Identification of Wilms' tumor 1-associating protein complex and its role in alternative splicing and the cell cycle.

Identification of Wilms' tumor 1-associating protein complex and its role in alternative splicing and the cell cycle.
Horiuchi K, Kawamura T, Iwanari H, Ohashi R, Naito M, Kodama T, Hamakubo T.
J Biol Chem. 2013 Oct 7. [Epub ahead of print]
PubMed
doi: 10.1074/jbc.M113.500397

Wilms’ tumor 1-associating protein (WTAP) is a putative splicing regulator which is thought to be required for cell cycle progression through the stabilization of cyclin A2 mRNA and mammalian early embryo development. WTAP was identified as a protein that specifically interacts with Wilms’ tumor 1. WTAP and WT1 are present together throughout the nucleoplasm as well as in speckles and colocalize in part with splicing factors. WTAP was also identified as as the mammalian homologue of the Drosophila female-lethal-2-D (fl(2)D). fl(2)D is required for the female developmental pathway because of its activation of female-specific patterns of alternative splicing on SXL and transformer pre-mRNA. However, the detailed molecular mechanism of WTAP in RNA processing is not well understood.

To further understand how WTAP acts in the context of the cellular machinery, we generated anti-WTAP monoclonal antibodies and identified the proteins which interact with WTAP using shotgun proteomics (Fig.1) in the following way. 1) To detect the intrinsic complex, we used three monoclonal antibodies against WTAP. The three clones recognize the N-terminal or C-terminal portions of the WTAP protein, which could compensate for any loss of a complex by one clone due to a covering of the epitope by binding proteins (lane1-3). 2) To capture the more transient protein-protein and/or RNA-dependent interactions, we also performed paraformaldehyde cross-linking and immunopurification (lane4 & 5). 3) We examined the interaction between WTAP and one of the interaction candidates, Hakai, which is a C3HC4-type RING finger containing E3 ubiquitin ligase. We generated stable cell lines expressing V5-tagged Hakai or mutant Hakai which does not interact with WTAP, and isolated the interacting protein using an anti-V5 antibody. From the results of serial proteomic analyses, it was determined that the components of the WTAP complex are enriched in proteins which are involved in post-transcriptional regulation, such as pre-mRNA splicing, mRNA stabilization, polyadenylation and/or mRNA export and that Virilizer, KIAA0853, Hakai, RBM15, BCLAF1, and THRAP3 are the major components of the WTAP complex. Among them, double knockdown of the SR-like proteins BCLAF1 and THRAP3 resulted in a decrease in the speckle localization of WTAP, whereas the nuclear speckles were intact. Depletion of the major components of the complex such as Virilizer homolog, KIAA0853, BCLAF1/THRAP3, Hakai and RBM15 resulted in reduced cell proliferation with G2-phase accumulation. Moreover, we found that the WTAP complex regulates alternative splicing of WTAP‘s own pre-mRNA and, thereby the expression of full length WTAP protein. Taken together, our findings suggest that WTAP localizes to nuclear speckles through the interaction of BCLAF1 or THRAP3 and thus takes part in posttranscriptional regulation.

Fig.1 Proteomic profile of the WTAP complexes.

• 1-3) immunopurified with anti-WTAP antibodies H1122, H1137 and Y6828
• 4) using with anti-viral protein antibody as negative control
• 5) immunopurified with H1122 from Hela cells
• 6-7) from Hela cells with paraformaldehyde treatment (-/+)
• 8-9) from V5-tagged Hakai or mutant Hakai stable cell lines

Fig.2 Hakai deletion mutants.
Hakai mutant lacking the RING finger did not interact with WTAP.

Fig.3 Confocal images of WTAP and SC35 in control or BCLAF1/THRAP3 siRNA-treated HUVECs. The depletion of BCLAF1 and THRAP3 resulted in a decrease nuclear speckle distribution of WTAP.

Fig.4 Model for splice site competition modulated by the WTAP complex
WTAP and its associating proteins localize to nuclear speckles through the interaction of BCLAF1/THRAP3, where WTAP may interact with spliceosome. Then the WTAP complex probably prevents 3’ splice of the exon and/or promotes intron retention and alternative polyadenylation. As a result, the WTAP complex leads to the generation of a truncated, non-functional isoform, thus regulating the expression of the full-length protein.