPubMedGoogle Scholar
  36. Shiraki T, Kondo S, Katayama S, Waki K, Kasukawa T, Kawaji H, Kodzius R, Watahiki A, Nakamura M, Arakawa T, Fukuda S, Sasaki D, Podhajska A, Harbers M, Kawai J, Carninci P, Hayashizaki Y (2003) Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage. Proc Natl Acad Sci USA 100:15776–15781CrossRefPubMedGoogle Scholar
  37. Steel LF, Telly DL, Leonard J, Rice BA, Monks B, Sawicki JA (1996) Elements in the murine c-mos messenger RNA 5′-untranslated region repress translation of downstream coding sequences. Cell Growth Differ 7:1415–1424PubMedGoogle Scholar
  38. Vattem KM, Wek RC (2004) Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc Natl Acad Sci USA 101:11269–11274CrossRefPubMedGoogle Scholar
  39. Verge V, Vonlanthen M, Masson JM, Trachsel H, Altmann M (2004) Localization of a promoter in the putative internal ribosome entry site of the Saccharomyces cerevisiae TIF4631 gene. RNA 10:277–286CrossRefPubMedGoogle Scholar
  40. Verna J, Lodder A, Lee K, Vagts A, Ballester R (1997) A family of genes required for maintenance of cell wall integrity and for the stress response in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 94:13804–13809CrossRefPubMedGoogle Scholar
  41. Vilela C, McCarthy JE (2003) Regulation of fungal gene expression via short open reading frames in the mRNA 5′ untranslated region. Mol Microbiol 49:859–867CrossRefPubMedGoogle Scholar
  42. Vilela C, Linz B, Rodrigues-Pousada C, McCarthy JE (1998) The yeast transcription factor genes YAP1 and YAP2 are subject to differential control at the levels of both translation and mRNA stability. Nucleic Acids Res 26:1150–1159CrossRefPubMedGoogle Scholar
  43. Vilela C, Ramirez CV, Linz B, Rodrigues-Pousada C, McCarthy JE (1999) Post-termination ribosome interactions with the 5′ UTR modulate yeast mRNA stability. EMBO J 18:3139–3152CrossRefPubMedGoogle Scholar
  44. Wang Z, Sachs MS (1997) Ribosome stalling is responsible for arginine-specific translational attenuation in Neurospora crassa. Mol Cell Biol 17:4904–4913PubMedGoogle Scholar
  45. Washburn MP, Koller A, Oshiro G, Ulaszek RR, Plouffe D, Deciu C, Winzeler E, Yates JR III (2003) Protein pathway and complex clustering of correlated mRNA and protein expression analyses in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 100:3107–3112CrossRefPubMedGoogle Scholar
  46. Wei CL, Ng P, Chiu KP, Wong CH, Ang CC, Lipovich L, Liu ET, Ruan Y (2004) 5′ Long serial analysis of gene expression (LongSAGE) and 3′ LongSAGE for transcriptome characterization and genome annotation. Proc Natl Acad Sci USA 101:11701–11706CrossRefPubMedGoogle Scholar
  47. Werner M, Feller A, Messenguy F, Pierard A (1987) The leader peptide of yeast gene CPA1 is essential for the translational repression of its expression. Cell 49:805–813CrossRefPubMedGoogle Scholar
  48. Wojda I, Alonso-Monge R, Bebelman JP, Mager WH, Siderius M (2003) Response to high osmotic conditions and elevated temperature in Saccharomyces cerevisiae is controlled by intracellular glycerol and involves coordinate activity of MAP kinase pathways. Microbiology 149:1193–1204CrossRefPubMedGoogle Scholar
  49. Zhang Z, Dietrich FS (2003) Verification of a new gene on Saccharomyces cerevisiae chromosome III. Yeast 20:731–738CrossRefPubMedGoogle Scholar
  50. Zhang Z, Dietrich FS (2005) Mapping of transcription start sites in Saccharomyces cerevisiae using 5′ SAGE. Nucleic Acids Res 33:2838–2851CrossRefPubMedGoogle Scholar
  51. Zhou W, Edelman GM, Mauro VP (2001) Transcript leader regions of two Saccharomyces cerevisiae mRNAs contain internal ribosome entry sites that function in living cells. Proc Natl Acad Sci USA 98:1531–1536CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  1. 1.Department of Molecular Genetics and MicrobiologyDuke University Medical CenterDurhamUSA

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