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. 1993 Nov 25;21(23):5316–5322. doi: 10.1093/nar/21.23.5316

Translational repression by the human iron-regulatory factor (IRF) in Saccharomyces cerevisiae.

C C Oliveira 1, B Goossen 1, N I Zanchin 1, J E McCarthy 1, M W Hentze 1, R Stripecke 1
PMCID: PMC310564  PMID: 8265343

Abstract

The regulation of the synthesis of ferritin and erythroid 5-aminolevulinate synthase in mammalian cells is mediated by the interaction of the iron regulatory factor (IRF) with a specific recognition site, the iron responsive element (IRE), in the 5' untranslated regions (UTRs) of the respective mRNAs. A new modular expression system was designed to allow reconstruction of this regulatory system in Saccharomyces cerevisiae. This comprised two components: a constitutively expressed reporter gene (luc; encoding luciferase) preceded by a 5' UTR including an IRE sequence, and an inducibly expressed cDNA encoding human IRF. Induction of the latter led to the in vivo synthesis of IRF, which in turn showed IRE-binding activity and also repressed translation of the luc mRNA bearing an IRE-containing 5' UTR. The upper stem-loop region of an IRE, with no further IRE-specific flanking sequences, sufficed for recognition and repression by IRF. Translational regulation of IRE-bearing mRNAs could also be demonstrated in cell-free yeast extracts. This work defines a minimal system for IRF/IRE translational regulation in yeast that requires no additional mammalian-specific components, thus providing direct proof that IRF functions as a translational repressor in vivo. It should be a useful tool as the basis for more detailed studies of eukaryotic translational regulation.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aziz N., Munro H. N. Iron regulates ferritin mRNA translation through a segment of its 5' untranslated region. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8478–8482. doi: 10.1073/pnas.84.23.8478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brown P. H., Daniels-McQueen S., Walden W. E., Patino M. M., Gaffield L., Bielser D., Thach R. E. Requirements for the translational repression of ferritin transcripts in wheat germ extracts by a 90-kDa protein from rabbit liver. J Biol Chem. 1989 Aug 15;264(23):13383–13386. [PubMed] [Google Scholar]
  3. Casey J. L., Koeller D. M., Ramin V. C., Klausner R. D., Harford J. B. Iron regulation of transferrin receptor mRNA levels requires iron-responsive elements and a rapid turnover determinant in the 3' untranslated region of the mRNA. EMBO J. 1989 Dec 1;8(12):3693–3699. doi: 10.1002/j.1460-2075.1989.tb08544.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Coccia E. M., Profita V., Fiorucci G., Romeo G., Affabris E., Testa U., Hentze M. W., Battistini A. Modulation of ferritin H-chain expression in Friend erythroleukemia cells: transcriptional and translational regulation by hemin. Mol Cell Biol. 1992 Jul;12(7):3015–3022. doi: 10.1128/mcb.12.7.3015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dandekar T., Stripecke R., Gray N. K., Goossen B., Constable A., Johansson H. E., Hentze M. W. Identification of a novel iron-responsive element in murine and human erythroid delta-aminolevulinic acid synthase mRNA. EMBO J. 1991 Jul;10(7):1903–1909. doi: 10.1002/j.1460-2075.1991.tb07716.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dix D. J., Lin P. N., McKenzie A. R., Walden W. E., Theil E. C. The influence of the base-paired flanking region on structure and function of the ferritin mRNA iron regulatory element. J Mol Biol. 1993 May 20;231(2):230–240. doi: 10.1006/jmbi.1993.1278. [DOI] [PubMed] [Google Scholar]
