Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 May;17(5):2698–2707. doi: 10.1128/mcb.17.5.2698

Coding elements in exons 2 and 3 target c-myc mRNA downregulation during myogenic differentiation.

N M Yeilding 1, W M Lee 1
PMCID: PMC232120  PMID: 9111340

Abstract

Downregulation in expression of the c-myc proto-oncogene is an early molecular event in differentiation of murine C2C12 myoblasts into multinucleated myotubes. During differentiation, levels of c-myc mRNA decrease 3- to 10-fold despite a lack of change in its transcription rate. To identify cis-acting elements that target c-myc mRNA for downregulation during myogenesis, we stably transfected C2C12 cells with mutant myc genes or chimeric genes in which various myc sequences were fused to the human beta-globin gene or to the bacterial chloramphenicol acetyltransferase (CAT) gene. Deletion of coding sequences from myc exon 2 or exon 3 abolished downregulation of myc mRNA during myogenic differentiation, while deletion of introns or sequences in the 5' or 3' untranslated regions (UTRs) did not, demonstrating that coding elements in both exons 2 and 3 are necessary for myc mRNA downregulation. Fusion of coding sequences from either myc exon 2 or 3 to beta-globin mRNA conferred downregulation onto the chimeric mRNA, while fusion of myc 3' UTR sequences or coding sequences from CAT or ribosomal protein L32 did not, demonstrating that coding elements in myc exons 2 and 3 specifically confer downregulation. These results present the apparent paradox that coding elements in either myc exon 2 or myc exon 3 are sufficient to confer downregulation onto beta-globin mRNA, but neither element alone was sufficient for myc mRNA downregulation, suggesting that some feature of beta-globin mRNA may potentiate the regulatory properties of myc exons 2 and 3. A similar regulatory function is not shared by all mRNAs because fusion of either myc exon 2 or myc exon 3 to CAT mRNA did not confer downregulation onto the chimeric mRNA, but fusion of the two elements together did. We conclude from these results that two myc regulatory elements, one exon 2 and one in exon 3, are required for myc mRNA downregulation. Finally, using a highly sensitive and specific PCR-based assay for comparing mRNA levels, we demonstrated that the downregulation mediated by myc exons 2 and 3 results in a decrease in cytoplasmic mRNA levels, but not nuclear mRNA levels, indicating that regulation is a postnuclear event.

Full Text

The Full Text of this article is available as a PDF (965.4 KB).

