Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Oct 15;88(20):8977–8981. doi: 10.1073/pnas.88.20.8977

Autoregulation of human thymidylate synthase messenger RNA translation by thymidylate synthase.

E Chu 1, D M Koeller 1, J L Casey 1, J C Drake 1, B A Chabner 1, P C Elwood 1, S Zinn 1, C J Allegra 1
PMCID: PMC52634  PMID: 1924359

Abstract

Thymidylate synthase (TS; 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) is essential for the de novo synthesis of thymidylate, a precursor of DNA. Previous studies have shown that the cellular level of this protein is regulated at both the transcriptional and posttranscriptional levels. The regulation of human TS mRNA translation was studied in vitro with a rabbit reticulocyte lysate system. The addition of purified human recombinant TS protein to in vitro translation reactions inhibited translation of TS mRNA. This inhibition was specific in that recombinant TS protein had no effect on the in vitro translation of mRNA for human chromogranin A, human folate receptor, preplacental lactogen, or total yeast RNA. The inclusion of dUMP, 5-fluoro-dUMP, or 5,10-methylene-tetrahydrofolate in in vitro translation reactions completely relieved the inhibition of TS mRNA translation by TS protein. Gel retardation assays confirmed a specific interaction between TS protein and its corresponding mRNA but not with unrelated mRNAs, including human placenta, human beta-actin, and yeast tRNA. These studies suggest that translation of TS mRNA is controlled by its own protein end product, TS, in an autoregulatory manner.

Full text

PDF
8977

Images in this article

8978Image
on p.8978
8979Image
on p.8979
8979Image
on p.8979
8979Image
on p.8979
8979Image
on p.8979
8980Image
on p.8980
8980Image
on p.8980

