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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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Rapamycin represses the translation of TOP mRNAs in an FKBP-12-dependent fashion.(A) HEK293 cells were amino acid-starved for 3 h and then refed for 3 h in the absence or presence of rapamycin (20 nM), FK506 (20 mM), or both. Cytoplasmic proteins were subjected to Western blot analysis. (B) HEK293 cells were amino acid-starved for 3 h (−AA), refed for 3 h (−AA→+AA) in the absence or presence of rapamycin (20 nM), FK506 (20 mM) or both. Cytoplasmic extracts were subjected to polysomal analysis.
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pone-0109410-g005: Rapamycin represses the translation of TOP mRNAs in an FKBP-12-dependent fashion.(A) HEK293 cells were amino acid-starved for 3 h and then refed for 3 h in the absence or presence of rapamycin (20 nM), FK506 (20 mM), or both. Cytoplasmic proteins were subjected to Western blot analysis. (B) HEK293 cells were amino acid-starved for 3 h (−AA), refed for 3 h (−AA→+AA) in the absence or presence of rapamycin (20 nM), FK506 (20 mM) or both. Cytoplasmic extracts were subjected to polysomal analysis.

Mentions: The relative resistance of amino acid-induced TOP mRNAs translation to raptor or rictor knockdown on the one hand (Fig. 3), and the apparent requirement for mTOR activity on the other (Fig. 4), posed a question whether FKBP12, which is known to mediate the inhibition of mTOR by rapamycin, is also involved in the translational repression of these mRNAs. To this end, we utilized FK506, a small molecule that competes with rapamycin for binding to FKBP12. Indeed FK506 can relieve mTOR inhibition, as monitored by phosphorylation of S6K1 and rpS6 (Fig. 5A), as well as the translational repression of TOP mRNAs (Fig. 5B).


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)

Rapamycin represses the translation of TOP mRNAs in an FKBP-12-dependent fashion.(A) HEK293 cells were amino acid-starved for 3 h and then refed for 3 h in the absence or presence of rapamycin (20 nM), FK506 (20 mM), or both. Cytoplasmic proteins were subjected to Western blot analysis. (B) HEK293 cells were amino acid-starved for 3 h (−AA), refed for 3 h (−AA→+AA) in the absence or presence of rapamycin (20 nM), FK506 (20 mM) or both. Cytoplasmic extracts were subjected to polysomal analysis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4206288&req=5

pone-0109410-g005: Rapamycin represses the translation of TOP mRNAs in an FKBP-12-dependent fashion.(A) HEK293 cells were amino acid-starved for 3 h and then refed for 3 h in the absence or presence of rapamycin (20 nM), FK506 (20 mM), or both. Cytoplasmic proteins were subjected to Western blot analysis. (B) HEK293 cells were amino acid-starved for 3 h (−AA), refed for 3 h (−AA→+AA) in the absence or presence of rapamycin (20 nM), FK506 (20 mM) or both. Cytoplasmic extracts were subjected to polysomal analysis.
Mentions: The relative resistance of amino acid-induced TOP mRNAs translation to raptor or rictor knockdown on the one hand (Fig. 3), and the apparent requirement for mTOR activity on the other (Fig. 4), posed a question whether FKBP12, which is known to mediate the inhibition of mTOR by rapamycin, is also involved in the translational repression of these mRNAs. To this end, we utilized FK506, a small molecule that competes with rapamycin for binding to FKBP12. Indeed FK506 can relieve mTOR inhibition, as monitored by phosphorylation of S6K1 and rpS6 (Fig. 5A), as well as the translational repression of TOP mRNAs (Fig. 5B).

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