Limits...
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.

Show MeSH

Related in: MedlinePlus

TSC2 or TSC1 deficiency rescues TOP mRNAs from translational repression in amino acid-starved cells.(A) TSC2+/+ and TSC2βˆ’/βˆ’ as well as TSC1+/+ and TSC1βˆ’/βˆ’ MEFs, were amino acid-starved for 16 h (βˆ’AA), amino acid-starved and then refed for 2 h, or amino acid staved during the last 16 h of 48 h serum starvation (βˆ’AA βˆ’serum). Subsequently cells were harvested and cytoplasmic extracts were prepared. These extracts were centrifuged through sucrose gradients and divided into polysomal (P) and subpolysomal (S) fractions. RNA from equivalent aliquots of these fractions was analyzed by Northern-blot hybridization with cDNAs for rpL32 mRNA (a TOP mRNA) and actin mRNA (a non TOP mRNA) (in the case of TSC2 also with cDNAs corresponding to rpS6 and tubulin). The radioactive signals were quantified, and the relative translational efficiency (% of the P signal relative to the P+S signals) of each mRNA is numerically presented beneath the autoradiograms as percentage of the mRNA engaged in polysomes. These figures are expressed as an average Β± SEM of the number of determinations in parenthesis, or the average with the individual values in parenthesis, if only two determinations are presented. (B) TSC2+/+, TSC2βˆ’/βˆ’ MEFs were untreated or amino acid-starved for 16 h and then harvested. The cytoplasmic proteins were subjected to Western blot analysis using the indicated antibodies.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4206288&req=5

pone-0109410-g001: TSC2 or TSC1 deficiency rescues TOP mRNAs from translational repression in amino acid-starved cells.(A) TSC2+/+ and TSC2βˆ’/βˆ’ as well as TSC1+/+ and TSC1βˆ’/βˆ’ MEFs, were amino acid-starved for 16 h (βˆ’AA), amino acid-starved and then refed for 2 h, or amino acid staved during the last 16 h of 48 h serum starvation (βˆ’AA βˆ’serum). Subsequently cells were harvested and cytoplasmic extracts were prepared. These extracts were centrifuged through sucrose gradients and divided into polysomal (P) and subpolysomal (S) fractions. RNA from equivalent aliquots of these fractions was analyzed by Northern-blot hybridization with cDNAs for rpL32 mRNA (a TOP mRNA) and actin mRNA (a non TOP mRNA) (in the case of TSC2 also with cDNAs corresponding to rpS6 and tubulin). The radioactive signals were quantified, and the relative translational efficiency (% of the P signal relative to the P+S signals) of each mRNA is numerically presented beneath the autoradiograms as percentage of the mRNA engaged in polysomes. These figures are expressed as an average Β± SEM of the number of determinations in parenthesis, or the average with the individual values in parenthesis, if only two determinations are presented. (B) TSC2+/+, TSC2βˆ’/βˆ’ MEFs were untreated or amino acid-starved for 16 h and then harvested. The cytoplasmic proteins were subjected to Western blot analysis using the indicated antibodies.

Mentions: The TSC1-TSC2 complex appears to mediate the translational repression of TOP mRNAs upon starvation for serum or oxygen [3], [18]. However, conflicting results regarding the role of this complex in transduction of amino acid signal to mTORC1 activity [41]–[44], rendered questionable its involvement also in transducing the signal of amino acid starvation to translational repression of TOP mRNAs. To directly address this issue, we monitored the translation efficiency of TOP mRNAs, as can be inferred from their relative polysomal association, when TSC2βˆ’/βˆ’ or TSC1βˆ’/βˆ’ MEFs were deprived of amino acids. Fig. 1A demonstrates that the translation of rpS6 mRNA was refractory to amino acid starvation in TSC2βˆ’/βˆ’ MEFs and rpL32 mRNAs in either of these cell lines, in contrast to the apparent sensitivity of these mRNAs in the wild-type counterparts. It is worth noting that the pronounce decrease in the translation efficiency of actin or tubulin mRNA upon amino acid starvation of TSC2+/+ and TSC2βˆ’/βˆ’ MEFs (Fig. 1A), relative to that observed in other cell types examined in this study, might simply reflect a cell type-specific response.


