Fig. 3

Structure and RNA binding properties of the SDN1 CTD RRM domain. a–c Electrophoretic mobility shift assays (EMSA) of wild-type SDN1 (a), SDN1 ΔC (b) and SDN1 RRM (c) with ³²P-labeled miR173. Assays were conducted at a series of protein concentrations: 0.5 µM, 1 µM, 5 µM, 10 µM, 50 µM, 100 µM, and 500 µM (from left to right). No Mg²⁺ was included to prevent catalytic activity. Scale bars represent standard deviations calculated from three biological replicates. d Plots showing the fraction of the protein-bound RNA at varying protein concentrations. The fraction of bound RNA was calculated from the densities of the free RNA bands in a, b and c. e Crystal structure of the SDN1 CTD E329A/E330A/E332A mutant. The N-terminal, first α helix connecting the RRM domain is colored in gray; the extended β4 and β5 in the RRM domain are shown in yellow. f Surface electrostatic potential of the SDN1 RRM domain showing that the antiparallel β sheet forms a positively charged surface. Red, −5.0 k_BT/e; Blue, + 5.0 k_BT/e. g Changes in chemical shifts for cross-peaks in the RRM spectra during NMR titration. Residues that show large chemical shift perturbations (Δδ_avg > 0.05 ppm) are colored in orange and indicated in orange dots in the diagram above. Residues of which cross peaks disappeared are indicated in pink dots in the diagram above. h The molecular surface of SDN1 RRM, onto which the above residues were mapped. This reveals that the antiparallel β sheet surface is responsible for RNA substrate binding. Residues that show large chemical shift changes or for which signals disappeared during titration are colored in orange or pink, respectively