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Nucleic Acids Research Advance Access published online on September 18, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm682
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

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

Ricardo Soto Rifo1,2, Emiliano P. Ricci1,2, Didier Décimo1,2, Olivier Moncorgé1,2 and Théophile Ohlmann1,2,*

1Inserm U 758, Lyon, F-69364 and 2Ecole Normale Supérieure de Lyon, Unité de Virologie Humaine, IFR 128, Lyon, F-69364, France

*To whom correspondence should be addressed. Tel: +33 472 728 953; Fax: +33 472 72 81 37; Email: tohlmann{at}ens-lyon.fr

Received July 19, 2007. Revised August 17, 2007. Accepted August 18, 2007.

Translation of most eukaryotic mRNAs involves the synergistic action between the 5' cap structure and the 3' poly(A) tail at the initiation step. The poly(A) tail has also been shown to stimulate translation of picornavirus internal ribosome entry sites (IRES)-directed translation. These effects have been attributed principally to interactions between eIF4G and poly(A)-binding protein (PABP) but also to the participation of PABP in other steps during translation initiation. As the rabbit reticulocyte lysate (RRL) does not recapitulate this cap/poly(A) synergy, several systems based on cellular cell-free extracts have been developed to study the effects of poly(A) tail in vitro but they generally exhibit low translational efficiency. Here, we describe that the non-nuclease-treated RRL (untreated RRL) is able to recapitulate the effects of poly(A) tail on translation in vitro. In this system, translation of a capped/polyadenylated RNA was specifically inhibited by either Paip2 or poly(rA), whereas translation directed by HCV IRES remained unaffected. Moreover, cleavage of eIF4G by FMDV L protease strongly stimulated translation directed by the EMCV IRES, thus recapitulating the competitive advantage that the proteolytic processing of eIF4G confers to IRES-driven RNAs.


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