Fig. 7. Effect of overexpressing 14-3-3ε on the length distribution of poly(A) tails in vivo. (A) Total RNA was purified from NIH 3T3 cells transfected with the indicated constructs and labeled at the 3′ end with [³²P]pCp. Total RNA from untranfected cells (mock) was also used as a control. We confirmed the quality of RNA by observing intact 28S and 18S rRNAs on the gels before and after the labeling. The labeled RNA was extensively digested with RNase A and RNase T1. The products were analyzed on a 5% sequencing gel. (B) RNA band intensities were determined by an image analyzer BAS-1500 (Fuji) and the sum of peak integration was normalized to that of control cells (mock). The relative intensity is represented as a ratio of the normalized intensity of the transfected cells to the intensity of control cells at a specific poly(A) length. The relative length distribution of poly(A) tails of mRNA isolated from cells transformed with GST poly(A) polymerase (PAP) (yellow). HA-14-3-3ε (red) or both GST–PAP and HA-14-3-3ε (blue) are shown. The black line represents a third-degree polynomial fit of each relative length distribution. The data are representative of three separate experiments.