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1.  miR-503 represses CUG-binding protein 1 translation by recruiting CUGBP1 mRNA to processing bodies 
Molecular Biology of the Cell  2012;23(1):151-162.
This study shows that microRNA-503 interacts with the CUG-binding protein 1 (CUGBP1) mRNA and represses its translation by recruiting the CUGBP1 mRNA to processing bodies.
microRNAs (miRNAs) and RNA-binding proteins (RBPs) jointly regulate gene expression at the posttranscriptional level and are involved in many aspects of cellular functions. The RBP CUG-binding protein 1 (CUGBP1) destabilizes and represses the translation of several target mRNAs, but the exact mechanism that regulates CUGBP1 abundance remains elusive. In this paper, we show that miR-503, computationally predicted to associate with three sites of the CUGBP1 mRNA, represses CUGBP1 expression. Overexpression of an miR-503 precursor (pre-miR-503) reduced the de novo synthesis of CUGBP1 protein, whereas inhibiting miR-503 by using an antisense RNA (antagomir) enhanced CUGBP1 biosynthesis and elevated its abundance; neither intervention changed total CUGBP1 mRNA levels. Studies using heterologous reporter constructs revealed a greater repressive effect of miR-503 through the CUGBP1 coding region sites than through the single CUGBP1 3′-untranslated region target site. CUGBP1 mRNA levels in processing bodies (P-bodies) increased in cells transfected with pre-miR-503, while silencing P-body resident proteins Ago2, RCK, or LSm4 decreased miR-503–mediated repression of CUGBP1 expression. Decreasing the levels of cellular polyamines reduced endogenous miR-503 levels and promoted CUGBP1 expression, an effect that was prevented by ectopic miR-503 overexpression. Repression of CUGBP1 by miR-503 in turn altered the expression of CUGBP1 target mRNAs and thus increased the sensitivity of intestinal epithelial cells to apoptosis. These findings identify miR-503 as both a novel regulator of CUGBP1 expression and a modulator of intestinal epithelial homoeostasis.
PMCID: PMC3248894  PMID: 22072795
2.  Competitive binding of CUGBP1 and HuR to occludin mRNA controls its translation and modulates epithelial barrier function 
Molecular Biology of the Cell  2013;24(2):85-99.
The present study shows that RNA-binding proteins CUGBP1 and HuR jointly regulate the translation of occludin and play a crucial role in the maintenance of tight junction integrity.
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.
PMCID: PMC3541967  PMID: 23155001
3.  Regulation of cyclin-dependent kinase 4 translation through CUG-binding protein 1 and microRNA-222 by polyamines 
Molecular Biology of the Cell  2011;22(17):3055-3069.
The findings presented indicate that polyamine-regulated CUG-binding protein 1 and microRNA-222 modulate cyclin-dependent kinase 4 (CDK4) translation at least in part by altering the recruitment of CDK4 mRNA to processing bodies.
The amino acid–derived polyamines are organic cations that are essential for growth in all mammalian cells, but their exact roles at the molecular level remain largely unknown. Here we provide evidence that polyamines promote the translation of cyclin-dependent kinase 4 (CDK4) by the action of CUG-binding protein 1 (CUGBP1) and microRNA-222 (miR-222) in intestinal epithelial cells. Both CUGBP1 and miR-222 were found to bind the CDK4 mRNA coding region and 3′-untranslated region and repressed CDK4 translation synergistically. Depletion of cellular polyamines increased cytoplasmic CUGBP1 abundance and miR-222 levels, induced their associations with the CDK4 mRNA, and inhibited CDK4 translation, whereas increasing the levels of cellular polyamines decreased CDK4 mRNA interaction with CUGBP1 and miR-222, in turn inducing CDK4 expression. Polyamine-deficient cells exhibited an increased colocalization of tagged CDK4 mRNA with processing bodies; this colocalization was abolished by silencing CUGBP1 and miR-222. Together, our findings indicate that polyamine-regulated CUGBP1 and miR-222 modulate CDK4 translation at least in part by altering the recruitment of CDK4 mRNA to processing bodies.
PMCID: PMC3164454  PMID: 21737690
4.  Smaug/SAMD4A Restores Translational Activity of CUGBP1 and Suppresses CUG-Induced Myopathy 
PLoS Genetics  2013;9(4):e1003445.
We report the identification and characterization of a previously unknown suppressor of myopathy caused by expansion of CUG repeats, the mutation that triggers Myotonic Dystrophy Type 1 (DM1). We screened a collection of genes encoding RNA–binding proteins as candidates to modify DM1 pathogenesis using a well established Drosophila model of the disease. The screen revealed smaug as a powerful modulator of CUG-induced toxicity. Increasing smaug levels prevents muscle wasting and restores muscle function, while reducing its function exacerbates CUG-induced phenotypes. Using human myoblasts, we show physical interactions between human Smaug (SMAUG1/SMAD4A) and CUGBP1. Increased levels of SMAUG1 correct the abnormally high nuclear accumulation of CUGBP1 in myoblasts from DM1 patients. In addition, augmenting SMAUG1 levels leads to a reduction of inactive CUGBP1-eIF2α translational complexes and to a correction of translation of MRG15, a downstream target of CUGBP1. Therefore, Smaug suppresses CUG-mediated muscle wasting at least in part via restoration of translational activity of CUGBP1.
Author Summary
Myotonic dystrophy type 1 (DM1) is the most common among the muscular dystrophies causing muscle weakness and wasting in adults, and it is triggered by expansion of an untranslated CUG repeat. To identify potential therapeutic approaches, we used a Drosophila DM1 model to screen for genes capable of suppressing CUG-induced toxicity. Here we report that increased levels of the smaug gene prevent muscle wasting and, perhaps more impressively, also prevent muscle dysfunction caused by the DM1 mutation. Smaug interacts genetically and physically with CUGBP1, an RNA–binding protein previously implicated in DM1. We used myoblasts from DM1 patients and control individuals to investigate how Smaug suppresses CUG-induced myopathy. We found that increased human SMAUG1 (a.k.a. SMAD4A) levels revert the abnormal accumulation of CUGBP1 in myoblasts nuclei and restore normal translation of at least one mRNA regulated by CUGBP1 in the cytoplasm. These findings demonstrate that manipulating Smaug activity protects against the effects of the DM1 mutation, and they also support the idea that restoring normal CUGBP1 function is a potential therapeutic approach.
PMCID: PMC3630084  PMID: 23637619
Experimental cell research  2008;314(11-12):2266-2278.
Differentiation of myocytes is impaired in patients with mytonic dystrophy type 1, DM1. CUG repeat binding protein, CUGBP1, is a key regulator of translation of proteins that are involved in muscle development and differentiation. In this paper, we present evidence that RNA-binding activity of CUGBP1 and its interactions with initiation translation complex eIF2 are differentially regulated during myogenesis by specific phosphorylation and that this regulation is altered in DM1. In normal myoblasts, Akt kinase phosphorylates CUGBP1 at Ser28 and increases interactions of CUGBP1 with cyclin D1 mRNA. During differentiation, CUGBP1 is phosphorylated by cyclinD3-cdk4/6 at Ser302, which increases CUGBP1 binding with p21 and C/EBPβ mRNAs. While cyclin D3 and cdk4 are elevated in normal myotubes; DM1 differentiating cells do not increase these proteins. In normal myotubes, CUGBP1 interacts with cyclin D3/cdk4/6 and eIF2; however, interactions of CUGBP1 with eIF2 are reduced in DM1 differentiating cells and correlate with impaired muscle differentiation in DM1. Ectopic expression of cyclin D3 in DM1 cells increases the CUGBP1-eIF2 complex, corrects expression of differentiation markers, myogenin and desmin, and enhances fusion of DM1 myoblasts. Thus, normalization of cyclin D3 might be a therapeutic approach to correct differentiation of skeletal muscle in DM1 patients.
PMCID: PMC2494712  PMID: 18570922
Myotonic Dystrophy 1; CUG repeats; CUGBP1-eIF2 complex; cyclin D3; differentiation
6.  CUG repeat binding protein (CUGBP1) interacts with the 5' region of C/EBPbeta mRNA and regulates translation of C/EBPbeta isoforms. 
Nucleic Acids Research  1999;27(22):4517-4525.
The transcription factor CCAAT/enhancer binding protein beta, C/EBPbeta, plays a significant role in the regulation of hepatocyte growth and differentiation. A single mRNA coding for C/EBPbeta produces several protein isoforms. Two pathways for generation of low molecular weight C/EBPbeta isoforms have been described: specific proteolytic cleavage and initiation of translation from different AUG codons of C/EBPbeta mRNA. A truncated C/EBPbeta isoform, LIP, is induced in rat livers in response to partial hepatectomy (PH) via the alternative translation mechanism. Here we present evidence that CUG repeat binding protein, CUGBP1, interacts with the 5' region of C/EBPbeta mRNA and regulates translation of C/EBPbeta isoforms. Two binding sites for CUGBP1 are located side by side between the first and second AUG codons of C/EBPbeta mRNA. One binding site is observed in an out of frame short open reading frame (sORF) that has been previously shown to regulate initiation of translation from different AUG codons of C/EBPbeta mRNA. Analysis of cytoplasmic and polysomal proteins from rat liver after PH showed that CUGBP1 is associated with polysomes that translate low molecular weight isoforms of C/EBPbeta. The binding activity of CUGBP1 to the 5' region of C/EBPbeta mRNA shows increased association with these polysomal fractions after PH. Addition of CUGBP1 into a cell-free translation system leads to increased translation of low molecular weight isoforms of C/EBPbeta. Our data demonstrate that CUGBP1 protein is an important component for the regulation of initiation from different AUG codons of C/EBPbeta mRNA.
PMCID: PMC148737  PMID: 10536163
7.  GSK3β is a new therapeutic target for myotonic dystrophy type 1 
Rare Diseases  2013;1:e26555.
Myotonic dystrophy type 1 (DM1), an incurable, neuromuscular disease, is caused by the expansion of CTG repeats within the 3′ UTR of DMPK on chromosome 19q. In DM1 patients, mutant DMPK transcripts deregulate RNA metabolism by altering CUG RNA-binding proteins. Several approaches have been proposed for DM1 therapy focused on specific degradation of the mutant CUG repeats or on correction of RNA-binding proteins, affected by CUG repeats. One such protein is CUG RNA-binding protein (CUGBP1). The ability of CUGBP1 to increase or inhibit translation depends on phosphorylation at Ser302, which is mediated by cyclin D3-CDK4. The mutant CUG repeats increase the levels of CUGBP1 protein and inhibit Ser302 phosphorylation, leading to the accumulation of CUGBP1 isoforms that repress translation (i.e., CUGBP1REP). Elevation of CUGBP1REP in DM1 is caused by increased GSK3β kinase, which reduces the cyclin D3-CDK4 pathway and subsequent phosphorylation of CUGBP1 at Ser302. In this review, we discuss our recent discovery showing that correction of GSK3β activity in the DM1 mouse model (i.e., HSALR mice) reduces DM1 muscle pathology. These findings demonstrate that GSK3β is a novel therapeutic target for treating DM1.
PMCID: PMC3927489  PMID: 25003008
CUG repeats; CUGBP1; GSK3β; TDZD-8; lithium; myotonic dystrophy type 1
8.  Heart-specific overexpression of CUGBP1 reproduces functional and molecular abnormalities of myotonic dystrophy type 1 
Human Molecular Genetics  2010;19(6):1066-1075.
Myotonic dystrophy type 1 (DM1) is caused by a CTG expansion within the 3′-untranslated region of the DMPK gene. The predominant mechanism of pathogenesis is a toxic gain of function of CUG repeat containing RNA transcribed from the expanded allele. The molecular mechanisms by which the RNA containing expanded repeats produce pathogenic effects include: sequestration of muscleblind-like 1 (MBNL1) protein and up-regulation of CUG binding protein 1 (CUGBP1). MBNL1 and CUGBP1 are RNA binding proteins that regulate alternative splicing transitions during development. Altered functions of these proteins in DM1 lead to misregulated splicing of their target genes, resulting in several features of the disease. The role of MBNL1 depletion in DM1 is well established through a mouse knock-out model that reproduces many disease features. Here we directly test the hypothesis that CUGBP1 up-regulation also contributes to manifestations of DM1. Using tetracycline-inducible CUGBP1 and heart-specific reverse tetracycline trans-activator transgenes, we expressed human CUGBP1 in adult mouse heart. Our results demonstrate that up-regulation of CUGBP1 is sufficient to reproduce molecular, histopathological and functional changes observed in a previously described DM1 mouse model that expresses expanded CUG RNA repeats as well as in individuals with DM1. These results strongly support a role for CUGBP1 up-regulation in DM1 pathogenesis.
PMCID: PMC2830830  PMID: 20051426
9.  The CUGBP2 Splicing Factor Regulates an Ensemble of Branchpoints from Perimeter Binding Sites with Implications for Autoregulation 
PLoS Genetics  2009;5(8):e1000595.
Alternative pre-mRNA splicing adjusts the transcriptional output of the genome by generating related mRNAs from a single primary transcript, thereby expanding protein diversity. A fundamental unanswered question is how splicing factors achieve specificity in the selection of target substrates despite the recognition of information-poor sequence motifs. The CUGBP2 splicing regulator plays a key role in the brain region-specific silencing of the NI exon of the NMDA R1 receptor. However, the sequence motifs utilized by this factor for specific target exon selection and its role in splicing silencing are not understood. Here, we use chemical modification footprinting to map the contact sites of CUGBP2 to GU-rich motifs closely positioned at the boundaries of the branch sites of the NI exon, and we demonstrate a mechanistic role for this specific arrangement of motifs for the regulation of branchpoint formation. General support for a branch site-perimeter–binding model is indicated by the identification of a group of novel target exons with a similar configuration of motifs that are silenced by CUGBP2. These results reveal an autoregulatory role for CUGBP2 as indicated by its direct interaction with functionally significant RNA motifs surrounding the branch sites upstream of exon 6 of the CUGBP2 transcript itself. The perimeter-binding model explains how CUGBP2 can effectively embrace the branch site region to achieve the specificity needed for the selection of exon targets and the fine-tuning of alternative splicing patterns.
