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. 1991 Dec;10(12):3887–3896. doi: 10.1002/j.1460-2075.1991.tb04958.x

Translation of a polycistronic mRNA in the presence of the cauliflower mosaic virus transactivator protein.

J Fütterer 1, T Hohn 1
PMCID: PMC453126  PMID: 1935908

Abstract

Polycistronic mRNAs containing an upstream beta-glucuronidase (GUS) and a downstream chloramphenicol acetyltransferase (CAT) reporter open reading frame (ORF) were expressed in transfected plant protoplasts. CAT expression could be strongly induced by coexpression of the cauliflower mosaic virus encoded translation transactivator. Transactivation was abolished when an upstream ORF overlapped the CAT ORF for a long distance. No specific sequence elements were required for transactivation but the presence of a short ORF upstream of the GUS ORF strongly enhanced the process. The inhibitory effect of additional presumed stem structures inserted into various regions of the reporter mRNAs indicates that both ORFs are translated by ribosomes that associate with the RNA at the 5' end and reach the ORFs by a linear migration mechanism.

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Selected References

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  1. Abastado J. P., Miller P. F., Jackson B. M., Hinnebusch A. G. Suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis for GCN4 translational control. Mol Cell Biol. 1991 Jan;11(1):486–496. doi: 10.1128/mcb.11.1.486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Angenon G., Uotila J., Kurkela S. A., Teeri T. H., Botterman J., Van Montagu M., Depicker A. Expression of dicistronic transcriptional units in transgenic tobacco. Mol Cell Biol. 1989 Dec;9(12):5676–5684. doi: 10.1128/mcb.9.12.5676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Azad A. A., Deacon N. J. The 3'-terminal primary structure of five eukaryotic 18S rRNAs determined by the direct chemical method of sequencing. The highly conserved sequences include an invariant region complementary to eukaryotic 5S rRNA. Nucleic Acids Res. 1980 Oct 10;8(19):4365–4376. doi: 10.1093/nar/8.19.4365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baim S. B., Sherman F. mRNA structures influencing translation in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1988 Apr;8(4):1591–1601. doi: 10.1128/mcb.8.4.1591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barkan A. Proteins encoded by a complex chloroplast transcription unit are each translated from both monocistronic and polycistronic mRNAs. EMBO J. 1988 Sep;7(9):2637–2644. doi: 10.1002/j.1460-2075.1988.tb03116.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Baughman G. A., Jacobs J. D., Howell S. H. Cauliflower mosaic virus gene VI produces a symptomatic phenotype in transgenic tobacco plants. Proc Natl Acad Sci U S A. 1988 Feb;85(3):733–737. doi: 10.1073/pnas.85.3.733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Baughman G., Howell S. H. Cauliflower mosaic virus 35 S RNA leader region inhibits translation of downstream genes. Virology. 1988 Nov;167(1):125–135. doi: 10.1016/0042-6822(88)90061-x. [DOI] [PubMed] [Google Scholar]
  8. Bonneville J. M., Sanfaçon H., Fütterer J., Hohn T. Posttranscriptional trans-activation in cauliflower mosaic virus. Cell. 1989 Dec 22;59(6):1135–1143. doi: 10.1016/0092-8674(89)90769-1. [DOI] [PubMed] [Google Scholar]
  9. Chevrier D., Vézina C., Bastille J., Linard C., Sonenberg N., Boileau G. Higher order structures of the 5'-proximal region decrease the efficiency of translation of the porcine pro-opiomelanocortin mRNA. J Biol Chem. 1988 Jan 15;263(2):902–910. [PubMed] [Google Scholar]
  10. Cone K. C., Steege D. A. Functional analysis of lac repressor restart sites in translational initiation and reinitiation. J Mol Biol. 1985 Dec 20;186(4):733–742. doi: 10.1016/0022-2836(85)90393-6. [DOI] [PubMed] [Google Scholar]
  11. Curran J., Kolakofsky D. Scanning independent ribosomal initiation of the Sendai virus X protein. EMBO J. 1988 Sep;7(9):2869–2874. doi: 10.1002/j.1460-2075.1988.tb03143.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Darlix J. L., Zuker M., Spahr P. F. Structure-function relationship of Rous sarcoma virus leader RNA. Nucleic Acids Res. 1982 Sep 11;10(17):5183–5196. doi: 10.1093/nar/10.17.5183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dixon L. K., Hohn T. Initiation of translation of the cauliflower mosaic virus genome from a polycistronic mRNA: evidence from deletion mutagenesis. EMBO J. 1984 Dec 1;3(12):2731–2736. doi: 10.1002/j.1460-2075.1984.tb02203.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Edery I., Petryshyn R., Sonenberg N. Activation of double-stranded RNA-dependent kinase (dsl) by the TAR region of HIV-1 mRNA: a novel translational control mechanism. Cell. 1989 Jan 27;56(2):303–312. doi: 10.1016/0092-8674(89)90904-5. [DOI] [PubMed] [Google Scholar]
