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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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Overexpression of RagBGTP can derepress TOP mRNA translation in amino acid-starved cells, but not in cells deprived of oxygen.(A) HEK293 cells were infected with lentiviral expression vectors encoding FLAG-RagB or FLAG-RagBGTP. 48 h post infection cells were subjected to selection by puromycin and 48 h later were either kept untreated (+), amino acid-starved for 8 h (−AA), amino acid starved during the last 3 h of 24 h serum starvation (–Ser/AA) or deprived of oxygen (−O2) for 16 h. Cells were harvested and their cytoplasmic proteins were subjected to Western blot analysis using the indicated antibodies. (B) Polysomal analysis of cytoplasmic extracts from cells treated as describe in (A).
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pone-0109410-g006: Overexpression of RagBGTP can derepress TOP mRNA translation in amino acid-starved cells, but not in cells deprived of oxygen.(A) HEK293 cells were infected with lentiviral expression vectors encoding FLAG-RagB or FLAG-RagBGTP. 48 h post infection cells were subjected to selection by puromycin and 48 h later were either kept untreated (+), amino acid-starved for 8 h (−AA), amino acid starved during the last 3 h of 24 h serum starvation (–Ser/AA) or deprived of oxygen (−O2) for 16 h. Cells were harvested and their cytoplasmic proteins were subjected to Western blot analysis using the indicated antibodies. (B) Polysomal analysis of cytoplasmic extracts from cells treated as describe in (A).

Mentions: Rag GTPases bind raptor and thus mediate amino acid signaling to mTORC1 [9]. Accordingly, expression of constitutively active mutant forms of RagA or B (GTP-bound) can protect mTORC1 activity in amino acid-deprived cells [9], [53]. However, the establishment of raptor as dispensable for amino acid- or oxygen-mediated translational activation of TOP mRNAs (Fig. 3B and [3]), raised a question regarding the role of Rag in this mode of regulation. To examine this issue, we measured the effect of FLAG-tagged wild type RagB or a mutant [RagB (Q99L)] that constitutively binds GTP (RagBGTP), on the translation efficiency of TOP mRNAs under stress conditions. Our results show that RagBGTP, but not wild type RagB, exerts complete, partial or no protective effect on rpS6 phosphorylation in cells that were amino acid-starved, starved for both serum and amino acids, or oxygen-deprived cells, respectively (Figs. 6A). Notably, RagBGTP exerts a similar stress-specific relief of the translational repression of rpL32 and rpS6 mRNAs (Fig. 6B), suggesting that RagB primarily mediates signals emanating from amino acids.


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)

Overexpression of RagBGTP can derepress TOP mRNA translation in amino acid-starved cells, but not in cells deprived of oxygen.(A) HEK293 cells were infected with lentiviral expression vectors encoding FLAG-RagB or FLAG-RagBGTP. 48 h post infection cells were subjected to selection by puromycin and 48 h later were either kept untreated (+), amino acid-starved for 8 h (−AA), amino acid starved during the last 3 h of 24 h serum starvation (–Ser/AA) or deprived of oxygen (−O2) for 16 h. Cells were harvested and their cytoplasmic proteins were subjected to Western blot analysis using the indicated antibodies. (B) Polysomal analysis of cytoplasmic extracts from cells treated as describe in (A).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4206288&req=5

pone-0109410-g006: Overexpression of RagBGTP can derepress TOP mRNA translation in amino acid-starved cells, but not in cells deprived of oxygen.(A) HEK293 cells were infected with lentiviral expression vectors encoding FLAG-RagB or FLAG-RagBGTP. 48 h post infection cells were subjected to selection by puromycin and 48 h later were either kept untreated (+), amino acid-starved for 8 h (−AA), amino acid starved during the last 3 h of 24 h serum starvation (–Ser/AA) or deprived of oxygen (−O2) for 16 h. Cells were harvested and their cytoplasmic proteins were subjected to Western blot analysis using the indicated antibodies. (B) Polysomal analysis of cytoplasmic extracts from cells treated as describe in (A).
Mentions: Rag GTPases bind raptor and thus mediate amino acid signaling to mTORC1 [9]. Accordingly, expression of constitutively active mutant forms of RagA or B (GTP-bound) can protect mTORC1 activity in amino acid-deprived cells [9], [53]. However, the establishment of raptor as dispensable for amino acid- or oxygen-mediated translational activation of TOP mRNAs (Fig. 3B and [3]), raised a question regarding the role of Rag in this mode of regulation. To examine this issue, we measured the effect of FLAG-tagged wild type RagB or a mutant [RagB (Q99L)] that constitutively binds GTP (RagBGTP), on the translation efficiency of TOP mRNAs under stress conditions. Our results show that RagBGTP, but not wild type RagB, exerts complete, partial or no protective effect on rpS6 phosphorylation in cells that were amino acid-starved, starved for both serum and amino acids, or oxygen-deprived cells, respectively (Figs. 6A). Notably, RagBGTP exerts a similar stress-specific relief of the translational repression of rpL32 and rpS6 mRNAs (Fig. 6B), suggesting that RagB primarily mediates signals emanating from amino acids.

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