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

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.

Fig 1. Depletion of Ccr4-Not complex components in Kc cells increases phosphorylation of 4E-BP and cell size upon insulin stimulation.

A. Increase in insulin-induced 4E-BP phosphorylation by RNAi knockdown of Ccr4-Not complex components. Kc cells were subjected to RNAi treatments against the indicated genes and stimulated with insulin 30 min before cell lysis. The lysates were analyzed by Western blotting using antibodies against anti-phospho 4E-BP and tubulin. B. Increased cell size caused by RNAi knockdown of Ccr4-Not complex components. Kc cells were treated as in A. The cell size (area) was measured from three independent experiments and normalized to control (EGFP RNAi). Data are presented as Means ± SEM.

Fig 2. Not1 depletion changes the mRNA level of 4E-BP.

Kc cells were treated with Not1 or mock (EGFP) RNAi and mRNA levels of the indicated genes were measured by qPCR and normalized to mock RNAi. Data are represented as Means ± SEM from three independent experiments. A. Increased 4E-BP mRNA levels after Not1 knockdown and insulin stimulation. Kc cells were insulin-stimulated 30 min before lysis. B. Increased 4E-BP mRNA levels after Not1 knockdown without concomitant stimulation. C. Combined effect of insulin stimulation and Not1 RNAi on 4E-BP mRNA levels.

Fig 3. Not1 reduction increases 4E-BP mRNA through the 3’UTR of 4E-BP gene.

A. Schematic illustration of the reporter constructs in which the 5’UTR or 3’UTR was exchanged with the counterpart of 4E-BP gene. B. Changes of the reporter mRNA levels by Not1 depletion. Cells were transfected with the reporter constructs and subjected to Not1 RNAi. After serum starvation overnight and 30 min 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.

Fig 4. 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).

Fig 5. 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.

Fig 6. Multi-functional regulation of 4E-BP gene expression by Ccr4-Not Complex.

This figure summarizes the current working hypothesis of how the Ccr4-Not complex regulates the expression of 4E-BP. The balance between transcriptional regulation and mRNA decay exerted by the Ccr4-Not complex determines—at least in part—the total protein levels of 4E-BP. Whether the transcriptional control by the complex is mediated by direct binding to the promoter or by indirectly controlling other factors such as transcription factors is not yet clear.