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Molecular and Cellular Biology, August 2007, p. 5746-5764, Vol. 27, No. 16
0270-7306/07/$08.00+0     doi:10.1128/MCB.02136-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Tuberous Sclerosis Complex Proteins 1 and 2 Control Serum-Dependent Translation in a TOP-Dependent and -Independent Manner{triangledown} ,{dagger}

Benoit Bilanges,1 Rhoda Argonza-Barrett,2 Marina Kolesnichenko,1 Christina Skinner,1 Manoj Nair,2 Michelle Chen,2 and David Stokoe1*

Cancer Research Institute, University of California, San Francisco, California,1 Agilent Technologies, Inc., Santa Clara, California2

Received 14 November 2006/ Returned for modification 30 January 2007/ Accepted 17 May 2007


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ABSTRACT
 
The tuberous sclerosis complex (TSC) proteins TSC1 and TSC2 regulate protein translation by inhibiting the serine/threonine kinase mTORC1 (for mammalian target of rapamycin complex 1). However, how TSC1 and TSC2 control overall protein synthesis and the translation of specific mRNAs in response to different mitogenic and nutritional stimuli is largely unknown. We show here that serum withdrawal inhibits mTORC1 signaling, causes disassembly of translation initiation complexes, and causes mRNA redistribution from polysomes to subpolysomes in wild-type mouse embryo fibroblasts (MEFs). In contrast, these responses are defective in Tsc1–/– or Tsc2–/– MEFs. Microarray analysis of polysome- and subpolysome-associated mRNAs uncovered specific mRNAs that are translationally regulated by serum, 90% of which are TSC1 and TSC2 dependent. Surprisingly, the mTORC1 inhibitor, rapamycin, abolished mTORC1 activity but only affected ~40% of the serum-regulated mRNAs. Serum-dependent signaling through mTORC1 and polysome redistribution of global and individual mRNAs were restored upon re-expression of TSC1 and TSC2. Serum-responsive mRNAs that are sensitive to inhibition by rapamycin are highly enriched for terminal oligopyrimidine and for very short 5' and 3' untranslated regions. These data demonstrate that the TSC1/TSC2 complex regulates protein translation through mainly mTORC1-dependent mechanisms and implicates a discrete profile of deregulated mRNA translation in tuberous sclerosis pathology.


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INTRODUCTION
 
Protein translation is controlled by multiple signaling pathways which can affect the rate of either global protein synthesis or a small subset of transcripts (16). Different mRNAs are translated at different rates depending on the activation of signal transduction pathways in response to changes in the extracellular environment. The phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway integrates signals from nutrients, energy status, and growth factors to regulate many processes, including cell growth and division, autophagy, protein translation, and metabolism. mTOR is a conserved Ser/Thr kinase first identified as a target of the immunosuppressant rapamycin (77). It is now known that mTOR forms two functionally distinct complexes: a rapamycin-sensitive mTORC1, composed of mTOR, raptor and GßL and a rapamycin-resistant mTORC2, composed of mTOR, mSIN1, Rictor, and GßL (13, 20, 28, 35, 61). mTORC1 is activated by growth factors in part through class Ia PI3K, and the importance of this pathway in protein translation has been shown through the use of inhibitors of PI3K (50). Deregulated protein translation through mTORC1 has been implicated in many human diseases, including tuberous sclerosis, the Peutz-Jeghers and Cowdens syndromes, and cancer (18).

Important effectors of PI3K mediating its effects on protein translation include protein kinase B (PKB/Akt) and the tuberous sclerosis complex protein 1 (TSC1)/TSC2 complex (also referred to as hamartin and tuberin, respectively). TSC1 and TSC2 form a complex that inhibits mTORC1 activity via inhibition of the small GTPase Rheb, a positive regulator of mTORC1. The TSC complex inhibits Rheb by decreasing its GTP levels via the GTPase-activating protein (GAP) activity of TSC2. Upon growth factor stimulation, TSC2 is phosphorylated by activated PKB/Akt at several sites which inhibit the ability of TSC2 to act as a Rheb GAP (reviewed in reference 44). PKB/Akt may also regulate mTORC1 activity by regulating AMPK phosphorylation of TSC2 (19). Moreover, mTORC1 activity is regulated by extracellular nutrients, although the signaling pathways involved and how they are coordinated with growth factors are just beginning to be unraveled (11).

Activated mTORC1 and mTORC2 have distinct downstream effectors (reviewed in reference 57). mTORC2 phosphorylates PKB/Akt on Ser473 to determine PKB/Akt substrate selectivity and seems to have a role in regulating the actin cytoskeleton and cell survival (28, 29, 67). In contrast, mTORC1 regulates growth through downstream effectors such as eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) and the ribosomal S6 kinases (S6K1 and S6K2). mTORC1-dependent phosphorylation of 4E-BP1 results in its dissociation from the initiation factor eIF4E that binds to the 5'-end cap of the mRNAs, thereby allowing the formation of translation initiation complexes crucial for protein synthesis. mTORC1-dependent phosphorylation of S6K1 at Thr389 is essential for S6K1 activation by creating a docking site for PDK1 (14). S6K1 phosphorylates the 40S ribosomal protein (RP) S6, the RNA processing protein SKAR, the initiation factor eIF4B, and elongation factor kinase eEF2K (71). Recently, Holz et al. identified direct interactions between mTORC1, S6K1, and its substrates and components of the translation preinitiation complex, thus providing new insights into how mTORC1 is connected to components of preinitiation apparatus (24).

In mammalian cells, mRNAs encoding for components of translational apparatus (RPs and initiation and elongation factors) are regulated at the translational level by mitogenic or nutritional stimuli. A structural feature common to such mRNAs is the presence of a 5'-terminal oligopyrimidine tract (5'TOP) within their 5' untranslated region (5'UTR). Interestingly, inhibition of mTORC1 by the macrolide drug rapamycin leads to inactivation of its downstream effectors and selectively suppresses mitogen-induced translation of 5'TOP containing mRNAs, such as eEF1A, eEF2, RpS6, and Rpl32. These mRNAs are redistributed from actively translated complexes (found in polysomes) into poorly translated complexes (found in small ribonucleoprotein particles) after rapamycin treatment (7, 32). The exact mechanism whereby mTORC1 regulates the translation of 5'TOP-containing mRNAs is still unclear as is the number and identity of regulated targets (51, 56, 69). However, in many cell types, rapamycin has only minor effects on the overall rate of protein synthesis (3, 23, 73, 74), suggesting additional mTORC1-independent pathways regulating translation.

Several studies demonstrate that pathways from multiple growth factors inhibit TSC1/TSC2 to regulate mTORC1 (66). Moreover, mammalian cells lacking Tsc1 or Tsc2 fail to downregulate mTORC1 function in response to growth factor deprivation, suggesting that growth factors control mTORC1 activation in a TSC1- and TSC2-dependent manner (26, 38, 79). To address whether mitogenic signals regulate translation in a TSC1/TSC2-dependent manner, we analyzed the distribution of mRNAs on polysomes/subpolysomes in wild-type (WT) and Tsc-deficient mouse embryo fibroblasts (MEFs). Using microarray analysis, we identify novel serum- and rapamycin-sensitive mRNAs translationally regulated in WT MEFs, as well as in Tsc1–/– and Tsc2–/– MEFs. This global analysis revealed three groups of mRNAs: those regulated by serum but not by rapamycin (which are mainly TSC dependent), those regulated by rapamycin but not by serum, and those regulated by both. This latter group is enriched for 5'TOP-containing mRNAs, which also possessed short 5'- and 3'UTRs. Since TSC is a disease caused (in the most part) by deregulated protein translation, identifying which subsets of mRNAs are translationally controlled by TSC1 or TSC2 signaling pathways is crucial for the discovery of new therapeutic targets.