Author Summary
Alternative splicing is a precisely controlled process that determines whether an exon will be included or skipped in the mature mRNA transcript. Factors that control alternative splicing bind to RNA sequence motifs in the exon or flanking introns and guide tissue and developmental specific splicing events. CUGBP2 is a dual functional regulator of alternative splicing that can cause inclusion or skipping of a target exon, depending on the context of its binding motifs. Previously, the mechanisms of regulation by this protein and the positional significance of its target motifs have not been characterized. In this study, the authors dissected the mechanism of exon skipping by CUGBP2 and demonstrate that a specific configuration of motifs at the perimeters of a functional reference point are intimately involved in this event. Furthermore, this mechanism of regulation is shown to have general significance because novel CUGBP2 target exons contain a similar arrangement of motifs. The most interesting of this group is an exon within the CUGBP2 transcript itself. This study underscores the importance of a functional reference point in the specificity of regulation by an alternative splicing factor and reveals a novel autoregulatory role for CUGBP2.
PMCID: PMC2715136  PMID: 19680430
10.  Elevation of RNA-binding protein CUGBP1 is an early event in an inducible heart-specific mouse model of myotonic dystrophy 
The Journal of Clinical Investigation  2007;117(10):2802-2811.
Myotonic dystrophy type 1 (DM1) is caused by a CTG trinucleotide expansion in the 3′ untranslated region (3′ UTR) of DM protein kinase (DMPK). The key feature of DM1 pathogenesis is nuclear accumulation of RNA, which causes aberrant alternative splicing of specific pre-mRNAs by altering the functions of CUG-binding proteins (CUGBPs). Cardiac involvement occurs in more than 80% of individuals with DM1 and is responsible for up to 30% of disease-related deaths. We have generated an inducible and heart-specific DM1 mouse model expressing expanded CUG RNA in the context of DMPK 3′ UTR that recapitulated pathological and molecular features of DM1 including dilated cardiomyopathy, arrhythmias, systolic and diastolic dysfunction, and misregulated alternative splicing. Combined in situ hybridization and immunofluorescent staining for CUGBP1 and CUGBP2, the 2 CUGBP1 and ETR-3 like factor (CELF) proteins expressed in heart, demonstrated elevated protein levels specifically in nuclei containing foci of CUG repeat RNA. A time-course study demonstrated that colocalization of MBNL1 with RNA foci and increased CUGBP1 occurred within hours of induced expression of CUG repeat RNA and coincided with reversion to embryonic splicing patterns. These results indicate that CUGBP1 upregulation is an early and primary response to expression of CUG repeat RNA.
PMCID: PMC1964514  PMID: 17823658
11.  Pathogenic mechanisms of myotonic dystrophy 
Biochemical Society transactions  2009;37(0 6):10.1042/BST0371281.
DM (myotonic dystrophy) is a dominantly inherited genetic disorder that is the most common cause of muscular dystrophy in adults affecting 1 in 8500 individuals worldwide. Different microsatellite expansions in two loci cause different forms of the disease that share similar features: DM1 (DM type 1) is caused by a tri- (CTG) nucleotide expansion within the DMPK (dystrophia myotonica protein kinase) 3′-untranslated region and DM2 (DM type 2) is caused by a tetra- (CCTG) nucleotide expansion within intron 1 of the ZNF9 (zinc finger 9) gene. The pathogenic mechanism of this disease involves the RNA transcribed from the expanded allele containing long tracts of (CUG)n or (CCUG)n. The RNA results in a toxic effect through two RNA-binding proteins: MBNL1 (muscleblind-like 1) and CUGBP1 (CUG-binding protein 1). In DM1, MBNL1 is sequestered on CUG repeat-containing RNA resulting in its loss-of-function, while CUGBP1 is up-regulated through a signalling pathway. The downstream effects include disrupted regulation of alternative splicing, mRNA translation and mRNA stability, which contribute to the multiple features of DM1. This review will focus on the RNA gain-of-function disease mechanism, the important roles of MBNL1 and CUGBP1 in DM1, and the relevance to other RNA dominant disorders.
PMCID: PMC3873089  PMID: 19909263
CUG repeat; CUG-binding protein 1 (CUGBP1); dystrophia myotonica protein kinase (DMPK); muscleblind-like 1 (MBNL1); myotonic dystrophy type 1 (DM1); RNA gain-of-function
12.  CUGBP1 overexpression in mouse skeletal muscle reproduces features of myotonic dystrophy type 1 
Human Molecular Genetics  2010;19(18):3614-3622.
The neuromuscular disease myotonic dystrophy type I (DM1) affects multiple organ systems with the major symptoms being severe muscle weakness, progressive muscle wasting and myotonia. The causative mutation in DM1 is a CTG repeat expansion in the 3′-untranslated region of the DM protein kinase (DMPK) gene. RNA transcribed from the expanded allele contains the expanded CUG repeats and leads to the nuclear depletion of Muscleblind-like 1 (MBNL1) and to the increased steady-state levels of CUG-binding protein 1 (CUGBP1). The pathogenic effects of MBNL1 depletion have previously been tested by the generation of MBNL1 knockout mice, but the consequence of CUGBP1 overexpression in adult muscle is not known. In a DM1 mouse model expressing RNA containing 960 CUG repeats in skeletal muscle, CUGBP1 up-regulation is temporally correlated with severe muscle wasting. In this study, we generated transgenic mice with doxycycline-inducible and skeletal muscle-specific expression of CUGBP1. Adult mouse skeletal muscle overexpressing CUGBP1 reproduces molecular and physiological defects of DM1 tissue. The results from this study strongly suggest that CUGBP1 has a major role in DM1 skeletal muscle pathogenesis.
PMCID: PMC2928132  PMID: 20603324
13.  Analysis of CUGBP1 Targets Identifies GU-Repeat Sequences That Mediate Rapid mRNA Decay ▿ †  
Molecular and Cellular Biology  2010;30(16):3970-3980.
CUG-repeat binding protein 1 (CUGBP1) mediates selective mRNA decay by binding to GU-rich elements (GREs) containing the sequence UGUUUGUUUGU found in the 3′ untranslated region (UTR) of short-lived transcripts. We used an anti-CUGBP1 antibody to immunoprecipitate CUGBP1 from HeLa cytoplasmic extracts and analyzed the associated transcripts using oligonucleotide microarrays. We identified 613 putative mRNA targets of CUGBP1 and found that the UGUUUGUUUGU GRE sequence and a GU-repeat sequence were both highly enriched in the 3′ UTRs of these targets. We showed that CUGBP1 bound specifically to the GU-repeat sequence and that insertion of this sequence into the 3′ UTR of a beta-globin reporter transcript conferred instability to the transcript. Based on these results, we redefined the GRE to include this GU-repeat sequence. Our results suggest that CUGBP1 coordinately regulates the mRNA decay of a network of transcripts involved in cell growth, cell motility, and apoptosis.
PMCID: PMC2916446  PMID: 20547756
14.  Conserved GU-Rich Elements Mediate mRNA Decay by Binding to CUG-Binding Protein 1 
Molecular cell  2008;29(2):263-270.
We used computational algorithms to find conserved sequences in the 3′ untranslated region (UTR) of transcripts that exhibited rapid decay in primary human T cells and found that the consensus sequence UGUUUGUUUGU, which we have termed a GU-rich element (GRE), was enriched in short-lived transcripts. Using a tet-off reporter system, we showed that insertion of GRE-containing sequences from c-jun, jun B, or TNF receptor 1B, but not mutated GRE sequences, into the 3′UTR of a β-globin transcript conferred instability on the otherwise stable β-globin transcript. CUG-binding protein 1 (CUGBP1) was identified as the major GRE-binding activity in cytoplasmic extracts from primary human T cells based on supershift and immunoprecipitation assays. siRNA-mediated knockdown of CUGBP1 in HeLa cells caused stabilization of GRE-containing transcripts, suggesting that CUGBP1 is a mediator of GRE-dependent mRNA decay. Overall, our results suggest that the GRE mediates coordinated mRNA decay by binding to CUGBP1.
PMCID: PMC2367162  PMID: 18243120
15.  Molecular Basis for Impaired Muscle Differentiation in Myotonic Dystrophy 
Molecular and Cellular Biology  2001;21(20):6927-6938.
Differentiation of skeletal muscle is affected in myotonic dystrophy (DM) patients. Analysis of cultured myoblasts from DM patients shows that DM myoblasts lose the capability to withdraw from the cell cycle during differentiation. Our data demonstrate that the expression and activity of the proteins responsible for cell cycle withdrawal are altered in DM muscle cells. Skeletal muscle cells from DM patients fail to induce cytoplasmic levels of a CUG RNA binding protein, CUGBP1, while normal differentiated cells accumulate CUGBP1 in the cytoplasm. In cells from normal patients, CUGBP1 up-regulates p21 protein during differentiation. Several lines of evidence show that CUGBP1 induces the translation of p21 via binding to a GC-rich sequence located within the 5′ region of p21 mRNA. Failure of DM cells to accumulate CUGBP1 in the cytoplasm leads to a significant reduction of p21 and to alterations of other proteins responsible for the cell cycle withdrawal. The activity of cdk4 declines during differentiation of cells from control patients, while in DM cells cdk4 is highly active during all stages of differentiation. In addition, DM cells do not form Rb/E2F repressor complexes that are abundant in differentiated cells from normal patients. Our data provide evidence for an impaired cell cycle withdrawal in DM muscle cells and suggest that alterations in the activity of CUGBP1 causes disruption of p21-dependent control of cell cycle arrest.
PMCID: PMC99869  PMID: 11564876
16.  Structural Insights into RNA Recognition by the Alternate-splicing Regulator CUG Binding Protein 1 
Structure(London, England:1993)  2010;18(10):1364-1377.
