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. 2000 Dec;6(12):1781–1790. doi: 10.1017/s1355838200001679

Picornavirus IRESes and the poly(A) tail jointly promote cap-independent translation in a mammalian cell-free system.

G Bergamini 1, T Preiss 1, M W Hentze 1
PMCID: PMC1370048  PMID: 11142378

Abstract

In eukaryotic cells, efficient translation of most cellular mRNAs requires the synergistic interplay between the m7GpppN cap structure and the poly(A) tail during initiation. We have developed and characterized a cell-free system from human HeLa cells that recapitulates this important feature, displaying more than one order of magnitude of translational synergism between the cap structure and the poly(A) tail. The stimulation of cap-dependent translation by the poly(A) tail is length-dependent, but not mediated by changes in mRNA stability. Using this system, we investigated the effect of the poly(A) tail on the translation of picornaviral RNAs, which are naturally polyadenylated but initiate translation via internal ribosome entry sites (IRESs). We show that translation driven by the IRESs of poliovirus (PV), encephalomyocarditis virus (EMCV), and hepatitis A virus is also significantly augmented by a poly(A) tail, ranging from an approximately 3-fold stimulation for the EMCV-IRES to a more than 10-fold effect for the PV IRES. These results raise interesting questions concerning the underlying molecular mechanism(s). The cell-free system described here should prove useful in studying these questions as well as providing a general biochemical tool to examine the translation initiation pathway in a more physiological setting.