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MATERIALS AND METHODS
 
Cell culture, constructs, and drug treatments. Immortalized littermate pairs of WT and null Tsc1 (Tsc1–/–p53+/+ and Tsc1+/+p53+/+) and Tsc2 (Tsc2–/–p53–/– and Tsc2+/+p53–/–) MEFs were generous gifts from D. Kwiatkowski (Brigham and Women's Hospital, Boston, MA) and were described previously (38, 78). Tsc2+/+p53–/– cells were used throughout the present study as WT MEFs, Tsc2–/– TP53–/– cells were used as Tsc2–/– MEFs, and Tsc1–/–p53+/+ cells were used as Tsc1–/– MEFs. Cells were grown in a humidifier incubator with 8% CO2 at 37°C in Dulbecco modified Eagle medium (DMEM) containing 4.5g of glucose (Life Technologies)/liter supplemented with 10% fetal bovine serum. Wortmannin and rapamycin (Calbiochem) were added to the appropriate media at the indicated times at 100 and 50 nM, respectively. For serum and/or nutrient starvation experiments, cells were seeded and maintained in DMEM plus 10% fetal calf serum (FCS). The following day, the cells were washed twice in Dulbecco's phosphate buffered saline (D-PBS) and maintained for the indicated time in DMEM alone, D-PBS alone, or D-PBS plus 10% dialyzed FCS. The human TSC1 full-length cDNA was subcloned into LXSP3 (Puro) retroviral vector. Retroviruses were generated by transient transfection of Phoenix packaging cells. Filtered supernatants containing virus were used to infect Tsc1–/– MEFs, followed by selection with 3 µg of puromycin/ml. We refer to Tsc1–/– MEFs expressing empty vector or WT TSC1 as Tsc1–/– (vector) or Tsc1–/– (Rev) MEFs, respectively. Tsc2–/– (vector) and Tsc2–/– (Rev) MEFs were provided by D. Kwiatkowski and were previously described (78).

Polysomal analysis and RNA preparation. The 4-h time point was selected for the microarray experiments. Cell extracts were prepared essentially as described previously (70). Briefly, after incubation with cycloheximide (CHX) at 90 µg/ml for 10 min, 2 x 106 cells were washed and treated with trypsin in presence of CHX (90 µg/ml). Cell pellets were washed twice in ice-cold PBS containing 90 µg of CHX/ml, resuspended in 150 µl of cold RSB (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 15 mM MgCl2) containing 100 µg of heparin/ml, and lysed in ice-cold lysis buffer (1.2% Triton X-100 and1.2% deoxycholate in RSB) on ice. Nuclei and cell debris were cleared out by centrifugation at 12,000 x g for 5 min in a microcentrifuge at 4°C. The supernatant was diluted with an equal volume of polysomal buffer (25 mM Tris-HCl [pH 7.4], 25 mM NaCl, 25 mM MgCl2, 0.05% Triton X-100, 0.14 M sucrose, 500 µg of heparin/ml) and layered over 12 ml of a 5 to 56% (wt/wt) sucrose gradient. The gradients were sedimented via centrifugation at 37,000 x g for 150 min at 4°C in a SW40 Ti rotor (Beckman). Twelve fractions of 1 ml each were collected, and RNAs were precipitated by a standard procedure, purified by using an RNeasy minikit (QIAGEN), and quantified by determining the absorbance at 260 nm. For microarrays, fractions 5 to 7 and 9 to 10 from three independent experiments were pooled for polysomal and subpolysomal RNA samples, respectively, and were reprecipitated with LiCl buffer (2 M LiCl, 20 mM Tris-HCl [pH 8.0]). RNA quality was monitored by using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Total RNAs, pooled from three independent experiments, were isolated according to the manufacturer instructions (Agilent Technologies). For Northern blot analysis, RNA was fractionated on formaldehyde-agarose gels and transferred to nylon Hybond N membrane (Amersham Biosciences). Northern blotting was performed essentially as recommended by the manufacturer. The TOP mRNA probe was mouse eEF1A. The non-TOP mRNA analyzed was mouse GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Samples were prepared by using products of reverse transcription-PCR (RT-PCR). Primers were designed according to sequences present in the GenBank/EBI data bank.

Immunoblotting. Cells were washed twice in cold PBS and harvested in protein lysis buffer containing 1% NP-40, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 1 mM dithiothreitol, phosphatase cocktail inhibitors (Sigma-Aldrich), and protease inhibitor cocktail (Boehringer Mannheim) (1 pill/10 ml of lysis buffer). Cell lysates were prepared as previously described (70). Equal amounts of total protein (30 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 4 to 20% gradient polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Immunostaining was performed with antibodies generated against phospho-Thr389 S6K, phospho-Ser235/236 S6, phospho-Ser240/244 S6, phospho-Ser422 eIF4B, phospho-Ser65 4E-BP1, phospho-Thr37/46 4E-BP1, phospho-Ser51 eIF2{alpha}, S6, and eIF4E (Cell Signaling, Beverley, MA); phospho-Ser246 PRAS40 (Biomol International, Plymouth, PA); eIF4G1 (Abcam, Inc., Cambridge, MA); and beta-actin (Sigma-Aldrich, Saint Louis, MO).

Microarray analysis. Microarray experiments were performed by using a mouse 22K (G4121A) oligonucleotide array (Agilent Technologies) according to the manufacturer's instructions. Briefly, polysomal, subpolysomal, or total RNA samples were tested on Agilent's Bioanalyzer 2100 for quality, amplified, labeled with cyanine 3 and cyanine 5 fluorescent dyes (2 µg of RNA per labeling reaction), and hybridized onto Agilent's 22K mouse oligonucleotide arrays (1 µg of labeled cRNA per channel for hybridization) using Agilent's reagents and protocols. Microarray data were analyzed with GeneSpring 7.2 software (Silicon Genetics) for the calculation of intensity ratios and dye-swap calculations as normalization methods. Features that showed large variability were filtered out by only including the features that had a standard variation of <0.7 between dye swap experiments across all experimental conditions. This left a subset of 7,055 genes in the data set. The normalized datasets were processed by calculating the simple log2 intensity ratios comparing polysome to subpolysomal values across control and starved samples. A twofold cutoff was chosen to identify polysome-associated RNAs.

Quantitative PCR analysis. Real-time PCR was performed in ABI 7500 (Applied Biosystems) as previously described (70). RNA samples from polysome and subpolysome fractions were normalized to GAPDH (the polysome/subpolysome ratio of GAPDH did not vary between the control and starved or treated samples).


