Dissection of protein and RNA regions required for SPEN binding to XIST A-repeat RNA

SPEN RRM 3 is required for binding to SRA RNA but not to XIST_A-full RNA. (A) Diagram of SPEN_{RRM 2–4} and the mutated RRM 3 protein construct SPEN_{RRM 2–4 mut3} used for RNA-binding studies. (B) Locations of mutations in the SPEN_{RRM 2–4 mut3} mutated RRM 3 construct which were mutated to alanine. (C) Quantification of the bound fraction of SPEN_{RRM 2–4} and SPEN_{RRM 2–4 mut3} with XIST_A-full, SRA, or BARD1 RNA from EMSA experiments, n = 3. (SHARP_{RRM 2–4}) or n = 4 (SHARP_{RRM 2–4 mut3}). Data are the mean ± SEM. (D) Comparison of binding affinities for SPEN_{RRM 2–4} and SPEN_{RRM 2–4 mut3} for XIST_A-full, SRA, or BARD1 RNA from replicate experiments, n = 3 (SPEN_{RRM 2–4}) or n = 4 (SPEN_{RRM 2–4 mut3}). (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. Data are the mean ± SEM.