Selected References

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

  1. Ayusawa D., Shimizu K., Koyama H., Kaneda S., Takeishi K., Seno T. Cell-cycle-directed regulation of thymidylate synthase messenger RNA in human diploid fibroblasts stimulated to proliferate. J Mol Biol. 1986 Aug 20;190(4):559–567. doi: 10.1016/0022-2836(86)90241-x. [DOI] [PubMed] [Google Scholar]
  2. Belfort M., Maley G., Pedersen-Lane J., Maley F. Primary structure of the Escherichia coli thyA gene and its thymidylate synthase product. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4914–4918. doi: 10.1073/pnas.80.16.4914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bernardi A., Spahr P. F. Nucleotide sequence at the binding site for coat protein on RNA of bacteriophage R17. Proc Natl Acad Sci U S A. 1972 Oct;69(10):3033–3037. doi: 10.1073/pnas.69.10.3033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beverley S. M., Ellenberger T. E., Cordingley J. S. Primary structure of the gene encoding the bifunctional dihydrofolate reductase-thymidylate synthase of Leishmania major. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2584–2588. doi: 10.1073/pnas.83.8.2584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carey J., Lowary P. T., Uhlenbeck O. C. Interaction of R17 coat protein with synthetic variants of its ribonucleic acid binding site. Biochemistry. 1983 Sep 27;22(20):4723–4730. doi: 10.1021/bi00289a017. [DOI] [PubMed] [Google Scholar]
  6. Carey J., Uhlenbeck O. C. Kinetic and thermodynamic characterization of the R17 coat protein-ribonucleic acid interaction. Biochemistry. 1983 May 24;22(11):2610–2615. doi: 10.1021/bi00280a003. [DOI] [PubMed] [Google Scholar]
  7. Chu E., Zinn S., Boarman D., Allegra C. J. Interaction of gamma interferon and 5-fluorouracil in the H630 human colon carcinoma cell line. Cancer Res. 1990 Sep 15;50(18):5834–5840. [PubMed] [Google Scholar]
  8. Chu F. K., Maley G. F., Maley F., Belfort M. Intervening sequence in the thymidylate synthase gene of bacteriophage T4. Proc Natl Acad Sci U S A. 1984 May;81(10):3049–3053. doi: 10.1073/pnas.81.10.3049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Conrad A. H., Ruddle F. H. Regulation of thymidylate synthetase activity in cultured mammalian cells. J Cell Sci. 1972 Mar;10(2):471–486. doi: 10.1242/jcs.10.2.471. [DOI] [PubMed] [Google Scholar]
  10. Danenberg P. V., Danenberg K. D. Effect of 5, 10-methylenetetrahydrofolate on the dissociation of 5-fluoro-2'-deoxyuridylate from thymidylate synthetase: evidence for an ordered mechanism. Biochemistry. 1978 Sep 19;17(19):4018–4024. doi: 10.1021/bi00612a022. [DOI] [PubMed] [Google Scholar]
  11. Davisson V. J., Sirawaraporn W., Santi D. V. Expression of human thymidylate synthase in Escherichia coli. J Biol Chem. 1989 Jun 5;264(16):9145–9148. [PubMed] [Google Scholar]
  12. Elwood P. C. Molecular cloning and characterization of the human folate-binding protein cDNA from placenta and malignant tissue culture (KB) cells. J Biol Chem. 1989 Sep 5;264(25):14893–14901. [PubMed] [Google Scholar]
  13. Galivan J. H., Maley F., Baugh C. M. Demonstration of separate binding sites for the folate coenzymes and deoxynucleotides with inactivated Lactobacillus casei thymidylate synthetase. Biochem Biophys Res Commun. 1976 Jul 26;71(2):527–534. doi: 10.1016/0006-291x(76)90819-6. [DOI] [PubMed] [Google Scholar]
  14. Garrett C. E., Coderre J. A., Meek T. D., Garvey E. P., Claman D. M., Beverley S. M., Santi D. V. A bifunctional thymidylate synthetase-dihydrofolate reductase in protozoa. Mol Biochem Parasitol. 1984 Apr;11:257–265. doi: 10.1016/0166-6851(84)90070-7. [DOI] [PubMed] [Google Scholar]
  15. Guan K. L., Weiner H. Influence of the 5'-end region of aldehyde dehydrogenase mRNA on translational efficiency. Potential secondary structure inhibition of translation in vitro. J Biol Chem. 1989 Oct 25;264(30):17764–17769. [PubMed] [Google Scholar]
  16. Hardy L. W., Finer-Moore J. S., Montfort W. R., Jones M. O., Santi D. V., Stroud R. M. Atomic structure of thymidylate synthase: target for rational drug design. Science. 1987 Jan 23;235(4787):448–455. doi: 10.1126/science.3099389. [DOI] [PubMed] [Google Scholar]
  17. Heidelberger C., Danenberg P. V., Moran R. G. Fluorinated pyrimidines and their nucleosides. Adv Enzymol Relat Areas Mol Biol. 1983;54:58–119. [PubMed] [Google Scholar]
  18. Helman L. J., Ahn T. G., Levine M. A., Allison A., Cohen P. S., Cooper M. J., Cohn D. V., Israel M. A. Molecular cloning and primary structure of human chromogranin A (secretory protein I) cDNA. J Biol Chem. 1988 Aug 15;263(23):11559–11563. [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Honess R. W., Bodemer W., Cameron K. R., Niller H. H., Fleckenstein B., Randall R. E. The A+T-rich genome of Herpesvirus saimiri contains a highly conserved gene for thymidylate synthase. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3604–3608. doi: 10.1073/pnas.83.11.3604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Horiuchi T., Macon K. J., Kidd V. J., Volanakis J. E. Translational regulation of complement protein C2 expression by differential utilization of the 5'-untranslated region of mRNA. J Biol Chem. 1990 Apr 25;265(12):6521–6524. [PubMed] [Google Scholar]
