Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1983 Oct;3(10):1792–1802. doi: 10.1128/mcb.3.10.1792

Delayed processing of dihydrofolate reductase heterogeneous nuclear RNA in amino acid-starved mouse fibroblasts.

M L Collins, J S Wu, C L Santiago, S L Hendrickson, L F Johnson
PMCID: PMC370041  PMID: 6646125

Abstract

We have studied the metabolism of dihydrofolate reductase (DHFR) RNA in cells synchronized in the G1 phase of the cell cycle by starvation for isoleucine and glutamine. The relative content and stability of DHFR mRNA and the relative rate of transcription of the DHFR gene are similar in starved and exponentially growing cells. However, the relative rate of labeling of DHFR mRNA is about three times lower in starved cells than in exponentially growing cells. When the starved cells are stimulated to reenter the cell cycle by feeding them with complete medium, the relative rate of labeling of DHFR mRNA increases about fourfold within 6 h. However, the relative rate of transcription of the DHFR gene changes very little during this period. Continuous labeling experiments show that starved cells convert DHFR heterogeneous nuclear RNA into cytoplasmic DHFR mRNA much more slowly than serum-limited or exponentially growing cells. Pulse-chase experiments show that DHFR mRNA sequences contained in DHFR heterogeneous nuclear RNA appear to be conserved in starved cells. In addition, the content of DHFR RNA sequences in the nuclei of starved cells is about three times greater than that in exponentially growing cells. Delayed processing of DHFR heterogeneous nuclear RNA is also observed when exponentially growing cells are treated with inhibitors of protein synthesis. Our results suggest that, although delayed processing leads to a decrease in the initial labeling rate of DHFR mRNA, it does not result in a decrease in the actual rate of production of the message.

