The product of an oculopharyngeal muscular dystrophy gene, poly(A

© 2001 Oxford University Press Human Molecular Genetics, 2001, Vol. 10, No. 11 1129–1139

The product of an oculopharyngeal muscular dystrophygene, poly(A)-binding protein 2, interacts with SKIP andstimulates muscle-specific gene expressionYeon-Jeong Kim1,2, Satoru Noguchi1, Yukiko K. Hayashi1,+, Toshifumi Tsukahara1,Takao Shimizu2 and Kiichi Arahata1

1Department of Neuromuscular Research, National Institute of Neuroscience, NCNP, 4-1-1 Ogawahigashi, Kodaira,Tokyo 187-8502, Japan and 2Department of Biochemistry and Molecular Biology, Faculty of Medicine,The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0031, Japan

Received 29 November 2000; Revised and Accepted 23 March 2001

Oculopharyngeal muscular dystrophy (OPMD) iscaused by short expansions of the GCG trinucleotiderepeat encoding the polyalanine tract of the poly(A)-binding protein 2 (PABP2). PABP2 binds to thegrowing poly(A) tail, stimulating its extension duringthe polyadenylation process, and limits the length ofthe newly synthesized poly(A) tail. Whereas PABP2is expressed ubiquitously, the clinical and patho-logical features of OPMD patients are restricted tothe skeletal muscle. To elucidate the possible role ofPABP2 in skeletal muscle, we established the stableC2 cell lines expressing human PABP2. Thesestable cell lines showed morphologically enhancedmyotube formation accompanied by an increasedexpression of myogenic factors, MyoD andmyogenin. In nuclear run-on assay, the transcriptionrate of the MyoD gene was significantly increased byPABP2 transfection. We found the N-terminal regionof PABP2 was responsible for the up-regulation ofthese myogenic factors. Furthermore, Ski-interactingprotein (SKIP) was isolated as a binding protein forPABP2 using the yeast two-hybrid system. Theinteraction of PABP2 and SKIP was confirmed byglutathione S-transferase-pulldown assay and immuno-precipitation. Confocal laser scanning showedPABP2 was co-localized with SKIP in nuclearspeckles. The reporter assays showed that PABP2co-operated with SKIP to synergistically activate E-box-mediated transcription through MyoD. Moreover,both PABP2 and SKIP were directly associated withMyoD to form a single complex. These findings suggestthat PABP2 and SKIP directly control the expressionof muscle-specific genes at the transcription level.

INTRODUCTION

Oculopharyngeal muscular dystrophy (OPMD) is a late-onsetdisease with autosomal dominant inheritance with progressive

swallowing difficulties (dysphagia), eyelid drooping (ptosis)and proximal limb weakness after the age of 50. Unique intra-nuclear inclusions in the skeletal muscle are a pathologicalhallmark of OPMD. A recent genetic study identified theresponsible mutation as a GCG repeat expansion encoding apolyalanine tract at the N-terminus of the poly(A)-bindingprotein 2 (PABP2) which causes OPMD (1).

PABP2 is an abundant nuclear protein which binds poly(A)with high affinity and specificity. In vitro analyses showed thatPABP2 stimulated poly(A) polymerase (PAP) in conjunctionwith cleavage and polyadenylation-specific factor (CPSF) toincrease the processivity of the polyadenylation reaction (2–4).Although the polyadenylation reaction is directly catalyzed byPAP, this enzyme by itself is almost inactive due to a lowaffinity with RNA (5). Processive and efficient poly(A) tailsynthesis requires both CPSF and PABP2 at the 3′-endprocessing complex (2,4). PABP2 is also involved in themechanism that limits the length of the newly synthesizedpoly(A) tail (4). In vitro reconstitution of the poly(A) tailsynthesis resulted in a rapid and processive synthesis of thepoly(A) tail to approximately 200–250 residues whichcorresponded to the length of the nascent poly(A) tail inmammals. The Poly(A) length restriction mechanism is prob-ably associated with the stoichiometric binding of multiplePABP2 to the nascent poly(A) tail, CPSF and PAP (4). Arecent study using electron microscopy provided evidence tosupport this stoichiometric binding (6).

Despite ubiquitous expression patterns of PABP2, theclinical and pathological phenotypes are restricted to theskeletal muscle, relatively specific to the levator palpebreasuperioris and pharyngeal muscle. Characterizing the functionalroles of PABP2 in skeletal muscle may contribute to elucidatingthe possible pathogenic mechanism in OPMD. To gain insight intothe possible roles of PABP2 in skeletal muscle, we establishedstable C2 cell lines expressing human PABP2. These stable celllines acquired the ability of enhanced myogenic differentiationcompared with their parent cell line. We demonstrate howPABP2 affects myogenic differentiation and elucidate itsfunctional role.

+To whom correspondence should be addressed. Tel: +81 42 341 2711; Fax: +81 42 346 1742; Email: [email protected]

1130 Human Molecular Genetics, 2001, Vol. 10, No. 11

RESULTS

Exogenous expression of human PABP2 promotesmyogenic differentiation in C2 cells

The mouse skeletal muscle cell line, C2, provides a good modelfor the study of myoblasts and their myogenic differentiation (7).Exposure of proliferating myoblasts to media lacking mitogeninduces withdrawal from the proliferative cell cycles, and cellsfuse to form multinucleated myotubes.

