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Reassessment of the role of TSC, mTORC1 and microRNAs in amino acids-meditated translational control of TOP mRNAs.

Patursky-Polischuk I, Kasir J, Miloslavski R, Hayouka Z, Hausner-Hanochi M, Stolovich-Rain M, Tsukerman P, Biton M, Mudhasani R, Jones SN, Meyuhas O - PLoS ONE (2014)

Bottom Line: However, we show here that titration of this microRNA failed to downregulate the basal translation efficiency of TOP mRNAs.Moreover, Drosha knockdown or Dicer knockout, which carries out the first and second processing steps in microRNAs biosynthesis, respectively, failed to block the translational activation of TOP mRNAs by amino acid or serum stimulation.Evidently, these results are questioning the positive role of microRNAs in this mode of regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Institute for Medical Research - Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.

ABSTRACT
TOP mRNAs encode components of the translational apparatus, and repression of their translation comprises one mechanism, by which cells encountering amino acid deprivation downregulate the biosynthesis of the protein synthesis machinery. This mode of regulation involves TSC as knockout of TSC1 or TSC2 rescued TOP mRNAs translation in amino acid-starved cells. The involvement of mTOR in translational control of TOP mRNAs is demonstrated by the ability of constitutively active mTOR to relieve the translational repression of TOP mRNA upon amino acid deprivation. Consistently, knockdown of this kinase as well as its inhibition by pharmacological means blocked amino acid-induced translational activation of these mRNAs. The signaling of amino acids to TOP mRNAs involves RagB, as overexpression of active RagB derepressed the translation of these mRNAs in amino acid-starved cells. Nonetheless, knockdown of raptor or rictor failed to suppress translational activation of TOP mRNAs by amino acids, suggesting that mTORC1 or mTORC2 plays a minor, if any, role in this mode of regulation. Finally, miR10a has previously been suggested to positively regulate the translation of TOP mRNAs. However, we show here that titration of this microRNA failed to downregulate the basal translation efficiency of TOP mRNAs. Moreover, Drosha knockdown or Dicer knockout, which carries out the first and second processing steps in microRNAs biosynthesis, respectively, failed to block the translational activation of TOP mRNAs by amino acid or serum stimulation. Evidently, these results are questioning the positive role of microRNAs in this mode of regulation.

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mTOR mediates amino acid-induced translational activation of TOP mRNAs.(A) Kinetics of the effect of rapamycin on mTORC1 activity. 293 cells were amino acid-starved for 2 h and then refed for the indicated time in the presence or absence of 20 nM rapamycin, after which cells were harvested. The cytoplasmic proteins were subjected to Western blot analysis with anti-rpS6 or anti-Phospho-rpS6 antibodies. The chemiluminescent signals of phospho rpS6 were quantified and normalized to those obtained for rpS6 within the same protein extract. The results are numerically presented relative to those obtained for amino acid-starved cells (time zero), which were arbitrarily set at 1. (B) Kinetics of the effect of rapamycin on polysomal association of TOP mRNAs. HEK293 cells were amino acid-starved for 3 h (time zero), and then refed in the absence (open symbols) or presence (filled symbols) of 20 nM rapamycin (rapa). At the indicated times cells were harvested and cytoplasmic extracts were subjected to polysomal analysis. The percentage of mRNA in polysomes at each time point is presented as an average of at least 2 measurements. (C) HEK293 cells were infected with viruses expressing HcRed (Red) shRNA or mTOR shRNA1. Cells were amino acid-starved for 3 h followed by 3 h amino acid stimulation on day 4 post-infection. The abundance of mTOR and its activity were monitored by Western blot analysis of cytoplasmic proteins with the indicated antibodies. (D) Cytoplasmic extracts from cells described in (C) were subjected to polysomal analysis. (E) and (F) HEK293 were transiently transfected with plasmid-based vectors expressing either wild-type (WT) mTOR or enhanced (En) mTOR. 48 h later cells were amino acid-starved for 3 h and harvested. Cytoplasmic proteins were subject to Western blot analysis (E) and cytoplasmic extracts to polysomal analysis (F). The percentage of mRNA in polysomes is presented as an average ± SEM of three experiments.
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pone-0109410-g002: mTOR mediates amino acid-induced translational activation of TOP mRNAs.(A) Kinetics of the effect of rapamycin on mTORC1 activity. 293 cells were amino acid-starved for 2 h and then refed for the indicated time in the presence or absence of 20 nM rapamycin, after which cells were harvested. The cytoplasmic proteins were subjected to Western blot analysis with anti-rpS6 or anti-Phospho-rpS6 antibodies. The chemiluminescent signals of phospho rpS6 were quantified and normalized to those obtained for rpS6 within the same protein extract. The results are numerically presented relative to those obtained for amino acid-starved cells (time zero), which were arbitrarily set at 1. (B) Kinetics of the effect of rapamycin on polysomal association of TOP mRNAs. HEK293 cells were amino acid-starved for 3 h (time zero), and then refed in the absence (open symbols) or presence (filled symbols) of 20 nM rapamycin (rapa). At the indicated times cells were harvested and cytoplasmic extracts were subjected to polysomal analysis. The percentage of mRNA in polysomes at each time point is presented as an average of at least 2 measurements. (C) HEK293 cells were infected with viruses expressing HcRed (Red) shRNA or mTOR shRNA1. Cells were amino acid-starved for 3 h followed by 3 h amino acid stimulation on day 4 post-infection. The abundance of mTOR and its activity were monitored by Western blot analysis of cytoplasmic proteins with the indicated antibodies. (D) Cytoplasmic extracts from cells described in (C) were subjected to polysomal analysis. (E) and (F) HEK293 were transiently transfected with plasmid-based vectors expressing either wild-type (WT) mTOR or enhanced (En) mTOR. 48 h later cells were amino acid-starved for 3 h and harvested. Cytoplasmic proteins were subject to Western blot analysis (E) and cytoplasmic extracts to polysomal analysis (F). The percentage of mRNA in polysomes is presented as an average ± SEM of three experiments.

