Neurobiology of Disease 46 (2012) 118–129

Contents lists available at SciVerse ScienceDirect

Neurobiology of Disease

j ourna l homepage: www.e lsev ie r .com/ locate /ynbd i

Nuclear speckles are involved in nuclear aggregation of PABPN1 and in thepathophysiology of oculopharyngeal muscular dystrophy

Rocío Bengoechea a, Olga Tapia a, Iñigo Casafont a, José Berciano b, Miguel Lafarga a,⁎, María T. Berciano a

a Department of Anatomy and Cell Biology and “Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)”, University of Cantabria (UC), Santander,Spainb Service of Neurology, University Hospital “Marqués de Valdecilla” (IFIMAV), University of Cantabria, CIBERNED and UC, Santander, Spain

⁎ Corresponding author at: Department of AnatomMedicine, Avd. Cardenal Herrera Oria s/n, 39011 San201903.

E-mail address: lafargam@unican.es (M. Lafarga).Available online on ScienceDirect (www.scienced

0969-9961/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.nbd.2011.12.052

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 June 2011Revised 19 December 2011Accepted 31 December 2011Available online 10 January 2012

Keywords:Oculopharyngeal muscular dystrophySkeletal myofibersIntranuclear inclusionsNuclear specklesPre-mRNA processingPoly(A)-binding protein nuclear 1Nuclear body biogenesisProteasome inhibition

Nuclear speckles are essential nuclear compartments involved in the assembly, delivery and recycling of pre-mRNA processing factors, and in the post-transcriptional processing of pre-mRNAs. Oculopharyngeal musculardystrophy (OPMD) is caused by a small expansion of the polyalanine tract in the poly(A)-binding protein nuclear1 (PABPN1). Aggregation of expanded PABPN1 into intranuclear inclusions (INIs) in skeletal muscle fibers is thepathological hallmark of OPMD. In this study what we have analyzed in muscle fibers of OPMD patients and inprimary cultures of humanmyoblasts are the relationships between nuclear speckles and INIs, and the contribu-tion of the former to the biogenesis of the latter. While nuclear speckles concentrate snRNP splicing factors andPABPN1 in control muscle fibers, they are depleted of PABPN1 and appear closely associated with INIs in musclefibers of OPMD patients. The induction of INI formation in human myoblasts expressing either wild type GFP-PABPN1 or expanded GFP-PABPN1-17ala demonstrates that the initial aggregation of PABPN1 proteins andtheir subsequent growth in INIs occurs at the edges of the nuclear speckles. Moreover, the growing of INIs grad-ually depletes PABPN1 proteins and poly(A) RNA from nuclear speckles, although the existence of these nuclearcompartments is preserved. Time-lapse experiments in cultured myoblasts confirm nuclear speckles as biogen-esis sites of PABPN1 inclusions. Given the functional importance of nuclear speckles in the post-transcriptionalprocessing of pre-mRNAs, the INI-dependentmolecular reorganization of these nuclear compartments inmusclefibers may cause a severe dysfunction in nuclear trafficking and processing of polyadenylated mRNAs, therebycontributing to the molecular pathophysiology of OPMD. Our results emphasize the potential importance of nu-clear speckles as nuclear targets of neuromuscular disorders.

© 2012 Elsevier Inc. All rights reserved.

Introduction

The cell nucleus is highly dynamic and its essential functions, suchas replication, transcription, DNA damage repair and RNA processing,are compartmentalized in distinct structures that concentrate specificfactors. Nuclear compartments include chromosome territories, dis-tributed in the chromatin domain (Cremer and Cremer, 2001), andnuclear speckles, nucleoli, Cajal bodies, PML bodies and orphan nucle-ar bodies, etc., which are located in the interchromatin regions(reviewed in, Misteli and Spector, 2011). The spatial organization ofthese nuclear compartments plays an essential role in genome func-tion and maintenance (Carmo-Fonseca, 2002; Cremer and Cremer,2001; Misteli and Spector, 2011).

y and Cell Biology, Faculty oftander, Spain. Fax: +34 942

irect.com).

rights reserved.

Within the nuclear environment, nuclear speckles are one of themost prominent nuclear compartments (Lamond and Spector, 2003;Spector and Lamond, 2011). They were initially described by Cajal,using silver staining procedures, as pale rounded or irregular areas ofhomogeneous texture in the neuronal nucleus (reviewed in Lafarga etal., 2009). Nuclear speckles or their ultrastructural equivalent, the inter-chromatin granule clusters, are enriched in components of the pre-mRNA splicingmachinery, particularly spliceosomal small nuclear ribo-nucleoprotein particles (snRNPs) and other non-snRNP protein splicingfactors such as SC35 and U2AF (Hall et al., 2006; Lamond and Spector,2003; Raska, 1995). They also contain several kinases, the PP1 phospha-tase, 3′-end processing factors, some transcription factors involved ingene expression driven by the RNA polymerase II, and a population ofpoly(A) RNA and noncoding RNA (Hall et al., 2006; Lamond andSpector, 2003; Spector and Lamond, 2011). Nuclear speckles are dy-namic structures involved in the storage, assembly and recycling ofpre-mRNA processing factors, and in the post-transcriptional proces-sing of pre-mRNAs (Hall et al., 2006; Melcák et al., 2000, 2001). Theysteadily exchange their components with the nucleoplasm and activetranscription factories, thereby facilitating the expression of multiple

119R. Bengoechea et al. / Neurobiology of Disease 46 (2012) 118–129

active genes, some of which associate directly with the edge of nuclearspeckles (Shopland et al., 2003).

The poly(A)-binding protein nuclear 1 (PABPN1) is an abundantnuclear protein that plays an essential role in the polyadenylation ofpre-mRNAs by increasing the processivity of poly(A) polymerase.PABPN1 binds with high affinity to poly(A) RNA, regulates the lengthof the poly(A) tail and, upon the completion of polyadenilation, ac-companies the poly(A) RNA released from transcription sites (for a re-view, see Calado and Carmo-Fonseca, 2000; Kühn and Wahle, 2004;Wahle, 1991). In mammalian cells, several reports provide evidencethat polyadenylation is required not only for the stability of mRNAsbut also for their nuclear exportation (Kühn andWahle, 2004).Withinthe nucleus, PABPN1 concentrates in nuclear speckles (Krause et al.,1994; Raz et al., 2011), a localization that is strictly dependent uponits binding to poly(A) tail of RNAs (Calado and Carmo-Fonseca, 2000).

In addition to the importance of PABPN1 for the processing of pre-mRNAs, the discovery thatmutations in the PABPN1 gene are responsiblefor oculopharyngeal muscular dystrophy (OPMD) links this protein witha human neuromuscular disorder (Brais et al., 1998). OPMD is a late-onset autosomal dominant myopathy caused by GCG expansions in thefirst exon of the PABPN1 gene. In the normal PABPN1 gene, (GCG)6 en-codes the first six alanines in a stretch of ten alanines. In OPMD patientsthis (GCG)6 repeat is expanded to (GCG)8–13, resulting in a stretch of12–17 alanines in the N-terminal domain of the mutated PABPN1(Abu-Baker and Rouleau, 2007; Brais, 2009; Brais et al., 1998). The path-ological hallmark of the OPMD is the presence of intranuclear inclusions(INIs) in skeletal myofibers composed of tubular filaments (Tomé andFardeau, 1980). In addition to expanded PABPN1 (expPABPN1), INIs inskeletal myofibers of OPMD patients contain ubiquitin, proteasomes,poly(A) RNAs, chaperones and arginine methyl transferases (Abe-Bakerand Rouleau, 2007; Calado et al., 2000; Tavanez et al., 2009).

The contribution of nuclear aggregates of expPABPN1 to the path-ogenesis of OPMD remains unclear. Previous experimental studieshave revealed that the overexpression of a soluble form ofexpPABPN1 promotes cell death and also suggests that the aggrega-tion of expPABPN1 into INIs seems to have a protective role(Messaed et al., 2007). Furthermore, we have previously reportedthat alanine expansions are not required for the nuclear aggregationof the wild type PABPN1 into INIs in oxytocin-producing neuronsunder normal physiological conditions (Berciano et al., 2004). How-ever, the retention of Hsp70 chaperones and arginine methyl trans-ferases at INIs in OPMD patients may lead to a cellular deficit inarginine methylation and chaperone activity, contributing to the mo-lecular pathophysiology of OPMD (Tavanez et al., 2009).

Previous studies in HeLa and U2OS cells have shown the concentra-tion of the endogenous PABPN1 in nuclear speckles (Krause et al.,1994; Raz et al., 2011) and also that the expression in these cells ofGFP-PABPN1 constructs with either wild type or expanded polyalaninestretch reproduces the localization of PABPN1 in speckles and inducesthe formation of INIs (Abu-Baker and Rouleau, 2007; Klein et al., 2008;Tavanez et al., 2005). The aim of this study is to investigate the nuclearcompartmentalization of INIs in muscle fibers of OPMD patients and inprimary cultures of human myoblasts expressing either GFP-PABPN1wt or GFP-PABPN1-17ala. More specifically, we have analyzedi) the dynamic relationship between INIs and nuclear speckles, ii) therole of speckles in thebiogenesis andmaintenance of INIs, iii) themolec-ular reorganization of speckles associatedwith the formation of INIs andits implication formRNA trafficking and processing, and iv) the differentaggregation rate of wild type and expanded PABPN1 into INIs.

Material and methods

Patients

Biopsy samples from vastus medialis (quadriceps) from two fe-male patients, aged 82 (patient1) and 62 years (patient 2), were

used. With informed consent, diagnostic genetic analysis was per-formed, both patients showing an expansion in the PABPN1 genewith GCG6/GCG11 genotype.

Teased skeletal myofibers and histology

Small bundles of skeletal myofibers, about 1 cm in length, fromOPMD-patients and control subjects were used for teased muscle fiberpreparations. The muscle fibers were dissociated from each other withfine forceps down to a single fiber on a siliconized slide. To facilitatethe adhesion of fibers to the slides, the preparations were frozen indry ice for 5 min. Then, the slides with adhered muscle fibers weredehydrated in 96° and 70° ethanol at 4 °C for 10 min, rinsed in PBSand sequentially treated with collagenase 0.25% at 37 °C for 20 min,0.5% Triton-X100 in PBS for 45 min and 0.01% Tween 20 in PBS for5 min and then processed for immunofluorescence.

Some muscle tissue fragments fixed with 3.7% paraformaldehydein PBS were dehydrated and embedded in diethylene glycol. Semithinsections, 1 μm thick, were processed for immunofluorescence as pre-viously described (Berciano et al., 2004).

Primary human skeletal muscle cultures

Muscle biopsies from three normal controls and one OPMD pa-tient were used for this study. They were taken from the vastus med-ialis muscle and washed in Hanks' Balanced Salt Solution buffer(Invitrogen, Spain) with Antibiotic–Antimycotic (Invitrogen, Spain).

Humanmuscle biopsies were minced and cultured in a monolayer.Briefly, muscle for culture was cut into small pieces and incubated for48 h in medium containing 1:1:1 of human plasma, fetal calf serumand M199 medium (Invitrogen, Spain). Then, connective tissue visi-ble under a dissecting microscope was removed and the muscle dis-sected into 2 mm pieces that were explanted into 35 mm Petridishes coated with 1:1 mixture of Fresh Human Plasma and gelatine.During the first 2 weeks, cultures were fed with medium containing56% Dulbecco's minimal essential medium (Invitrogen, Spain), 19%M199 medium, 15% fetal bovine serum (FBS), 10 g/ml Insulin, 2 mMGlutamine, 1% Antibiotic–Antimycotic (Invitrogen, Spain), 10 ng/mlEpidermal Growth Factor (Peprotech, UK) and 25 ng/ml FibroblastGrowth Factor (Peprotech, UK).

To obtain highly purified myoblasts, primary cultures were sortedfor the early surface marker CD56+ by immunomagnetic selection.Cultured cells (107) were mixed with 80 μl of PBS adding 20 μl ofCD56+-coated microbeads (Miltenyi Biotec, Germany) and incubatedat 4–8 °C for 15 min. Unbound microbeads were removed by washingcells in excess PBS buffer followed by centrifugation at 1800 rpm for10 min. The cell pellet was resuspended in 500 μl of PBS buffer at aconcentration of 108 cells/500 μl before being separated on a midi-MACS cell separator (Miltenyi Biotec, Germany). CD56-positive cellswere seeded at 2000 cells/cm2 using the culture medium for the myo-blast stage containing 15% of FBS and without growth factors. For theproteasome modification experiments, control and OPMD myoblastscultured for three weeks were treated with the proteasome inhibitorbortezomib (Millennium Pharmaceuticals Inc, Cambridge, MA), at aconcentration of 50 nM, for 24 h, 48 h and 72 h.

Immunofluorescence and confocal microscopy

For light immunocytochemistry teased skeletal myofibers, 1 μmthick semithin sections and cultured myoblasts were processed forimmunofluorescence. Then, the samples were sequentially treatedwith 0.5% Triton X-100 in PBS for 15 min, 0.1 M glycine in PBS con-taining 1% bovine serum albumin (BSA) for 30 min and 0.01%Tween 20 in PBS for 5 min. The samples were incubated for 3 h withthe primary antibody containing 1% BSA at room temperature,washed with 0.01% Tween 20 in PBS, incubated for 45 min in the

120 R. Bengoechea et al. / Neurobiology of Disease 46 (2012) 118–129

specific secondary antibody conjugated with FITC, TexasRed or Cy5(Jackson, USA), washed in PBS and mounted with the antifading me-dium Vectashield (Vector, USA). Some samples were stained withphalloidin-FITC (Sigma, UK) for the demonstration of actin filaments.

The following primary antibodies were used in this study: rabbitpolyclonal serum anti-PABPN1 (Krause et al., 1994); mouse polyclonalserum raised against recombinant human PABPN1 (Berciano et al.,2004); mouse monoclonal antibodies anti-2,2,7-trimethylguanosinecap (TMG-cap) of spliceosomal snRNPs (Oncogene Research, USA),rabbit polyclonal anti-desmin (DAKO) and human autoimmuneserum anti-Sm complex of spliceosomal snRNPs.

Samples were examined with a LSM 510 laser scanning microscope(Zeiss, Germany) equipped with an argon (488 nm), HeNe (543 nm)and HeNe (633 nm) ion lasers and using a 63× immersion oil objective(1.4 NA). For double labeling experiments, images of the same confocalplane were sequentially recorded and pseudocolor images were gener-ated and superimposed. TIFF images were transferred to Adobe Photo-shop 7.0 software (Adobe Systems Inc.) for presentation. The surfacearea of the PABPN1-positive INIs and the fluorescence intensity ofPABPN1 signal in the nuclear speckles were estimated by using theImage J software for image analysis (NIH, Bethesda, Maryland, USA;http://rsb.info.nih.gov/ij/).

In situ hybridization for poly(A) RNAs

In situ hybridization was performed on cultured myoblasts fixedwith 3.7% paraformaldehyde following the procedure previously de-scribed (Berciano et al., 2004). We used a biotinylated 2′-O-alkyl oli-goribonucleotide poly (U) containing 20 tandem uridine residuesprovided by A. I. Lamond (University of Dundee, Scotland) or a bioti-nylated poly(dT) probe as previously described (Berciano et al.,2004). Briefly, samples were rinsed in 6× SSPE (0.9 M NaCl, 0.06 MNaH2PO4, 6 mM EDTA, pH 7.4) containing 0.01% Tween 20 and incu-bated for 30 min in 10 μl of 0.5 mg/ml tRNA, 6× SSPE and 5× Den-hardt's. Hybridization was performed by adding 10 μl of the poly(dT) or poly (U) probe diluted to 2 pM/μl in 6× SSPE, 5× Denhardt's.Hybridization was carried out for 1 h at room temperature in a humidchamber. After washing (3×15 min each in 6× SSPE; 2× for 5 mineach in 4× SSC, 0.1% Tween 20) the hybridization signal was detectedwith FITC-avidin. All samples were mounted with Vectashield (Vec-tor, USA). Some samples were processed for double labeling experi-ments combining poly(A) RNA detection with immunofluorescencewith either anti-PABPN1 or anti-TMG-cap antibodies. All sampleswere mounted with Vectashield (Vector, USA).

Immunogold electron microscopy

For electron microscopy immunocytochemistry, muscle fragmentsfrom control and OPMD patients were dehydrated in increasing con-centrations of methanol (at −20 °C) and embedded in Lowicryl K4Mat −20 °C. Ultrathin sections were sequentially incubated with 0.1 Mglycine in PBS (15 min), 1% normal goat serum in PBS (5 min), andthe primary antibodies (rabbit polyclonal and mouse polyclonalanti-PABPN1 indicated above) diluted in PBS containing 0.1 M glycineand BSA (1 h at room temperature). After washing, the sections wereincubated with the specific secondary antibody conjugated with10 nm gold particles (BioCell, UK) diluted 1.25 in BSA in PBS(45 min at room temperature). After washing, the sections werestained with uranyl acetate and citrate lead. As controls, sectionswere treated as described but without the primary antibody.

Expression of fusion protein

The GFP-PABPN1-wt construct, which contains the cDNA encod-ing PABPN1, and the GFP-PABPN1-ala17 construct, encoding aPABPN1 mutant with a stretch of 17 alanines instead of 10, have

been previously described (Calado et al., 2000: Tavanez et al., 2005).Both DNA constructs were verified by sequencing. Plasmids were pu-rified using the Qiagen plasmid DNA midi kit. Primary cultures ofmyoblasts were transferred into a 24-well plate 24 h after isolationand transfected using microporation (Microporator MP100, DigitalBio, Labtech, France) with either pEGFP-wtPABPN1 or pEGFP-expPABPN1 plasmids using a standard program: 1 pulse of 1200 V,30 ms with 0.5 μg of plasmid for 5.105 cells. Transfection efficiencywas monitored at 24 h by analyzing GFP-positive cells under a fluo-rescent microscope.

Live-cell imaging in real time

Live-cell imaging was performed using a Nikon TI microscope(Nikon Instruments, Inc, Japan) with a CFI Plan Fluor 40× (0.6 NA)objective UV equipped with an autofocus system and camera ORCAR2 (Hamamatsu, Japan). After 8 h post-transfection with eitherGFP-PABPN1wt or GFP-PABPN1-17ala constructs, myoblasts wereplated onto glass-bottomed 35 mM μ-dishes (Ibidi, Germany) and,prior to imaging, the cell culture medium was replaced with freshphenol red-free medium. Cells were maintained at 37 °C in atemperature-controlled microscope stage incubator (OKOLAB,Italy). Cells with reduced expression levels of fusion proteins and ini-tial labeling of nuclear speckles were selected for live-cell imaging.Images were captured every 15 min for 24 h and analyzed usingthe Nikon software for live-cell imaging (Nikon Instruments, Inc,Japan) and Image J (NIH, USA). Three transfection experimentswith either wild type or expanded PABPN1 constructs wereperformed.

Protein extraction and Western blot analysis

After 24 h post-transfection, HEK-293 cells were harvested incold Buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2)supplemented with 1 μg/ml leupeptin, 1 μg/ml aprotinin and0.5 mM phenylmethylsulfonyl fluoride (PMSF) and containing 2.5%Nonidet P-40 for 30 min on ice. The lysate was centrifuged at3500 rpm for 4 min at 4 °C. Supernatants were taken as a cytosolicfraction. Pellets (nuclei) were incubated on ice in Buffer C (20 mMHEPES, pH 7.9, 0.45 M NaCl, 1 mM EDTA) supplemented with 1 μg/ml leupeptin, 1 μg/ml aprotinin and 0.5 mM PMSF for 20 min. Cellu-lar debris was removed by centrifugation at 14,000 rpm for 10 min at4 °C, and the supernatant was taken as a nuclear fraction. Equalamounts of nuclear proteins were separated by electrophoresis on10% SDS-PAGE before being transferred to nitrocellulose mem-branes. The membranes were immunodetected using a rabbit mono-clonal antibody directed against PABP2 (1:5000) (Epitomics, USA),and a rabbit IgG fraction antibody directed against GFP (1:1000)(Invitrogen). Immunoblots were developed using Li-Cor IRDye 800and 680 (Rockland)-labeled antibodies with the Odyssey infraredimaging system (Li-Cor).

Results

Organization of PABPN1 nuclear inclusions in OPMD patients

Genetic analysis showed that the mutation responsible for OPMDin both patients was the expansion from (GCG)6 to (GCG)11. The sub-cellular distribution of PABPN1 was investigated in teased myofibersof muscle biopsies from controls and OPMD patients. Double labelingfor polymerized actin with phalloidin-FITC and immunostaining forPABPN1 in control myofibers revealed a diffuse nucleoplasmic pat-tern of PABPN1 in addition to being concentrated in nuclear speckles(Fig. 1A). Myofibers of the OPMD patients showed the typical pres-ence of one or, more frequently, a few INIs intensely stained withthe polyclonal anti-PABPN1 in some myonuclei of both patients

Fig. 1. A–D. Confocal images of PABPN1 immunolabeling in teased muscle fibers from control (A) and OPMD patients. A–C. Double staining for PABPN1 and actin (Phalloidin-FITC) ofa control myonucleus containing PABPN1-positive nuclear speckles in addition to a diffuse nucleoplasmic signal (A), and two OPMD myonuclei harboring INIs Scale bar=5 μm. D.Large PABPN1-positive INIs (arrows) coexist with several smaller nuclear microfoci (arrowheads) of PABPN1 immunoreactivity (Scale bar=3). E–G. Immunogold electron micros-copy localization of PABPN1 in INIs from OPMD patients. Scale bar=50 nm. E. Small size INI. F. The INI appears as a cleared nucleoplasmic area surrounded by chromatin domains.Gold particles of PABPN1 immunoreactivity are concentrated within the INI composed of a network of tubular filaments. G. Detail of an INI showing the combination of dense amor-phous aggregates and interconnecting tubular filaments. Gold particles decorate both components. Scale bars: E=200nm, F=175nm, G=90nm. P1 and P2=patients 1 and 2.