To examine the possible role of PABP2 in skeletal muscle,C2 cells were transfected with full-length human PABP2cDNA under the control of the cytomegalovirus promoter. Thehuman PABP2 was Flag-epitope tagged at the C-terminus(Flag-PABP2) to distinguish it from the endogenous (mouse)PABP2. G418-resistant clones were selected and screened byexamining human PABP2 expression using RT–PCR andwestern blot analyses. Twenty-eight G418-resistant clonesexpressing human PABP2 were obtained. Clones S1 and S7,which showed similar morphology to their parent cells duringthe growth phase were chosen for further characterization. Wedetermined the expression rate of transfected human PABP2relative to endogenous PABP2 using quantitative RT–PCR.The mRNA expression levels of exogenous (human) PABP2 inclones S1 and S7 were 14.6 and 28.9% of their endogenousPABP2 levels, respectively (data not shown). However, theseclones showed 3-fold greater mRNA expression of the endogenousPABP2 with respect to their parent cells (data not shown).

Parental C2 cells and the stable clones were cultured toconfluence, and then induced to fuse into myotubes asdescribed in Materials and Methods. S1 and S7 showedaccelerated morphological differentiation compared with theparental C2 (Fig. 1). In cultures approaching confluence,myotubes began to be observed in S7 cultured in growthmedium [Fig. 1, GM (day 0)]. Two days after transferring todifferentiation medium, large myotubes were observed in theS1 and S7 clones, but not in parental C2 [Fig. 1, DM (day 2)].In parental C2, myotubes were observed 3 days after culture indifferentiation medium [Fig. 1, DM (day 3)]. Thus, exogenousexpression of human PABP2 in C2 cells leads to enhancedmorphological differentiation.

The protein expression of the exogenous PABP2 in thestable cell lines was characterized by immunofluorescence andwestern blotting using anti-Flag polyclonal antibody. Flag-PABP2was detected more prominently in the nuclei of myotubes thanmyoblasts by immunofluorescence (Fig. 1). No staining couldbe seen in parental C2 (Fig. 1). Specific bands for Flag-PABP2(∼50 kDa) were detected in S1 and S7 by western blot analysis(Fig. 2A). Consistent with the result of immunofluorescenceanalysis, expression of Flag-PABP2 was increased in differentiatedmyotubes relative to myoblasts in western blot analysis(Fig. 2A). The bands of ∼75 kDa that the anti-Flag polyclonalantibody detected in all lanes were unknown but probably non-specific because they are present in the C2 lanes as well(Fig. 2A).

The mRNAs for MyoD and myogenin were up-regulated inthe stable cells expressing exogenous PABP2

Myogenesis is a result of a multiple transcription process ofmuscle-specific genes by the myogenic factors (basic helix–loop–helix transcription factors). Previous studies have demonstratedthat expressions of myogenic factors, MyoD and myogenin,are induced as an early event in myogenic differentiationin vitro (8), and that their actions as transcriptional regulatorsare required for terminal myoblast differentiation in vivo (9–11).

We examined the mRNA expression levels of myogenicfactors, such as MyoD and myogenin, in the stable clonesusing quantitative RT–PCR. At the confluence stage in thegrowth medium, S1 and S7 expressed 4.2- and 8-fold greaterMyoD mRNA compared with parental C2, respectively(Fig. 2B). The protein level of MyoD was also higher in S1 andS7 than in parental C2 at this stage (Fig. 2A, GM). Themaximal differences of MyoD expression among C2, S1 andS7 were observed after 1 day of culture in the differentiationmedium. At this stage, S1 and S7 showed 6.4- and 12-foldhigher expression levels of MyoD mRNA compared with theirparent cells, respectively (Fig. 2B, DM1). The expression levelof myogenin mRNA during the growth stage and confluencephase was extremely low in each clone (Fig. 2B, Gr and Co).After culture for 1 day in the differentiation medium, the expres-

Figure 1. Enhanced myotube formation in stable C2 cells expressing human PABP2. Phase contrast and immunofluorescence images were taken from the cellscultured in growth medium (GM, day 0) and 2 and 3 days in differentiation medium (DM, day 2 and day 3). Anti-Flag polyclonal antibody was used to detecthuman Flag-PABP2. Scale bar, 50 µm.

Human Molecular Genetics, 2001, Vol. 10, No. 11 1131

sion levels of myogenin mRNA in S1 and S7 were 25- and 37-fold higher than in parental C2, respectively (Fig. 2B, DM1).The myogenin protein was also up-regulated during the differ-entiation (Fig. 2A). One of the muscle-specific mRNAs,myosin light chain 1a (MLC1a), was highly expressed in S1and S7 compared with parental C2 during the differentiation(Fig. 2B). The expression levels of Myf-5 mRNA were notsignificantly changed between the stable clones and parentalC2 (Fig. 2B).

Overexpression of PABP2 results in an increase in thetranscription rate of the MyoD gene

To clarify the up-regulation mechanism of MyoD, weperformed nuclear run-on assay to measure the transcriptionrate of the MyoD gene from the C2 cells transiently transfectedwith PABP2 expression plasmid. The transcription rate of theMyoD gene relative to the glyceraldehyde-3-phosphatedehydrogenase (GAPDH) gene showed a 2.5-fold increase inthe PABP2 transfection (Fig. 3A). In agreement with the resultof the nuclear run-on assay, northern blot analysis showed thatthe mRNA expression of MyoD was increased by the transfectionof PABP2 expression plasmid (Fig. 3B). Thus, MyoD mRNAis specifically up-regulated by PABP2 transfection.