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Selected References

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  1. Andino R., Böddeker N., Silvera D., Gamarnik A. V. Intracellular determinants of picornavirus replication. Trends Microbiol. 1999 Feb;7(2):76–82. doi: 10.1016/s0966-842x(98)01446-2. [DOI] [PubMed] [Google Scholar]
  2. Anthony D. D., Merrick W. C. Eukaryotic initiation factor (eIF)-4F. Implications for a role in internal initiation of translation. J Biol Chem. 1991 Jun 5;266(16):10218–10226. [PubMed] [Google Scholar]
  3. Belsham G. J., Sonenberg N. RNA-protein interactions in regulation of picornavirus RNA translation. Microbiol Rev. 1996 Sep;60(3):499–511. doi: 10.1128/mr.60.3.499-511.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blyn L. B., Towner J. S., Semler B. L., Ehrenfeld E. Requirement of poly(rC) binding protein 2 for translation of poliovirus RNA. J Virol. 1997 Aug;71(8):6243–6246. doi: 10.1128/jvi.71.8.6243-6246.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Borman A. M., Bailly J. L., Girard M., Kean K. M. Picornavirus internal ribosome entry segments: comparison of translation efficiency and the requirements for optimal internal initiation of translation in vitro. Nucleic Acids Res. 1995 Sep 25;23(18):3656–3663. doi: 10.1093/nar/23.18.3656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Borman A. M., Kean K. M. Intact eukaryotic initiation factor 4G is required for hepatitis A virus internal initiation of translation. Virology. 1997 Oct 13;237(1):129–136. doi: 10.1006/viro.1997.8761. [DOI] [PubMed] [Google Scholar]
  7. Borman A. M., Kirchweger R., Ziegler E., Rhoads R. E., Skern T., Kean K. M. elF4G and its proteolytic cleavage products: effect on initiation of protein synthesis from capped, uncapped, and IRES-containing mRNAs. RNA. 1997 Feb;3(2):186–196. [PMC free article] [PubMed] [Google Scholar]
  8. Borman A. M., Le Mercier P., Girard M., Kean K. M. Comparison of picornaviral IRES-driven internal initiation of translation in cultured cells of different origins. Nucleic Acids Res. 1997 Mar 1;25(5):925–932. doi: 10.1093/nar/25.5.925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Castagnetti S., Hentze M. W., Ephrussi A., Gebauer F. Control of oskar mRNA translation by Bruno in a novel cell-free system from Drosophila ovaries. Development. 2000 Mar;127(5):1063–1068. doi: 10.1242/dev.127.5.1063. [DOI] [PubMed] [Google Scholar]
  10. Cohen J. I., Ticehurst J. R., Feinstone S. M., Rosenblum B., Purcell R. H. Hepatitis A virus cDNA and its RNA transcripts are infectious in cell culture. J Virol. 1987 Oct;61(10):3035–3039. doi: 10.1128/jvi.61.10.3035-3039.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Créancier L., Morello D., Mercier P., Prats A. C. Fibroblast growth factor 2 internal ribosome entry site (IRES) activity ex vivo and in transgenic mice reveals a stringent tissue-specific regulation. J Cell Biol. 2000 Jul 10;150(1):275–281. doi: 10.1083/jcb.150.1.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Deshpande A. K., Chatterjee B., Roy A. K. Translation and stability of rat liver messenger RNA for alpha 2 mu-globulin in Xenopus oocyte. The role of terminal poly(A). J Biol Chem. 1979 Sep 25;254(18):8937–8942. [PubMed] [Google Scholar]
  13. Drummond D. R., Armstrong J., Colman A. The effect of capping and polyadenylation on the stability, movement and translation of synthetic messenger RNAs in Xenopus oocytes. Nucleic Acids Res. 1985 Oct 25;13(20):7375–7394. doi: 10.1093/nar/13.20.7375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fortes P., Inada T., Preiss T., Hentze M. W., Mattaj I. W., Sachs A. B. The yeast nuclear cap binding complex can interact with translation factor eIF4G and mediate translation initiation. Mol Cell. 2000 Jul;6(1):191–196. [PubMed] [Google Scholar]
  15. Galili G., Kawata E. E., Smith L. D., Larkins B. A. Role of the 3'-poly(A) sequence in translational regulation of mRNAs in Xenopus laevis oocytes. J Biol Chem. 1988 Apr 25;263(12):5764–5770. [PubMed] [Google Scholar]
  16. Gallie D. R., Tanguay R. L., Leathers V. The tobacco etch viral 5' leader and poly(A) tail are functionally synergistic regulators of translation. Gene. 1995 Nov 20;165(2):233–238. doi: 10.1016/0378-1119(95)00521-7. [DOI] [PubMed] [Google Scholar]
  17. Gallie D. R. The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency. Genes Dev. 1991 Nov;5(11):2108–2116. doi: 10.1101/gad.5.11.2108. [DOI] [PubMed] [Google Scholar]