Mentions: Rapamycin is a widely used tool for establishing the role of mTOR in many biological processes. However, while this drug inhibited mTORC1 activity with a half-time of about 2 min (Fig. 2A and [45]), it repressed the translation of rpL32 mRNA much more slowly, reaching its maximal effect after 2 h (Fig. 2B). This and previous conflicting reports on the translational repression of TOP mRNAs by rapamycin [2], [18], [20], prompted us to verify the role of mTOR in signaling toward these mRNAs. mTOR knockdown, using lentivirus expressing mTOR shRNA, resulted in downregulation of both mTORC1 activity, as can be judged by the phosphorylation status of S6K and translational activation of rpL32 mRNA upon amino acid stimulation (Figs. 2C and 2D).


Reassessment of the role of TSC, mTORC1 and microRNAs in amino acids-meditated translational control of TOP mRNAs.

Patursky-Polischuk I, Kasir J, Miloslavski R, Hayouka Z, Hausner-Hanochi M, Stolovich-Rain M, Tsukerman P, Biton M, Mudhasani R, Jones SN, Meyuhas O - PLoS ONE (2014)

mTOR mediates amino acid-induced translational activation of TOP mRNAs.(A) Kinetics of the effect of rapamycin on mTORC1 activity. 293 cells were amino acid-starved for 2 h and then refed for the indicated time in the presence or absence of 20 nM rapamycin, after which cells were harvested. The cytoplasmic proteins were subjected to Western blot analysis with anti-rpS6 or anti-Phospho-rpS6 antibodies. The chemiluminescent signals of phospho rpS6 were quantified and normalized to those obtained for rpS6 within the same protein extract. The results are numerically presented relative to those obtained for amino acid-starved cells (time zero), which were arbitrarily set at 1. (B) Kinetics of the effect of rapamycin on polysomal association of TOP mRNAs. HEK293 cells were amino acid-starved for 3 h (time zero), and then refed in the absence (open symbols) or presence (filled symbols) of 20 nM rapamycin (rapa). At the indicated times cells were harvested and cytoplasmic extracts were subjected to polysomal analysis. The percentage of mRNA in polysomes at each time point is presented as an average of at least 2 measurements. (C) HEK293 cells were infected with viruses expressing HcRed (Red) shRNA or mTOR shRNA1. Cells were amino acid-starved for 3 h followed by 3 h amino acid stimulation on day 4 post-infection. The abundance of mTOR and its activity were monitored by Western blot analysis of cytoplasmic proteins with the indicated antibodies. (D) Cytoplasmic extracts from cells described in (C) were subjected to polysomal analysis. (E) and (F) HEK293 were transiently transfected with plasmid-based vectors expressing either wild-type (WT) mTOR or enhanced (En) mTOR. 48 h later cells were amino acid-starved for 3 h and harvested. Cytoplasmic proteins were subject to Western blot analysis (E) and cytoplasmic extracts to polysomal analysis (F). The percentage of mRNA in polysomes is presented as an average ± SEM of three experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4206288&req=5