The N-terminal region of PABP2 (PABP2-N) is responsiblefor the up-regulation of MyoD and myogenin mRNAs

PABP2 contains an acidic N-terminus, highly conserved RNP-typeRNA-binding domain known as RNA recognition motifs(RRMs) and a basic C-terminus (12). To determine whichregion of PABP2 is involved in the up-regulation of MyoD andmyogenin, we transiently transfected the expression plasmids

encoding either the full-length or truncated PABP2 (Fig. 4A)in C2 cells and measured the expression levels of MyoD andmyogenin mRNAs using quantitative RT–PCR. The proteinsexpressed in each transfected sample were confirmed bywestern blot analysis (Fig. 4B). At 24 h after transfection, cellshad almost reached confluence and no marked change wasobserved in the measured mRNA levels in each transfectedsample (Fig. 4C). At 48 and 72 h after transfection, the mRNAlevels of MyoD and myogenin showed a 2-fold increase in thecells transfected with full-length PABP2 (PABP2-FL) andPABP2-N expression plasmids relative to the mock control(Fig. 4C). However, the cells transfected with the RNA-binding

Figure 2. Western blot and quantitative RT–PCR analyses of the stable C2 cells expressing human PABP2. (A) Cell lysates were prepared from myoblasts ingrowth medium (GM) and myotubes 2 days after the culture in differentiation medium (DM). Approximately 30 µg of protein from each cell lysate was analyzed bywestern blotting using the indicated antibodies. (B) Total RNA was isolated from parental and stable C2 cells at the growth (Gr) and confluent (Co) stages (∼50 and100% confluence, respectively), and during 3 days after switching to differentiation medium (DM). Total cDNA contents were normalized by the amount of β-actincDNA. The ratio of target cDNA/β-actin cDNA amounts from parental C2 at the confluent stage was arbitrarily set at 1. The values are derived from triplicates.Error bars indicate the SD of the means.

Figure 3. The effect of PABP2 transfection on MyoD gene transcription. C2cells were transiently transfected with 8 µg of PABP2 expression plasmid orcontrol vector plasmid in a 100 mm culture dish. Twenty-four hours aftertransfection the culture medium was changed to a differentiation medium. Afteran additional 48 h incubation, cells were harvested for nuclear run-on assay(A) and northern blot analysis (B).


domain of PABP2 (RRMs) showed no notable increase in theamount of the target mRNAs compared with the mock controls(Fig. 4C). In addition, MLC1a expression was activated 2-foldby transient expression of PABP2-FL and PABP2-N relative tothe mock control (Fig. 4C).

PABP2 interacts with a nuclear protein Ski-interactingprotein (SKIP)

To identify proteins that interact with the N-terminal region ofPABP2 (amino acids 1–145), we performed yeast two-hybridscreening. The yeast-expressing N-terminal region of PABP2was transformed with a human skeletal muscle cDNA librarywhich consisted of clones carrying cDNA fused to the GAL4activation domain. Finally, several positive clones wereobtained from 3 × 106 leucine and tryptophan auxotrophictransformants. The restriction enzyme digestion and sequenceanalysis resulted in the identification of four kinds ofindependent cDNAs encoding SKIP (13), DnaJ-like heat shockprotein 40 (14), human SWI-SNF component BRG1 (15) andan unknown protein containing zinc-finger motifs. Since SKIPwas found in eight overlapping clones including a full-lengthopen reading frame, it was used for additional studies.

To verify the interaction between PABP2 and SKIP, aglutathione S-transferase (GST)-pulldown assay was carriedout. For the GST-pulldown assay, GST-fusion proteins werepurified from Escherichia coli (BL21) harboring expressionplasmids encoding PABP2-FL (GST-PABP2-FL), its N-terminalregion (GST-PABP2-N) and C-terminal region which containedthe RNA recognition motifs (GST-RRMs). The purified fusion

proteins were incubated with cell lysates from COS-7 cells tran-siently transfected with the expression plasmid for SKIP fused tothe Myc-epitope tag (Myc-SKIP). The GST-pulldown assayrevealed that Myc-SKIP was specifically bound to GST-PABP2-FL and GST-PABP2-N, but not to GST-RRMs orGST alone (Fig. 5A). Furthermore, the interaction betweenPABP2 and SKIP was also confirmed by immunoprecipitation.For the immunoprecipitation assay, COS-7 cells were transientlyco-transfected with the expression plasmids encoding Myc-SKIPand Flag-PABP2. The protein complex was immunoprecipitatedfrom the cell lysates using anti-Flag polyclonal antibodyrecognizing Flag-PABP2. Immunoprecipitates were analyzed bywestern blotting using anti-Myc monoclonal antibody. As shownin Figure 5B, Myc-SKIP was specifically co-immunoprecipitatedwith Flag-PABP2 from the co-transfected COS-7 lysate. Ascontrols, Myc-SKIP was not detected in the immune complexfrom the COS-7 lysates transfected with the expressionplasmids encoding either Myc-SKIP or Flag-PABP2 (Fig. 5B).These results clearly indicate that PABP2 interacts with SKIPin mammalian cells.

SKIP is co-localized with PABP2 in nuclear speckles

Intranuclear localization of PABP2 and SKIP in HeLa cellswas examined under a confocal microscope. A previous studyreported that PABP2 was localized in the nuclear speckles ofHeLa cells (16). The splicing factor, SC-35, was used for themarker of the nuclear speckles (16,17). As shown in Figure 6A,transiently expressed Flag-PABP2 was co-localized in nuclearspeckles with SC-35. This is thought to be a similar pattern to

Figure 4. The N-terminal region of PABP2 is responsible for the up-regulation of MyoD and myogenin mRNAs. (A) Structures of the full-length and truncatedPABP2 cDNA used in this experiment. (B) The protein expressions in the C2 cells transiently transfected with indicated expression plasmids were verified bywestern blot analysis using anti-Flag polyclonal antibody. Cells were directly dissolved in SDS-sample buffer and subjected to western blot analysis. (C) QuantitativeRT–PCR resulting from C2 cells transiently transfected with 2 µg of expression plasmids encoding full-length or truncated PABP2 cDNA is shown. Approximately5 × 105 cells were plated on 60 mm dishes in growth medium 24 h prior to transfection. The cells almost reached confluence 24 h after transfection. At this time,the growth medium was replaced with a differentiation medium. The ratio of target cDNA/β-actin cDNA amounts from the mock controls at 24 h after transfectionwas arbitrarily set at 1. The values are derived from two sets of independently transfected triplicates. Error bars indicate the SD of the means.


that for endogenous PABP2. In HeLa cells co-transfected withthe Flag-PABP2 and Myc-SKIP expression plasmids these twoproteins were strongly co-localized in nuclear speckles (Fig. 6A).No staining was detected in non-transfected cells (data notshown). This finding also supports the suggestion that thesetwo proteins interact with each other in the nuclear speckles.

