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. 2013 Jan;24(2):85-99.
doi: 10.1091/mbc.E12-07-0531. Epub 2012 Nov 14.

Competitive binding of CUGBP1 and HuR to occludin mRNA controls its translation and modulates epithelial barrier function

Ting-Xi Yu  1 , Jaladanki N Rao, Tongtong Zou, Lan Liu, Lan Xiao, Miao Ouyang, Shan Cao, Myriam Gorospe, Jian-Ying Wang
Affiliations

Competitive binding of CUGBP1 and HuR to occludin mRNA controls its translation and modulates epithelial barrier function

Ting-Xi Yu et al. Mol Biol Cell. 2013 Jan.

Abstract

RNA-binding proteins CUG-binding protein 1 (CUGBP1) and HuR are highly expressed in epithelial tissues and modulate the stability and translation of target mRNAs. Here we present evidence that CUGBP1 and HuR jointly regulate the translation of occludin and play a crucial role in the maintenance of tight junction (TJ) integrity in the intestinal epithelial cell monolayer. CUGBP1 and HuR competed for association with the same occludin 3'-untranslated region element and regulated occludin translation competitively and in opposite directions. CUGBP1 overexpression decreased HuR binding to occludin mRNA, repressed occludin translation, and compromised the TJ barrier function, whereas HuR overexpression inhibited CUGBP1 association with occludin mRNA and promoted occludin translation, thereby enhancing the barrier integrity. Repression of occludin translation by CUGBP1 was due to the colocalization of CUGBP1 and tagged occludin RNA in processing bodies (P-bodies), and this colocalization was prevented by HuR overexpression. These findings indicate that CUGBP1 represses occludin translation by increasing occludin mRNA recruitment to P-bodies, whereas HuR promotes occludin translation by blocking occludin mRNA translocation to P-bodies via the displacement of CUGBP1.

