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. 2005 Aug;25(15):6454–6463. doi: 10.1128/MCB.25.15.6454-6463.2005

FIG. 1.

FIG. 1.

TTP binds to AU2 of β4GalT1 3′UTR, but is released upon TNF-α stimulation. (A) The expression of β4GalT1 mRNA is higher in HeLa TO cells that lack the expression of TTP but normal in HeLa TO cells lacking BRF1 expression. Stable HeLa TO knockdown cell lines for both TTP and BRF1 were generated as described in Materials and Methods; a scrambled (scr) control was included to monitor nonspecific effects. Quantitative real-time PCR analysis was performed on mRNA isolated from resting cells to determine the relative expression level of β4GalT1 mRNA. (B) The half-life of β4GalT1 mRNA is prolonged in TTP but not BRF1 knockdown HeLa TO cells. The half-life of β4GalT1 mRNA was determined by quantitative real-time PCR analysis of mRNA isolated from resting cells at different times after addition of ActD. (C) Overexpression of TTP accelerates the decay of chimeric mRNA containing the wild-type (wt) β4GalT1 3′UTR, but not the AU2 mutant. TTP was overexpressed in HeLa TO cells cotransfected to express chimeric mRNA containing either the wt β4GalT1 3′UTR or the AU2 mutant. At 24 h after transfection, the half-life of chimeric mRNAs was determined by quantitative real-time PCR analysis on mRNA isolated at different times after addition of doxycycline. The graphs (relative chimeric mRNA expression versus time after addition of DOX) for the determination of the half-lives are available in Fig. S1 in the supplemental material. The overexpression of His-tagged TTP was checked by Western blotting with an anti-His antibody as described in Materials and Methods. (D) TNF-α induces the release of binding of TTP from β4GalT1 mRNA. HUVECs, stimulated with 100 U/ml TNF-α as indicated, were treated with formaldehyde to preserve protein complexes bound to mRNA by reversible cross-links. Anti-TTP immunocomplexes were analyzed for the presence of β4GalT1 mRNA by quantitative real-time PCR. (E) TTP binds AU2. RNA/immunoprecipitation assays were performed on HeLa TO cells expressing chimeric mRNAs containing either the wt β4GalT1 3′UTR or the AU2 mutant as described in panel D.