RNA CUG Repeats Sequester CUGBP1 and Alter Protein Levels and Activity of CUGBP1*

Nikolai A. TimchenkoDagger , Zong-Jin Cai§, Alana L. WelmDagger , Sita Reddy, Tetsuo Ashizawa||, and Lubov T. Timchenko§**

From the Departments of Dagger  Pathology and Huffington Center on Aging, § Medicine, Section of Cardiovascular Sciences, and || Neurology, Baylor College of Medicine, Houston, Texas 77030 and the  Department of Biochemistry and Molecular Biology, University of Southern California, School of Medicine, Los Angeles, California 90033

Received for publication, July 6, 2000, and in revised form, October 10, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

An RNA CUG triplet repeat binding protein, CUGBP1, regulates splicing and translation of various RNAs. Expansion of RNA CUG repeats in the 3'-untranslated repeat of the mutant myotonin protein kinase (DMPK) mRNA in myotonic dystrophy (DM) is associated with alterations in binding activity of CUGBP1. To investigate whether CUGBP1 is directly affected by expansion of CUG repeats in DM tissues, we examined the intracellular status of CUGBP1 in DM patients as well as in cultured cells over expressing RNA CUG repeats. The analysis of RNA·protein complexes showed that, in control tissues, the majority of CUGBP1 is free of RNA, whereas in DM patients the majority of CUGBP1 is associated with RNA containing CUG repeats. Similarly to DM patients, overexpression of RNA CUG repeats in cultured cells results in the re-allocation of CUGBP1 from a free state to the RNA·protein complexes containing CUG repeats. CUG repeat-dependent translocation of CUGBP1 into RNA·protein complexes is associated with increased levels of CUGBP1 protein and its binding activity. Experiments with cyclohexamide-dependent block of protein synthesis showed that the half-life of CUGBP1 is increased in cells expressing CUG repeats. Alteration of CUGBP1 in DM is accompanied by alteration in translation of a transcription factor CCAAT/enhancer-binding protein beta  (C/EBPbeta ), which has been previously described to be a target of CUGBP1. Analysis of C/EBPbeta isoforms in DM patients with altered levels of CUGBP1 showed that translation of a dominant negative isoform, LIP, is induced by CUGBP1. Results of this paper demonstrate that the expansion of CUG repeats in DM affects RNA-binding proteins and leads to alteration in RNA processing.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The mutation leading to myotonic dystrophy (DM)1 (1) is an expanded CTG trinucleotide repeat located in the 3'-untranslated region (UTR) of the myotonin protein kinase (DMPK) gene (2, 3). It was initially suggested that alterations in expression of DMPK protein may cause DM pathogenesis (4, 5). However, both DMPK knockout and transgenic mice overexpressing DMPK did not develop the complete DM phenotype (6, 7). Given these observations, several new hypotheses for DM pathogenesis have been proposed. One hypothesis suggests that CTG repeats might affect transcription of gene(s) that are located upstream or downstream of these CTG elements (8, 9). Two genes adjacent to DMPK have been identified and investigated (8-10). Deletion of DMAHP in mice showed that reduction of DMAHP is associated with the increased rate of cataracts but not myotonia or cardiac abnormalities (10). An alternative hypothesis suggests that DM pathogenesis is due to expression of mutant DMPK mRNA (11). This hypothesis proposes that mutant DMPK mRNA has a dominant effect on RNA metabolism (11). Following this RNA-based hypothesis, we suggested that expanded RNA CUG repeats in DMPK mRNA may serve as binding sites for specific CUG triplet repeat RNA-binding proteins (12-17). We also proposed that the dramatic increase of (CUG)n repeats in DM patients might affect expression of these RNA-binding proteins (14, 17). The RNA-based hypothesis for DM pathogenesis has been recently proved by Thornton and colleagues (18), showing that overexpression of RNA CUG repeats in transgenic mice induces myopathy and myotonia.

A number of RNA-binding proteins with specific binding activity to CUG repeats have been identified (12, 13, 16, 19, 20). These proteins include CUGBP1, ETR-3, and Brunol 1 and recently identified a group of expansion binding proteins that preferentially bind to double-stranded structures containing CUG repeats (20). All of these proteins are likely to be involved in DM pathogenesis, because their activities are altered in DM patients (13-15, 20, 21). CUGBP1 was discovered in 1996 and has been characterized in detail (12-15, 17, 21, 22). CUGBP1 is localized in both nuclei and cytoplasm. The nuclear function of CUGBP1 seems to be associated with splicing (21). It has been demonstrated that cardiac troponin T (cTnT) is a target of CUGBP1 (21) and that splicing of cTnT is altered in hearts of DM patients. CUGBP1 is homologous to Brunol proteins that play a role in translation (19). This suggests that CUGBP1 may play a role in translation of RNA. In agreement with this suggestion, a significant portion of CUGBP1 has been found to localize in the cytoplasm (22). It has been recently shown that CUGBP1 is associated with polysomes and is involved in the regulation of translation of C(U/C)G-containing mRNA (22). This function of CUGBP1 may play an important role in regulation of cell growth and proliferation by controlling activities of transcription factors and cell cycle proteins. A member of the CCAAT/Enhancer Binding Protein family, C/EBPbeta , is encoded by an intronless gene, but a single mRNA of C/EBPbeta can produce several isoforms with different transcriptional activities (Ref. 23 and see Fig. 5A). Three major isoforms of C/EBPbeta are produced by alternative translation: full-length (FL, 38 kDa), liver activator protein (LAP, 35 kDa) and liver inhibitory protein (LIP, 20 kDa). CUGBP1 has been shown to regulate translation of a dominant negative isoform of C/EBPbeta , liver-enriched transcriptional inhibitory protein (LIP) (22). The ability of CUGBP1 to regulate translation of C(U/C)G-containing mRNAs suggests that altered expression of CUGBP1 in DM may lead to changes in translation of C(U/C)G-containing mRNAs.