  18. Gebauer F., Corona D. F., Preiss T., Becker P. B., Hentze M. W. Translational control of dosage compensation in Drosophila by Sex-lethal: cooperative silencing via the 5' and 3' UTRs of msl-2 mRNA is independent of the poly(A) tail. EMBO J. 1999 Nov 1;18(21):6146–6154. doi: 10.1093/emboj/18.21.6146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gingras A. C., Svitkin Y., Belsham G. J., Pause A., Sonenberg N. Activation of the translational suppressor 4E-BP1 following infection with encephalomyocarditis virus and poliovirus. Proc Natl Acad Sci U S A. 1996 May 28;93(11):5578–5583. doi: 10.1073/pnas.93.11.5578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Haller A. A., Semler B. L. Linker scanning mutagenesis of the internal ribosome entry site of poliovirus RNA. J Virol. 1992 Aug;66(8):5075–5086. doi: 10.1128/jvi.66.8.5075-5086.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hellen C. U., Wimmer E. Translation of encephalomyocarditis virus RNA by internal ribosomal entry. Curr Top Microbiol Immunol. 1995;203:31–63. doi: 10.1007/978-3-642-79663-0_2. [DOI] [PubMed] [Google Scholar]
  22. Hruby D. E., Roberts W. K. Encephalomyocarditis virus RNA: variations in polyadenylic acid content and biological activity. J Virol. 1976 Aug;19(2):325–330. doi: 10.1128/jvi.19.2.325-330.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hunt S. L., Hsuan J. J., Totty N., Jackson R. J. unr, a cellular cytoplasmic RNA-binding protein with five cold-shock domains, is required for internal initiation of translation of human rhinovirus RNA. Genes Dev. 1999 Feb 15;13(4):437–448. doi: 10.1101/gad.13.4.437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hunt S. L., Jackson R. J. Polypyrimidine-tract binding protein (PTB) is necessary, but not sufficient, for efficient internal initiation of translation of human rhinovirus-2 RNA. RNA. 1999 Mar;5(3):344–359. doi: 10.1017/s1355838299981414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Iizuka N., Najita L., Franzusoff A., Sarnow P. Cap-dependent and cap-independent translation by internal initiation of mRNAs in cell extracts prepared from Saccharomyces cerevisiae. Mol Cell Biol. 1994 Nov;14(11):7322–7330. doi: 10.1128/mcb.14.11.7322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Imataka H., Gradi A., Sonenberg N. A newly identified N-terminal amino acid sequence of human eIF4G binds poly(A)-binding protein and functions in poly(A)-dependent translation. EMBO J. 1998 Dec 15;17(24):7480–7489. doi: 10.1093/emboj/17.24.7480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jackson R. J., Hunt S. L., Gibbs C. L., Kaminski A. Internal initiation of translation of picornavirus RNAs. Mol Biol Rep. 1994 May;19(3):147–159. doi: 10.1007/BF00986957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jang S. K., Wimmer E. Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosomal entry site and involvement of a cellular 57-kD RNA-binding protein. Genes Dev. 1990 Sep;4(9):1560–1572. doi: 10.1101/gad.4.9.1560. [DOI] [PubMed] [Google Scholar]
  29. Joachims M., Van Breugel P. C., Lloyd R. E. Cleavage of poly(A)-binding protein by enterovirus proteases concurrent with inhibition of translation in vitro. J Virol. 1999 Jan;73(1):718–727. doi: 10.1128/jvi.73.1.718-727.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kerekatte V., Keiper B. D., Badorff C., Cai A., Knowlton K. U., Rhoads R. E. Cleavage of Poly(A)-binding protein by coxsackievirus 2A protease in vitro and in vivo: another mechanism for host protein synthesis shutoff? J Virol. 1999 Jan;73(1):709–717. doi: 10.1128/jvi.73.1.709-717.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lamphear B. J., Kirchweger R., Skern T., Rhoads R. E. Mapping of functional domains in eukaryotic protein synthesis initiation factor 4G (eIF4G) with picornaviral proteases. Implications for cap-dependent and cap-independent translational initiation. J Biol Chem. 1995 Sep 15;270(37):21975–21983. doi: 10.1074/jbc.270.37.21975. [DOI] [PubMed] [Google Scholar]
  32. Le H., Tanguay R. L., Balasta M. L., Wei C. C., Browning K. S., Metz A. M., Goss D. J., Gallie D. R. Translation initiation factors eIF-iso4G and eIF-4B interact with the poly(A)-binding protein and increase its RNA binding activity. J Biol Chem. 1997 Jun 27;272(26):16247–16255. doi: 10.1074/jbc.272.26.16247. [DOI] [PubMed] [Google Scholar]
  33. Lie Y. S., Macdonald P. M. In vitro translation extracts prepared from Drosophila ovaries and embryos. Biochem Biophys Res Commun. 2000 Apr 13;270(2):473–481. doi: 10.1006/bbrc.2000.2453. [DOI] [PubMed] [Google Scholar]
  34. Meerovitch K., Svitkin Y. V., Lee H. S., Lejbkowicz F., Kenan D. J., Chan E. K., Agol V. I., Keene J. D., Sonenberg N. La autoantigen enhances and corrects aberrant translation of poliovirus RNA in reticulocyte lysate. J Virol. 1993 Jul;67(7):3798–3807. doi: 10.1128/jvi.67.7.3798-3807.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Michel Y. M., Poncet D., Piron M., Kean K. M., Borman A. M. Cap-Poly(A) synergy in mammalian cell-free extracts. Investigation of the requirements for poly(A)-mediated stimulation of translation initiation. J Biol Chem. 2000 Oct 13;275(41):32268–32276. doi: 10.1074/jbc.M004304200. [DOI] [PubMed] [Google Scholar]