At 48 h after transfection ∼5% of transfected HeLa cellsshowed some large aggregates with strong staining by anti-Flag antibody in the nucleus, indicating that they contain ahigh concentration of Flag-PABP2 (Fig. 6B). These aggregatesappeared round in shape with variable size, and SKIP andSC-35 were strongly associated with them (Fig. 6B). Thesepresumably appear to be the result of overexpression ofPABP2. Although it is not known whether these aggregates aresimilar to inclusions observed in OPMD muscle, we found thatthe nuclear distributions of SC-35 and SKIP were changed bythe aggregate formation.

PABP2 and SKIP co-operate E-box-mediated transcriptionin the presence of MyoD

Myogenic factors control the expression of muscle-specificgenes by binding to a specific DNA sequence (CANNTG),called E-box, in their promoter region. To evaluate the effect ofPABP2 and SKIP on E-box-mediated transcription, luciferase

reporter assays were performed using C3H10T1/2 cells.C3H10T1/2 is a mouse non-muscle fibroblastic cell line.Exogenous expression of myogenic factors in C3H10T1/2cells is sufficient to convert them into a muscle-specificlineage (18,19). As a model system for muscle-specific tran-scription, we used the MyoD-responsive reporter plasmid(4RE-tk-luc), which contains a firefly luciferase genecontrolled by four tandem E-boxes from muscle creatinekinase enhancer upstream of the thymidine kinase basalpromoter (20). C3H10T1/2 cells were transiently co-trans-fected with the reporter plasmid and the expression plasmidsencoding PABP2, SKIP and MyoD. This reporter plasmid wasactivated to 7-fold greater luciferase activity by MyoD expression(Fig. 7). In the absence of MyoD, the expression of PABP2 andSKIP failed to activate the reporter over the basal levels(Fig. 7A). In the presence of MyoD, the effect of SKIP and

Figure 5. Interaction of PABP2 and SKIP. (A) GST-pulldown assay shows theinteraction between GST-PABP2 and Myc-SKIP. To construct the expressionplasmids for GST-fusion proteins, previously described PABP2 cDNAfragments (Fig. 4A) were used. The indicated fusion proteins were expressed inE.coli (BL21), purified, and then incubated with the lysate from COS-7 cellstransfected with the Myc-SKIP expression plasmid. The associated proteinswere recovered with glutathione-Sepharose resin, and then subjected to westernblot (WB) analysis using anti-Myc monoclonal antibody. Five percent of celllysates were loaded as the Input. (B) Myc-SKIP is co-immunoprecipitated withFlag-PABP2. Cell lysates from COS-7 transfected with the indicated expressionplasmids were immunoprecipitated using the anti-Flag polyclonal antibody.Immunoprecipitates (IP) were analyzed by western blot using anti-Mycmonoclonal antibody for Myc-SKIP and anti-Flag M2 monoclonal antibody forFlag-PABP2. The arrowhead indicates the immunoglobulin heavy chain.

Figure 6. Nuclear localization of PABP2 and SKIP. HeLa cells were transfectedwith the expression plasmids for Flag-PABP2 and/or Myc-SKIP. A singleconfocal scan is shown. Scale bar, 5 µm. (A) Flag-PABP2 (green) localized innuclear speckles (top). Cells were counter-stained with endogenous SC-35 (red).Co-transfected HeLa cells (bottom) show that Flag-PABP2 (green) is co-localizedwith Myc-SKIP in the nuclear speckles (red). (B) Intranuclear aggregatesformation in HeLa cells transiently transfected with Flag-PABP2 expressionplasmid. Approximately 5% of Flag-PABP2 transiently transfected cells show theaggregate bodies at 48 h after transfection. The nuclear distribution of SC-35 andMyc-SKIP were changed and were strongly associated with aggregates byPABP2 expression.


PABP2 on MyoD-dependent transactivation was characterizedat various amounts of expression plasmids. MyoD-responsiveluciferase activity was greatly activated by PABP2 transfectionin a dose-dependent manner (Fig. 7A). The cells transfectedwith SKIP expression plasmid showed a similar result, butmore efficient luciferase transactivation was observedcompared with cells transfected with PABP2 expressionplasmid of the same dose (Fig. 7A). The transfections ofPABP2-FL or PABP2-N activated reporter activity in thepresence of MyoD (Fig. 7B).

To evaluate the synergistic effect of PABP2 and SKIP on thereporter gene, the cells were co-transfected with expressionplasmids encoding the truncated PABP2 and SKIP. We deter-mined the amount of PABP2 and SKIP expression plasmidsfor transfection as a 2-fold transactivation on the reporter in thepresence of MyoD. This is consistent with the result ofquantitative RT–PCR from C2 cells as described previously

(Fig. 4C). In the presence of MyoD, the transfection of PABP2synergistically stimulated the luciferase activity with itsbinding partner, SKIP (Fig. 7B). We observed an approximately6-fold synergistic transactivation (Fig. 7B). Moreover, asimilar transactivation in the cells transfected with PABP2-Nwas also observed (Fig. 7B). In contrast, few synergisticactivities were observed in the cell co-transfected with theexpression plasmid for RRMs and SKIP (Fig. 7B).