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RESULTS
 
Either growth factors or nutrients are sufficient for mTORC1 activity in Tsc1–/– and Tsc2–/– MEFs. The TSC/mTORC1 pathway is important for the regulation of mRNA translation and integrates various signals such as nutrients, growth factors, energy, and stress to regulate cell growth and cell proliferation. To assess the role of TSC1 and TSC2 in mTORC1 signaling, we compared the effects of serum starvation, nutrient starvation, or serum and nutrient starvation on mTORC1 activity in WT, Tsc1–/–, and Tsc2–/– MEFs. Throughout the present study, we define mTORC1 activity through both its ability to phosphorylate its defined substrates (S6K [Thr-389] and 4EBP1 [mobility shift and Ser-65]) and its ability to be inhibited by rapamycin. In addition, we used the phosphorylation of RP S6 (at Ser235/236 and Ser240/244) as an additional measure for the activity of S6K. As shown in Fig. 1, under normal growth conditions (DMEM plus 10% FCS) WT MEFs displayed strong mTORC1 activity that is sensitive to rapamycin. However, depletion of either growth factors (FCS) or nutrients (DMEM) in these cells rapidly decreased the mTORC1 signaling. Withdrawal of either growth factors or nutrients also reduced the phosphorylation of 4E-BP1, as indicated by the appearance of faster mobility forms. In contrast, Tsc1–/– and Tsc2–/– MEFs maintained elevated mTORC1 activity upon 4 h growth factor or nutrient depletion (Fig. 1). In the absence of nutrients but in the presence of dialyzed FCS (D-PBS+dFCS) in Tsc1–/– or Tsc2–/– MEFs, S6K and 4E-BP1 phosphorylation is reduced but with delayed kinetics compared to WT cells. In contrast, the withdrawal of both growth factors and nutrients (D-PBS) dramatically reduced the mTORC1/S6K/4E-BP1 signaling in these cells. Elevated S6K and S6 phosphorylation and 4E-BP1 mobility shift in Tsc1- and Tsc2-null cells was always inhibited by rapamycin, thus confirming a dependence on mTORC1. However, wortmannin was only effective at inhibiting mTORC1 activity induced by growth factors but not nutrients, confirming the importance of PI3K in mitogen-mediated but not nutrient-mediated signaling to mTORC1.


Figure 1
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  		FIG. 1. The TSC1/TSC2 complex is critical for growth factor and nutrient signaling to mTORC1. (A) WT, Tsc1–/–, and Tsc2–/– MEFs were cultured in the presence of DMEM and 10% FCS (Nutr.+FCS). Cells were then transferred to conditions containing either nutrient alone (Nutr. Only [DMEM]), serum alone 10% dialyzed FCS (dFCS) in D-PBS (No Nutr.+dFCS), or devoid of both nutrient and serum (No Nutr. No FCS [D-PBS]) for the indicated times. Lysates were then prepared and separated by SDS-10% PAGE and Western blotted with antibodies to phospho-S6K (T389), phospho-S6 (S235/236 and S240/244), total S6, and 4E-BP1. Wortmannin (100 nM) and rapamycin (50 nM) were added 4 h prior to cell harvesting as indicated.

Serum depletion inhibits the assembly of translation initiation complexes. mTORC1 integrates nutrient and growth factor signals to regulate the translation initiation of mRNAs important for cell growth (54). To determine whether mTORC1 inhibition mediated by serum and/or nutrient withdrawal was correlated with inhibition of translation initiation, we next examined the consequences on the maintenance of cap-dependent translation. mTORC1 regulates cap-dependent translation in part through the phosphorylation of 4E-BP1. Unphosphorylated 4E-BP1 inhibits eIF4E by preventing its binding to eIF4G and subsequent association with capped mRNAs. 4E-BP1 phosphorylation at Ser65 is correlated with the dissociation of 4E-BP1 from eIF4E (46, 75). Active and inactive translational complexes can be determined by measuring the levels of 4E-BP1 associated with eIF4E precipitated through its binding to the capped mRNA mimic methyl7-GTP Sepharose. Figure 2 shows that in the presence of growth factors and nutrients (DMEM+FCS) in both WT MEFs and Tsc1–/– and Tsc2–/– MEFs, low levels of 4E-BP1 were associated with methyl7-GTP Sepharose (Fig. 2, lanes 1, 13, and 25). This closely correlated with the levels of phosphorylation of 4E-BP1 detected with either specific antibodies (Ser65) or with total 4E-BP1 mobility. Deprivation of either growth factors (FCS) or nutrients (DMEM) resulted in increased levels of 4E-BP1 bound to eIF4E in WT MEFs but not in the Tsc-null cells (in Fig. 2, compare lanes 4 and 10 to lanes 16 and 22 and to lanes 28 and 34). However, the withdrawal of both nutrients and growth factors (No Nutr No FCS) strongly increased the amount of 4E-BP1 bound to eIF4E in WT, Tsc1–/–, and Tsc2–/– MEFs. Rapamycin strongly enhanced the association of hypophosphorylated 4E-BP1 to methyl7-GTP Sepharose under all of the conditions tested in all three MEFs, demonstrating the contribution of mTORC1 in the assembly of translation initiation complexes (Fig. 2, compare lanes 7, 19, and 31). Taken together, these data demonstrate that, in the absence of TSC1 or TSC2, either growth factors or nutrients are sufficient for mTORC1 activation and protein translation initiation.


Figure 2
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  		FIG. 2. The TSC1/TSC2 complex is critical for maintaining translation initiation complexes. MEFs were cultured as described in Fig. 1. Wortmannin or rapamycin was added at the time of media transfer. Methyl7-GTP Sepharose was used to precipitate translation initiation complexes. Associated proteins were detected by Western blotting with antibodies to eIF4E and 4E-BP1.

Serum depletion reduces polysome formation in WT MEFs but not in Tsc1–/– and Tsc2–/– MEFs. mRNA translation is a tightly controlled process taking place on ribosomes. Actively translated mRNAs are distributed to polyribosomes (or polysomes), and inactive mRNAs are associated with subpolysome and monosome fractions. We therefore investigated whether the inhibition of cap-dependent translation upon serum withdrawal had consequences on the distribution of RNA from polysomes to subpolysomes. To address this question, we isolated polysomal mRNA from total cellular mRNA by fractionation through a 5 to 56% (wt/wt) sucrose gradient. To confirm the correct identification of the polysomal peaks, we analyzed the polysomal localization of a well-characterized TOP mRNA, eEF1{alpha}, known to be redistributed from polysome complexes into subpolysomal fractions after serum starvation (7). eEF1{alpha} demonstrated robust translocation from polysomes to subpolysomes in response to serum deprivation (see Fig. S1 in the supplemental material), suggesting that the polysomal/subpolysomal fractions were accurately isolated in our experiment and therefore suitable for further analysis. We observed a redistribution of polysome-associated RNAs into subpolysomes in response to a 4-h serum starvation in WT MEFs (Fig. 3). However, in Tsc1–/– and Tsc2–/– MEFs, serum withdrawal did not significantly affect the polysome-subpolysome profile (Fig. 3). This redistribution of RNA from polysomes to subpolysomes in response to serum starvation in WT MEFs precisely correlated with the effects observed on cap-dependent translation shown in Fig. 2. Altogether, these results are consistent with the regulation of mTORC1 signaling and protein translation showing serum dependence in WT MEFs and serum independence in Tsc1–/– and Tsc2–/– MEFs.


Figure 3
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  		FIG. 3. The TSC1/TSC2 complex is critical for regulating mRNA association with polysomes. Polysome profile of WT, Tsc1–/–, and Tsc2–/– MEFs. Cells were cultured in DMEM in presence or absence of 10% FCS for 4 h. Cell lysates were prepared and placed on top of a 5 to 56% (wt/wt) sucrose gradient. After centrifugation, 12 fractions were collected, and RNA was isolated from each fraction and quantitated by using absorbance at 260 nm. The RNA content in each fraction is displayed as a percentage relative to the total RNA measured across the gradient. Polysomes are represented by fractions 4 to 7, and subpolysomes/free RNA are represented by fractions 9 to 11. The results are expressed as means ± the standard deviations (SD) for three independent experiments.