Selected References

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

  1. Armelin H. A., Armelin M. C., Kelly K., Stewart T., Leder P., Cochran B. H., Stiles C. D. Functional role for c-myc in mitogenic response to platelet-derived growth factor. Nature. 1984 Aug 23;310(5979):655–660. doi: 10.1038/310655a0. [DOI] [PubMed] [Google Scholar]
  2. Bello-Fernandez C., Cleveland J. L. c-myc transactivates the ornithine decarboxylase gene. Curr Top Microbiol Immunol. 1992;182:445–452. doi: 10.1007/978-3-642-77633-5_56. [DOI] [PubMed] [Google Scholar]
  3. Bello-Fernandez C., Packham G., Cleveland J. L. The ornithine decarboxylase gene is a transcriptional target of c-Myc. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7804–7808. doi: 10.1073/pnas.90.16.7804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bentley D. L., Groudine M. A block to elongation is largely responsible for decreased transcription of c-myc in differentiated HL60 cells. Nature. 1986 Jun 12;321(6071):702–706. doi: 10.1038/321702a0. [DOI] [PubMed] [Google Scholar]
  5. Bernstein P. L., Herrick D. J., Prokipcak R. D., Ross J. Control of c-myc mRNA half-life in vitro by a protein capable of binding to a coding region stability determinant. Genes Dev. 1992 Apr;6(4):642–654. doi: 10.1101/gad.6.4.642. [DOI] [PubMed] [Google Scholar]
  6. Blanchard J. M., Piechaczyk M., Dani C., Chambard J. C., Franchi A., Pouyssegur J., Jeanteur P. c-myc gene is transcribed at high rate in G0-arrested fibroblasts and is post-transcriptionally regulated in response to growth factors. Nature. 1985 Oct 3;317(6036):443–445. doi: 10.1038/317443a0. [DOI] [PubMed] [Google Scholar]
  7. Blau H. M., Pavlath G. K., Hardeman E. C., Chiu C. P., Silberstein L., Webster S. G., Miller S. C., Webster C. Plasticity of the differentiated state. Science. 1985 Nov 15;230(4727):758–766. doi: 10.1126/science.2414846. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Cesarman E., Dalla-Favera R., Bentley D., Groudine M. Mutations in the first exon are associated with altered transcription of c-myc in Burkitt lymphoma. Science. 1987 Nov 27;238(4831):1272–1275. doi: 10.1126/science.3685977. [DOI] [PubMed] [Google Scholar]
  10. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Coppola J. A., Cole M. D. Constitutive c-myc oncogene expression blocks mouse erythroleukaemia cell differentiation but not commitment. Nature. 1986 Apr 24;320(6064):760–763. doi: 10.1038/320760a0. [DOI] [PubMed] [Google Scholar]
  12. Dani C., Blanchard J. M., Piechaczyk M., El Sabouty S., Marty L., Jeanteur P. Extreme instability of myc mRNA in normal and transformed human cells. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7046–7050. doi: 10.1073/pnas.81.22.7046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dani C., Mechti N., Piechaczyk M., Lebleu B., Jeanteur P., Blanchard J. M. Increased rate of degradation of c-myc mRNA in interferon-treated Daudi cells. Proc Natl Acad Sci U S A. 1985 Aug;82(15):4896–4899. doi: 10.1073/pnas.82.15.4896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dean M., Levine R. A., Campisi J. c-myc regulation during retinoic acid-induced differentiation of F9 cells is posttranscriptional and associated with growth arrest. Mol Cell Biol. 1986 Feb;6(2):518–524. doi: 10.1128/mcb.6.2.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dony C., Kessel M., Gruss P. Post-transcriptional control of myc and p53 expression during differentiation of the embryonal carcinoma cell line F9. Nature. 1985 Oct 17;317(6038):636–639. doi: 10.1038/317636a0. [DOI] [PubMed] [Google Scholar]
  16. Dudov K. P., Perry R. P. The gene family encoding the mouse ribosomal protein L32 contains a uniquely expressed intron-containing gene and an unmutated processed gene. Cell. 1984 Jun;37(2):457–468. doi: 10.1016/0092-8674(84)90376-3. [DOI] [PubMed] [Google Scholar]
  17. Eilers M., Schirm S., Bishop J. M. The MYC protein activates transcription of the alpha-prothymosin gene. EMBO J. 1991 Jan;10(1):133–141. doi: 10.1002/j.1460-2075.1991.tb07929.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Freytag S. O. Enforced expression of the c-myc oncogene inhibits cell differentiation by precluding entry into a distinct predifferentiation state in G0/G1. Mol Cell Biol. 1988 Apr;8(4):1614–1624. doi: 10.1128/mcb.8.4.1614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Galaktionov K., Chen X., Beach D. Cdc25 cell-cycle phosphatase as a target of c-myc. Nature. 1996 Aug 8;382(6591):511–517. doi: 10.1038/382511a0. [DOI] [PubMed] [Google Scholar]
  20. Graves R. A., Pandey N. B., Chodchoy N., Marzluff W. F. Translation is required for regulation of histone mRNA degradation. Cell. 1987 Feb 27;48(4):615–626. doi: 10.1016/0092-8674(87)90240-6. [DOI] [PubMed] [Google Scholar]
  21. Greenberg M. E., Ziff E. B. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature. 1984 Oct 4;311(5985):433–438. doi: 10.1038/311433a0. [DOI] [PubMed] [Google Scholar]
  22. Griep A. E., Westphal H. Antisense Myc sequences induce differentiation of F9 cells. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6806–6810. doi: 10.1073/pnas.85.18.6806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Grosso L. E., Pitot H. C. Transcriptional regulation of c-myc during chemically induced differentiation of HL-60 cultures. Cancer Res. 1985 Feb;45(2):847–850. [PubMed] [Google Scholar]
  24. Heikkila R., Schwab G., Wickstrom E., Loke S. L., Pluznik D. H., Watt R., Neckers L. M. A c-myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from G0 to G1. 1987 Jul 30-Aug 5Nature. 328(6129):445–449. doi: 10.1038/328445a0. [DOI] [PubMed] [Google Scholar]
  25. Henriksson M., Lüscher B. Proteins of the Myc network: essential regulators of cell growth and differentiation. Adv Cancer Res. 1996;68:109–182. doi: 10.1016/s0065-230x(08)60353-x. [DOI] [PubMed] [Google Scholar]
