Figure 4

Figure 4 Disruption of Mettl16 invokes the alteration of m⁶A in cell cycle genes.

(A) Schematic of Mettl16-WT and two Mettl16 mutants, including exclusive cytoplasmic Mettl16 (del-NLS) and catalytic-dead Mettl16 (PP180/181AA). (B, C) qPCR (B) and WISH (C) analysis showing the decreased expression of HSPC marker cmyb in mettl16-deficient zebrafish at 4 dpf was restored by Mettl16-WT, but not Mettl16-del-NLS and Mettl16-PP180/181AA. n ≥ 10 per group, performed with three biological replicates (B). Numbers at the bottom right indicate the number of embryos with similar staining patterns among all embryos examined, n = 3 independent experiments (C). Scale bars, 100 μm (C). (D) Quantification of WISH in (C). ‘n’ indicates the number of individuals analyzed for cmyb⁺ cells in the CHT at 4 dpf, respectively. (E) m⁶A enrichment in RNAs in the CHT of siblings and mettl16-deficient zebrafish at 3 dpf. The m⁶A/A level of siblings was set to one. n ≥ 20 per group, performed with three biological replicates. (F) Volcano plots reveal the differential m⁶A peaks of total RNAs in mettl16^−/− mutants compared with siblings at 3 dpf. The significantly increased and decreased m⁶A peaks are highlighted in red and blue, respectively (P ≤ 0.05, |log₂FC | ≥1). Statistical analysis was performed by a hypergeometric test. (G) Gene ontology analysis of the m⁶A peaks that decrease upon mettl16 knockout. Statistical analysis was performed by a hypergeometric test. (H) Pie chart depicting the annotations of the m⁶A peaks that decrease upon mettl16 knockout. (I) Pie chart depicting the percent of the significantly decreased m⁶A peaks which have a UACAGAGAA highly similar motif upon mettl16 knockout. (J) Heatmap showing differentially expressed genes in mettl16^−/− mutants compared with siblings at 3 dpf. (K) GSEA plot showing the general downregulation of genes involved in the regulation of cell proliferation in mettl16^−/− mutants compared with siblings. Statistical analysis was performed by permutation test using GSEA software. Data information: In (B, D, E), data were represented as mean ± SEM, *adjusted P < 0.05, ** adjusted P < 0.01, **** adjusted P < 0.0001, n.s. non-significant, one-way ANOVA analysis with post hoc test of Tukey’s multiple comparison correction (B, D), Student’s t-test (E). Source data are available online for this figure.