Fig 1: 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.
Fig 2: Schematic representation of the role of the MACOM and the MAC. The MACOM can regulate gene expression in two ways: either on its own (MAC-independent functions) or by interacting with MAC components (m6A methylation). Flacc (Zc3h13) is a novel component of the MACOM that stabilizes the interaction between Fl(2)d and Nito (Wtap and Rbm15) proteins, thereby ensuring deposition of m6A to targeted transcripts.
Fig 3: Zc3h13/Flacc interacts with the m6A machinery. (A) TAP-LC-MS/MS of endogenously Flag-Avi-tagged Rbm15 mESCs. Parental cells were used as background control, and proteins were purified in the presence of 350 mM NaCl. Highlighted in the volcano plot are enriched proteins previously identified as Mettl3 interactors (red) as well as Zc3h13 (green). (B) Heat map comparing relative label-free quantification (LFQ) intensities of selected Mettl3-bound proteins across increasing NaCl concentrations. Statistical analysis was done with Perseus (see the Materials and Methods for details). MS raw data were deposited in ProteomeXchange. (C,D) Stable isotope labeling of amino acids in cell culture (SILAC) coupled to MS analysis using Nito-Myc as bait. Scatter plot of normalized forward versus inverted reverse experiments plotted on a log2 scale. The threshold was set to a 1.5-fold enrichment (red dashed line). Proteins in the top right quadrant of C are enriched in both replicates. Gene ontology (GO) term analysis (Tyanova et al. 2016) for enriched proteins is shown in D. (E) Coimmunoprecipitation experiments were carried out with lysates prepared from S2R+ cells transfected with FlagMyc-Flacc and HA-Nito. In control lanes, S2R+ cells were transfected with FlagMyc alone and an identical HA-containing protein. Extracts were immunoprecipitated with Myc antibody and immunoblotted using Flag and HA antibodies. Two percent of input was loaded. The same experiment was repeated in the presence of RNaseT1. Nito and Flacc interact with each other in an RNA-independent manner. (F) Table representing orthologous proteins of the m6A–METTL complex (MAC) and the m6A-METTL-associated complex (MACOM) in mice and flies.
Fig 4: Zc3h13 stabilizes the interaction between RBM15 and WTAP. (A,B) Comparison of TAP-LC-MS/MS of endogenously Flag-Avi-tagged Rbm15 mESCs in either a wild-type or a Zc3h13 knockout background. Rbm15 and associated proteins were purified in the presence of 350 mM NaCl. (A) Volcano plot showing enriched proteins in wild-type cells (right) versus Zc3h13 knockout cells (left). (B) Table of spectral counts, unique peptides, and percentage coverage of TAP-LC-MS/MS data in A. (C) Split luciferase NanoBiT assay examining the interaction of mouse Rbm15 and Wtap. (Left) Scheme representing luciferase reconstitution upon transfection of large (LgBit) and small (SmBit) NanoLuc subunit fusions and the interaction of Rbm15 and WTAP. (Right) Comparison of Rbm15–Wtap NanoBiT NanoLuc signal in wild-type and Zc3h13 and Mettl3 knockout cells. The mean of three independent experiments, three transfections each, is shown. Errors bars indicate SD. (*) P = 0.026, calculated using two-tailed Student's t-test.
Fig 5: The effects of RNA‐binding motif protein 15 (RBM15) on the m6A modification level and stability of LINC01087. (A) Total m6A modification levels in lung adenocarcinoma (LUAD) cells and human bronchial epithelial cells were determined using the methylated RNA binding protein immunoprecipitation (Me‐RIP) assay. (B) The binding relationship between RBM15 and LINC01087 was determined using the RNA immunoprecipitation (RIP) assay. RBM15 antibody was used for RIP assay to isolate RBM15‐bonded RNAs, followed by real‐time quantitative polymerase chain reaction (RT‐qPCR) using LINC01087 specific primers. (C) Levels of m6A modification in LINC01087 after RBM15 knockdown in LUAD cells were determined with the MeRIP assay. (D) m6A modification sites in LINC01087 were predicted using the SRAMP database. (E) Schematic diagram showing m6A modification site and synonymous mutation in LINC01087. (F) The expression level of LINC01087 in LUAD cells cotransfected with sh‐RBM15 and LINC01087‐WT/LINC01087‐MUT was determined by RT‐qPCR. (A–C) Mut, adenine residues substituted by cytosine. (G) Levels of m6A modification in LINC01087 in LUAD cells cotransfected with sh‐RBM15 and LINC01087‐WT or LINC01087‐MUT were determined using the Me‐RIP assay. (H) LINC01087 mRNA stability in LUAD cells after RBM15 knockdown was determined with the RNA stability assay. Data are presented as means ± SD. N = 3/group. All data were obtained from three independent replicates. *p <0.05; **p <0.01; ***p <0.001.
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