1. Volume 132, Issue 3, Pages 1055-1065 (March 2007)
Functional Antagonism Between RNA Binding Proteins HuR and CUGBP2 Determines the Fate of COX-2 mRNA Translation 
Sripathi M. Sureban, Nabendu Murmu, Pavel Rodriguez, Randal May, Reeti Maheshwari, Brian K. Dieckgraefe, Courtney W. Houchen, Shrikant Anant 
Gastroenterology 
Volume 132, Issue 3, Pages (March 2007)
DOI: /j.gastro
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2. Figure 1
CUGBP2 and HuR bind with equal affinities to COX-2 3′UTR. (A) COX-2 3′UTR showing the presence of AUUUA sequences. Top panel: the numbers below indicate the boundaries of the regions responsible for regulating mRNA stability and translation. The black boxes specify the region responsible for both mRNA stability and translation, the grey boxes show the region responsible only for the stability of COX-2 mRNA. *AUUUA sequence. Bottom panel: the AUUUA sequences are present throughout the 3′UTR but are clustered in the first 60 nt of the UTR. There are 6 repeats of the AU-rich sequences located in the first 60 nt, which is grouped in 3 sets of AU-rich sequences (denoted I–III). (B) CUGBP2 and HuR bind to COX-2 3′UTR with similar affinities. The left panel shows the saturation kinetics of binding of recombinant GST/HuR fusion protein and the right panel shows recombinant GST/CUGBP2 binding to the COX-2 3′UTR. The data are plotted as the fraction of RNA bound (mean ± SD) to the indicated amount of the respective proteins. Both the proteins show similar affinities to COX-2 3′UTR. There was no statistical difference in the RNA binding affinities between the 2 proteins. Each point represents data from the 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
CUGBP2 competes with HuR to bind the COX-2 3′UTR. (A) SDS gel representation of UV cross-linked recombinant GST/CUGBP2 and GST/HuR protein binding with 32 P-labeled cRNA containing 1–60 nt of COX-2 3′UTR. Varying concentrations of recombinant GST/HuR and GST/CUGBP2 were mixed with 32 P-labeled COX-2 3′UTR RNA and UV cross-linked. The mixture was separated on an SDS gel and autoradiographed. The gel picture shows binding of the sample mixture containing CUGBP2 and HuR to COX-2 3′UTR RNA. The sample in the first lane contained CUGBP2 alone, and the subsequent samples had decreasing amounts of CUGBP2 and increasing amounts of HuR, with the last sample containing only HuR. (B) Radioactivity count of the individual samples considering CUGBP2 (gray column) and HuR (black column) alone as 100% binding to the COX-2 3′UTR RNA, which are the positive controls. With the decrease in CUGBP2 and increase in HuR, the binding shifts from CUGBP2 to HuR. At 50% of the total RNA bound, the protein mixture consisted of 0.47 pmol CUGBP2 and 0.89 pmol HuR, indicating that CUGBP2 competes with HuR in binding to COX-2 3′UTR RNA (a, P < .01; b, P < .6).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
Nuclear-cytoplasmic shuttling of CUGBP2 and HuR. HCT-116 cells, transfected with N-terminal FLAG-tagged CUGBP2 or N-terminal FLAG-tagged HuR (as positive control for shuttling) was fused with untransfected NIH-3T3 cells in the presence of polyethylene glycol. Cycloheximide was added to the cells before fusion to prevent de novo protein synthesis. (A, D) Presence of the respective proteins. (B, E) Distinct punctuated staining of NIH-3T3 mouse nuclei (indicated by an arrow) with Hoechst 33342; the HCT-116 cells stain uniform blue. (C, F) Merged images. Similar to HuR, CUGBP2 in the both the human and mouse nuclei.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

5. Figure 4
CUGBP2 and HuR heterodimerize. (A) GST pull-down assay. In vitro translated 35 S-labeled HuR was incubated with recombinant GST/HuR and GST/CUGBP2 proteins, and purified over a column containing glutathione sepharose beads. Total input protein (t), eluted sample from column incubated with the 35 S-labeled HuR alone used as negative control (−), and eluted sample (p) and the unbound sample (s) from the recombinant proteins were separated on an SDS–polyacrylamide gel electrophoresis and autoradiographed. The gel picture shows the presence of 35 S-HuR in the samples bound with recombinant GST/HuR, indicating that HuR homodimerizes, and also in the sample containing recombinant GST/CUGBP2, indicating that HuR heterodimerizes with CUGBP2. The supernatant samples did not show the presence of 35 S-HuR. Representative of 3 experiments. (B) Yeast 2-hybrid assay. HuR and CUGBP2 cDNAs were cloned in both the bait and test plasmids for expression as fusion protein with the Gal4 DNA binding domain and Gal4 DNA activation domain, respectively. As a positive control, p53 and SV40 T large antigen (RecT), 2 proteins known to interact, were expressed with the Gal4 DNA binding and activation domains, respectively. Presence of an interaction results in growth of the cell, and also in blue colonies in the presence of X-α-Gal owing to transcription of the MEL1 gene encoding α-galactosidase. Both HuR and CUGBP2 homodimerized and heterodimerized to form blue colonies. On the other hand, these proteins did not interact with the RecT or p53 proteins, showing specificity of the interaction in the analyses (negative control). (C) Immunofluorescence analysis. Flag-tagged HuR and myc-tagged CUGBP2 were co-expressed in HCT-116 cells and stained with Cy3 (red for myc tag) and Cy5 (green for FLAG tag). Overlapping red and green labeling are in yellow. The nucleus is stained with 4,6-diamidino-2-phenylindole. The colocalization of CUGBP2 and HuR in the nucleus is shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 5
