Abstract
We have studied the regulation of expression of the asparagine synthetase (AS) gene in ts11 cells, a mutant of BHK hamster cells which encodes a temperature-sensitive AS and therefore does not produce endogenous asparagine at 39.5 degrees C. Incubation of ts11 cells at the nonpermissive temperature drastically increases the level of AS mRNA, and the stimulation of AS mRNA expression is effectively suppressed by the addition of asparagine to the medium. We show here that regulation of AS gene expression involves cis-acting elements which are contained in the mRNA as well as in the 5' genomic region. When a plasmid containing the human AS cDNA under the control of the human AS promoter region was stably transfected into ts11 cells, the expression of human AS RNAs was regulated as that of the endogenous hamster transcripts, indicating that this construct contained all cis elements necessary for regulation. Expression of the AS cDNA in ts11 cells under the control of a constitutive foreign promoter was also regulated by the concentration of asparagine, and this regulation required translation. When we introduced by mutagenesis a number of stop codons in the AS cDNA, the mutant mRNAs with short open reading frames were expressed at low levels that were not increased by asparagine deprivation. Inhibition of protein and RNA synthesis also prevented down-regulation of AS mRNA levels by high concentrations of asparagine. In a parallel series of experiments, we showed that an AS DNA fragment including the promoter and first exon can also regulate RNA expression in response to asparagine concentration. Furthermore, similar increases in the levels of AS RNAs are produced not only by asparagine deprivation in ts11 cells but also by deprivation of human and wild-type BHK cells of leucine, isoleucine, or glutamine. Thus, regulation of AS gene expression is a response to amino acid starvation through mechanisms which appear to involve both changes in RNA stability and change in the rates of transcription initiation or elongation.
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Selected References
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- Andrulis I. L., Chen J., Ray P. N. Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells. Mol Cell Biol. 1987 Jul;7(7):2435–2443. doi: 10.1128/mcb.7.7.2435. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Andrulis I. L., Hatfield G. W., Arfin S. M. Asparaginyl-tRNA aminoacylation levels and asparagine synthetase expression in cultured Chinese hamster ovary cells. J Biol Chem. 1979 Nov 10;254(21):10629–10633. [PMC free article] [PubMed] [Google Scholar]
- Barker G. F., Beemon K. Nonsense codons within the Rous sarcoma virus gag gene decrease the stability of unspliced viral RNA. Mol Cell Biol. 1991 May;11(5):2760–2768. doi: 10.1128/mcb.11.5.2760. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brawerman G. mRNA decay: finding the right targets. Cell. 1989 Apr 7;57(1):9–10. doi: 10.1016/0092-8674(89)90166-9. [DOI] [PubMed] [Google Scholar]
- Cheng J., Fogel-Petrovic M., Maquat L. E. Translation to near the distal end of the penultimate exon is required for normal levels of spliced triosephosphate isomerase mRNA. Mol Cell Biol. 1990 Oct;10(10):5215–5225. doi: 10.1128/mcb.10.10.5215. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Daar I. O., Maquat L. E. Premature translation termination mediates triosephosphate isomerase mRNA degradation. Mol Cell Biol. 1988 Feb;8(2):802–813. doi: 10.1128/mcb.8.2.802. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gong S. S., Basilico C. A mammalian temperature-sensitive mutation affecting G1 progression results from a single amino acid substitution in asparagine synthetase. Nucleic Acids Res. 1990 Jun 25;18(12):3509–3513. doi: 10.1093/nar/18.12.3509. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Greco A., Gong S. S., Ittmann M., Basilico C. Organization and expression of the cell cycle gene, ts11, that encodes asparagine synthetase. Mol Cell Biol. 1989 Jun;9(6):2350–2359. doi: 10.1128/mcb.9.6.2350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Greco A., Ittmann M., Basilico C. Molecular cloning of a gene that is necessary for G1 progression in mammalian cells. Proc Natl Acad Sci U S A. 1987 Mar;84(6):1565–1569. doi: 10.1073/pnas.84.6.1565. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hope I. A., Struhl K. GCN4 protein, synthesized in vitro, binds HIS3 regulatory sequences: implications for general control of amino acid biosynthetic genes in yeast. Cell. 1985 Nov;43(1):177–188. doi: 10.1016/0092-8674(85)90022-4. [DOI] [PubMed] [Google Scholar]
