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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 interacts with Not1 independently of RNA. (A) T-REx-Hela cells were stably transfected with either the GS tag alone or GS-TTP, and treated with doxycycline for 16 h to induce expression of the tagged proteins. Cytoplasmic lysates were prepared from ∼2 × 108 cells, and tagged proteins were purified using streptavidin sepharose. Proteins were eluted with biotin, resolved on a 5–20% gradient polyacrylamide gel and visualized by colloidal Coomassie staining. (B) HEK293 cells were transiently transfected with Flag-Not1 together with either YFP or YFP–TTP. After 1 day, cytoplasmic lysates (input) were prepared for IP with GFP-binder. Western blot analysis was carried out with antibodies against the Flag and YFP tags. (C) HEK293 cells were transiently transfected with TTP-myc together with either Flag alone or Flag-Not1. Cytoplasmic lysates were used for IP with Flag antibody, and western blots analysis was carried out with antibodies against the myc and Flag tags. (D) HEK293 cells were transiently transfected with Flag-Not1 together with YFP, YFP–TTP or the zinc-finger mutant YFP–TTP-M1,2. IP and western blot analysis was carried out as in panel B, except that RNase A was added during IP where indicated. (E) HeLa cells were transiently transfected with a β-globin reporter gene containing the ARE of TNFα in its 3′-UTR. In addition, cells were transfected with YFP, YFP–TTP or the zinc-finger mutant YFP–TTP–M1,2. RNA and protein were extracted from both the input and IP samples. Western blot analysis was carried out with an antibody against YFP, and globin-ARE as well as nucleolin mRNA were detected by northern blot analysis.
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Figure 1: TTP interacts with Not1 independently of RNA. (A) T-REx-Hela cells were stably transfected with either the GS tag alone or GS-TTP, and treated with doxycycline for 16 h to induce expression of the tagged proteins. Cytoplasmic lysates were prepared from ∼2 × 108 cells, and tagged proteins were purified using streptavidin sepharose. Proteins were eluted with biotin, resolved on a 5–20% gradient polyacrylamide gel and visualized by colloidal Coomassie staining. (B) HEK293 cells were transiently transfected with Flag-Not1 together with either YFP or YFP–TTP. After 1 day, cytoplasmic lysates (input) were prepared for IP with GFP-binder. Western blot analysis was carried out with antibodies against the Flag and YFP tags. (C) HEK293 cells were transiently transfected with TTP-myc together with either Flag alone or Flag-Not1. Cytoplasmic lysates were used for IP with Flag antibody, and western blots analysis was carried out with antibodies against the myc and Flag tags. (D) HEK293 cells were transiently transfected with Flag-Not1 together with YFP, YFP–TTP or the zinc-finger mutant YFP–TTP-M1,2. IP and western blot analysis was carried out as in panel B, except that RNase A was added during IP where indicated. (E) HeLa cells were transiently transfected with a β-globin reporter gene containing the ARE of TNFα in its 3′-UTR. In addition, cells were transfected with YFP, YFP–TTP or the zinc-finger mutant YFP–TTP–M1,2. RNA and protein were extracted from both the input and IP samples. Western blot analysis was carried out with an antibody against YFP, and globin-ARE as well as nucleolin mRNA were detected by northern blot analysis.

Mentions: In order to identify proteins that associate with TTP, we generated a Tet-inducible vector expressing GS-tagged TTP. This tag encodes for protein G and a streptavidin-binding peptide (42). Stably transfected HeLa T-REx cells were generated with inducible expression of GS-TTP, and the fusion protein was affinity-purified on streptavidin sepharose. Due to difficulties with TEV cleavage, we could not perform a two-step purification as intended initially. Upon elution with biotin, co-purifying proteins were visualized by Coomassie staining and compared to proteins co-purifying with the GS tag alone (Figure 1A). Proteins from both purifications were identified by mass spectrometry. If a protein was identified by three or more peptides, and if the peptide number was ≥3-fold higher in the GS-TTP purification compared with the GS purification, it was considered a potential candidate associated with TTP (Supplemental Table S2). Among previously known interacting partners (46), we found 14-3-3 adaptor proteins (40,47) to co-purify with wild-type TTP (Figure 1A), but not with a non-phosphorylatable S52A/S178A mutant of TTP (data not shown). Interestingly, five of the seven isoforms of 14-3-3 could be identified in our mass spec analysis, suggesting that the interaction of phospho-TTP with 14-3-3 is not isoform-specific.Figure 1.


