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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

TTP C-terminal domain interacts with Not1–Caf1a and induces mRNA deadenylation. (A) Schematic representation of the murine TTP fragments used in this study. N, N-terminal domain; Z, zinc-finger domain; C, C-terminal domain; aa, amino acids. (B) HEK293 cells were transiently transfected with YFP-tagged TTP constructs as indicated together with Flag-Not1. Cytoplasmic lysates (input) were prepared after 24 h for IP with GFP-binder. Inputs were divided and one half was treated with RNase I during IP. Western blot analysis was carried out with antibodies against YFP, Flag and endogenous Caf1a. To control RNase I efficiency, RNA was isolated from unbound fractions. Ribosomal and tRNA was visualized by ethidium bromide staining, nucleolin mRNA was visualized on a northern blot. (C) HeLa cells were transiently transfected with MS2cp-TTP-C or MS2cp-TTP-N together with the Tet-Off transactivator and a pTet-Off-driven β-globin reporter gene that contains six MS2-binding sites (bs) in its 3′ UTR. In addition, empty vector or dominant negative Caf1a-AA was co-transfected. Reporter gene transcription was blocked specifically by addition of doxycycline 20 h after transfection, and total RNA was isolated after the time intervals indicated. Globin-MS2bs 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-MS2bs 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-MS2bs mRNA was quantified and normalized to nucleolin mRNA. Average values ± SE were obtained from three biological repeat experiments and plotted as percentage of the initial time point.
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Figure 5: TTP C-terminal domain interacts with Not1–Caf1a and induces mRNA deadenylation. (A) Schematic representation of the murine TTP fragments used in this study. N, N-terminal domain; Z, zinc-finger domain; C, C-terminal domain; aa, amino acids. (B) HEK293 cells were transiently transfected with YFP-tagged TTP constructs as indicated together with Flag-Not1. Cytoplasmic lysates (input) were prepared after 24 h for IP with GFP-binder. Inputs were divided and one half was treated with RNase I during IP. Western blot analysis was carried out with antibodies against YFP, Flag and endogenous Caf1a. To control RNase I efficiency, RNA was isolated from unbound fractions. Ribosomal and tRNA was visualized by ethidium bromide staining, nucleolin mRNA was visualized on a northern blot. (C) HeLa cells were transiently transfected with MS2cp-TTP-C or MS2cp-TTP-N together with the Tet-Off transactivator and a pTet-Off-driven β-globin reporter gene that contains six MS2-binding sites (bs) in its 3′ UTR. In addition, empty vector or dominant negative Caf1a-AA was co-transfected. Reporter gene transcription was blocked specifically by addition of doxycycline 20 h after transfection, and total RNA was isolated after the time intervals indicated. Globin-MS2bs 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-MS2bs 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-MS2bs mRNA was quantified and normalized to nucleolin mRNA. Average values ± SE were obtained from three biological repeat experiments and plotted as percentage of the initial time point.

Mentions: TTP contains in its central part a tandem CCCH zinc-finger domain that is required for binding of AREs (13), whereas the N-terminal domain of TTP was found to stimulate mRNA decay by recruiting components of the general mRNA decay machinery (20). To further understand the interaction of TTP with Not1 and Caf1, we expressed the N- and C-terminal domains of TTP, schematically depicted in Figure 5A, in HEK293 cells. By co-IP analysis we found that the N-terminal domain of TTP (amino acid 1–94) does not interact with either Caf1a or Flag-Not1 (Figure 5B, lane 10). The NZ fragment (amino acid 1–176) comprising the N-terminus, the zing-finger domain and a small part of the C-terminal domain showed only a very weak interaction with Flag-Not1 or Caf1a (lane 12). In contrast, the C-terminal domain (amino acid 152–319) was able to co-IP Flag-Not1 and Caf1a as efficiently as full-length TTP (compare lanes 8 and 14). Treatment of the samples during IP with RNase I, which also cleaves RNA within a poly(A) tail, did not abrogate the interaction with either Flag-Not1 or Caf1a (lanes 9 and 15). Efficient degradation of RNA is shown in the bottom panels of Figure 5B for rRNA and nucleolin as an abundant mRNA. Since TTP–Not1/Caf1a interactions are technically challenging to detect, a repeat IP is shown in Supplementary Figure S1A and B. From these results we concluded that Not1 and Caf1a primarily interact with the C-terminal domain of TTP in an RNA-independent manner. We further tested the interaction of TTP with Ccr4a and again found the C-terminal domain of TTP to associate with HA-Ccr4a (Supplementary Figure S1C).Figure 5.


