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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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Caf1 interacts with TTP and mediates deadenylation of globin-ARE-mRNA. (A) HeLa cells were transiently transfected with YFP, YFP–TTP or TTP–M1,2. Cytoplasmic lysates (input) were prepared after 24 h for IP with GFP-binder. Inputs were divided and one half was treated with RNase A during IP. Western blot analysis was carried out with antibodies against YFP and endogenous Caf1a. (B) HeLa cells were transiently transfected with TTP, the Tet-Off transactivator and a pTet-Off-driven β-globin reporter gene containing the ARE of TNF-α in its 3′-UTR. Cells were co-transfected with either empty vector, Caf1a-wt or dominant negative Caf1a-AA. Reporter gene transcription was blocked specifically by addition of doxycycline 20 h after transfection, and total RNA was isolated after the indicated time intervals. Globin-ARE and nucleolin mRNA were detected by northern blot analysis. dT indicates that the RNA was treated with oligo-dT and RNase H to generate deadenylated mRNA. In the middle 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. In the bottom panel, the overall signal intensity of globin-ARE mRNA was quantified and normalized to nucleolin mRNA. Average values ± SE were obtained from three biological repeat experiments and plotted as % of the initial time point.
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Figure 4: Caf1 interacts with TTP and mediates deadenylation of globin-ARE-mRNA. (A) HeLa cells were transiently transfected with YFP, YFP–TTP or TTP–M1,2. Cytoplasmic lysates (input) were prepared after 24 h for IP with GFP-binder. Inputs were divided and one half was treated with RNase A during IP. Western blot analysis was carried out with antibodies against YFP and endogenous Caf1a. (B) HeLa cells were transiently transfected with TTP, the Tet-Off transactivator and a pTet-Off-driven β-globin reporter gene containing the ARE of TNF-α in its 3′-UTR. Cells were co-transfected with either empty vector, Caf1a-wt or dominant negative Caf1a-AA. Reporter gene transcription was blocked specifically by addition of doxycycline 20 h after transfection, and total RNA was isolated after the indicated time intervals. Globin-ARE and nucleolin mRNA were detected by northern blot analysis. dT indicates that the RNA was treated with oligo-dT and RNase H to generate deadenylated mRNA. In the middle 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. In the bottom panel, the overall signal intensity of globin-ARE mRNA was quantified and normalized to nucleolin mRNA. Average values ± SE were obtained from three biological repeat experiments and plotted as % of the initial time point.

Mentions: In yeast, Ccr4 and Caf1 were found to be the major cytoplasmic deadenylases (35), whereas in trypanosomes, Drosophila and human cells, Caf1 appears to be the predominant deadenylase (27,49). Previously, we have shown that in human HT1080 fibrosarcoma cells, Caf1 is of particular importance for the rapid degradation of an ARE-containing mRNA (27). To further establish the link between Caf1 and AMD, we examined whether TTP could co-IP with endogenous Caf1a in HEK293 cells. Indeed, we found that YFP-tagged TTP associates with Caf1a (Figure 4A, lane 6), whereas YFP alone did not (lane 4). Importantly, addition of RNase A to the IP did not reduce the interaction of YFP-TTP with Caf1a (lane 7). Given that YFP-TTP-M1,2 also co-IPs with Caf1a (lanes 8 and 9), we concluded that TTP associates with the Caf1 deadenylase in an RNA-independent manner.Figure 4.


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)

Caf1 interacts with TTP and mediates deadenylation of globin-ARE-mRNA. (A) HeLa cells were transiently transfected with YFP, YFP–TTP or TTP–M1,2. Cytoplasmic lysates (input) were prepared after 24 h for IP with GFP-binder. Inputs were divided and one half was treated with RNase A during IP. Western blot analysis was carried out with antibodies against YFP and endogenous Caf1a. (B) HeLa cells were transiently transfected with TTP, the Tet-Off transactivator and a pTet-Off-driven β-globin reporter gene containing the ARE of TNF-α in its 3′-UTR. Cells were co-transfected with either empty vector, Caf1a-wt or dominant negative Caf1a-AA. Reporter gene transcription was blocked specifically by addition of doxycycline 20 h after transfection, and total RNA was isolated after the indicated time intervals. Globin-ARE and nucleolin mRNA were detected by northern blot analysis. dT indicates that the RNA was treated with oligo-dT and RNase H to generate deadenylated mRNA. In the middle 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. In the bottom panel, the overall signal intensity of globin-ARE mRNA was quantified and normalized to nucleolin mRNA. Average values ± SE were obtained from three biological repeat experiments and plotted as % of the initial time point.
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Figure 4: Caf1 interacts with TTP and mediates deadenylation of globin-ARE-mRNA. (A) HeLa cells were transiently transfected with YFP, YFP–TTP or TTP–M1,2. Cytoplasmic lysates (input) were prepared after 24 h for IP with GFP-binder. Inputs were divided and one half was treated with RNase A during IP. Western blot analysis was carried out with antibodies against YFP and endogenous Caf1a. (B) HeLa cells were transiently transfected with TTP, the Tet-Off transactivator and a pTet-Off-driven β-globin reporter gene containing the ARE of TNF-α in its 3′-UTR. Cells were co-transfected with either empty vector, Caf1a-wt or dominant negative Caf1a-AA. Reporter gene transcription was blocked specifically by addition of doxycycline 20 h after transfection, and total RNA was isolated after the indicated time intervals. Globin-ARE and nucleolin mRNA were detected by northern blot analysis. dT indicates that the RNA was treated with oligo-dT and RNase H to generate deadenylated mRNA. In the middle 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. In the bottom panel, the overall signal intensity of globin-ARE mRNA was quantified and normalized to nucleolin mRNA. Average values ± SE were obtained from three biological repeat experiments and plotted as % of the initial time point.
Mentions: In yeast, Ccr4 and Caf1 were found to be the major cytoplasmic deadenylases (35), whereas in trypanosomes, Drosophila and human cells, Caf1 appears to be the predominant deadenylase (27,49). Previously, we have shown that in human HT1080 fibrosarcoma cells, Caf1 is of particular importance for the rapid degradation of an ARE-containing mRNA (27). To further establish the link between Caf1 and AMD, we examined whether TTP could co-IP with endogenous Caf1a in HEK293 cells. Indeed, we found that YFP-tagged TTP associates with Caf1a (Figure 4A, lane 6), whereas YFP alone did not (lane 4). Importantly, addition of RNase A to the IP did not reduce the interaction of YFP-TTP with Caf1a (lane 7). Given that YFP-TTP-M1,2 also co-IPs with Caf1a (lanes 8 and 9), we concluded that TTP associates with the Caf1 deadenylase in an RNA-independent manner.Figure 4.

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