121R. Bengoechea et al. / Neurobiology of Disease 46 (2012) 118–129

(Figs. 1B and C), as has been previously reported (Calado et al.,2000). Additionally, INI-containing myonuclei showed a diffuse nu-cleoplasmic distribution of PABPN1 excluding irregular areas freeof immunoreactivity (Figs. 1B and C). Intranuclear PABPN1 aggre-gates had an irregular shape and variable size, ranging from smallfoci of 0.1 μm2 to large well defined INIs, up to 7 μm2 (Figs. 1D and2J).

By immunoelectron microscopy with anti-PABPN1 antibodies, theultrastructural counterparts of small foci detected with immunofluo-rescence were seen to correspond to small interchromatin domainscomposed of scattered tubular filaments interspersed with smallmasses of chromatin (Fig. 1E). As far as large inclusions were

concerned, their fine structure consisted of a network of loosely ar-ranged tubular filaments distributed in a cleared interchromatin do-main, as initially reported by Tomé and Fardeau (1980), which weredecorated with immunogold particles (Fig. 1F). Interestingly, highmagnification analysis revealed that the majority of large INIs wereorganized as regular arrays of small dense masses of amorphous ma-terial interconnected by the characteristic short tubular filaments thatirradiated from the amorphous material (Fig. 1G). Both components,tubular filaments and amorphous material, were decorated withgold particles (Fig. 1G). The immunolabeling of the amorphousdense material indicates the existence of an unstructured fraction ofPABPN1 that is not assembled into tubular filaments.

Fig. 2. A–L. Confocal images of double immunolabeling experiments for PABPN1 and TMG-cap, a marker of snRNP splicing factors, in skeletal muscle fibers from control (A–C) andOPMD patients (D–L). A–C. In control fibers PABPN1 and snRNPs colocalized in typical nuclear speckles. D–L. Muscle fibers of OPMD patients contain typical INIs intensely immu-nolabeled for PABPN1 that appear closely associated with nuclear speckles which, though immunoreactive for snRNPs, are free of, or have only a weak, PABPN1 immunofluores-cence. Scale bar=5 μm. M. Immunogold electron microscopy of a OPMD patient's muscle fiber showing a typical INI decorated with gold particles of PABPN1 immunoreactivityand associated with an interchromatin granule cluster (IGC). Note the paucity of gold particles in the IGC. N. Interchromatin granule cluster (IGC) of a control myonucleus decoratedwith numerous gold particles of PABPN1 immunoreactivity. Scale bar=200 nm.

122 R. Bengoechea et al. / Neurobiology of Disease 46 (2012) 118–129

Mutated PABPN1-positive INIs associated with nuclear speckles ofsplicing factors in myonuclei of OPMD patients

Since both endogenous wild type PABPN1 and the fusion proteinGFP-PABPN1wt co-localize with the splicing factor SC35 in nuclearspeckles of human HeLa, A549-tTA and U2OS cells (Calado et al.,2000; Krause et al., 1994; Raz et al., 2011; Sasseville et al., 2006),

we investigated whether this co-localization is preserved in myonu-clei from both control and OPMD patients, and the possible spatialrelationship between INIs and nuclear speckles. Co-staining forTMG-cap of snRNAs, a component of spliceosomal snRNPs thatserves as a marker of nuclear speckles (Lamond and Spector, 2003),and PABPN1 in myonuclei from human controls revealed that bothmolecular components concentrate and co-localize in typical nuclear

123R. Bengoechea et al. / Neurobiology of Disease 46 (2012) 118–129

speckles, with a weaker signal apparent throughout the nucleoplasmexcluding the nucleolus (Figs. 2A–C). In myonuclei from OPMD pa-tients the speckle pattern of PABPN1 immunostaining disappeared,although typical nuclear speckles enriched in snRNP splicing factorswere well preserved (Figs. 2D–L). Moreover, INIs from OPMD pa-tients did not concentrate snRNP splicing factors, but appearedclosely associated with nuclear speckles intensely immunolabeledfor these factors (Figs. 2F, I and L). Quantitative analysis revealedthat 86.7%±7.4% (M±SD, n=92) of INIs from both patients werephysically associated with nuclear speckles immunolabeled forsnRNP splicing factors. Immunogold electron microscopy of myonu-clei from OPMD patients confirmed the direct spatial association be-tween interchromatin granule clusters, the ultrastructuralequivalent of nuclear speckles at electron microscopy level(Lamond and Spector, 2003), and INIs (Fig. 2M). As shown inFig. 2M, whereas the INI appeared clearly delineated and specificallydecorated with numerous gold particles of PABPN1 immunoreactiv-ity, only a few scattered gold particles were found in the associatedinterchromatin granule cluster, indicating that expPABPN1 was notconcentrated in this nuclear domain. In contrast, interchromatingranule clusters were clearly decorated with numerous gold parti-cles in control myonuclei (Fig. 2N).

The formation of INIs in human myoblasts expressing GFP-PABPN1wt orGFP-PABPN1-17ala occurs in nuclear speckles, which are progressivelydepleted of PABPN1 proteins and poly(A) RNA

Since PABPN1 inclusions constitute a pathological hallmark ofOMPD, our aim was to further investigate the dynamic interrelation-ships between nuclear speckles and intranuclear PABPN1 inclusionsin human myoblasts, the main target cells of OPMD pathology(reviewed in Brais, 2009). To do this, we performed transfection ex-periments in primary human myoblasts with GFP-PABPN1wt andGFP-expPABPN1 constructs (Calado et al., 2000c; Tavanez et al.,2005), the latter encoding a PABPN1 mutant with a stretch of 17 ala-nines instead of 10, an expansion that has been described in OPMDpatients (Brais et al., 1998). The integrity of both fusion proteinswas confirmed by Western blotting using anti-GFP and anti-PABPN1antibodies (Fig. 3A).

The myoblast phenotype in transfected cell expressing PABPN1constructs was confirmed with immunostaining for desmin, an inter-mediate filament protein of muscle fibers (Fig. 3B). Double labelingfor the Sm complex proteins of spliceosomal snRNPs, as a marker ofnuclear speckles, and poly(A) RNA in myoblasts expressing low levelsof either GFP-PABPN1wt or GFP-PABPN1-17ala constructs revealedthat, in the absence of PABPN1-positive INIs, both fusion proteinswere concentrated in typical nuclear speckles enriched in Sm com-plex proteins and poly(A) RNA (Figs. 3C–J). Additionally, we observeda diffuse nuclear co-localization of spliceosomal snRNPs and ectopicPABPN1 proteins throughout the nucleus (Figs. 3C, D, G and H), sup-porting that a fraction of these molecular components is engaged inco-transcriptional pre-mRNA processing at active transcription sites.