In this paper, we present evidence showing that RNA CUG repeats directly affect expression and activity of CUGBP1. Experiments in tissue culture demonstrate that endogenous CUGBP1 is titrated from a free pool by overexpression of transcripts with long CUG repeats. Formation of CUGBP1·CUG RNA complexes is accompanied by increased stability of CUGBP1 protein and subsequent elevation of CUGBP1. Similar to results obtained in tissue culture, analysis of DM patients showed that protein levels of CUGBP1 are increased in DM patients and the majority of CUGBP1 is detectable within RNA·protein complexes containing CUG repeats. Sequestration of CUGBP1 in DM patients is accompanied by altered translation of CUGBP1-dependent mRNAs.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transfection of Plasmid DNA Expressing CTG Repeats and Analysis of CUGBP1 Protein Levels-- Long CTG repeats (170 and 500 repeats) were synthesized as described previously (24), fused with a human beta -actin promoter, and cloned into the pSV2 vector. The resulting plasmid included the sequence of the human beta -actin gene, consisting of 3 kb of 5'-flanking sequence followed by 78 bp of 5'-UTR and 832 bp of intron I. CTG repeat sequences were cloned between the beta -actin promoter and the SV40 polyadenylation signal. Cos7 cells were grown in Dulbecco's modified Eagle's medium. For transfections, plasmid DNA (18 µg/15-cm dish) was mixed with LipofectAMINE or Lipofectin and added to cultured cells. Control transfections were performed with vector DNA (BS). The efficiency of transfection was evaluated by using plasmid DNA containing beta -galactosidase. After transfection, cells with empty vector and CTG-containing plasmids were used for protein and RNA extraction.

Northern Analysis-- To analyze the level of RNA (CUG)170-500 expression in cultured cells, total RNA was purified from transfected cells with TRI reagent. The integrity of RNA has been verified by gel electrophoresis. To evaluate the level of CUG expression, RNA was transferred onto a Z-probe membrane and hybridized with a 32P-(CAG)8 probe. RNA containing 170 CUG repeats was detected after 12 h of exposure. RNA containing 500 CUG repeats was detected after 36 h of exposure because of lower efficiency of RNA expression from the construct containing 500 CTG repeats. No signals for CUG-containing RNAs were detected in control cells transfected with BS after 36 h of exposure.

Determination of Half-life for CUGBP1 Protein-- Cos7 or HT1080 cultured cells were transfected with wild type (empty) vector or with plasmid expressing 170 CUG repeats. Cyclohexamide (CHX, 10 µg/ml) was added, and proteins were isolated 2, 4, and 8 h after CHX addition. CUGBP1 levels were examined by Western assay with monoclonal antibodies to CUGBP1 (13). The block of protein synthesis by CHX was verified by analysis of p21 protein half-life in HT1080 cells. In experiments presented in the manuscript, the p21 half-life in both control and CUG-expressing cells was 40-60 min and agreed with our previous estimate (25). Densitometric analysis of CUGBP1 protein levels indicated that CUGBP1 half-life is ~3 h in control cells.

UV Cross-linking Assay-- RNA oligomer CUG8 was labeled with [gamma -32P]ATP and T4 kinase. Cytoplasmic proteins were incubated with the probe, treated with UV light, and analyzed by polyacrylamide gel electrophoresis containing SDS. Where indicated, unlabeled RNA competitor (100 ng) was added prior to protein addition. To verify the concentration of proteins used in UV cross-linking assay, the membrane was stained with Coomassie Blue after the UV cross-link analysis. All gels presented in this paper were equally loaded.

Western Analysis-- Proteins from DM tissues (14, 21) were isolated as described previously (12-15). Protein extracts from normal controls were purchased from CLONTECH Co. 50 or 100 µg of protein was loaded on a 10-12% polyacrylamide gel and transferred onto a nitrocellulose filter (Bio-Rad). The filter was blocked with 10% dry milk/2% bovine serum albumin prepared in TTBS buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween-20) for 1 h at room temperature. Primary antibodies to CUGBP1 or to C/EBPbeta (C-19, Santa Cruz Biotechnology) were added, and the filter was incubated for 1 h, washed, and then incubated with secondary antibody for 1 h. Immunoreactive proteins were detected using the ECL method. After detection of the protein of interest, the membrane was stripped and reprobed with anti-beta -actin. For quantitative analysis, the intensity of the signals was determined on the Alpha Imager 2000 gel documentation and analysis system. Protein levels were calculated as a ratio to control protein. In the case of C/EBPbeta , the ratios LAP/FL or LIP/FL were calculated.

To study CUGBP1 expression in cultured cells, whole cell protein extracts were prepared from transfected cells as described (13) and analyzed by Western assay with monoclonal (13) or polyclonal antibodies against CUGBP1 (16). To verify protein loading, membranes were stripped and reprobed with antibodies against beta -actin. The intensity of CUGBP1 was determined on the Alpha Imager 2000 documentation and analysis system, and the amount of CUGBP1 was calculated as a ratio to beta -actin.

Semi-quantitative RT-PCR-- Total RNA was extracted from heart tissues and from transfected cells with TRI reagent (Molecular Research Center). mRNA was extracted with a poly(A) Quick kit (Stratagene). Normal control poly(A) and total RNA were from CLONTECH Co. Total RNA (1 µg) and mRNA (100 ng) were used for the RT reaction with M-Mul (Stratagene) and oligo(dT) primers. The RT product (2 µl) was then used for PCR, with two sets of primers: one set for the CUGBP1 gene and another one for the TBP gene (internal control). A PCR assay (50 µl) contained 20 pmol of each primer; 50 mM KCl; 10 mM Tris-HCl, pH 9.0; 0.1% Triton X-100; 1.5 mM MgCl2; 200 µM each of dATP, dCTP, dTTP, and dGTP; 100 µCi of [alpha -32P]dCTP; and 5 units of Taq polymerase (Promega). Amplification was performed in a Robocycler (Stratagene) under the following conditions: denaturation was at 94 °C for 90 s, annealing at 54 °C for 90 s, and extension at 72 °C for 150 s. The optimal number of cycles when PCR was linear (with heteroduplex formation) was 19 cycles. The PCR products were separated by 10% polyacrylamide gel electrophoresis. Intensities of PCR products were quantified by PhosphorImager scans (Molecular Dynamics) using the ImageQuaNT version 1.1 image analysis program (Molecular Dynamics). The CUGBP1 level was calculated as a ratio of the peak area for TBP. Primer sequences for RT-PCR were as follows: CUGBP1, 5'-CCAGACAACCAGATCTTGATGCT-3' and 5'-AGGTTTCATCTGTATAGGGTGATG-3'; TBP, 5'-CCAGGAAATAACTCTGGCTCATAAC-3', and 5'-AGTGAAGAACAGTCCAGACTGGCAG-3.