Identification of specific mRNAs translationally regulated by FCS in WT, Tsc1–/–, and Tsc2–/– MEFs. To identify specific mRNAs translationally regulated by serum in WT, Tsc1–/–, and Tsc2–/– MEFs, we subjected subpolysomal and polysomal RNAs to microarray analysis with Agilent 22k mouse 60-mer oligonucleotide arrays. The experimental approach is shown in Fig. 4A and is described in the figure legend. As shown in Fig. 4B, the withdrawal of 10% FCS in WT MEFs increased the number of RNAs showing a polysome/subpolysome ratio of <0.5, from 38 to 102 RNAs, whereas fewer mRNAs displayed this property in Tsc1–/– and Tsc2–/– MEFs. This correlates with the global polysomal profile and the inhibition of the mTORC1 signaling observed in Fig. 3. Figure S2 in the supplemental material illustrates how the nine polysome-associated mRNAs in WT MEFs in presence of DMEM+FCS shifted toward the subpolysome population upon FCS withdrawal and the 102 subpolysome-associated mRNAs of WT MEFs cultured in DMEM shifted toward the polysome population of WT MEFs cultured in DMEM plus FCS.


Figure 4
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  		FIG. 4. Translational profiling of WT, Tsc1–/–, and Tsc2–/– MEFs upon serum starvation. (A) Overview of the experimental design. This figure represents the experimental design, illustrated by the condition DMEM plus 10% FCS. Cell lysates prepared from MEFs grown in DMEM in the presence of 10% FCS were layered on top of a 5 to 56% (wt/wt) sucrose gradient (left panel). Subpolysomal (SP) and polysomal (P) RNA fractions from three independent experiments were isolated and separately pooled. The purified pooled subpolysome RNA fractions were then labeled with the fluorescent dye Cy3 (plain arrow), and the purified pooled polysomal RNA fractions were labeled with Cy5 (plain arrow). Samples labeled with the opposite dye (Cy5 and Cy3) configurations were hybridized on a single array. To compensate for labeling dye bias, a duplicate hybridization of the same samples with dye reversal (dashed arrow) was performed. Fluorescence intensities were imported and analyzed with GeneSpring (version 7.2; Silicon Genetics). The normalized datasets were processed by calculating the log2 intensity ratios comparing polysome to subpolysome values (Cy3 versus Cy5) across control (DMEM plus 10% FCS) and starved samples (DMEM). (B) Scatter plot representing the polysome-associated mRNAs (y axis) and subpolysome-associated mRNAs (x axis). The log2 intensity values comparing polysome to subpolysome are plotted for each condition as indicated above each plot. mRNA datum points above or below a twofold cutoff are represented outside of the green lines. The number of mRNAs displaying >2-fold polysome or subpolysome association is indicated above or below each green lines. (C) The polysome/subpolysome ratios for the starved condition (DMEM), log2[P(DMEM)/SP(DMEM)], are plotted against the polysome-subpolysome ratio for the control condition, log2[P(DMEM+FCS)/SP(DMEM+FCS)], for every mRNA. Green lines represent a twofold cutoff. mRNAs that show no change in their polysome association are displayed on the red diagonal, whereas mRNAs outside the green lines indicate those that shift >2-fold toward either the polysomal or subpolysomal fractions. mRNAs that shift >2-fold toward the polysome in WT MEFs in response to serum are indicated with white dots in all three scatter plots. (D) Venn diagram representing the comparison of mRNAs upregulated (twofold) by serum in WT, Tsc1–/–, and Tsc2–/– MEFs. The overlap between the mRNAs regulated in each condition is displayed by the superposition of the circle. The number of mRNAs regulated in each condition is indicated in each circle. (E) Gene ontology analysis of the mRNAs regulated by serum in a TSC1- and TSC2-dependent manner.

We next analyzed which mRNAs most dramatically shift between polysomes to subpolysomes upon serum withdrawal in WT, Tsc1–/–, and Tsc2–/– MEFs. To investigate this, we compared the ratio of every mRNA in polysome (P) and subpolysome (SP) fractions between control (DMEM+FCS) and serum-starved (DMEM) cells. The ratio of the Cy5 intensity and Cy3 intensity (representing the polysome/subpolysome ratio) reflects its relative polysome distribution. Therefore, mRNAs redistributing from polysomes to subpolysomes upon serum deprivation in WT MEFs that do not change under these conditions in Tsc1–/– and Tsc2–/– MEFs likely represent the RNAs translationally regulated by serum in a TSC1- and TSC2-dependent manner. We used an arbitrary cutoff value of twofold redistribution across polysomes under different conditions to designate translational regulation, so the numbers of mRNAs changing are not absolute, but different cutoff values showed similar trends. Figure 4C displays the change in polysome association of each mRNA when cultured in DMEM+FCS compared to DMEM alone. In WT MEFs, 175 mRNAs were preferentially associated with polysomes in the presence of serum, whereas only 40 and 42 mRNAs were more associated with polysomes in Tsc1–/– and Tsc2–/– MEFs, respectively, under the same conditions (Fig. 4C). Importantly, 160 of 175 mRNAs were translationally regulated by serum in WT MEFs but not in Tsc1–/– and Tsc2–/– MEFs (indicated by the white dots in Fig. 4C), demonstrating that ca. 90% of serum-dependent mRNA translation is mediated through the TSC1/TSC2 complex in MEFs (Fig. 4D).

Serum regulates ribosome biogenesis in a TSC1 and TSC2-dependent manner. Table 1 lists the top 30 RNAs putatively regulated in a serum-sensitive fashion in a TSC1- and TSC2-dependent manner. Two predominant biological groups were significantly enriched (with P < = 4 x 10–11) in the list of 175 serum-regulated genes by gene ontology analysis, namely, protein biosynthesis and ribosome biogenesis. Fifty different RPs and eleven translation initiation and elongation factors were in this group, suggesting that many of the serum-regulated RNAs are involved in ribosome biogenesis or encode components of the translational machinery (see Table S1 in the supplemental material). Some of the mRNAs that we identified have previously been shown to be regulated by serum, amino acids, and/or rapamycin, e.g., eEF1a, eEF1b, and eEF2 and RPs such as Rpl32 and Rps6 (7, 25, 31, 80). However, we also identified many additional regulated mRNAs, and other gene ontology categories were also represented, such as genes involved in metabolism, transport, transcription and development (Fig. 4E; also see Table S1 in the supplemental material).


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  		TABLE 1. mRNAs translationally repressed upon serum starvation (top 30) in WT MEFs

To confirm our microarray data, we validated some of the genes by TaqMan analysis as represented in Fig. 5 (right panel). Approximately, 80% of the specific probes (of 19 tested) were validated by TaqMan, although three genes (Tnrc6b, Ddx6, and Rpl21) were not. In general, the fold shift from subpolysome to polysome in response to serum measured by TaqMan followed relatively closely the fold shift calculated by the microarray analysis (Fig. 5, left panel). The entire TaqMan results completed for each individual target are shown in Fig. S3 in the supplemental material.


Figure 5
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  		FIG. 5. Quantitative RT-PCR (qRT-PCR) analysis of mRNAs regulated by serum in TSC1- and TSC2-dependent manner. MEFs were grown in DMEM in the presence or in absence of 10% FCS for 4 h. RNA was isolated from 5 to 56% (wt/wt) sucrose gradient and purified. Polysome and subpolysome fraction were separately pooled and amplified by qRT-PCR with specific primers. Each gene expression was quantified relative to GAPDH and the shift was calculated by dividing the polysome/subpolysome ratio in DMEM+FCS by the polysome/subpolysome ratio in DMEM.

The transcriptional response to serum withdrawal was also analyzed in WT, Tsc1–/–, and Tsc2–/– MEFs using the same Agilent 22k microarrays. Although 84 genes were found to be transcriptionally downregulated (>2-fold) upon serum depletion in WT MEFs, only 7 genes overlapped with our list of 175 mRNAs translationally regulated by serum (data not shown). We also analyzed the total mRNA levels of the 19 genes validated in the TaqMan analysis shown in Fig. 5. Overall, the transcriptional profile of these genes was not dramatically affected by serum deprivation, although two genes were downregulated >2-fold (c-Jun and Rpl36a), and one gene was upregulated >2-fold (p57Kip1) (see Fig. S4 in the supplemental material).