As occurred in OPMD myofibers, the INIs induced in cultured myo-blasts expressing either GFP-PABPN1wt or GFP-PABPN1-17ala did notconcentrate snRNP splicing factors over the nucleoplasmic level andremained closely associated with typical nuclear speckles, immunola-beled for snRNPs, in all stages of their formation (Figs. 4A–R). Thus,both nuclear microfoci of 0.1 to 0.2 μm2, identified as precursors of INIs,and small rounded INIs (0.3 to 0.75 μm2) appeared as sharply definedrounded structures located at the edge of nuclear speckles and containingthe highest accumulation of PABPN1 proteins (Figs. 4A–L). Interestingly,nuclear speckles concentrated PABPN1 and Smcomplex proteins inmyo-blasts containing precursors of inclusions and small INIs (Figs. 4A–L). Incontrast, nuclear speckles failed to accumulate PABPN1 fusion proteinsin myoblasts harboring irregularly shaped and large-size INIs, althoughthe speckles remained closely associated with INIs (Figs. 4M–R). This

suggests that the continuous growth of INIs progressively depletes nucle-ar speckles of PABPN1 proteins. Nextwe estimated the proportion of pre-INIs and INIs associated with nuclear speckles in myoblasts expressingeither GFP-PABPN1wt (n=60) or GFP-PABPN1-17ala (n=60) andimmunostained for the Sm complex. Approximately 99% of pre-INIswere associated with nuclear speckles in both myoblasts expressingwild-type and expanded PABPN1 (99.1±1.7 and 98.9±2.0, respectively,values are mean±SD). Moreover, all INIs remained associated with nu-clear speckles.

We next investigated whether the progressive PABPN1 depletionfrom nuclear speckles in myoblasts containing INIs was accompaniedby the loss of snRNAs and poly(A) RNAs, two categories of RNAenriched in nuclear speckles (Hall et al., 2006; Lamond and Spector,2003). We performed immunolabeling for the TMG-cap of snRNAsin combination with in situ hybridization for poly(A) RNA with thepoly(dT) probe. The highest accumulation of poly(A) RNA wasdetected in INIs in all stages of their formation (Figs. 5A–X), in agree-ment with the previous localization of poly(A) RNA in INIs of bothOPMD patients and rat supraoptic neurons (Calado et al., 2000;Berciano et al., 2004). In myoblasts containing INIs typical nuclearspeckles enriched in spliceosomal snRNAs were preserved (Fig. 5),but the intensity of the fluorescent signals for poly(A) RNAs andGFP-tagged fusion proteins in these speckles progressively dimin-ished as the growth of INIs proceeded (Figs. 5A–X). In fact, the specklepattern of both poly(A) RNA and PABPN1 proteins, detected in myo-blasts free of INIs (Figs. 3C–J), tended to disappear in myoblasts con-taining large INIs (Figs. 5Q–X). To determine whether the progressiveloss of PABPN1 proteins from nuclear speckles correlates with the INIsize, the surface areas of INIs were plotted against fluorescence inten-sities of GFP-PABPN1wt and GFP-PABPN1-17ala proteins measuredin nuclear speckles. As shown in Fig. 5Y, there was a linear negativerelationship between these two parameters, with correlation coeffi-cients (r2) of 0.756 (GFP-PABPN1wt) and 0.748 (GFP-PABPN1-17ala). This confirms the existence of a significant inverse correlation(pb0.05) between the size of INIs and concentration of PABPN1 pro-teins within nuclear speckles. Moreover, differences in the aggrega-tion process of the wild-type and expanded PABPN1 into INIs weredetected. Thus, at 12 h and 24 h post-transfection, GFP-PABPN1-17ala was more prone to aggregation in the majority of myoblaststhan was its wild-type counterpart (Fig. 5Z). Since previous studieshave demonstrated the involvement the ubiquitin-proteasome sys-tem in the nuclear aggregation of PABPN1 (Anvar et al., 2011; Abu-Baker et al., 2003; Brais, 2009), we investigated whether the expan-sion of the polyalanine tract facilitates protein aggregation inuntransfected OPMD myoblasts treated with the proteasome inhibi-tor bortezomib. Interestingly, approximately 7% of untreated myo-blasts from OPMD exhibited PABPN1-positive INIs after threeweeks of culture, whereas INIs were absent in control myoblasts(Figs. 6E and I). Bortezomib treatment (50 nM) for 24 h, 48 h and72 h caused accumulation of both expanded and wild-type PABPN1in INIs, but the fraction of cells with INIs was significantly higher inOPMD myoblasts (Fig. 6). Importantly, the pattern of formationof INIs associated with nuclear speckles was similar to thatshown in myoblasts transfected with the PABPN1 constructs. Thus,the pre-INIs appeared at the edge of PABPN1-positive nuclearspeckles (Figs. 6B, C and F), whereas this speckle pattern of PABPN1proteins tended to disappear in myoblasts harboring large INIs(Fig. 6H).

To further confirm the nuclear speckles as biogenesis sites of INIs,we performed time-lapse experiments in myoblasts expressing eitherGFP-PABPN1wt or GFP-PABPN1-17ala. GFP fluorescence was moni-tored in real-time microscopy for 24 h. As a starting point for imagingwe selected cells with a weak labeling of nuclear speckles and lackingin INIs. As is shown in Fig. 7, both fusion proteins aggregate into INIsat the nuclear speckles, but the myoblasts expressing the expandedPABPN1 exhibited higher aggregation kinetics than the myoblasts

Fig. 3. A. Western blot analysis of the expression of PABPN1 proteins in HeLa cells transfected with either GFP-PABPN1wt (10ala) or GFP-PABPN1-17ala constructs detected withrabbit polyclonal antibodies anti-PABPN1 and anti-GFP. B. A myoblast transfected with the GFP-PABPN1wt construct and immunolabeled for desmin shows a nuclear speckle dis-tribution pattern of this fusion protein. C–J. Combination of immunolabeling for the Sm complex of spliceosomal snRNPs and in situ hybridization for poly(A) RNAs in myoblastsexpressing GFP-PABPN1wt (C–F) or GFP-PABPN1-17ala (G–J). Note the co-localization of the fusion proteins and poly(A) RNA in nuclear speckles intensely immunostained for spli-ceosomal snRNPs, as well as the absence of PABPN1-positive INIs. Scale bar=5 μm.

124 R. Bengoechea et al. / Neurobiology of Disease 46 (2012) 118–129

expressing the wild type protein. The average appearance times ofpre-INIs, visualized as bright microfoci at the nuclear speckles, in cul-tured myoblasts were 7.8 h±0.7 h and 15.0 h±1.5 h for GFP-PABPN1-17ala (n=30) and GFP-PABPN1wt (n=30), respectively(Fig. 7 and Video 1; pb0.001, ANOVA single factor analysis). The pro-gressive growth and fusion of small nuclear foci resulted in the for-mation of INIs, which were more prominent (at 24 h post-transfection) in myoblasts transfected with the GFP-PABPN1-17alaconstruct (Fig. 7 and Video 1, supplementary material). Interestingly,the formation of INIs was accompanied by the progressive disappear-ance of the nuclear speckle labeling for both PABPN1 fusion proteins,resulting in a diffuse fluorescence throughout the nucleoplasm(Fig. 7).

Discussion

Our study in OPMD skeletal myofibers demonstrates the existenceof microfoci of PABPN1 immunoreactivity, in addition to the largerPABPN1-positive INIs previously described (Calado et al., 2000). Wepropose that these microfoci correspond to precursors of INIs at an

initial stage of PABPN1 aggregation. Curiously, the presence in matureINIs of small dense masses of amorphous material immunoreactivefor PABPN1, together with the typical tubular filaments (Calado etal., 2000; Tomé and Fardeau, 1980), indicate that a fraction of mutantPABPN1 is not assembled into tubular filaments. What is noteworthy,however, is the common and specific spatial association of INIs withnuclear speckles in the OPMDmuscle fibers, suggesting the participa-tion of nuclear speckles in the biogenesis of INIs. We have previouslyobserved a similar association in supraoptic neurons expressing nor-mal PABPN1 under physiological conditions (Villagrá et al., 2008), in-dicating that the specific spatial relationship between INIs andnuclear speckles is independent of the polyalanine expansion.