CUG binding protein 1 (CUGBP1) regulates multiple aspects of nuclear and cytoplasmic mRNA processing, with implications for onset of myotonic dystrophy. CUGBP1 harbors three RRM domains and preferentially targets UGU-rich mRNA elements. We report on crystal structures of CUGBP1 RRM1 and tandem RRM1/2 domains bound to RNAs containing tandem UGU(U/G) elements. Both RRM1 in RRM1-RNA and RRM2 in RRM1/2-RNA complexes use similar principles to target UGU(U/G) elements, with recognition mediated by face-to-edge stacking and water-mediated hydrogen bonding networks. The UG step adopts a left-handed Z-RNA conformation, with the syn guanine recognized through Hoogsteen edge-protein backbone hydrogen-bonding interactions. NMR studies on the RRM1/2-RNA complex establish that both RRM domains target tandem UGUU motifs in solution, while filter-binding assays identify a preference for recognition of GU over AU or GC steps. We discuss the implications of CUGBP1-mediated targeting and sequestration of UGU(U/G) elements on pre-mRNA alternative-splicing regulation, translational regulation and mRNA decay.
PMCID: PMC3381513  PMID: 20947024
17.  Increased steady state levels of CUGBP1 in Myotonic Dystrophy 1 are due to PKC-mediated hyper-phosphorylation 
Molecular cell  2007;28(1):68-78.
The genetic basis of myotonic dystrophy type 1 (DM1) is a CTG expansion in the 3' untranslated region (UTR) of DMPK. The pathogenic mechanism involves an RNA gain of function in which the repeat containing transcripts accumulate in nuclei and alter the functions of RNA binding proteins such as CUG binding protein 1 (CUGBP1). CUGBP1 levels are increased in DM1 myoblasts, heart and skeletal muscle tissues and in some DM1 mouse models. However, the molecular mechanisms for increased CUGBP1 in DM1 are unclear. Here we demonstrate that expression of DMPK-CUG repeat RNA results in hyper-phosphorylation and stabilization of CUGBP1. CUGBP1 is hyper-phosphorylated in DM1 tissues, cells, and a DM1 mouse model. Activation of PKC is required for CUGBP1 hyper-phosphorylation in DM1 cells and PKCα and βII directly phosphorylate CUGBP1 in vitro. These results indicate that inappropriate activation of the PKC pathway contributes to the pathogenic effects of a non-coding RNA.
PMCID: PMC2083558  PMID: 17936705
18.  The role of CUGBP1 in age-dependent changes of liver functions 
Ageing research reviews  2012;11(4):442-449.
Aging liver is characterized by alterations of liver biology and by a reduction of many functions which are important for the maintenance of body homeostasis. The main dysfunctions include appearance of enlarged hepatocytes, impaired liver regeneration after partial hepatectomy (PH), development of hepatic steatosis, reduction of secretion of proteins and alterations in the hepatic sinusoid. RNA binding proteins are involved in the regulation of gene expression in all tissues including regulation of biological processes in the liver. This review is focused on the role of a conserved, multi-functional RNA-binding protein, CUGBP1, in the development of aging phenotype in the liver. CUGBP1 has been identified as a protein which binds to RNA CUG repeats expanded in Myotonic Dystrophy type 1 (DM1). CUGBP1 is highly expressed in the liver and regulates translation of proteins which are critical for maintenance of liver functions. In livers of young mice, CUGBP1 forms complexes with eukaryotic translation initiation factor eIF2 and supports translation of C/EBPβ and HDAC1 proteins, which are involved in liver growth, differentiation and liver cancer. Aging changes several signaling pathways which lead to the elevation of the CUGBP1-eIF2α complex and to an increase of translation of C/EBPβ and HDAC1. These proteins form multi-protein complexes with additional transcription factors and with chromatin remodeling proteins causing epigenetic alterations of gene expression in livers of old mice. It appears that CUGBP1-mediated translational elevation of HDAC1 is one of the key events in the epigenetic changes in livers of old mice, leading to the development of age- associated dysfunctions of the liver. This review will also discuss a possible role of CUGBP1 in liver dysfunction in patients affected with DM1.
PMCID: PMC3610417  PMID: 22446383
Aging; liver; CUGBP1; C/EBP; chromatin; epigenetic
19.  RNA Binding Protein CUGBP2/CELF2 Mediates Curcumin-Induced Mitotic Catastrophe of Pancreatic Cancer Cells 
PLoS ONE  2011;6(2):e16958.
Curcumin inhibits the growth of pancreatic cancer tumor xenografts in nude mice; however, the mechanism of action is not well understood. It is becoming increasingly clear that RNA binding proteins regulate posttranscriptional gene expression and play a critical role in RNA stability and translation. Here, we have determined that curcumin modulates the expression of RNA binding protein CUGBP2 to inhibit pancreatic cancer growth.
