N6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells

Abstract

N(6)-methyladenosine (m(6)A) has been identified as the most abundant internal modification of messenger RNA in eukaryotes. m(6)A modification is involved in cell fate determination in yeast and embryo development in plants. Its mammalian function remains unknown but thousands of mammalian mRNAs and long non-coding RNAs (lncRNAs) show m(6)A modification and m(6)A demethylases are required for mammalian energy homeostasis and fertility. We identify two proteins, the putative m(6)A MTase, methyltransferase-like 3 (Mettl3; ref. ), and a related but uncharacterized protein Mettl14, that function synergistically to control m(6)A formation in mammalian cells. Knockdown of Mettl3 and Mettl14 in mouse embryonic stem cells (mESCs) led to similar phenotypes, characterized by lack of m(6)A RNA methylation and lost self-renewal capability. A large number of transcripts, including many encoding developmental regulators, exhibit m(6)A methylation inversely correlated with mRNA stability and gene expression. The human antigen R (HuR) and microRNA pathways were linked to these effects. This gene regulatory mechanism operating in mESCs through m(6)A methylation is required to keep mESCs at their ground state and may be relevant to thousands of mRNAs and lncRNAs in various cell types.

Figure 1. Mettl3 and Mettl14 are required for m⁶A formation in vitro and in vivo

A. Schematic drawing showing predicted MTase domains of mouse Mettl3 (Accession: NP_062695.2) and Mettl14 (Accession: NP_964000.2) proteins. Numbers represent amino acid numbers. B. RT-qPCR (left) and western blot (right) showing Mettl3 (left panel) and Mettl14 (right panel) expression in mESC clones stably expressing shRNAs targeting each. GapDH serves as a loading control in the western blot. Scr: scramble; shM3, shRNA against Mettl3; shM14, shRNA against Mettl14. The original gel is shown in Supplementary Fig.5. C. Ethidium bromide staining (left panel) and m⁶A immunoblot (right panel) of DNA-free, rRNA-free Poly(A)+ RNA from Mettl3 kd, Mettl14 kd, and control cells. D. Measurement of percentage of m⁶A/A ratio by MS. E. Flag, Mettl3 or Mettl14 western immunoblots of proteins purified from lysates of HEK293 cells overexpressing flag-tagged Mettl3, Mettl14, or luciferase using M2 beads. Luc, luciferase; M3, Mettl3; M14, Mettl14. The original gel is shown in Supplementary Fig.5. F. CPM counts of RNA probes extracted after an in vitro methylation assay. G. CPM counts of excised m⁶A spots from digested RNA probes used in the methylation assay. Error bars from panels B and F-G represent means ± SEM from 3 separate experiments, expect M3 in panel G where n=7. One-tailed Student’s t-test, *P< 0.05, **P < 0.01, ***P < 0.001, ****P<0.0001 vs. scramble control. Scr: scramble control.

Figure 2. Mettl3 and Mettl14 interact and regulate each other’s stability

A. meRIP-seq analysis of a Sox17 transcript showing loss of m⁶A methylation in both Mettl3 kd and Mettl14 kd cells. Yellow highlighting, peak location. Scr: scramble; shM3, shRNA against Mettl3; shM14, shRNA against Mettl14. B. Venn diagram showing overlap of Mettl3 and Mettl14 targets. C. Mettl3 or Mettl14 western immunoblots of endogenous proteins co-IP’d using antibodies against Mettl3 or Mettl14, or IgG in mESC lysates. M3, Mettl3; M14, Mettl14. The original gel is shown in Supplementary Fig.5. D. Western blot analysis of Mettl3 and Mettl14 protein levels in Mettl3 kd and Mettl14 kd mESCs, respectively. The original gel is shown in Supplementary Fig.5. E. RT-qPCR analysis of Mettl3 and Mettl14 RNA levels in Mettl3 kd and Mettl14 kd mESCs, respectively. F. GO analysis of 4395 shared Mettl3 and Mettl14 targets. Error bars from panel E represent means ± SEM from 3 separate experiments. One-tailed Student’s t-test, **P < 0.01, ***P < 0.001 vs. scramble control. Scr: scramble.

