Although 5-methylcytosine (m5C) is a widespread modification in RNAs, its regulation and biological role in pathological conditions (such as cancer) remain unknown. Here, we provide the single-nucleotide resolution landscape of messenger RNA m5C modifications in human urothelial carcinoma of the bladder (UCB). We identify numerous oncogene RNAs with hypermethylated m5C sites causally linked to their upregulation in UCBs and further demonstrate YBX1 as an m5C ‘reader’ recognizing m5C-modified mRNAs through the indole ring of W65 in its cold-shock domain. YBX1 maintains the stability of its target mRNA by recruiting ELAVL1. Moreover, NSUN2 and YBX1 are demonstrated to drive UCB pathogenesis by targeting the m5C methylation site in the HDGF 3′ untranslated region. Clinically, a high coexpression of NUSN2, YBX1 and HDGF predicts the poorest survival. Our findings reveal an unprecedented mechanism of RNA m5C-regulated oncogene activation, providing a potential therapeutic strategy for UCB.
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Deep-sequencing (RNA-Seq, RNA-BisSeq and PAR-CLIP-Seq) data that support the findings of this study have been deposited in the Gene Expression Omnibus database under accession number GSE133671, and also the Genome Sequence Archive under the accession number CRA001050 linked to the project PRJCA000975. The coordinates and structure factors have been deposited in the Protein Data Bank under accession code 6A6L. The data for human UCB, kidney renal clear cell carcinoma, prostate adenocarcinoma, pancreatic adenocarcinoma, breast invasive carcinoma, liver hepatocellular carcinoma and uterine corpus endometrial carcinoma were derived from the TCGA Research Network (http://cancergenome.nih.gov/). The source data for Figs. 1b,d,f, 3j,l, 4d,f, 5d,e, 6a–i, 7d,f and Supplementary Figs. 1g, 5c,e–i, 6c,f–h have been provided in Supplementary Table 10. All other data supporting the findings of this study are available from the corresponding author on reasonable request.
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This work was supported by grants from the National Key R&D Program of China (grant no. 2017YFC1309001 to D.X.), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no. XDA16000000 to Y.-G.Y.), the National Natural Science Foundation of China (grant nos 31625016 to Y.-G.Y., 81672530 to F.-J.Z., 31870741 to Y.H. and 31670824 to B.-F.S.), the National Key R&D Program of China Stem Cell and Translational Research (grant no. 2018YFA0109700 to Y.Y.), the Youth Innovation Promotion Association of CAS (grant nos 2016097 to B.-F.S. and 2018133 to Y.Y.), Shanghai Municipal Science and Technology Major Project (grant no. 2017SHZDZX01 to Y.-G.Y.), the NSFC consulting grant (grant no. 91753000 to Y.-G.Y.). We thank the staff from BL18U1 at the Shanghai Synchrotron Radiation Facility and the staff from the National Facility for Protein Science in Shanghai, Zhangjiang Laboratory for their technical support and assistance. We thank the staff from the Nanjing Biomedical Research Institute of Nanjing University for their assistance with data collection. We thank X. Ding and M. Zhang at the Laboratory of Proteomics core facility at the Institute of Biophysics, CAS for their technical support for the LC-MS analysis, the TCGA research network for providing the data analysed in this manuscript and the BIG CAS genomic platform for sequencing.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Supplementary Figure 1 Overview of m5C patterns and m5C-related pathways in tumour and normal samples.
a-d, RNA categories (a), mRNA region distributions (b), metagene profiles (c) and logo plot (d) of m5C sites in tumour and normal samples. The shaded area represents the range from minimal to maximal values (c). e, Heatmap showing the methylation level change of differential m5C sites (4,126 hypermethylated and 1,154 hypomethylated) between tumour and normal samples. f, The distribution of m5C differential levels (tumour relative to normal). g, UHPLC-MS/MS showing the percentage of mRNA m5C in five paired tumour and adjacent normal samples. T: tumour; N: adjacent normal. Data represent mean ± s.d. (n = 3 independent experiments). The P values were determined by a two-sided unpaired Student's t-test. h, Canonical pathway analysis of hypermethylated mRNAs (n = 2,041) in tumours. i, Heatmap showing the tendency of 103 hypermethylated genes in tumours involving 6 canonical cancer-related pathways (JAK/STAT, ERBB, PI3K/AKT, VEGF, EMT and ERK/MAPK). j, Canonical pathway analysis of hypomethylated mRNAs (n = 504) in tumours. k, Heatmap showing the methylation level change of differential m5C sites (185 sites: higher methylation; 90 sites: lower methylation) in MIBC relative to NMIBC. l, Canonical pathway analysis of mRNAs with higher methylated m5C sites (n = 185, MIBC relative to NMIBC). m, Heatmap showing the methylation tendency of 36 metastasis-related genes in MIBC relative to NMIBC. n, Heatmap showing the methylation level change of differential m5C sites (109 sites: higher methylation; 104 sites: lower methylation) in LM relative to non-LM UCBs. o, Heatmap showing the methylation tendency of 22 metastasis-related genes in LM relative to non-LM UCBs. Heatmaps (e, i, k, m-o) showing the z-score of m5C level. Colours from blue to red indicate low to high. 65 samples (36 UCB and 29 adjacent normal tissues) were used for a-f, h-j; 36 UCB samples (24 MIBC and 12 NMIBC) were used for k-m; 36 samples (15 LM and 21 non-LM) were used for n, o. All P values in h, j and l were calculated by one-sided Fisher’s exact test via IPA software. Source data for g are provided in Supplementary Table 10.