Fractionation and Analysis of CUGBP1·RNA Complexes using HPLC-based Size Exclusion Chromatography-- Cytoplasm from Cos7 cells or from tissues of DM patients was fractionated by size exclusion chromatography on an SEC-400 column (BioLogic HR, Bio-Rad). Standard protein molecular weight markers were run in parallel. 300-µl fractions were collected and used for further analysis of CUGBP1 protein and its binding activity. The presence of total RNA in HPLC fractions was examined by agarose gel electrophoresis followed by EtBr staining and by slot hybridization of gel filtration fractions with 18 S rRNA-specific probe (29). The position of CUG repeat-containing mRNA within the fractions was determined by slot hybridization with a CAG8 DNA probe labeled by gamma -32P in a kinase reaction. The conditions for slot hybridization are as follows. 100 µl of each fraction was denatured with 50% formamide and blotted onto the membrane. The membrane was preincubated with a hybridization mixture (40% formamide: 4× SSC/5%SDS) for 1 h. 32P-Labeled CAG8 probe was added and incubated for overnight under the same conditions. The membrane was washed with 2× SSC at room temperature for 2 h and exposed to x-ray film (BioMax). The binding activity of CUGBP1 was examined using gel-shift and UV cross-linking assays with CUG8 probe as described above. 5 µl of each fraction was used in these assays. Western analysis of the fractions was performed as described above.

Generation of Stable Clones and Analysis of Protein Expression-- CUGBP1-stable clones were generated using an inducible LacSwitch mammalian system as described in our earlier study (25). The coding region of CUGBP1 was cloned into pOP-13 vector under a Rous sarcoma virus promoter that is regulated by Lac-Repressor. Human fibroblasts were stably transfected with Lac-Repressor and pOP-13-CUGBP1-antisense plasmids. Clones resistant to hygromycin and to G418 were selected and analyzed for the CUGBP1 expression after addition of IPTG. Several clones showed 2- to 5-fold reduction of CUGBP1 protein by expression of antisense CUGBP1 mRNA. One clone showed 6- to 8-fold reduction of CUGBP1 and was selected for further studies. The paper represents data obtained with this clone. Expression of antisense CUGBP1 was induced by addition of 10 mM IPTG, and proteins were isolated 4, 8, and 24 h after IPTG addition. CUGBP1 protein levels were determined by Western analysis with monoclonal as well as with polyclonal antibodies to CUGBP1 as described (13, 16). C/EBPbeta isoforms were analyzed by Western blotting with polyclonal (C-19, Santa Cruz Biotechnology) antibodies. Ratios of LIP/LAP and LAP/FL isoforms were calculated by densitometry.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Majority of CUGBP1 in DM Heart Extracts Is Associated with RNA Containing CUG Repeats-- CUGBP1 has been shown to interact with CUG repeats, and its activity is altered in DM patients (13, 15, 21). To examine whether CUG repeats can directly affect CUGBP1, we initially investigated the status of CUGBP1 in DM cells containing the mutant DMPK transcripts with a large expansion of CUG repeats (15, 21) and in control tissues with a normal size of CUG repeats within DMPK mRNA. Cardiac proteins were fractionated by size exclusion chromatography and analyzed by gel-shift assay with a CUG8 probe. Normal control hearts contain a majority of CUGBP1 located in fractions with molecular mass corresponding to the size of free CUGBP1 (51 kDa). With the sensitivity of our gel-shift assays, little or no CUGBP1 is detectable in high molecular mass fractions that contain RNA (Fig. 1A). Additionally, we performed slot hybridization with a short CAG8 probe (see "Experimental Procedures") that specifically hybridizes to CUG repeats. Under conditions of our experiments, RNA CUG repeats were not detected in any size exclusion fractions from control hearts. On the contrary, gel-shift analysis of fractions from DM tissue showed a remarkably different profile. We found that the majority of CUGBP1 is observed in high molecular weight fractions of DM extracts. Free CUGBP1 is detectable in low molecular weight fractions only after long exposure (data not shown). Slot hybridization of the DM fractions with the CAG8 probe revealed the presence of CUG repeat-containing RNAs that are colocalized with CUGBP1. The detection of CUG repeat-containing RNAs in DM cells and not in control cells is consistent with CUG repeat expansion in the DMPK gene. Under conditions of hybridization, short CUG repeats or interrupted CUG repeats are not detectable. Western analysis of size exclusion chromatography fractions confirmed that CUGBP1 is present in high molecular mass complexes in cardiac DM cells (Fig. 1B). Reprobing the membranes with antibodies to alpha -actin showed a similar position of alpha -actin in fractions from DM and normal hearts, indicating that global differences in the fractionation procedure did not take place. It is necessary to note that the sensitivity of Western analysis after gel filtration was significantly reduced, and we were able to detect CUGBP1 only in fractions with higher amounts of the protein, whereas gel-shift detected CUGBP1 in several fractions. The above results show that the majority of CUGBP1 is associated with RNA in DM heart extracts, whereas in control samples, CUGBP1 is not bound to RNA. Furthermore, RNA·CUGBP1 complexes in DM extracts colocalized with CUG-containing RNAs. Under sensitivity of slot hybridization with the short CAG probe (24 nucleotides), normal DMPK transcripts with six CUG repeats were not detectable. In DM extracts, CUG-containing RNA was easily detectable probably due to an increased number of CUG repeats within the mutant DMPK mRNA. These data suggested that the majority of CUGBP1 is bound to expanded CUG repeats within the mutant DMPK mRNA. To directly examine this suggestion, we performed an analysis of CUGBP1·RNA complexes in cultured cells and a direct effect of RNA CUG repeats on CUGBP1 expression.



