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Not1 mediates recruitment of the deadenylase Caf1 to mRNAs targeted for degradation by tristetraprolin.

Sandler H, Kreth J, Timmers HT, Stoecklin G - Nucleic Acids Res. (2011)

Bottom Line: In the cytoplasm, the complex is required for messenger RNA (mRNA) turnover through its two associated deadenylases, Ccr4 and Caf1.Here, we provide evidence that human Not1 in the cytoplasm associates with the C-terminal domain of tristetraprolin (TTP), an RNA binding protein that mediates rapid degradation of mRNAs containing AU-rich elements (AREs).Not1 shows extensive interaction through its central region with TTP, whereas binding of Caf1 is restricted to a smaller central domain within Not1.

View Article: PubMed Central - PubMed

Affiliation: Helmholtz Junior Research Group Posttranscriptional Control of Gene Expression, German Cancer Research Center, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

ABSTRACT
The carbon catabolite repressor protein 4 (Ccr4)-Negative on TATA (Not) complex controls gene expression at two levels. In the nucleus, it regulates the basal transcription machinery, nuclear receptor-mediated transcription and histone modifications. In the cytoplasm, the complex is required for messenger RNA (mRNA) turnover through its two associated deadenylases, Ccr4 and Caf1. Not1 is the largest protein of the Ccr4-Not complex and serves as a scaffold for other subunits of the complex. Here, we provide evidence that human Not1 in the cytoplasm associates with the C-terminal domain of tristetraprolin (TTP), an RNA binding protein that mediates rapid degradation of mRNAs containing AU-rich elements (AREs). Not1 shows extensive interaction through its central region with TTP, whereas binding of Caf1 is restricted to a smaller central domain within Not1. Importantly, Not1 is required for the rapid decay of ARE-mRNAs, and TTP can recruit the Caf1 deadenylase only in presence of Not1. Thus, cytoplasmic Not1 provides a platform that allows a specific RNA binding protein to recruit the Caf1 deadenylase and thereby trigger decay of its target mRNAs.

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Related in: MedlinePlus

Not1 is required for ARE-mediated mRNA deadenylation and decay. (A) HeLa cells were transfected with two independent control siRNAs or two siRNAs targeting Not1. After 48 h, cells were transfected again with the same siRNAs together with a plasmid encoding TTP, a pTet-Off-driven β-globin reporter gene containing the ARE of TNF-α in its 3′ UTR, and the Tet-Off transactivator. Reporter gene transcription was blocked specifically by addition of doxycycline 24 h after the second transfection, and RNA was isolated after the time intervals indicated. Globin-ARE and nucleolin mRNA were detected by northern blot analysis; a high and low exposure is provided for the globin-ARE signal. The size of the mRNA was determined by comparison to an RNA marker. In the bottom panel, deadenylation was visualized by quantifying the signal intensity of globin-ARE mRNA along the length of the signal and plotting it as a function of mRNA size. (B) Quantification of the globin-ARE reporter mRNA decay in panel A and repeat experiments. Signal intensities of globin-ARE mRNA were normalized to nucleolin mRNA and represented as % of the initial value. Results from the two control and the two Not1 siRNA transfections were combined. The graph shows average values ± SE from eight (si-control) and six (si-Not1) biological repeat experiments. (C) HeLa cells were transfected twice with siRNAs as in panel A. Not1 mRNA levels were quantified by real-time PCR using nucleolin mRNA for normalization. (D) HEK293 cells were first transfected with Flag-Not1, and 1 day later with the siRNAs indicated. After one additional day, cytoplasmic lysates were prepared and analyzed by western blotting using antibodies against Flag and 14-3-3.
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Figure 2: Not1 is required for ARE-mediated mRNA deadenylation and decay. (A) HeLa cells were transfected with two independent control siRNAs or two siRNAs targeting Not1. After 48 h, cells were transfected again with the same siRNAs together with a plasmid encoding TTP, a pTet-Off-driven β-globin reporter gene containing the ARE of TNF-α in its 3′ UTR, and the Tet-Off transactivator. Reporter gene transcription was blocked specifically by addition of doxycycline 24 h after the second transfection, and RNA was isolated after the time intervals indicated. Globin-ARE and nucleolin mRNA were detected by northern blot analysis; a high and low exposure is provided for the globin-ARE signal. The size of the mRNA was determined by comparison to an RNA marker. In the bottom panel, deadenylation was visualized by quantifying the signal intensity of globin-ARE mRNA along the length of the signal and plotting it as a function of mRNA size. (B) Quantification of the globin-ARE reporter mRNA decay in panel A and repeat experiments. Signal intensities of globin-ARE mRNA were normalized to nucleolin mRNA and represented as % of the initial value. Results from the two control and the two Not1 siRNA transfections were combined. The graph shows average values ± SE from eight (si-control) and six (si-Not1) biological repeat experiments. (C) HeLa cells were transfected twice with siRNAs as in panel A. Not1 mRNA levels were quantified by real-time PCR using nucleolin mRNA for normalization. (D) HEK293 cells were first transfected with Flag-Not1, and 1 day later with the siRNAs indicated. After one additional day, cytoplasmic lysates were prepared and analyzed by western blotting using antibodies against Flag and 14-3-3.

