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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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micorRNAs are dispensable for serum-induced translational activation of TOP mRNAs.(A) Total RNA was prepared from Dicer+/+ and Dicer−/− MEFs and the relative abundance of the indicated miRs was assessed by quantitative PCR. The abundance of each miR in Dicer−/− MEFs was normalized to that in Dicer+/+ MEFs, which was arbitrarily set at one. (B) Dicer+/+ and Dicer−/− MEFs were seeded in 96-well plates at a density of 4×103 cells per well. Cells were either untreated (Con) or serum starved (−Ser) for the indicated amount of time. Proliferation was monitored by the methylene blue staining protocol [68]. Absorbance measured 24 h after platting, set arbitrarily at 1, and measured at later time points (average ± SEM [n = 5] for each time point) was normalized to that value (note that the error bars are are smaller than the size of the symbols). (C) Dicer+/+ and Dicer−/− MEFs were either untreated (Control), serum starved (−Serum) for 48 h or serum starved for 48 h and then serum refed for 3 h (−Serum→+Serum), or treated with 50 nM Torin 1 for 2 h. Cells were harvested and subjected to polysomal analysis using the indicated probes.
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pone-0109410-g008: micorRNAs are dispensable for serum-induced translational activation of TOP mRNAs.(A) Total RNA was prepared from Dicer+/+ and Dicer−/− MEFs and the relative abundance of the indicated miRs was assessed by quantitative PCR. The abundance of each miR in Dicer−/− MEFs was normalized to that in Dicer+/+ MEFs, which was arbitrarily set at one. (B) Dicer+/+ and Dicer−/− MEFs were seeded in 96-well plates at a density of 4×103 cells per well. Cells were either untreated (Con) or serum starved (−Ser) for the indicated amount of time. Proliferation was monitored by the methylene blue staining protocol [68]. Absorbance measured 24 h after platting, set arbitrarily at 1, and measured at later time points (average ± SEM [n = 5] for each time point) was normalized to that value (note that the error bars are are smaller than the size of the symbols). (C) Dicer+/+ and Dicer−/− MEFs were either untreated (Control), serum starved (−Serum) for 48 h or serum starved for 48 h and then serum refed for 3 h (−Serum→+Serum), or treated with 50 nM Torin 1 for 2 h. Cells were harvested and subjected to polysomal analysis using the indicated probes.

Mentions: The relative inefficient translation of rpL32 mRNA in Drosha knockdown cells (Fig. 7F) could have reflected the requirement for one or more miRs for efficient basal translation of TOP mRNAs, or alternatively, a side effect of the infection by the respective lentivirus. In order to distinguish between these two options, we took advantage of a hemangiosarcoma cell line that had been derived from mouse deficient for Dicer, the cytoplasmic RNase that conducts the second processing step of miRs [55]. These cells, indeed, exhibited an extensive decrease in the abundance of representative miRs (Fig. 8A), yet showed an increase, rather than a decrease, in the basal translation efficiency of both rpL32 and rpS6 mRNAs. These results clearly show that if miRs play a role in this mode of regulation, it is a negative one, rather than a positive one. Consistently, TOP mRNAs were downregulated by serum starvation of Dicer−/− cells to a lesser extent than in their Dicer+/+ counterparts and underwent complete recovery following serum refeeding (Fig. 8B). Evidently, the translational repression of TOP mRNAs under serum starvation results from mitotic arrest [56]. However, the relative resistance of Dicer−/− cells to serum starvation cannot be ascribed to an acquired resistance to this stress, as serum starvation had a similar inhibitory effect on the proliferation of both cell genotypes (Fig. 8C). Moreover, it cannot be attributed to the conversion of the mTOR in Dicer−/− cell to a constitutively active one, as it was readily inhibited by Torin 1 (Fig. 8B). Notably, Dicer−/− cells, like iRapKO and iRicKO MEFs, exhibit an inherent resistance to prolonged amino acid starvation (data not shown), and therefore could not be used for a study of the translational behavior of their TOP mRNAs under amino acid deficiency. Collectively, our results with both Drosha knockdown and Dicer knockout, indicate that miRs are not required for efficient translation of TOP mRNAs or for their translational activation following stimulation by either amino acid or serum.


