FIG. 1.
PTEN represses RNA Pol III transcription via its lipid phosphatase activity independently of p53. (A) PTEN expression in PTEN null cell lines decreases RNA Pol III transcription. Prostate PC-3 (p53−/−) and LnCap (p53+/+) and glioblastoma A172 (p53−/−) and U87 (p53+/+) cell lines were transiently cotransfected with a tRNA gene reporter and an expression vector for PTEN or PTEN-G129E, a lipid phosphatase-defective mutant. Total RNA was isolated, and RNase protection assays were performed to determine the amounts of transcript. The change in transcription is calculated based on reporter gene activity in cells transfected with an empty vector control. (B) Expression of PTEN and PTEN-G129E in U87 cells. Total protein was isolated from cells transfected as described above (A) and subjected to immunoblot analysis using antibodies against PTEN or β-actin. Representative autoradiographs are shown. (C) Targeting PTEN to the nucleus prevents repression of RNA Pol III transcription. U87 cells were transfected with an expression vector for PTEN or PTEN-NLS. (Left) Total RNA was isolated and reverse transcribed. Quantitative real-time PCR analysis was performed to quantify the level of transcription of endogenous precursor tRNALeu, 7SL RNA, and U6 RNA. For quantification, transcript levels were each normalized to values obtained for GAPDH transcripts. The change in transcription is calculated based on the level of transcripts in cells transfected with an empty vector. Graphs represent determinations from at least three biologically independent samples (means ± standard errors [SE]). (Right) Total protein was isolated from transfected cells and subjected to immunoblot analysis using antibodies against PTEN or β-actin. Representative autoradiographs are shown.