Methodology/Principal Findings
In this study, we show that curcumin treated tumor xenografts have a significant reduction in tumor volume and angiogenesis. Curcumin inhibited the proliferation, while inducing G2-M arrest and apoptosis resulting in mitotic catastrophe of various pancreatic cancer cells. This was further confirmed by increased phosphorylation of checkpoint kinase 2 (Chk2) protein coupled with higher levels of nuclear cyclin B1 and Cdc-2. Curcumin increased the expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) mRNA, but protein levels were lower. Furthermore, curcumin increased the expression of RNA binding proteins CUGBP2/CELF2 and TIA-1. CUGBP2 binding to COX-2 and VEGF mRNA was also enhanced, thereby increasing mRNA stability, the half-life changing from 30 min to 8 h. On the other hand, silencer-mediated knockdown of CUGBP2 partially restored the expression of COX-2 and VEGF even with curcumin treatment. COX-2 and VEGF mRNA levels were reduced to control levels, while proteins levels were higher.
Curcumin inhibits pancreatic tumor growth through mitotic catastrophe by increasing the expression of RNA binding protein CUGBP2, thereby inhibiting the translation of COX-2 and VEGF mRNA. These data suggest that translation inhibition is a novel mechanism of action for curcumin during the therapeutic intervention of pancreatic cancers.
PMCID: PMC3037932  PMID: 21347286
20.  CUGBP2 directly interacts with U2 17S snRNP components and promotes U2 snRNA binding to cardiac troponin T pre-mRNA 
Nucleic Acids Research  2009;37(13):4275-4286.
CUGBP2 (ETR-3/NAPOR/BRUNOL3) promotes inclusion of cardiac troponin T (cTNT) exon 5 via binding between positions 21 and 74 of the downstream intron. The molecular mechanism by which CUGBP2 activates cTNT exon 5 inclusion is unknown. Our results suggest that CUGBP2 promotes exon inclusion by a novel mechanism in which CUGBP2 directly interacts with components of the activated U2 snRNP and enhances binding of U2 snRNP to the branch site located upstream of the exon. Using an in vitro splicing assay, we show that recombinant CUGBP2 enhances complex A formation of a cTNT pre-mRNA. Enhanced complex A assembly requires both the upstream and downstream introns consistent with dual requirements for the downstream CUGBP2-binding site and an upstream branch site for U2 snRNP binding. We also show that CUGBP2 enhances binding of U2 snRNA to the cTNT pre-mRNA consistent with enhanced complex A assembly. Purification of CUGBP2-interacting proteins using tandem affinity purification leads to the demonstration that the core 17S U2 snRNP components, SF3b145 and SF3b49 bind directly to CUGBP2. We conclude that CUGBP2 activates exon inclusion by forming direct interactions with components of the 17S snRNP complex and recruits and/or stabilizes binding of U2 snRNP.
PMCID: PMC2715230  PMID: 19443441
21.  Far Upstream Element Binding Protein 1 Activates Translation of p27Kip1 mRNA Through Its Internal Ribosomal Entry Site 
The cyclin dependent kinase inhibitor p27 plays an important role in controlling the eukaryotic cell cycle by regulating progression through G1 and entry into S phase. It is often elevated during differentiation and under conditions of cellular stress. In contrast, it is commonly downregulated in cancer cells and its levels are generally inversely correlated with favorable prognosis. The cellular levels of p27 are regulated, in part, by translational control mechanisms. The 5′-untranslated region (5′-UTR) of the p27 mRNA harbors an internal ribosome entry site (IRES) which may facilitate synthesis of p27 in certain conditions. In this study, Far Upstream Element (FUSE) Binding Protein 1 (FBP1) was shown to directly bind to the human p27 5′-UTR and to promote IRES activity. An eight-nucleotide element downstream of a U-rich region within the 5′-UTR was important for FBP1 binding and p27 IRES activity. Overexpression of FBP1 enhanced endogenous p27 levels and stimulated translation initiation. In contrast, repression of FBP1 by siRNA transfection downregulated endogenous p27 protein levels. Using rabbit reticulocyte lysates, FBP1 stimulated p27 mRNA translation in vitro. The central domain of FBP1, containing four K homology motifs, was required for p27 5′-UTR RNA binding and the N terminal domain was important for translational activation. These findings indicate that FBP1 is a novel activator of p27 translation upon binding to the 5′-UTR.
PMCID: PMC3206725  PMID: 21855647
p27Kip1; cell cycle; IRES; FBP; 5′-untranslated region
22.  CUGBP1 and MBNL1 preferentially bind to 3′ UTRs and facilitate mRNA decay 
Scientific Reports  2012;2:209.
CUGBP1 and MBNL1 are developmentally regulated RNA-binding proteins that are causally associated with myotonic dystrophy type 1. We globally determined the in vivo RNA-binding sites of CUGBP1 and MBNL1. Interestingly, CUGBP1 and MBNL1 are both preferentially bound to 3′ UTRs. Analysis of CUGBP1- and MBNL1-bound 3′ UTRs demonstrated that both factors mediate accelerated mRNA decay and temporal profiles of expression arrays supported this. Role of CUGBP1 on accelerated mRNA decay has been previously reported, but the similar function of MBNL1 has not been reported to date. It is well established that CUGBP1 and MBNL1 regulate alternative splicing. Screening by exon array and validation by RT-PCR revealed position dependence of CUGBP1- and MBNL1-binding sites on the resulting alternative splicing pattern. This study suggests that regulation of CUGBP1 and MBNL1 is essential for accurate control of destabilization of a broad spectrum of mRNAs as well as of alternative splicing events.
