Human Pumilio 1 binding to modified RNAs in vitro was consistent with λ-dynamics predictions. (A) Electrophoretic mobility shift assays (EMSAs) were used to estimate the binding affinity in vitro. RNA oligos without or with the designated RNA modifications were incubated with increasing concentrations (0–1000 nM) of recombinant human Pumilio 1 (hPUM1) RNA-binding domain, run on a polyacrylamide gel, and imaged for carboxyfluorescein (FAM) fluorescence. A sample without protein served as an unbound RNA control. The lower band corresponds to unbound RNA. The upper band corresponds to RNA bound to the recombinant protein. The binding affinity can be estimated by calculating the protein concentration at the binding inflection point. Shown are representative gels from hPUM1 EMSA binding experiments. (B) EMSAs were performed at least three times, and calculated binding dissociation constants (Kd) were reported with their mean and standard deviation. (*) P < 0.05; (**) P < 0.01. See Supplemental Figure S1 for full statistical analyses and Materials and Methods for more experimental details.
