FIGURE 4.

Heterologous reporter constructs. (A) The pTRE-d2EGFP and a series of derivative pTRE-d2EGFP-3′UTR reporter plasmids were transfected into HeLa Tet-Off cells. Twenty-four hours following transfection, doxycycline was added to block transcription of the reporter mRNAs. Residual d2EGFP transcript levels were monitored as a function of time using qRT-PCR. Each point represents the mean (±SEM) determined from three independent experiments. The mean half-lives of the heterologous transcripts were 5.6 ± 0.5, 2.0 ± 0.9, 1.5 ± 0.3, 4.8 ± 0.7, and 5.5 ± 0.8 for control, DDB2, GDF15, TP53I3, and CRYAB, respectively. (B) Sequence analysis of 3′ UTRs led to the identification of known destabilizing elements. The mean number of U-, AU-, and GU-rich other miscellaneous elements (see Supplemental Table 4) per 3′ UTR is presented for the seven stable and the 22 unstable p53-induced transcripts. The mean number of these elements per 3′ UTR and the mean number of elements per kilobase of 3′ UTR is presented. Each point represents the mean (±SEM) determined for seven stable and 22 unstable transcripts. The indicated P-values were determined by a Student's t-test.