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. 2011 Mar 8;39(12):5215–5231. doi: 10.1093/nar/gkr086

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© The Author(s) 2011. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 8. — Free poly(A) RNA is sufficient to restore a microRNA response in nuclease-treated RRL. (A) Translation of 0.46 fmol of Luc (black bars), and Luc-451X6 (gray bars) was carried out for 60 min in untreated RRL (URRL) or nuclease-treated RRL alone (RRL) or in the presence of 5 pmol of capped/polyadenylated Firefly-coding competitor mRNAs under control of the β-globin 5′UTR (+/+) or uncapped/non-polyadenylated mRNAs under the control of the HCV and CrPV IRESes. (B) Translation of 0.46 fmol of Luc (black bars), and Luc-451X6 (gray bars) was carried out for 1 h in untreated RRL (URRL) or nuclease-treated RRL alone (RRL) or in the presence of 1.2 pmol of free poly(A) [poly(A)] (GE Healthcare), 27 µM of free cap analog (Cap analog) (New England Biolabs) or 1.2 pmol of free poly(A) together with 27 µM of cap analog [Cap analog + poly(A)]. Total luciferase activity (left panel) as well as luciferase activity normalized against Luc expression (right panel) are presented. (C) Translation of 0.46 fmol of Globin, EMCV and HCV RNAs bearing no target sites (No Target, black bars) or 6 target-sites for miR-451 (451X6, gray bars), was carried out in RRL (left panel) and RRL in the presence of free poly(A) RNA (right panel) for 60 min. Error bars correspond to three independent experiments.