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Figures

FIGURE 1:
FIGURE 1:
CUGBP1 overexpression inhibits occludin mRNA translation via its 3′-UTR. (A) Representative immunoblots of CUGBP1 and occludin proteins. Cells were transfected with the vector expressing CUGBP1 or control empty vector; protein levels were measured by Western immunoblotting analysis at various times after the transfection by using specific antibody against CUGBP1 or occludin. (B) Levels of occludin mRNA in cells treated as described in panel (A). Total RNA was harvested, and the levels of occludin mRNA were measured by Q-PCR analysis. Data were normalized to GAPDH mRNA levels, and values are shown as the means ± SEM of data from triplicate experiments. (C) Newly translated occludin protein as measured by l-[35S]methionine and l-[35S]cysteine/IP assays in cells transfected with the CUGBP1 expression vector or control vector for 48 h. Top, representative immunoblots of newly synthesized occludin; bottom, quantitative analysis of the immunoblotting signals as measured by densitometry. Values are mean ± SEM of data from three separate experiments. * p < 0.05 compared with cells transfected with control vector. (D) Polysomal profiles in cells described in (C): a) cells transfected with control vector; b) cells transfected with the CUGBP1 expression vector. Nuclei were pelleted, and the resulting supernatants were fractionated through a 10-50% linear sucrose gradient. (E) Distributions of occludin (top) and GAPDH (bottom) mRNAs in each gradient fraction prepared from cells described in panel (D). Total RNA was isolated from the different fractions, and the levels of occludin and GAPDH mRNAs were measured by Q-PCR analysis and plotted as a percentage of the total occludin or GAPDH mRNA levels in that sample. Three independent experiments were performed and showed similar results. (F) Changes in occludin translation efficiency as measured by occludin 3′-UTR-luciferase reporter assays. Top, schematic of plasmids: control (pGL3-Luc); and chimeric firefly luciferase-occludin 3′-UTR (Luc-Occl-3′-UTR). Bottom, levels of occludin translation. The Luc-Occl-3′-UTR or pGL3-Luc (negative control) was cotransfected with a Renilla luciferase reporter. Luciferase values were normalized to luciferase mRNA levels to calculate the translation efficiencies and expressed as means ± SEM of data from three separate experiments. *p < 0.05 compared with cells transfected with control vector. (G) Cellular distribution of occludin and ZO-1 in cells described in (C). After the transfection for 48 h, cells were fixed, permeabilized, incubated with the antibody against occludin or ZO-1, and then with anti-IgG conjugated with FITC. Original magnification, 1000×. Three experiments were performed that showed similar results.
FIGURE 2:
FIGURE 2:
CUGBP1 silencing enhances occludin translation and promotes the epithelial barrier function. (A) Representative immunoblots of CUGBP1 and occludin proteins. After cells were transfected with either siRNA targeting the CUGBP1 mRNA CR (siCUGBP1) or C-siRNA for different times, whole-cell lysates were harvested for Western blot analysis. (B) Levels of occludin mRNA as measured by Q-PCR analysis in cells described in panel (A). The data were normalized to GAPDH mRNA levels and shown as the means ± SEM of data from triplicate experiments. (C) Newly translated occludin protein in cells transfected with siCUGBP1 or C-siRNA for 48 h as measured by l-[35S]methionine and l-[35S]cysteine/IP assays. Top, representative immunoblots of newly synthesized occludin; bottom, quantitative analysis of the immunoblotting signals as measured by densitometry. Values are means ± SEM of data from three separate experiments. * p < 0.05 compared with cells transfected with C-siRNA. (D) Changes in occludin translation efficiency as measured by using pGL3-Luc-Occl-3′-UTR reporter assays in cells described in panel (C). Twenty-four hours after cells were transfected with the Luc-Occl-3′-UTR or pGL3-Luc, the levels of luciferase activity were examined and normalized to the mRNA levels to calculate the translation efficiencies. Values were expressed as means ± SEM of data from three separate experiments; * p < 0.05 compared with cells transfected with C-siRNA. (E) Changes in TEER (left panel) and FITC–dextran paracellular permeability (right panel) in cells transfected with siCUGBP1 alone or cotransfected with siCUGBP1 and siRNA targeting occludin (siOccludin). TEER assays were performed on 12-mm Transwell filters 48 h after transfection as described in Materials and Methods; paracellular permeability was assayed by using the membrane-impermeable trace molecule FITC–dextran that was added to the insert medium. Values are the means ± SEM of data from six samples. *,+ p < 0.05 compared with cells transfected with C-siRNA or siCUGBP1, respectively. (F) Changes in TEER and FITC–dextran paracellular permeability in cells transfected with the CUGBP1 expression vector or cotransfected with CUGBP1 and occludin expression vectors. *, # p < 0.05 compared with cells transfected with control vector (vector) or CUGBP1 expression vector alone, respectively. (G) Changes in the levels of CUGBP1 and occludin proteins in cells described in (E) and (F).
FIGURE 3:
FIGURE 3:
CUGBP1 binds the occludin mRNA 3′-UTR via GRE. (A) Association of endogenous CUGBP1 with endogenous occludin mRNA. After IP of RNA-protein complexes from cell lysates using either anti-CUGBP1 antibody (Ab) or control IgG1, RNA was isolated and measured by RT-PCR (top) and Q-PCR (bottom) analyses. Low-level amplification of GAPDH (housekeeping mRNA, which is not a CUGBP1 target) served as negative controls. Values are the means ± SEM from triplicate samples. (B) Representative CUGBP1, HuR, and TIAR immunoblots using the pull-down materials by biotinylated transcripts of the occludin 5′-UTR, CR, or 3′-UTR. Top panel, schematic representation of the biotinylated transcripts used in this study. Cytoplasmic lysates were incubated with 6 μg of biotinylated occludin 5′-UTR, CR, or 3′-UTR, and the resulting RNP complexes were pulled down by using streptavidin-coated beads. The presence of CUGBP1, HuR, or TIAR in the pull-down material was assayed by Western blotting. β-Actin in the pull-down material was also examined and served as a negative control. (C) Binding of CUGBP1 or HuR to different fractions of 3′-UTR of the occludin mRNA. Top panel, schematic representation of the occludin 3′-UTR biotinylated transcripts. After incubation of cytoplasmic lysates with the full-length (FL) or various fractions (F) of the occludin 3′-UTR, the resulting RNP complexes were pulled down, and the abundance of HuR and β-actin proteins in the pull-down material was examined. (D) Changes in binding of occludin 3′-UTR to CUGBP1 and HuR after deletion mutation of the F4. Top panel, schematic representation of the biotinylated transcripts of mutated occludin 3′-UTR used in this study.
