FIG. 4.
The PI3K/Akt/mTOR signal transduction pathway regulates RNA Pol III transcription. (A) Inhibiting the PI3K/Akt/mTOR pathway represses RNA Pol III transcription. LN18 cells were transfected with the tRNA gene reporter and expression vectors for dominant negative mutants of PI3K (PI3K-DN) or Akt (Akt-DN) or were treated with 0.5 μM wortmannin (6 h) or 100 nM rapamycin for 3 h prior to harvesting of the cells. Total RNA was isolated, and RNase protection assays were performed. The change in transcription is calculated based on reporter gene activity in untreated cells transfected with an empty vector control. (B) Inhibiting mTOR decreases RNA Pol III transcription in vitro. Nuclear extracts were prepared from LN18 cells following treatment with 100 nM rapamycin for 3 h. Transcription assays were carried out using the tRNA reporter gene as a template and 10 or 20 μg of nuclear extract. (C) Inhibiting mTOR decreases endogenous tRNA but not U6 RNA transcription. MCF-10A and U87 cells were treated with 100 nM rapamycin for 3 h prior to harvesting of cells. Total RNA was isolated and reverse transcribed. Quantitative real-time PCR analysis was performed to quantify the level of transcription of endogenous precursor tRNALeu 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 treated with dimethyl sulfoxide. (D) Activating the PI3K pathway induces RNA Pol III transcription in LnCap cells. LnCap cells were transfected with the tRNA gene reporter, treated with wortmannin, or cotransfected with RasV12C40, a constitutively activated Ras specific for PI3K activation, as designated. Total RNA was isolated, and RNase protection assays were performed. The change in transcription is calculated based on reporter gene activity in untreated cells transfected with an empty vector control. (E) RNA Pol III transcription is induced by the activation of PI3K in primary rat hepatocytes. Primary rat hepatocytes were cotransfected the tRNA gene reporter and RasV12 or RasV12C40 expression vectors and/or were treated with 0.5 μM wortmannin for 6 h prior to harvesting of the cells. Total RNA was isolated, and RNase protection assays were performed. The change in transcription is calculated based on reporter gene activity in untreated cells transfected with an empty vector control. (F) Expression of constitutively activated S6K1 alleviates PTEN-mediated repression of RNA Pol III-dependent transcription. U87 cells were transfected with an expression vector for PTEN and with either constitutively activated S6K1 (S6K1-E389) or an empty vector. Total RNA was isolated and reverse transcribed. Quantitative real-time PCR analysis was performed to quantify the level of transcription of endogenous precursor tRNALeu and 7SL 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. All graphs represent determinations from at least three biologically independent samples (means ± SE).