m 6 A RNA methylation orchestrates transcriptional dormancy during developmental pausing

Abstract

Embryos across metazoan lineages can enter reversible states of developmental pausing, or diapause, in response to adverse environmental conditions. The molecular mechanisms that underlie this remarkable dormant state remain largely unknown. Here we show that m ⁶ A RNA methylation by Mettl3 is required for developmental pausing in mice by maintaining dormancy of paused embryonic stem cells and blastocysts. Mettl3 enforces transcriptional dormancy via two interconnected mechanisms: i) it promotes global mRNA destabilization and ii) suppresses global nascent transcription by specifically destabilizing the mRNA of the transcriptional amplifier and oncogene N-Myc, which we identify as a critical anti-pausing factor. Our findings reveal Mettl3 as a key orchestrator of the crosstalk between transcriptomic and epitranscriptomic regulation during pausing, with implications for dormancy in stem cells and cancer.

Fig. 1.. The m6A methyltransferase Mettl3 is essential for paused pluripotency.

(A) Screening of RNA modifications by mass spectrometry in paused ESCs, normalized to FBS-grown ESCs. Data are mean ± SD (n=3). Ratio paired Student’s t-tests **P < 0.01. (B) Dot blot showing that the increase of m⁶A in paused ESCs is abrogated in Mettl3^−/−. (C) Growth curves showing that Mettl3^−/− ESCs fail to suppress proliferation in paused conditions. Data are mean ± SEM (n=3). Linear regression test **P < 0.01, ***P < 0.001. (D) Mettl3 loss leads to the premature death of mouse blastocysts cultured ex vivo in paused conditions. Log-rank test **P < 0.01, ****P < 0.0001. (E) Quantification of recovered (live) embryos at E3.5 (control) and at Equivalent Days of Gestation (EDG) 8.5 following hormonal diapause, showing that Mettl3^TCP−/− embryos are impaired at undergoing hormonal diapause. χ2 test *P < 0.05.

Fig. 2.. Mettl3 regulates hypotranscription in paused pluripotency.

(A) Total RNA per cell in Mettl3^+/+ and Mettl3^−/− ESCs, in FBS and paused conditions. Data are mean ± SD, (n=3). Paired Student’s t-tests **P < 0.01. (B,C) Nascent transcription by EU incorporation, quantified by median fluorescence intensity (MFI) relative to Mettl3^+/+ FBS in each experiment. Data are mean ± SD (n=4). Paired Student’s t-tests **P < 0.01. (D,E) Immunofluorescence and nuclear signal quantification of EU incorporation in ex vivo paused blastocysts, showing increased nascent transcription in Mettl3^TCP−/−. Data are mean ± SD. One-way ANOVA with Dunnett’s multiple comparison test ****P < 0.0001. (F) Heatmap of gene expression for all genes expressed in ESCs, showing defective hypotranscription in paused Mettl3^−/− ESCs. (G,H) Gene set enrichment analysis (GSEA) of gene expression changes in paused ESCs, using the “GO biological processes” (G) and “hallmarks” collections (H). Scatter plots of the normalized enrichment scores (NES), with Spearman correlation coefficient (ρ) with representative pathways showing defective hypotranscription in Mettl3^−/− (red dots) highlighted. pre-ranked gene set enrichment analysis with FDR correction *P < 0.05, **P < 0.01, ****P < 0.0001.

Fig. 3.. Mettl3-mediated m6A promotes RNA instability during pausing.

(A) PCA plot showing that paused ESCs have distinct m⁶A profiles (n=3). (B,C) MeRIP-seq shows increased m⁶A in paused ESCs (n=3, gain/loss: adjusted P < 0.05 and absolute fold-change >1.5). (D) Heatmaps of Mettl3 ChIP-seq signal in FBS-grown and paused ESCs, showing increased Mettl3 binding in paused ESCs (n=2). (E) Overlap between targets of m⁶A and Mettl3, identifying all target genes or genes with increased levels of m⁶A and Mettl3 (log₂FC > 0) in paused ESCs. P-value by hypergeometric test. (F) Metagene profiles of m⁶A and Mettl3 peaks. (G) RNAs with increased m⁶A in pausing, as defined in (A), are significantly more downregulated than RNAs with decreased m⁶A in paused Mettl3^+/+ (but not Mettl3^−/−) ESCs. Student’s t-tests **P < 0.01. (H) Differences in expression (log₂FC paused/FBS) between exonic and intronic RNA-seq data indicate a global decrease in RNA stability in Mettl3^+/+ (but not Mettl3^−/−) ESCs upon pausing. P-value by paired Student’s t-test.

Fig. 4.. Mettl3 regulates pausing via m6A-mediated destabilization of Mycn mRNA.

(A) Top putative anti-pausing factors ranked by Spearman correlation (ρ) with the m⁶A machinery. (B) Increased N-Myc in Mettl3^−/− ESCs by RNA-seq (n=3) and western blot (n=4). (C) Immunofluorescence and nuclear signal quantification of N-Myc in ex vivo paused blastocysts. (D) Median log₂ fold-changes for “hallmark” gene sets in Myc/Mycn DKO ESCs versus diapaused embryos or paused ESCs, with spearman correlation (ρ). (E) Blocking Myc signaling partially restored the decreases in proliferation (n=4) and total RNA (n=5) in paused Mettl3^−/− ESCs. (F) Increased Mycn mRNA stability in paused Mettl3^−/− ESCs (n=3). t_1/2: half-life. (G) Mycn m⁶A site reduces transcript stability in paused ESCs (n=5). (H-K) Site-specific demethylation of Mycn leads to increased Mycn mRNA stability (H, n=3). increased expression (I, n=7 by RT-qPCR and n=3 by western blot), increased total RNA (J, n=6) and higher proliferation (K, n=5). (L) Model for the regulation of paused pluripotency by m⁶A RNA methylation. All data are mean ± SD, except times series which are mean ± SEM. P-values by two-tailed paired Student’s t-tests (B,C,G), linear regression test (E left,F,H,K), one-way ANOVA with Dunnett’s multiple comparison tests (E right,I,J). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.