Fig 2: FMRP and FXR1P regulate RNA editing.a, Western blot of ADAR1, ADAR2, FMRP and FXR1P proteins in the nuclear and cytoplasmic fractions of HeLa cells. Cell fractionation was confirmed by Western blotting of ß-tubulin and U1–70K as marker proteins. Control cells and cells with stable knockdown of FMR1 or FXR1 were used. Experiment was repeated twice with similar results. b, Co-IP experiments with or without RNase A in HeLa cells between ADAR1, ADAR2, FMRP and FXR1P. Endogenous proteins were targeted except for ADAR2 (where a FLAG-tagged ADAR2 was expressed). Experiment was repeated 3 times with similar results. c, Shortest distance between FMRP or FXR1P eCLIP peaks and turquoise editing sites resulted from the WGCNA analysis (orange). Ten sets of random control sites (gray) are depicted for comparison (see Methods). Number of editing sites (N) is shown (see Methods for P value calculation). d, Experimental testing of an RNA editing site in the TEAD1 gene for its dependence on ADAR1, ADAR2, FMRP or FXR1P. Control HeLa cells or cells with stable knockdown of one of these proteins were used to express a minigene that contains the editing site. Editing levels were measured by Sanger sequencing. Example sequencing traces are shown for each sample with the targeted editing site underlined. Boxplots include three biological replicates. Overall P value (shown above plot) was calculated by one-way ANOVA. Individual comparison P values were calculated by one-tailed Student’s t-test. e, Similar as d, for an editing site in the EEF2K gene. f. similar as d, but displaying editing levels of the TEAD1 editing site in minigenes with the wild-type sequence or different versions of mutants introduced to predicted FMRP binding motifs (see Supplementary Fig. 20). Wild-type HeLa cells were used to express these minigenes. g, Similar as f, for the editing site in the EEF2K gene (see Supplementary Fig. 20). h, RNA editing levels in control HeLa cells and cells with stable knockdown of ADAR1, ADAR2, FMR1 or FXR1. Hyper-editing sites in three genes were tested. Error bars represent standard errors of three biological replicates. i, Editing levels in control HeLa cells in the same hyper-edited genes as in h, but with mutations introduced at predicted FXR1 binding motifs (see Supplementary Fig. 23c-e). Error bars are standard errors of three biological replicates. j, Editing changes at six differential editing sites in ASD induced by control shRNA (shControl) or shRNA knockdown of ADAR1, ADAR2, FMR1, and FXR1 in two neuroblastoma cell lines, KELLY and SK-N-BE(2) (see Supplementary Fig. 24). Boxplots show editing changes against shRNA control over the six editing sites (n=6 editing sites). P-values calculated using two-tailed t-test. Boxplot definitions for d-g, j: center=median, lower hinge=25th percentile, upper hinge=75th percentile, min and max extend to observations at most 1.5 * IQR.