Supplementary Figure 3: Direct and unbiased detection of protein–RNA interactions within the PRC2-AEBP2 complex.
From: RNA exploits an exposed regulatory site to inhibit the enzymatic activity of PRC2
a, Evidence of UV cross-linking, analyzed using 18% SDS–PAGE and visualized by Coomassie blue and silver staining. Mw: Molecular weight marker; Pre: input before adding LysC or ArgC protease; Inp: input; FT: flow-through; EL: eluate. Scatterplots (bottom) indicate intensities identified by MS/MS for each of the peptides in the input (x-axis) and eluate (y-axis) in four independent RBDmap experiments (in assorted colors). Although the recovered peptides were obtained in quantities below the detection limit of SDS–PAGE (EL lanes in all gels), they were detected by MS/MS only in the +UV sample, indicating the stringency of the purification process. b, PRC2-AEBP2 mutants were evaluated by 10% SDS–PAGE and gel filtration chromatography (Sephacryl S-400 HR resin). c, Fluorescence anisotropy used to quantify the affinity of the mutants to G4 24 RNA. The resulting dissociation constant (Kd), Hill coefficients and the derived ΔΔG are indicated together with details of the mutated amino acids in EZH2 and EED. Error bars in (c) represent standard deviation based on three independent experiments that were performed on different days. Standard errors are indicated in (d) when applicable. e-g, The impaired RNA-binding activity of the mutants and their position on the surface of PRC2 is represented in a ΔΔG heat map using the PRC2-AEBP2 structure (regulatory and substrate peptides are colored in magenta and black respectively). h, Bar plot represents the relative HMTase activities of PRC2-AEBP2 mutations toward the H3 substrate compared to the wild-type, which is indicated as a dashed gray line. Error bars indicate standard deviations as measured across three independent experiments. P values were determined using unpaired two-tailed Student’s t-test; *, P < 0.05. i,j, Representative Coomassie blue-stained SDS–PAGE and the corresponding radiograms used for the HMTase assays are in Fig. 3g and Supplemental Fig. 3h. k, The affinity of PRC2-AEBP2 to 32P-radiolabeled G4 256 RNA was quantified using EMSA. Data points represent three-fold dilutions of PRC2-AEBP2 starting from 50 nM. l, Quantification was done by fitting the EMSA data to an equilibrium binding curve. Error bars indicate standard deviation based on three independent experiments that were performed on different days. The resulting dissociation constant (Kd), Hill coefficient and standard errors are indicated.