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 1.   CUGBP1 is associated with RNA in DM hearts. A, cytoplasm from control and DM hearts was fractionated on size exclusion chromatography as described under "Experimental Procedures" and analyzed by gel-shift assay with the CUG8 probe. The position of CUGBP1 is shown. In cytoplasm from DM hearts, CUGBP1·CUG8 complex migrates as a doublet band. CAG-slot hybridization of the fractions with CAG8 probe (see "Experimental Procedures"). The unlabeled arrow shows unknown RNA-binding protein that interacts with CUG repeats (12). B, Western assay of the fractions. 40 µl of each fraction was loaded on denatured gel electrophoresis, transferred onto the membrane, and probed with antibodies to CUGBP1. The membranes were stripped and reprobed with antibodies to alpha -actin.

Overexpression of CUG Repeats in Cos7 Cells Induces Association of CUGBP1 with Large RNA·Protein Complexes-- Gel filtration analysis of CUGBP1 in several cultured cells indicated a quite different status of CUGBP1 protein. We observed that, in HeLa cells, the majority of CUGBP1 is associated with endogenous RNA (data not shown). In contrast, the majority of CUGBP1 in Cos7 cells was not associated with endogenous RNA transcripts (Fig. 2). Therefore, these cells were used for investigation of the direct effect of CUG repeats on CUGBP1 protein and activity. Given the observation that, in DM patients, CUGBP1 is colocalized with RNA transcripts containing CUG repeats (Fig. 1), we first examined whether CUG repeats cause translocation of CUGBP1 into high molecular weight RNA·protein complexes. Cos7 cells were transfected with a plasmid expressing 170 CUG repeats or with empty vector. Proteins were isolated 24 h after transfection, fractionated by gel filtration, and analyzed by gel-shift assay with CUG probe. Fig. 2 shows a CUG binding profile of gel filtration fractions. In the cells transfected by empty vector, CUGBP1 is located in fractions containing proteins with molecular masses of 30-80 kDa. In cells expressing CUG repeats, CUGBP1 is shifted to fractions containing RNA·protein complexes with molecular masses ranging from 200 to 500 kDa. To examine whether CUG transcripts cause the shift of CUGBP1 into RNA·protein complexes, gel filtration fractions were analyzed by slot hybridization with CAG probe. Fig. 2 (CAG) shows that, in Cos7 cells, endogenous transcripts with CUG repeats are not detectable and that CUG repeats expressed from the plasmid colocalize with shifted CUGBP1. These data indicate that CUG repeats titrate endogenous CUGBP1 and forms CUG·CUGBP1 complexes. It is necessary to note that, under conditions optimal for protein purification, we observed slight degradation of RNA that, in turn, causes a relatively broad range of shift for CUGBP1. However, the majority of shifted CUGBP1 is localized in fractions containing CUG repeats. In addition to formation of RNA·protein complexes, we reproducibly observed overall increased RNA binding activity of CUGBP1 in cells overexpressing CUG repeats (Fig. 2). This observation was unexpected and prompted us to investigate the effect of CUG repeats on protein levels of CUGBP1.



View larger version (55K):
[in this window]
[in a new window]
 
Fig. 2.   Overexpression of RNA CUG repeats in Cos7 cells leads to sequestration of CUGBP1 from free pool. Cos7 cells were transfected with empty vector (control) or with vector expressing RNA transcripts with 170 CUG repeats. Proteins were isolated 24 h after transfection and analyzed by gel-shift assay with CUG8 probe. C, homogenous CUGBP1 control showing the position of CUGBP1·RNA complexes. The bottom part of each fractionation (CAG) shows slot hybridization of gel filtration fractions with the CAG8 probe.

RNA CUG Repeats Induce CUGBP1 Protein Levels-- Given the increase of CUGBP1 binding activity by expression of CUG repeats, we suggest that CUG repeats may affect protein levels or activity of CUGBP1. To test this suggestion, we examined the effect of large RNA CUG repeats on the expression of CUGBP1 in cultured cells. Cos7 cells were transfected with empty vector (control) or with plasmids expressing transcripts with 170 or 500 CUG repeats. First, we analyzed whether RNA CUG repeats are expressed in cultured cells after transfection. Northern hybridization (Fig. 3A) shows that both RNAs containing 170 and 500 CUG repeats were expressed in Cos7 cells, although the efficiency of expression for CUG170 was significantly higher than for CUG500. The difference in CUG170 and CUG500 expression was not due to different efficiency of transfection that was identical for both plasmids. Lower expression for plasmid with 500 CTG repeats might be associated with a negative effect of long CTG expansion on the transcriptional rate. Both CUG-containing RNAs migrate as diffused bands probably due to triplet repeat instability of long repeats (1). RNA expressed from the construct containing 170 CUG repeats revealed an additional transcript of smaller size, which could be a product of the degradation of a major transcript or product of the CTG170 construct where the CTG repeat was contracted. Western blotting analysis with antibodies against CUGBP1 (Fig. 3B) showed that CUGBP1 is induced in cells expressing RNA CUG repeats. Normalization of CUGBP1 levels as a ratio to beta -actin showed a 2- to 3-fold induction of CUGBP1 by expression of 170 CUG repeats and a 4- to 5-fold induction by expression of 500 CUG repeats (Fig. 3B, graph at bottom). Elevation of CUGBP1 in cells expressing CUG repeats was reproducibly observed in experiments using both monoclonal and polyclonal antibodies to CUGBP1 for Western analysis. The relative induction of CUGBP1 protein correlated with the number of CUG repeats within the RNA, rather than with the levels of RNA expression. Thus, these experiments showed that overexpression of CUG repeats increases CUGBP1 protein levels in cultured cells. Similar induction of CUGBP1 is observed in DM patients with large expansion of CUG repeats (see below).



