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. 2012 May 24;40(15):7541–7551. doi: 10.1093/nar/gks471

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© The Author(s) 2012. 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/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Effects of uORFs in leader RNAs on the translation of reporter RNAs associated with leader RNAs via dsRNA bridges. (A) Schematic diagram of leader and reporter RNAs. The leader RNA contained uORFs encoding oligopeptides [36 or 18 amino acids (a.a.)] preceded by sequences with a strong (ATT ATG G), adequate (TTT ATG G) or weak (TTT ATG T) Kozak context, or no AUG (ATT ACA G) (23). The 5′ terminus of the leader RNA was G-capped, and the 3′-end contained a sequence complementary to the 5′ sequence in the reporter RNA. The reporter RNA is the same as that described in Figure 1A. The uORFs are located 70 nt downstream of the cap structure and 30 nt upstream of the complementary sequence. The spacer regions were originated from the human β-globin gene (left panel). RNA complexes containing both leader and reporter RNAs covalently connected by a stem-loop structure (i.e. within a single molecule) were constructed to serve as control mRNAs (right panel). (B) The RNA annealing process and in vitro translation experiments were performed as described in Materials and Methods, except that the reaction time for in vitro translation was 15 min instead of 60 min. Reporter translational efficiencies were determined by measuring firefly luciferase activity after pre-annealing leader and reporter RNAs (lanes 1–5 and 10–13), or with omission of the pre-annealing step (lanes 6–9 and 14–17). The relative luciferase activity in each translation mixture is shown. The luciferase activity of the reaction without leader RNA was set to unity to permit comparisons. The error bars indicate the standard deviations in six independent experiments. (C) The luciferase activities in the reaction mixtures were further analyzed after normalization to the luciferase activity in the reaction mixture containing leader RNAs with no AUG. Specifically, the luciferase activities in lanes 2–5 were normalized to that in lane 5 in panel (B), and the luciferase activities in lanes 10–13 were normalized to that in lane 13 in panel (B). (D) The translation of reporter RNAs containing both leader and reporter RNAs in a single RNA molecule was monitored by measuring luciferase activity in reaction mixtures. Relative translational efficiency is depicted after normalization to luciferase activity in the reaction mixture containing no uORF (ATT ACA G). Lanes 1–3 and 5–7 depict relative translational efficiencies of mRNAs containing uORFs encoding oligopeptides of 36 and 18 amino acids, respectively.

Figure 3. — Effects of uORFs in leader RNAs on the translation of reporter RNAs associated with leader RNAs via dsRNA bridges. (A) Schematic diagram of leader and reporter RNAs. The leader RNA contained uORFs encoding oligopeptides [36 or 18 amino acids (a.a.)] preceded by sequences with a strong (ATT ATG G), adequate (TTT ATG G) or weak (TTT ATG T) Kozak context, or no AUG (ATT ACA G) (23). The 5′ terminus of the leader RNA was G-capped, and the 3′-end contained a sequence complementary to the 5′ sequence in the reporter RNA. The reporter RNA is the same as that described in Figure 1A. The uORFs are located 70 nt downstream of the cap structure and 30 nt upstream of the complementary sequence. The spacer regions were originated from the human β-globin gene (left panel). RNA complexes containing both leader and reporter RNAs covalently connected by a stem-loop structure (i.e. within a single molecule) were constructed to serve as control mRNAs (right panel). (B) The RNA annealing process and in vitro translation experiments were performed as described in Materials and Methods, except that the reaction time for in vitro translation was 15 min instead of 60 min. Reporter translational efficiencies were determined by measuring firefly luciferase activity after pre-annealing leader and reporter RNAs (lanes 1–5 and 10–13), or with omission of the pre-annealing step (lanes 6–9 and 14–17). The relative luciferase activity in each translation mixture is shown. The luciferase activity of the reaction without leader RNA was set to unity to permit comparisons. The error bars indicate the standard deviations in six independent experiments. (C) The luciferase activities in the reaction mixtures were further analyzed after normalization to the luciferase activity in the reaction mixture containing leader RNAs with no AUG. Specifically, the luciferase activities in lanes 2–5 were normalized to that in lane 5 in panel (B), and the luciferase activities in lanes 10–13 were normalized to that in lane 13 in panel (B). (D) The translation of reporter RNAs containing both leader and reporter RNAs in a single RNA molecule was monitored by measuring luciferase activity in reaction mixtures. Relative translational efficiency is depicted after normalization to luciferase activity in the reaction mixture containing no uORF (ATT ACA G). Lanes 1–3 and 5–7 depict relative translational efficiencies of mRNAs containing uORFs encoding oligopeptides of 36 and 18 amino acids, respectively.