WTAP regulates the nuclear speckle localization of METTL3 and METTL14. (A) The graph represents the quantification of three independent dot-blot experiments (raw data were included in Supplementary information, Figure S3A). The y-axis represents the relative intensity of dots relative to that of the control group. P values were calculated using a two-tailed t-test. Error bars represent SD. (B-D) After transfection (48 h) with the indicated fluorescence FAM-labeled siRNA, HeLa cells were fixed and immunostained with the indicated antibodies. DNA was visualized by DAPI. Scale bar, 10 μm. Supportive data were included in Supplementary information, Figure S3.

The WMM complex plays essential roles during zebrafish embryogenesis. (A) Embryos injected with individual (WTAP-, METTL3-) or combined (METTL3+WTAP) MOs. The double knockdown showed more severe morphological defects, compared to other groups, at 24 hpf. Red arrows mark head and eyes, while blue arrows mark brain ventricle. The curve of the notochord is labeled by the double dashed lines. (B) Expression of somite marker myod in the morphants at 24 hpf. myod expression was increased in WTAP-, METTL3-, or double morphants. (C) Increased apoptosis (TUNEL assay) was observed in embryos injected with individual or combined MOs. (D) Overexpression of full-length or N-terminal but not C-terminal zebrafish WTAP mRNA prevented apoptosis in zebrafish WTAP-MO embryos. Note that there is auto-fluorescence on the yolk. Anterior to the left and dorsal is up. Scale bar, 250 μm. Supportive data were included in Supplementary information, Figures S5F and S6.

Expression pattern and functional assay of WTAP and METTL3 in zebrafish embryos (A) Whole-mount in situ hybridization (WISH) shows ubiquitous expression of WTAP and METTL3 during embryogenesis, respectively. (B) To test the efficiency of WTAP- and METTL3-MOs one-cell embryos were injected with 25 pg of pWTAP-GFP plasmid DNA alone or in combination with WTAP MO (left panel) or METTL3 MO (right panel). (C) To test the expression of the neuro-ectoderm marker “goosecoid” and mesoderm marker “no tail” in embryos injected with individual or a combination of WTAP and METTL3 MOs. Red bracket indicates the length and blue bracket the width of “no tail” expression in the notochord. (D) HeLa cells were treated with siControl, siMETTL3 or siWTAP RNAi for 48h, then stained with annexin V-FITC and PI and subjected to apoptotic analyses by flow cytometry. (E-F) Western blots showing knock down efficiency of siMETTL3 (E) and siWTAP (F) in cells used in the apoptotic assay shown in (D). (G) Zebrafish embryos were treated with control, WTAP- and METTL3-MOs as indicated. mRNA was extracted and 50 ng of mRNA was spotted onto a nylon membrane and m6A levels were detected with anti-m6A antibody.