Supplementary Figure 2 Correlation of m5C level with the oncogenic gene expression in bladder cancer samples.
a, Scatter plot showing the correlation of m5C methylation with gene expression levels for MLLT11 transcription factor 7 cofactor (MLLT11) and proteasome 26S subunit, non-ATPase 3 (PSMD3) in 65 tissue samples. The centre line and the shaded regions means the regression line and 95% confidence interval, respectively. Spearman's correlation coefficient and P value was calculated using the function “cor.test” in the statistical language R. b, c, Boxplot showing the distribution of m5C methylation level (b) and the mRNA expression (c) of MLLT11 and PSMD3 in tumours compared to matched normal samples. The P values were determined by a two-sided paired Wilcoxon signed rank test. d, Kaplan-Meier analysis indicating the correlation of the mRNA expression of MLLT11 and PSMD3 with poor DFS rates in UCB patients from TCGA analysis. The P values were determined by a two-sided log-rank test. e, Scatter plot showing the correlation of m5C methylation levels for HDGF, F11R, RAB11FIP4, MLLT11 and PSMD3 with NSUN2 gene expression level in 65 tissue samples. The centre line and the shaded regions means the regression line and 95% confidence interval, respectively. Spearman’s correlation coefficient and P value was calculated using the function “cor.test” in the statistical language R. In boxplots of b and c, central bars represent medians, box edges represent the first and third interquartile ranges. 22 pairs of cancerous and non-cancerous bladder tissues were used for b and c.
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.
a, Western blotting showing the NSUN2 and YBX1 knockdown efficiency and Myc-ELAVL1 overexpression in NSUN2 or YBX1 knockdown HeLa cells. ACTIN was used as loading control (n = 3 independent experiments). b, PAR-CLIP assay of RNAs pulled down by Myc-ELAVL1 in NSUN2 or YBX1 deficient T24 cells (n = 2 independent experiments). c, Western blotting displaying the NSUN2 and YBX1 knockdown efficiency and Myc-ELAVL1 overexpression in NSUN2 or YBX1 knockdown T24 cells. ACTIN was used as loading control (n = 2 independent experiments). d, Western blotting showing the levels of YBX1, Flag-YBX1, siYBX1-insensitive Flag-YBX1-WT and Flag-YBX1-W65A in control and YBX1 knockdown HeLa cells used for PAR-CLIP. ACTIN was used as loading control (n = 4 independent experiments). e, Overlap of m5C-containing genes with YBX1 and ELAVL1 binding targets. Unprocessed gels for a-d are provided in Supplementary Figs. 8–10.