The presence of INIs in skeletal myofibers is a pathological hall-mark of the OPMD, although there is controversy over the contribu-tion of nuclear aggregates of expPABPN1 to the pathogenesis of thedisease (Abu-Baker and Rouleau, 2007; Brais, 2009; Mankodi et al.,2012). OPMD is an autosomal dominant disorder in which the mutantallele of PABPN1 replaces the normal allele of PABPN1, suggesting adominant-negative effect of the mutant PABPN1. In this regard, a re-cent molecular genetic study on muscle tissue of OPMD patients has

Fig. 4. Immunostaining for the SM complex in myoblasts expressing either GFP-PABPN1wt or GFP-PABPN1-17ala and containing INIs. A–F. The expression of both wild type andexpanded PABPN1 induces the initial aggregation of these fusion proteins in nuclear spots, precursor of INIs, located at the edge of nuclear speckles. These latter containPABPN1 protein and accumulated spliceosomal snRNPs. G–L. At a more advanced stage of PABPN1 aggregation, rounded INIs appear closely associated with nuclear speckles.M–R. Myoblasts containing irregular and larger INIs, where the association between inclusions and nuclear speckles is preserved, but the latter do not concentrate PABPN1 proteins.Scale bar=5 μm.

125R. Bengoechea et al. / Neurobiology of Disease 46 (2012) 118–129

shown a significantly higher amount of RT-PCR products of thePABPN1 mRNA transcribed from the mutant allele than from the nor-mal allele (Schröder et al. 2011). Thus, the loss of function of the nor-mal allele of PABPN1 and/or the gain of function of the mutant allele

could be involved in the pathogenesis of OPMD (Apponi et al.,2011). Moreover, these gene dysfunctions may affect the essentialrole of PABPN1 in pre-mRNA processing and export, cellular mecha-nisms that require the participation of nuclear speckles (Hall et al.,

Fig. 5. A–X. Double labeling experiments for TMG-cap and poly(A) RNA in myoblasts expressing either GFP-PABPN1wt or GFP-PABPN1-17ala and containing PABPN1 aggregates.Note the progressive reduction of the PABPN1 and poly(A) RNA signals in nuclear speckles in parallel with the increase in the INI size. Scale bar=5 μm. Y. Quantitative analysis offluorescence intensity of PABPN1 proteins in nuclear speckles and INI size indicates a negative relationship between these two parameters. Z. At 24 h and 48 h post-transfection, asignificant increase in the proportion of transfected myoblasts containing INIs is observed when GFP-PABPN1-17ala is expressed, in comparison to GFP-PABPN1wt expression(n=50, for each of transfected groups of myoblasts). Values are mean±SD. p*b0.001, **pb0.001 versus myoblasts expressing GFP-PABPN1wt, ANOVA single factor analysis.

126 R. Bengoechea et al. / Neurobiology of Disease 46 (2012) 118–129

2006; Melcák et al., 2001; Politz et al., 2006) commonly associatedwith INIs in OPMD myonuclei (present results). Accordingly, deple-tion of PABPN1 in mouse myoblast impacts dramatically uponmRNA biogenesis and export, and leads to defects in cell proliferationand differentiation (Apponi et al., 2010).

Previous studies have shown that the nuclear targeting of mutatedPABPN1 is essential to form aggregates and to confer OPMD-associated cellular toxicity (Abu-Baker et al., 2005; Abu-Baker andRouleau, 2007; Brais, 2009). Within the nucleus, wild type PABPN1concentrates in nuclear speckles in HeLa and U2OS cells (Krause et

Fig. 6. Effects of proteasome inhibition with bortezomib on the nuclear accumulation of PABPN1 in control (A–D) and OPMD (E–H) myoblasts. Immunostaining with the anti-PABPN1 antibody. Whereas the untreated control exhibits the typical PABPN1 distribution in nuclear speckles diffusely throughout the nucleoplasm (A), a reduced fraction ofOPMD myoblasts shows INIs (arrowhead in E). Treatment with bortezomib induces the formation of pre-INIs associated with nuclear speckles (arrowheads) in both control (B–D) and OPMD myoblasts (F–H). Note in H that the formation of large INIs is associated with the disappearance of the nuclear speckle pattern of PABPN1 immunostaining. I. Pro-portion of control and OPMD myoblasts containing pre-INIs and INIs at different time points of bortezomib treatment (n=100 for each group of myoblasts). Values are mean±SD. p*b0.001versus control myoblasts at each time point studied, ANOVA single factor analysis.

Fig. 7. A. Time-lapse experiment in a myoblast expressing either GFP-PABPN1-17ala or GFP-PABPN1wt constructs. As a starting point for imaging we select the initial expression offusion proteins in nuclear speckles (0 h). PABPN1 pre-INIs are detected at 8 h and 14 h in myoblasts expressing GFP-PABPN1-17ala and GFP-PABPN1wt, respectively. Note that theformation of these pre-INIs is associated with nuclear speckles (arrowheads). At 24 h post-transfection, INIs are more prominent in myoblasts expressing the expanded PABPN1.Scale bar=5 μm. B. Distribution of the appearance times of pre-INIs in myoblast expressing GFP-PABPN1-17ala (n=30) and GFP-PABPN1wt (n=30).

127R. Bengoechea et al. / Neurobiology of Disease 46 (2012) 118–129

128 R. Bengoechea et al. / Neurobiology of Disease 46 (2012) 118–129

al., 1994; Messaed et al., 2007; Raz et al., 2011; Tavanez et al., 2005), alocalization dependent on its binding to poly(A) RNA (Calado andCarmo-Fonseca, 2000). Our study extends this finding to nuclearspeckles of normal skeletal muscle fibers and human cultured myo-blasts. In the latter cells expressing either PABPN1wt or PABPN1-17ala, we demonstrated that both fusion proteins completely co-localized with poly(A) RNA in nuclear speckles before the formationof INIs, supporting that most, if not all, wild type and mutantPABPN1 proteins resident in nuclear speckles are bound to thepoly(A) tail of polyadenylated RNAs. The complete overlap ofexpPABPN1 and speckles observed in myoblasts differs from resultspreviously published by Messaed et al. (2007) in HeLa cells expres-sing GFP-expPABPN1 (18-ala). In their study, expPABPN1 only par-tially overlap with nuclear speckles, suggesting the existence ofnuclear domains of exclusive distribution for either expPABPN1 orSC35, a marker of nuclear speckles (Hall et al., 2006). This apparentdiscrepancy may be due to cell-specific differences in the organiza-tion of nuclear speckles and expression patterns of PABPN1 proteins.