pone-0109410-g002: mTOR mediates amino acid-induced translational activation of TOP mRNAs.(A) Kinetics of the effect of rapamycin on mTORC1 activity. 293 cells were amino acid-starved for 2 h and then refed for the indicated time in the presence or absence of 20 nM rapamycin, after which cells were harvested. The cytoplasmic proteins were subjected to Western blot analysis with anti-rpS6 or anti-Phospho-rpS6 antibodies. The chemiluminescent signals of phospho rpS6 were quantified and normalized to those obtained for rpS6 within the same protein extract. The results are numerically presented relative to those obtained for amino acid-starved cells (time zero), which were arbitrarily set at 1. (B) Kinetics of the effect of rapamycin on polysomal association of TOP mRNAs. HEK293 cells were amino acid-starved for 3 h (time zero), and then refed in the absence (open symbols) or presence (filled symbols) of 20 nM rapamycin (rapa). At the indicated times cells were harvested and cytoplasmic extracts were subjected to polysomal analysis. The percentage of mRNA in polysomes at each time point is presented as an average of at least 2 measurements. (C) HEK293 cells were infected with viruses expressing HcRed (Red) shRNA or mTOR shRNA1. Cells were amino acid-starved for 3 h followed by 3 h amino acid stimulation on day 4 post-infection. The abundance of mTOR and its activity were monitored by Western blot analysis of cytoplasmic proteins with the indicated antibodies. (D) Cytoplasmic extracts from cells described in (C) were subjected to polysomal analysis. (E) and (F) HEK293 were transiently transfected with plasmid-based vectors expressing either wild-type (WT) mTOR or enhanced (En) mTOR. 48 h later cells were amino acid-starved for 3 h and harvested. Cytoplasmic proteins were subject to Western blot analysis (E) and cytoplasmic extracts to polysomal analysis (F). The percentage of mRNA in polysomes is presented as an average ± SEM of three experiments.
Mentions: Rapamycin is a widely used tool for establishing the role of mTOR in many biological processes. However, while this drug inhibited mTORC1 activity with a half-time of about 2 min (Fig. 2A and [45]), it repressed the translation of rpL32 mRNA much more slowly, reaching its maximal effect after 2 h (Fig. 2B). This and previous conflicting reports on the translational repression of TOP mRNAs by rapamycin [2], [18], [20], prompted us to verify the role of mTOR in signaling toward these mRNAs. mTOR knockdown, using lentivirus expressing mTOR shRNA, resulted in downregulation of both mTORC1 activity, as can be judged by the phosphorylation status of S6K and translational activation of rpL32 mRNA upon amino acid stimulation (Figs. 2C and 2D).

Bottom Line: However, we show here that titration of this microRNA failed to downregulate the basal translation efficiency of TOP mRNAs.Moreover, Drosha knockdown or Dicer knockout, which carries out the first and second processing steps in microRNAs biosynthesis, respectively, failed to block the translational activation of TOP mRNAs by amino acid or serum stimulation.Evidently, these results are questioning the positive role of microRNAs in this mode of regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Institute for Medical Research - Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.

ABSTRACT
TOP mRNAs encode components of the translational apparatus, and repression of their translation comprises one mechanism, by which cells encountering amino acid deprivation downregulate the biosynthesis of the protein synthesis machinery. This mode of regulation involves TSC as knockout of TSC1 or TSC2 rescued TOP mRNAs translation in amino acid-starved cells. The involvement of mTOR in translational control of TOP mRNAs is demonstrated by the ability of constitutively active mTOR to relieve the translational repression of TOP mRNA upon amino acid deprivation. Consistently, knockdown of this kinase as well as its inhibition by pharmacological means blocked amino acid-induced translational activation of these mRNAs. The signaling of amino acids to TOP mRNAs involves RagB, as overexpression of active RagB derepressed the translation of these mRNAs in amino acid-starved cells. Nonetheless, knockdown of raptor or rictor failed to suppress translational activation of TOP mRNAs by amino acids, suggesting that mTORC1 or mTORC2 plays a minor, if any, role in this mode of regulation. Finally, miR10a has previously been suggested to positively regulate the translation of TOP mRNAs. However, we show here that titration of this microRNA failed to downregulate the basal translation efficiency of TOP mRNAs. Moreover, Drosha knockdown or Dicer knockout, which carries out the first and second processing steps in microRNAs biosynthesis, respectively, failed to block the translational activation of TOP mRNAs by amino acid or serum stimulation. Evidently, these results are questioning the positive role of microRNAs in this mode of regulation.

Show MeSH
Related in: MedlinePlus