We next investigated whether PABP2 and SKIP exist in asingle complex with MyoD because the transactivation forMyoD-responsive reporter might be caused by protein–proteininteraction. Immunoprecipitation assay was performed usingthe anti-MyoD polyclonal antibody in C3H10T1/2 cells transientlyco-transfected with the indicated expression plasmids. Westernblot analysis showed that PABP2 or SKIP as well as bothPABP2 and SKIP could be associated with MyoD (Fig. 8).These associations with MyoD were specific because omissionof the anti-MyoD antibody or MyoD protein did not result inthe co-precipitation of SKIP and PABP2 (Fig. 8).

DISCUSSION

In the present study, we demonstrate the possible involvementof PABP2 in the expression of muscle-specific genes and skel-etal myogenesis. We established stable C2 cell lines expressinghuman PABP2. A cell line constitutively expressing humanPABP2 showed accelerated morphological differentiationaccompanied by enhanced mRNA expression of MyoD as wellas the differentiation markers, myogenin and MLC1a.Although the cytomegalovirus promoter tends to induce veryhigh levels of expression of transgenes, our stable cell linesshowed only 14.6 and 28.9% of the exogenous PABP2 expression.On the other hand, the entire mRNA expression levels ofPABP2 were approximately 3.5-fold higher than their parentcells for unknown reasons. In addition, the exogenous PABP2protein was easily detected by western blotting during themyogenic differentiation. Therefore, the enhanced myotubeformation of the stable cell lines may be due to the over-expression of PABP2.

The poly(A) length of mRNA has functional implications forits turnover, translation and export. In general, regulation ofpolyadenylation is distinct from the nuclear reaction and maydepend on several specific factors and a cis-acting sequence

Figure 7. PABP2 co-operates with SKIP to stimulate E-box-mediatedtranscription through MyoD. (A) C3H10T1/2 cells were transiently transfectedwith reporter plasmid (4RE-tk-luc) and expression plasmids encoding PABP2,SKIP and MyoD as indicated. The increasing amounts (0.2, 0.4, 0.6 and 0.8 µg,respectively) of expression plasmids for PABP2 and SKIP were used for trans-fection. (B) The expression plasmids encoding full-length or truncated forms ofPABP2 were used as described in Figure 4A. The amount of PABP2 and SKIPexpression plasmids used for transfections was 0.4 and 0.2 µg, respectively. Thetotal amount of DNA in each transfection was kept constant by addition of pUC18 DNA. The reporter activity from the cells transfected with reporter plasmidalone was assigned a value of 1. The values are derived from two sets ofindependently transfected triplicates. Error bars indicate the SD of the means.

Figure 8. PABP2 and SKIP associate with MyoD. C3H10T1/2 cells weretransiently transfected with 1 µg of MyoD and SKIP expression plasmids and2 µg of PABP2 expression plasmid in 60 mm culture plates. The cell lysates wereimmunoprecipitated using anti-MyoD polyclonal antibody. Immunoprecipitates(IP) were analyzed by western blotting using anti-Myc monoclonal antibody,anti-Flag monoclonal antibody (M2) and anti-MyoD monoclonal antibody. Thearrowheads indicate the immunoglobulin heavy chains.


element (21). PABP2 acts to limit the overall length of newlysynthesized poly(A) tail to 200–250 residues (4). PABP2 canshuttle between the nucleus to the cytoplasm suggesting itsinvolvement in mRNA export (22). Chen et al. (23) reportedthat the influenza virus NS1A protein inhibited PABP2function during 3′-end processing which is responsible for theblock in the nuclear export of cellular mRNAs (23). Therefore,the enhanced differentiation observed in the stable cell linesmight be caused by specifically promoted mRNA export ofMyoD due to the PABP2 overexpression. However, wewonder why mRNA of MyoD is specifically exported. One ofthe possible explanations is overexpressed PABP2 whichpresumably affects the transcription of the MyoD gene. In thepresent study, the result of nuclear run-on assays indicates thathigh levels of the MyoD transcription occur in the nucleusdepending on the transfection of PABP2 expression plasmid.In addition, nuclear run-on assay as well as Northern blotanalysis showed no notable change of the expression level ofGAPDH mRNA by the PABP2 transfection. These findingssuggest that MyoD mRNA is specifically regulated at thetranscription level by the PABP2 transfection.

We also demonstrated that PABP2-N played a crucial role inthe up-regulation of MyoD mRNA. One muscle-specific mRNA,MLC1a, was increased by the tranfections of expression plasmidsencoding PABP2-N as well as PABP2-FL, suggesting that theN-terminal region of PABP2 contributed to activating muscle-specific gene expression via the up-regulation of myogenicfactors. In addition, we identified that PABP2 interacted withSKIP. SKIP was originally identified as a protein that interactswith both the cellular and viral forms of the oncoprotein Ski(13). SKIP is highly homologous to Bx42, a Drosophila melano-gaster nuclear protein involved in ecdysone-stimulated geneexpression (24). A recent study demonstrated that NcoA-62functions as a co-activator protein involved in vitamin D-mediatedtranscription (25). SKIP has a nucleotide sequence virtuallyidentical to the cDNA termed NcoA-62 (25). This protein alsoshows a transactivation activity as a co-activator for theretinoic acid-, estrogen- and glucorticoid receptor-mediatedtranscription pathways (25). Several lines of evidence havebeen reported suggesting that skeletal myogenesis and muscle-specific transcription are positively regulated by hormone-relatedfactors including the receptors of retinoic acid (26–29) andinsulin-like growth factors (30–32), and by several onco-proteins (33–36). These are directly or indirectly related to thetranscriptional machinery mediated by myogenic factors.Through the reporter assay, we showed that transient transfectionof PABP2 results in transactivation for E-box-mediatedtranscription in the presence of MyoD. SKIP co-operated withPABP2 to stimulate the E-box-mediated transcription in thepresence of MyoD. Moreover, we identified that SKIP andPABP2 were associated in a single complex with MyoD. Thisfinding suggests that the co-operation by PABP2 and SKIP tostimulate MyoD-dependent transcription may depend on theirability to form such a complex and provides evidence thatPABP2 may function as a potential co-factor of transcription or itsrelated protein in the skeletal muscle. This is a function ofPABP2 distinguishable from that involved in the polyadenyla-tion process.

