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. 2013 Nov 15;288(46):33292-302.
doi: 10.1074/jbc.M113.500397. Epub 2013 Oct 7.

Identification of Wilms' Tumor 1-associating Protein Complex and Its Role in Alternative Splicing and the Cell Cycle

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Free PMC article

Identification of Wilms' Tumor 1-associating Protein Complex and Its Role in Alternative Splicing and the Cell Cycle

Keiko Horiuchi et al. J Biol Chem. .
Free PMC article

Abstract

Wilms' tumor 1-associating protein (WTAP) is a putative splicing regulator that is thought to be required for cell cycle progression through the stabilization of cyclin A2 mRNA and mammalian early embryo development. To further understand how WTAP acts in the context of the cellular machinery, we identified its interacting proteins in human umbilical vein endothelial cells and HeLa cells using shotgun proteomics. Here we show that WTAP forms a novel protein complex including Hakai, Virilizer homolog, KIAA0853, RBM15, the arginine/serine-rich domain-containing proteins BCLAF1 and THRAP3, and certain general splicing regulators, most of which have reported roles in post-transcriptional regulation. The depletion of these respective components of the complex resulted in reduced cell proliferation along with G2/M accumulation. Double knockdown of the serine/arginine-rich (SR)-like proteins BCLAF1 and THRAP3 by siRNA resulted in a decrease in the nuclear speckle localization of WTAP, whereas the nuclear speckles were intact. Furthermore, we found that the WTAP complex regulates alternative splicing of the WTAP pre-mRNA by promoting the production of a truncated isoform, leading to a change in WTAP protein expression. Collectively, these findings show that the WTAP complex is a novel component of the RNA processing machinery, implying an important role in both posttranscriptional control and cell cycle regulation.

Keywords: Alternative Splicing; Cell Cycle; Nuclear RNA; Protein Complexes; Proteomics.