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)

TTP C-terminal domain interacts with Not1–Caf1a and induces mRNA deadenylation. (A) Schematic representation of the murine TTP fragments used in this study. N, N-terminal domain; Z, zinc-finger domain; C, C-terminal domain; aa, amino acids. (B) HEK293 cells were transiently transfected with YFP-tagged TTP constructs as indicated together with Flag-Not1. Cytoplasmic lysates (input) were prepared after 24 h for IP with GFP-binder. Inputs were divided and one half was treated with RNase I during IP. Western blot analysis was carried out with antibodies against YFP, Flag and endogenous Caf1a. To control RNase I efficiency, RNA was isolated from unbound fractions. Ribosomal and tRNA was visualized by ethidium bromide staining, nucleolin mRNA was visualized on a northern blot. (C) HeLa cells were transiently transfected with MS2cp-TTP-C or MS2cp-TTP-N together with the Tet-Off transactivator and a pTet-Off-driven β-globin reporter gene that contains six MS2-binding sites (bs) in its 3′ UTR. In addition, empty vector or dominant negative Caf1a-AA was co-transfected. Reporter gene transcription was blocked specifically by addition of doxycycline 20 h after transfection, and total RNA was isolated after the time intervals indicated. Globin-MS2bs 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-MS2bs 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-MS2bs mRNA was quantified and normalized to nucleolin mRNA. Average values ± SE were obtained from three biological repeat experiments and plotted as percentage of the initial time point.
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Figure 5: TTP C-terminal domain interacts with Not1–Caf1a and induces mRNA deadenylation. (A) Schematic representation of the murine TTP fragments used in this study. N, N-terminal domain; Z, zinc-finger domain; C, C-terminal domain; aa, amino acids. (B) HEK293 cells were transiently transfected with YFP-tagged TTP constructs as indicated together with Flag-Not1. Cytoplasmic lysates (input) were prepared after 24 h for IP with GFP-binder. Inputs were divided and one half was treated with RNase I during IP. Western blot analysis was carried out with antibodies against YFP, Flag and endogenous Caf1a. To control RNase I efficiency, RNA was isolated from unbound fractions. Ribosomal and tRNA was visualized by ethidium bromide staining, nucleolin mRNA was visualized on a northern blot. (C) HeLa cells were transiently transfected with MS2cp-TTP-C or MS2cp-TTP-N together with the Tet-Off transactivator and a pTet-Off-driven β-globin reporter gene that contains six MS2-binding sites (bs) in its 3′ UTR. In addition, empty vector or dominant negative Caf1a-AA was co-transfected. Reporter gene transcription was blocked specifically by addition of doxycycline 20 h after transfection, and total RNA was isolated after the time intervals indicated. Globin-MS2bs 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-MS2bs 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-MS2bs mRNA was quantified and normalized to nucleolin mRNA. Average values ± SE were obtained from three biological repeat experiments and plotted as percentage of the initial time point.
Mentions: TTP contains in its central part a tandem CCCH zinc-finger domain that is required for binding of AREs (13), whereas the N-terminal domain of TTP was found to stimulate mRNA decay by recruiting components of the general mRNA decay machinery (20). To further understand the interaction of TTP with Not1 and Caf1, we expressed the N- and C-terminal domains of TTP, schematically depicted in Figure 5A, in HEK293 cells. By co-IP analysis we found that the N-terminal domain of TTP (amino acid 1–94) does not interact with either Caf1a or Flag-Not1 (Figure 5B, lane 10). The NZ fragment (amino acid 1–176) comprising the N-terminus, the zing-finger domain and a small part of the C-terminal domain showed only a very weak interaction with Flag-Not1 or Caf1a (lane 12). In contrast, the C-terminal domain (amino acid 152–319) was able to co-IP Flag-Not1 and Caf1a as efficiently as full-length TTP (compare lanes 8 and 14). Treatment of the samples during IP with RNase I, which also cleaves RNA within a poly(A) tail, did not abrogate the interaction with either Flag-Not1 or Caf1a (lanes 9 and 15). Efficient degradation of RNA is shown in the bottom panels of Figure 5B for rRNA and nucleolin as an abundant mRNA. Since TTP–Not1/Caf1a interactions are technically challenging to detect, a repeat IP is shown in Supplementary Figure S1A and B. From these results we concluded that Not1 and Caf1a primarily interact with the C-terminal domain of TTP in an RNA-independent manner. We further tested the interaction of TTP with Ccr4a and again found the C-terminal domain of TTP to associate with HA-Ccr4a (Supplementary Figure S1C).Figure 5.

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