  23. Jenh C. H., Geyer P. K., Johnson L. F. Control of thymidylate synthase mRNA content and gene transcription in an overproducing mouse cell line. Mol Cell Biol. 1985 Oct;5(10):2527–2532. doi: 10.1128/mcb.5.10.2527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jenh C. H., Rao L. G., Johnson L. F. Regulation of thymidylate synthase enzyme synthesis in 5-fluorodeoxyuridine-resistant mouse fibroblasts during the transition from the resting to growing state. J Cell Physiol. 1985 Jan;122(1):149–154. doi: 10.1002/jcp.1041220122. [DOI] [PubMed] [Google Scholar]
  25. Klausner R. D., Harford J. B. cis-trans models for post-transcriptional gene regulation. Science. 1989 Nov 17;246(4932):870–872. doi: 10.1126/science.2683086. [DOI] [PubMed] [Google Scholar]
  26. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Lorenson M. Y., Maley G. F., Maley F. The purification and properties of thymidylate synthetase from chick embryo extracts. J Biol Chem. 1967 Jul 25;242(14):3332–3344. [PubMed] [Google Scholar]
  29. Lu Y. Z., Aiello P. D., Matthews R. G. Studies on the polyglutamate specificity of thymidylate synthase from fetal pig liver. Biochemistry. 1984 Dec 18;23(26):6870–6876. doi: 10.1021/bi00321a091. [DOI] [PubMed] [Google Scholar]
  30. Matthews D. A., Villafranca J. E., Janson C. A., Smith W. W., Welsh K., Freer S. Stereochemical mechanism of action for thymidylate synthase based on the X-ray structure of the covalent inhibitory ternary complex with 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate. J Mol Biol. 1990 Aug 20;214(4):937–948. doi: 10.1016/0022-2836(90)90347-O. [DOI] [PubMed] [Google Scholar]
  31. Moldave K. Eukaryotic protein synthesis. Annu Rev Biochem. 1985;54:1109–1149. doi: 10.1146/annurev.bi.54.070185.005333. [DOI] [PubMed] [Google Scholar]
  32. Mueller P. P., Hinnebusch A. G. Multiple upstream AUG codons mediate translational control of GCN4. Cell. 1986 Apr 25;45(2):201–207. doi: 10.1016/0092-8674(86)90384-3. [DOI] [PubMed] [Google Scholar]
  33. Navalgund L. G., Rossana C., Muench A. J., Johnson L. F. Cell cycle regulation of thymidylate synthetase gene expression in cultured mouse fibroblasts. J Biol Chem. 1980 Aug 10;255(15):7386–7390. [PubMed] [Google Scholar]
  34. Parkin N. T., Cohen E. A., Darveau A., Rosen C., Haseltine W., Sonenberg N. Mutational analysis of the 5' non-coding region of human immunodeficiency virus type 1: effects of secondary structure on translation. EMBO J. 1988 Sep;7(9):2831–2837. doi: 10.1002/j.1460-2075.1988.tb03139.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Perryman S. M., Rossana C., Deng T. L., Vanin E. F., Johnson L. F. Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme. Mol Biol Evol. 1986 Jul;3(4):313–321. doi: 10.1093/oxfordjournals.molbev.a040400. [DOI] [PubMed] [Google Scholar]
  36. Rode W., Scanlon K. J., Moroson B. A., Bertino J. R. Regulation of thymidylate synthetase in mouse leukemia cells (L1210). J Biol Chem. 1980 Feb 25;255(4):1305–1311. [PubMed] [Google Scholar]
  37. Romaniuk P. J., Lowary P., Wu H. N., Stormo G., Uhlenbeck O. C. RNA binding site of R17 coat protein. Biochemistry. 1987 Mar 24;26(6):1563–1568. doi: 10.1021/bi00380a011. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schuetz J. D., Gorse K. M., Goldman I. D., Westin E. H. Transient inhibition of DNA synthesis by 5-fluorodeoxyuridine leads to overexpression of dihydrofolate reductase with increased frequency of methotrexate resistance. J Biol Chem. 1988 Jun 5;263(16):7708–7712. [PubMed] [Google Scholar]
  41. Spears C. P., Shahinian A. H., Moran R. G., Heidelberger C., Corbett T. H. In vivo kinetics of thymidylate synthetase inhibition of 5-fluorouracil-sensitive and -resistant murine colon adenocarcinomas. Cancer Res. 1982 Feb;42(2):450–456. [PubMed] [Google Scholar]
  42. Swain S. M., Lippman M. E., Egan E. F., Drake J. C., Steinberg S. M., Allegra C. J. Fluorouracil and high-dose leucovorin in previously treated patients with metastatic breast cancer. J Clin Oncol. 1989 Jul;7(7):890–899. doi: 10.1200/JCO.1989.7.7.890. [DOI] [PubMed] [Google Scholar]
  43. Takeishi K., Kaneda S., Ayusawa D., Shimizu K., Gotoh O., Seno T. Nucleotide sequence of a functional cDNA for human thymidylate synthase. Nucleic Acids Res. 1985 Mar 25;13(6):2035–2043. doi: 10.1093/nar/13.6.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Theil E. C. Regulation of ferritin and transferrin receptor mRNAs. J Biol Chem. 1990 Mar 25;265(9):4771–4774. [PubMed] [Google Scholar]
  45. Walden W. E., Daniels-McQueen S., Brown P. H., Gaffield L., Russell D. A., Bielser D., Bailey L. C., Thach R. E. Translational repression in eukaryotes: partial purification and characterization of a repressor of ferritin mRNA translation. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9503–9507. doi: 10.1073/pnas.85.24.9503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Waterhouse P., Khokha R., Denhardt D. T. Modulation of translation by the 5' leader sequence of the mRNA encoding murine tissue inhibitor of metalloproteinases. J Biol Chem. 1990 Apr 5;265(10):5585–5589. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES