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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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Not1 associates with TTP and Caf1a differentially and is required for the interaction between TTP and Caf1a. (A) Schematic representation of the human Not1 fragments used in this study; aa, amino acids. (B) HEK293 cells were transiently transfected with Flag-tagged Not1 constructs as indicated together with YFP-TTP. Cytoplasmic lysates (input) were prepared after 24 h for IP with Flag antibody. Western blot analysis was carried out with antibodies against YFP, Flag and endogenous Caf1a. HC stands for IgG heavy chain. (C) HEK293 cells were transiently transfected with YFP-tagged Not1 constructs as indicated together with TTP-myc. Cytoplasmic lysates (input) were prepared after 24 h for IP with GFP-binder. Western blot analysis was carried out with antibodies against YFP, myc and endogenous Caf1a. (D) HeLa cells were transfected simultaneously with either two control siRNAs or two siRNAs targeting Not1. After 48 h, cells were transfected with the same siRNAs again together with YFP or YFP–TTP. Cytoplasmic lysates (input) were prepared 24 h later for IP with GFP-binder. Western blot analysis was carried out with antibodies against YFP and endogenous Caf1a.
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Figure 6: Not1 associates with TTP and Caf1a differentially and is required for the interaction between TTP and Caf1a. (A) Schematic representation of the human Not1 fragments used in this study; aa, amino acids. (B) HEK293 cells were transiently transfected with Flag-tagged Not1 constructs as indicated together with YFP-TTP. Cytoplasmic lysates (input) were prepared after 24 h for IP with Flag antibody. Western blot analysis was carried out with antibodies against YFP, Flag and endogenous Caf1a. HC stands for IgG heavy chain. (C) HEK293 cells were transiently transfected with YFP-tagged Not1 constructs as indicated together with TTP-myc. Cytoplasmic lysates (input) were prepared after 24 h for IP with GFP-binder. Western blot analysis was carried out with antibodies against YFP, myc and endogenous Caf1a. (D) HeLa cells were transfected simultaneously with either two control siRNAs or two siRNAs targeting Not1. After 48 h, cells were transfected with the same siRNAs again together with YFP or YFP–TTP. Cytoplasmic lysates (input) were prepared 24 h later for IP with GFP-binder. Western blot analysis was carried out with antibodies against YFP and endogenous Caf1a.

Mentions: To further examine the interactions between TTP, Not1 and Caf1a, we divided the human Not1 cDNA, which encodes for a 2376-amino acid long protein, into several fragments (Figure 6A). The most N-terminal fragment of Not1 encompassing amino acid 1–700 did not co-IP with TTP, whereas a longer N-terminal fragment covering amino acid 1–997 did interact with TTP (Figure 6B, lanes 7 and 8). Importantly, neither of these two fragments showed an interaction with Caf1a. This result suggests that the Not1 region between amino acid 700 and 997 is important for the interaction with TTP, and that TTP can associate with Not1 independently of Caf1.Figure 6.


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 associates with TTP and Caf1a differentially and is required for the interaction between TTP and Caf1a. (A) Schematic representation of the human Not1 fragments used in this study; aa, amino acids. (B) HEK293 cells were transiently transfected with Flag-tagged Not1 constructs as indicated together with YFP-TTP. Cytoplasmic lysates (input) were prepared after 24 h for IP with Flag antibody. Western blot analysis was carried out with antibodies against YFP, Flag and endogenous Caf1a. HC stands for IgG heavy chain. (C) HEK293 cells were transiently transfected with YFP-tagged Not1 constructs as indicated together with TTP-myc. Cytoplasmic lysates (input) were prepared after 24 h for IP with GFP-binder. Western blot analysis was carried out with antibodies against YFP, myc and endogenous Caf1a. (D) HeLa cells were transfected simultaneously with either two control siRNAs or two siRNAs targeting Not1. After 48 h, cells were transfected with the same siRNAs again together with YFP or YFP–TTP. Cytoplasmic lysates (input) were prepared 24 h later for IP with GFP-binder. Western blot analysis was carried out with antibodies against YFP and endogenous Caf1a.
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Figure 6: Not1 associates with TTP and Caf1a differentially and is required for the interaction between TTP and Caf1a. (A) Schematic representation of the human Not1 fragments used in this study; aa, amino acids. (B) HEK293 cells were transiently transfected with Flag-tagged Not1 constructs as indicated together with YFP-TTP. Cytoplasmic lysates (input) were prepared after 24 h for IP with Flag antibody. Western blot analysis was carried out with antibodies against YFP, Flag and endogenous Caf1a. HC stands for IgG heavy chain. (C) HEK293 cells were transiently transfected with YFP-tagged Not1 constructs as indicated together with TTP-myc. Cytoplasmic lysates (input) were prepared after 24 h for IP with GFP-binder. Western blot analysis was carried out with antibodies against YFP, myc and endogenous Caf1a. (D) HeLa cells were transfected simultaneously with either two control siRNAs or two siRNAs targeting Not1. After 48 h, cells were transfected with the same siRNAs again together with YFP or YFP–TTP. Cytoplasmic lysates (input) were prepared 24 h later for IP with GFP-binder. Western blot analysis was carried out with antibodies against YFP and endogenous Caf1a.
Mentions: To further examine the interactions between TTP, Not1 and Caf1a, we divided the human Not1 cDNA, which encodes for a 2376-amino acid long protein, into several fragments (Figure 6A). The most N-terminal fragment of Not1 encompassing amino acid 1–700 did not co-IP with TTP, whereas a longer N-terminal fragment covering amino acid 1–997 did interact with TTP (Figure 6B, lanes 7 and 8). Importantly, neither of these two fragments showed an interaction with Caf1a. This result suggests that the Not1 region between amino acid 700 and 997 is important for the interaction with TTP, and that TTP can associate with Not1 independently of Caf1.Figure 6.

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