CUGBP2 inhibits, whereas HuR enhances, in vitro COX-2 mRNA translation. (A) Luciferase transcript containing the full-length murine COX-2 3′UTR. (B) The Luc/COX-2 3′UTR mRNA (black columns) and the control Luc mRNA (gray columns) were subjected to in vitro translation in the presence of increasing concentrations of recombinant GST/HuR, and the luciferase activity was measured. With increasing concentrations of HuR there was a corresponding increase in the translation of the Luc/COX-2 3′UTR mRNA, but not the control Luc mRNA (*P < .01). (C) With increasing concentrations of CUGBP2 as indicated, there was greater suppression of the Luc/COX-2 3′UTR mRNA (black columns). No effects were observed with the control Luc mRNA (gray columns; *P < .01). (D) Luc/COX-2 3′UTR mRNA (black columns) was in vitro translated in the presence of increasing concentrations of CUGBP2 and a corresponding decreasing concentration of HuR. Translation in the presence of HuR alone was considered as 100%. There was a consistent decrease in the translation with the increasing concentration of CUGBP2. The 50% reduction in translation was observed in a protein mixture that contained 0.47 pmol CUGBP2 and 0.89 pmol HuR, suggesting a competitive advantage for CUGBP2 over HuR in regulating the luciferase/COX-2 mRNA translation (*P < .01). Gray columns, control Luc mRNA. (E) Luc/COX-2 3′UTR mRNA (black columns) translation was performed in the presence of 0.75 pmol HuR, and increasing concentrations of CUGBP2 (0–0.6 pmol). Significant inhibition (*P < .01) was observed beginning with 25 ng CUGBP2. Gray columns, control Luc mRNA. These results are from 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

7. Figure 6
CUGBP2 competes with HuR in regulating ARE-driven mRNA translation in vivo. (A) HT-29 cells were cotransfected with HA epitope–tagged CUGBP2, FLAG epitope–tagged HuR, and firefly luciferase gene containing the full-length 2.2-kb COX-2 3′UTR (Luc/COX-2 3′UTR). Cells also were subjected to 12 Gy γ-irradiation where indicated (lanes 6–9). Total cell lysates were prepared and immunoprecipitated with anti-HA and anti-FLAG antibodies to immunoprecipitate CUGBP2 and HuR, respectively. As a negative control, a normal rabbit IgG (nrs) preparation was used. RNA bound to the immunoprecipitates were isolated and subjected to semiquantitative reverse-transcription polymerase chain reaction for luciferase gene. The luciferase mRNA was found in anti-HA and anti-FLAG immunoprecipitates, but not with NRS, showing in vivo binding of the 2 proteins to the COX-2 3′UTR. Moreover, in cells that were not subjected to the radiation treatment, more of the Luc/COX-2 3′UTR mRNA was bound to HuR, whereas in cells also subjected to 12 Gy γ-radiation (12 Gy), the majority of the transcript was bound to CUGBP2. (B) Total cell lysates from the transfections were subjected to Western blot analyses for COX-2. HuR alone increased COX-2 protein levels (lane 3), but this was inhibited in the presence of cotransfected CUGBP2 (lane 4). Furthermore, after radiation, COX-2 protein levels did not increase even with HuR overexpression (lane 7), and there was a further decrease in COX-2 levels in cells expressing both HuR and CUGBP2 (lane 8). Actin was used as control for protein loading. (C) The total cell lysates from the transfections also were subjected to an in vitro luciferase activity assay. In the unirradiated samples, cells expressing FLAG epitope–tagged HuR showed increased luciferase activity, whereas those that expressed HA epitope–tagged CUGBP2 showed a decreased luciferase activity when compared with the controls. When cells expressed both proteins, there was no effect. After radiation there was an overall decrease in the luciferase activity. In contrast, in cells subjected to 12 Gy γ-irradiation, HuR did not have any effect on translation. Furthermore, expression of HA-tagged CUGBP2 enhanced the translation inhibition effect (a, P < .01 when comparing irradiated with unirradiated cells; b, P < .01 when comparing FLAG-HuR–transfected with untransfected cells; c, P < .01 when comparing HA-CUGBP2–transfected with untransfected cells; d, P = .5 when comparing cells transfected with both with untransfected cells). ■, No IR; ▩, 12 Gy.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions