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, 18 (4), 423-34

mTOR-dependent Activation of the Transcription Factor TIF-IA Links rRNA Synthesis to Nutrient Availability

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mTOR-dependent Activation of the Transcription Factor TIF-IA Links rRNA Synthesis to Nutrient Availability

Christine Mayer et al. Genes Dev.

Abstract

In cycling cells, transcription of ribosomal RNA genes by RNA polymerase I (Pol I) is tightly coordinated with cell growth. Here, we show that the mammalian target of rapamycin (mTOR) regulates Pol I transcription by modulating the activity of TIF-IA, a regulatory factor that senses nutrient and growth-factor availability. Inhibition of mTOR signaling by rapamycin inactivates TIF-IA and impairs transcription-initiation complex formation. Moreover, rapamycin treatment leads to translocation of TIF-IA into the cytoplasm. Rapamycin-mediated inactivation of TIF-IA is caused by hypophosphorylation of Se 44 (S44) and hyperphosphorylation of Se 199 (S199). Phosphorylation at these sites affects TIF-IA activity in opposite ways, for example, phosphorylation of S44 activates and S199 inactivates TIF-IA. The results identify a new target formTOR-signaling pathways and elucidate the molecular mechanism underlying mTOR-dependent regulation of RNA synthesis.

Figures

Figure 1.
Figure 1.
Rapamycin treatment impairs Pol I transcription by inactivating TIF-IA. (A) Northern blot. 45S pre-rRNA levels were monitored in 15 μg of total RNA from untreated Hela cells or cells that were treated with 20 or 80 nM rapamycin for 60 min. Western blots demonstrating that the amount of Pol I, TIF-IB/SL1, TIF-IA, and UBF remained unaffected by rapamycin treatment are shown below. (B) Complementation of transcriptional activity in nuclear extracts from rapamycin-treated cells. Transcription reactions contained nuclear extracts (30 μg) from untreated (NE) or rapamycin-treated (NERapa) FM3A cells. (Lanes 3–6) Fractions containing partially purified Pol I transcription factors (Schnapp and Grummt 1996) were added to NERapa-containing reactions. (C) TIF-IA from rapamycin-treated cells is transcriptionally inactive. Twenty nanograms and 40 ng of TIF-IA immunopurified from untreated (lanes 2,3) and rapamycin-treated NIH3T3 cells (lanes 4,5) were added to transcription reactions containing nuclear extracts (30 μg protein) from density-arrested cells. A silver-stained SDS–polyacrylamide gel showing the amount of TIF-IA added to the transcription assays is shown below. (D) Exogenous mTOR and S6K restore transcriptional activity in nuclear extracts from rapamycin-treated cells. Reactions were supplemented with 50 ng of TIF-IA (lane 1), 10, 20, and 30 ng of HA-mTOR (lanes 3–5), 100, 150, and 200 ng of GST–S6K1 (lanes 7–9) or the same amounts of GST–S6K1dd (lanes 10–12).
Figure 2.
Figure 2.
Rapamycin treatment alters the phosphorylation pattern of TIF-IA. (A) Two-dimensional tryptic phosphopeptide maps of TIF-IA labeled in vivo. HEK293T cells overexpressing Flag-tagged TIF-IA or TIF-IAS44A, respectively, were labeled for 2 h with [32P]orthophosphate in the absence or presence of 20 nM rapamycin. TIF-IA was immunopurified and subjected to two-dimensional tryptic phosphopeptide mapping. (B) Nuclear extracts from rapamycin-treated cells fail to phosphorylate TIF-IA at S44. Immobilized TIF-IA was incubated with [γ-32P]ATP and nuclear extract from untreated, rapamycin-treated, or amino acid-starved cells as indicated. The tryptic phosphopeptide map of in vitro phosphorylated TIF-IA was compared with the phosphorylation pattern of TIF-IA labeled in vivo.
Figure 3.
Figure 3.
Phosphorylation of S44 activates, whereas phosphorylation at S199 inactivates TIF-IA. (A) Transcriptional activity of TIF-IAS44A in vitro and in vivo. (Lanes 1–5) To assay transcriptional activity in vitro, TIF-IA and TIF-IAS44A were immunopurified from HEK293T cells, and assayed for their ability to rescue Pol I transcription in a nuclear extract from density-arrested cells. (Lanes 6–12) To monitor TIF-IA activity in vivo, NIH3T3 cells were cotransfected with 2.5 μg of Pol I reporter plasmid (pMr1930-BH) and 0.25, 0.5, or 1 μg of pcDNA3.1-TIF-IA or TIF-IAS44A, and transcripts from the reporter plasmid were analyzed on Northern blots. (B) Transcriptional activity of S199 point mutants in vitro and in vivo. (Lanes 1–7) Flag-tagged wild-type TIF-IA and the indicated mutants were immunopurified from HEK293T cells, and 30 and 60 ng were assayed for their capability to restore transcriptional activity of nuclear extract from density-arrested FM3A cells. The amount and purity of TIF-IA were estimated on silver-stained SDS–polyacrylamide gels (data not shown). (Lanes 8–12) NIH3T3 cells were cotransfected with the Pol I reporter plasmid pMr1930-BHand 0.5 or 1 μg of the expression vector encoding TIF-IA or TIF-IAS199A, respectively, and transcripts from the reporter plasmid were monitored on Northern blots.