Mentions: Next, we determined whether Not1 is required for AMD. We first reduced Not1 expression by transfection of two different siRNAs in HeLa cells, and subsequently transfected TTP together with a β-globin reporter gene that contains the ARE of TNFα in its 3′-UTR and is driven by a tetracycline-sensitive promoter. Degradation of the reporter mRNA was measured after blocking its transcription with doxycycline. In the presence of two different control siRNAs, the globin-ARE mRNA showed very rapid deadenylation and subsequent degradation (Figure 2A, left panels). In contrast, both siRNAs directed against Not1 caused a strong stabilization of the globin-ARE mRNA (right panels). By analyzing the reporter mRNA size, we noticed that the globin-ARE mRNA remained above the 800 nt mark after Not1 knock down, whereas it was shortened to ∼700 nt in the control conditions. A lower exposure of the globin-ARE mRNA signal in the second panel of Figure 2A clearly shows the deadenylated mRNA species that is present in the control knock downs, but absent in the Not1 knock downs. The difference was visualized by plotting the signal intensity as a function of mRNA size in the panels below of the northern blots of Figure 2A. This result indicated that Not1 is primarily required for full deadenylation of the reporter mRNA. Quantification of the overall mRNA signals in three biological repeat experiments showed that knocking down Not1 increases the half-life of globin-ARE mRNA from 0.5 to 1.2 h (Figure 2B). By quantitative PCR, we determined that the two siRNAs reduced Not1 mRNA expression levels to ∼10% and 30%, respectively (Figure 2C). Knock down efficiency was also confirmed at the protein level, as both siRNAs reduced Flag-Not1 levels below the detection limit of our western blot analysis (Figure 2D). Taken together, we concluded from these experiments that Not1 is required for AMD of a reporter mRNA containing a class II ARE.Figure 2.


Not1 mediates recruitment of the deadenylase Caf1 to mRNAs targeted for degradation by tristetraprolin.

Sandler H, Kreth J, Timmers HT, Stoecklin G - Nucleic Acids Res. (2011)

