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. 1994 Sep;14(9):5898–5909. doi: 10.1128/mcb.14.9.5898

Proteins binding to 5' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells.

R Stripecke 1, C C Oliveira 1, J E McCarthy 1, M W Hentze 1
PMCID: PMC359116  PMID: 8065323

Abstract

We demonstrate that a bacteriophage protein and a spliceosomal protein can be converted into eukaryotic translational repressor proteins. mRNAs with binding sites for the bacteriophage MS2 coat protein or the spliceosomal human U1A protein were expressed in human HeLa cells and yeast. The presence of the appropriate binding protein resulted in specific, dose-dependent translational repression when the binding sites were located in the 5' untranslated region (UTR) of the reporter mRNAs. Neither mRNA export from the nucleus to the cytoplasm nor mRNA stability was demonstrably affected by the binding proteins. The data thus reveal a general mechanism for translational regulation: formation of mRNA-protein complexes in the 5' UTR controls translation initiation by steric blockage of a sensitive step in the initiation pathway. Moreover, the findings establish the basis for novel strategies to study RNA-protein interactions in vivo and to clone RNA-binding proteins.

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Selected References

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

  1. Bag J., Sarkar S. Studies on a nonpolysomal ribonucleoprotein coding for myosin heavy chains from chick embryonic muscles. J Biol Chem. 1976 Dec 10;251(23):7600–7609. [PubMed] [Google Scholar]
  2. Boelens W. C., Jansen E. J., van Venrooij W. J., Stripecke R., Mattaj I. W., Gunderson S. I. The human U1 snRNP-specific U1A protein inhibits polyadenylation of its own pre-mRNA. Cell. 1993 Mar 26;72(6):881–892. doi: 10.1016/0092-8674(93)90577-d. [DOI] [PubMed] [Google Scholar]
  3. Boelens W., Scherly D., Jansen E. J., Kolen K., Mattaj I. W., van Venrooij W. J. Analysis of in vitro binding of U1-A protein mutants to U1 snRNA. Nucleic Acids Res. 1991 Sep 11;19(17):4611–4618. doi: 10.1093/nar/19.17.4611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brunel C., Caillet J., Lesage P., Graffe M., Dondon J., Moine H., Romby P., Ehresmann C., Ehresmann B., Grunberg-Manago M. Domains of the Escherichia coli threonyl-tRNA synthetase translational operator and their relation to threonine tRNA isoacceptors. J Mol Biol. 1992 Oct 5;227(3):621–634. doi: 10.1016/0022-2836(92)90212-3. [DOI] [PubMed] [Google Scholar]
  5. Carey J., Cameron V., de Haseth P. L., Uhlenbeck O. C. Sequence-specific interaction of R17 coat protein with its ribonucleic acid binding site. Biochemistry. 1983 May 24;22(11):2601–2610. doi: 10.1021/bi00280a002. [DOI] [PubMed] [Google Scholar]
  6. Casey J. L., Hentze M. W., Koeller D. M., Caughman S. W., Rouault T. A., Klausner R. D., Harford J. B. Iron-responsive elements: regulatory RNA sequences that control mRNA levels and translation. Science. 1988 May 13;240(4854):924–928. doi: 10.1126/science.2452485. [DOI] [PubMed] [Google Scholar]
  7. Chen-Kiang S., Lavery D. J. Preparation of precursors to mRNA from mammalian cell nuclei. Methods Enzymol. 1989;180:69–82. doi: 10.1016/0076-6879(89)80093-x. [DOI] [PubMed] [Google Scholar]
  8. Chu E., Voeller D., Koeller D. M., Drake J. C., Takimoto C. H., Maley G. F., Maley F., Allegra C. J. Identification of an RNA binding site for human thymidylate synthase. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):517–521. doi: 10.1073/pnas.90.2.517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dabeva M. D., Warner J. R. Ribosomal protein L32 of Saccharomyces cerevisiae regulates both splicing and translation of its own transcript. J Biol Chem. 1993 Sep 15;268(26):19669–19674. [PubMed] [Google Scholar]
  10. Ding D., Lipshitz H. D. Localized RNAs and their functions. Bioessays. 1993 Oct;15(10):651–658. doi: 10.1002/bies.950151004. [DOI] [PubMed] [Google Scholar]
  11. Gait M. J., Karn J. RNA recognition by the human immunodeficiency virus Tat and Rev proteins. Trends Biochem Sci. 1993 Jul;18(7):255–259. doi: 10.1016/0968-0004(93)90176-n. [DOI] [PubMed] [Google Scholar]