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 interacts with Not1 independently of RNA. (A) T-REx-Hela cells were stably transfected with either the GS tag alone or GS-TTP, and treated with doxycycline for 16 h to induce expression of the tagged proteins. Cytoplasmic lysates were prepared from ∼2 × 108 cells, and tagged proteins were purified using streptavidin sepharose. Proteins were eluted with biotin, resolved on a 5–20% gradient polyacrylamide gel and visualized by colloidal Coomassie staining. (B) HEK293 cells were transiently transfected with Flag-Not1 together with either YFP or YFP–TTP. After 1 day, cytoplasmic lysates (input) were prepared for IP with GFP-binder. Western blot analysis was carried out with antibodies against the Flag and YFP tags. (C) HEK293 cells were transiently transfected with TTP-myc together with either Flag alone or Flag-Not1. Cytoplasmic lysates were used for IP with Flag antibody, and western blots analysis was carried out with antibodies against the myc and Flag tags. (D) HEK293 cells were transiently transfected with Flag-Not1 together with YFP, YFP–TTP or the zinc-finger mutant YFP–TTP-M1,2. IP and western blot analysis was carried out as in panel B, except that RNase A was added during IP where indicated. (E) HeLa cells were transiently transfected with a β-globin reporter gene containing the ARE of TNFα in its 3′-UTR. In addition, cells were transfected with YFP, YFP–TTP or the zinc-finger mutant YFP–TTP–M1,2. RNA and protein were extracted from both the input and IP samples. Western blot analysis was carried out with an antibody against YFP, and globin-ARE as well as nucleolin mRNA were detected by northern blot analysis.
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Related In: Results  -  Collection

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Figure 1: TTP interacts with Not1 independently of RNA. (A) T-REx-Hela cells were stably transfected with either the GS tag alone or GS-TTP, and treated with doxycycline for 16 h to induce expression of the tagged proteins. Cytoplasmic lysates were prepared from ∼2 × 108 cells, and tagged proteins were purified using streptavidin sepharose. Proteins were eluted with biotin, resolved on a 5–20% gradient polyacrylamide gel and visualized by colloidal Coomassie staining. (B) HEK293 cells were transiently transfected with Flag-Not1 together with either YFP or YFP–TTP. After 1 day, cytoplasmic lysates (input) were prepared for IP with GFP-binder. Western blot analysis was carried out with antibodies against the Flag and YFP tags. (C) HEK293 cells were transiently transfected with TTP-myc together with either Flag alone or Flag-Not1. Cytoplasmic lysates were used for IP with Flag antibody, and western blots analysis was carried out with antibodies against the myc and Flag tags. (D) HEK293 cells were transiently transfected with Flag-Not1 together with YFP, YFP–TTP or the zinc-finger mutant YFP–TTP-M1,2. IP and western blot analysis was carried out as in panel B, except that RNase A was added during IP where indicated. (E) HeLa cells were transiently transfected with a β-globin reporter gene containing the ARE of TNFα in its 3′-UTR. In addition, cells were transfected with YFP, YFP–TTP or the zinc-finger mutant YFP–TTP–M1,2. RNA and protein were extracted from both the input and IP samples. Western blot analysis was carried out with an antibody against YFP, and globin-ARE as well as nucleolin mRNA were detected by northern blot analysis.
Mentions: In order to identify proteins that associate with TTP, we generated a Tet-inducible vector expressing GS-tagged TTP. This tag encodes for protein G and a streptavidin-binding peptide (42). Stably transfected HeLa T-REx cells were generated with inducible expression of GS-TTP, and the fusion protein was affinity-purified on streptavidin sepharose. Due to difficulties with TEV cleavage, we could not perform a two-step purification as intended initially. Upon elution with biotin, co-purifying proteins were visualized by Coomassie staining and compared to proteins co-purifying with the GS tag alone (Figure 1A). Proteins from both purifications were identified by mass spectrometry. If a protein was identified by three or more peptides, and if the peptide number was ≥3-fold higher in the GS-TTP purification compared with the GS purification, it was considered a potential candidate associated with TTP (Supplemental Table S2). Among previously known interacting partners (46), we found 14-3-3 adaptor proteins (40,47) to co-purify with wild-type TTP (Figure 1A), but not with a non-phosphorylatable S52A/S178A mutant of TTP (data not shown). Interestingly, five of the seven isoforms of 14-3-3 could be identified in our mass spec analysis, suggesting that the interaction of phospho-TTP with 14-3-3 is not isoform-specific.Figure 1.

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