Modulation of RNA-binding properties of the RNA helicase UPF1 by its activator UPF2

RNA-binding properties of UPF2_S and its impact on binding of UPF1 to RNA. (A) Fluorescence anisotropy competition assay to determine how addition of UPF2_S affects binding of UPF1 to 6-FAM-U₁₂ RNA. Titration of increasing amounts of UPF2_S into a constant amount of UPF1–U₁₂ RNA mixture results in partial displacement of UPF1 from RNA. The data points and error bars of this and all other fluorescence anisotropy experiments are the mean and standard deviation of at least two independent experiments. (B) Fluorescence anisotropy assay to determine the dissociation constant (K_D) of the UPF2_S–U₁₂ RNA interaction. The error associated with the K_D is its standard deviation (SD). UPF2_S binds U₁₂ RNA with a modest affinity, which is an order of magnitude lower than the RNA-binding affinity of UPF1 (∼50 nM). (C) Analytical SEC analysis of binding of UPF2_S to U₁₅ RNA. Top and bottom panels show the overlay of chromatograms of UPF2_S without (blue traces) and with RNA (black traces), and the corresponding PAGE analyses of peak fractions of each run, respectively. Peak 3 at 1.9 mL corresponds to the U₁₅ RNA. UPF2_S does not form a stable complex with RNA.