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Inhibitory effect of myb7 uORF on downstream gene expression in homologous (rice) and heterologous (tobacco) systems

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Abstract

The rice myb7 mRNA contains in its long leader an upstream open reading frame (uORF) putatively coding for a 40 amino acid peptide. uORFs have been found in the leader of mRNAs encoding transcriptional factors or other proteins involved in cellular growth and development. They are thought to translationally regulate the expression of downstream ORFs. Here, we showed the ability of the myb7 uORF to inhibit the expression of downstream reporter genes both in homologous (rice) and heterologous (tobacco) systems. This effect seems partially related to its translation, as indicated by the comparison with the mutagenized uORF. In both systems most of the inhibitory effect was due to the presence of the intercistronic region, in disagreement with the Kozak model. Moreover, replacing the uORF or the intercistronic region with a different one, we demonstrated that the inhibitory effect strictly depends on their co-presence. Finally, in vitro assays showed that the myb7 uORF is translated and inhibits the downstream ORF translation.

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References

  • Alderete, J.P., Jarrahian, S. and Geballe, A.P. 1999. Translational effects of mutation and polymorphisms in a repressive upstream open reading frame of the human cytomegalovirus UL4 gene. J. Virol. 73: 8330–8337.

    Google Scholar 

  • Bilang, R., Klöti, A., Schrott, M. and Potrykus, I. 1994. PEG-mediated direct gene transfer and electroporation In: S.B. Gelvin and R.A. Schilperoort (Eds.) Plant Molecular Biology Manual, Kluwer Academic Publishers, Dordrecht/Boston/London, pp. A1: 1-16.

    Google Scholar 

  • Child, S.J., Miller, M.K. and Geballe, A.P. 1999. Translational control by an upstream open reading frame in the HER-2/neu transcript. J. Biol. Chem. 274: 24335–24341.

    Google Scholar 

  • Christensen, H. A. and Quail, P.H. 1996. Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res. 5: 213–218.

    Google Scholar 

  • Christou P. 1994. Rice Biotechnology and Genetic Engineering. Technomic Publishing Co., Lancaster, USA.

    Google Scholar 

  • D'Amico, L., Valvasina, B., Daminati, M.G., Fabbrini, M.S., Nitti, G., Bollini, R., Ceriotti, A. and Vitale, A. 1992. Bean homologs of the mammalian glucose-regulated proteins: induction by tunicamycin and interaction with newly synthesized seed storage proteins in the endoplasmic reticulum. Plant J. 2: 443–455.

    Google Scholar 

  • Delbecq, P., Werner, M., Feller, A., Filipkowski, R.K., Messenguy, F. and Piérard, A. 1994. A segment of mRNA encoding the leader peptide of the CPA1 gene confers repression by arginine on a heterologous yeast gene transcript. Mol. Cell. Biol. 14: 2378–2390.

    Google Scholar 

  • Fromm, M., Taylor, L.P. and Walbot, V. 1985. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc. Natl. Acad. Sci. USA 82: 5824–5828.

    Google Scholar 

  • Giovinazzo, G., Manzocchi, L.A., Bianchi, M.W., Coraggio, I. and Viotti, A. 1992. Functional analysis of the regulatory region of a zein gene in transiently transformed protoplasts. Plant Mol. Biol. 19: 257–263.

    Google Scholar 

  • Grant, C.M. and Hinnebusch, A.G. 1994. Effect of sequence context at stop codons on efficiency of reinitiation in gcn4 translational control. Mol. Cell. Biol. 14: 606–618.

    Google Scholar 

  • Gray, N.K. and Wickens, M. 1998. Control of translation initiation in animals. Annu. Rev. Cell Dev. Biol. 14: 399–458.

    Google Scholar 

  • Jefferson R.A. 1987. Assaying chimeric genes in plants: the Gus gene fusion system. Plant Mol. Biol. Rep. 5: 387–405.

    Google Scholar 

  • Jefferson R.A., Kavanagh T.A. and Bevan M.W. 1987. Gus fusions: ?-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901–3907.

    Google Scholar 

  • Kemper, E.L., da Silva, M.J. and Arruda, P. 1996. Effect of microprojectile bombardment parameters and osmotic treatment on particle penetration and tissue damage in transiently transformed cultured immature maize (Zea mays L.) embryos. Plant Sci. 121: 85–93.

    Google Scholar 

  • Kozak, M., 1987. Effects of intercistronic length on the efficiency of reinitiation by eukaryotic ribosomes. Mol. Cell. Biol. 7: 3438–3445.

    Google Scholar 

  • Kozak, M., 1989. The scanning model for translation: an update. J. Cell Biol. 108: 229–241.

    Google Scholar 

  • Kozak, M., 1999. Inititiation of translation in prokaryotes and eukaryotes. Gene 234: 187–208.

    Google Scholar 

  • Krieg, P.A. and Melton, D.A. 1984. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucl. Acids Res. 12: 7057–7070.

    Google Scholar 

  • Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

    Google Scholar 

  • Lincoln, A.J., Monczak, Y., Williams, S.C. and Johnson, P.F. 1998. Inhibition of CCAAT/enhancer-binding protein ? and ? translation by upstream open reading frames. J. Biol. Chem. 273: 9552–9560.

    Google Scholar 

  • Locatelli, F., Bracale, M., Magaraggia, F., Faoro, F., Manzocchi, L.A. and Coraggio, I. 2000. The product of the rice myb7 unspliced mRNA dimerizes with the maize leucine zipper Opaque2 and stimulates its activity in a transient expression assay. J. Biol. Chem. 275: 17619–17625.