Our immunofluorescence study also demonstrated the co-localization of PABP2 and SKIP in nuclear speckles. Thenuclear speckles are highly enriched in pre-mRNA splicing

factors (37–40) including the 3′-end-processing factors andpoly(A) RNA (16,41). These correspond to the interchromatingranule clusters and perichromatin fibrils imaged by electronmicroscopy (42). Several recent findings suggest that mostsplicing and 3′-end processing occurs co-transcriptionally onperichromatin fibrils, and interchromatin granule clustersrepresent reservoirs, from which pre-mRNA processing factorsare recruited to the nascent transcript (40,43,44). The nucleardistribution of PABP2 shows a unique pattern compared withmajor 3′-end processing factors including cleavage stimulationfactor (CstF), CPSF and PAP. A previous immunofluorescencestudy has shown that PABP2 is concentrated in nuclearspeckles (16); however, other components of the 3′-endprocessing machinery (CPSF, CstF, PAP) do not concentratein speckles (45). Although these components are found intranscription sites, they show different nuclear distributionsfrom PABP2 under physiological condition (45). These differencesin nuclear distributions indicate that PABP2 may differ func-tionally from other 3′-end processing components. We showed thatthe exogenous PABP2 in the stable cell lines was accumulated inthe nuclei during myogenic differentiation. It appears that themuscle-specific gene expression during myogenesis requiresthe accumulation of PABP2.

In the skeletal muscle from OPMD patients, unique intranuclearfilamentous inclusions are found in ∼2.5% of nuclei by electronmicroscopy (46,47). It appears that increased repeat expansioncauses abnormal aggregation of PABP2 and the formation ofthe filamentous nuclear inclusions in skeletal muscle (48–52).These observations raise the possibility that the nuclearfilament inclusions formed by the GCG repeat expansion ofPABP2 could exert a gain of toxic function to accelerate celldeath. We transfected PABP2 containing expanded repeats ofGCG (nine GCG repeats) and compared the frequency ofaggregate formation with normal PABP2 (six GCG repeats).However, significant differences in frequency and distributionwere not observed (data not shown). Nevertheless, we foundthat ∼5% of PABP2 transfected HeLa cells contained largeaggregates that altered the spatial distribution of nuclearspeckles. Similar aggregates were observed with a highfrequency in COS-1 cells transfected with ataxin-1 containingan expansion of the polyglutamine tract, which is responsiblefor spinocerebellar ataxia type 1 (SCA1) (53). The aggregatesobserved in the COS-1 cells transfected with expanded ataxin-1altered the distribution of the nuclear matrix-associate structuresand suggested their involvement in the pathogenesis of SCA1(53). PABP2 is also known as a nuclear matrix-associatedprotein (54) and localized in the nuclear speckles. The nuclearspeckles play an important role in splicing and processing ofpre-mRNA, and their altered distribution may be associatedwith OPMD pathogenesis. Further studies should be needed toclarify the pathophysiological mechanism of OPMD whosemuscles contain inclusions with accumulation of PABP2 (52).

MATERIALS AND METHODS

Plasmids

The coding region of PABP2 was amplified from the first strandcDNA for human skeletal muscle by PCR. The cDNA fragmentsencoding the N- and C-terminus of PABP2 were amplifiedseparately using the following primers: N-terminal fragment, 5′-


GATGGCGGCGGCGGCGGCGGCG-3′ (sense) and 5′-GCTT-CTCTACCTCGTTCTGATAGCT-3′ (antisense); C-terminalfragment, 5′-CCCGGAGCTGGAAGCTATCAA-3′ (sense)and 5′-TTACGTAAGGGGAATACCATGATGT-3′ (antisense).For Flag-epitope (DYKDDDDK) tagging, the antisensesequence against the Flag-epitope was added to the 5′-end ofthe C-terminus antisense primer which was used for PCRamplification. The amplified product was subcloned intopGEM-T Easy vector (Promega) and sequenced. Basically,these subclones were used to generate the expressionconstructs of PABP2 for mammalian cells, yeasts and E.coli.To construct the PABP2 expression plasmid for mammaliancells, the EcoRI-XhoI fragment of the N-terminus and theXhoI-NotI fragment of the C-terminus were fused intoEcoRI-NotI sites of pCI-neo (Promega) to generate pCI-PABP2-FL. The expression plasmid for the truncated form ofPABP2, pCI-RRMs (amino acids 127–306), was derived frompCI-PABP2-FL by XhoI digestion and self-ligation. TheEcoRI-XhoI fragment of the N-terminus was subcloned intoEcoRI-SalI sites to generate pCI-PABP2-N (amino acids 1–145).The expression plasmids for GST-fusion proteins wereconstructed in the same manner as described above using thepGEX-6P-2 vector (Amersham Pharmacia Biotech). Full-length SKIP cDNA was isolated from a human skeletal musclecDNA library (Clontech) using yeast two-hybrid screening.The SKIP cDNA insert was excised from the yeast plasmid,pACT2 (Clontech), and then subcloned into EcoRI-XhoI sitesof pMyc-CMV (Clontech) to generate pCMV-Myc-SKIP.MyoD cDNA was amplified by RT–PCR from C2 cells andsubcloned into pRc/RSV (Invitrogen) to generate pRSV-MyoD.The luciferase reporter plasmid (4RE-tk-luc) was generated asdescribed previously (20).

Cell culture and stable transfectants

C3H10T1/2, HeLa and COS-7 cells were maintained inDulbecco’s modified Eagle’s medium (DMEM; Sigma)supplemented with 10% fetal bovine serum. All cultures weregrown at 37°C in a humidified atmosphere containing 5% CO2.