Not1 is required for ARE-mediated mRNA deadenylation and decay. (A) HeLa cells were transfected with two independent control siRNAs or two siRNAs targeting Not1. After 48 h, cells were transfected again with the same siRNAs together with a plasmid encoding TTP, a pTet-Off-driven β-globin reporter gene containing the ARE of TNF-α in its 3′ UTR, and the Tet-Off transactivator. Reporter gene transcription was blocked specifically by addition of doxycycline 24 h after the second transfection, and RNA was isolated after the time intervals indicated. Globin-ARE and nucleolin mRNA were detected by northern blot analysis; a high and low exposure is provided for the globin-ARE signal. The size of the mRNA was determined by comparison to an RNA marker. In the bottom panel, deadenylation was visualized by quantifying the signal intensity of globin-ARE mRNA along the length of the signal and plotting it as a function of mRNA size. (B) Quantification of the globin-ARE reporter mRNA decay in panel A and repeat experiments. Signal intensities of globin-ARE mRNA were normalized to nucleolin mRNA and represented as % of the initial value. Results from the two control and the two Not1 siRNA transfections were combined. The graph shows average values ± SE from eight (si-control) and six (si-Not1) biological repeat experiments. (C) HeLa cells were transfected twice with siRNAs as in panel A. Not1 mRNA levels were quantified by real-time PCR using nucleolin mRNA for normalization. (D) HEK293 cells were first transfected with Flag-Not1, and 1 day later with the siRNAs indicated. After one additional day, cytoplasmic lysates were prepared and analyzed by western blotting using antibodies against Flag and 14-3-3.
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Figure 2: Not1 is required for ARE-mediated mRNA deadenylation and decay. (A) HeLa cells were transfected with two independent control siRNAs or two siRNAs targeting Not1. After 48 h, cells were transfected again with the same siRNAs together with a plasmid encoding TTP, a pTet-Off-driven β-globin reporter gene containing the ARE of TNF-α in its 3′ UTR, and the Tet-Off transactivator. Reporter gene transcription was blocked specifically by addition of doxycycline 24 h after the second transfection, and RNA was isolated after the time intervals indicated. Globin-ARE and nucleolin mRNA were detected by northern blot analysis; a high and low exposure is provided for the globin-ARE signal. The size of the mRNA was determined by comparison to an RNA marker. In the bottom panel, deadenylation was visualized by quantifying the signal intensity of globin-ARE mRNA along the length of the signal and plotting it as a function of mRNA size. (B) Quantification of the globin-ARE reporter mRNA decay in panel A and repeat experiments. Signal intensities of globin-ARE mRNA were normalized to nucleolin mRNA and represented as % of the initial value. Results from the two control and the two Not1 siRNA transfections were combined. The graph shows average values ± SE from eight (si-control) and six (si-Not1) biological repeat experiments. (C) HeLa cells were transfected twice with siRNAs as in panel A. Not1 mRNA levels were quantified by real-time PCR using nucleolin mRNA for normalization. (D) HEK293 cells were first transfected with Flag-Not1, and 1 day later with the siRNAs indicated. After one additional day, cytoplasmic lysates were prepared and analyzed by western blotting using antibodies against Flag and 14-3-3.
Mentions: Next, we determined whether Not1 is required for AMD. We first reduced Not1 expression by transfection of two different siRNAs in HeLa cells, and subsequently transfected TTP together with a β-globin reporter gene that contains the ARE of TNFα in its 3′-UTR and is driven by a tetracycline-sensitive promoter. Degradation of the reporter mRNA was measured after blocking its transcription with doxycycline. In the presence of two different control siRNAs, the globin-ARE mRNA showed very rapid deadenylation and subsequent degradation (Figure 2A, left panels). In contrast, both siRNAs directed against Not1 caused a strong stabilization of the globin-ARE mRNA (right panels). By analyzing the reporter mRNA size, we noticed that the globin-ARE mRNA remained above the 800 nt mark after Not1 knock down, whereas it was shortened to ∼700 nt in the control conditions. A lower exposure of the globin-ARE mRNA signal in the second panel of Figure 2A clearly shows the deadenylated mRNA species that is present in the control knock downs, but absent in the Not1 knock downs. The difference was visualized by plotting the signal intensity as a function of mRNA size in the panels below of the northern blots of Figure 2A. This result indicated that Not1 is primarily required for full deadenylation of the reporter mRNA. Quantification of the overall mRNA signals in three biological repeat experiments showed that knocking down Not1 increases the half-life of globin-ARE mRNA from 0.5 to 1.2 h (Figure 2B). By quantitative PCR, we determined that the two siRNAs reduced Not1 mRNA expression levels to ∼10% and 30%, respectively (Figure 2C). Knock down efficiency was also confirmed at the protein level, as both siRNAs reduced Flag-Not1 levels below the detection limit of our western blot analysis (Figure 2D). Taken together, we concluded from these experiments that Not1 is required for AMD of a reporter mRNA containing a class II ARE.Figure 2.

Bottom Line: In the cytoplasm, the complex is required for messenger RNA (mRNA) turnover through its two associated deadenylases, Ccr4 and Caf1.Here, we provide evidence that human Not1 in the cytoplasm associates with the C-terminal domain of tristetraprolin (TTP), an RNA binding protein that mediates rapid degradation of mRNAs containing AU-rich elements (AREs).Not1 shows extensive interaction through its central region with TTP, whereas binding of Caf1 is restricted to a smaller central domain within Not1.

View Article: PubMed Central - PubMed

Affiliation: Helmholtz Junior Research Group Posttranscriptional Control of Gene Expression, German Cancer Research Center, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

ABSTRACT
The carbon catabolite repressor protein 4 (Ccr4)-Negative on TATA (Not) complex controls gene expression at two levels. In the nucleus, it regulates the basal transcription machinery, nuclear receptor-mediated transcription and histone modifications. In the cytoplasm, the complex is required for messenger RNA (mRNA) turnover through its two associated deadenylases, Ccr4 and Caf1. Not1 is the largest protein of the Ccr4-Not complex and serves as a scaffold for other subunits of the complex. Here, we provide evidence that human Not1 in the cytoplasm associates with the C-terminal domain of tristetraprolin (TTP), an RNA binding protein that mediates rapid degradation of mRNAs containing AU-rich elements (AREs). Not1 shows extensive interaction through its central region with TTP, whereas binding of Caf1 is restricted to a smaller central domain within Not1. Importantly, Not1 is required for the rapid decay of ARE-mRNAs, and TTP can recruit the Caf1 deadenylase only in presence of Not1. Thus, cytoplasmic Not1 provides a platform that allows a specific RNA binding protein to recruit the Caf1 deadenylase and thereby trigger decay of its target mRNAs.

Show MeSH
Related in: MedlinePlus