Figure 4.
Figure 4.
Cdk2/cyclin E-mediated phosphorylation at Ser 44 activates TIF-IA. (A) Cdk2/cyclin E phosphorylates S44 (phosphopeptide b). GST–TIF-IA was phosphorylated with immunopurified Cdk2/cyclin E in vitro and subjected to tryptic phosphopeptide mapping. To prove that the labeled phosphopeptides that represent spots b and b′, TIF-IA labeled in vitro was mixed with TIF-IA purified from metabolically labeled HEK293T cells, and tryptic phosphopeptides were analyzed by two-dimensional electrophoresis and chromatography. (B) A synthetic peptide harboring the target sequence for Cdk2/cyclin E competes for phosphorylation at S44. TIF-IA was incubated with [γ-32P]ATP and nuclear extract from growing cells in the absence or presence (10 μM) of a synthetic Cdk2/cyclin E target peptide (YGRKKRRQRRRGPVKRRLDL, Calbiochem). (C) Phosphorylation by Cdk2/cyclin E is required for TIF-IA activity. Thirty nanograms and 90 ng of Flag-tagged TIF-IA from untreated (lanes 2,3) or rapamycin-treated (lanes 4–7) HeLa cells were assayed for their capability to restore transcription of nuclear extracts from rapamycin-treated cells. (Lanes 6,7) TIF-IA was phosphorylated with immunopurified Cdk2/cyclin E in the presence of ATPγS (100 μM) before adding to the transcription reactions.
Figure 5.
Figure 5.
PP2A counteracts S44 phosphorylation by Cdk2/cyclin E. (A) Dephosphorylation by PP2A inactivates TIF-IA. Immobilized Flag-tagged TIF-IA was incubated with 0.003 units of either PP2A (Upstate Biochemicals) or calf intestine alkaline phosphatase (CIAP, Roche Molecular Biochemicals) for 30 min at 30°C in the supplied phosphatase buffer. Thirty nanograms and 90 ng of untreated (lanes 2,3), PP2A-treated (lanes 4,5), and CIAP-treated (lanes 6,7) TIF-IA were assayed for their capability to activate transcription in nuclear extracts from rapamycin-treated cells. (B) Phosphatase inhibitors alleviate rapamycin-mediated inactivation of TIF-IA. TIF-IA was purified from cells that were treated for 1 h with either okadaic acid (1 μM), rapamycin (20 nM), or calyculin A (50 nM), in the absence or presence of rapamycin as indicated. Thirty nanograms (even numbers) and 90 ng (odd numbers except 1) of TIF-IA were assayed for their capability to activate transcription of nuclear extracts from rapamycin-treated cells. (C) Inhibition of PP2A/PP1 in vivo alleviates rapamycin-dependent hypophosphorylation of S44. Hela cells overexpressing Flag-tagged TIF-IA were labeled for 2 h with [32P]orthophosphate in the absence (left) or presence (middle) of rapamycin. (Right) Metabolic labeling was performed in the presence of both rapamycin (20 nM) and calyculin (50 nM). Radiolabeled TIF-IA was immunopurified and subjected to two-dimensional tryptic phosphopeptide mapping.
Figure 6.
Figure 6.
Phosphorylation of S199 impairs the interaction of TIF-IA with Pol I. (A) Rapamycin treatment inhibits the interaction between TIF-IA and Pol I. Lysates from mock- or rapamycin-treated HeLa cells expressing Flag-tagged TIF-IA were incubated with anti-Flag antibodies, and immunoprecipitated TIF-IA and Pol I were visualized on Western blots using anti-Flag, anti-RPA116, or anti-PAF67 antibodies. (B) A mutant that mimics phosphorylation of TIF-IA at S199 fails to interact with Pol I and TIF-IB. Flag-tagged TIF-IA or the indicated point mutants were overexpressed in HEK293T cells and immunoprecipitated with anti-Flag antibodies. Coprecipitated Pol I and TIF-IB/SL1 were visualized on Western blots with anti-RPA116 and anti-TAFI110 antibodies.
Figure 7.
Figure 7.
mTOR controls the nucle(ol)ar localization of TIF-IA. (A) Rapamycin treatment mediates translocation of TIF-IA into the cytoplasm. HeLa cells expressing Flag-tagged TIF-IA were incubated with 20 nM rapamycin for 2 h and immunostained with Cy3-conjugated anti-Flag antibodies. Fluorescence and merged images with phase contrast from the same view are shown. (B) TIF-IA accumulates in the cytoplasm after rapamycin treatment. The levels of TIF-IA and Pol I (RPA116) were analyzed by Western blotting in whole-cell extracts (lanes 1,2), nuclear extract (lanes 3,4), and the respective S-100 fraction (lanes 5,6) from untreated or rapamycin-treated FM3A cells. (C) Rapamycin-treatment inactivates both nuclear and cytoplasmic TIF-IA. Transcription reactions contained 50 μg of nuclear extract from growing (lanes 1–3) or rapamycin-treated (lanes 4–6) cells. The assays were supplemented with 50 μg of the respective S-100 fraction (lanes 2,5) or 3 ng of cellular TIF-IA (lanes 3,6).

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