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. 1999 Sep;5(9):1149–1157. doi: 10.1017/s1355838299990325

A stable hairpin preceded by a short open reading frame promotes nonlinear ribosome migration on a synthetic mRNA leader.

M Hemmings-Mieszczak 1, T Hohn 1
PMCID: PMC1369838  PMID: 10496216

Abstract

The regulation of cauliflower mosaic virus (CaMV) pregenomic 35S RNA translation occurs via nonlinear ribosome migration (ribosome shunt) and is mediated by an elongated hairpin structure in the leader. The replacement of the viral leader by a series of short, low-energy stems in either orientation supports efficient ribosomal shunting, showing that the stem per se, and not its sequence, is recognized by the translation machinery. The requirement for cis-acting sequences from the unstructured terminal regions of the viral leader was analyzed: the 5'-terminal polypyrimidine stretch and the short upstream open reading frame (uORF) A stimulate translation, whereas the 3'-flanking region seems not to be essential. Based on these results, an artificial leader was designed with a stable stem flanked by unstructured sequences derived from parts of the 5'- and 3'-proximal regions of the CaMV 35S RNA leader. This artificial leader is shunt-competent in translation assays in vivo and in vitro, indicating that a low-energy stem, broadly used as a device to successfully interfere with ribosome scanning, can efficiently support translation, if preceded by a short uORF. The synthetic 140-nt leader can functionally replace the CaMV 35S RNA 600-nt leader, thus implicating the universal role that nonlinear ribosome scanning could play in translation initiation in eukaryotes.

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Selected References

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  1. Andjelković M., Alessi D. R., Meier R., Fernandez A., Lamb N. J., Frech M., Cron P., Cohen P., Lucocq J. M., Hemmings B. A. Role of translocation in the activation and function of protein kinase B. J Biol Chem. 1997 Dec 12;272(50):31515–31524. doi: 10.1074/jbc.272.50.31515. [DOI] [PubMed] [Google Scholar]
  2. Belsham G. J. Dual initiation sites of protein synthesis on foot-and-mouth disease virus RNA are selected following internal entry and scanning of ribosomes in vivo. EMBO J. 1992 Mar;11(3):1105–1110. doi: 10.1002/j.1460-2075.1992.tb05150.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Curran J., Kolakofsky D. Scanning independent ribosomal initiation of the Sendai virus Y proteins in vitro and in vivo. EMBO J. 1989 Feb;8(2):521–526. doi: 10.1002/j.1460-2075.1989.tb03406.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dominguez D. I., Ryabova L. A., Pooggin M. M., Schmidt-Puchta W., Fütterer J., Hohn T. Ribosome shunting in cauliflower mosaic virus. Identification of an essential and sufficient structural element. J Biol Chem. 1998 Feb 6;273(6):3669–3678. doi: 10.1074/jbc.273.6.3669. [DOI] [PubMed] [Google Scholar]
  6. Franck A., Guilley H., Jonard G., Richards K., Hirth L. Nucleotide sequence of cauliflower mosaic virus DNA. Cell. 1980 Aug;21(1):285–294. doi: 10.1016/0092-8674(80)90136-1. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Fütterer J., Kiss-László Z., Hohn T. Nonlinear ribosome migration on cauliflower mosaic virus 35S RNA. Cell. 1993 May 21;73(4):789–802. doi: 10.1016/0092-8674(93)90257-q. [DOI] [PubMed] [Google Scholar]
  9. Fütterer J., Potrykus I., Bao Y., Li L., Burns T. M., Hull R., Hohn T. Position-dependent ATT initiation during plant pararetrovirus rice tungro bacilliform virus translation. J Virol. 1996 May;70(5):2999–3010. doi: 10.1128/jvi.70.5.2999-3010.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Geballe A. P., Morris D. R. Initiation codons within 5'-leaders of mRNAs as regulators of translation. Trends Biochem Sci. 1994 Apr;19(4):159–164. doi: 10.1016/0968-0004(94)90277-1. [DOI] [PubMed] [Google Scholar]
  11. Gray N. K., Hentze M. W. Iron regulatory protein prevents binding of the 43S translation pre-initiation complex to ferritin and eALAS mRNAs. EMBO J. 1994 Aug 15;13(16):3882–3891. doi: 10.1002/j.1460-2075.1994.tb06699.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hemmings-Mieszczak M., Steger G., Hohn T. Alternative structures of the cauliflower mosaic virus 35 S RNA leader: implications for viral expression and replication. J Mol Biol. 1997 Apr 18;267(5):1075–1088. doi: 10.1006/jmbi.1997.0929. [DOI] [PubMed] [Google Scholar]
  13. Hemmings-Mieszczak M., Steger G., Hohn T. Regulation of CaMV 35 S RNA translation is mediated by a stable hairpin in the leader. RNA. 1998 Jan;4(1):101–111. [PMC free article] [PubMed] [Google Scholar]
  14. Hinnebusch A. G. Translational control of GCN4: an in vivo barometer of initiation-factor activity. Trends Biochem Sci. 1994 Oct;19(10):409–414. doi: 10.1016/0968-0004(94)90089-2. [DOI] [PubMed] [Google Scholar]
  15. Kaminski A., Howell M. T., Jackson R. J. Initiation of encephalomyocarditis virus RNA translation: the authentic initiation site is not selected by a scanning mechanism. EMBO J. 1990 Nov;9(11):3753–3759. doi: 10.1002/j.1460-2075.1990.tb07588.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kozak M. A consideration of alternative models for the initiation of translation in eukaryotes. Crit Rev Biochem Mol Biol. 1992;27(4-5):385–402. doi: 10.3109/10409239209082567. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Kozak M. Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc Natl Acad Sci U S A. 1986 May;83(9):2850–2854. doi: 10.1073/pnas.83.9.2850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. 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]
  23. Pilipenko E. V., Gmyl A. P., Maslova S. V., Svitkin Y. V., Sinyakov A. N., Agol V. I. Prokaryotic-like cis elements in the cap-independent internal initiation of translation on picornavirus RNA. Cell. 1992 Jan 10;68(1):119–131. doi: 10.1016/0092-8674(92)90211-t. [DOI] [PubMed] [Google Scholar]
  24. Pooggin M. M., Hohn T., Fütterer J. Forced evolution reveals the importance of short open reading frame A and secondary structure in the cauliflower mosaic virus 35S RNA leader. J Virol. 1998 May;72(5):4157–4169. doi: 10.1128/jvi.72.5.4157-4169.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Scheper G. C., Voorma H. O., Thomas A. A. Basepairing with 18S ribosomal RNA in internal initiation of translation. FEBS Lett. 1994 Oct 3;352(3):271–275. doi: 10.1016/0014-5793(94)00975-9. [DOI] [PubMed] [Google Scholar]
  26. Schmidt-Puchta W., Dominguez D., Lewetag D., Hohn T. Plant ribosome shunting in vitro. Nucleic Acids Res. 1997 Jul 15;25(14):2854–2860. doi: 10.1093/nar/25.14.2854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  28. Schärer-Hernández N., Hohn T. Nonlinear ribosome migration on cauliflower mosaic virus 35S RNA in transgenic tobacco plants. Virology. 1998 Mar 15;242(2):403–413. doi: 10.1006/viro.1998.9038. [DOI] [PubMed] [Google Scholar]
  29. Stripecke R., Oliveira C. C., McCarthy J. E., Hentze M. W. Proteins binding to 5' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells. Mol Cell Biol. 1994 Sep;14(9):5898–5909. doi: 10.1128/mcb.14.9.5898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yueh A., Schneider R. J. Selective translation initiation by ribosome jumping in adenovirus-infected and heat-shocked cells. Genes Dev. 1996 Jun 15;10(12):1557–1567. doi: 10.1101/gad.10.12.1557. [DOI] [PubMed] [Google Scholar]

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