C2 cells were cultured in growth medium (DMEMcontaining 15% fetal bovine serum) at low cell-density tosustain a proliferative state. To induce differentiation, cellswere plated at a density of 104 cells/cm2 into collagen coatedculture plates (IWAKI) in growth medium. After 2 days incu-bation the cells were almost confluent and the growth mediumwas changed to a differentiation medium (DMEM containing5% horse serum) and then cells were cultured for 3 days.

To establish the C2-derived stable transfectants, C2 cellswere plated at 5 × 105 cells in 60 mm dishes for 24 h prior totransfection. The cells were transfected with an expressionplasmid for C-terminally Flag-epitope tagged PABP2 cDNAusing lipofectamine reagent (Gibco BRL Life Technologies).Following incubation for 24 h, cells were selected in thepresence of 800 µg/ml G418 (Gibco BRL Life Technologies)in growth medium. The resistant clones were pooled 7 daysafter incubation and replated in 96-well culture plates at anextremely low density to avoid cross-contamination. After anadditional 7 days incubation, G418-resistant clones weresubcultured and maintained in growth medium containing400 µg/ml G418. The expression of Flag-tagged PABP2 was

confirmed by RT–PCR and western blot analysis from eachindividual clone.

Quantitative RT–PCR

Quantitative RT–PCR was performed by the method ofKinoshita et al. (55) with slight modification. Total RNAswere isolated from the growth and differentiation phases of C2cells using TRIzol reagent (Gibco BRL Life Technologies).First-strand cDNA was synthesized from 2 µg of total RNA byreverse transcription at 37°C for 1 h. The reaction mixture wasdiluted in water to 100 µl, and used for PCR amplificationusing primers that are specific to MyoD, myogenin, Myf-5 andMLC1a. The length of the PCR products was in the 250–400 bprange. The fluorescent reporter dye, NED, was covalentlylinked to the 5′-end of the sense primers. β-actin was used asthe internal standard of the cDNA content. The sense primer ofβ-actin was labeled with HEX amidite. Amplification was firstperformed with β-actin primers to normalize the amount ofcDNA present in each preparation, and the normalized valueswere used to adjust for the amount of templates used in thePCR reaction with the gene-specific primers. The PCRreaction was performed in a final volume of 25 µl containing10 pmol of each primer, 0.2 mM dNTP and 1.2 U of Tag DNApolymerase (Promega) under the following conditions;denaturation at 95°C for 30 s, primer annealing at 56°C for30 s, and primer extension at 72°C for 40 s. Appropriatecycling numbers for each primer pair were pre-determined toensure that PCRs were in a linear concentration range. Onemicroliter of the amplified products was loaded for capillaryelectrophoresis using an ABI PRISM 310 genetic analyzer(Perkin-Elmer Applied Biosystem) and quantified usingGeneScan Analysis software (Perkin-Elmer Applied Biosystem).

Nuclear run-on assay and northern blot analysis

For the nuclear run-on experiment, nuclei from the control andPABP2 transfected cells were isolated using a previouslyreported procedure (56). The nuclei were resuspended in200 ml of transcription buffer (10 mM Tris pH 8.0, 150 mMKCl, 3 mM MgCl2, 20% glycerol, 2 mM DTT) and subjectedto in vitro transcription by adding 1 mM each of ATP, GTP,CTP and 200 µCi of [α-32P]UTP (Amersham PharmaciaBiotech) to label nascent transcripts for 30 min at 30°C.Following DNase I and proteinase K treatment, radiolabeledRNA was extracted using TRIzol reagent. The cDNA fragment(5 µg of MyoD, 2 µg of GAPDH) and 5 µg of linearizedpUC18 plasmid DNA were denatured with NaOH, slot-blottedon positively charged nylon membrane (Hybond N+, AmershamPharmacia Biotech), and then hybridized overnight at 68°Cwith 1 × 107 c.p.m. of labeled transcripts in ExpressHybhybridization solution (Clontech). After hybridization, filterswere stringently washed and exposed to an imaging plate forquantification of radioactivity using an image analyzer, BAS-2500 (FUJIFIRM)

For northern blot analysis, 10 µg of each total RNA was sizefractionated on 1.2% formaldehyde-agarose gel and trans-ferred to positively charged nylon membrane with an alkalinecondition. The filters were hybridized for 2 h at 68°C with 32P-radiolabeled MyoD or GAPDH cDNA fragments inExpressHyb hybridization solution (Clontech).


Yeast two-hybrid screening

The cDNA fragment encoding PABP2-N (amino acids 1–145)was subcloned into a yeast two-hybrid vector, pAS2-1(Clontech) containing the GAL4 DNA-binding domain, whichwas used as bait. Yeast strain AH109 (Clontech) was trans-formed using the lithium acetate transformation method (57).Yeast two-hybrid screening was performed by transforming ahuman skeletal muscle cDNA library (Clontech) fused to theactivation domain into the AH109 expressing bait protein.Approximately 3 × 106 transformants were plated on selectionmedium lacking leucine, tryptophan, histidine and adenine,and then cultured at 30°C for 5 days. To reduce backgroundtransformants, primary screened yeast colonies were collectedand replated on selection medium containing 15 mM 3-amino-1, 2, 4-triazole (Sigma). The positive clones were confirmed bya β-galactosidase filter-lift assay.

