Wilms' tumor 1-associating protein regulates G2/M transition through stabilization of cyclin A2 mRNA

Abstract

Wilms' tumor 1-associating protein (WTAP) has been reported to be a ubiquitously expressed nuclear protein. Although a relation to splicing factors has been postulated, its actual physiological function still remains to be elucidated. To investigate the role of WTAP, we generated WTAP-knockout mice and performed small interfering RNA (siRNA)-mediated knockdown analyses in primary cultured cells. In DNA microarrays using human umbilical vein endothelial cells, WTAP-targeted siRNA treatment resulted in markedly reduced expression of cell-cycle-related genes. siRNA-mediated WTAP knockdown down-regulated the stability of cyclin A2 mRNA through a nine-nucleotide essential sequence in cyclin A2 mRNA 3' UTR. WTAP knockdown induced G2 accumulation, which is partially rescued by adenoviral overexpression of cyclin A2. Moreover, WTAP-null mice exhibited proliferative failure with death resulting at approximately embryonic day 6.5, an etiology almost identical to cyclin A2-null mice. Collectively, these findings establish WTAP as an essential factor for the stabilization of cyclin A2 mRNA, thereby regulating G2/M cell-cycle transition.

Fig. 1.

WTAP siRNA-treated cells display growth inhibition in G₂/M phase. (A) The specificity of anti-WTAP polyclonal antibody. COS7 cells transiently transfected with a pcDNA3.1(−)-WTAP-myc-His or empty vector were subjected to Western blot analysis by using anti-WTAP or -myc antibody. Arrows indicate the band corresponding to WTAP (≈50 kDa), detected by an anti-myc antibody. (B) Efficient reduction of WTAP protein was confirmed by Western blots in WTAP siRNA-transfected HUVEC. HUVEC were transfected with WTAP siRNA or control siRNA. Total proteins were extracted 72 h after transfection. (C) Cell growth rates in WTAP siRNA or control siRNA-treated HUVEC. Cell numbers were determined by using a hemacytometer. (D) Cell-cycle analysis was carried out by using siRNA-treated HUVEC. Forty-eight hours after siRNA transfection, cells were harvested and stained with propidium iodide and then analyzed for DNA content with a FACScalibur. ∗, P < 0.05; ∗∗, P < 0.001 vs. contol.

Fig. 2.

WTAP knockdown leads to the reduction of cyclin A2 mRNA and protein levels because of destabilization of cyclin A2 mRNA. (A) Quantitative real-time PCR analysis of cyclin A2 expression. Cyclin A2 mRNA levels were decreased to 10% that of controls 24 h after siRNA transfection. Values are WTAP RNAi vs. control RNAi samples normalized to cyclophilin mRNA levels. (B) Western blot analysis of WTAP and cyclin A2. Cells were harvested 72 h after siRNA transfection. (C) The effect of WTAP knockdown on cyclin A2 promoter activity was studied by using a cyclin A2 promoter-luciferase reporter assay. The full promoter region of cyclin A2, corresponding to −737 to +108 bp (start site, +1), was inserted into the pGL3-basic plasmid. (D) Actinomycin D (AMD) was added at 24 h after siRNA transfection, and total RNA was prepared at each indicated time point. The remaining cyclin A2 mRNA was measured by quantitative real-time PCR and normalized to rpL32 mRNA, which has a half-life of >25 h. (E) Influence of the 3′UTR of cyclin A2 mRNA on WTAP-mediated mRNA stability. A luciferase-cyclin A2 3′UTR chimeric plasmid was constructed by subcloning in the 3′UTR fragment of cyclin A2 immediately downstream of the firefly luciferase ORF in a pGL3-control vector. At 12 h after siRNA transfection, HUVEC were transiently transfected with the chimeric luciferase-cyclin A2 3′UTR plasmid. Eighteen hours later, dual-luciferase assays were performed. Relative luciferase activity (firefly/Renilla) was normalized to the relative basal luciferase activity obtained from a pGL3-control vector. (F) Responsible region for WTAP-mediated mRNA stability was analyzed by using deletion constructs of cyclin A2 mRNA 3′UTR. *, P < 0.05; **, P < 0.001 vs. control.

Fig. 3.

Influence of adenovirus-mediated expression of cyclin A2 on cell-cycle profile in WTAP siRNA or control siRNA-treated HUVEC. HUVEC were infected with adeno-control or adeno-cyclin A2 at a multiplicity of infection of 20. (A) Forty-eight hours after siRNA transfection, cells were harvested and subjected to the Western blots for WTAP and cyclin A2. (B) FACS analysis. ∗, P < 0.05 vs. contol.

Fig. 4.

WTAP-null mice exhibited proliferative failure, with death resulting at approximately E6.5. (A) Generation of WTAP-null mice. Retroviral integration and disruption of the WTAP gene. VICTR 48 integrated in the intron between exons 1 and 2. NEO, a functional fusion between the β-gal and neomycin resistance genes; pA, polyadenylation sequence; SV40tpA, SV40 triple polyadenylation sequence; PGK, phosphoglycerate kinase-1 promoter; BTK, Burton's tyrosine kinase. (B) PCR genotyping of embryos from WTAP+/− intercrosses by using three primers, A, B, and LTR, to simultaneously amplify both WT (341 bp) and mutated (mut, 255 bp) alleles. ∗, Nonspecific band.

Fig. 5.

Morphology of embryos from WTAP+/− heterozygous matings. (A and B) Heterozygous WTAP+/− embryo at E8.5 (A) and homozygous WTAP−/− littermate (B). Homozygous WTAP−/− embryos are small and present no embryo body. (C–E) Representative H&E stainings of E6.5 normal (C) and abnormal (D and E) embryos cross-sectioned. (E) Some abnormal embryos have a dilated proamniotic canal. (F–I) Representative H&E and WTAP staining of E6.5 embryos sagittally sectioned . (F) Normal embryos exhibit defined layers of cells from embryonic ectoderm and endoderm. (H) Abnormal embryos are smaller and present no defined layers. No specific WTAP stainings were observed in morphologically abnormal embryos (I, compare with G), confirming that these abnormal embryos are homozygous null mutants. (Scale bars: A and B, 500 μm; C–E, 200 μm; F and H, 100 μm.)

Fig. 6.

Cell-cycle-dependent expression of WTAP. (A) Western blots of WTAP and cyclin A2 of the G₁, S, or G₂/M phase. For synchronization studies, HUVEC were serum-starved for 36 h, then released by medium exchange. The cell-cycle profile was monitored by FACS analysis, and proteins were collected at each time point. (B) Relative protein expression of WTAP was normalized to β-actin expression. (C) EMSA using cyclin A2 mRNA 3′UTR full and the indicated concentrations of either GST or GST-WTAP. Arrowheads indicate the RNA–protein complexes.