TRAP1 binds RNA in living cells. (A) RNA-binding activity of TRAP1-FLAG-HA in HeLa FITR cells. RNA copurified with TRAP1 was dephosphorylated and ligated at the 3′-end to an infrared preA-L3-IR800-biotin DNA adaptor in order to be visualized at 800 nm (top panel). Representative western blot to confirm equal IP efficiencies (bottom panel). Asterisk indicates the expected molecular mass of TRAP1. (B) eGFP-based RNA-binding assay. Relative green fluorescence signal of RNA-bound fraction over input from cells expressing either TRAP1-eGFP or eGFP proteins (n = 5). Significance was assessed by unpaired Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001). Error bars represent SEM. (C) RNA-binding activity of TRAP1 in PEA1 cells. Protein–RNA complexes are visualized by ligation of a biotinylated oligo to bound RNA and then detected with an HRP-linked streptavidin (left panel). Representative western blot to confirm equal IP efficiencies (right panel). (D) Assessment of RNA-binding by OOPS. HeLa TRAP1-FLAG-HA cells were treated with either antimycin A 1 μM (4 h) or emetine 100 μg/mL (15 min), or grown in media without glucose or glutamine for 4 h. At the end of treatment, cells were UV cross-linked and subjected to OOPS, and both inputs and OOPS eluates were analyzed by western blot. TIAR and H3 were used as positive and negative controls, respectively.