  15. Fajardo J. E., Shatkin A. J. Translation of bicistronic viral mRNA in transfected cells: regulation at the level of elongation. Proc Natl Acad Sci U S A. 1990 Jan;87(1):328–332. doi: 10.1073/pnas.87.1.328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Freier S. M., Kierzek R., Jaeger J. A., Sugimoto N., Caruthers M. H., Neilson T., Turner D. H. Improved free-energy parameters for predictions of RNA duplex stability. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9373–9377. doi: 10.1073/pnas.83.24.9373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fromm M., Taylor L. P., Walbot V. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5824–5828. doi: 10.1073/pnas.82.17.5824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fütterer J., Gordon K., Bonneville J. M., Sanfaçon H., Pisan B., Penswick J., Hohn T. The leading sequence of caulimovirus large RNA can be folded into a large stem-loop structure. Nucleic Acids Res. 1988 Sep 12;16(17):8377–8390. doi: 10.1093/nar/16.17.8377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fütterer J., Gordon K., Pfeiffer P., Sanfaçon H., Pisan B., Bonneville J. M., Hohn T. Differential inhibition of downstream gene expression by the cauliflower mosaic virus 35S RNA leader. Virus Genes. 1989 Sep;3(1):45–55. doi: 10.1007/BF00301986. [DOI] [PubMed] [Google Scholar]
  20. Fütterer J., Gordon K., Sanfaçon H., Bonneville J. M., Hohn T. Positive and negative control of translation by the leader sequence of cauliflower mosaic virus pregenomic 35S RNA. EMBO J. 1990 Jun;9(6):1697–1707. doi: 10.1002/j.1460-2075.1990.tb08293.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gordon K., Pfeiffer P., Fütterer J., Hohn T. In vitro expression of cauliflower mosaic virus genes. EMBO J. 1988 Feb;7(2):309–317. doi: 10.1002/j.1460-2075.1988.tb02814.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Gowda S., Wu F. C., Scholthof H. B., Shepherd R. J. Gene VI of figwort mosaic virus (caulimovirus group) functions in posttranscriptional expression of genes on the full-length RNA transcript. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9203–9207. doi: 10.1073/pnas.86.23.9203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Guilley H., Dudley R. K., Jonard G., Balàzs E., Richards K. E. Transcription of Cauliflower mosaic virus DNA: detection of promoter sequences, and characterization of transcripts. Cell. 1982 Oct;30(3):763–773. doi: 10.1016/0092-8674(82)90281-1. [DOI] [PubMed] [Google Scholar]
  25. Herman R. C. Alternatives for the initiation of translation. Trends Biochem Sci. 1989 Jun;14(6):219–222. doi: 10.1016/0968-0004(89)90030-3. [DOI] [PubMed] [Google Scholar]
  26. Hinnebusch A. G. Novel mechanisms of translational control in Saccharomyces cerevisiae. Trends Genet. 1988 Jun;4(6):169–174. doi: 10.1016/0168-9525(88)90023-6. [DOI] [PubMed] [Google Scholar]
  27. Horvath C. M., Williams M. A., Lamb R. A. Eukaryotic coupled translation of tandem cistrons: identification of the influenza B virus BM2 polypeptide. EMBO J. 1990 Aug;9(8):2639–2647. doi: 10.1002/j.1460-2075.1990.tb07446.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hull R., Sadler J., Longstaff M. The sequence of carnation etched ring virus DNA: comparison with cauliflower mosaic virus and retroviruses. EMBO J. 1986 Dec 1;5(12):3083–3090. doi: 10.1002/j.1460-2075.1986.tb04614.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jackson R. J., Howell M. T., Kaminski A. The novel mechanism of initiation of picornavirus RNA translation. Trends Biochem Sci. 1990 Dec;15(12):477–483. doi: 10.1016/0968-0004(90)90302-r. [DOI] [PubMed] [Google Scholar]
  30. Jang S. K., Kräusslich H. G., Nicklin M. J., Duke G. M., Palmenberg A. C., Wimmer E. A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol. 1988 Aug;62(8):2636–2643. doi: 10.1128/jvi.62.8.2636-2643.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Jefferson R. A., Burgess S. M., Hirsh D. beta-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8447–8451. doi: 10.1073/pnas.83.22.8447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kaufman R. J., Murtha P., Davies M. V. Translational efficiency of polycistronic mRNAs and their utilization to express heterologous genes in mammalian cells. EMBO J. 1987 Jan;6(1):187–193. doi: 10.1002/j.1460-2075.1987.tb04737.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kozak M. Bifunctional messenger RNAs in eukaryotes. Cell. 1986 Nov 21;47(4):481–483. doi: 10.1016/0092-8674(86)90609-4. [DOI] [PubMed] [Google Scholar]
