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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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The kinase activity of mTOR is essential for translational control of TOP mRNAs.(A) HEK293 cells were transfected with vectors expressing mTOR-wt, mTOR-rr or mTOR-rr-kd, two days later the cells were amino acid-starved for 3 h followed by 3 h refeeding without or with 20 nM rapamycin. Cytoplasmic proteins derived from the cells were subjected to Western blot analysis with the indicated antibodies. B) Cytoplasmic extracts derived from cells treated as described in (A), were subjected to polysomal analysis. C) HEK293 cells were amino acid-starved for 3 h, or amino acid-starved for 3 h followed by 3 h refeeding without or with 50 nM Torin1. Cytoplasmic proteins derived from the cells were subjected to Western blot analysis with the indicated antibodies. D) Cytoplasmic extracts derived from cells treated as described in (C) were subjected to polysomal analysis and the percentage of mRNA in polysomes is presented as an average ± SEM of three experiments.
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pone-0109410-g004: The kinase activity of mTOR is essential for translational control of TOP mRNAs.(A) HEK293 cells were transfected with vectors expressing mTOR-wt, mTOR-rr or mTOR-rr-kd, two days later the cells were amino acid-starved for 3 h followed by 3 h refeeding without or with 20 nM rapamycin. Cytoplasmic proteins derived from the cells were subjected to Western blot analysis with the indicated antibodies. B) Cytoplasmic extracts derived from cells treated as described in (A), were subjected to polysomal analysis. C) HEK293 cells were amino acid-starved for 3 h, or amino acid-starved for 3 h followed by 3 h refeeding without or with 50 nM Torin1. Cytoplasmic proteins derived from the cells were subjected to Western blot analysis with the indicated antibodies. D) Cytoplasmic extracts derived from cells treated as described in (C) were subjected to polysomal analysis and the percentage of mRNA in polysomes is presented as an average ± SEM of three experiments.

Mentions: One plausible explanation for the apparent ability of mTOR to regulate TOP mRNA translation in an either mTORC1- or mTORC2-independent fashion might be its role as a scaffold protein, rather than an active kinase. Indeed, such a role has previously been proposed for regulation of dystrophin gene expression by mTOR [48]. Two complementary experimental approaches were used to directly address this possibility: a) HEK293 cells were transfected with wild type mTOR [49], rapamycin-resistant mTOR mutant (mTOR-rr) that contains S2035I substitution, or catalytically inactive version of mTOR-rr that contains an additional D2338A substitution (mTOR-rr-kd) [50]. Cells were amino acid-starved and then were refed in the absence or presence of rapamycin. The results show that overexpression of mTOR-rr, but not mTOR-rr-kd or wild type mTOR, can rescue both mTORC1 activity (Fig. 4A) and the translation efficiency of TOP mRNAs (Fig. 4B). b) HEK293 cells were amino acid-starved and then were refed with or without Torin1, a selective ATP-competitive mTOR inhibitor that can block all examined mTOR activities [3], [51]. Indeed, this inhibitor fully suppresses the amino acid-induced translational activation of rpL32 mRNA, to the same degree (about 50% in polysomes) as do rapamycin and mTOR knockdown (compare Fig. 4D with Figs. 2B and 2D). Taken together, these results clearly attest to the positive regulatory role of mTOR catalytic activity in amino acid-induced translational activation of TOP mRNAs.


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)

The kinase activity of mTOR is essential for translational control of TOP mRNAs.(A) HEK293 cells were transfected with vectors expressing mTOR-wt, mTOR-rr or mTOR-rr-kd, two days later the cells were amino acid-starved for 3 h followed by 3 h refeeding without or with 20 nM rapamycin. Cytoplasmic proteins derived from the cells were subjected to Western blot analysis with the indicated antibodies. B) Cytoplasmic extracts derived from cells treated as described in (A), were subjected to polysomal analysis. C) HEK293 cells were amino acid-starved for 3 h, or amino acid-starved for 3 h followed by 3 h refeeding without or with 50 nM Torin1. Cytoplasmic proteins derived from the cells were subjected to Western blot analysis with the indicated antibodies. D) Cytoplasmic extracts derived from cells treated as described in (C) were subjected to polysomal analysis and the percentage of mRNA in polysomes is presented as an average ± SEM of three experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4206288&req=5

pone-0109410-g004: The kinase activity of mTOR is essential for translational control of TOP mRNAs.(A) HEK293 cells were transfected with vectors expressing mTOR-wt, mTOR-rr or mTOR-rr-kd, two days later the cells were amino acid-starved for 3 h followed by 3 h refeeding without or with 20 nM rapamycin. Cytoplasmic proteins derived from the cells were subjected to Western blot analysis with the indicated antibodies. B) Cytoplasmic extracts derived from cells treated as described in (A), were subjected to polysomal analysis. C) HEK293 cells were amino acid-starved for 3 h, or amino acid-starved for 3 h followed by 3 h refeeding without or with 50 nM Torin1. Cytoplasmic proteins derived from the cells were subjected to Western blot analysis with the indicated antibodies. D) Cytoplasmic extracts derived from cells treated as described in (C) were subjected to polysomal analysis and the percentage of mRNA in polysomes is presented as an average ± SEM of three experiments.
Mentions: One plausible explanation for the apparent ability of mTOR to regulate TOP mRNA translation in an either mTORC1- or mTORC2-independent fashion might be its role as a scaffold protein, rather than an active kinase. Indeed, such a role has previously been proposed for regulation of dystrophin gene expression by mTOR [48]. Two complementary experimental approaches were used to directly address this possibility: a) HEK293 cells were transfected with wild type mTOR [49], rapamycin-resistant mTOR mutant (mTOR-rr) that contains S2035I substitution, or catalytically inactive version of mTOR-rr that contains an additional D2338A substitution (mTOR-rr-kd) [50]. Cells were amino acid-starved and then were refed in the absence or presence of rapamycin. The results show that overexpression of mTOR-rr, but not mTOR-rr-kd or wild type mTOR, can rescue both mTORC1 activity (Fig. 4A) and the translation efficiency of TOP mRNAs (Fig. 4B). b) HEK293 cells were amino acid-starved and then were refed with or without Torin1, a selective ATP-competitive mTOR inhibitor that can block all examined mTOR activities [3], [51]. Indeed, this inhibitor fully suppresses the amino acid-induced translational activation of rpL32 mRNA, to the same degree (about 50% in polysomes) as do rapamycin and mTOR knockdown (compare Fig. 4D with Figs. 2B and 2D). Taken together, these results clearly attest to the positive regulatory role of mTOR catalytic activity in amino acid-induced translational activation of TOP mRNAs.

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