Also of note is the fact that the formation of INIs and their associationwith nuclear speckles in human myoblasts transfected with the GFP-PABPN1 constructs occur under conditions of very low expression ofboth fusion proteins. This reasonably rules out the formation of INIs asan artifact of over-expression. Indeed, wild type PABPN1 forms INIs insupraoptic (Berciano et al., 2004) and sensory ganglion neurons(unpublished results) under physiological conditions. Although bothGFP-PABPN1wt and GFP-PABPN1-17ala fusion proteins aggregate intoINIs in human myoblasts, our quantitative analysis of the proportionof cells containing INIs and the time-lapse study in living myoblasts in-dicate that the expansion of the polyalanine tract enhances the aggrega-tion kinetics of PABPN1 into INIs. Similarly, the inhibition of proteasomein untransfected myoblasts from OPMD results in a higher accumula-tion of expanded PABPN1 into INIs, as compared with wild type myo-blasts. Interestingly, the bortezomib-induced formation of INIs occursat the nuclear speckles, providing additional evidence for the involve-ment of nuclear speckles in the biogenesis of PABPN1 inclusions.Taken together, these results also support the view that the expansionof the polyalanine tract in mutant PABPN1 facilitates protein aggrega-tion (Abu-Baker and Rouleau, 2007; Bao et al., 2004; Fan et al., 2001)and underlie the involvement of the UPS in OPMD (Anvar et al., 2011;Abu-Baker et al., 2003).

Our results in human myoblasts transfected with the GFP-PABPN1constructs show that the edges of nuclear speckles are the biogenesissites of INIs, although expanded PABPN1 exhibits faster aggregationkinetics than the wild type. This suggests that the nuclear speckle sur-face provides the optimal environment for PABPN1 aggregation.Speckles have the highest nuclear concentration of poly(A) RNA andPABPN1, presumably as PABPN1/poly(A) complexes, and this macro-molecular crowding may provide the environment needed for oligo-merization of PABPN1 proteins by increasing the rate and specificityof molecular interactions. Furthermore, the specific formation ofINIs at the periphery of nuclear speckles suggests that the nucleationmechanism may be related to the organization of genome aroundspeckles. More specifically, it is well known that nuclear specklesare formed in close proximity to certain protein-coding genes, a local-ization that may keep splicing factors concentrated near nascent tran-scripts (Hall et al., 2006; Rino and Carmo-Fonseca, 2009). In thiscontext, we propose that mRNA transcripts of active genes localizedat the edge of nuclear speckles may be implicated in the initial nucle-ation of INIs. In agreement with this view, recent live-cell imagingstudies provide compelling evidence that specific RNA transcriptscan function as a scaffold that recruits proteins which interact withRNA to assemble nuclear structures such as nuclear speckles, para-speckles, histone locus bodies and nuclear stress bodies (Mao et al.,2011; Shevtsov and Dundr, 2011).

Importantly, our results indicate that nuclear speckles have a re-duced concentration of mutant PABPN1 in INI-containing myonuclei

of OPMD patients in comparison with nuclear speckles of controlmyofibers. This finding is consistent with the nuclear redistributionof PABPN1 to INIs observed in HeLa and U2OS cells expressingPABPN1 constructs (Tavanez et al., 2005; Raz et al., 2011). It also sup-ports our view that the formation of INIs in myonuclei of OPMD pa-tients produces a depletion of mutant PABPN1 in speckles, but doesnot affect the existence of these splicing factor compartments,which maintain the ability to recruit snRNP splicing factors. In fact,typical nuclear speckles immunolabeled for snRNPs are preserved inall stages of the formation of INIs in our cellular model of culturedmyoblasts. Interestingly, in addition to PABPN1 proteins, the growthof INIs in cultured myoblasts depletes poly(A) RNA in nuclearspeckles. We believe that growing INIs retain poly(A) RNA andPABPN1, which would otherwise be trafficking in nuclear speckles.Indeed, photobleaching studies in living cells have shown that nucle-ar speckles concentrate a population of mobile poly(A) RNAs in con-tinuous flux with the nucleoplasm (Molenaar et al., 2004; Politz etal., 2006), suggesting that a fraction of these polyadenylated RNAsare involved in post-transcriptional processing of pre-mRNAs beforetheir nuclear export (reviewed in Hall et al., 2006; Melcák et al.,2000, 2001). In this context, we propose that the dynamic aggrega-tion of poly(A) RNA/PABPN1 complexes into growing INIs graduallydepletes the nuclear speckles of these molecular components, there-by interfering with the normal trafficking and post-transcriptionalprocessing of polyadenylated mRNAs in speckles.

Given the great importance of nuclear speckles in nuclear physiol-ogy (Hall et al., 2006; Melcák et al., 2000; Spector and Lamond, 2011),their dysfunction may disturb pre-mRNA processing in skeletal myo-fibers contributing to the molecular pathophysiology of OPMD. Inter-estingly, a dysfunction of nuclear speckles has also beendemonstrated in myotonic dystrophy type I (DMI) caused by the mu-tation in the DMPK (dystrophia myotonica protein kinase) gene(Harley et al., 1992). The DMPK gene locus localizes at the edge of nu-clear speckles and the normal DMPK mRNA exhibits a transcriptiondependent accumulation in nuclear speckles (Smith et al., 2007). InDMI, the intranuclear path of DMPKmRNA is altered andmutant tran-scripts do not enter nuclear speckles, but rather accumulate in intra-nuclear granules (Smith et al., 2007). Taken together, these studieslead us to view nuclear speckles as new cellular targets in the molec-ular pathophysiology of neuromuscular disorders.

There is some evidence that supports that the INIs in OPMD have aprotective role, and cell death has been shown to be mediated by thesoluble form of expanded PABPN1 (Messaed et al., 2007; Messaed andRouleau, 2009). However, our results are consistent with a scenario inwhich a loss-of-function mechanism, mediated by the depletion ofPABPN1 from nuclear speckles and consequent dysfunction of pre-mRNA processing, contributes to OPMD pathogenesis. In support ofthis view, a previous study has demonstrated that the depletion ofPABPN1 using siRNA in mouse myoblast impacts dramatically uponmRNA biogenesis and export (Apponi et al., 2010).

In conclusion, the molecular reorganization of nuclear specklesand their implication in biogenesis and maintenance of PABPN1 in-clusions in skeletal myofibers may cause a severe dysfunction in nu-clear trafficking, processing and export of polyadenylated mRNAs,which contributes to the molecular pathology of OPMD. Our observa-tions emphasize the potential importance of nuclear speckles as nu-clear targets of neuromuscular disorders.

Supplementary data to this article can be found online at doi:10.1016/j.nbd.2011.12.052.

Acknowledgments

The authors wish to thank R. García-Ceballos, Saray Pereda andFidel Madrazo for technical assistance, and Dr Jon Infante for provid-ing clinical data from patient 2. We are also grateful to Prof. E. Wahle(University of Halle, Germany) for generously providing anti-PABPN1

129R. Bengoechea et al. / Neurobiology of Disease 46 (2012) 118–129

rabbit serum and Prof. M. Carmo-Fonseca (Institute of MolecularMedicine, Lisbon) for mouse polyclonal anti-PABPN1 antibody. Thiswork was supported by the following grants: “Dirección General deInvestigación” (BFU2008-00175 and BFU2011-23983), “Centro deInvestigación Biomédica en Red sobre Enfermedades Neurodegenera-tivas” (CIBERNED; CB06/05/0037) Spain, and “Fundación Marqués deValdecilla” from Santander, Spain.

References

Abu-Baker, A., et al., 2003. Involvement of the ubiquitin-proteasome pathway andmolecularchaperones in oculopharingeal muscular dystrophy. Hum. Mol. Genet. 12, 2609–2623.

Abu-Baker, A., et al., 2005. Cytoplasmic targeting of mutant poly(A)-binding proteinnuclear 1 suppresses protein aggregation and toxicity in oculopharyngeal muscu-lar dystrophy. Traffic 6, 766–779.

