Fig 2: WTAP regulates alternative splicing of SLC2A6 via rG4 sequences.A, G4NN scores of the included exons (n = 75) by KD of WTAP complex and nondifferentially spliced exons (n = 978). Upstream and downstream exon sequences and 100 nucleotides of 5'ss or 3'ss of flanking introns of the regulated exons were used for the analysis. Boxes indicate interquartile range (IQR), whiskers extend to 1.5 times IQR. Outliers, which lie beyond the extremes of the whiskers, were excluded. Significant differences were evaluated using Welch’s ANOVA followed by Games–Howell post hoc pairwise comparison (*p < 0.01, **p < 0.001, ***p < 0.0001). Bottom, the number of exons/introns with >0.5 G4NN score and p-values (Fisher’s exact test) are indicated. B, G4NN scores linked to the decreased retained introns (n = 90) by KD of WTAP complex and nonaffected introns (n = 1000) are indicated; 100 nucleotides of 5'ss or 3'ss of intron, upstream exon, and downstream exon. C, the interaction of WTAP and SLC2A6 RNA was determined using RNA Coimmunoprecipitation (RIP) and quantitative RT-PCR. HUVECs were cross-linked with 1% formaldehyde, and the protein/RNA complexes cross-linked to WTAP were immunoprecipitated with anti-WTAP (H1122) antibody or anti-viral gp64 protein antibody (as control). RNA was extracted from the immunoprecipitants (IPs) following reverse cross-linking and used for RT-PCR analysis (left). Western blot analysis of WTAP protein in IPs. 5% input and 10% IP were loaded. Middle, representative gel image of RT-PCR from the RIP samples using SLC2A6-or GAPDH-specific primers. Right, the interaction of WTAP with SCL6A6 transcripts was determined using RIP-qPCR. GAPDH was used as a negative control. Horizontal black lines represent the average of five independent experiments; p-value versus control IgG (t test). D, minigene splicing assays showing that rG4 sequences influence exon inclusion. Left, diagram of the exon/intron structure of the genomic region included in the SLC2A6 minigene and RT-PCR analysis of minigene-derived transcripts isolated from HUVEC transfected with the indicated constructs (WT, WT sequence; mt1, a mutation in G4 of intron 8; mt2, a mutation in G4 of intron 7; mt1+mt2, double mutations of G4 both in downstream and upstream introns). The positions of different spliced products are indicated by drawing, on the right side of the gel image. Right, quantification of the percentage of exon 8 inclusion for five independent biological replicates. Significant differences were evaluated using one-way ANOVA/Tukey HSD post hoc test (*p < 0.01 **p < 0.001, ***p < 0.0001). E, model: Inhibitory effect of the WTAP complex on splice site recognition. Exon skipping, the WTAP complex is recruited to splice sites via the interaction between rG4-binding proteins and rG4 sequences located in the flanking introns of the alternative exon (or recruited to other region via other RNA binding protein of WTAP complex) and interferes with the splice sites recognition through rG4 sequences, thereby leading to exon skipping. In the case of SLC2A6, rG4s promote exon inclusion possibly via the structure or other RBPs of the splicing enhancers, which WTAP inhibits or competes with. Another possibility is that the WTAP complex may sequester rG4 binding proteins, which are involved in splice site recognition. Exon skipping by the WTAP complex results in generating truncated/nonfunctional isoform, thus regulates the expression of the full-length protein. Intron retention, the WTAP complex is recruited to the last exon and inhibits splicing inside the last exon, as well as promotes polyadenylation. WTAP/CBLL1 would promote polyadenylation by recruiting the 3'end-processing factors such as CPSFs. Splicing in the last exon changes the C-terminus structure of the protein or affects mRNA stability and thus protein expression.

Fig 3: Immunoprecipitation of CBLL1 complexes using high-affinity monoclonal antibodies.A, domains and unique amino acid repeat in the CBLL1 protein. It contains a RING finger and phosphor-tyrosine binding motif at the N-terminus, and unique proline-rich repeats that span most of the C-terminal region. Specific monoclonal antibodies were generated using the antigen in the C-terminal region indicated with a bold line. B, the specificity of anti-CBLL1 antibodies. Whole lysates of HUVECs were subjected to immunoblot analysis using Y6018 and Y6037 antibodies. The arrow indicates the band corresponding to CBLL1, which is efficiently immunoprecipitated by each antibody. C, the SYPRO Ruby stain of the immunopurified CBLL1 and its interacting proteins from HUVECs. D, the proteomic profile of CBLL1-interacting proteins. The values represent the total spectrum counts (SPC). E, network of physical relationships (edges) of the top 56 identified proteins isolated with at least one unique peptide by the anti-CBLL1 antibodies, Y6016 and Y6037, but not with the negative control antiviral protein antibody (gp64). The illustration and enrichment analysis were performed using Cytoscape version 3.8.0.

Fig 4: WTAP complex major components promote intron retention, polyadenylation, and exon skipping. Predicted AS changes by KD of WTAP complex were validated using RT-PCR: (A) intron retention, (B) polyadenylation and splicing, and (D) exon skipping. Predicted AS changes by KD of WTAP complex were validated using RT-PCR. Left, representative gel image of RT-PCR. The PCR products were stained with SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher). The drawing right side of the image indicates the corresponding spliced/unspliced products. Rectangle, thin rectangle, and line correspond to an exon, 3' UTR, and introns, respectively. Right, dot plot of the percent of exon inclusion (Percent spliced-in; PSI) or intron retention (Percent intron retention; PIR) calculated using the molarity data from the Bioanalyzer. The horizontal lines represent the average from three independent experiments performed in HUVEC, except for MSL1, five independent experiments, and EXD3, four independent experiments. The x-axis represents the following: 1. Control siRNA, 2. WTAP KD, 3. VIRMA KD, 4. ZC3H13 KD, 5. CBLL1 KD, 6. BCLAF1/THRAP3 KD, 7. RBM15/RBM15B KD, 8. METTL3 KD, and 9. METTL14 KD. **p < 0.01, *p < 0.05 versus control siRNA-treated samples (t test). C, ratios of exon inclusion to skip and inclusion to alternative polyadenylated products in WTAP complex KD samples relative to the values in control samples, as determined using nested-qPCR from three independent biological replicates. The horizontal lines represent the average. cont, control siRNA; inc, inclusion; pA, alternative polyadenylation.