Studying RNA-binding domains by dual fluorescence RNA-binding assay. (A) Schematic representation of the PABP–eGFP fusion protein and its deletion mutants. (B,C) eGFP, PABP–eGFP, PABP–NTD–eGFP, and PABP–CTD–eGFP cell lines were induced for 16 h and irradiated with 254-nm UV light. Control samples were RNase-treated and recombinant proteins were immunoprecipitated with GFP-multiTrap. Co-immunoprecipitated RNAs were hybridized with oligo(DT) coupled with WellRED D4. The graphs represent relative green (eGFP) and far red (WellRED D4) fluorescence levels (B) and their ratios normalized to RNase-treated samples (C). The graphs were generated from three biological and three technical replicates (total of nine immunoprecipitations). (D) Schematic representation of the eGFP-fused RBP motifs of ALY and SDAD1. After incubation of the respective cell lines for 16 h with or without tetracycline, cells were lysed in loading buffer. Expression levels and molecular size of eGFP fusion proteins were determined by Western blotting. (E,F) The eGFP fusion proteins were induced in HeLa Flp-In cells for 16 h by addition of tetracyclin. After lysis, control samples were RNase-treated and recombinant proteins were immunoprecipitated with GFP-multiTrap. Co-immunoprecipitated RNAs were hybridized with oligo(DT) coupled with WellRED D4. The graphs represent relative green (eGFP) and far red (WellRED D4) fluorescence levels (E) and their ratio normalized to RNase-treated samples (F). The graphs were generated from two biological and three technical replicates (total of six immunoprecipitations). (G) Schematic representation of interactome capture. (H) eGFP fusion proteins were expressed in HeLa Flp-In cells for 16 h and irradiated with 254-nm UV light. After lysis, protein–RNA complexes were isolated with oligo(DT) and eluted in a low salt buffer at 55°C. Green fluorescence (eGFP) was measured in a plate reader and represented in a graph. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. Horizontal red and green lines represent averaged eGFP or Tred background signal, respectively, extracted from control samples.