Rapamycin inhibits mTORC1 signaling in WT, Tsc1–/–, and Tsc2–/– MEFs. We next investigated whether inhibition of mTORC1 activity, using the mTORC1 specific inhibitor rapamycin, would affect the same mRNAs as regulated by serum (described above). We performed a similar analysis on WT, Tsc1–/–, and Tsc2–/– MEFs cultured in presence of 50 nM rapamycin for 4 h. As shown in Fig. 6A, rapamycin caused inactivation of mTORC1, as judged by dephosphorylation of S6 at Ser235/236 and Ser240/244 and by the appearance of faster-migrating species corresponding to the hypophosphorylated 4E-BP1.


Figure 6
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  		FIG. 6. Rapamycin decreases mTORC1 signaling and polysome formation. (A) WT, Tsc1–/–, and Tsc2–/– MEFs were grown in DMEM+FCS, and 4 h prior to harvesting the cells were transferred into appropriate media as indicated. When indicated, rapamycin was added at the same time as the media transfer. Proteins were separated by SDS-PAGE and identified by Western blotting with specific antibodies. (B) In parallel experiments, cell lysates were prepared and layered on top of a 5 to 56% (wt/wt) sucrose gradient as described in the legend for Fig. 3. This graph represents the mean of three independent experiments ± the SD.

Since mTORC1 signaling was maintained in Tsc1–/– and Tsc2–/– MEFs upon serum starvation and showed comparable activity to WT MEFs in the presence of serum, we compared the effects of rapamycin on polysomal profiles and microarray analysis under these same conditions to identify specific mRNAs translationally regulated by rapamycin. Rapamycin had a minor effect on polysome distribution in WT MEFs cultured in the presence of FCS (Fig. 6B; also see Fig. S5 in the supplemental material). However, rapamycin caused a more significant decrease in the amount of polysome-associated mRNAs in both Tsc1–/– and Tsc2–/– MEFs (fractions 5 to 7 in Fig. 6B). This decrease was also accompanied by a significant increase of the amount of subpolysome-associated RNAs (fractions 9 and 10 in Fig. 6B). We purified polysome-associated mRNAs and subpolysome-associated mRNAs from sucrose gradients and hybridized them to Agilent 22k mouse 60-mer oligonucleotide arrays using a dye reversal as a control (as described in the previous analysis). Figure 7A illustrates that 113 mRNAs showed a >2-fold association with polysomes in WT MEFs in DMEM+FCS compared to treatment with rapamycin. Although the majority of mRNAs repressed by rapamycin were also repressed after serum withdrawal (73 of 113), there were also many mRNAs regulated only by rapamycin but not by serum (40 of 113) (Fig. 7B). Conversely, there were also many mRNAs regulated by serum but not by rapamycin (102 mRNAs found) in WT MEFs, suggesting that these are regulated in an mTORC1-independent manner (Fig. 7B). A total of 86% (88 of 102) of these serum-sensitive and rapamycin-insensitive mRNAs in the WT MEFs are not serum regulated in the Tsc1- and Tsc2-null cells. For these mRNAs, the lack of polysomal redistribution in response to serum withdrawal could either be due to the fact that the mRNA is constitutively associated with polysomes or constitutively associated with subpolysomes. Thirty-three of these eighty-eight mRNAs are constitutively polysome-associated in the Tsc1–/– and Tsc2–/– MEFs in absence of serum (indicated as "H" in Table S1 in the supplemental material). In contrast, 85% (62 of 73) of the serum- and rapamycin-sensitive mRNAs are constitutively polysome associated in the Tsc1- and Tsc2-null cells in the absence of serum (indicated as "H" in Table S2 in the supplemental material). Of the 33 constitutively polysome-associated mRNAs in the Tsc1–/– and Tsc2–/– MEFs, only 7 were resistant to rapamycin in both WT and null MEFs (indicated by an asterisk in Table S1 in the supplemental material), suggesting that only a small number of mRNAs are translated in a TSC1- and TSC2-dependent but rapamycin-independent manner.


Figure 7
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  		FIG. 7. Rapamycin represses translation of specific mRNAs in WT MEFs. (A) The left-hand scatter plot is the same scatter plot shown in Fig. 4C. The right-hand plot shows the polysome/subpolysome ratio of rapamycin-treated WT MEFs, log2[P(DMEM+FCS+Rap)/SP(DMEM+FCS+Rap)], plotted against the polysome/subpolysome ratio for the WT MEFs growing in the presence of 10% FCS, log2[P(DMEM+FCS)/SP(DMEM+FCS)], for every mRNA. Green lines represent a twofold cutoff as shown in Fig. 2. (B) Comparison of mRNAs translationally regulated by serum (red and yellow) and mRNAs translationally regulated by rapamycin (green and yellow) in WT MEFs. The overlap between the serum-sensitive mRNAs and the rapamycin-sensitive mRNAs is displayed in yellow. The number of mRNAs regulated in each condition is indicated in each circle. (C) Representation of k means cluster analysis of 498 genes regulated by rapamycin and serum in WT, Tsc1–/–, and Tsc2–/– MEFs. Genes are represented vertically, and experimental conditions are displayed horizontally. Blue indicates mRNAs that do not shift upon treatment, whereas red indicates mRNAs that shift upon either serum or rapamycin treatment. D, DMEM; D+F, DMEM + 10%FCS; D+Rap, DMEM+rapamycin; D+F+Rap, DMEM+FCS+rapamycin. On the right of the tree, the "Rap +S" group represents mRNAs regulated by both rapamycin and serum, whereas the "S" group represents mRNAs regulated by serum only. (D) Global comparison of mRNAs translationally repressed by rapamycin in WT MEFs (113 mRNAs) cultured in DMEM+FCS and in Tsc1–/– (341 mRNAs) and Tsc2–/– (219 mRNAs) MEFs cultured in DMEM alone. The number of mRNAs regulated in each condition is indicated in each circle. (E) Gene ontology analysis of the mRNAs repressed by both rapamycin and serum in WT MEFs in a TSC1- and TSC2-dependent manner.

Rapamycin represses the translation of specific mRNAs. To examine whether a specific translational signature would emerge across the different conditions, we performed hierarchical clustering on all genes regulated by serum and/or rapamycin in all three MEFs. The cluster algorithm separates the experimental conditions into two distinct groups that tightly correlated with the mTORC1 activity status of these samples (Fig. 7C). One cluster (cluster L) is represented by a set of mRNAs affected by low mTORC1 activity (see the figure legend for details); all rapamycin-treated samples, as well as serum-starved WT MEFs, are grouped in this category. The other cluster (cluster H) is represented by high mTORC1 activity (Fig. 7C). Both Tsc2–/– and Tsc1–/– MEFs displayed very closely related translational profiles when cultured either in the presence or in the absence of FCS, whereas WT MEFs grown in DMEM+FCS defined a distinct group within the high mTORC1 activity cluster. A similar cluster pattern was observed when all filtered mRNAs from the gene list present in the microarray were used to perform this analysis (data not shown).

