RNA CUG Repeats Sequester CUGBP1 and Alter Protein Levels and
Activity of CUGBP1*
Nikolai A.
Timchenko
,
Zong-Jin
Cai§,
Alana L.
Welm
,
Sita
Reddy¶,
Tetsuo
Ashizawa
, and
Lubov T.
Timchenko§**
From the Departments of
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 |
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
(C/EBP
), which has been previously described to be a target of CUGBP1. Analysis of C/EBP
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 |
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/EBP
, is encoded by an
intronless gene, but a single mRNA of C/EBP
can produce several
isoforms with different transcriptional activities (Ref. 23 and see
Fig. 5A). Three major isoforms of C/EBP
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/EBP
, 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 |
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
-actin promoter, and cloned into the pSV2 vector. The resulting
plasmid included the sequence of the human
-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
-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
-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 [
-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/EBP
(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-
-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/EBP
, 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
-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
-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
[
-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
-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/EBP
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 |
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
-actin showed a similar position of
-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.

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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 -actin.
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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.

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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.
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|
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
-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).

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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
-actin, and the levels of CUGBP1 protein were calculated as the
ratio to -actin. The bottom part shows the summary of
three independent experiments.
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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.

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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.
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CUGBP1 Is Induced in DM and Correlates with Aberrant Translation of
C/EBP
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/EBP
isoforms (22). A single C/EBP
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/EBP
mRNA (sORF, Fig.
5A) and is able to induce translation of low molecular
weight C/EBP
isoforms in liver and in cell free translation systems
(23). Importantly, CUGBP1 induces the production of the dominant
negative LIP isoform of C/EBP
, 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/EBP
mRNA,
Western analysis of cardiac tissues was performed. In normal cardiac
tissues, the full-length (FL) isoform of C/EBP
is the major product
of translation. However, DM hearts, which contain high levels of
CUGBP1, show quite a different pattern of C/EBP
isoforms (Fig.
5B). The lower molecular weight isoforms of C/EBP
, 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/EBP
and with a mutant C/EBP
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/EBP
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/EBP
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/EBP
protein (26).

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|
Fig. 5.
Translation of C/EBP
isoforms is altered in DM hearts with elevated levels of
CUGBP1. A, diagram showing structure of C/EBP
mRNA. Positions of ATG codons for full-length (FL), LAP,
and LIP isoforms of C/EBP are shown on the top.
DBD, DNA-binding domain; sORF, out of frame short
open reading frame, the 5'-region of C/EBP mRNA that interacts
with CUGBP1 and controls translation of C/EBP isoforms.
B, Western analysis of CUGBP1 and C/EBP expression in
control and DM hearts. Liv, liver proteins serve as a
positive control for C/EBP 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/EBP .
Positions of the full-length C/EBP (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/EBP mRNA.
Positions of LAP and FL C/EBP isoforms are shown.
|
|
To confirm that CUGBP1 regulates C/EBP
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
-actin (loading control). Analysis of C/EBP
isoforms showed that production of truncated isoforms of C/EBP
, 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/EBP
isoforms. Because the
various C/EBP
isoforms have different capacities in activation of
promoters, the increased production of low molecular weight C/EBP
isoforms in DM hearts can potentially affect expression of downstream
targets of C/EBP
.

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Fig. 6.
CUGBP1 regulates C/EBP
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 -action after reprobing the membrane. B,
the ratio of C/EBP isoforms is altered in cells with reduced levels
of CUGBP1. Nuclear proteins were analyzed by Western assay with
antibodies to C/EBP (C19). The ratio LIP/FL was
calculated by densitometric analysis of two independent
experiments.
|
|
 |
DISCUSSION |
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/EBP
mRNA and is able to induce translation of a dominant negative isoform of C/EBP
, LIP (22, 26). Analysis of C/EBP
proteins in DM hearts showed that expression of two low molecular weight isoforms of C/EBP
, 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/EBP
:
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/EBP
.
Experiments with stable clones expressing antisense CUGBP1 mRNA
revealed that CUGBP1 controls the ratio of C/EBP
isoforms. Although
our data suggest that CUGBP1 directly regulates C/EBP
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/EBP
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/EBP
, CCAAT/enhancer-binding protein
;
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-
-D-galactopyranoside;
HPLC, high
pressure liquid chromatography;
dsRNA, double-stranded RNA.
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