  7. Gerstel B., Tuite M. F., McCarthy J. E. The effects of 5'-capping, 3'-polyadenylation and leader composition upon the translation and stability of mRNA in a cell-free extract derived from the yeast Saccharomyces cerevisiae. Mol Microbiol. 1992 Aug;6(16):2339–2348. doi: 10.1111/j.1365-2958.1992.tb01409.x. [DOI] [PubMed] [Google Scholar]
  8. Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
  9. Goossen B., Caughman S. W., Harford J. B., Klausner R. D., Hentze M. W. Translational repression by a complex between the iron-responsive element of ferritin mRNA and its specific cytoplasmic binding protein is position-dependent in vivo. EMBO J. 1990 Dec;9(12):4127–4133. doi: 10.1002/j.1460-2075.1990.tb07635.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Goossen B., Hentze M. W. Position is the critical determinant for function of iron-responsive elements as translational regulators. Mol Cell Biol. 1992 May;12(5):1959–1966. doi: 10.1128/mcb.12.5.1959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hentze M. W., Argos P. Homology between IRE-BP, a regulatory RNA-binding protein, aconitase, and isopropylmalate isomerase. Nucleic Acids Res. 1991 Apr 25;19(8):1739–1740. doi: 10.1093/nar/19.8.1739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hentze M. W., Caughman S. W., Rouault T. A., Barriocanal J. G., Dancis A., Harford J. B., Klausner R. D. Identification of the iron-responsive element for the translational regulation of human ferritin mRNA. Science. 1987 Dec 11;238(4833):1570–1573. doi: 10.1126/science.3685996. [DOI] [PubMed] [Google Scholar]
  13. Hentze M. W., Rouault T. A., Harford J. B., Klausner R. D. Oxidation-reduction and the molecular mechanism of a regulatory RNA-protein interaction. Science. 1989 Apr 21;244(4902):357–359. doi: 10.1126/science.2711187. [DOI] [PubMed] [Google Scholar]
  14. Hirling H., Emery-Goodman A., Thompson N., Neupert B., Seiser C., Kühn L. C. Expression of active iron regulatory factor from a full-length human cDNA by in vitro transcription/translation. Nucleic Acids Res. 1992 Jan 11;20(1):33–39. doi: 10.1093/nar/20.1.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jobling S. A., Cuthbert C. M., Rogers S. G., Fraley R. T., Gehrke L. In vitro transcription and translational efficiency of chimeric SP6 messenger RNAs devoid of 5' vector nucleotides. Nucleic Acids Res. 1988 May 25;16(10):4483–4498. doi: 10.1093/nar/16.10.4483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kennedy M. C., Mende-Mueller L., Blondin G. A., Beinert H. Purification and characterization of cytosolic aconitase from beef liver and its relationship to the iron-responsive element binding protein. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11730–11734. doi: 10.1073/pnas.89.24.11730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Klausner R. D., Rouault T. A., Harford J. B. Regulating the fate of mRNA: the control of cellular iron metabolism. Cell. 1993 Jan 15;72(1):19–28. doi: 10.1016/0092-8674(93)90046-s. [DOI] [PubMed] [Google Scholar]
  18. Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Köhrer K., Domdey H. Preparation of high molecular weight RNA. Methods Enzymol. 1991;194:398–405. doi: 10.1016/0076-6879(91)94030-g. [DOI] [PubMed] [Google Scholar]
  20. Leer R. J., van Raamsdonk-Duin M. M., Hagendoorn M. J., Mager W. H., Planta R. J. Structural comparison of yeast ribosomal protein genes. Nucleic Acids Res. 1984 Sep 11;12(17):6685–6700. doi: 10.1093/nar/12.17.6685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Leibold E. A., Guo B. Iron-dependent regulation of ferritin and transferrin receptor expression by the iron-responsive element binding protein. Annu Rev Nutr. 1992;12:345–368. doi: 10.1146/annurev.nu.12.070192.002021. [DOI] [PubMed] [Google Scholar]
  22. Lindahl L., Hinnebusch A. Diversity of mechanisms in the regulation of translation in prokaryotes and lower eukaryotes. Curr Opin Genet Dev. 1992 Oct;2(5):720–726. doi: 10.1016/s0959-437x(05)80132-7. [DOI] [PubMed] [Google Scholar]