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)

micorRNAs are dispensable for serum-induced translational activation of TOP mRNAs.(A) Total RNA was prepared from Dicer+/+ and Dicer−/− MEFs and the relative abundance of the indicated miRs was assessed by quantitative PCR. The abundance of each miR in Dicer−/− MEFs was normalized to that in Dicer+/+ MEFs, which was arbitrarily set at one. (B) Dicer+/+ and Dicer−/− MEFs were seeded in 96-well plates at a density of 4×103 cells per well. Cells were either untreated (Con) or serum starved (−Ser) for the indicated amount of time. Proliferation was monitored by the methylene blue staining protocol [68]. Absorbance measured 24 h after platting, set arbitrarily at 1, and measured at later time points (average ± SEM [n = 5] for each time point) was normalized to that value (note that the error bars are are smaller than the size of the symbols). (C) Dicer+/+ and Dicer−/− MEFs were either untreated (Control), serum starved (−Serum) for 48 h or serum starved for 48 h and then serum refed for 3 h (−Serum→+Serum), or treated with 50 nM Torin 1 for 2 h. Cells were harvested and subjected to polysomal analysis using the indicated probes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4206288&req=5

pone-0109410-g008: micorRNAs are dispensable for serum-induced translational activation of TOP mRNAs.(A) Total RNA was prepared from Dicer+/+ and Dicer−/− MEFs and the relative abundance of the indicated miRs was assessed by quantitative PCR. The abundance of each miR in Dicer−/− MEFs was normalized to that in Dicer+/+ MEFs, which was arbitrarily set at one. (B) Dicer+/+ and Dicer−/− MEFs were seeded in 96-well plates at a density of 4×103 cells per well. Cells were either untreated (Con) or serum starved (−Ser) for the indicated amount of time. Proliferation was monitored by the methylene blue staining protocol [68]. Absorbance measured 24 h after platting, set arbitrarily at 1, and measured at later time points (average ± SEM [n = 5] for each time point) was normalized to that value (note that the error bars are are smaller than the size of the symbols). (C) Dicer+/+ and Dicer−/− MEFs were either untreated (Control), serum starved (−Serum) for 48 h or serum starved for 48 h and then serum refed for 3 h (−Serum→+Serum), or treated with 50 nM Torin 1 for 2 h. Cells were harvested and subjected to polysomal analysis using the indicated probes.
Mentions: The relative inefficient translation of rpL32 mRNA in Drosha knockdown cells (Fig. 7F) could have reflected the requirement for one or more miRs for efficient basal translation of TOP mRNAs, or alternatively, a side effect of the infection by the respective lentivirus. In order to distinguish between these two options, we took advantage of a hemangiosarcoma cell line that had been derived from mouse deficient for Dicer, the cytoplasmic RNase that conducts the second processing step of miRs [55]. These cells, indeed, exhibited an extensive decrease in the abundance of representative miRs (Fig. 8A), yet showed an increase, rather than a decrease, in the basal translation efficiency of both rpL32 and rpS6 mRNAs. These results clearly show that if miRs play a role in this mode of regulation, it is a negative one, rather than a positive one. Consistently, TOP mRNAs were downregulated by serum starvation of Dicer−/− cells to a lesser extent than in their Dicer+/+ counterparts and underwent complete recovery following serum refeeding (Fig. 8B). Evidently, the translational repression of TOP mRNAs under serum starvation results from mitotic arrest [56]. However, the relative resistance of Dicer−/− cells to serum starvation cannot be ascribed to an acquired resistance to this stress, as serum starvation had a similar inhibitory effect on the proliferation of both cell genotypes (Fig. 8C). Moreover, it cannot be attributed to the conversion of the mTOR in Dicer−/− cell to a constitutively active one, as it was readily inhibited by Torin 1 (Fig. 8B). Notably, Dicer−/− cells, like iRapKO and iRicKO MEFs, exhibit an inherent resistance to prolonged amino acid starvation (data not shown), and therefore could not be used for a study of the translational behavior of their TOP mRNAs under amino acid deficiency. Collectively, our results with both Drosha knockdown and Dicer knockout, indicate that miRs are not required for efficient translation of TOP mRNAs or for their translational activation following stimulation by either amino acid or serum.

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