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 3.   Protein levels of CUGBP1 are increased in cultured cells expressing CUG repeats. A, expression of RNA CUG repeats from DNA constructs in COS7 cells. Cells were transfected with DNA constructs, total RNA was isolated and analyzed by hybridization with the CAG8 probe (upper section). For the detection of RNA transcripts with 500 CUG repeats (lane 3), 3-fold longer exposure was necessary. The bottom section shows staining of the same gel with ethidium bromide. Positions of 28 S and 18 S rRNAs are shown. B, CUGBP1 protein levels in cells expressing CUG repeats. Whole cell protein extracts were isolated from cells expressing 170 and 500 CUG repeats. The number of CUG repeats is shown on the top. CUGBP1 was determined by Western analysis with antibodies against CUGBP1. The same membrane was reprobed with antibodies against beta -actin, and the levels of CUGBP1 protein were calculated as the ratio to beta -actin. The bottom part shows the summary of three independent experiments.

The Stability of CUGBP1 Protein Is Increased in Cells Expressing RNA CUG Repeats-- The elevation of CUGBP1 protein levels in cultured cells expressing large CUG repeats prompted us to investigate the mechanism responsible for this elevation. First, we examined CUGBP1 mRNA levels in control cells and in cells expressing CUG repeats. Results of semi-quantitative RT-PCR showed little or no differences in CUGBP1 mRNA levels (Fig. 5A). These data suggested that CUG repeats affect protein levels of CUGBP1. Therefore, the half-life of CUGBP1 was determined in Cos7 cells using cyclohexamide (CHX) to block protein synthesis. Cells were transfected with a vector expressing CUG repeats (170 repeats) or with empty vector (control), and CUGBP1 protein levels were determined 2, 4, and 8 h after CHX addition. Fig. 4B shows a reproducible result of Western analysis of CUGBP1 in these cells at different time points after CHX addition. We found that CUGBP1 levels are significantly reduced by 4 h after CHX treatment in control cells. However, in cells expressing RNA CUG repeats, CUGBP1 protein levels begin to be reduced only at the last time point (8 h). Densitometric analysis of three experiments showed that the half-life of CUGBP1 in control cells is ~3 h, whereas the half-life of CUGBP1 is longer than 8 h in the presence of CUG repeats. These data suggest that overexpression of CUG repeats in cells increases the stability of CUGBP1 protein.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4.   Half-life of CUGBP1 is increased in cells expressing CUG repeats. A, levels of CUGBP1 mRNA are not affected by CUG repeats. Semi-quantitative RT-PCR was performed with primers specific for CUGBP1 and TBP (see "Experimental Procedures"). CUGBP1 levels are shown below as ratio to TBP. B, Cos7 cells were transfected with empty vector and with vector expressing 500 CUG repeats. Protein synthesis was blocked by CHX, proteins were isolated at different time points after CHX addition (indicated on the top) and analyzed by Western assay with monoclonal antibodies to CUGBP1. To equilibrate detection of CUGBP1, 100 µg of cytoplasm from control and 25 µg from CUG expressing cells were used for Western assay. Levels of CUGBP1 were calculated as a percentage of the 0 time point. A summary of three independent experiments is shown. Half-life of CUGBP1 is ~3 h in control cells.

CUGBP1 Is Induced in DM and Correlates with Aberrant Translation of C/EBPbeta mRNA-- Because protein levels of CUGBP1 are induced in cultured cells by overexpression of CUG repeats, we examined whether CUGBP1 levels are affected in DM by large CUG expansion within the mutant DMPK mRNA. Western assay showed that, in agreement with results in tissue culture, CUGBP1 is also elevated in DM heart (Fig. 5B). In agreement with the increase of CUGBP1 protein, RNA binding activity of CUGBP1 is also elevated in DM cells as is shown by UV cross-linking assay (Fig. 5C.) This activity is specific to CUG repeats, because cold CUG oligomer competes for the binding. Thus Western and UV cross-linking analyses showed that protein levels and RNA binding activity of CUGBP1 are elevated in DM patients. We have previously shown that CUGBP1 is involved in the regulation of translation of C/EBPbeta isoforms (22). A single C/EBPbeta mRNA produces several protein isoforms through the initiation of translation from different in-frame AUG codons (Fig. 5A) (23). CUGBP1 interacts with the 5'-region of C/EBPbeta mRNA (sORF, Fig. 5A) and is able to induce translation of low molecular weight C/EBPbeta isoforms in liver and in cell free translation systems (23). Importantly, CUGBP1 induces the production of the dominant negative LIP isoform of C/EBPbeta , which has been shown to inhibit C/EBP-mediated transcription (22, 26). To examine whether the increased levels of CUGBP1 in DM may affect translation of C/EBPbeta mRNA, Western analysis of cardiac tissues was performed. In normal cardiac tissues, the full-length (FL) isoform of C/EBPbeta is the major product of translation. However, DM hearts, which contain high levels of CUGBP1, show quite a different pattern of C/EBPbeta isoforms (Fig. 5B). The lower molecular weight isoforms of C/EBPbeta , LAP and LIP, are induced in DM hearts relative to the controls. Densitometric analysis showed a 4- to 5-fold induction of LAP, calculated as a ratio of LAP/FL. LIP is not detectable in normal hearts, but can be detected with a long exposure in DM cells expressing high levels of CUGBP1 (Fig. 5B, bottom part). To examine directly whether CUGBP1 from DM heart induces production of C/EBP-truncated isoforms, CUGBP1 was immunoprecipitated and added into a cell-free translation system in reticulocyte lysate that was programmed with wild type C/EBPbeta and with a mutant C/EBPbeta containing a mutation in the third AUG codon (26). Fig. 5D shows that CUGBP1 from DM patients induces translation of LIP, whereas CUGBP1 from control patients does not affect the LIP/LAP ratio. The alterations in C/EBPbeta isoforms are due to translational regulation, because CUGBP1 is not able to induce LIP from the ATG3 mutant. We also did not detect proteolytic cleavage of FL C/EBPbeta in protein extracts from DM heart (Fig. 5D and data not shown). Thus, these observations show that levels of CUGBP1 are induced in DM and that CUGBP1 from DM patients is able to induce LIP production in a cell-free translation system via translational mechanism. The effect of CUGBP1 on the LAP/FL ratio was difficult to examine, because RL translates very weak FL C/EBPbeta protein (26).



