FIG. 5.

Effect of TTP on steady-state levels of MLP-Plk3 fusion mRNA. A portion of the Plk3 3′-UTR (bp 2049 to 2365 of GenBank accession number NM_013807.2) was fused 3′ of the coding region of MLP to create the MLP-Plk3 fusion transcript for cotransfection experiments with TTP DNA. (A) The three potential TTP binding sites within the 3′-UTR of the Plk3 transcript are underlined. (B) Various amounts of TTP DNA were cotransfected into HEK293 cells along with 0.5 μg of the plasmid coding for the MLP-Plk3 fusion transcript. Total plasmid DNA was kept constant (5 μg) by the addition of the pBS(+) vector. RNA was isolated 48 h after DNA was introduced into the cells. A total of 10 μg of total cellular RNA was loaded per gel lane. The results of a Northern blot from such an experiment probed for MLP-Plk3 and TTP are shown at the top and bottom, respectively. Cells were transfected with the pBS(+) vector alone (mock) (lane 1), MLP-Plk3 DNA alone (lane 2), MLP-Plk3 DNA plus various amounts of TTP DNA (lanes 3 to 7), and the TTP mutant C124R (lane 8). The native MLP transcript migrates above the MLP-Plk3 fusion transcript, as indicated. (C) The amount of MLP-Plk3 mRNA shown by Northern blotting was quantified using a phosphorimager and ImageQuant software. The means represent the amounts of MLP-Plk3 mRNA normalized against endogenous MLP, and error bars represent the SD derived from between 3 and 12 experiments (arbitrary units). Comparisons of MLP-Plk3 transcripts in the absence of TTP and various amounts of TTP were determined by one-way ANOVA and the Bonferroni multiple-comparisons test (* represents a P value of <0.05, and ** represents a P value of <0.01).