Figures

FIGURE 1.
FIGURE 1.
Immunopurification of the WTAP binding proteins using three anti-WTAP monoclonal antibodies. A, three specific monoclonal antibodies against WTAP were generated. The recognition region of each antibody is indicated with a heavy line. B, specificity of anti-WTAP antibodies. Whole lysates of HUVECs were subjected to immunoblot analysis using H1122 and H1137 antibodies, whereas nuclear extracts were used for Y6828. The arrow indicates the band corresponding to WTAP, which is undetectable in WTAP siRNA-treated HUVECs. The asterisks represent nonspecific or cross-reactive bands. C, immunopurified (IP) WTAP and its interacting proteins from HUVECs were stained with SYPRO Ruby and immunoblotted with an anti-WTAP polyclonal antibody. D, Venn diagram of the number of proteins isolated with at least three unique peptides by the anti-WTAP antibodies H1122, H1137, and Y6828 but not with the negative control anti-viral protein antibody K7124. The overlapping proteins that were isolated by all three of the anti-WTAP antibodies are indicated. E, gene ontology analysis of the proteins isolated with at least three unique peptides by the anti-WTAP antibodies H1122, H1137, and Y6828 but not the anti-gp64 antibody. The significant enrichment of functional biological processes is shown along with the p value.
FIGURE 2.
FIGURE 2.
Proteomic profile of the WTAP complexes. A, the domains and unique amino acid repeats in the Hakai protein. Various Hakai deletion mutants are shown. The interaction of these mutants with WTAP was assessed by means of co-immunoprecipitation (IP) experiments. B, the cellular localization of Hakai-V5 and Hakai-delRING-V5. The nucleus was stained with TO-PRO-3 reagent. Bar, 10 μm. C, immunoprecipitation of Hakai complexes from the tetracycline-inducible HEK293 stable cell lines expressing Hakai-V5 or Hakai-delRING-V5. Immunopurified Hakai and its interacting proteins were resolved by SDS-PAGE and stained with SYPRO Ruby solution. The efficiency of immunoprecipitation was determined by Western blot. D, the proteomic profile of the WTAP complexes. This list shows the proteins isolated using the H1122, H1137, or Y6828 antibodies with a unique peptide number of ≥3 from HUVEC extracts or from PFA(+) HeLa cell extracts but not with the negative control antibody K7124 from HUVEC extracts or the anti-V5 antibody from the Hakai-delRING-V5 sample. The complete list of the identified proteins is presented in supplemental Table S1. The values represent the unweighted spectrum count (SPC) level divided by the molecular weight (MW) in kDa to determine the relative quantity of the immunopurified proteins. Hierarchical clustering was performed using JMP 7 software (SAS Institute, Cary, NC). E, immunopurification of the proteins cross-linked to WTAP. HeLa cells were cross-linked with paraformaldehyde, and the proteins cross-linked to WTAP were immunopurified with the H1122 antibody. Interacting proteins were resolved by SDS-PAGE and stained with SYPRO Ruby solution.
FIGURE 3.
FIGURE 3.
The effect of the depletion of each component of the WTAP complex on cell proliferation. A, the efficiency of RNAi was confirmed by Western blot using whole cell extracts except for the detection of the Virilizer and KIAA0853 proteins, for which nuclear extract was used to detect the endogenous proteins. α-Tublin or nucleoporin was used as a loading control. B, effect of the depletion of Hakai, Virilizer, KIAA0853, RBM15, or BCLAF1/THRAP3 on cell proliferation. The values are the average of six independent experiments. C, the cell cycle profile was analyzed using FACScalibur. Forty-eight hours after siRNA transfection, cells were harvested and stained with propidium iodide for DNA content determination. The values are the average ± S.D. (error bars), n = 10. For B and C, **, p < 0.01; *, p < 0.05 versus control (Tukey-Kramer post hoc test).
FIGURE 4.
FIGURE 4.
The intracellular localization of the WTAP complex. A, immunofluorescence analysis of HUVEC using anti-WTAP (rabbit polyclonal antibody), anti-BCLAF1, anti-THRAP3, anti-Virilizer, anti-KIAA0853, anti-Hakai, anti-RBM15, and anti-SC35 antibodies. WTAP, BCLAF1, THRAP3, Virilizer, KIAA0853, and RBM15 are all partially co-localized with SC35 in nuclear speckles and are also present in the nucleoplasm. B, the intracellular localization of WTAP (Y6828), BCLAF1, and THRAP3. WTAP is co-localized with BCLAF1 and THRAP3 in nuclear speckles and the nucleoplasm. C, the nuclear speckle localization of WTAP became dispersed upon knockdown of BCLAF1/THRAP3. HUVECs were treated with BCLAF1/THRAP3 siRNAs or control siRNA and, after 48 h, immunostained with an anti-WTAP antibody (Y6828) together with anti-BCLAF1 or anti-THRAP3 antibodies. The WTAP signal became evidently more dispersed in BCLAF1/THRAP3 knockdown cells compared with the signal in control cells. D, confocal images of WTAP (rabbit polyclonal antibody) and SC35 in control or BCLAF1/THRAP3 siRNA-treated HUVECs. The DNA is counterstained with DAPI (blue). E, the quantification of the colocalization coefficient between WTAP and SC35. The values are the average of 20 independent single-cell images. *, p < 0.05 (t test). Error bars, S.D. Bar, 10 μm.
FIGURE 5.
FIGURE 5.
The interaction of WTAP and the noncoding RNA MALAT1. A, a representative gel image of RT-PCR from the RIP samples using MALAT1- or GAPDH-specific primers (left). The interaction of WTAP with MALAT1 was determined by RIP-quantitative PCR (right). GAPDH was used as a negative control. The values are the average of five independent experiments; *, p < 0.05 (t test). Error bars, S.D. B, RNA-FISH and immunostaining were performed using a probe against MALAT1 and an anti-SC35 antibody in the control or BCLAF1/THRAP3 siRNA-treated HUVECs. Bar, 10 μm. C, the quantification of the colocalization coefficient between MALAT1 and SC35. The values are the average of 20 independent single-cell images. Error bars, S.D.
FIGURE 6.
FIGURE 6.
The WTAP complex autoregulates the alternative splicing of WTAP pre-mRNA. A, Western blot analysis of the WTAP protein. In the course of the depletion of the WTAP complex proteins, the protein level of WTAP was increased compared with control cells. α-Tublin was used as a loading control. We did not detect the shorter isoform of the endogenous WTAP protein despite the application of several specific antibodies. B, the effect of the depletion of each protein in the WTAP complex on the WTAP transcript was determined by RNase protection assay. The region used for the probe is indicated. **, p < 0.01; *, p < 0.05 (t test), n = 4. C, representative gel image of RT-PCR from the RIP samples using WTAP-specific primers (top). The interaction of WTAP with WTAP pre-mRNA was determined by RIP-quantitative PCR (bottom). GAPDH was used as a negative control (the same data as in Fig. 5A). The values are the average of five independent experiments; *, p < 0.05 (t test). Error bars, S.D. IB, immunoblot.

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