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Beta-Catenin stabilizes cyclooxygenase-2 mRNA by interacting with AU-rich elements of 3'-UTR.

Lee HK, Jeong S - Nucleic Acids Res. (2006)

Bottom Line: Expression of COX-2 mRNA is regulated by various cytokines, growth factors and other signals. beta-Catenin, a key transcription factor in the Wnt signal pathway, activates transcription of COX-2.Here we found that COX-2 mRNA was also substantially stabilized by activating beta-catenin in NIH3T3 and 293T cells.Taken together, we provided evidences for beta-catenin as an RNA-binding factor and a regulator of stabilization of COX-2 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, BK21 Graduate Program for RNA Biology, Institute of Nanosensor and Biotechnology, Dankook University, Seoul 140-714, Republic of Korea.

ABSTRACT
Cyclooxygenase-2 (COX-2) mRNA is induced in the majority of human colorectal carcinomas. Transcriptional regulation plays a key role in COX-2 expression in human colon carcinoma cells, but post-transcriptional regulation of its mRNA is also critical for tumorigenesis. Expression of COX-2 mRNA is regulated by various cytokines, growth factors and other signals. beta-Catenin, a key transcription factor in the Wnt signal pathway, activates transcription of COX-2. Here we found that COX-2 mRNA was also substantially stabilized by activating beta-catenin in NIH3T3 and 293T cells. We identified the beta-catenin-responsive element in the proximal region of the COX-2 3'-untranslated region (3'-UTR) and showed that beta-catenin interacted with AU-rich elements (ARE) of 3'-UTR in vitro and in vivo. Interestingly, beta-catenin induced the cytoplasmic localization of the RNA stabilizing factor, HuR, which may bind to beta-catenin in an RNA-mediated complex and facilitate beta-catenin-dependent stabilization of COX-2 mRNA. Taken together, we provided evidences for beta-catenin as an RNA-binding factor and a regulator of stabilization of COX-2 mRNA.

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Mapping of the β-catenin-dependent stabilizing element in the F1 UTR. (A) Schematic representation of the different F1 UTR probes. ARE sequences are indicated as boxes and mutant sequences are shown by underlining and in italics. (B) RNA substrates were incubated with cytoplasmic extracts from NIH3T3 cells, and the reactions were stopped by adding stop buffer at the indicated times. Processed RNA was resolved on a 7 M urea/5% acrylamide gel and visualized by autoradiography. (C) RNA substrates were incubated with cytoplasmic extracts from either vector (control) or β-catenin-expressing NIH3T3 cells. mRNA degradation was examined as in (B).
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fig3: Mapping of the β-catenin-dependent stabilizing element in the F1 UTR. (A) Schematic representation of the different F1 UTR probes. ARE sequences are indicated as boxes and mutant sequences are shown by underlining and in italics. (B) RNA substrates were incubated with cytoplasmic extracts from NIH3T3 cells, and the reactions were stopped by adding stop buffer at the indicated times. Processed RNA was resolved on a 7 M urea/5% acrylamide gel and visualized by autoradiography. (C) RNA substrates were incubated with cytoplasmic extracts from either vector (control) or β-catenin-expressing NIH3T3 cells. mRNA degradation was examined as in (B).

Mentions: Since the F1 region of the COX-2 UTR contains multiple copies of ARE, we dissected the F1 sequence. AREs are classified into three groups according to features of their sequence and their RNA decay characteristics (36,37). The F1 region of the COX-2 3′-UTR contains both class I ARE with one to three scattered copies of the AUUUA motif and class II ARE with multiple overlapping copies of that motif. To further define the β-catenin-responsive sequences in the F1 region, we further divided the 145 nt of F1 into 40 nt fragments (Figure 3A) and evaluated their degradation rates. We found that both the F1-1 (class II) and F1-2 (class I) fragments were unstable, whereas the F1-3 (non-ARE) fragment was as stable as the control GAPDH mRNA (Figure 3B). We next tested which RNA substrates responded to β-catenin-induced stabilization. As shown in Figure 3C, strong expression of β-catenin stabilized F1-2 rather than F1-1. Furthermore, mutation of the UUU residues of AUUUA to CGC in F1-2 (F1-2 MT) completely abolished the ARE-induced instability. These in vitro decay data indicate that β-catenin is responsible for stabilization of the class I ARE region of the F1 UTR.


