UV Cross-Linking and RNA-Binding Assay

Trichomonad cytoplasmic extracts and the recombinant proteins TvACTN3r, the TvACTN3 domains (DIr, DIIr, and DIIIr), hIRP-1r (used as a positive control), and BSA (1 μg) (used as a negative control) were incubated with 200,000 cpm (10–15 ng) ³²P-labeled RNA probes for 30 min at 4°C in 25 μL reaction buffer (10 mM HEPES-KOH [pH 7.4], 3 mM MgCl₂, 5% (v/v) glycerol, 100 mM KCl, 20 U RNasin, and 5 μg yeast tRNA; Invitrogen). After RNA-binding, the reaction mixture was placed on ice and irradiated with a UV-lamp (UVP; 800,000 μJ/cm²) for 15 min. Unprotected RNA was digested for 30 min at 25°C with RNase A (10 μg) and RNase T1 (20 U). RPCs were resolved on 10% SDS-PAGE gels. The gels were stained with Coomassie Brilliant Blue (CBB) and dried, and radioactive bands were visualized by autoradiography in a FLA 5000 phosphoimager (Fujifilm, Co, Tokyo Japan) with a Software MultiGauge V3.0 [22 (link)]. These experiments were independently performed at least three times, with similar results.