Stage-specific requirement for METTL3-dependent m6A epitranscriptomic regulation during myogenesis

Abstract

The regulatory role of N⁶-methyladenosine (m⁶A) modification in skeletal muscle myogenesis and muscle homeostasis remains poorly characterized, particularly regarding the functional significance of methyltransferase-like 3 (METTL3), the catalytic subunit of the m⁶A methyltransferase complex (MTC), in myogenic regulation. Through systematic investigation of m⁶A epitranscriptomic remodeling during myogenesis, we demonstrate that METTL3-mediated m⁶As orchestrates myoblast fusion processes in both differentiation and regeneration contexts. Notably, we observed marked induction of Mettl3 expression post-injury, accompanied by substantial transcriptomic alterations in myogenesis-related pathways. High-resolution m⁶A mapping revealed distinct dynamic patterns of METTL3-regulated m⁶As during differentiation, exhibiting dichotomous regulation across target transcripts. Mechanistically, we identified myogenic fusion factors Mymx and Mymk as direct targets of METTL3, showing concomitant upregulation of both transcript abundance and m⁶A deposition during myogenesis. This study provides comprehensive multi-omics resources delineating the mechanistic landscape of METTL3-regulated m⁶As in myogenic programming, establishing METTL3 as a critical regulatory node governing myoblast fusion dynamic.

Fig. 1. Identification and integrative analysis of DEGs during skeletal muscle regeneration.

a Timeline characterizing the skeletal muscle repair model. b Representative immunohistochemistry of METTL3 from TA muscles at 1-, 3-, 5-, and 10-days following CTX-induced skeletal muscle injury (n = 3). Scale bars, 100 μm. c Quantification of anti-METTL3 staining intensity in immunohistochemistry from panel (b). d GSEA analysis on the entire set of DEGs between the third day post-injury and pre-injury conditions. e GSEA analysis on the entire set of DEGs between the first- and third-day post-injury. f GSEA analysis on the entire set of DEGs between day 3 and day 5 post-injury. g GSEA analysis on the entire set of DEGs between day 3 and day 10 post-injury. Data presented as means ± SEM. ns. not significant, *P < 0.05, **P < 0.01, and ***P < 0.001, by two-sided Student’s t test.

Fig. 2. Comparative analysis of gene expression levels on the third day post-injury relative to those at other time points.

a Volcano plot of DEGs between the first- and third-day post-injury. b Volcano plot of DEGs between the third day post-injury and pre-injury conditions. c Volcano plot of DEGs between day 3 and day 5 post-injury. d Volcano plot of DEGs between day 3 and day 10 post-injury. e Venn diagram showing the proportion of DEGs between the four comparison groups. f KEGG and GO analysis of intersection of the overlapping genes. g The Sankey map illustrates the GO pathway of TOP genes enrichment.

Fig. 3. The impact of Mettl3 overexpression on myoblast differentiation and fusion.

a Immunoblotting analysis of MyHC during myoblasts differentiation. b Immunoblotting analysis of METTL3 during myoblasts differentiation. c Immunoblotting analysis of METTL3 and MyHC in Mettl3-overexpressing cells and GFP-overexpressing cells. GFP-overexpressing cells were used as negative controls. d Representative immunofluorescent staining of Mettl3 overexpressed cells and wildtype cells on the fourth day post-differentiation (n = 3). Red indicated MyHC; blue indicated DAPI staining of nuclei. The merged images were shown. Scale bars, 50 μm. e Differentiation index was quantified from representative immunofluorescent images. The differentiation index is defined as the percentage of MyHC-positive cells relative to the total number of nuclei. The fusion index is defined as the ratio of the number of myotubes, characterized by MyHC-positive cells containing at least two nuclei, to the total number of nuclei in the field. f GO analysis of DEGs (up-regulated or down-regulated) in Mettl3-overexpressing cells. g Volcano plot of DEGs between Mettl3-overexpressing cells and GFP-overexpressing cells on the fourth day post-differentiation. Data presented as means ± SEM. ns., not significant, *P < 0.05, **P < 0.01, and ***P < 0.001, by two-sided Student’s t test, GAPDH was used as the internal control.

Fig. 4. Inhibition of Mettl3 expression affected the expression of genes related to myoblast fusion.

a Immunoblotting analysis demonstrating METTL3 expression in Mettl3 knockdown cells compared to the control group. b Immunoblotting analysis demonstrating MyHC expression in Mettl3 knockdown cells compared to the control group. c Representative immunofluorescent staining of Mettl3 knockdown cells compared to the control group on the third day post-differentiation (n = 3). Red indicated MyHC; blue indicated DAPI staining of nuclei. The merged images were shown. Scale bars, 50 μm. d Differentiation index was quantified from representative immunofluorescent images. The differentiation index is defined as the percentage of MyHC-positive cells relative to the total number of nuclei. The fusion index is defined as the ratio of the number of myotubes, characterized by MyHC-positive cells containing at least two nuclei, to the total number of nuclei in the field. e RT-qPCR analysis showing the expression levels of genes associated with myoblast fusion in Mettl3 knockdown cells relative to the control group on the third day post-differentiation. Data presented as means ± SEM. ns. not significant, *P < 0.05, **P < 0.01, and ***P < 0.001, by two-sided Student’s t test, GAPDH was used as the internal control.

