Figure 4
From: Mettl3-mediated m6A regulates spermatogonial differentiation and meiosis initiation
Mettl3-mediated m6A regulates alternative splicing of genes involved in spermatogenesis. (A) Schematics and statistics of the five types of alternative splicing events in Mettl3Ctrl and Mettl3cKO testes at P6 and P12. The differential alternative splicing events were identified by rMATS and the FDR = 0.05 were used as cutoff. Each group sample included two replicates. (B) The logo plot for bases adjacent to m6A sites. All 12 516 putative m6A residues were used to enrich the m6A motif and the plot was produced by WebLogo. (C) Distribution of m6A sites across the length of mRNA transcripts. (D) Transcriptome-wide distribution of m6A sites. Pie charts showing the percentage of m6A sites in distinct RNA sequence types: 5′ UTR, CDS, Stop codon and 3′ UTR. (E) The average m6A site frequency per gene in all genes and in differentially spliced genes in Mettl3Ctrl testes at P12. (F) The cumulative frequency curve for the alternative exon inclusion ratio in Mettl3Ctrl and Mettl3cKO testes at P12. The exon inclusion level was calculated by alternative exon coverage divided by constitutive exon coverage and transformed by log2. The exons containing m6A sites in Mettl3cKO testes showed lower coverage level than in Mettl3Ctrl testes (P < 0.01, Wilcoxon signed-rank paired test). No significant difference was exhibited between exons without m6A modification. (G, H) Distribution of RNA-seq reads of Cdk11b (G) and Syce2 (H) in Mettl3Ctrl and Mettl3cKO testes samples, the alternative exon of each gene was marked in grey and shadow. The m6A site location was marked as a red triangle. The exon skipping event was validated by RT-PCR (right panel). (I) The interaction network showing genes involved in spermatogenesis regulation. The genes containing alternative exons which had been validated by RT-PCR were marked in red. The regulation relationships were produced by Pathway Studio; the positive, negative regulation among these genes was marked as arrow, T-head respectively, while the regulation with unknown effects between two genes was connected directly.