In addition, microarray analysis of the Tsc1–/– and Tsc2–/– MEFs cultured in DMEM (in the absence of FCS) showed that 341 and 219 mRNAs, respectively, shift >2-fold to the subpolysome fractions in response to rapamycin treatment. The Venn diagram illustrated in Fig. 7D shows that most of the 113 mRNAs suppressed by rapamycin in WT MEFs were also regulated in Tsc1–/– and Tsc2–/– MEFs. Although many mRNAs were coregulated by rapamycin across the three different MEFs, there were also mRNAs that appear to be regulated uniquely in cells in one or two genotypes. Of the 76 mRNAs regulated by rapamycin in all three cell types, 41 encode for RPs, and 9 encode for translation initiation or elongation factors, therefore enriching the class of genes involved in ribosome biogenesis to 66% in this group (Fig. 7E). Tables S2 and S3 in the supplemental material display the identity of mRNAs translationally repressed by rapamycin in WT, Tsc1–/–, and Tsc2–/– MEFs. Moreover, we showed that there are more mRNAs that shift from polysome to subpolysome fractions in response to rapamycin in the serum-starved Tsc-null MEFs than in the serum-fed WT MEFs (Fig. 6B and 7D). Therefore, we believe that the overlap between the Tsc1–/– and Tsc2–/– MEFs in this group (Fig. 7D) represents a group of high confidence mTORC1-specific translational targets since these are not complicated by serum effects. The identities of the additional 82 genes are provided in Table S4 in the supplemental material.

We confirmed our microarray data using TaqMan analysis as shown in Fig. 8A; see also Fig. S6 in the supplemental material. We found that the correlation between the two analyses was generally consistent except for a few mRNAs such as Rpl4, Arid5b, and Ddx6. Although the regulation by rapamycin measured by TaqMan was often weaker than the regulation observed by microarray, the general trend was similar, with some mRNAs being regulated only by serum and not by rapamycin, some being regulated by rapamycin and not serum, and some being regulated by both.


Figure 8
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  		FIG. 8. Validation by TaqMan and Western blot in WT MEFs. (A) Change of the polysome/subpolysome ratio of WT MEFs when cells were cultured in DMEM+FCS compared to DMEM alone or DMEM+FCS+rapamycin. WT cells were grown in DMEM + 10% FCS in the presence or in the absence of rapamycin (50 nM) for 4 h or in absence of serum for 4 h. RNA was isolated from 5 to 56% (wt/wt) sucrose gradients and purified. Each polysome and subpolysome fraction was separately pooled and amplified by qRT-PCR with specific primers. Each gene expression was quantified relative to GAPDH, and the shift was calculated by dividing the polysome/subpolysome ratio in DMEM+FCS by the polysome/subpolysome ratio in DMEM or by the polysome/subpolysome ratio in DMEM+FCS+rapamycin. (B) Western blot analysis of proteins translationally regulated by serum. WT, Tsc1–/–, and Tsc2–/– MEFs were serum starved overnight and then stimulated with 10% FCS or 10% FCS+Rap (50 nM) for the indicated time. Cell lysates were prepared separated by SDS-PAGE and blotted with specific antibodies.

Since we have identified a subset of mRNAs that were redistributed from the polysome to the subpolysome upon serum starvation or upon rapamycin treatment, we assessed whether such translational regulation would be detectable at the protein level. To address this, we examined protein expression by SDS-PAGE with specific antibodies based on their availability. Four proteins were tested and analyzed by Western blotting. To minimize effects of protein stability confounding our analyses, we starved cells of serum overnight and restimulated them with 10% FCS in the absence or presence of rapamycin at the indicated times (Fig. 8B; see also Fig. S7 in the supplemental material). Consistent with the microarray results, steady-state levels of eIF4B, p57Kip2, c-Jun, and Tpt1 were all increased by FCS in WT MEFs. eIF4B, p57Kip2, and Tpt1 expression required 4 h of serum stimulation to show increases in protein levels, and this increase was prevented by rapamycin. These proteins do not show increased levels in response to serum in Tsc1–/– and Tsc2–/– MEFs, but their expression is still decreased in response to rapamycin treatment, a finding consistent with the polysome analysis. Longer treatment (8 to 24 h) with rapamycin is required to visualize decreased protein levels, possibly due to the long half-lives of these proteins. c-Jun was regulated differently in that its expression was increased earlier after serum stimulation in WT, Tsc1–/–, and Tsc2–/– MEFs and was not inhibited by rapamycin in WT MEFs. c-Jun mRNA was also transcriptionally regulated by serum in the MEFs of all three genotypes (see Fig. S4 in the supplemental material), indicating that transcriptional regulation appears to be dominant over translational regulation for this protein. Conversely, p57kip2 mRNA levels were actually increased upon serum withdrawal, and yet the p57kip2 protein levels increased upon serum stimulation in a rapamycin-dependent manner, showing that in this case translation is dominant over transcription.

Re-expression of TSC1 or TSC2 in Tsc-deficient cells restores mTORC1 regulation by serum and the translation of specific mRNAs. Because we used nonisogenic Tsc-deficient cells in our study, we were concerned that the effects we observed were independent of their genetic status. To address this issue, we reintroduced WT TSC1 into the Tsc1–/– MEFs and WT TSC2 into the Tsc2–/– MEFs using retroviral expression. TSC1 deficiency reduces the steady-state levels of TSC2 (10, 78), and this was rescued upon re-expression of TSC1 (Fig. 9A). Restoration of TSC1 and TSC2 expression into TSC-deficient cells also resensitized mTORC1 activity to serum withdrawal, suggesting that most of the serum effects signal through TSC1/TSC2 in our model system. Upstream signaling to mTORC1 was barely affected in the reconstituted cells, as shown by the similar effect of serum depletion on the phosphorylation of PRAS40, a PKB target known to be regulated by growth factors (37, 60). Consistent with the restoration of mTORC1 regulation by serum, the global polysomal profile of these revertant cells displayed redistribution from polysomes to subpolysomes upon serum depletion to an extent similar to that of WT MEFs (Fig. 9B). We also performed TaqMan analysis on the mRNAs previously identified as being TSC dependent in the Tsc (Vector) and Tsc (Rev) cells (Fig. 9C). Most of the mRNAs tested displayed a significant redistribution from polysomes to subpolysomes upon serum starvation in revertant cells (Rev) compared to the control cells (Vector), suggesting that these mRNAs are regulated by serum in a TSC1/TSC2-dependent manner.


Figure 9
Figure 9
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  		FIG. 9. Serum regulates mTORC1 activity and the translation of specific mRNAs in a TSC1- and TSC2-dependent manner. (A) Cells of the indicated genotype were serum starved for 4 h, and cell lysates were subjected to immunoblot analysis with the indicated antibodies. (B) In parallel experiments, cell lysates were prepared and layered on top of a 5 to 56% (wt/wt) sucrose gradient, as described in the legend for Fig. 3C. The results are representative of two independent experiments. (C) Reexpression of either Tsc1 or Tsc2 in null MEFs restores the translation of specific mRNAs by serum. The experimental details were the same as for Fig. 8A.

