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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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Knockdown of miRs fails to suppress translational activation of TOP mRNAs.(A) MDA-MB-231 cells were infected with lentivirus expressing either anti-miR 10b sponge or anti-miR-BART 1–5p sponge (control). The fluorescent signals of MICB and GFP were analyzed by FACS. The mean intensity of MICB or GFP in the control miR–transduced cells was arbitrarily set up to be 1, and the relative increase in the MICB expression or the decrease in the GFP fluorescence in sponge-10b–transduced cells was calculated accordingly (individual numbers are presented within the bars). (B) MDA-MB-231 cells infected with lentiviruses described in (A) were transiently transfected with Dual luciferase PsiCheck2 reporter vectors. These vectors contained within the 3′ UTR of the Renilla luciferase either a fragment from the 3′-UTR of NCOR2 that bears miR-10a/10b binding site (designated miR-10), or a negative control with a mutated miR-10a/10b seed region (designated miR-10mut). The Renilla to Firefly activity ratio (R/FF) was calculated for each sample and the average obtained for the miR-10b sponge-infected cells was normalized to that obtained for the control sponge-infected cells, which arbitrarily was set at 1.0. (*) p<0.001 versus miR-10a transfected cells (n = 8). (C) MDA-MB-231 cells that were either kept uninfected (None), expressed anti-miR 10b sponge or control sponge (Control) were kept untreated and cytoplasmic extracts from these cells were subjected to polysomal analysis. (D) and (F) RKO cells infected with Sin 18, an empty lentiviral vector (EV), or by lentivirus expressing shDrosha RNA. Cells were either untreated [Control in (F)], starved for serum for 19 h and during the last 3 h also for amino acids and then either kept without serum and amino acids [− in (D); −AA in (F)] or refed for just amino acid for additional 2 h [+ in (D); −AA→+AA in (F)]. Similarly infected cells were serum starved for 48 h [−in (D); –Serum in (F)] or serum starved for 48 h and then serum refed for 3 h [+ in (D); –Ser →+Ser in (F)]. Cells were harvested and subjected to Western blot analysis with the indicated antibodies (D) or subjected to polysomal analysis (F). (E) Total RNA was prepared from RKO cells infected with either empty lentiviral vector (EV) or lentivirus expressing shDrosha RNA. The abundance of each of the indicated miRs in Drosha knockdown cells was normalized to that in cells infected with empty vector, which was arbitrarily set at one.
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pone-0109410-g007: Knockdown of miRs fails to suppress translational activation of TOP mRNAs.(A) MDA-MB-231 cells were infected with lentivirus expressing either anti-miR 10b sponge or anti-miR-BART 1–5p sponge (control). The fluorescent signals of MICB and GFP were analyzed by FACS. The mean intensity of MICB or GFP in the control miR–transduced cells was arbitrarily set up to be 1, and the relative increase in the MICB expression or the decrease in the GFP fluorescence in sponge-10b–transduced cells was calculated accordingly (individual numbers are presented within the bars). (B) MDA-MB-231 cells infected with lentiviruses described in (A) were transiently transfected with Dual luciferase PsiCheck2 reporter vectors. These vectors contained within the 3′ UTR of the Renilla luciferase either a fragment from the 3′-UTR of NCOR2 that bears miR-10a/10b binding site (designated miR-10), or a negative control with a mutated miR-10a/10b seed region (designated miR-10mut). The Renilla to Firefly activity ratio (R/FF) was calculated for each sample and the average obtained for the miR-10b sponge-infected cells was normalized to that obtained for the control sponge-infected cells, which arbitrarily was set at 1.0. (*) p<0.001 versus miR-10a transfected cells (n = 8). (C) MDA-MB-231 cells that were either kept uninfected (None), expressed anti-miR 10b sponge or control sponge (Control) were kept untreated and cytoplasmic extracts from these cells were subjected to polysomal analysis. (D) and (F) RKO cells infected with Sin 18, an empty lentiviral vector (EV), or by lentivirus expressing shDrosha RNA. Cells were either untreated [Control in (F)], starved for serum for 19 h and during the last 3 h also for amino acids and then either kept without serum and amino acids [− in (D); −AA in (F)] or refed for just amino acid for additional 2 h [+ in (D); −AA→+AA in (F)]. Similarly infected cells were serum starved for 48 h [−in (D); –Serum in (F)] or serum starved for 48 h and then serum refed for 3 h [+ in (D); –Ser →+Ser in (F)]. Cells were harvested and subjected to Western blot analysis with the indicated antibodies (D) or subjected to polysomal analysis (F). (E) Total RNA was prepared from RKO cells infected with either empty lentiviral vector (EV) or lentivirus expressing shDrosha RNA. The abundance of each of the indicated miRs in Drosha knockdown cells was normalized to that in cells infected with empty vector, which was arbitrarily set at one.

Mentions: The location of the sponge sequence downstream of the GFP open reading frame enabled us to assess the sponge activity. Thus, the sequestration of the relevant miRNA in MDA-MB-231 cells indeed led to reduced GFP fluorescence intensity (Fig. 7A). Moreover, miR-10b has been implicated in downregulation of the stress-induced cell surface molecule, MICB (MHC class I chain related gene B) [38], and therefore, titrating out this miR resulted in increased expression of MICB (Fig. 7A). Next, we set to examine the ability of miR-10b sponge to derepress the expression of a luciferase encoded by an mRNA that contained in its 3′ untranslated region (3′ UTR) the wild-484 bp-long 3′ UTR from NCOR2 mRNA (designated as miR-10a) [39]. To this end, MDA-MB-231 cells were transiently transfected with miR-10a luciferase reporter. This reporter construct showed a small (about 19%), yet statistically significant, increase in luciferase activity expressing mir-10b sponge, but not a control sponge. Moreover, a reporter containing NCOR2 3′ UTR with mutated miR-10a binding motif (designated miR-10a mut), failed to respond to the expression of the miR-10b sponge (Fig. 7B).


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)

Knockdown of miRs fails to suppress translational activation of TOP mRNAs.(A) MDA-MB-231 cells were infected with lentivirus expressing either anti-miR 10b sponge or anti-miR-BART 1–5p sponge (control). The fluorescent signals of MICB and GFP were analyzed by FACS. The mean intensity of MICB or GFP in the control miR–transduced cells was arbitrarily set up to be 1, and the relative increase in the MICB expression or the decrease in the GFP fluorescence in sponge-10b–transduced cells was calculated accordingly (individual numbers are presented within the bars). (B) MDA-MB-231 cells infected with lentiviruses described in (A) were transiently transfected with Dual luciferase PsiCheck2 reporter vectors. These vectors contained within the 3′ UTR of the Renilla luciferase either a fragment from the 3′-UTR of NCOR2 that bears miR-10a/10b binding site (designated miR-10), or a negative control with a mutated miR-10a/10b seed region (designated miR-10mut). The Renilla to Firefly activity ratio (R/FF) was calculated for each sample and the average obtained for the miR-10b sponge-infected cells was normalized to that obtained for the control sponge-infected cells, which arbitrarily was set at 1.0. (*) p<0.001 versus miR-10a transfected cells (n = 8). (C) MDA-MB-231 cells that were either kept uninfected (None), expressed anti-miR 10b sponge or control sponge (Control) were kept untreated and cytoplasmic extracts from these cells were subjected to polysomal analysis. (D) and (F) RKO cells infected with Sin 18, an empty lentiviral vector (EV), or by lentivirus expressing shDrosha RNA. Cells were either untreated [Control in (F)], starved for serum for 19 h and during the last 3 h also for amino acids and then either kept without serum and amino acids [− in (D); −AA in (F)] or refed for just amino acid for additional 2 h [+ in (D); −AA→+AA in (F)]. Similarly infected cells were serum starved for 48 h [−in (D); –Serum in (F)] or serum starved for 48 h and then serum refed for 3 h [+ in (D); –Ser →+Ser in (F)]. Cells were harvested and subjected to Western blot analysis with the indicated antibodies (D) or subjected to polysomal analysis (F). (E) Total RNA was prepared from RKO cells infected with either empty lentiviral vector (EV) or lentivirus expressing shDrosha RNA. The abundance of each of the indicated miRs in Drosha knockdown cells was normalized to that in cells infected with empty vector, which was arbitrarily set at one.
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Related In: Results  -  Collection

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

pone-0109410-g007: Knockdown of miRs fails to suppress translational activation of TOP mRNAs.(A) MDA-MB-231 cells were infected with lentivirus expressing either anti-miR 10b sponge or anti-miR-BART 1–5p sponge (control). The fluorescent signals of MICB and GFP were analyzed by FACS. The mean intensity of MICB or GFP in the control miR–transduced cells was arbitrarily set up to be 1, and the relative increase in the MICB expression or the decrease in the GFP fluorescence in sponge-10b–transduced cells was calculated accordingly (individual numbers are presented within the bars). (B) MDA-MB-231 cells infected with lentiviruses described in (A) were transiently transfected with Dual luciferase PsiCheck2 reporter vectors. These vectors contained within the 3′ UTR of the Renilla luciferase either a fragment from the 3′-UTR of NCOR2 that bears miR-10a/10b binding site (designated miR-10), or a negative control with a mutated miR-10a/10b seed region (designated miR-10mut). The Renilla to Firefly activity ratio (R/FF) was calculated for each sample and the average obtained for the miR-10b sponge-infected cells was normalized to that obtained for the control sponge-infected cells, which arbitrarily was set at 1.0. (*) p<0.001 versus miR-10a transfected cells (n = 8). (C) MDA-MB-231 cells that were either kept uninfected (None), expressed anti-miR 10b sponge or control sponge (Control) were kept untreated and cytoplasmic extracts from these cells were subjected to polysomal analysis. (D) and (F) RKO cells infected with Sin 18, an empty lentiviral vector (EV), or by lentivirus expressing shDrosha RNA. Cells were either untreated [Control in (F)], starved for serum for 19 h and during the last 3 h also for amino acids and then either kept without serum and amino acids [− in (D); −AA in (F)] or refed for just amino acid for additional 2 h [+ in (D); −AA→+AA in (F)]. Similarly infected cells were serum starved for 48 h [−in (D); –Serum in (F)] or serum starved for 48 h and then serum refed for 3 h [+ in (D); –Ser →+Ser in (F)]. Cells were harvested and subjected to Western blot analysis with the indicated antibodies (D) or subjected to polysomal analysis (F). (E) Total RNA was prepared from RKO cells infected with either empty lentiviral vector (EV) or lentivirus expressing shDrosha RNA. The abundance of each of the indicated miRs in Drosha knockdown cells was normalized to that in cells infected with empty vector, which was arbitrarily set at one.
Mentions: The location of the sponge sequence downstream of the GFP open reading frame enabled us to assess the sponge activity. Thus, the sequestration of the relevant miRNA in MDA-MB-231 cells indeed led to reduced GFP fluorescence intensity (Fig. 7A). Moreover, miR-10b has been implicated in downregulation of the stress-induced cell surface molecule, MICB (MHC class I chain related gene B) [38], and therefore, titrating out this miR resulted in increased expression of MICB (Fig. 7A). Next, we set to examine the ability of miR-10b sponge to derepress the expression of a luciferase encoded by an mRNA that contained in its 3′ untranslated region (3′ UTR) the wild-484 bp-long 3′ UTR from NCOR2 mRNA (designated as miR-10a) [39]. To this end, MDA-MB-231 cells were transiently transfected with miR-10a luciferase reporter. This reporter construct showed a small (about 19%), yet statistically significant, increase in luciferase activity expressing mir-10b sponge, but not a control sponge. Moreover, a reporter containing NCOR2 3′ UTR with mutated miR-10a binding motif (designated miR-10a mut), failed to respond to the expression of the miR-10b sponge (Fig. 7B).

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