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)

TSC2 or TSC1 deficiency rescues TOP mRNAs from translational repression in amino acid-starved cells.(A) TSC2+/+ and TSC2βˆ’/βˆ’ as well as TSC1+/+ and TSC1βˆ’/βˆ’ MEFs, were amino acid-starved for 16 h (βˆ’AA), amino acid-starved and then refed for 2 h, or amino acid staved during the last 16 h of 48 h serum starvation (βˆ’AA βˆ’serum). Subsequently cells were harvested and cytoplasmic extracts were prepared. These extracts were centrifuged through sucrose gradients and divided into polysomal (P) and subpolysomal (S) fractions. RNA from equivalent aliquots of these fractions was analyzed by Northern-blot hybridization with cDNAs for rpL32 mRNA (a TOP mRNA) and actin mRNA (a non TOP mRNA) (in the case of TSC2 also with cDNAs corresponding to rpS6 and tubulin). The radioactive signals were quantified, and the relative translational efficiency (% of the P signal relative to the P+S signals) of each mRNA is numerically presented beneath the autoradiograms as percentage of the mRNA engaged in polysomes. These figures are expressed as an average Β± SEM of the number of determinations in parenthesis, or the average with the individual values in parenthesis, if only two determinations are presented. (B) TSC2+/+, TSC2βˆ’/βˆ’ MEFs were untreated or amino acid-starved for 16 h and then harvested. The cytoplasmic proteins were subjected to Western blot analysis using the indicated antibodies.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0109410-g001: TSC2 or TSC1 deficiency rescues TOP mRNAs from translational repression in amino acid-starved cells.(A) TSC2+/+ and TSC2βˆ’/βˆ’ as well as TSC1+/+ and TSC1βˆ’/βˆ’ MEFs, were amino acid-starved for 16 h (βˆ’AA), amino acid-starved and then refed for 2 h, or amino acid staved during the last 16 h of 48 h serum starvation (βˆ’AA βˆ’serum). Subsequently cells were harvested and cytoplasmic extracts were prepared. These extracts were centrifuged through sucrose gradients and divided into polysomal (P) and subpolysomal (S) fractions. RNA from equivalent aliquots of these fractions was analyzed by Northern-blot hybridization with cDNAs for rpL32 mRNA (a TOP mRNA) and actin mRNA (a non TOP mRNA) (in the case of TSC2 also with cDNAs corresponding to rpS6 and tubulin). The radioactive signals were quantified, and the relative translational efficiency (% of the P signal relative to the P+S signals) of each mRNA is numerically presented beneath the autoradiograms as percentage of the mRNA engaged in polysomes. These figures are expressed as an average Β± SEM of the number of determinations in parenthesis, or the average with the individual values in parenthesis, if only two determinations are presented. (B) TSC2+/+, TSC2βˆ’/βˆ’ MEFs were untreated or amino acid-starved for 16 h and then harvested. The cytoplasmic proteins were subjected to Western blot analysis using the indicated antibodies.
Mentions: The TSC1-TSC2 complex appears to mediate the translational repression of TOP mRNAs upon starvation for serum or oxygen [3], [18]. However, conflicting results regarding the role of this complex in transduction of amino acid signal to mTORC1 activity [41]–[44], rendered questionable its involvement also in transducing the signal of amino acid starvation to translational repression of TOP mRNAs. To directly address this issue, we monitored the translation efficiency of TOP mRNAs, as can be inferred from their relative polysomal association, when TSC2βˆ’/βˆ’ or TSC1βˆ’/βˆ’ MEFs were deprived of amino acids. Fig. 1A demonstrates that the translation of rpS6 mRNA was refractory to amino acid starvation in TSC2βˆ’/βˆ’ MEFs and rpL32 mRNAs in either of these cell lines, in contrast to the apparent sensitivity of these mRNAs in the wild-type counterparts. It is worth noting that the pronounce decrease in the translation efficiency of actin or tubulin mRNA upon amino acid starvation of TSC2+/+ and TSC2βˆ’/βˆ’ MEFs (Fig. 1A), relative to that observed in other cell types examined in this study, might simply reflect a cell type-specific response.

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