Supplementary Figure 3: YBX1 serves as a specific m5C reader.
From: 5-methylcytosine promotes pathogenesis of bladder cancer through stabilizing mRNAs
a, Nuclear and cytoplasmic fractions of 293T cells were analysed by western blotting. PARP1 and β-Tubulin serve as nuclear and cytoplasmic markers, respectively. b, SDS-PAGE showing the purified wild-type (WT) and mutant (W65F) YBX1 CSD proteins as stained by coomassie brilliant blue. Positions of molecular markers are indicated on the left in kDa. c-e, Comparison of different binding modes for m6A RNA, 5mC DNA, and m5C RNA. c, Recognition of m6A by the YTH domain of YTHDC1.d, Recognition of 5mC by the MBD domain of MBD4. e, Recognition of 5mC by the Zinc-finger domain of Klf4. f, Sequence alignment of YBX1 CSD homologs, including human (hs, homo sapiens, P67809), mouse (mm, mus musculus, P62960), zebrafish (zf, danio rerio, A1A605), frog (xt, xenopus laevis, P21573), chick (gg, gallus, Q06066), fly (dm, drosophila melanogaster, O46173), and silkworm (bm, bombyx mori, Q6F6B1). Identical residues are shaded in red. Secondary structural elements of YBX1 CSD are displayed above. The residues involving π-π stacking interactions are marked by red triangles. N67 and N70 that contact m5C5 through hydrogen bond interactions are indicated with brown diamond. g, h, Western blotting showing the NSUN2 knockdown efficiency and Flag-YBX1 overexpression in NSUN2-depleted HeLa (g) and T24 (h) cells. ACTIN serves as loading control. i, Western blotting showing the levels of NSUN2, Flag-YBX1, siNSUN2-Insensitive Myc-NSUN2 wild-type (Myc-NSUN2-WT) and double-mutant (Myc-NSUN2-DM) in control and NSUN2 knockdown HeLa cells used for PAR-CLIP. ACTIN was used as loading control. j, Overlap of m5C-containing genes with YBX1 and ALYREF binding targets, respectively. The experiments were performed twice independently with similar results. k, Cumulative distribution of m5C levels in input and YBX1-RIP mRNA samples in T24 cells (m5C sites in input: n = 22,104; m5C sites in YBX1-RIP: n = 5,496. Experiments were repeated three times independently with similar results for a, g, h and i. The P value was calculated by a two-sided unpaired Wilcoxon and Mann–Whitney test. Unprocessed gels for a, g, h and i are provided in Supplementary Figs. 8–10.