  23. McCarthy J. E., Gualerzi C. Translational control of prokaryotic gene expression. Trends Genet. 1990 Mar;6(3):78–85. doi: 10.1016/0168-9525(90)90098-q. [DOI] [PubMed] [Google Scholar]
  24. Melefors O., Goossen B., Johansson H. E., Stripecke R., Gray N. K., Hentze M. W. Translational control of 5-aminolevulinate synthase mRNA by iron-responsive elements in erythroid cells. J Biol Chem. 1993 Mar 15;268(8):5974–5978. [PubMed] [Google Scholar]
  25. Melefors O., Hentze M. W. Translational regulation by mRNA/protein interactions in eukaryotic cells: ferritin and beyond. Bioessays. 1993 Feb;15(2):85–90. doi: 10.1002/bies.950150203. [DOI] [PubMed] [Google Scholar]
  26. Müllner E. W., Kühn L. C. A stem-loop in the 3' untranslated region mediates iron-dependent regulation of transferrin receptor mRNA stability in the cytoplasm. Cell. 1988 Jun 3;53(5):815–825. doi: 10.1016/0092-8674(88)90098-0. [DOI] [PubMed] [Google Scholar]
  27. Müllner E. W., Rothenberger S., Müller A. M., Kühn L. C. In vivo and in vitro modulation of the mRNA-binding activity of iron-regulatory factor. Tissue distribution and effects of cell proliferation, iron levels and redox state. Eur J Biochem. 1992 Sep 15;208(3):597–605. doi: 10.1111/j.1432-1033.1992.tb17224.x. [DOI] [PubMed] [Google Scholar]
  28. Nielsen D. A., Shapiro D. J. Preparation of capped RNA transcripts using T7 RNA polymerase. Nucleic Acids Res. 1986 Jul 25;14(14):5936–5936. doi: 10.1093/nar/14.14.5936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Oliveira C. C., van den Heuvel J. J., McCarthy J. E. 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. 1993 Aug;9(3):521–532. doi: 10.1111/j.1365-2958.1993.tb01713.x. [DOI] [PubMed] [Google Scholar]
  30. Patino M. M., Walden W. E. Cloning of a functional cDNA for the rabbit ferritin mRNA repressor protein. Demonstration of a tissue-specific pattern of expression. J Biol Chem. 1992 Sep 15;267(26):19011–19016. [PubMed] [Google Scholar]
  31. Rothenberger S., Müllner E. W., Kühn L. C. The mRNA-binding protein which controls ferritin and transferrin receptor expression is conserved during evolution. Nucleic Acids Res. 1990 Mar 11;18(5):1175–1179. doi: 10.1093/nar/18.5.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rouault T. A., Hentze M. W., Caughman S. W., Harford J. B., Klausner R. D. Binding of a cytosolic protein to the iron-responsive element of human ferritin messenger RNA. Science. 1988 Sep 2;241(4870):1207–1210. doi: 10.1126/science.3413484. [DOI] [PubMed] [Google Scholar]
  33. Rouault T. A., Stout C. D., Kaptain S., Harford J. B., Klausner R. D. Structural relationship between an iron-regulated RNA-binding protein (IRE-BP) and aconitase: functional implications. Cell. 1991 Mar 8;64(5):881–883. doi: 10.1016/0092-8674(91)90312-m. [DOI] [PubMed] [Google Scholar]
  34. Tuite M. F., Plesset J. mRNA-dependent yeast cell-free translation systems: theory and practice. Yeast. 1986 Mar;2(1):35–52. doi: 10.1002/yea.320020103. [DOI] [PubMed] [Google Scholar]
  35. Vega Laso M. R., Zhu D., Sagliocco F., Brown A. J., Tuite M. F., McCarthy J. E. Inhibition of translational initiation in the yeast Saccharomyces cerevisiae as a function of the stability and position of hairpin structures in the mRNA leader. J Biol Chem. 1993 Mar 25;268(9):6453–6462. [PubMed] [Google Scholar]
  36. Weiss G., Goossen B., Doppler W., Fuchs D., Pantopoulos K., Werner-Felmayer G., Wachter H., Hentze M. W. Translational regulation via iron-responsive elements by the nitric oxide/NO-synthase pathway. EMBO J. 1993 Sep;12(9):3651–3657. doi: 10.1002/j.1460-2075.1993.tb06039.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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