Beta-Catenin stabilizes cyclooxygenase-2 mRNA by interacting with AU-rich elements of 3'-UTR.

Lee HK, Jeong S - Nucleic Acids Res. (2006)

Mapping of the β-catenin-dependent stabilizing element in the F1 UTR. (A) Schematic representation of the different F1 UTR probes. ARE sequences are indicated as boxes and mutant sequences are shown by underlining and in italics. (B) RNA substrates were incubated with cytoplasmic extracts from NIH3T3 cells, and the reactions were stopped by adding stop buffer at the indicated times. Processed RNA was resolved on a 7 M urea/5% acrylamide gel and visualized by autoradiography. (C) RNA substrates were incubated with cytoplasmic extracts from either vector (control) or β-catenin-expressing NIH3T3 cells. mRNA degradation was examined as in (B).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1636482&req=5

fig3: Mapping of the β-catenin-dependent stabilizing element in the F1 UTR. (A) Schematic representation of the different F1 UTR probes. ARE sequences are indicated as boxes and mutant sequences are shown by underlining and in italics. (B) RNA substrates were incubated with cytoplasmic extracts from NIH3T3 cells, and the reactions were stopped by adding stop buffer at the indicated times. Processed RNA was resolved on a 7 M urea/5% acrylamide gel and visualized by autoradiography. (C) RNA substrates were incubated with cytoplasmic extracts from either vector (control) or β-catenin-expressing NIH3T3 cells. mRNA degradation was examined as in (B).
Mentions: Since the F1 region of the COX-2 UTR contains multiple copies of ARE, we dissected the F1 sequence. AREs are classified into three groups according to features of their sequence and their RNA decay characteristics (36,37). The F1 region of the COX-2 3′-UTR contains both class I ARE with one to three scattered copies of the AUUUA motif and class II ARE with multiple overlapping copies of that motif. To further define the β-catenin-responsive sequences in the F1 region, we further divided the 145 nt of F1 into 40 nt fragments (Figure 3A) and evaluated their degradation rates. We found that both the F1-1 (class II) and F1-2 (class I) fragments were unstable, whereas the F1-3 (non-ARE) fragment was as stable as the control GAPDH mRNA (Figure 3B). We next tested which RNA substrates responded to β-catenin-induced stabilization. As shown in Figure 3C, strong expression of β-catenin stabilized F1-2 rather than F1-1. Furthermore, mutation of the UUU residues of AUUUA to CGC in F1-2 (F1-2 MT) completely abolished the ARE-induced instability. These in vitro decay data indicate that β-catenin is responsible for stabilization of the class I ARE region of the F1 UTR.

Bottom Line: Expression of COX-2 mRNA is regulated by various cytokines, growth factors and other signals. beta-Catenin, a key transcription factor in the Wnt signal pathway, activates transcription of COX-2.Here we found that COX-2 mRNA was also substantially stabilized by activating beta-catenin in NIH3T3 and 293T cells.Taken together, we provided evidences for beta-catenin as an RNA-binding factor and a regulator of stabilization of COX-2 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, BK21 Graduate Program for RNA Biology, Institute of Nanosensor and Biotechnology, Dankook University, Seoul 140-714, Republic of Korea.

ABSTRACT
Cyclooxygenase-2 (COX-2) mRNA is induced in the majority of human colorectal carcinomas. Transcriptional regulation plays a key role in COX-2 expression in human colon carcinoma cells, but post-transcriptional regulation of its mRNA is also critical for tumorigenesis. Expression of COX-2 mRNA is regulated by various cytokines, growth factors and other signals. beta-Catenin, a key transcription factor in the Wnt signal pathway, activates transcription of COX-2. Here we found that COX-2 mRNA was also substantially stabilized by activating beta-catenin in NIH3T3 and 293T cells. We identified the beta-catenin-responsive element in the proximal region of the COX-2 3'-untranslated region (3'-UTR) and showed that beta-catenin interacted with AU-rich elements (ARE) of 3'-UTR in vitro and in vivo. Interestingly, beta-catenin induced the cytoplasmic localization of the RNA stabilizing factor, HuR, which may bind to beta-catenin in an RNA-mediated complex and facilitate beta-catenin-dependent stabilization of COX-2 mRNA. Taken together, we provided evidences for beta-catenin as an RNA-binding factor and a regulator of stabilization of COX-2 mRNA.

Show MeSH
Related in: MedlinePlus