  36. Nomoto A., Detjen B., Pozzatti R., Wimmer E. The location of the polio genome protein in viral RNAs and its implication for RNA synthesis. Nature. 1977 Jul 21;268(5617):208–213. doi: 10.1038/268208a0. [DOI] [PubMed] [Google Scholar]
  37. Otero L. J., Ashe M. P., Sachs A. B. The yeast poly(A)-binding protein Pab1p stimulates in vitro poly(A)-dependent and cap-dependent translation by distinct mechanisms. EMBO J. 1999 Jun 1;18(11):3153–3163. doi: 10.1093/emboj/18.11.3153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Parsley T. B., Towner J. S., Blyn L. B., Ehrenfeld E., Semler B. L. Poly (rC) binding protein 2 forms a ternary complex with the 5'-terminal sequences of poliovirus RNA and the viral 3CD proteinase. RNA. 1997 Oct;3(10):1124–1134. [PMC free article] [PubMed] [Google Scholar]
  39. Pause A., Méthot N., Svitkin Y., Merrick W. C., Sonenberg N. Dominant negative mutants of mammalian translation initiation factor eIF-4A define a critical role for eIF-4F in cap-dependent and cap-independent initiation of translation. EMBO J. 1994 Mar 1;13(5):1205–1215. doi: 10.1002/j.1460-2075.1994.tb06370.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Pestova T. V., Hellen C. U., Shatsky I. N. Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry. Mol Cell Biol. 1996 Dec;16(12):6859–6869. doi: 10.1128/mcb.16.12.6859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pestova T. V., Shatsky I. N., Hellen C. U. Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes. Mol Cell Biol. 1996 Dec;16(12):6870–6878. doi: 10.1128/mcb.16.12.6870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Preiss T., Hentze M. W. Dual function of the messenger RNA cap structure in poly(A)-tail-promoted translation in yeast. Nature. 1998 Apr 2;392(6675):516–520. doi: 10.1038/33192. [DOI] [PubMed] [Google Scholar]
  44. Sachs A. B., Sarnow P., Hentze M. W. Starting at the beginning, middle, and end: translation initiation in eukaryotes. Cell. 1997 Jun 13;89(6):831–838. doi: 10.1016/s0092-8674(00)80268-8. [DOI] [PubMed] [Google Scholar]
  45. Scheper G. C., Voorma H. O., Thomas A. A. Eukaryotic initiation factors-4E and -4F stimulate 5' cap-dependent as well as internal initiation of protein synthesis. J Biol Chem. 1992 Apr 15;267(11):7269–7274. [PubMed] [Google Scholar]
  46. Spector D. H., Baltimore D. Requirement of 3'-terminal poly(adenylic acid) for the infectivity of poliovirus RNA. Proc Natl Acad Sci U S A. 1974 Aug;71(8):2983–2987. doi: 10.1073/pnas.71.8.2983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Stanway G. Structure, function and evolution of picornaviruses. J Gen Virol. 1990 Nov;71(Pt 11):2483–2501. doi: 10.1099/0022-1317-71-11-2483. [DOI] [PubMed] [Google Scholar]
  48. Stripecke R., Hentze M. W. Bacteriophage and spliceosomal proteins function as position-dependent cis/trans repressors of mRNA translation in vitro. Nucleic Acids Res. 1992 Nov 11;20(21):5555–5564. doi: 10.1093/nar/20.21.5555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Svitkin Y. V., Ginevskaya V. A., Ugarova T. Y., Agol V. I. A cell-free model of the encephalomyocarditis virus-induced inhibition of host cell protein synthesis. Virology. 1978 Jun 1;87(1):199–203. doi: 10.1016/0042-6822(78)90172-1. [DOI] [PubMed] [Google Scholar]
  50. Svitkin Y. V., Gradi A., Imataka H., Morino S., Sonenberg N. Eukaryotic initiation factor 4GII (eIF4GII), but not eIF4GI, cleavage correlates with inhibition of host cell protein synthesis after human rhinovirus infection. J Virol. 1999 Apr;73(4):3467–3472. doi: 10.1128/jvi.73.4.3467-3472.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Svitkin Y. V., Meerovitch K., Lee H. S., Dholakia J. N., Kenan D. J., Agol V. I., Sonenberg N. Internal translation initiation on poliovirus RNA: further characterization of La function in poliovirus translation in vitro. J Virol. 1994 Mar;68(3):1544–1550. doi: 10.1128/jvi.68.3.1544-1550.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tarun S. Z., Jr, Sachs A. B. A common function for mRNA 5' and 3' ends in translation initiation in yeast. Genes Dev. 1995 Dec 1;9(23):2997–3007. doi: 10.1101/gad.9.23.2997. [DOI] [PubMed] [Google Scholar]
  53. Tarun S. Z., Jr, Sachs A. B. Association of the yeast poly(A) tail binding protein with translation initiation factor eIF-4G. EMBO J. 1996 Dec 16;15(24):7168–7177. [PMC free article] [PubMed] [Google Scholar]
  54. Walter B. L., Nguyen J. H., Ehrenfeld E., Semler B. L. Differential utilization of poly(rC) binding protein 2 in translation directed by picornavirus IRES elements. RNA. 1999 Dec;5(12):1570–1585. doi: 10.1017/s1355838299991483. [DOI] [PMC free article] [PubMed] [Google Scholar]

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