Immunofluorescence analysis

Immunofluorescence analysis was performed on C2 myoblastsand myotubes grown on collagen coated culture plates. Phos-phate-buffered saline (PBS) was used to wash cells extensivelyat room temperature before fixation and after each step of thedescribed procedures. Cells were fixed and permeabilized in3% paraformaldehyde, 0.3% Triton X-100 in PBS for 5 min,followed by incubation with 3% paraformaldehyde for 15 min.The fixed cells were blocked for 1 h using 5% normal goatserum in PBS, and then incubated for 1 h at room temperaturewith 1:500 rabbit anti-Flag polyclonal antibody (ZYMEDLaboratories) recognizing Flag-PABP2. Subsequently, cellswere incubated with 1:100 FITC-conjugated goat anti-rabbitantibody (Tago) for 1 h. Samples were photographed using anOlympus AX 70 microscope (Olympus Optical).

To determine intranuclear localization, HeLa cells werecultured on coverslips and transfected with the expressionplasmid for Flag-PABP2 and/or Myc-SKIP. Cells were fixed at48 h after transfection and incubated with 1:500 rabbit anti-Flagpolyclonal antibody and/or mouse anti-Myc monoclonal antibody(Clontech). The secondary antibodies, 1:100 FITC-conjugatedgoat anti-rabbit antibody and rhodamine-conjugated anti-mouse antibody (Tago) were used. Samples were observedunder a confocal laser-scanning microscope (Leica). For local-ization of the SC-35 protein, a monoclonal antibody againstSC-35 was used as the culture supernatant at a 1:100 dilution.

Luciferase assay

C3H10T1/2 cells were transiently co-transfected with 0.6 µgof reporter plasmid (4RE-tk-luc), 0.3 µg of MyoD construct(pRSV-MyoD), 0.02 µg of pRL-CMV (Promega) and variousamounts of each expression construct per well in 6-well plates. TheCMV promoter-driven sea-pansy luciferase plasmid, pRL-CMV,was used as an internal control to normalize firefly luciferaseactivity. The total amount of plasmid DNA was adjusted to2 µg by addition of pUC18 plasmid DNA. The growth mediumwas changed to a differentiation medium 24 h after transfection,and 48 h later the cells were harvested for luciferase assay. Theluciferase activity was assayed using a dual-luciferase assaysystem (Promega) according to the manufacturer’s instructionwith a TD-20/20 luminometer (Turner Designs).

Preparation of cell lysate and western blot

Cell lysates were prepared from C2, COS-7 and C3H10T1/2cells for western blot analysis and protein interaction assay.After washing with PBS, pelleted cells were incubated in fivevolumes of buffer (10 mM Tris pH 8.0, 10 mM NaCl, 2 mMMgCl2) supplemented with a mixture of protease inhibitor(1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/mlpepstatin and 5 µg/ml aprotinin) for 10 min on ice. The detergent,Igepal CA-630 (Sigma) was added to 0.5% and incubation wasallowed to continue for 10 min. Cells were pelleted by centri-fugation, and resuspended in extraction buffer (20 mM HEPESpH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 50 mM NaF, 0.2 mMEDTA, 0.5 mM DTT, 20% glycerol) supplemented with themixture of protease inhibitors, and incubated for 1 h with slowrotation at 4°C. The lysate was centrifuged at 15 000 g for20 min and the supernatant was recovered.

The cell lysates were analyzed by SDS–PAGE and trans-ferred to polyvinylidene difluoride filter using a trans-blotelectrophoretic transfer cell (Bio-Rad). Filters were blockedfor non-specific binding in Tris-buffered saline containing0.1% Tween-20 with 5% powdered skimmed milk for 1 h andthen incubated with primary antibody for 1 h at room temperature.The following primary antibodies were used; 1:500 anti-Flagpolyclonal antibody (ZYMED), 1:500 anti-MyoD polyclonalantibody (C-20; Santa Cruz Biotechnology) and anti-myogenin polyclonal antibody (M-255; Santa Cruz Biotech-nology). The filters were incubated with 1:2000 horseradishperoxidase-conjugated secondary antibody (Tago) for 1 h.Immunoreactive bands were visualized using an enhancedchemiluminescence system (Amersham Pharmacia Biotech).

GST-pulldown assay and immunoprecipitation

Approximately 200 µg of protein from cell lysate was dilutedin the same volume of buffer (50 mM Tris pH 7.5, 0.2% IgepalCA-630) for the GST-pulldown assay and immunoprecipitation.For the GST-pulldown assay, the diluted cell lysate wasincubated with 1 µg of purified GST-fusion protein and 30 µlof glutathione-Sepharose 4B (Amersham Pharmacia Biotech)for 2 h at 4°C with gentle rocking. The resins were washed fivetimes with 1 ml of ice-cold wash buffer (20 mM Tris pH 7.5,150 mM NaCl, 1 mM EDTA, 0.1% Igepal CA-630) and boiledin 20 µl of SDS–PAGE sample buffer for 5 min. Elutedproteins were analyzed by western blot using an anti-Mycmonoclonal antibody (Clontech).

For immunoprecipitation the diluted cell lysate was incubatedwith 2 µg of anti-Flag polyclonal antibody or anti-MyoD poly-clonal antibody (M-318; Santa Cruz Biotechnology) and 30 µlof protein G–Sepharose for 2 h at 4°C with slow rotation.Washing conditions and elution of associated proteins wereidentical to those of the GST-pulldown assay. To detectassociated proteins by western blotting, the following anti-bodies were used; 1:1000 anti-Flag M2 monoclonal antibody(Sigma) for Flag-PABP2, 1:1000 anti-Myc monoclonal anti-body for Myc-SKIP, 1:100 anti-MyoD monoclonal antibody(Novocastra).

ACKNOWLEDGEMENTS

The authors thank Dr C. Akazawa for helpful advice andencouragement. This work was supported by Research Grant


and Grants-in-Aid for Scientific Research for Center ofExcellence (COE) from the Ministry of Health and Welfare,Japan and from the Core Research for Evolutional Science andTechnology, Japan Science and Technology. Y.-J.K. wassupported by a scholarship from the Kambayashi scholarshipfoundation.

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The product of an oculopharyngeal muscular dystrophy gene, poly(A