Full text

PDF
1792

Selected References

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

  1. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beltz G. A., Flint S. J. Inhibition of HeLa cell protein synthesis during adenovirus infection. Restriction of cellular messenger RNA sequences to the nucleus. J Mol Biol. 1979 Jun 25;131(2):353–373. doi: 10.1016/0022-2836(79)90081-0. [DOI] [PubMed] [Google Scholar]
  3. Chang A. C., Nunberg J. H., Kaufman R. J., Erlich H. A., Schimke R. T., Cohen S. N. Phenotypic expression in E. coli of a DNA sequence coding for mouse dihydrofolate reductase. Nature. 1978 Oct 19;275(5681):617–624. doi: 10.1038/275617a0. [DOI] [PubMed] [Google Scholar]
  4. Chen-Kiang S., Nevins J. R., Darnell J. E., Jr N-6-methyl-adenosine in adenovirus type 2 nuclear RNA is conserved in the formation of messenger RNA. J Mol Biol. 1979 Dec 15;135(3):733–752. doi: 10.1016/0022-2836(79)90174-8. [DOI] [PubMed] [Google Scholar]
  5. Gudewicz T. M., Morhenn V. B., Kellems R. E. The effect of polyoma virus, serum factors, and dibutyryl cyclic AMP on dihydrofolate reductase synthesis, and the entry of quiescent cells into S phase. J Cell Physiol. 1981 Jul;108(1):1–8. doi: 10.1002/jcp.1041080102. [DOI] [PubMed] [Google Scholar]
  6. Hendrickson S. L., Wu J. S., Johnson L. F. Cell cycle regulation of dihydrofolate reductase mRNA metabolism in mouse fibroblasts. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5140–5144. doi: 10.1073/pnas.77.9.5140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hendrickson S., Johnson L. F. Evidence for highly stable nuclear poly(A) in cultured mammalian cells. Biochim Biophys Acta. 1978 Feb 16;517(2):287–295. doi: 10.1016/0005-2787(78)90195-8. [DOI] [PubMed] [Google Scholar]
  8. Herman R. C., Penman S. Multiple decay rates of heterogeneous nuclear RNA in HeLa cells. Biochemistry. 1977 Jul 26;16(15):3460–3465. doi: 10.1021/bi00634a026. [DOI] [PubMed] [Google Scholar]
  9. Holley R. W., Kiernan J. A. Control of the initiation of DNA synthesis in 3T3 cells: low-molecular weight nutrients. Proc Natl Acad Sci U S A. 1974 Aug;71(8):2942–2945. doi: 10.1073/pnas.71.8.2942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Johnson L. F., Fuhrman C. L., Wiedemann L. M. Regulation of dihydrofolate reductase gene expression in mouse fibroblasts during the transition from the resting to growing state. J Cell Physiol. 1978 Dec;97(3 Pt 2 Suppl 1):397–306. doi: 10.1002/jcp.1040970314. [DOI] [PubMed] [Google Scholar]
  11. Kellems R. E., Alt F. W., Schimke R. T. Regulation of folate reductase synthesis in sensitive and methotrexate-resistant sarcoma 180 cells. In vitro translation and characterization of folate reductase mRNA. J Biol Chem. 1976 Nov 25;251(22):6987–6993. [PubMed] [Google Scholar]
  12. Lehrach H., Diamond D., Wozney J. M., Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry. 1977 Oct 18;16(21):4743–4751. doi: 10.1021/bi00640a033. [DOI] [PubMed] [Google Scholar]
  13. Leys E. J., Kellems R. E. Control of dihydrofolate reductase messenger ribonucleic acid production. Mol Cell Biol. 1981 Nov;1(11):961–971. doi: 10.1128/mcb.1.11.961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mariani B. D., Slate D. L., Schimke R. T. S phase-specific synthesis of dihydrofolate reductase in Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4985–4989. doi: 10.1073/pnas.78.8.4985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mayrand S., Pederson T. Heat shock alters nuclear ribonucleoprotein assembly in Drosophila cells. Mol Cell Biol. 1983 Feb;3(2):161–171. doi: 10.1128/mcb.3.2.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Medrano E. E., Pardee A. B. Prevalent deficiency in tumor cells of cycloheximide-induced cycle arrest. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4123–4126. doi: 10.1073/pnas.77.7.4123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Setzer D. R., McGrogan M., Nunberg J. H., Schimke R. T. Size heterogeneity in the 3' end of dihydrofolate reductase messenger RNAs in mouse cells. Cell. 1980 Nov;22(2 Pt 2):361–370. doi: 10.1016/0092-8674(80)90346-3. [DOI] [PubMed] [Google Scholar]
  18. Strohman R. C., Moss P. S., Micou-Eastwood J., Spector D., Przybyla A., Paterson B. Messenger RNA for myosin polypeptides: isolation from single myogenic cell cultures. Cell. 1977 Feb;10(2):265–273. doi: 10.1016/0092-8674(77)90220-3. [DOI] [PubMed] [Google Scholar]
  19. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tobey R. A., Ley K. D. Regulation of initiation of DNA synthesis in Chinese hamster cells. I. Production of stable, reversible G1-arrested populations in suspension culture. J Cell Biol. 1970 Jul;46(1):151–157. doi: 10.1083/jcb.46.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wiedemann L. M., Johnson L. F. Regulation of dihydrofolate reductase synthesis in an overproducing 3T6 cell line during transition from resting to growing state. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2818–2822. doi: 10.1073/pnas.76.6.2818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wu J. S., Johnson L. F. Regulation of dihydrofolate reductase gene transcription in methotrexate-resistant mouse fibroblasts. J Cell Physiol. 1982 Feb;110(2):183–189. doi: 10.1002/jcp.1041100212. [DOI] [PubMed] [Google Scholar]
  24. Wu J. S., Wiedemann L. M., Johnson L. F. Inhibition of dihydrofolate reductase gene expression following serum withdrawal or db-cAMP addition in methotrexate-resistant mouse fibroblasts. Exp Cell Res. 1982 Sep;141(1):159–169. doi: 10.1016/0014-4827(82)90078-7. [DOI] [PubMed] [Google Scholar]

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

RESOURCES