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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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Regulation of 4E-BP translation by Ccr4-Not complex is not detected.A. Schematic of reporter constructs that were used for protein level assessment. B. Effect of Not1 reduction on the protein levels of the reporter constructs. Cells were under Not1 RNAi and transfection with the indicated constructs. After serum starvation overnight and insulin stimulation for 30 min, cells were lysed and subjected to Western blotting analysis using antibodies against GFP and tubulin. The GFP protein levels (normalized to tubulin) were densitometrically quantified (Image J) from two independent experiments and normalized to untreated (no RNAi/insulin). Means ± SEM are shown. A representative blot is shown. C. Relative mRNA and protein levels of the reporter constructs normalized to pAAA.
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pone.0113902.g005: Regulation of 4E-BP translation by Ccr4-Not complex is not detected.A. Schematic of reporter constructs that were used for protein level assessment. B. Effect of Not1 reduction on the protein levels of the reporter constructs. Cells were under Not1 RNAi and transfection with the indicated constructs. After serum starvation overnight and insulin stimulation for 30 min, cells were lysed and subjected to Western blotting analysis using antibodies against GFP and tubulin. The GFP protein levels (normalized to tubulin) were densitometrically quantified (Image J) from two independent experiments and normalized to untreated (no RNAi/insulin). Means ± SEM are shown. A representative blot is shown. C. Relative mRNA and protein levels of the reporter constructs normalized to pAAA.

Mentions: Translational inhibition by Ccr4-Not complex has been implicated. Poly(A) tails are considered to have a role in the initiation of translation by recruiting poly(A) binding proteins that interact with eukaryotic initiation factor 4G bound to 5’ cap of the mRNA [35,36,37]. Poly(A) shortening by Ccr4-Not complex can be connected to translational repression. In addition, Ccr4-Not complex has been shown to interact with decapping factors [38,39,40]. To investigate the possible effects of Not1 on the translation of the 4E-BP gene, we determined the protein levels of the reporter constructs (Fig. 5A). Importantly, since all reporter constructs produce EGFP proteins, any effects of the Ccr4-Not1 complex on the 4E-BP protein (e.g. degradation via ubiquitination) are eliminated. Thus, significant differences between the EGFP protein levels and the EGFP mRNA levels of each reporter construct can be interpreted as Not1-specific alterations in the translation of the 4E-BP mRNA. Cell lysates were analyzed by Western blotting using a GFP antibody (Fig. 5B). Not1 knockdown increased the EGFP protein levels of the construct carrying the 4E-BP 3’UTR (pAAE), but decreased the EGFP protein levels of the construct containing the 4E-BP promoter region and 5’UTR (pEEA), which is reminiscent of the corresponding mRNA levels. Indeed, comparative analysis between the appropriate EGFP mRNA and protein levels showed that the relative differences between the constructs are mostly identical (Fig. 5C). The same experiments were performed in the absence of insulin (S1A and B Fig.), which did not make differences between the protein and mRNA levels of the reporter constructs. Thus, our study did not identify 4E-BP-specific translation control by Ccr4-Not complex.


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

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

Regulation of 4E-BP translation by Ccr4-Not complex is not detected.A. Schematic of reporter constructs that were used for protein level assessment. B. Effect of Not1 reduction on the protein levels of the reporter constructs. Cells were under Not1 RNAi and transfection with the indicated constructs. After serum starvation overnight and insulin stimulation for 30 min, cells were lysed and subjected to Western blotting analysis using antibodies against GFP and tubulin. The GFP protein levels (normalized to tubulin) were densitometrically quantified (Image J) from two independent experiments and normalized to untreated (no RNAi/insulin). Means ± SEM are shown. A representative blot is shown. C. Relative mRNA and protein levels of the reporter constructs normalized to pAAA.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4368434&req=5

pone.0113902.g005: Regulation of 4E-BP translation by Ccr4-Not complex is not detected.A. Schematic of reporter constructs that were used for protein level assessment. B. Effect of Not1 reduction on the protein levels of the reporter constructs. Cells were under Not1 RNAi and transfection with the indicated constructs. After serum starvation overnight and insulin stimulation for 30 min, cells were lysed and subjected to Western blotting analysis using antibodies against GFP and tubulin. The GFP protein levels (normalized to tubulin) were densitometrically quantified (Image J) from two independent experiments and normalized to untreated (no RNAi/insulin). Means ± SEM are shown. A representative blot is shown. C. Relative mRNA and protein levels of the reporter constructs normalized to pAAA.
Mentions: Translational inhibition by Ccr4-Not complex has been implicated. Poly(A) tails are considered to have a role in the initiation of translation by recruiting poly(A) binding proteins that interact with eukaryotic initiation factor 4G bound to 5’ cap of the mRNA [35,36,37]. Poly(A) shortening by Ccr4-Not complex can be connected to translational repression. In addition, Ccr4-Not complex has been shown to interact with decapping factors [38,39,40]. To investigate the possible effects of Not1 on the translation of the 4E-BP gene, we determined the protein levels of the reporter constructs (Fig. 5A). Importantly, since all reporter constructs produce EGFP proteins, any effects of the Ccr4-Not1 complex on the 4E-BP protein (e.g. degradation via ubiquitination) are eliminated. Thus, significant differences between the EGFP protein levels and the EGFP mRNA levels of each reporter construct can be interpreted as Not1-specific alterations in the translation of the 4E-BP mRNA. Cell lysates were analyzed by Western blotting using a GFP antibody (Fig. 5B). Not1 knockdown increased the EGFP protein levels of the construct carrying the 4E-BP 3’UTR (pAAE), but decreased the EGFP protein levels of the construct containing the 4E-BP promoter region and 5’UTR (pEEA), which is reminiscent of the corresponding mRNA levels. Indeed, comparative analysis between the appropriate EGFP mRNA and protein levels showed that the relative differences between the constructs are mostly identical (Fig. 5C). The same experiments were performed in the absence of insulin (S1A and B Fig.), which did not make differences between the protein and mRNA levels of the reporter constructs. Thus, our study did not identify 4E-BP-specific translation control by Ccr4-Not complex.

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