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 5.   Translation of C/EBPbeta isoforms is altered in DM hearts with elevated levels of CUGBP1. A, diagram showing structure of C/EBPbeta mRNA. Positions of ATG codons for full-length (FL), LAP, and LIP isoforms of C/EBPbeta are shown on the top. DBD, DNA-binding domain; sORF, out of frame short open reading frame, the 5'-region of C/EBPbeta mRNA that interacts with CUGBP1 and controls translation of C/EBPbeta isoforms. B, Western analysis of CUGBP1 and C/EBPbeta expression in control and DM hearts. Liv, liver proteins serve as a positive control for C/EBPbeta isoforms. C, proteins isolated from control heart; DM, proteins isolated from heart of DM patient. The position of CUGBP1 is shown by an arrow. The same membrane was stripped and reprobed with antibodies to C/EBPbeta . Positions of the full-length C/EBPbeta (FL), LAP, and LIP isoforms are shown by arrows. Longer exposure (bottom part) of the same membrane shows induction of LIP in DM heart. The ratio of LAP/FL isoforms is shown below as a summary of three independent experiments. C, UV cross-linking assay with CUG probe. Proteins were incubated with the probe, treated with UV, and separated by denaturing gel electrophoresis. CUGBP1 incubation with CUGBP1 purified from HeLa cells by HPLC. Con, control heart; DM, proteins from DM patient. CUG8, cold CUG oligomer was added into the binding reaction. D, CUGBP1 induces LIP via translational mechanism. CUGBP1 was immunoprecipitated from control (C) and DM heart and added into reticulocyte lysate programmed with wild type (WT) or ATG3 mutant (ATG3) C/EBPbeta mRNA. Positions of LAP and FL C/EBPbeta isoforms are shown.

To confirm that CUGBP1 regulates C/EBPbeta isoforms, we generated stable clones containing antisense CUGBP1 mRNA under Lac-Repressor control as described in our earlier study (25). Among several clones, we identified one clone that showed significant reduction of endogenous CUGBP1 in response to expression of antisense CUGBP1 mRNA. Fig. 6 shows that expression of antisense CUGBP1 mRNA after IPTG addition leads to 6- to 8-fold reduction of CUGBP1 as a ratio to beta -actin (loading control). Analysis of C/EBPbeta isoforms showed that production of truncated isoforms of C/EBPbeta , LAP and LIP, is reduced in cells with lower levels of CUGBP1. Thus, both DM tissues and cultured cells showed that alterations in CUGBP1 levels lead to changes in the translation of C/EBPbeta isoforms. Because the various C/EBPbeta isoforms have different capacities in activation of promoters, the increased production of low molecular weight C/EBPbeta isoforms in DM hearts can potentially affect expression of downstream targets of C/EBPbeta .



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 6.   CUGBP1 regulates C/EBPbeta isoforms in cultured cells. A, expression of CUGBP1 in stable antisense clone. Antisense CUGBP1 mRNA was induced by addition of 5, 10, and 15 mM IPTG. Proteins were isolated 18 h after IPTG addition and analyzed by Western blotting with monoclonal antibodies to CUGBP1. Levels of CUGBP1 were calculated as the ratio to beta -action after reprobing the membrane. B, the ratio of C/EBPbeta isoforms is altered in cells with reduced levels of CUGBP1. Nuclear proteins were analyzed by Western assay with antibodies to C/EBPbeta (C19). The ratio LIP/FL was calculated by densitometric analysis of two independent experiments.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The RNA-based mechanism for DM pathology has been suggested by several observations from several different investigators (11-21, 27). This RNA-based hypothesis suggests that overexpression of CUG repeats in mutant DMPK mRNA may affect specific CUG-binding proteins in DM patients (12-17, 20, 21). Although there are several putative pathways by which CUG repeats may affect RNA-binding proteins, the most simple mechanism is the direct association of CUG-binding proteins with CUG repeats, leading to sequestration of the protein from the free pool. CUGBP1 was identified as a protein that specifically binds to the 3'-UTR of DMPK mRNA (12, 13) and is affected in DM tissues with a large expansion of CUG repeats (13-15, 21). In this report, we analyzed the direct effect of RNA CUG repeats on CUGBP1 expression. Initially, we attempted to apply indirect immunofluorescent staining and hybridization in situ to investigate whether CUGBP1 and DMPK mRNA are colocalized; however, the combination of these two methods was found to be nonapplicable for these studies. Therefore, we developed a new HPLC-based approach that allows detection of CUGBP1 when it is bound to RNA, as well as when it exists as a free protein. Our data demonstrated that, in DM patients, CUGBP1 is observed in high molecular weight RNA·protein complexes containing CUG repeats, whereas in normal controls, the majority of CUGBP1 is free of RNA. This observation supports the hypothesis that expansion of CUG repeats in DM patients sequesters CUGBP1 protein. To further address this hypothesis by experimentation, we studied the direct effect of CUG repeats on CUGBP1 in cultured cells. Gel filtration analysis of CUGBP1·RNA complexes in several types of cultured cells showed that HeLa cells are not appropriate for studies of CUGBP1 sequestration, because the majority of CUGBP1 in HeLa cells is associated with endogenous transcripts and there is little or no CUGBP1 free of RNA (data not shown).

Among several cell lines, Cos7 cells contain CUGBP1 free of RNA. Therefore, Cos7 cells were used for studies of direct effect of CUG repeats on CUGBP1 protein. Overexpression of transcripts containing long CUG repeats in Cos7 cells leads to titration of CUGBP1 and to formation of CUGBP1·CUG RNA complexes. This also led to an overall increase of RNA binding activity of CUGBP1. Increasing RNA binding activity of CUGBP1 in cells overexpressing CUG repeats was unexpected and prompted us to investigate the effect of CUG repeats on protein levels of CUGBP1. Experiments with CHX block of protein synthesis showed that half-life of CUGBP1 is increased in cells expressing transcripts with CUG repeats. Thus, experiments in cultured cells demonstrated that CUG repeats titrate CUGBP1 out of the free pool and increase CUGBP1 protein levels by stabilization of CUGBP1.

