Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase

Abstract

The methyltransferase like 3 (METTL3)-containing methyltransferase complex catalyzes the N6-methyladenosine (m6A) formation, a novel epitranscriptomic marker; however, the nature of this complex remains largely unknown. Here we report two new components of the human m6A methyltransferase complex, Wilms' tumor 1-associating protein (WTAP) and methyltransferase like 14 (METTL14). WTAP interacts with METTL3 and METTL14, and is required for their localization into nuclear speckles enriched with pre-mRNA processing factors and for catalytic activity of the m6A methyltransferase in vivo. The majority of RNAs bound by WTAP and METTL3 in vivo represent mRNAs containing the consensus m6A motif. In the absence of WTAP, the RNA-binding capability of METTL3 is strongly reduced, suggesting that WTAP may function to regulate recruitment of the m6A methyltransferase complex to mRNA targets. Furthermore, transcriptomic analyses in combination with photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) illustrate that WTAP and METTL3 regulate expression and alternative splicing of genes involved in transcription and RNA processing. Morpholino-mediated knockdown targeting WTAP and/or METTL3 in zebrafish embryos caused tissue differentiation defects and increased apoptosis. These findings provide strong evidence that WTAP may function as a regulatory subunit in the m6A methyltransferase complex and play a critical role in epitranscriptomic regulation of RNA metabolism.

Figure 1

WTAP interacts with METTL3 and METTL14. (A) 293T cells were transfected with Flag-WTAP and Myc-METTL3 constructs as indicated. Forty-eight hours later, cells were lysed and the lysates were subjected to immunoprecipitation using anti-Myc (Myc-IP) followed by immunoblotting with the anti-Flag antibodies. (B) 293T cells were treated with control siRNA (siCTRL) or siRNA targeting WTAP (siWTAP) for 48 h. Then cells were lysed and the lysates were subjected to IP using anti-WTAP. The immunoprecipitated samples were analyzed by immunoblotting with the anti-METTL3 antibodies. (C) Purified recombinant His-WTAP proteins were mixed with either GST or GST-METTL3 proteins as indicated, pulled down with GST beads, and subjected to immunoblotting with the indicated antibodies. (D) 293T cells were co-transfected with Myc-METTL3 and Flag-WTAP full-length (-FL), N-terminal (-N) or C-terminal (-C) constructs as indicated. Forty-eight hours later, cells were lysed and the lysates were subjected to Myc-IP followed by immunoblotting with the anti-Flag antibodies. (E) 293T cells were transfected with Flag-WTAP and HA-METTL14 constructs as indicated. Forty-eight hours later cells were lysed and the lysates were subjected to HA-IP followed by immunoblotting with the anti-Flag antibodies. (F) 293T cells were co-transfected with HA-METTL14 and Flag-WTAP full-length (-FL), N-terminal (-N) or C-terminal (-C) constructs as indicated. Forty-eight hours later, cells were lysed and the lysates were subjected to HA-IP followed by immunoblotting with the anti-Flag antibodies. Supportive data were included in Supplementary Information, Figures S1 and S2.

Figure 2

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.

Figure 3

METTL3 and WTAP bind m6A consensus sequence in mRNA and affect gene expression and alternative splicing. (A) Percentage of various RNA species bound by WTAP and METTL3 based on PAR-CLIP analyses. The WTAP- and METTL3-binding clusters were identified by PARalyzer algorithm. Annotation of clusters was based on the human Ensembl gtf file (version 72, hg19). The majority of binding sites of WTAP and METTL3 were located in mRNA. (B) Pie chart depicting the distribution of binding clusters in mRNA based on PAR-CLIP sequence clusters for WTAP and METTL3 after normalization of the overall length of the different transcript regions. Length of these different transcript regions was extracted from Ensembl annotations and the distribution percentage of clusters in these regions were normalized by their length. (C) Enriched sequence motif analysis of binding clusters indentified by PAR-CLIP. Upper panel, WTAP-binding motif (P = 1e-14); middle panel, METTL3-binding motif (P = 1e-13); lower panel, binding motif obtained when only genes found in both WTAP- and METTL3-binding clusters were included (P = 1e-19). Binding motifs were computed by the HOMER program. (D) Venn diagram of the overlapping genes with binding clusters of WTAP and METTL3 in the PAR-CLIP samples. (E) Percentage of WTAP/METTL3 clusters in CDS and UTR regions overlapped with m6A sites. (F) HeLa cells were transfected with siCTRL or siWTAP and Myc-METTL3 for 48 h as indicated. The cell lysates were then subjected to PAR-CLIP using anti-Myc. The pulled down RNA products in the RNA-METTL3 complex were labeled by Biotin and detected by Biotin chemiluminescent nucleic acid kit. (G) Percentage of WTAP- (711 multi-isoform and 41 single-isoform) and METTLE3- (3 155 multi-isoform and 192 single-isoform) binding mRNAs derived from single-isoform or multi-isoform genes and the reference Ensembl genes of human (P = 2.2e-16, Fisher test). (H) Percentage of constitutively or alternatively spliced exons adjacent to intronic binding clusters of WTAP (left), METTL3 (middle), overlap of WTAP and METTL3 (right). Supportive data were included in Supplementary information, Figures S4 and S5.

Figure 4

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.

Figure 5

A schematic model of the WMM complex function. The WMM complex mediates methylation of internal adenosine residues in eukaryotic mRNA, forming N6-methyladenosine. WTAP binds to the m6A consensus RRACH motif of mRNA and recruits catalytic subunits METTL3 and METTL14. Then the METTL3-METTL14 complex carries out m6A methyltransferase activity in the m6A motif.