Figure 2. An RNA aptamer targeting eIF1A for inactivation negatively affects both cap- and IRES-driven translation in lysate.

(A) Sequence and predicted secondary structure of the α-eIF1A aptamer used in this study. (B) Top: Results of pull-down experiment with the HCV IRES (similar to Figure 1), showing substantial depletion of eIF1A with aptamer treatment compared to negative control antisense (AS) aptamer RNA. Bottom: Pull-down experiments with several IRES RNAs, with blotting to detect eIFs that should not be removed or depleted by the aptamer. (C) ³⁵S-Met labeling of translation products in RRL using a dual-reporter mRNA template. The aptamer treatment reduces protein levels over time from IRES-driven (CAT) and cap-driven (Rluc) messages. The effect of apatamer (+ apt) addition is shown compared to the addition of no RNA or a randomized 40 nucleotide RNA (+ N40). (D) Diagram of monocistronic reporters used in the experiments of panels (E–H). (E) Results of translation assays in RRL using reporter RNAs. The level of each RNA in untreated lysate is set at 100% and the effect of aptamer treatment is reported as a percentage of that for each RNA. Capped (Cap) RNA is the positive control and the CrPV IRES is the negative control for a requirement for eIF1A. (F) Identical to panel (E) but assayed in HeLa cell extract. (G) Translation assays in RRL using reporter RNAs with the HCV, CSFV, and SPV-1 IRESs. Cap is included as a control. (H) Identical to panel (G) but assayed in HeLa cell extract. In panels (E–H), error bars represent averages ±SEM of ≥3 independent experiments. Statistical significance shown by: *p<0.05, **p<0.01, ***p<0.001.

DOI: http://dx.doi.org/10.7554/eLife.21198.004

Figure 2—figure supplement 1. Validation of α-eIF1A aptamer.

(A) Binding profiles of the eIF1A aptamer using surface plasmon resonance. The aptamer RNA (apt) or the ‘N40’ random sequence RNA was immobilized on a sensor chip for Biacore2000 analysis. eIF1A solution was prepared and was injected at time 0 for 120 s. From the fitting of these sensorgrams by the instrument software BIAevaluation, the dissociation constant (Kd) of the aptamer-eIF1A interaction was estimated as 5.2 nM. (B) Pulldown assay of translation factors bound to eIF1A. The following samples were loaded on each lane: lane 1, 1 μl of HeLa cell extract; lane 2, coprecipitated sample by Ni-NTA agarose; lane 3, coprecipitated sample by Ni-NTA agarose with His-CAT; and lane 4, coprecipitated sample by Ni-NTA agarose with 6XHis-eIF1A. Separated and transferred proteins are detected by Western blotting using antibodies against eIF3 p66, eIF3 p110, rpS6, eIF2α and eIF2β (from upper to lower). These proteins bound with eIF1A specifically. (C) Pull-down assay from HeLa cell extracts using Ni-NTA agarose and 6XHis-eIF1A, with 0.3, 1, 3 or 10 μM N40 random RNA (N40) or 0.3, 1, 3 or 10 μM aptamer (labeled). Two components of eIF3, p66 and p110, plus rpS6, eIF2α and eIF2β bound to eIF1A. When aptamer was added to this pull-down reaction, the precipitation of eIF3 components and rpS6 was reduced, suggesting that the aptamer blocked the interaction of eIF1A with eIF3 and rpS6.

DOI: http://dx.doi.org/10.7554/eLife.21198.005