Fig. 5. m⁶A profiles in Mettl3-overexpressing myoblasts during differentiation.

a Schematic of the phenotypic analysis and m⁶A-seq procedure. b Venn diagram of peaks enriched in GFP-overexpressing cells and Mettl3-overexpressing cells. c GO analysis of genes encoding mRNAs with common m⁶As in GFP-overexpressing cells during differentiation. d GO analysis of genes encoding mRNAs with common m⁶As in Mettl3-overexpressing cells and GFP-overexpressing cells at GM stages. e GO analysis of genes encoding mRNAs with common m⁶As in Mettl3-overexpressing cells and GFP-overexpressing cells on the fourth day post-differentiation. f GO analysis of genes encoding mRNAs with specific m⁶As in GFP-overexpressing cells during differentiation. g GO analysis of genes encoding mRNAs with specific m⁶As in Mettl3-overexpressing cells and GFP-overexpressing cells prior to differentiation induction. h GO analysis of genes encoding mRNAs with specific m⁶As in Mettl3-overexpressing cells and GFP-overexpressing cells on the fourth day post-differentiation.

Fig. 6. Features of METTL3 regulated lncRNA m⁶A alterations during differentiation.

a Venn diagram of lncRNA peaks enriched in GFP-overexpressing cells and Mettl3-overexpressing cells. b Metagene profiles of enrichment of all m⁶A peaks across lncRNAs transcriptome in Mettl3-overexpressing cells and GFP-overexpressing cells before and after induced differentiation. c Pie charts represent the proportion of m⁶A peaks in the three regions of lncRNAs before and after induced differentiation. d Histogram represents the relative enrichment of m⁶A peaks in the three regions of lncRNAs before and after induced differentiation.

Fig. 7. METTL3 regulates skeletal muscle fusion in an m⁶A-dependent manner.

a Venn diagram showing the number of overlapping DEGs between the four comparison groups during skeletal muscle regeneration (big circles) and the number of differentially expressed transcripts that also contain m⁶As in Mettl3-overexpressing cells and GFP-overexpressing cells before and after induced differentiation (small circles). b Heatmap of differentially expressed transcripts with m⁶As in Mettl3-overexpressing cells on the fourth day post-differentiation. c Heatmap of differentially expressed transcripts with m⁶As in Mettl3-overexpressing cells prior to differentiation induction. d Integrative Genomics View (IGV) of input and immunoprecipitation overlays on the Mymk gene from the MeRIP-seq data set for Mettl3-overexpressing cells and GFP-overexpressing cells before and after induced differentiation. e IGV of input and immunoprecipitation overlays on the Mymx gene from the MeRIP-seq data set for Mettl3-overexpressing cells and GFP-overexpressing cells before and after induced differentiation. f IGV of input and immunoprecipitation overlays on the Tnni1 gene from the MeRIP-seq data set for Mettl3-overexpressing cells and GFP-overexpressing cells before and after induced differentiation. g IGV of input and immunoprecipitation overlays on the Tnni2 gene from the MeRIP-seq data set for Mettl3-overexpressing cells and GFP-overexpressing cells before and after induced differentiation. h IGV of input and immunoprecipitation overlays on the Tnnc2 gene from the MeRIP-seq data set for Mettl3-overexpressing cells and GFP-overexpressing cells before and after induced differentiation. i IGV of input and immunoprecipitation overlays on the Chrng gene from the MeRIP-seq data set for Mettl3-overexpressing cells and GFP-overexpressing cells before and after induced differentiation.

Fig. 8. Methylated RIP–qPCR analysis to examine m⁶A and expression levels of mRNAs with METTL3-regulated m⁶As.

a RT-qPCR analysis of Mymk expression in immunoprecipitated RNAs during differentiation. b RT-qPCR analysis of Mymx expression in immunoprecipitated RNAs during differentiation. c RT-qPCR analysis of Tnni1 expression in immunoprecipitated RNAs during differentiation. d RT-qPCR analysis of Tnni2 expression in immunoprecipitated RNAs during differentiation. e RT-qPCR analysis of Chrng expression in immunoprecipitated RNAs during differentiation. f RT-qPCR analysis of Tnnc2 expression in immunoprecipitated RNAs during differentiation. IgG Immunoprecipitation was used as negative control. g RT-qPCR analysis of Mymk expression during myoblasts differentiation. h RT-qPCR analysis of Mymx expression during myoblasts differentiation. i RT-qPCR analysis of Mymk expression from TA muscles at 1-, 3-, 5- and 10-days following CTX-induced skeletal muscle injury. j RT-qPCR analysis of Mymx expression from TA muscles at 1-, 3-, 5- and 10-days following CTX-induced skeletal muscle injury. Data presented as means ± SEM. ns. not significant, *P < 0.05, **P < 0.01, and ***P < 0.001, by two-sided Student’s t test.