Figure 3. Mettl3 or Mettl14 knockdown mESCs lose self-renewal capability

A. Phase contrast microscopy showing colony morphology of kd versus control mESC cells. Scr: scramble; shM3, shRNA against Mettl3; shM14, shRNA against Mettl14. B. Growth curve assessing cell proliferation kinetics of kd versus control cells. P values are generated by two-way ANOVA. C. Quantification of AP-positive colonies. D. Heat map analysis based on microarray comparison of gene expression in kd and control cells. E. RT-qPCR analysis of pluripotency (left) and differentiation (right) genes in kd versus control cells. F. GSEA analysis on enrichment of developmental regulators (left) and pluripotency-related genes (right) in Mettl3 kd versus control cells. A False Discovery Rate (FDR) of <0.178 was calculated for bivalent genes and FDR<0 for pluripotency-related genes. Note that FDR<0.25 is statistically significant for GSEA analysis: www.broadinstitute.org/gsea/doc/GSEAUserGuideFrame.html. Scale bars, 100 μm. Error bars from panels B-C and E represent mean ± SEM from 3 separate experiments. One-tailed Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P<0.0001 vs. scramble control. Scr: scramble.

Figure 4. m⁶A modification regulates mRNA stability

A. Enrichment of up- or down- regulated Mettl3 and Mettl14 targets in kd versus control cells. Arrows indicate up- or down regulation of gene expression. Scr: scramble; shM3, shRNA against Mettl3; shM14, shRNA against Mettl14. B. Comparison of cumulative transcript abundance in cells treated with Actinomycin D for 0, 4, and or 8 hrs by microarray analysis. RNAs from 0 hr serves as base-line. KS test, p< 2.2e-16 comparing the changes in relative RNA levels from 4 to 8 hrs between Mettl targets and non-targets in both Mettl3 kd and Mettl14 kd cells. C. Enrichment of bivalent and pluripotency (Oct4 PPI) genes in shared Mettl3 and Mettl14 targets. D. meRIP-qPCR of specific bivalent and pluripotency genes (grey area). E. Enrichment of Mettl3 and Mettl14 targets showing increased RNA stability in kd cells among bivalent and pluripotency genes. F. Measurement of a percentage of m⁶A/A ratio by MS in undifferentiated (day 0), day6, and day 12 differentiated mESCs. G. RT-qPCR of Mettl3, Mettl14, and Rex1 expression in undifferentiated and differentiated mESCs. H. meRIP-qPCR of specific bivalent (gray area) and pluripotency genes in mESCs during differentiation. Error bars from panels D and G-H represent means ± SEM from 3 separate experiments. One-tailed Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. scramble control. Scr: scramble.

Figure 5. The HuR-microRNA pathway functions in m⁶A methylation-mediated RNA stability

A. Enrichment of genes showing increased RNA stability following Mettl3 or Mettl14 kd among all shared Mettl3 and Mettl14 targets and among targets with and without canonical HuR binding sites in their 3′-UTR regions. M3, Mettl3; M14, Mettl14. B. Left panel, denaturing PolyAcrylamide Gel Electrophoresis (PAGE) showing the size of fragmented, DNAse-treated, rRNA-free mRNAs extracted from scramble and kd cells; mid-panel, non-denaturing PAGE showing differential binding of HuR to RNAs probes from left panel. Scr: scramble control, shM14, shRNA against Mettl14, shM3, shRNA against Mettl3. NP: no protein added. Right panel, non-denaturing PAGE showing differential binding of HuR to RNA probes enlisted in C. C. RNA probes with the canonical UUUUU HuR binding site locatd either next to A (0A) or m⁶A (0m⁶A) or separated by a 12-nucleotide spacer (12A and 12m⁶A). D. HuR RIP-qPCR of Pou5f1, Igfbp3, and Otx2 from kd versus control cells. E. RT-qPCR of Pou5f1, Igfbp3, and Otx2 in Actinomycin D-treated kd cells. In the case of Igfbp3, cells are also treated with and without siRNA targeting HuR. P values are generated using two-way ANOVA. F. RT-qPCR of HuR, Igfbp3, Otx2, and Pou5f1 in mESC with depleted HuR. shHuR#1-3: shRNAs against HuR. G. Argonaute 2 (Ago2) RIP–qPCR of Igfbp3 and Pou5f1 RNAs from kd versus control cells. H. Enrichment of genes showing increased RNA stability and expression among shared Mettl3 and Mettl14 targets. Targets are classified by whether they display Ago2 binding sites. I. Model: In wildtype mESCs, Mettl3 and Mettl14 methylate RNA synergistically and m⁶A methylation on some transcripts, particularly those encoding developmental regulators, blocks HuR binding, resulting in transcript destabilization. In Mettl3 and Mettl14 kd cells, loss of m⁶A allows HuR-mRNA interaction and attenuation of microRNA targeting, enhancing stability of transcripts especially those encoding developmental regulators, and promoting loss of the mESC ground state. A potentially methylated A is shown in red. Error bars from panels D-G represent means ± SEM from 3 separate experiments. One-tailed Student’s t-test, *P < 0.05, ** p< 0.01, ****P<0.0001 vs. scramble control. Scr: scramble.