    Google Scholar 

  • Loguercio, L.L., Zhang, J.Q. and Wilkins, T.A. 1999. Differential regulation of six novel myb-domain genes defines two distinct expression patterns in allotetraploid cotton (Gossypium hirsutum L.). Mol. Gen. Genet. 261: 660–671.

    Google Scholar 

  • Lohmer, S., Maddaloni, M., Motto, M., Salamini, F. and Thompson, R.D. 1993. Translation of the mRNA of the maize transcriptional activator opaque-2 is inhibited by upstream open reading frames present in the leader sequence. Plant Cell 5: 65–73.

    Google Scholar 

  • Luehrsen, K.R. and Walbot, V. 1994. The impact of AUG start codon context on maize gene expression in vivo. Plant Cell Rep. 13: 454–458.

    Google Scholar 

  • Magaraggia, F., Solinas, G., Valle, G., Giovinazzo, G. and Coraggio, I. 1997. Maturation and translation mechanisms involved in the expression of a myb gene of rice. Plant Mol. Biol. 35: 1003–1008.

    Google Scholar 

  • Mathews, D.H., Sabina, J., Zuker, M. and Turner, D.H. 1999. Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288: 911–940.

    Google Scholar 

  • Mize, G.J., Ruan, H., Low, J.J. and Morris, D.R. 1998. The inhibitory upstream open reading frame from mammalian Sadenosylmethionine decarboxylase mRNA has a strict sequence specificity in critical positions. J. Biol. Chem. 273: 32500–32505.

    Google Scholar 

  • Morris, D.R. and Geballe, A.P. 2000. Upstream open reading frames as regulators of mRNA translation. Mol. Cell. Biol. 20: 8635–8642.

    Google Scholar 

  • Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15: 473–497.

    Google Scholar 

  • Nagy, J.I. and Maliga, P. 1976. Callus induction and plant regeneration from mesophyll protoplasts of Nicotiana sylvestris. Z. Pflanzenphysiol. 78: 453–544.

    Google Scholar 

  • Oliveira, C.C. and McCarthy, J.E.G. 1995. The relationship between eukaryotic translation and mRNA stability. J. Biol. Chem. 270: 8936–8943.

    Google Scholar 

  • Parola, A.L. and Kobilka, B.K. 1994. The peptide product of a 5' leader cistron in the ?2 adrenergic receptor mRNA inhibits receptor synthesis. J. Biol. Chem. 269: 4497–4505.

    Google Scholar 

  • Potrykus, I. and Shillito, R.D. 1986. Protoplasts: isolation, culture, plant regeneration. Meth. Enzymol. 118: 549–578.

    Google Scholar 

  • Reynolds, K., Zimmer, A.M. and Zimmer A. 1996. Regulation of RAR ?2 mRNA expression: evidence for an inhibitory peptide encoded in the 5'-untranslated region. J. Cell Biol. 134: 827–835.

    Google Scholar 

  • Ruan, H., Hill, J.R., Fatemie-Nainie, S. and Morris, D.R. 1994. Cell-specific translational regulation of S-adenosylmethionine decarboxylase mRNA. J. Biol. Chem. 269: 17905–17910.

    Google Scholar 

  • Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Plainview, NY.

    Google Scholar 

  • Vilela, C., Ramirez, C.V., Linz, B., Rodrigues-Pousada, C. and McCarthy, J.E.G. 1999. Post-termination ribosome interactions with the 5' UTR modulate yeast mRNA stability. EMBO J. 18: 3139–3152.

    Google Scholar 

  • Vivier, M.A., Sollitti, P. and Pretorius, I.S. 1999. Functional analysis of multiple AUG codons in the transcripts of the STA2 glucoamylase gene from Saccharomyces cerevisiae. Mol. Gen. Genet. 261: 11–20.

    Google Scholar 

  • Wang, L. and Wessler, S.R. 1998. Inefficient reinitiation is responsible for upstream open reading frame-mediated translational repression of the maize R gene. Plant Cell 10: 1733–1746.

    Google Scholar 

  • Wang, L. and Wessler, S.R. 2001. Role of mRNA secondary structure in translational repression of the maize transcriptional activator Lc. Plant Physiol. 125: 1380–1387.

    Google Scholar 

  • Wang, Z., Gaba, A., and Sachs, M.S. 1999. A highly conserved mechanism of regulated ribosome stalling mediated by fungal arginine attenuator peptides that appears independent of the charging status of arginyl-tRNAs. J. Biol. Chem. 274: 37565–37574.

    Google Scholar 

  • Wang, Z. and Sachs, M.S. 1997. Ribosome stalling is responsible for arginine-specific translational attenuation in Neurospora crassa. Mol. Cell. Biol. 17: 4904–4913.

    Google Scholar 

  • Werner, M., Feller, A., Messenguy, F. and 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 

  • Widholm, J.M. 1972. The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cell. Stain Technol. 47: 189–194.

    Google Scholar 

  • Zuker, M., Mathews, D.H. and Turner, D.H. 1999. Algorithms and thermodynamics for RNA secondary structure prediction: a practical guide. In: J. Barciszewski and B.F.C. Clark (Eds.) RNA Biochemistry and Biotechnology, NATO ASI Series, Kluwer Academic Publishers, Dordrecht/Boston/London, pp. 11–43.

    Google Scholar 

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Locatelli, F., Magnani, E., Vighi, C. et al. Inhibitory effect of myb7 uORF on downstream gene expression in homologous (rice) and heterologous (tobacco) systems. Plant Mol Biol 48, 309–318 (2002). https://doi.org/10.1023/A:1013340004348

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