Serum regulates ribosome biogenesis and a subset of TOP mRNAs in a TSC1- and TSC2-dependent manner. Translation efficiency is determined at the posttranscriptional level, and this regulation is mostly mediated by cis-acting elements located in the 5'- and 3'UTRs (52). Table 1 shows that serum starvation in WT MEFs strongly affected mRNAs encoding RPs, suggesting that a common motif in their 5'UTRs may be functionally relevant. A common feature of RNAs encoding components of the translational machinery is the presence in their 5'UTRs of a TOP motif. This motif is currently known to be associated with mTORC1-dependent translation regulation (25). However, there are mRNAs regulated by mTORC1 that do not contain TOP motifs (48, 76), and also mRNAs containing this motif that are not regulated by mTORC1 (17). Therefore, to examine whether the 175 serum-regulated mRNAs contained a TOP motif or a specific functional sequence within their 5'UTRs, we retrieved from current databases all 5'UTR sequences, when available, of these mRNAs. The structural feature of 5'TOP motifs is defined as initiating with a C residue followed by a stretch of 4 to 14 uninterrupted pyrimidine residues. In our study, although 33% of the serum-translationally regulated mRNAs were TOP containing mRNAs, the majority do not contain this motif, suggesting that motifs other than TOP are important for translational regulation of these mRNAs (Fig. 10A, top left pie chart). In comparison, the frequency of TOP motifs in mRNAs that are not regulated by mTORC1 was 10% (data not shown). Rapamycin-sensitive mRNAs consisted of 47% TOP mRNAs, but the highest proportion of 5'TOP mRNAs (55%) were those regulated by both serum and rapamycin (Fig. 10A, bottom middle pie chart). Among the 102 mRNAs regulated only by serum and not by rapamycin (Fig. 10A, bottom left pie chart), only 18% were TOP mRNAs, and the mRNAs regulated by rapamycin only and not by serum (40 mRNAs) showed a slight enrichment for TOP motifs (33%). Altogether, this suggests that 5'TOP motifs are greatly enriched in the group of mRNAs that are regulated by both growth factors and mTORC1. However, it also suggests that non-TOP containing mRNAs are regulated by TSC1 and TSC2 but in a mTORC1-independent manner. Interestingly, we also found that the mRNAs regulated by both serum and rapamycin contain a short 5'UTR and 3'UTR (medians of 41 and 64 nucleotides, respectively), whereas mRNAs regulated by either serum alone or rapamycin alone have a longer 5'UTR (medians of 119 and 100, respectively) and 3'UTR (medians of 477 and 531, respectively) (Fig. 10B). These 5' and 3'UTR lengths are similar to those of 160 randomly chosen nonregulated mRNAs (98 and 755 nucleotides, respectively). Altogether, this suggests that this is relevant for the function of these mRNAs and might be implicated in growth-associated translational regulation.


Figure 10
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  		FIG. 10. Comparison of the 5'UTRs. For each mRNAs found regulated by serum or rapamycin, 5'UTRs were retrieved from database resources and analyzed for the presence of a 5'TOP motif. The serum-regulated mRNAs and rapamycin-regulated mRNAs are compared as shown in the middle panel with the Venn diagram. For each subclass displayed on the Venn diagram, 5'TOP motifs were quantified and reported in the pie chart. (B) Analysis of 5'UTR and 3'UTR lengths. UTR sequences were obtained from UC Santa Cruz genome browser (www.genome.ucsc.edu). The average {Delta}G values for each UTR were obtained by inputting each sequence into the secondary structure prediction program mfold and averaging the {Delta}G values of the six most favorable structures computed.


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DISCUSSION
 
In this study, we identify mRNAs whose translation is regulated by growth factor signal transduction pathways acting through the TSC1/TSC2 complex and mTORC1 by using polysome profiling and microarray analysis. In agreement with previous reports (26, 30, 38, 45, 64, 68), we found that mTORC1 regulation by growth factors requires an intact TSC1/TSC2 complex, since cells deficient for Tsc1 or Tsc2 fail to downregulate mTORC1 function in response to serum deprivation, and rescue experiments restore mTORC1 sensitivity to serum withdrawal. Both growth factor and nutrient inputs are necessary for the full activation of mTORC1 in WT MEFs, whereas in MEFs lacking either Tsc1 or Tsc2 either input maintains partial or full mTORC1 activity. We observed a strong correlation between the changes in mTORC1 signaling and the overall RNA polysome association, as seen by the decreases in RNA polysome association upon serum depletion in WT MEFs and the constitutive polysome association in the Tsc-null MEFs. Almost all mRNAs (90%) found associated with polysomes in response to serum depend on an intact TSC1/TSC2 complex, suggesting a critical role of TSC proteins in serum-mediated protein translation. However, TSC1 and TSC2 may also play a role independently of each other since some mRNAs appear to be serum sensitive in either a TSC2-dependent or a TSC1-dependent manner (Fig. 4D; also see Table S5 in the supplemental material). If confirmed, these findings might contribute to the differences in tuberous sclerosis phenotypes seen in patients expressing mutated Tsc1 versus Tsc2 (12). An alternative explanation could be that some of the mRNAs differentially regulated between the TSC1-null and the TSC2-null MEFs could be p53 dependent, since these cells differ in their p53 status (78).

Clues as to the importance of TOR proteins in the regulation of protein translation came initially from studies in yeast; treatment of Saccharomyces cerevisiae infection with rapamycin causes a rapid and dramatic decrease in global translation (9, 58). In contrast, when rapamycin is added to mammalian cells, a more complex effect on translation is observed. Global translation rates are decreased to a small extent, in a somewhat cell type-specific manner (3, 50), whereas the translation of specific mRNAs is reduced much more dramatically. In our study using WT MEFs cultured in the presence of DMEM+FCS, 1.6% mRNAs showed a strong translational repression (>2-fold) after the addition of rapamycin. This finding is very similar to that of a study showing 2.2% of mRNAs in Jurkat T cells translationally regulated by rapamycin (17). Many of these were RPs and other components of the translational apparatus. Consistent with this, 66% of rapamycin-regulated mRNAs in our analysis consisted of the translation apparatus, suggesting that a major function of mTORC1 is to prepare cells for increased protein translation. In contrast, RPs, initiation and elongation factors, and RNA processing-related proteins only represented 56 of 175 (33%) of the serum-regulated mRNAs. Interestingly, we also observed that rapamycin increased the translation of some mRNAs (Fig. 7A), suggesting that mTORC1 activity may also translationally repress a subset of mRNAs in MEFs. This phenomenon has also been previously noted, with increased translation of mos and Cdc25 seen upon treatment of Xenopus oocytes with rapamycin (63). However, the mechanism and significance of this observation remains speculative. Surprisingly, we also noticed that rapamycin treatment caused global polysomal redistribution in Tsc1–/– and Tsc2–/– MEFs but not in WT MEFs. Therefore, the overlap of the rapamycin-regulated mRNAs between the Tsc1–/– and Tsc2–/– MEFs (Fig. 7D; also see Fig. S4 in the supplemental material) represents a group of high confidence mTORC1-specific translational targets, since these are not complicated by serum effects. Previous studies reported that dysregulation of signaling pathways leading to activation of mTOR sensitizes cells to antiproliferative effects of rapamycin in vivo (47, 53), possibly due to the phenomenon referred to as "oncogene addiction." Based on this observation, one might predict that TSC-defective cells would likewise be particularly sensitive to rapamycin.

Consistent with the notion of the "negative feedback loop" mediated by S6K (21, 65, 78), we also observed that both Tsc1–/– and Tsc2–/– MEFs have reduced PKB/Akt activity. This feedback loop might contribute to the lack of effect of serum on polysomal redistribution of mRNAs in the Tsc–/– MEFs. However, we still observed an inhibitory effect on PKB/Akt phosphorylation and its substrate PRAS40 upon serum starvation, suggesting that the effects observed in our study are at least partially caused by PI3K signaling in these cells. Interestingly, two recent studies showed that unphosphorylated PRAS40 binds and inhibits mTORC1, which is relieved upon phosphorylation by PKB/Akt (60, 72). However, in the absence of TSC1/TSC2, elevated RhebGTP likely overcomes this inhibitory effect (60; our observations). We also cannot rule out the possibility that other pathways, such as ERK-p90RSK signaling, play a role in a serum-regulated translation via TSC1/TSC2 (41) or in an mTORC1-independent manner (55). A number of mechanisms have been proposed to account for growth factor-independent regulation of mTORC1 activity by nutrients. These include signaling through AMP-dependent protein kinase (regulated by cellular AMP levels) (27), REDD1 (in response to hypoxia) (4), and class III PI3Ks (for sensing the presence of amino acids) (6, 49). It is tempting to speculate that one or more of these nutrient inputs could account for the regulation of the ~40 serum-independent but mTORC1-dependent mRNAs discovered in our screen. Although the majority of mRNAs repressed by rapamycin were also repressed after serum withdrawal (73 of 113 [72 of which are TSC1 and TSC2 dependent]), there are many mRNAs (102 of 175) that are regulated by serum but not by rapamycin. As detailed in Results and in Table S1 in the supplemental material, only 7 of these 102 mRNAs were constitutively associated with polysomes in the presence or absence of serum in cells lacking TSC1 or TSC2 and resistant to rapamycin in cells of all genotypes. These mRNAs are likely regulated in a TSC-dependent but mTORC1-independent manner.

