Biolayer interferometry

The biolayer interferometry system (BLItz, Pall ForteBio) was used to evaluate molecular interactions and to calculate binding affinities. To calculate the affinity of protein binding to RNA, a 5′ biotinylated poly(U) RNA of 15 residues (Sigma‐Aldrich) at a concentration of 50–100 μg/ml was immobilized on Streptavidin biosensors (ForteBio). The streptavidin biosensors were hydrated for 10 min before use in a buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM β‐mercaptoethanol, and 0.5 mg/ml BSA. At least three increasing concentrations (0.25–20 μM) of each prey protein in a 4 μl volume were utilized to estimate the binding affinity. The prey proteins were diluted in the same hydration buffer that was used throughout the experiment containing 0.5 mg/ml BSA in order to eliminate non‐specific binding of the prey protein to the Streptavidin biosensor.

To assess the binding of GST‐Mex67_(528–599), GST‐Mex67 UBA_(545–599), or GST‐Mex67∆UBA_(481–544) constructs to Mip6 RRM3/4_(313–480), the bait protein at a concentration of 50 μg/ml was immobilized on anti‐GST biosensors (ForteBio). Different concentrations of the prey protein were then used to evaluate the association and dissociation steps. BSA (0.5 mg/ml) was used in the buffer throughout the experiment to reduce non‐specific binding. Curve fitting, and association (Ka) and dissociation (Kd) constant calculations were done using BLItz Pro 1.2 software. The duration of the loading, association, and dissociation steps were adjusted for each experiment depending on the saturation of the signal obtained.