FIG. 2.

Second RRM of GRSF-1 is required for mRNA binding in vitro. (A) Coomassie blue-stained denaturing 12% polyacrylamide gel of equal moles (1.25 × 10⁻¹¹ mol) of wild-type and mutant GST-GRSF-1 fusion proteins purified by glutathione affinity chromatography from E. coli. (B) UV cross-linking analysis of thrombin-cleaved GRSF-1 (rGRSF-1) and intact wild-type (Wt) and mutant GST-GRSF-1 fusion proteins (1.25 × 10⁻¹¹ mol) incubated with radiolabeled NP 5′ UTR RNA (10⁶ dpm) in 5 mM HEPES (pH 7.6)-25 mM KCl-2 mM MgCl₂-5% glycerol-100 mM NaCl-2 mM dithiothreitol-20 U of RNasin (Promega) at 30°C for 15 min. Samples were then exposed to UV light (1.0 J) in a Stratalinker (Stratagene), followed by incubation with 10 mg of RNase A per ml and 2 U of RNase T₁ at 37°C for 30 min. Samples were suspended in 1× Laemmli buffer and separated by SDS-10% PAGE, and the gel was dried and visualized with a Storm 850 phosphorimager (Molecular Dynamics). These data are representative of two independent experiments.