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. 2007 Aug;27(16):5746-64.
doi: 10.1128/MCB.02136-06. Epub 2007 Jun 11.

Tuberous sclerosis complex proteins 1 and 2 control serum-dependent translation in a TOP-dependent and -independent manner

Benoit Bilanges  1 , Rhoda Argonza-Barrett, Marina Kolesnichenko, Christina Skinner, Manoj Nair, Michelle Chen, David Stokoe
Affiliations

Tuberous sclerosis complex proteins 1 and 2 control serum-dependent translation in a TOP-dependent and -independent manner

Benoit Bilanges et al. Mol Cell Biol. 2007 Aug.

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 approximately 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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Figures

FIG. 1.
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.
FIG. 2.
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.
FIG. 3.
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.
FIG. 4.
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.
FIG. 5.
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.
FIG. 6.
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.
FIG. 7.
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.
FIG. 8.
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.
FIG. 9.
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.
FIG. 9.
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.
FIG. 10.
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 ΔG values for each UTR were obtained by inputting each sequence into the secondary structure prediction program mfold and averaging the ΔG values of the six most favorable structures computed.
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