Figure 2

Examination of the RNA-binding activity of RBP7910 using EMSAs. (A) Gel mobility shift assays show the binding of 40 nM of the recombinant protein to increasing concentrations of different RNA substrates. The wedges show the increasing concentrations of ³²P-labeled RNAs (0.1–1.2 nM, gA6[14]; 5–30 nM, pre-edited CYb; and 0.5–10 nM edited CYb), and shifted bound protein-RNA complexes are marked with black triangles. Bound and free RNA concentrations from the experiments shown in panel A were used to estimate the binding activity of RBP7910 to each RNA substrate (left panel). The saturation binding curve was obtained using none-linear regression analyses of five individual experiments for each substrate. Calculated Kd ± SD values in nanomolar units are shown for each labeled RNA substrate. (B) Competition assays verified the better affinity of the protein to gA6[14] to other guides and mRNAs. Competition assays were done by incubation of a fixed concentration of purified protein and labeled gA6[14] in the absence and presence of increasing concentrations of unlabeled competitors (gCYb RNA variants, pre-edited, and edited CYb mRNAs). Asterisk indicates the input labeled RNA in the absence of the protein and the white star shows the labeled RNA with protein in the absence of the competitor RNA. Numbers above the panels indicate the fold excess of the unlabeled RNA competitors and numbers below of each panel is the shift percentage in the presence of competitor RNAs normalized to the shift in the absence of a competitor \((whitestar)\) . The name of unlabeled RNA substrate used for each assay is indicated above each panel along with the complete sequence under each panel.