  34. Kozak M. Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs. Mol Cell Biol. 1989 Nov;9(11):5134–5142. doi: 10.1128/mcb.9.11.5134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kozak M. Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol Cell Biol. 1987 Oct;7(10):3438–3445. doi: 10.1128/mcb.7.10.3438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  37. Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Liu C. C., Simonsen C. C., Levinson A. D. Initiation of translation at internal AUG codons in mammalian cells. Nature. 1984 May 3;309(5963):82–85. doi: 10.1038/309082a0. [DOI] [PubMed] [Google Scholar]
  39. Mason W. S., Taylor J. M., Hull R. Retroid virus genome replication. Adv Virus Res. 1987;32:35–96. doi: 10.1016/s0065-3527(08)60474-1. [DOI] [PubMed] [Google Scholar]
  40. Miller P. F., Hinnebusch A. G. Sequences that surround the stop codons of upstream open reading frames in GCN4 mRNA determine their distinct functions in translational control. Genes Dev. 1989 Aug;3(8):1217–1225. doi: 10.1101/gad.3.8.1217. [DOI] [PubMed] [Google Scholar]
  41. Pain V. M. Initiation of protein synthesis in mammalian cells. Biochem J. 1986 May 1;235(3):625–637. doi: 10.1042/bj2350625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Peabody D. S., Berg P. Termination-reinitiation occurs in the translation of mammalian cell mRNAs. Mol Cell Biol. 1986 Jul;6(7):2695–2703. doi: 10.1128/mcb.6.7.2695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Peabody D. S., Subramani S., Berg P. Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants. Mol Cell Biol. 1986 Jul;6(7):2704–2711. doi: 10.1128/mcb.6.7.2704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Pelletier J., Sonenberg N. Insertion mutagenesis to increase secondary structure within the 5' noncoding region of a eukaryotic mRNA reduces translational efficiency. Cell. 1985 Mar;40(3):515–526. doi: 10.1016/0092-8674(85)90200-4. [DOI] [PubMed] [Google Scholar]
  45. Pelletier J., Sonenberg N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature. 1988 Jul 28;334(6180):320–325. doi: 10.1038/334320a0. [DOI] [PubMed] [Google Scholar]
  46. Pietrzak M., Shillito R. D., Hohn T., Potrykus I. Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector. Nucleic Acids Res. 1986 Jul 25;14(14):5857–5868. doi: 10.1093/nar/14.14.5857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Rhoads R. E. Cap recognition and the entry of mRNA into the protein synthesis initiation cycle. Trends Biochem Sci. 1988 Feb;13(2):52–56. doi: 10.1016/0968-0004(88)90028-x. [DOI] [PubMed] [Google Scholar]
  48. Sanfaçon H., Hohn T. Proximity to the promoter inhibits recognition of cauliflower mosaic virus polyadenylation signal. Nature. 1990 Jul 5;346(6279):81–84. doi: 10.1038/346081a0. [DOI] [PubMed] [Google Scholar]
  49. Schwartz S., Felber B. K., Fenyö E. M., Pavlakis G. N. Env and Vpu proteins of human immunodeficiency virus type 1 are produced from multiple bicistronic mRNAs. J Virol. 1990 Nov;64(11):5448–5456. doi: 10.1128/jvi.64.11.5448-5456.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sedman S. A., Gelembiuk G. W., Mertz J. E. Translation initiation at a downstream AUG occurs with increased efficiency when the upstream AUG is located very close to the 5' cap. J Virol. 1990 Jan;64(1):453–457. doi: 10.1128/jvi.64.1.453-457.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sedman S. A., Good P. J., Mertz J. E. Leader-encoded open reading frames modulate both the absolute and relative rates of synthesis of the virion proteins of simian virus 40. J Virol. 1989 Sep;63(9):3884–3893. doi: 10.1128/jvi.63.9.3884-3893.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sedman S. A., Mertz J. E. Mechanisms of synthesis of virion proteins from the functionally bigenic late mRNAs of simian virus 40. J Virol. 1988 Mar;62(3):954–961. doi: 10.1128/jvi.62.3.954-961.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Spanjaard R. A., van Duin J. Translational reinitiation in the presence and absence of a Shine and Dalgarno sequence. Nucleic Acids Res. 1989 Jul 25;17(14):5501–5507. doi: 10.1093/nar/17.14.5501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Thomas K. R., Capecchi M. R. Introduction of homologous DNA sequences into mammalian cells induces mutations in the cognate gene. Nature. 1986 Nov 6;324(6092):34–38. doi: 10.1038/324034a0. [DOI] [PubMed] [Google Scholar]
  55. Wang F., Petti L., Braun D., Seung S., Kieff E. A bicistronic Epstein-Barr virus mRNA encodes two nuclear proteins in latently infected, growth-transformed lymphocytes. J Virol. 1987 Apr;61(4):945–954. doi: 10.1128/jvi.61.4.945-954.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Williams N. P., Mueller P. P., Hinnebusch A. G. 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. 1988 Sep;8(9):3827–3836. doi: 10.1128/mcb.8.9.3827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]

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