a, Western blotting showing NSUN2/YBX1 expression in NSUN2- or YBX1-knockdown or control UCB cells. GAPDH serves as protein loading control. b, Western blotting showing NSUN2 expression in NSUN2-knockdown UCB cells, which stably expressing NSUN2-WT, NSUN2- C321A, or NSUN2-DM. GAPDH was used as loading control. c, NSUN2 WT, not the C321A or DM mutant could restore the decreased colony formation ability of NSUN2-depleted cells. Top: representative images of crystal violet staining of cells; Bottom: histograms of colony numbers. Data are mean ± s.d. (n = 3 independent experiments). d, Western blotting showing YBX1 expression in YBX1-knockdown UCB cells, which stably expressing YBX1-WT or YBX1-W65A mutant. ACTIN was used as loading control. e, YBX1 WT, not the W65A mutant could restore the decreased colony formation ability of YBX1-depleted cells. Top: representative images of crystal violet staining of cells; Bottom: histograms of colony numbers. Data are mean ± s.d. (n = 3 independent experiments). f, NSUN2 WT, not the C321A or DM mutant could restore the decreased cell invasion ability of NSUN2-depleted cells tested by transwell assay. Top: representative images; Bottom: histograms of invasive cell numbers. Scale bar, 100 µm. Data are mean ± s.d. (n = 3 independent experiments). g, Inverted invasion assay showing the rescued cell invasive capacity of NSUN2-depleted UCB cells by reconstitution of NSUN2 WT compared to that of C321A or DM mutant. Data are mean ± s.d. (n = 5 independent experiments). h, YBX1 WT, not the W65A mutant could restore the decreased cell invasion ability of YBX1-depleted cells tested by transwell assay (n = 3 independent experiments). Top: representative images; Bottom: histograms of invasive cell numbers. Scale bar, 100 µm. i, Inverted invasion assay showing the rescued cell invasive in YBX1-knockdown UCB cells by reconstitution of YBX1 WT in relative to W65A mutant. Data are mean ± s.d. (n = 5 independent experiments). The precise P values (two-sided unpaired Student's t-test) and source data for c, e-i are provided in Supplementary Table 10. The experiments were performed twice independently with similar results for a, b and d. Unprocessed gels for a, b and d are provided in Supplementary Figs. 8–10.
a,b, Spearman’s correlation analysis demonstrating positive correlation between the expression level of NSUN2 (a) or YBX1 (b) and HDGF mRNA expression in UCB tissues from SYSUCC cohorts and TCGA, respectively. The centre line and the shaded regions indicate the regression line and 95% confidence interval, respectively. Spearman’s correlation coefficient and P value were calculated using the function “cor.test” in the statistical language R. c, Reduced HDGF mRNA half-life by silencing NSUN2 or YBX1 in T24 cells. Data are mean ± s.d. (n = 3 independent experiments). d, Western blotting showing the protein expression of NSUN2 and HDGF in NSUN2-knockdown UCB cells, which stably expressing NSUN2-WT, NSUN2- C321A, or NSUN2-DM. GAPDH was used as loading control. e, Western blotting showing the protein expression of YBX1 and HDGF in YBX1-knockdown UCB cells, which stably expressing YBX1-WT or YBX1-W65A. ACTIN was used as loading control. f, Relative luciferase mRNA expression of luciferase reporter gene with HDGF-m5C WT site (HDGF-WT) or mutant site (HDGF-Mut) in control, NSUN2-knockdown (top) or YBX1-knockdown (bottom) T24 cells. Data are mean ± s.d. (n = 3 independent experiments). g, Relative luciferase activity of luciferase reporter gene with HDGF-m5C WT site (HDGF-WT) or mutant site (HDGF-Mut) in control or NSUN2-knockdown (top) or YBX1-knockdown (bottom) T24 cells (n = 3 independent experiments). h, Knockdown of HDGF through injection of shRNA lentivirus strongly inhibited the subcutaneously growth of PDX tumour in vivo. Left: statistical analysis of xenograft tumour weight of each group. Right: volume-tracking plots. Data are mean ± s.d. (n = 5 animals). All P values were determined by a two-sided unpaired Student's t-test. The experiments were performed twice independently with similar results for d and e. Unprocessed gels for d and e are provided in Supplementary Figs. 8–10. The precise P values (two-sided unpaired Student’s t-test) and source data for c, f-h are presented in Supplementary Table 10.
Supplementary Figure 7 NSUN2/YBX1/HDGF regulatory axis plays a critical role in the pathogenesis of solid tumour.
a-f, Left: Spearman’s correlation analysis demonstrating positive correlation between YBX1 and HDGF mRNA expression levels in Kidney Renal Clear Cell Carcinoma, Prostate Adenocarcinoma, Pancreatic Adenocarcinoma, Breast Invasive Carcinoma, Liver Hepatocellular Carcinoma, Uterine Corpus Endometrial Carcinoma tissues from TCGA analysis, respectively. The centre line and the shaded regions indicate the regression line and 95% confidence interval, respectively. Spearman’s correlation coefficient and P value were calculated using the function “cor.test” in the statistical language R. Right: Kaplan-Meier analysis indicates the correlation of higher co-expression of NSUN2-YBX1-HDGF with poorer DFS rates in the indicated cancer patients. P values were determined by a two-sided log-rank test.
The black sections indicate blot results shown in the indicated figures.
Supplementary Figs. 1–10 and legends for Supplementary Tables 1–10
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Chen, X., Li, A., Sun, BF. et al. 5-methylcytosine promotes pathogenesis of bladder cancer through stabilizing mRNAs. Nat Cell Biol 21, 978–990 (2019). https://doi.org/10.1038/s41556-019-0361-y
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