- Kern F. G., Basilico C. Transcription from the polyoma late promoter in cells stably transformed by chimeric plasmids. Mol Cell Biol. 1985 Apr;5(4):797–807. doi: 10.1128/mcb.5.4.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kinniburgh A. J., Maquat L. E., Schedl T., Rachmilewitz E., Ross J. mRNA-deficient beta o-thalassemia results from a single nucleotide deletion. Nucleic Acids Res. 1982 Sep 25;10(18):5421–5427. doi: 10.1093/nar/10.18.5421. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kruijer W., Cooper J. A., Hunter T., Verma I. M. Platelet-derived growth factor induces rapid but transient expression of the c-fos gene and protein. Nature. 1984 Dec 20;312(5996):711–716. doi: 10.1038/312711a0. [DOI] [PubMed] [Google Scholar]
- Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lawn R. M., Efstratiadis A., O'Connell C., Maniatis T. The nucleotide sequence of the human beta-globin gene. Cell. 1980 Oct;21(3):647–651. doi: 10.1016/0092-8674(80)90428-6. [DOI] [PubMed] [Google Scholar]
- Linial M., Gunderson N., Groudine M. Enhanced transcription of c-myc in bursal lymphoma cells requires continuous protein synthesis. Science. 1985 Dec 6;230(4730):1126–1132. doi: 10.1126/science.2999973. [DOI] [PubMed] [Google Scholar]
- Luckow B., Schütz G. CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res. 1987 Jul 10;15(13):5490–5490. doi: 10.1093/nar/15.13.5490. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McKnight S. L., Kingsbury R. Transcriptional control signals of a eukaryotic protein-coding gene. Science. 1982 Jul 23;217(4557):316–324. doi: 10.1126/science.6283634. [DOI] [PubMed] [Google Scholar]
- Müller R., Bravo R., Burckhardt J., Curran T. Induction of c-fos gene and protein by growth factors precedes activation of c-myc. Nature. 1984 Dec 20;312(5996):716–720. doi: 10.1038/312716a0. [DOI] [PubMed] [Google Scholar]
- Okayama H., Berg P. High-efficiency cloning of full-length cDNA. Mol Cell Biol. 1982 Feb;2(2):161–170. doi: 10.1128/mcb.2.2.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pardee A. B. G1 events and regulation of cell proliferation. Science. 1989 Nov 3;246(4930):603–608. doi: 10.1126/science.2683075. [DOI] [PubMed] [Google Scholar]
- Shyu A. B., Belasco J. G., Greenberg M. E. Two distinct destabilizing elements in the c-fos message trigger deadenylation as a first step in rapid mRNA decay. Genes Dev. 1991 Feb;5(2):221–231. doi: 10.1101/gad.5.2.221. [DOI] [PubMed] [Google Scholar]
- Strijker R., Fritz D. T., Levinson A. D. Adenovirus VAI-RNA regulates gene expression by controlling stability of ribosome-bound RNAs. EMBO J. 1989 Sep;8(9):2669–2675. doi: 10.1002/j.1460-2075.1989.tb08407.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Takeshita K., Forget B. G., Scarpa A., Benz E. J., Jr Intranuclear defect in beta-globin mRNA accumulation due to a premature translation termination codon. Blood. 1984 Jul;64(1):13–22. [PubMed] [Google Scholar]
- Urlaub G., Mitchell P. J., Ciudad C. J., Chasin L. A. Nonsense mutations in the dihydrofolate reductase gene affect RNA processing. Mol Cell Biol. 1989 Jul;9(7):2868–2880. doi: 10.1128/mcb.9.7.2868. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Yanofsky C. Transcription attenuation. J Biol Chem. 1988 Jan 15;263(2):609–612. [PubMed] [Google Scholar]
- Yen T. J., Gay D. A., Pachter J. S., Cleveland D. W. Autoregulated changes in stability of polyribosome-bound beta-tubulin mRNAs are specified by the first 13 translated nucleotides. Mol Cell Biol. 1988 Mar;8(3):1224–1235. doi: 10.1128/mcb.8.3.1224. [DOI] [PMC free article] [PubMed] [Google Scholar]