The mechanism(s) accounting for the serum-dependent, TSC1/TSC2-dependent, but rapamycin-independent mRNAs is currently not clear. Interestingly, several studies have identified an mTORC1-independent function of TSC in mammalian cells (5, 33, 34, 59). Whether these processes mediate the mTORC1-independent regulation of translation by TSC is not known. One possibility is that TSC1/TSC2 signals to the second mTOR complex, mTORC2 (containing Rictor/mAVO3), which is rapamycin insensitive (61, 62). Consistent with this hypothesis, it was shown that Rheb might mediate signals to the actin cytoskeleton via mTORC2 in a rapamycin-insensitive manner (15, 29). Determining whether global or specific mRNA translation is compromised in cells lacking Rictor/mAVO3 would help to address this issue.

Most mammalian RP mRNAs harbor a TOP motif in their 5'UTRs that mediates translational control in response to appropriate growth conditions. Individual 5'TOP-containing mRNAs, e.g., eEF1A, eEF2, S6, rpL32, and rpS19, have been shown to be translationally inhibited by rapamycin (7, 32, 80). In the present study, these mRNAs, together with 37 others, were inhibited by rapamycin in all three MEFs, giving a representation of 55% 5'TOP mRNAs in this group of 76 mRNAs. We also found that the 5' and 3'UTRs of mRNAs regulated by both serum and rapamycin contain a shorter sequence compared to the other groups. This striking structural feature may be relevant for the function of these mRNAs. Based on a closed loop model, one can speculate that these mRNAs get recruited more rapidly, leading to an increase of ribosomal proteins under appropriate conditions. It is also clear from our studies that the presence of a 5'TOP motif does not de facto stipulate serum- or rapamycin-induced regulation, since ca. 10% of these nonregulated mRNAs examined were found to contain 5'TOP motifs, at least as defined by the National Center for Biotechnology Information and UC Santa Cruz databases. Importantly, we cannot exclude that the translational regulation of TOP mRNAs may also be dependent on their 3'UTRs, as was recently suggested (40). This offers an attractive model in which miRNAs could also play a role since these noncoding RNAs bind to the target 3'UTRs and function as translational repressors (22).

It is still unclear by which mechanism mTORC1 regulates TOP-containing mRNAs. Until recently, it was thought that the recruitment of TOP-containing mRNAs into polysomes was the result of activation of S6K1 and subsequent phosphorylation of S6. However, recent studies suggest that TOP regulation does not depend on S6K or S6 phosphorylation (2, 51, 56, 69). Nevertheless, in cells lacking PDK1, which have no S6K activity, the translation of certain mRNAs was compromised, which could be restored by expressing an activated form of PKB/Akt (70). It would be informative to address the role of S6K in translational regulation directly by analyzing the polysome profiles of S6K1/2 knockout cells.

Moreover, it is thought that eIF4E stimulates, as a consequence of mTORC1 activation, a subset of mRNAs whose 5'UTRs contain an extensive secondary structure, such as cyclin D1, FGF2, VEGF, HIF-1{alpha}, ODC, and c-myc (36, 43). It is not clear why these targets were not identified as mTORC1 regulated in our analysis. However, two recent studies performing a translational profiling to identify eIF4E targets also failed to identify c-myc, cyclin D1, or HIF-1{alpha} (39, 42). Several factors—such as the use of different microarray platforms, experimental design, statistical analyses, and different time points—may explain these discrepancies.

As previously discussed, some of the regulated mRNAs found in the present study have functions, in addition to ribosome biogenesis, in translation initiation. A striking observation was that components of the preinitiation complex (five subunits of eIF3), S6K substrates (eIF4B and S6), and alpha-4 protein were all translationally repressed in response to serum starvation, emphasizing the critical role of translation initiation in regulating protein synthesis. We also found that Tpt1 (for tumor protein, translationally controlled 1) was repressed by serum starvation and rapamycin treatment in a TSC1- and TSC2-dependent manner. TPT1 was initially identified as a serum-inducible mRNA. TPT1 acts as a guanine nucleotide dissociation inhibitor on the translation elongation factor eEF-1A, therefore implicating TPT1 in an elongation step of protein translation (8). Another interesting target we found in our analysis is p57kip2: p57kip2 mRNA levels were transcriptionally increased upon serum withdrawal, and yet p57kip2 protein levels increase upon serum stimulation in a rapamycin-dependent manner. This suggests that for this protein, translational regulation is dominant over transcriptional regulation. In addition, increased expression of p57kip2 might contribute to the premature senescence seen in Tsc2–/– MEFs (78).

The present study therefore increases our understanding of how signal transduction pathways impact on cis-acting regulatory sequences to control protein translation in response to changes in the extracellular environment. Since tuberous sclerosis is a disease likely caused (at least in part) by increased translation of particular mTOR-dependent mRNAs, the identification of these is expected to provide new therapeutic avenues for this disease. Future experiments will be required to dissect the role of individual targets played in the biological processes induced by growth factors and nutrients, especially during cancer development. Since signaling pathways stimulated by these stimuli have been strongly implicated in sporadic human tumorigenesis, the proteins that are translated in response to this likely play a role in malignant progression and could be novel therapeutic targets. Although mTORC1 can currently be inhibited by rapamycin, these agents will be ineffective in tumors showing deregulation downstream of this protein, such as the overexpression of eIF4G and S6K seen in breast cancer (1). The appreciation that mTORC1 is directly inhibited by TSC1/TSC2 has led to the rapid application of rapamycin for the treatment of tuberous sclerosis. However, certain TSC patients are resistant to such treatment; therefore, the identification of the rapamycin-insensitive mRNAs found in the present study may provide additional therapeutic targets to overcome this problem.


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ACKNOWLEDGMENTS
 
We thank David Kwiatkowski for kindly providing WT, Tsc1-null, and Tsc2-null MEFs and TSC2 reconstituted Tsc2-null MEFs. We are grateful to the Genome Analysis Core for TaqMan analysis. We thank Mike Fried, Pablo Rodriguez-Viciana, Tanja Tamgüney, Christina Spevak, and Clodagh O'Shea for critical and helpful comments on the manuscript.

This study was supported by Grants from the U.S. Department of Defense Tuberous Sclerosis Research Program (TS030017 and TS050054).


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FOOTNOTES
 
* Corresponding author. Mailing address: 2340 Sutter Street, San Francisco, CA 94115. Phone: (415) 502-2598. Fax: (415) 502-3179. E-mail: dstokoe{at}cc.ucsf.edu Back

{triangledown} Published ahead of print on 11 June 2007. Back

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/. Back


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Molecular and Cellular Biology, August 2007, p. 5746-5764, Vol. 27, No. 16
0270-7306/07/$08.00+0     doi:10.1128/MCB.02136-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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