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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 mRNA decay induced by tethering of TTP. (A) HeLa cells were transiently transfected with MS2cp, MS2cp-TTP or the zinc-finger mutant MS2cp-TTP-M1,2 that does not bind mRNA by itself. In addition, a pTet-Off-driven β-globin reporter gene that contains six MS2-binding sites (bs) in its 3′ UTR together with the Tet-Off transactivator were transfected into all cells. Transcription of the reporter gene was turned off by treating cells with doxycycline. RNA was isolated after the indicated time intervals. Globin-MS2bs and nucleolin mRNA levels were detected by northern blot analysis. The bottom graph shows quantification of the globin-MS2bs mRNA. Average values ± SE from three biological repeat experiments are represented. (B) 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 MS2cp-TTP-M1,2, the pTet-Off-globin-MS2bs reporter gene and the Tet-Off transactivator; 24 h later, cells were treated with doxycycline and total RNA was isolated after the indicated time intervals. Globin-MS2bs and nucleolin mRNA levels were detected by northern blot analysis. For quantification, signal intensities of globin-MS2bs mRNA were normalized to nucleolin mRNA and represented as percentage of the initial value. The bottom graph shows average values ± SE from four biological repeat experiments.
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Figure 3: Not1 is required for mRNA decay induced by tethering of TTP. (A) HeLa cells were transiently transfected with MS2cp, MS2cp-TTP or the zinc-finger mutant MS2cp-TTP-M1,2 that does not bind mRNA by itself. In addition, a pTet-Off-driven β-globin reporter gene that contains six MS2-binding sites (bs) in its 3′ UTR together with the Tet-Off transactivator were transfected into all cells. Transcription of the reporter gene was turned off by treating cells with doxycycline. RNA was isolated after the indicated time intervals. Globin-MS2bs and nucleolin mRNA levels were detected by northern blot analysis. The bottom graph shows quantification of the globin-MS2bs mRNA. Average values ± SE from three biological repeat experiments are represented. (B) 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 MS2cp-TTP-M1,2, the pTet-Off-globin-MS2bs reporter gene and the Tet-Off transactivator; 24 h later, cells were treated with doxycycline and total RNA was isolated after the indicated time intervals. Globin-MS2bs and nucleolin mRNA levels were detected by northern blot analysis. For quantification, signal intensities of globin-MS2bs mRNA were normalized to nucleolin mRNA and represented as percentage of the initial value. The bottom graph shows average values ± SE from four biological repeat experiments.

Mentions: Since several ARE-binding proteins including TTP, BRF1, KSRP and AUF1 participate in AMD (51), we then tested whether an mRNA whose decay is mediated by TTP alone would also require Not1 for degradation. For this purpose, we chose a tethering approach whereby TTP is forced to bind an mRNA lacking an ARE through the tight interaction of the MS2 bacteriophage coat protein (cp) with its cognate binding site (bs) in the MS2 RNA. MS2cp-TTP fusion proteins were expressed in HeLa cells together with a β-globin reporter mRNA containing six repeats of the MS2bs in its 3′-UTR. Figure 3A shows that tethering of MS2cp-TTP causes rapid degradation of the mRNA, whereas the mRNA remains stable after tethering of MS2cp alone. Tethering of the TTP-M1,2 mutant, which does not bind RNA, was as efficient as tethering of wt TTP (Figure 3A). This confirms that TTP functions by recruiting components of the RNA decay machinery to the bound RNA (16,20). We then tested the effect of knocking down Not1 by transfecting two siRNAs (S021 and S034) simultaneously. As seen with the ARE-containing mRNA above, we observed that the mRNA tethered to MS2cp-TTP-M1,2 was stabilized by knock down of Not1 (Figure 3B). Quantification of four biological repeat experiments showed an increase in the mRNA half-life from 1.6 h to >6 h. Thus, mRNA decay mediated by binding of TTP alone is clearly dependent on the deadenylase scaffold protein Not1.Figure 3.


