Post-transcriptional regulation in higher eukaryotes: The role of the reporter gene in controlling expression (original) (raw)
Summary
We have investigated whether reporter genes influence cytoplasmic regulation of gene expression in tobacco and Chinese hamster ovary (CHO) cells. Two genes, uidA encoding β-glucuronidase (GUS) from Eseherichia coli and Luc, encoding firefly luciferase (LUC), were used to analyze the ability of a cap, polyadenylated tail, and the 5′- and 3′-untranslated regions (UTR) from tobacco mosaic virus (TMV) to regulate expression. The regulation associated with the 5′ cap structure and the TMV 5′-UTR, both of which enhance translational efficiency, was reporter gene-independent. The poly(A) tail and the TMV 3′-UTR, which is functionally equivalent to a poly(A) tail, increase translational efficiency as well as mRNA stability. The regulation associated with these 3′ ends was highly reporter gene-dependent; their effect on GUS expression was almost an order of magnitude greater than that on LUC expression. In tobacco, the tenfold reporter gene effect on poly(A) tail or TMV 3′-UTR function could not be explained by a differential impact on mRNA stability; GUS and LUC mRNA half-life increased only twofold when either the poly(A) tail or TMV 3′-UTR was present. In CHO cells, however, GUS mRNA was stabilized to a greater extent by a poly(A) tail or the TMV 3′-UTR than was LUC mRNA.
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References
- Aziz N, Munro HN (1987) Iron regulates ferritin mRNA translation through a segment of its 5′ untranslated region. Proc Natl Acad Sci USA 84:8478–8482
Google Scholar - van Belkum A, Abrahams JP, Pleij CWA, Bosch L (1985) Five pseudoknots are present at the 204 nucleotides long 3′ noncoding region of tobacco mosaic virus RNA. Nucleic Acids Res 13:7673–7686
Google Scholar - Brawerman G (1981) The role of the poly(A) sequences in mammalian messenger RNA. CRC Crit Rev Biochem 10:1–38
Google Scholar - Brawerman G (1987) Determinants of messenger RNA stability. Cell 48:5–6
Google Scholar - Buchman AR, Berg P (1988) Comparison of intron-dependent and intron-independent gene expression. Mol Cell Biol 8:4395–4405
Google Scholar - Callis J, Fromm M, Walbot V (1987a) Introns increase gene expression in cultured maize cells. Genes Dev 1:1183–1200
Google Scholar - Callis J, Fromm M, Walbot V (1987b) Expression of mRNA electroporated in plant and animal cells. Nucleic Acids Res 15:5823–5831
Google Scholar - Casey JL, Hentze MW, Koeller DM, Caughman SW, Rouault TA, Klausner RD, Harford JB (1988) Iron-responsive elements: regulatory RNA sequences that control mRNA levels and translation. Science 240:924–928
Google Scholar - Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159
Google Scholar - Cullen BR (1988) Expression of a cloned human interleukin-2 cDNA is enhanced by the substitution of a heterologous mRNA leader region. DNA 7:645–650
Google Scholar - Fromm ME, Callis J, Taylor LP, Walbot V (1987) Electroporation of DNA and RNA into plant protoplasts. Methods Enzymol 153:351–366
Google Scholar - Gallie DR, Walbot V (1990) RNA pseudoknot domain of tobacco mosaic virus can functionally substitute for a poly(A) tail in plant and animal cells. Genes Dev 4:1149–1157
Google Scholar - Gallie DR, Sleat DE, Watts JW, Turner PC, Wilson TMA (1987a) The 5′-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo. Nucleic Acids Res 15:3257–3273
Google Scholar - Gallie DR, Sleat DW, Watts JW, Turner PC, Wilson TMA (1987b) A comparison of eukaryotic viral 5′-leader sequences as enhancers of mRNA expression in vivo. Nucleic Acids Res 15:8693–8711
Google Scholar - Gallie DR, Walbot V, Hershey JWB (1988) The ribosomal fraction mediates the translational enhancement associated with the 5'leader of tobacco mosaic virus. Nucleic Acids Res 16:8675–8694
Google Scholar - Gallie DR, Lucas WJ, Walbot V (1989) Visualizing mRNA expression in plant protoplasts: factors influencing efficient mRNA uptake and translation. Plant Cell 1:301–311
Google Scholar - Geballe AP, Spaete RR, Mocarski ES (1986) A _cis_-acting element within the 5′ leader of a cytomegalovirus β transcript determines kinetic class. Cell 46:865–872
Google Scholar - Haenni A-L, Joshi S, Chapeville F (1982) tRNA-like structures in the genomes of RNA viruses. Prog Nucleic Acids Res Mol Biol 27:85–104
Google Scholar - Hall TC (1979) Transfer RNA-like structures in viral genomes. Int Rev Cytol 60:1–26
Google Scholar - Jefferson RA, Burgess SM, Hirsh D (1986) β-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci USA 83:8447–8451
Google Scholar - Jobling SA, Gehrke L (1987) Enhanced translation of chimaeric messenger RNAs containing a plant viral untranslated leader region. Nature 325:622–625
Google Scholar - Jones TR, Cole MD (1987) Rapid cytoplasmic turnover of c-myc mRNA: requirement of the 3′ untranslated sequences. Mol Cell Biol 7:4513–4521