  12. Gold L. Posttranscriptional regulatory mechanisms in Escherichia coli. Annu Rev Biochem. 1988;57:199–233. doi: 10.1146/annurev.bi.57.070188.001215. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  16. Gray N. K., Quick S., Goossen B., Constable A., Hirling H., Kühn L. C., Hentze M. W. Recombinant iron-regulatory factor functions as an iron-responsive-element-binding protein, a translational repressor and an aconitase. A functional assay for translational repression and direct demonstration of the iron switch. Eur J Biochem. 1993 Dec 1;218(2):657–667. doi: 10.1111/j.1432-1033.1993.tb18420.x. [DOI] [PubMed] [Google Scholar]
  17. Green S., Issemann I., Sheer E. A versatile in vivo and in vitro eukaryotic expression vector for protein engineering. Nucleic Acids Res. 1988 Jan 11;16(1):369–369. doi: 10.1093/nar/16.1.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gross K. W., Jacobs-Lorena M., Baglioni C., Gross P. R. Cell-free translation of maternal messenger RNA from sea urchin eggs. Proc Natl Acad Sci U S A. 1973 Sep;70(9):2614–2618. doi: 10.1073/pnas.70.9.2614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hall K. B., Stump W. T. Interaction of N-terminal domain of U1A protein with an RNA stem/loop. Nucleic Acids Res. 1992 Aug 25;20(16):4283–4290. doi: 10.1093/nar/20.16.4283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Hentze M. W., Keim S., Papadopoulos P., O'Brien S., Modi W., Drysdale J., Leonard W. J., Harford J. B., Klausner R. D. Cloning, characterization, expression, and chromosomal localization of a human ferritin heavy-chain gene. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7226–7230. doi: 10.1073/pnas.83.19.7226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hinnebusch A. G. Involvement of an initiation factor and protein phosphorylation in translational control of GCN4 mRNA. Trends Biochem Sci. 1990 Apr;15(4):148–152. doi: 10.1016/0968-0004(90)90215-w. [DOI] [PubMed] [Google Scholar]
  23. Jackson R. J. Cytoplasmic regulation of mRNA function: the importance of the 3' untranslated region. Cell. 1993 Jul 16;74(1):9–14. doi: 10.1016/0092-8674(93)90290-7. [DOI] [PubMed] [Google Scholar]
  24. Kambach C., Mattaj I. W. Intracellular distribution of the U1A protein depends on active transport and nuclear binding to U1 snRNA. J Cell Biol. 1992 Jul;118(1):11–21. doi: 10.1083/jcb.118.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kingsman S. M., Cousens D., Stanway C. A., Chambers A., Wilson M., Kingsman A. J. High-efficiency yeast expression vectors based on the promoter of the phosphoglycerate kinase gene. Methods Enzymol. 1990;185:329–341. doi: 10.1016/0076-6879(90)85029-n. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Kozak M. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol. 1987 Aug 20;196(4):947–950. doi: 10.1016/0022-2836(87)90418-9. [DOI] [PubMed] [Google Scholar]
  28. Kozak M. Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc Natl Acad Sci U S A. 1986 May;83(9):2850–2854. doi: 10.1073/pnas.83.9.2850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Leibold E. A., Munro H. N. Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5' untranslated region of ferritin heavy- and light-subunit mRNAs. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2171–2175. doi: 10.1073/pnas.85.7.2171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lowary P. T., Uhlenbeck O. C. An RNA mutation that increases the affinity of an RNA-protein interaction. Nucleic Acids Res. 1987 Dec 23;15(24):10483–10493. doi: 10.1093/nar/15.24.10483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Merrick W. C. Mechanism and regulation of eukaryotic protein synthesis. Microbiol Rev. 1992 Jun;56(2):291–315. doi: 10.1128/mr.56.2.291-315.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Oliveira C. C., Goossen B., Zanchin N. I., McCarthy J. E., Hentze M. W., Stripecke R. Translational repression by the human iron-regulatory factor (IRF) in Saccharomyces cerevisiae. Nucleic Acids Res. 1993 Nov 25;21(23):5316–5322. doi: 10.1093/nar/21.23.5316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Ostareck-Lederer A., Ostareck D. H., Standart N., Thiele B. J. Translation of 15-lipoxygenase mRNA is inhibited by a protein that binds to a repeated sequence in the 3' untranslated region. EMBO J. 1994 Mar 15;13(6):1476–1481. doi: 10.1002/j.1460-2075.1994.tb06402.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Peabody D. S., Ely K. R. Control of translational repression by protein-protein interactions. Nucleic Acids Res. 1992 Apr 11;20(7):1649–1655. doi: 10.1093/nar/20.7.1649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Peabody D. S. Translational repression by bacteriophage MS2 coat protein expressed from a plasmid. A system for genetic analysis of a protein-RNA interaction. J Biol Chem. 1990 Apr 5;265(10):5684–5689. [PubMed] [Google Scholar]
  39. Pelletier J., Sonenberg N. Insertion mutagenesis to increase secondary structure within the 5' noncoding region of a eukaryotic mRNA reduces translational efficiency. Cell. 1985 Mar;40(3):515–526. doi: 10.1016/0092-8674(85)90200-4. [DOI] [PubMed] [Google Scholar]
  40. Philippe C., Eyermann F., Bénard L., Portier C., Ehresmann B., Ehresmann C. Ribosomal protein S15 from Escherichia coli modulates its own translation by trapping the ribosome on the mRNA initiation loading site. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4394–4398. doi: 10.1073/pnas.90.10.4394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Picard D., Salser S. J., Yamamoto K. R. A movable and regulable inactivation function within the steroid binding domain of the glucocorticoid receptor. Cell. 1988 Sep 23;54(7):1073–1080. doi: 10.1016/0092-8674(88)90122-5. [DOI] [PubMed] [Google Scholar]
  42. Sachs A. B. Messenger RNA degradation in eukaryotes. Cell. 1993 Aug 13;74(3):413–421. doi: 10.1016/0092-8674(93)80043-e. [DOI] [PubMed] [Google Scholar]
  43. Scherly D., Boelens W., van Venrooij W. J., Dathan N. A., Hamm J., Mattaj I. W. Identification of the RNA binding segment of human U1 A protein and definition of its binding site on U1 snRNA. EMBO J. 1989 Dec 20;8(13):4163–4170. doi: 10.1002/j.1460-2075.1989.tb08601.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schäfer M., Kuhn R., Bosse F., Schäfer U. A conserved element in the leader mediates post-meiotic translation as well as cytoplasmic polyadenylation of a Drosophila spermatocyte mRNA. EMBO J. 1990 Dec;9(13):4519–4525. doi: 10.1002/j.1460-2075.1990.tb07903.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Siegel V., Walter P. Each of the activities of signal recognition particle (SRP) is contained within a distinct domain: analysis of biochemical mutants of SRP. Cell. 1988 Jan 15;52(1):39–49. doi: 10.1016/0092-8674(88)90529-6. [DOI] [PubMed] [Google Scholar]
  46. Sillekens P. T., Habets W. J., Beijer R. P., van Venrooij W. J. cDNA cloning of the human U1 snRNA-associated A protein: extensive homology between U1 and U2 snRNP-specific proteins. EMBO J. 1987 Dec 1;6(12):3841–3848. doi: 10.1002/j.1460-2075.1987.tb02721.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Spedding G., Draper D. E. Allosteric mechanism for translational repression in the Escherichia coli alpha operon. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4399–4403. doi: 10.1073/pnas.90.10.4399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Stripecke R., Hentze M. W. Bacteriophage and spliceosomal proteins function as position-dependent cis/trans repressors of mRNA translation in vitro. Nucleic Acids Res. 1992 Nov 11;20(21):5555–5564. doi: 10.1093/nar/20.21.5555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. Walden W. E., Patino M. M., Gaffield L. Purification of a specific repressor of ferritin mRNA translation from rabbit liver. J Biol Chem. 1989 Aug 15;264(23):13765–13769. [PubMed] [Google Scholar]
  51. Witherell G. W., Gott J. M., Uhlenbeck O. C. Specific interaction between RNA phage coat proteins and RNA. Prog Nucleic Acid Res Mol Biol. 1991;40:185–220. doi: 10.1016/s0079-6603(08)60842-9. [DOI] [PubMed] [Google Scholar]
  52. Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. van Gelder C. W., Gunderson S. I., Jansen E. J., Boelens W. C., Polycarpou-Schwarz M., Mattaj I. W., van Venrooij W. J. A complex secondary structure in U1A pre-mRNA that binds two molecules of U1A protein is required for regulation of polyadenylation. EMBO J. 1993 Dec 15;12(13):5191–5200. doi: 10.1002/j.1460-2075.1993.tb06214.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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