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 mRNA decay induced by tethering of TTP. (A) HeLa cells were transiently transfected with MS2cp, MS2cp-TTP or the zinc-finger mutant MS2cp-TTP-M1,2 that does not bind mRNA by itself. In addition, a pTet-Off-driven β-globin reporter gene that contains six MS2-binding sites (bs) in its 3′ UTR together with the Tet-Off transactivator were transfected into all cells. Transcription of the reporter gene was turned off by treating cells with doxycycline. RNA was isolated after the indicated time intervals. Globin-MS2bs and nucleolin mRNA levels were detected by northern blot analysis. The bottom graph shows quantification of the globin-MS2bs mRNA. Average values ± SE from three biological repeat experiments are represented. (B) 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 MS2cp-TTP-M1,2, the pTet-Off-globin-MS2bs reporter gene and the Tet-Off transactivator; 24 h later, cells were treated with doxycycline and total RNA was isolated after the indicated time intervals. Globin-MS2bs and nucleolin mRNA levels were detected by northern blot analysis. For quantification, signal intensities of globin-MS2bs mRNA were normalized to nucleolin mRNA and represented as percentage of the initial value. The bottom graph shows average values ± SE from four biological repeat experiments.
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Figure 3: Not1 is required for mRNA decay induced by tethering of TTP. (A) HeLa cells were transiently transfected with MS2cp, MS2cp-TTP or the zinc-finger mutant MS2cp-TTP-M1,2 that does not bind mRNA by itself. In addition, a pTet-Off-driven β-globin reporter gene that contains six MS2-binding sites (bs) in its 3′ UTR together with the Tet-Off transactivator were transfected into all cells. Transcription of the reporter gene was turned off by treating cells with doxycycline. RNA was isolated after the indicated time intervals. Globin-MS2bs and nucleolin mRNA levels were detected by northern blot analysis. The bottom graph shows quantification of the globin-MS2bs mRNA. Average values ± SE from three biological repeat experiments are represented. (B) 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 MS2cp-TTP-M1,2, the pTet-Off-globin-MS2bs reporter gene and the Tet-Off transactivator; 24 h later, cells were treated with doxycycline and total RNA was isolated after the indicated time intervals. Globin-MS2bs and nucleolin mRNA levels were detected by northern blot analysis. For quantification, signal intensities of globin-MS2bs mRNA were normalized to nucleolin mRNA and represented as percentage of the initial value. The bottom graph shows average values ± SE from four biological repeat experiments.
Mentions: Since several ARE-binding proteins including TTP, BRF1, KSRP and AUF1 participate in AMD (51), we then tested whether an mRNA whose decay is mediated by TTP alone would also require Not1 for degradation. For this purpose, we chose a tethering approach whereby TTP is forced to bind an mRNA lacking an ARE through the tight interaction of the MS2 bacteriophage coat protein (cp) with its cognate binding site (bs) in the MS2 RNA. MS2cp-TTP fusion proteins were expressed in HeLa cells together with a β-globin reporter mRNA containing six repeats of the MS2bs in its 3′-UTR. Figure 3A shows that tethering of MS2cp-TTP causes rapid degradation of the mRNA, whereas the mRNA remains stable after tethering of MS2cp alone. Tethering of the TTP-M1,2 mutant, which does not bind RNA, was as efficient as tethering of wt TTP (Figure 3A). This confirms that TTP functions by recruiting components of the RNA decay machinery to the bound RNA (16,20). We then tested the effect of knocking down Not1 by transfecting two siRNAs (S021 and S034) simultaneously. As seen with the ARE-containing mRNA above, we observed that the mRNA tethered to MS2cp-TTP-M1,2 was stabilized by knock down of Not1 (Figure 3B). Quantification of four biological repeat experiments showed an increase in the mRNA half-life from 1.6 h to >6 h. Thus, mRNA decay mediated by binding of TTP alone is clearly dependent on the deadenylase scaffold protein Not1.Figure 3.

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