  26. Herrick D. J., Ross J. The half-life of c-myc mRNA in growing and serum-stimulated cells: influence of the coding and 3' untranslated regions and role of ribosome translocation. Mol Cell Biol. 1994 Mar;14(3):2119–2128. doi: 10.1128/mcb.14.3.2119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jones T. R., Cole M. D. Rapid cytoplasmic turnover of c-myc mRNA: requirement of the 3' untranslated sequences. Mol Cell Biol. 1987 Dec;7(12):4513–4521. doi: 10.1128/mcb.7.12.4513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kaczmarek L., Hyland J. K., Watt R., Rosenberg M., Baserga R. Microinjected c-myc as a competence factor. Science. 1985 Jun 14;228(4705):1313–1315. doi: 10.1126/science.4001943. [DOI] [PubMed] [Google Scholar]
  29. Kelly K., Cochran B. H., Stiles C. D., Leder P. Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell. 1983 Dec;35(3 Pt 2):603–610. doi: 10.1016/0092-8674(83)90092-2. [DOI] [PubMed] [Google Scholar]
  30. Knight E., Jr, Anton E. D., Fahey D., Friedland B. K., Jonak G. J. Interferon regulates c-myc gene expression in Daudi cells at the post-transcriptional level. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1151–1154. doi: 10.1073/pnas.82.4.1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Krystal G., Birrer M., Way J., Nau M., Sausville E., Thompson C., Minna J., Battey J. Multiple mechanisms for transcriptional regulation of the myc gene family in small-cell lung cancer. Mol Cell Biol. 1988 Aug;8(8):3373–3381. doi: 10.1128/mcb.8.8.3373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lavenu A., Pistoi S., Pournin S., Babinet C., Morello D. Both coding exons of the c-myc gene contribute to its posttranscriptional regulation in the quiescent liver and regenerating liver and after protein synthesis inhibition. Mol Cell Biol. 1995 Aug;15(8):4410–4419. doi: 10.1128/mcb.15.8.4410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Levine R. A., McCormack J. E., Buckler A., Sonenshein G. E. Transcriptional and posttranscriptional control of c-myc gene expression in WEHI 231 cells. Mol Cell Biol. 1986 Nov;6(11):4112–4116. doi: 10.1128/mcb.6.11.4112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Makino R., Akiyama K., Yasuda J., Mashiyama S., Honda S., Sekiya T., Hayashi K. Cloning and characterization of a c-myc intron binding protein (MIBP1). Nucleic Acids Res. 1994 Dec 25;22(25):5679–5685. doi: 10.1093/nar/22.25.5679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. McMaster G. K., Carmichael G. G. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4835–4838. doi: 10.1073/pnas.74.11.4835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mechti N., Piechaczyk M., Blanchard J. M., Marty L., Bonnieu A., Jeanteur P., Lebleu B. Transcriptional and post-transcriptional regulation of c-myc expression during the differentiation of murine erythroleukemia Friend cells. Nucleic Acids Res. 1986 Dec 22;14(24):9653–9666. doi: 10.1093/nar/14.24.9653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Nepveu A., Marcu K. B. Intragenic pausing and anti-sense transcription within the murine c-myc locus. EMBO J. 1986 Nov;5(11):2859–2865. doi: 10.1002/j.1460-2075.1986.tb04580.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Philipp A., Schneider A., Väsrik I., Finke K., Xiong Y., Beach D., Alitalo K., Eilers M. Repression of cyclin D1: a novel function of MYC. Mol Cell Biol. 1994 Jun;14(6):4032–4043. doi: 10.1128/mcb.14.6.4032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pistoi S., Roland J., Babinet C., Morello D. Exon 2-mediated c-myc mRNA decay in vivo is independent of its translation. Mol Cell Biol. 1996 Sep;16(9):5107–5116. doi: 10.1128/mcb.16.9.5107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Prochownik E. V., Kukowska J., Rodgers C. c-myc antisense transcripts accelerate differentiation and inhibit G1 progression in murine erythroleukemia cells. Mol Cell Biol. 1988 Sep;8(9):3683–3695. doi: 10.1128/mcb.8.9.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Samaniego F., Chin J., Iwai K., Rouault T. A., Klausner R. D. Molecular characterization of a second iron-responsive element binding protein, iron regulatory protein 2. Structure, function, and post-translational regulation. J Biol Chem. 1994 Dec 9;269(49):30904–30910. [PubMed] [Google Scholar]
  43. Schiavi S. C., Wellington C. L., Shyu A. B., Chen C. Y., Greenberg M. E., Belasco J. G. Multiple elements in the c-fos protein-coding region facilitate mRNA deadenylation and decay by a mechanism coupled to translation. J Biol Chem. 1994 Feb 4;269(5):3441–3448. [PubMed] [Google Scholar]
  44. Stone J., de Lange T., Ramsay G., Jakobovits E., Bishop J. M., Varmus H., Lee W. Definition of regions in human c-myc that are involved in transformation and nuclear localization. Mol Cell Biol. 1987 May;7(5):1697–1709. doi: 10.1128/mcb.7.5.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wisdom R., Lee W. The protein-coding region of c-myc mRNA contains a sequence that specifies rapid mRNA turnover and induction by protein synthesis inhibitors. Genes Dev. 1991 Feb;5(2):232–243. doi: 10.1101/gad.5.2.232. [DOI] [PubMed] [Google Scholar]
  46. Wisdom R., Lee W. Translation of c-myc mRNA is required for its post-transcriptional regulation during myogenesis. J Biol Chem. 1990 Nov 5;265(31):19015–19021. [PubMed] [Google Scholar]
  47. Yeilding N. M., Rehman M. T., Lee W. M. Identification of sequences in c-myc mRNA that regulate its steady-state levels. Mol Cell Biol. 1996 Jul;16(7):3511–3522. doi: 10.1128/mcb.16.7.3511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yen T. J., Machlin P. S., Cleveland D. W. Autoregulated instability of beta-tubulin mRNAs by recognition of the nascent amino terminus of beta-tubulin. Nature. 1988 Aug 18;334(6183):580–585. doi: 10.1038/334580a0. [DOI] [PubMed] [Google Scholar]
  49. Yokoyama K., Imamoto F. Transcriptional control of the endogenous MYC protooncogene by antisense RNA. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7363–7367. doi: 10.1073/pnas.84.21.7363. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES