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. 2019 Jan 30;47(5):2244–2262. doi: 10.1093/nar/gkz032

Figure 6.

Figure 6.

IDH1 binds to GA/AU-rich single-stranded RNA. (A) GA- and AU-rich RNA motifs are enriched in IDH1’s RNA targets in all sequences or in the 3′ UTR regions, respectively. (B–E) EMSA analysis of direct binding of purified wild-type to various fluorescence-labeled RNA/DNA probes as indicated. (B) EMSA with the 5 × GAA ssRNA probe containing the overall motif #1. (C) EMSA with ssRNA probes with 2–5 copies of GAA(A) nucleotides. (D) Binding isotherms for IDH1-RNA interactions. Each data point is shown as mean ± SD (n = 3 biological replicates). Error bars represent standard deviations (SD), some of which are too small to be visualized. (E) Summary of RNA-binding activities of IDH1 toward various ssRNA probes used above. Dissociation constants (Kd) are shown. N.D., not detected. GAA and AUUU units are underlined. (F) Relative m6A level of IDH1 RNA targets in ESCs was determined by HPLC-MS/MS following FBioRIP assays. Data are shown as mean ± SD, n = 3. (G) Comparison of RNA-binding activities of wild-type (WT) and oncogenic R132H mutant of IDH1 to the 5× GAA ssRNA probe as shown in the panel (B).