FIGURE 4:
FIGURE 4:
Changes in the levels of occludin 3′-UTR luciferase reporter activity after deletion of CUGBP1-binding sites. (A) Activity of various pLuc-occludin 3′-UTR luciferase reporters with or without CUGBP1-binding site in cells overexpressing CUGBP1. Top panels, schematic of firefly luciferase reporter constructs containing full-length (FL) or different fragments (F) of the occludin 3′-UTR. BS, CUGBP1-binding site. Twenty-four hours after the cells were transfected with either CUGBP1 expression vector or control vector, the cells were further transfected with each of various occludin 3′-UTR luciferase reporter constructs and a Renilla luciferase control reporter. The levels of firefly and Renilla luciferase activities were assayed 24 h later. The results were normalized to the Renilla luciferase activity and are shown as the means ± SEM of data from three separate experiments. * p < 0.05 compared with cells transfected with control vector. (B) Activity of various pLuc-occludin 3′-UTR luciferase reporters after CUGBP1 silencing. Cells were initially transfected with either siCUGBP1 or C-siRNA for 24 h and then with each of various occludin 3′-UTR luciferase reporter constructs in cotransfection with the Renilla luciferase reporter. * p < 0.05 compared with cells transfected with C-siRNA. (C) Effect of deletion of specific CUGBP1-binding site (schematic) in occludin 3′-UTR on luciferase reporter activity after CUGBP1 overexpression or CUGBP1 silencing. * p < 0.05 compared with cells transfected with control vector or C-siRNA.
FIGURE 5:
FIGURE 5:
HuR represses occludin mRNA/CUGBP1 association and prevents CUGBP1-induced repression of occludin translation. (A) Representative immunoblots of CUGBP1, HuR, and occludin. After cells were transfected with the CUGBP1 or HuR expression vector alone or cotransfected with both for 48 h, whole-cell lysates were harvested for Western blot analysis. (B) Changes in binding of the occludin mRNA to CUGBP1 and HuR as detected by RNP-IP/Q-PCR analysis: a) cells overexpressing HuR; b) cells overexpressing CUGBP1. Values were means ± SEM from triplicate samples. * p < 0.05 compared with cells transfected with control vector. (C) Effect of GST-HuR added to the binding reaction on association of HuR or CUGBP1 with the occludin 3′-UTR: a) GST-HuR fusion protein identified by anti-GST antibody (left) or recognized by anti-HuR antibody (right); b) protein input in the binding reaction mixture; c) interactions of HuR and CUGBP1 with the occludin 3′-UTR. Various concentrations of GST-HuR were used; the levels of binding complexes were detected by pull-down assays. Three independent experiments were performed showing similar results. (D) Effect of GST-CUGBP1 on association of HuR or CUGBP1 with the occludin 3′-UTR: a) protein input in the binding reaction mixture; b) bindings of HuR and CUGBP1 to the occludin 3′-UTR. (E) Effect of increasing the levels of HuR on occludin translation as measured by occludin 3′-UTR-luciferase reporter assays in cells overexpressing CUGBP1 as described in (A). Values were expressed as means ± SEM of data from three separate experiments. * p < 0.05 compared with cells transfected with control vector.
FIGURE 6:
FIGURE 6:
CUGBP1 promotes interaction of the occludin mRNA with components of P-bodies. (A) Fluorescence analysis of CUGBP1 colocalization with PB-resident proteins Ago2 and RCK. Red (top), antibody detecting CUGBP1; green (middle), antibodies detecting Ago2 and RCK; yellow (bottom), merging of the two signals. (B) Physical interaction of CUGBP1 with PB component proteins. IP of protein complexes were performed on intact lysates (–) or lysates that had been incubated with both RNase A and RNase T1 (+), using IgG or antibody against CUGBP1 (top) or Ago2 (bottom). The levels of CUGBP1, Ago2, RCK, and hDCP1 were examined by Western blot analysis. (C) Effect of CUGBP1 on occludin mRNA interaction with components of PBs. After cells were transfected with the CUGBP1 expression vector or control vector for 48 h, the association of occludin mRNA with Ago2 or RCK was measured by RNP IP using anti-Ago2 (top) or RCK (bottom) antibodies, which was followed by Q-PCR analysis. Values are means ± SEM of data from three separate experiments. * p < 0.05 compared with cells transfected with control vector. (D) Effect of silencing Ago2 and RCK on occludin expression in cells overexpressing CUGBP1. Cells were transfected with CUGBP1 expression vector or cotransfected with the CUGBP1 and specific siRNA targeting Ago2 (siAgo2) or RCK (siRCK); 48 h later, the levels of CUGBP1, Ago2, RCK, and occludin proteins were assessed by Western blot analysis. Equal loading was monitored by blotting GAPDH.
FIGURE 7:
FIGURE 7:
HuR inhibits CUGBP1-induced occludin mRNA recruitment to P-bodies. (A) Interaction of occludin mRNA with components of P-bodies in cells overexpressing CUGBP1 or HuR alone, or both. After cells were transfected with the HuR or CUGBP1 expression vector alone or they were cotransfected with both HuR and CUGBP1 vectors for 48 h, the associations of occludin mRNA with Ago2 (left) and RCK (right) were measured by RNP-IP using anti-Ago2 or RCK antibodies, or control IgG followed by Q-PCR analysis. Values are means ± SEM of data from three separate experiments. *,+ p < 0.05 compared with cells transfected with control vector or cells transfected with the CUGBP1 expression vector, respectively. (B) Schematic of the plasmids used for the visualization of occludin mRNA. pMS2 and pMS2-occludin expressed MS2 and MS2-occludin RNAs, each containing 24 tandem MS2 hairpins; pMS2-YFP expressed a fusion fluorescent protein (MS2-YFP) capable of detecting MS2-containing RNA.. (C) Images of occludin mRNA colocalization with P-bodies: a) cells transfected with pMS2 alone; b) cells transfected with control vector and pMS2-occludin; c) cells transfected with CUGBP1 expression vector and pMS2-occludin; d) cells cotransfected with HuR and CUGBP1 expression vectors and with pMS2-occludin. Using confocal microscopy, MS2 and MS2-occludin mRNA were visualized using MS2-YFP (green fluorescence); red, Ago2 (P-body marker) signals; yellow, colocalized red and green signals. Three experiments were performed that showed similar results. (D) Quantification of the number of merged signals indicative of colocalization of MS2-occludin RNA and Ago2 signals in cells described in (C). Values are the means ± SEM of data from six samples. * p < 0.05 compared with cells transfected with control vector; + p < 0.05 compared with cells transfected with the CUGBP1 expression vector.
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