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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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Raptor and rictor are dispensable for translational activation of TOP mRNAs by amino acids.(A) HEK293 cells were infected with viruses expressing Red shRNA, raptor shRNA (Rap) or rictor shRNAs (Ric). The abundance of raptor or rictor, as well the phosphorylation status of direct and indirect substrates of the respective complexes, mTORC1 and mTORC2 (left and right, respectively), was monitored by Western blot analysis. (B) HEK293 cells infected with viruses expressing HcRed, raptor or rictor shRNA were amino acid starved for 3 h (−AA) or starved and then refed for 3 h (−AA→+AA). Cytoplasmic extracts from these cells were subjected to polysomal analysis and the data are presented as described in the legend to Fig. 2F. Numbers above bars are individual values, when only two measurements were performed.
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pone-0109410-g003: Raptor and rictor are dispensable for translational activation of TOP mRNAs by amino acids.(A) HEK293 cells were infected with viruses expressing Red shRNA, raptor shRNA (Rap) or rictor shRNAs (Ric). The abundance of raptor or rictor, as well the phosphorylation status of direct and indirect substrates of the respective complexes, mTORC1 and mTORC2 (left and right, respectively), was monitored by Western blot analysis. (B) HEK293 cells infected with viruses expressing HcRed, raptor or rictor shRNA were amino acid starved for 3 h (−AA) or starved and then refed for 3 h (−AA→+AA). Cytoplasmic extracts from these cells were subjected to polysomal analysis and the data are presented as described in the legend to Fig. 2F. Numbers above bars are individual values, when only two measurements were performed.

Mentions: Establishing the role of mTOR in amino acid-mediated translational activation of TOP mRNAs has further underscored the discrepancy between the promptness and sensitivity of mTORC1 response to rapamycin and the relative rapamycin resistance exhibited by TOP mRNAs (Fig. 2 and [20]). To examine whether mTORC1 is involved at all in amino acid-induced translational activation of these mRNAs, we knocked down its core constituent, raptor, in HEK293 cells. This silencing, indeed, led to a pronounced decrease in raptor level and mTORC1 activity, as exemplified by the hypophosphorylation of S6K1 and rpS6 (Figure 3A). However, despite this efficient elimination of raptor, the translational activation of rpL32 mRNA by amino acids was unaffected (Fig. 3B).


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)

Raptor and rictor are dispensable for translational activation of TOP mRNAs by amino acids.(A) HEK293 cells were infected with viruses expressing Red shRNA, raptor shRNA (Rap) or rictor shRNAs (Ric). The abundance of raptor or rictor, as well the phosphorylation status of direct and indirect substrates of the respective complexes, mTORC1 and mTORC2 (left and right, respectively), was monitored by Western blot analysis. (B) HEK293 cells infected with viruses expressing HcRed, raptor or rictor shRNA were amino acid starved for 3 h (−AA) or starved and then refed for 3 h (−AA→+AA). Cytoplasmic extracts from these cells were subjected to polysomal analysis and the data are presented as described in the legend to Fig. 2F. Numbers above bars are individual values, when only two measurements were performed.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4206288&req=5

pone-0109410-g003: Raptor and rictor are dispensable for translational activation of TOP mRNAs by amino acids.(A) HEK293 cells were infected with viruses expressing Red shRNA, raptor shRNA (Rap) or rictor shRNAs (Ric). The abundance of raptor or rictor, as well the phosphorylation status of direct and indirect substrates of the respective complexes, mTORC1 and mTORC2 (left and right, respectively), was monitored by Western blot analysis. (B) HEK293 cells infected with viruses expressing HcRed, raptor or rictor shRNA were amino acid starved for 3 h (−AA) or starved and then refed for 3 h (−AA→+AA). Cytoplasmic extracts from these cells were subjected to polysomal analysis and the data are presented as described in the legend to Fig. 2F. Numbers above bars are individual values, when only two measurements were performed.
Mentions: Establishing the role of mTOR in amino acid-mediated translational activation of TOP mRNAs has further underscored the discrepancy between the promptness and sensitivity of mTORC1 response to rapamycin and the relative rapamycin resistance exhibited by TOP mRNAs (Fig. 2 and [20]). To examine whether mTORC1 is involved at all in amino acid-induced translational activation of these mRNAs, we knocked down its core constituent, raptor, in HEK293 cells. This silencing, indeed, led to a pronounced decrease in raptor level and mTORC1 activity, as exemplified by the hypophosphorylation of S6K1 and rpS6 (Figure 3A). However, despite this efficient elimination of raptor, the translational activation of rpL32 mRNA by amino acids was unaffected (Fig. 3B).

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