Analysis of RNA·protein complexes demonstrated that the formation of high molecular weight RNA protein complexes in Cos7 cells mimics the situation observed in DM patients (compare Figs. 1 and 2). Because the number of CUG repeats is dramatically increased in the mutant DMPK transcripts, we suggested that this increase might also lead to alterations in CUGBP1 protein levels. Similarly, the levels of CUGBP1 are also elevated in DM tissues that express the mutant DMPK mRNA with an increased number of CUG repeats. These data suggest that, similar to events in cultured cells, CUGBP1 protein is stabilized in DM tissues, perhaps due to increased association with the mutant DMPK mRNA. The elucidation of the pathway for stabilization of CUGBP1 by CUG repeats requires additional experiments. Theoretically, one may suggest that CUGBP1 is protected from degradation when it exists as a CUGBP1·CUG RNA complex. Another suggestion is based on the observations that long CUG repeats can form hairpin structures (27, 28), which in turn are able to activate dsRNA-dependent kinase (27). In this scenario, overexpression of CUG repeats may activate dsRNA-dependent kinase, leading to alterations in downstream targets, possibly including enzymes that are involved in degradation of CUGBP1.

Given the observation that cytoplasmic CUGBP1 is associated with polysomes (22), we examined whether accumulation of CUGBP1 in DM hearts affects translation of mRNAs containing CUGBP1 binding sites. We have previously described that CUGBP1 binds to the 5'-region of C/EBPbeta mRNA and is able to induce translation of a dominant negative isoform of C/EBPbeta , LIP (22, 26). Analysis of C/EBPbeta proteins in DM hearts showed that expression of two low molecular weight isoforms of C/EBPbeta , LAP and LIP, are induced in DM hearts containing high levels of CUGBP1. There are two mechanisms that have been described to generate low molecular weight isoforms of C/EBPbeta : alternative translation initiation (22, 26), and specific proteolytic cleavage (29). CUGBP1 induces LIP production in liver and in cell free systems through the alternative translation mechanism (22, 26). In DM hearts, this also appears to be the case, because the protease activity that generates LIP is not detectable in these tissues (Fig. 5D and data not shown). These data demonstrate that elevation of CUGBP1 in DM hearts is accompanied by alterations in translation of at least one known cytoplasmic CUGBP1 target: C/EBPbeta . Experiments with stable clones expressing antisense CUGBP1 mRNA revealed that CUGBP1 controls the ratio of C/EBPbeta isoforms. Although our data suggest that CUGBP1 directly regulates C/EBPbeta isoforms in DM tissues, we do not rule out that other RNA-binding proteins can also be affected in DM and can contribute to alterations in processes such as translation. It is also possible that overexpression of CUG repeats in DM tissues leads to the activation of the dsRNA-dependent kinase (27), which in turn, may result in phosphorylation of components of the translational machinery. Further studies are necessary to elucidate this mechanism. Induced expression of a dominant negative C/EBPbeta isoform, LIP, in DM tissues may affect different signal transduction pathways. It has been recently shown by Calkhoven et al. (30) that 3T3-L1 cells expressing LIP have an increased rate of proliferation and do not differentiate. The authors conclude that LIP/FL ratio is important for regulation of cell growth and differentiation. Thus, increased expression of LIP in DM may contribute to some of the abnormalities observed in DM tissues (1).

The results from this paper show that expansion of CUG repeats in DM patients leads to accumulation of CUGBP1 that is associated with these RNA transcripts. The formation of DMPK mRNA-(CUGBP1)n complexes may create a pool of bound CUGBP1 that may be released from the complexes by regulation of the affinity of CUGBP1 to the DMPK mRNA or by overexpression of other RNAs with binding sites for CUGBP1. These potential scenarios may take place during different stages of development and/or cell differentiation in DM tissues inducing DM specific features.


    ACKNOWLEDGEMENTS

We thank Anne-Sophie Lia-Baldini for the excellent performance of semi-quantitative RT-PCR.


    FOOTNOTES

* This work was supported by National Institute of Health Grants AR10D44387 (to L. T. T.), AG16392 (to L. T. T.), AG00756-01 (to N. A. T.), and GM55188-01 (to N. A. T.) and by grants from the Muscular Dystrophy Association (to L. T. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Section of Cardiovascular Sciences, Dept. of Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-6911; Fax: 713-798-3142; E-mail: lubovt@bcm.tmc.edu.

Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M005960200


    ABBREVIATIONS

The abbreviations used are: DM, myotonic dystrophy; UTR, untranslated repeat; DMPK, myotonin protein kinase; C/EBPbeta , CCAAT/enhancer-binding protein beta ; FL, full-length; LAP, liver activator protein; LIP, liver inhibitory protein; kb, kilobase(s); bp, base pair(s); BS, vector DNA; CHX, cyclohexamide; RT-PCR, reverse transcription-polymerase chain reaction; TBP, TATA box binding protein; IPTG, isopropyl-1-thio-beta -D-galactopyranoside; HPLC, high pressure liquid chromatography; dsRNA, double-stranded RNA.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Harper, P. S. (1989) Myotonic Dystrophy , 2nd Ed. , Saunders, London/Philadelphia
2. Aslanidis, C., Jansen, J., Amemiya, C., Shutler, G., Mahadevan, M., Tsilfidis, C., Chen, C., Alleman, J., Wormskamp, N. G., Vooijs, M., Buxton, J., Johnson, K., Sweets, H. J. M., Lennon, G. G., Carrano, A. V., Korneluk, R. G., Wieringa, B., and de Jong, P. J. (1992) Nature 355, 548-551[CrossRef][Medline] [Order article via Infotrieve]
3. Fu, Y.-H., Pizzuti, A., Fenwick, R. G., Jr., King, J., Rajnarayan, S., Dunne, P. W., Dubel, J., Nasser, G. A., Ashizawa, T., de Jong, P., Wieringa, B., Korneluk, R. G., Perryman, M. B., Epstein, H. F., and Caskey, C. T. (1992) Science 255, 1256-1258[Medline] [Order article via Infotrieve]
4. Fu, Y.-H., Friedman, D. L., Richards, S., Pearlman, J. A., Gibbs, R. A., Pizzuti, A., Ashizawa, T., Perryman, M. B., Scarlato, G., Fenwick, R. G., Jr., and Caskey, C. T. (1993) Science 260, 235-238[Medline] [Order article via Infotrieve]
5. Sabourin, L. A., Mahadevan, M. S., Narang, M., Lee, D. S. C., Surh, L. C., and Korneluk, R. G. (1993) Nat. Genet. 4, 233-238[Medline] [Order article via Infotrieve]
6. Jansen, G., Croenen, P. J. T. A., Bachner, D., Jap, P. H. K., Coerwinkel, M., Oerlemans, F., van den Brock, W., Gohlsch, B., Pette, D., Plomp, J. J., Molenaar, P. C., Nederhoff, M. G. J., van Echteld, C. J. A., Dekker, M., Berns, A., Hameister, H., and Wieringa, B. (1996) Nat. Genet. 13, 316-324[Medline] [Order article via Infotrieve]
7. Reddy, S., Smith, D. B. J., Rich, M. M., Leferovich, J. M., Reily, P., Davis, B. M., Tran, K., Rayburn, H., Bronson, R., Cros, D., Balise-Gordon, R. J., and Housman, D. (1996) Nat. Genet. 13, 325-335[Medline] [Order article via Infotrieve]
8. Boucher, C. A., King, S. K., Carey, N., Krahe, R., Winshester, C. L., Rahman, S., Creavin, T., Meghji, P., Bailey, M. E., and Chartier, F. L. (1995) Hum. Mol. Genet. 4, 1919-1925[Abstract]
9. Jansen, G., Bachner, D., Coerwinkel, M., Wormskamp, N., Hameister, H., and Wieringa, B. (1995) Hum. Mol. Genet. 4, 843-852[Abstract]
10. Klesert, T. R., Cho, D. H., Clark, J. I., Maylie, J., Adelman, J., Snider, L., Yuen, E. C., Soriano, P., and Tapscott, S. J. (2000) Nat. Genet. 25, 105-109[CrossRef][Medline] [Order article via Infotrieve]
11. Wang, J., Pegoraro, E., Menegazzo, E., Gennarelli, M., Hoop, R. C., Angelini, C., and Hoffman, E. P. (1995) Hum. Mol. Genet. 4, 599-606[Abstract]
12. Timchenko, L. T., Timchenko, N. A., Caskey, C. T., and Roberts, R. (1996) Hum. Mol. Genet. 5, 115-121[Abstract/Free Full Text]
13. Timchenko, L. T., Miller, J., Timchenko, N. A., DeVore, D. R., Datar, K. V., Lin, L., Roberts, R., Caskey, C. T., and Swanson, M. S. (1996) Nucleic Acids Res. 24, 4407-4414[Abstract/Free Full Text]
14. Caskey, C. T., Swanson, M. S., and Timchenko, L. T. (1996) Cold Spring Harbor Symp. Quant. Biol. 61, 607-614[Medline] [Order article via Infotrieve]
15. Roberts, R., Timchenko, N. A., Miller, J. W., Reddy, S., Caskey, C. T., Swanson, M. S., and Timchenko, L. T. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 13221-13226[Abstract/Free Full Text]
16. Lu, X., Timchenko, N. A., and Timchenko, L. T. (1999) Hum. Mol. Genet. 8, 53-60[Abstract/Free Full Text]
17. Timchenko, L. T. (1999) Am. J. Hum. Genet. 64, 360-364[CrossRef][Medline] [Order article via Infotrieve]
18. Mankodi, A., Logigian, E., Callahan, L., McClain, C., White, R., Henderson, D., Krym, M., and Thornton, C. A. (2000) Science 289, 1769-1773[Abstract/Free Full Text]
19. Good, P. J., Chen, Q., Warner, S. J., and Herring, D. C. (2000) J. Biol. Chem. 275, 28563-28592
20. Miller, J. W., Urbinati, C. R., Teng-Umnuay, P., Stenberg, M. G., Byrne, B. J., Thornton, C. A., and Swanson, M. S. (2000) EMBO J. 19, 4439-4448[Abstract/Free Full Text]
21. Philips, A. V, Timchenko, L. T., and Cooper, T. (1998) Science 280, 737-741[Abstract/Free Full Text]
22. Timchenko, N. A., Welm, A. L., Lu, X., and Timchenko, L. T. (1999) Nucleic Acids Res. 27, 4517-4525[Abstract/Free Full Text]
23. Descombes, P., and Schibler, U. (1991) Cell 67, 569-579[Medline] [Order article via Infotrieve]
24. Ordway, J. M., and Detloff, P. J. (1996) BioTechniques 21, 609-612[Medline] [Order article via Infotrieve]
25. Timchenko, N. A, Wilde, M., Nakanishi, M., Smith, J. R., and Darlington, G. J. (1996) Genes Dev. 10, 804-815[Abstract]
26. Welm, A. L., Mackey, S. L., Timchenko, L. T., Darlington, G. J., and Timchenko, N. A. (2000) J. Biol. Chem. 275, 27406-27413[Abstract/Free Full Text]
27. Tian, B., White, R. G., Xia, T., Welle, S., Turner, D. H., Mathews, M. B., and Thornton, C. A. (2000) RNA (N. Y.) 6, 79-87[Abstract/Free Full Text]
28. Michalowski, S., Miller, J. W., Urbinati, C. R., Paliouras, M., Swanson, M. S., and Griffith, J. (1999) Nucleic Acids Res. 27, 3534-3542[Abstract/Free Full Text]
29. Welm, A. L., Timchenko, N. A., and Darlington, G. J. (1999) Mol. Cell. Biol. 19, 1695-1704[Abstract/Free Full Text]
30. Calkhoven, C. F., Muller, C., and Luetz, A. (2000) Genes Dev. 14, 1920-1932[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.