Nucleic Acids Research, 2003, Vol. 31, No. 2 722-733
© 2003 Oxford University Press
Comparison of the capacity of different viral internal ribosome entry segments to direct translation initiation in poly(A)-dependent reticulocyte lysates
Unité de Régulation de la Traduction Eucaryote et Virale, CNRS URA 1966, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France
*To whom correspondence should be addressed at present address: Cytoskeleton and Cell Motility Laboratory, School of Biological Sciences, University of Exeter, Perry Road, Exeter, UK. Tel: +44 1392 263 263; Fax: +44 1392 264 668; Email: a.m.borman{at}exeter.ac.uk
Polyadenylation stimulates translation of capped eukaryotic mRNAs and those carrying picornaviral internal ribosome entry segments (IRESes) in vivo. Rabbit reticulocyte lysates (RRL) reproduce poly(A)-mediated translation stimulation in vitro after partial depletion of ribosomes and ribosome-associated factors. Here, we have evaluated the effects of varying different parameters (extent of extract depletion, cleavage of eIF4G, concentrations of KCl, MgCl2 and programming mRNA) on IRES-driven translation efficiency and poly(A)-dependency in ribosome-depleted RRL. For comparison, the study included a standard capped, polyadenylated mRNA. Dramatic differences were observed in the abilities of the different IRESes to direct translation in ribosome-depleted extracts. While the hepatitis A virus IRES was incapable of driving translation in physiological conditions in depleted RRL, mRNAs carrying the foot-and-mouth disease virus and hepatitis C virus IRESes were translated significantly better than a standard cellular mRNA in the same conditions. Indeed, the capacities of these IRESes to direct translation in ribosome-depleted RRL were similar to those reported previously in certain cell lines. Both the abilities of the IRESes to drive translation and their individual salt optima in ribosome-depleted extracts suggest that these elements have dramatically different affinities for some component(s) of the canonical translation machinery. Finally, using poliovirus as an example, we show that the ribosome-depleted system is well suited to the study of the translational capacity of naturally occurring IRES variants.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  B. Zhang, G. Morace, V. Gauss-Muller, and Y. Kusov Poly(A) binding protein, C-terminally truncated by the hepatitis A virus proteinase 3C, inhibits viral translation Nucleic Acids Res., September 27, 2007; 35(17): 5975 - 5984. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. S. Rifo, E. P. Ricci, D. Decimo, O. Moncorge, and T. Ohlmann Back to basics: the untreated rabbit reticulocyte lysate as a competitive system to recapitulate cap/poly(A) synergy and the selective advantage of IRES-driven translation Nucleic Acids Res., September 25, 2007; 35(18): e121 - e121. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Wang, M. Weaver, and N. S. Magnuson Cryptic promoter activity in the DNA sequence corresponding to the pim-1 5'-UTR Nucleic Acids Res., April 20, 2005; 33(7): 2248 - 2258. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Koloteva-Levine, D. Pinchasi, I. Pereman, A. Zur, M. Brandeis, and O. Elroy-Stein The Apc5 Subunit of the Anaphase-Promoting Complex/Cyclosome Interacts with Poly(A) Binding Protein and Represses Internal Ribosome Entry Site-Mediated Translation Mol. Cell. Biol., May 1, 2004; 24(9): 3577 - 3587. [Abstract] [Full Text] [PDF]
|
|