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Multi-functional regulation of 4E-BP gene expression by the Ccr4-Not complex.

Okada H, Schittenhelm RB, Straessle A, Hafen E - PLoS ONE (2015)

Bottom Line: Examination of the gene expression mechanism using reporter swap constructs reveals that Not1 depletion increases reporter mRNAs with the 3'UTR of 4E-BP gene, but decreases the ones with the 4E-BP promoter region, suggesting that Ccr4-Not complex regulates both degradation and transcription of 4E-BP mRNA.These results indicate that the Ccr4-Not complex controls expression of a single gene at multiple levels and adjusts the magnitude of the total effect.Thus, our study reveals a novel regulatory mechanism of a key component of the mTOR signaling pathway at the level of gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Systems Biology, Swiss Federal Institute of Technology (ETH) Zurich, Wolfgang Pauli Str. 16, 8093, Zürich, Switzerland.

ABSTRACT
The mechanistic target of rapamycin (mTOR) signaling pathway is highly conserved from yeast to humans. It senses various environmental cues to regulate cellular growth and homeostasis. Deregulation of the pathway has been implicated in many pathological conditions including cancer. Phosphorylation cascades through the pathway have been extensively studied but not much is known about the regulation of gene expression of the pathway components. Here, we report that the mRNA level of eukaryotic translation initiation factor (eIF) subunit 4E-binding protein (4E-BP) gene, one of the key mTOR signaling components, is regulated by the highly conserved Ccr4-Not complex. RNAi knockdown of Not1, a putative scaffold protein of this protein complex, increases the mRNA level of 4E-BP in Drosophila Kc cells. Examination of the gene expression mechanism using reporter swap constructs reveals that Not1 depletion increases reporter mRNAs with the 3'UTR of 4E-BP gene, but decreases the ones with the 4E-BP promoter region, suggesting that Ccr4-Not complex regulates both degradation and transcription of 4E-BP mRNA. These results indicate that the Ccr4-Not complex controls expression of a single gene at multiple levels and adjusts the magnitude of the total effect. Thus, our study reveals a novel regulatory mechanism of a key component of the mTOR signaling pathway at the level of gene expression.

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Not1 reduction decreases 4E-BP mRNA through the promoter region of 4E-BP gene.A. Schematic of reporter constructs in which the promoter region, 5’UTR and/or 3’UTR were swapped with the counterpart(s) of 4E-BP gene. (see 3B). B. Changes of the reporter mRNA levels by Not1 depletion. Cells were subjected to Not1 RNAi and transfection with same amounts of reporter constructs. After serum starvation overnight and 30min insulin stimulation, mRNA levels of reporter constructs were measured by qPCR and normalized to untreated (no RNAi/insulin). Data are represented by Means ± SEM from three independent experiments. (see 3B). C. Changes of the reporter mRNA levels by Not1 depletion in the absence of insulin stimulation. The experiment was performed as in B except the absence of insulin addition. D. Combined effect of insulin stimulation and Not1 depletion on the reporter mRNA levels. The mRNA levels of the indicated constructs were normalized to untreated (no RNAi/insulin).
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pone.0113902.g004: Not1 reduction decreases 4E-BP mRNA through the promoter region of 4E-BP gene.A. Schematic of reporter constructs in which the promoter region, 5’UTR and/or 3’UTR were swapped with the counterpart(s) of 4E-BP gene. (see 3B). B. Changes of the reporter mRNA levels by Not1 depletion. Cells were subjected to Not1 RNAi and transfection with same amounts of reporter constructs. After serum starvation overnight and 30min insulin stimulation, mRNA levels of reporter constructs were measured by qPCR and normalized to untreated (no RNAi/insulin). Data are represented by Means ± SEM from three independent experiments. (see 3B). C. Changes of the reporter mRNA levels by Not1 depletion in the absence of insulin stimulation. The experiment was performed as in B except the absence of insulin addition. D. Combined effect of insulin stimulation and Not1 depletion on the reporter mRNA levels. The mRNA levels of the indicated constructs were normalized to untreated (no RNAi/insulin).

Mentions: To examine the possibility whether the Ccr4-Not complex is also involved in the transcription of 4E-BP, we created another series of reporter constructs containing various combinations of 4E-BP gene regulatory regions including its promoter region (Fig. 4A). The presence of the promoter region of the 4E-BP gene (pEEA and pEAA) caused a reduction in the mRNA levels upon Not1 depletion irrespectively of the 5’UTR suggesting that the Ccr4-Not complex positively regulates the transcription of 4E-BP (Fig. 4B). The reporter constructs pEAE and pEEE, which contain both the 4E-BP promoter region and the 3’ UTR, showed mRNA levels in between those of the 4E-BP promoter and the 4E-BP 3’UTR alone, indicating that the Ccr4-Not complex controls both the transcription and decay of 4E-BP mRNA. The same experiments in the absence of insulin stimulation resulted in very similar levels of reporter mRNAs (Fig. 4C) suggesting that activation of insulin signaling is not necessary for the transcriptional reduction of 4E-BP mRNA. To more carefully examine effects of Not1 depletion and insulin addition separately, the changes of the (representative) reporter mRNA levels induced by Not1 depletion and/or insulin stimulation were compared by normalizing to Untr. (no treatment) (Fig. 4D). A moderate increase of control pAAA mRNA by Not1 depletion is observed but it is not sure whether this increase is due to cellular stress from long-term double-stranded RNA (dsRNA) incubation or due to real Not1 RNAi effect on the control construct. However, the much larger increase in pAAE (4E-BP 3’UTR) mRNA and the decrease in pEEA (4E-BP promoter + 5’UTR) mRNA confirmed a 4E-BP-specific mRNA regulation (mRNA decay and transcription) by the complex. The effect of insulin addition was undetectable, which further supports the notion that insulin signaling does not affect the functions of the complex. The exception is that insulin addition reduced pEEA mRNA level that reflects the insulin-induced FOXO-mediated transcriptional reduction of 4E-BP. In summary, Ccr4-Not complex affects both the generation and clearance of 4E-BP mRNA and consequently causes a reducing effect on mRNA as a total.


Multi-functional regulation of 4E-BP gene expression by the Ccr4-Not complex.

Okada H, Schittenhelm RB, Straessle A, Hafen E - PLoS ONE (2015)

Not1 reduction decreases 4E-BP mRNA through the promoter region of 4E-BP gene.A. Schematic of reporter constructs in which the promoter region, 5’UTR and/or 3’UTR were swapped with the counterpart(s) of 4E-BP gene. (see 3B). B. Changes of the reporter mRNA levels by Not1 depletion. Cells were subjected to Not1 RNAi and transfection with same amounts of reporter constructs. After serum starvation overnight and 30min insulin stimulation, mRNA levels of reporter constructs were measured by qPCR and normalized to untreated (no RNAi/insulin). Data are represented by Means ± SEM from three independent experiments. (see 3B). C. Changes of the reporter mRNA levels by Not1 depletion in the absence of insulin stimulation. The experiment was performed as in B except the absence of insulin addition. D. Combined effect of insulin stimulation and Not1 depletion on the reporter mRNA levels. The mRNA levels of the indicated constructs were normalized to untreated (no RNAi/insulin).
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pone.0113902.g004: Not1 reduction decreases 4E-BP mRNA through the promoter region of 4E-BP gene.A. Schematic of reporter constructs in which the promoter region, 5’UTR and/or 3’UTR were swapped with the counterpart(s) of 4E-BP gene. (see 3B). B. Changes of the reporter mRNA levels by Not1 depletion. Cells were subjected to Not1 RNAi and transfection with same amounts of reporter constructs. After serum starvation overnight and 30min insulin stimulation, mRNA levels of reporter constructs were measured by qPCR and normalized to untreated (no RNAi/insulin). Data are represented by Means ± SEM from three independent experiments. (see 3B). C. Changes of the reporter mRNA levels by Not1 depletion in the absence of insulin stimulation. The experiment was performed as in B except the absence of insulin addition. D. Combined effect of insulin stimulation and Not1 depletion on the reporter mRNA levels. The mRNA levels of the indicated constructs were normalized to untreated (no RNAi/insulin).
Mentions: To examine the possibility whether the Ccr4-Not complex is also involved in the transcription of 4E-BP, we created another series of reporter constructs containing various combinations of 4E-BP gene regulatory regions including its promoter region (Fig. 4A). The presence of the promoter region of the 4E-BP gene (pEEA and pEAA) caused a reduction in the mRNA levels upon Not1 depletion irrespectively of the 5’UTR suggesting that the Ccr4-Not complex positively regulates the transcription of 4E-BP (Fig. 4B). The reporter constructs pEAE and pEEE, which contain both the 4E-BP promoter region and the 3’ UTR, showed mRNA levels in between those of the 4E-BP promoter and the 4E-BP 3’UTR alone, indicating that the Ccr4-Not complex controls both the transcription and decay of 4E-BP mRNA. The same experiments in the absence of insulin stimulation resulted in very similar levels of reporter mRNAs (Fig. 4C) suggesting that activation of insulin signaling is not necessary for the transcriptional reduction of 4E-BP mRNA. To more carefully examine effects of Not1 depletion and insulin addition separately, the changes of the (representative) reporter mRNA levels induced by Not1 depletion and/or insulin stimulation were compared by normalizing to Untr. (no treatment) (Fig. 4D). A moderate increase of control pAAA mRNA by Not1 depletion is observed but it is not sure whether this increase is due to cellular stress from long-term double-stranded RNA (dsRNA) incubation or due to real Not1 RNAi effect on the control construct. However, the much larger increase in pAAE (4E-BP 3’UTR) mRNA and the decrease in pEEA (4E-BP promoter + 5’UTR) mRNA confirmed a 4E-BP-specific mRNA regulation (mRNA decay and transcription) by the complex. The effect of insulin addition was undetectable, which further supports the notion that insulin signaling does not affect the functions of the complex. The exception is that insulin addition reduced pEEA mRNA level that reflects the insulin-induced FOXO-mediated transcriptional reduction of 4E-BP. In summary, Ccr4-Not complex affects both the generation and clearance of 4E-BP mRNA and consequently causes a reducing effect on mRNA as a total.

Bottom Line: Examination of the gene expression mechanism using reporter swap constructs reveals that Not1 depletion increases reporter mRNAs with the 3'UTR of 4E-BP gene, but decreases the ones with the 4E-BP promoter region, suggesting that Ccr4-Not complex regulates both degradation and transcription of 4E-BP mRNA.These results indicate that the Ccr4-Not complex controls expression of a single gene at multiple levels and adjusts the magnitude of the total effect.Thus, our study reveals a novel regulatory mechanism of a key component of the mTOR signaling pathway at the level of gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Systems Biology, Swiss Federal Institute of Technology (ETH) Zurich, Wolfgang Pauli Str. 16, 8093, Zürich, Switzerland.

ABSTRACT
The mechanistic target of rapamycin (mTOR) signaling pathway is highly conserved from yeast to humans. It senses various environmental cues to regulate cellular growth and homeostasis. Deregulation of the pathway has been implicated in many pathological conditions including cancer. Phosphorylation cascades through the pathway have been extensively studied but not much is known about the regulation of gene expression of the pathway components. Here, we report that the mRNA level of eukaryotic translation initiation factor (eIF) subunit 4E-binding protein (4E-BP) gene, one of the key mTOR signaling components, is regulated by the highly conserved Ccr4-Not complex. RNAi knockdown of Not1, a putative scaffold protein of this protein complex, increases the mRNA level of 4E-BP in Drosophila Kc cells. Examination of the gene expression mechanism using reporter swap constructs reveals that Not1 depletion increases reporter mRNAs with the 3'UTR of 4E-BP gene, but decreases the ones with the 4E-BP promoter region, suggesting that Ccr4-Not complex regulates both degradation and transcription of 4E-BP mRNA. These results indicate that the Ccr4-Not complex controls expression of a single gene at multiple levels and adjusts the magnitude of the total effect. Thus, our study reveals a novel regulatory mechanism of a key component of the mTOR signaling pathway at the level of gene expression.

Show MeSH
Related in: MedlinePlus