Google Scholar - Kozak M (1986) Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc Natl Acad Sci USA 83:2850–2854
Google Scholar - Kozak M (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol 196:947–950
Google Scholar - Levine BJ, Chodchoy N, Marzluff WF, Skoultchi AI (1987) Coupling of replication type histone mRNA levels to DNA synthesis requires the stem-loop sequence at the 3′ end of the mRNA. Proc Natl Acad Sci USA 84:6189–6193
Google Scholar - Logan J, Shenk T (1984) Adenovirus tripartite leader sequence enhances translation of mRNAs late after infection. Proc Natl Acad Sci USA 81:3655–3659
Google Scholar - Lutcke HA, Chow KC, Mickel FS, Moss KA, Kern HF, Scheele GA (1987) Selection of AUG initiation codons differs in plants and animals. EMBO J 6:43–48
Google Scholar - Malim MH, Hauber J, Le S-Y, Maizel JV, Cullen BR (1989) The HIV-1 _rev trans_-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature 338:254–257
Google Scholar - Mandeles S (1968) Location of unique sequences in tobacco mosaic virus ribonucleic acid. J Biol Chem 243:3671–3674
Google Scholar - Melton DA, Kreig PA, Rebagliati MR, Maniatis T, Zinn K, Green MR (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 12:7035–7056
Google Scholar - Mueller PP, Hinnebusch AG (1986) Multiple upstream AUG codons mediate translational control of GCN4. Cell 45:201–207
Google Scholar - Mullner EW, Kuhn LC (1988) A stem-loop in the 3′ untranslated region mediates iron-dependent regulation of transferrin receptor mRNA stability in the cytoplasm. Cell 53:815–825
Google Scholar - Pandey NB, Marzluff WF (1987) The stem-loop structure at the 3′ end of histone mRNA is necessary and sufficient for regulation of histone mRNA stability. Mol Cell Biol 7:4557–4559
Google Scholar - Pelletier J, Sonenberg N (1985) Insertion mutagenesis to increase secondary structure within the 5′ noncoding region of a eukaryotic mRNA reduces translational efficiency. Cell 40:515–526
Google Scholar - Pelletier J, Sonenberg N (1988) Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 334:320–325
Google Scholar - Rhoads RE (1988) Cap recognition and the entry of mRNA into the protein synthesis initiation cycle. Trends Biol Sci 13:52–56
Google Scholar - Sachs AB, Kornberg RD (1985) Nuclear polyadenylate-binding protein. Mol Cell Biol 5:1993–1996
Google Scholar - Sachs AB, Davis RW, Kornberg RD (1987) A single domain of yeast poly(A)-binding protein is necessary and sufficient for mRNA binding and cell viability. Mol Cell Biol 7:3268–3276
Google Scholar - Schenborn ET, Mierendorf RC (1985) A novel transcription property of SP6 and T7 RNA polymerase: dependence on template structure. Nucleic Acids Res 13:6223–6236
Google Scholar - Shaw G, Kamen R (1986) A conserved AU sequence from the 3′ untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46:659–667
Google Scholar - Werner M, Feller A, Messenguy F, Pierard A (1987) The leader peptide of yeast gene cpa1 is essential for the translational repression of its expression. Cell 49:805–813
Google Scholar - de Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S (1987) Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7:725–737
Google Scholar - Williams NP, Meuller PP, Hinnebusch AG (1988) The positive regulatory function of the 5′-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences. Mol Cell Biol 8:3827–3836
Google Scholar - Wilson T, Treisman R (1988) Removal of poly(A) and consequent degradation of c-fos mRNA facilitated by 3′ AU-rich sequences. Nature 336:396–399
Google Scholar - Wolin S, Walter P (1988) Ribosome pausing and stacking during translation of a eukaryotic mRNA. EMBO J 7:3559–3569
Google Scholar - Wreschner DH, Rechavi G (1988) Differential mRNA stability to reticulocyte ribonucleases correlates with 3′ non-coding (U)nA sequences. Eur J Biochem 172:333–340
Google Scholar
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Author notes
- Daniel R. Gallie
Present address: Department of Biochemistry, University of California, 92521, Riverside, CA, USA - John N. Feder
Present address: Howard Hughes Medical Institute and Department of Physiology and Biochemistry, University of California, 94143, San Francisco, CA, USA
Authors and Affiliations
- Department of Biological Sciences, Stanford University, 94305-5020, Stanford, CA, USA
Daniel R. Gallie, John N. Feder, Robert T. Schimke & Virginia Walbot
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- Daniel R. Gallie
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Communicated by H. Saedler
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Gallie, D.R., Feder, J.N., Schimke, R.T. et al. Post-transcriptional regulation in higher eukaryotes: The role of the reporter gene in controlling expression.Molec. Gen. Genet. 228, 258–264 (1991). https://doi.org/10.1007/BF00282474
- Received: 27 February 1991
- Issue Date: August 1991
- DOI: https://doi.org/10.1007/BF00282474