Purification of eIF4F and cap binding assay

Highly purified eIF4F was purified from rabbit reticulocytes as previously described (16). A graphical representation of the purification schema is shown in Figure ​Figure1A.1A. For purification of eIF4E:eIF4G complexes from E. coli, pET28-eIF4E, pET28-eIF4G (90–651), or pET28-eIF4G (90–1129) were transformed into Rosetta2 pLysS cells, grown in 2XYT media until OD reached 0.6 and then induced with 1 mM IPTG for 4 h. Aliquots of bacteria were spun down (1 ml for eIF4E and 10 ml for eIF4G) and frozen at –80°C. For purification, frozen aliquots were thawed on ice and resuspended in 20 mM Tris [pH 8.0], 150 mM NaCl, 0.5 mg/ml lysozyme. Cells were further disrupted by sonication and lysate was clarified by centrifugation. 10% NP-40 was added to supernatant to a final concentration of 0.5%. Individual lysates or combined lysates were incubated with m⁷GTP-agarose (Jena Biosciences) for 2 h and washed 3× with 20 mM Tris [pH 8.0], 150 mM NaCl, 0.5% NP-40. Cap competition assays were performed with small RNAs as described previously (4).

G4-tiRNAs directly target eIF4F. (A) Strategy of purification of eIF4F adapted from (16). (B) Purified human eIF4F is sensitive to TOG containing tiRNAs in a G-quadruplex dependent manner. Purified eIF4F was bound to m⁷GTP-agarose and challenged with indicated small RNAs. 5′tiRNA^Ala efficiently disrupted the eIF4F complex, but 5′tiRNA^Ala(daG), which cannot form a G4 efficiently had reduced activity. (C) Schematic of eIF4G indicating domains required for interaction with other proteins (orange), protease cleavage sites (red lines), sites of truncations (black lines). RNA Binding region (red bar) and HEAT1 repeat (green bar) are indicated. (D) Recombinant eIF4G containing the RNA binding region is sensitive to 5′tiRNA^Ala. eIF4E and truncation of eIF4G were expressed and purified from E. coli, bound to m⁷GTP-agarose and challenged with indicated RNAs. 5′tiRNA^Ala could disrupt eIF4F when eIF4G retained the RNA binding regions (90–1129). This disruption was dependent upon G4 formation as 5′tiRNA^Ala(daG) is incapable of disrupting eIF4F.