PMCID: PMC3250574  PMID: 22355723
23.  GSK3β mediates muscle pathology in myotonic dystrophy 
The Journal of Clinical Investigation  2012;122(12):4461-4472.
Myotonic dystrophy type 1 (DM1) is a complex neuromuscular disease characterized by skeletal muscle wasting, weakness, and myotonia. DM1 is caused by the accumulation of CUG repeats, which alter the biological activities of RNA-binding proteins, including CUG-binding protein 1 (CUGBP1). CUGBP1 is an important skeletal muscle translational regulator that is activated by cyclin D3–dependent kinase 4 (CDK4). Here we show that mutant CUG repeats suppress Cdk4 signaling by increasing the stability and activity of glycogen synthase kinase 3β (GSK3β). Using a mouse model of DM1 (HSALR), we found that CUG repeats in the 3′ untranslated region (UTR) of human skeletal actin increase active GSK3β in skeletal muscle of mice, prior to the development of skeletal muscle weakness. Inhibition of GSK3β in both DM1 cell culture and mouse models corrected cyclin D3 levels and reduced muscle weakness and myotonia in DM1 mice. Our data predict that compounds normalizing GSK3β activity might be beneficial for improvement of muscle function in patients with DM1.
PMCID: PMC3533547  PMID: 23160194
24.  Calreticulin Interacts with C/EBPα and C/EBPβ mRNAs and Represses Translation of C/EBP Proteins 
Molecular and Cellular Biology  2002;22(20):7242-7257.
We previously identified an RNA binding protein, CUGBP1, which binds to GCN repeats located within the 5′ region of C/EBPβ mRNAs and regulates translation of C/EBPβ isoforms. To further investigate the role of RNA binding proteins in the posttranscriptional control of C/EBP proteins, we purified additional RNA binding proteins that interact with GC-rich RNAs and that may regulate RNA processing. In HeLa cells, the majority of GC-rich RNA binding proteins are associated with endogenous RNA transcripts. The separation of these proteins from endogenous RNA identified several proteins in addition to CUGBP1 that specifically interact with the GC-rich 5′ region of C/EBPβ mRNA. One of these proteins was purified to homogeneity and was identified as calreticulin (CRT). CRT is a multifunctional protein involved in several biological processes, including interaction with and regulation of rubella virus RNA processing. Our data demonstrate that both CUGBP1 and CRT interact with GCU repeats within myotonin protein kinase and with GCN repeats within C/EBPα and C/EBPβ mRNAs. GCN repeats within these mRNAs form stable SL structures. The interaction of CRT with SL structures of C/EBPβ and C/EBPα mRNAs leads to inhibition of translation of C/EBP proteins in vitro and in vivo. Deletions or mutations abolishing the formation of SL structures within C/EBPα and C/EBPβ mRNAs lead to a failure of CRT to inhibit translation of C/EBP proteins. CRT-dependent inhibition of C/EBPα is sufficient to block the growth-inhibitory activity of C/EBPα. This finding further defines the molecular mechanism for posttranscriptional regulation of the C/EBPα and C/EBPβ proteins.
PMCID: PMC139801  PMID: 12242300
25.  Polypyrimidine Tract-Binding Protein Enhances the Internal Ribosomal Entry Site-Dependent Translation of p27Kip1 mRNA and Modulates Transition from G1 to S Phase 
Molecular and Cellular Biology  2005;25(4):1283-1297.
The p27Kip1 protein plays a critical role in the regulation of cell proliferation through the inhibition of cyclin-dependent kinase activity. Translation of p27Kip1 is directed by an internal ribosomal entry site (IRES) in the 5′ nontranslated region of p27Kip1 mRNA. Here, we report that polypyrimidine tract-binding protein (PTB) specifically enhances the IRES activity of p27Kip1 mRNA through an interaction with the IRES element. We found that addition of PTB to an in vitro translation system and overexpression of PTB in 293T cells augmented the IRES activity of p27Kip1 mRNA but that knockdown of PTB by introduction of PTB-specific small interfering RNAs (siRNAs) diminished the IRES activity of p27Kip1 mRNA. Moreover, the G1 phase in the cell cycle (which is maintained in part by p27Kip1) was shortened in cells depleted of PTB by siRNA knockdown. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced differentiation in HL60 cells was used to examine PTB-induced modulation of p27Kip1 protein synthesis during differentiation. The IRES activity of p27Kip1 mRNA in HL60 cells was increased by TPA treatment (with a concomitant increase in PTB protein levels), but the levels of p27Kip1 mRNA remained unchanged. Together, these data suggest that PTB modulates cell cycle and differentiation, at least in part, by enhancing the IRES activity of p27Kip1 mRNA.
PMCID: PMC548013  PMID: 15684381

Results 1-25 (871193)