Abu-Baker, A., Rouleau, G.A., 2007. Oculopharyngeal muscular dystrophy: recent ad-vances in the understanding of the molecular pathogenic mechanisms and treat-ment strategies. Biochem. Biophys. Acta 1172, 173–185.

Anvar, S.Y., et al., 2011. Deregulation of the ubiquitin-proteasome system is the predomi-nantmolecular pathology inOPMDanimalmodels andpatients. SkeletalMuscle 1, 15.

Apponi, L.H., et al., 2010. Loss of nuclear poly(A)-binding protein 1 causes defects inmyogenesis and mRNA biogenesis. Hum. Mol. Genet. 19, 1058–1065.

Apponi, L.H., et al., 2011. RNA-binding protein and gene regulation in myogenesis.Trends Pharmacol. Sci. 32, 652–658.

Bao, Y.P., et al., 2004. Congo red, doxycycline, and HSP70 overexpression reduce aggre-gate formation and cell death in cell models of oculopharyngeal muscular dystro-phy. J. Med. Genet. 41, 47–51.

Berciano, M.T., et al., 2004. Oculopharyngeal muscular dystrophy-like nuclear inclu-sions are present in normal magnocellular neurosecretory neurons of the hypo-thalamus. Hum. Mol. Genet. 13, 829–838.

Brais, B., et al., 1998. Short GCG expansions in the PABP2 gene cause oculopharyngealmuscular dystrophy. Nat. Genet. 18, 164–167.

Brais, B., 2009. Oculopharyngeal muscular dystrophy: a polyalanine myopathy. Curr.Neurol. Neurosci. Rep. 9, 76–82.

Calado, A., Carmo-Fonseca, M., 2000. Localization of poly(A)-binding protein 2 (PABP2)in nuclear speckles is independent of import into the nucleus and requires bindingto poly(A) RNA. J. Cell Sci. 113, 2309–2318.

Calado, A., et al., 2000. Nuclear inclusions in oculopharyngeal muscular distrophy con-sist of poly (A) binding protein 2 aggregates which sequester poly(A) RNA. Hum.Mol. Genet. 9, 2321–2328.

Carmo-Fonseca, M., 2002. The contribution of nuclear compartmentalization to generegulation. Cell 108, 513–521.

Cremer, T., Cremer, C., 2001. Chromosome territories, nuclear architecture and generegulation in mammalian cells. Nat. Rev. Genet. 2, 292–301.

Fan, X., et al., 2001. Oligomerization of polyalanine expanded PABPN1 facilitates nucle-ar protein aggregation that is associated with cell death. Hum. Mol. Genet. 10,2341–2351.

Hall, L.L., et al., 2006. Molecular anatomy of a speckle. Anat. Record. 288, 664–675.Harley, H.G., et al., 1992. Expansion of an unstable DNA region and phenotypic varia-

tion in myotonic dystrophy. Nature 355, 545–546.Klein, A.F., et al., 2008. PABPN1 polyalanine tract deletion and long expansions modify

its aggregation pattern and expression. Exp. Cell Res. 314, 1652–1666.Krause, S., et al., 1994. Immunodetection of poly(A)-binding protein II in the cell nucleus.

Exp. Cell Res. 214, 75–82.

Kühn, U., Wahle, E., 2004. Structure and function of poly(A) binding proteins. Biochim.Biophys. Acta 1678, 67–84.

Lafarga, M., et al., 2009. Cajal's contribution to the knowledge of the neuronal cell nu-cleus. Chromosoma 118, 437–443.

Lamond, A.I., Spector, D.L., 2003. Nuclear speckles: a model for nuclear organelles. Nat.Rev. Mol. Cell Biol. 4, 605–612.

Mankodi, A., et al., 2012. Progressive myopathy in an inducible mouse model of oculo-pharyngeal muscular dystrophy. Neurobiol. Dis. 45, 539–546.

Mao, Y.S., et al., 2011. Direct visualization of the co-transcriptional assembly of a nuclearbody by noncoding RNAs. Nat. Cell Biol. 13, 95–101.

Melcák, I., et al., 2000. Nuclear pre-mRNA compartmentalization: trafficking of re-leased transcripts to splicing factor reservoirs. Mol. Biol. Cell 11, 497–510.

Melcák, I., et al., 2001. Prespliceosomal assembly on microinjected precursor mRNAtakes place in nuclear speckles. Mol. Bio. Cell 12, 393–406.

Messaed, C., et al., 2007. Soluble expanded PABPN1 promotes cell death in oculophar-yngeal muscular dystrophy. Neurobiol. Dis. 26, 546–557.

Messaed, C., Rouleau, A.G., 2009. Molecular mechanisms underlying polyalanine diseases.Neurobiol. Dis. 34, 397–405.

Misteli, T., Spector, D.L., 2011. The Nucleus. Cold Sping Harbor Lab. Press, Cold SpringHarbor, New York.

Molenaar, C., et al., 2004. Poly(A)+RNAs roam the cell nucleus and pass through speckledomains in transcriptionally active and inactive cells. J. Cell Biol. 165, 191–202.

Politz, J.C., et al., 2006. Rapid, diffusional shuttling of poly(A) RNA between nuclearspeckles and the nucleoplasm. Mol. Biol. Cell 17, 1239–1249.

Raska, I., 1995. Nuclear ultrastructures associated with the RNA synthesis and processing.J. Cell. Biochem. 59, 11–26.

Raz, V., et al., 2011. Reversible aggregation of PABPN1 pre-inclusion structures. Nucleus2, 208–218.

Rino, J., Carmo-Fonseca, M., 2009. The spliceosome: a self-organized macromolecularmachine in the nucleus? Trends Cell Biol. 19, 375–384.

Sasseville, A., et al., 2006. The dynamism of PABPN1 nuclear inclusions during cellcycle. Neurobiol. Dis. 23, 621–629.

Schröder, J.M., et al., 2011. Oculopharyngeal muscle dystrophy: fine structure andmRNA expression levels of PABPN1. Clin. Neuropathol. 30, 94–103.

Shevtsov, S.P., Dundr, M., 2011. Nucleation of nuclear bodies by RNA. Nat. Cell Biol. 13,167–173.

Shopland, L.S., et al., 2003. Clustering of multiple specific genes and gene-rich R-bandsaround SC-35 domains: evidence for local euchromatic neighborhoods. J. Cell Biol.162, 981–990.

Smith, K.P., et al., 2007. Defining early steps in mRNA transport: mutant mRNA in myo-tonic dystrophy type I is blocked at entry into SC-35 domains. J. Cell Biol. 178,951–964.

Spector, D.L., Lamond, A.I., 2011. Nuclear speckles. Cold Spring Harb. Perspect. Biol. 3,a000646.

Tavanez, J.P., et al., 2005. In vivo aggregation properties of the nuclear poly(A)-bindingprotein PABPN1. RNA 11, 752–762.

Tavanez, J.P., et al., 2009. Hsp70 chaperones and type I PRMTs are sequestered at intra-nuclear inclusions caused by polyalanine expansions in PABPN1. PLoS One 4,e6418.

Tomé, F.M.S., Fardeau, M., 1980. Nuclear inclusions in oculopharyngeal dystrophy. ActaNeuropathol. 49, 85–87.

Villagrá, N.T., et al., 2008. Nuclear compartmentalization and dynamics of thepoly(A)-binding protein nuclear 1 (PABPN1) inclusions in supraoptic neuronsunder physiological and osmotic stress conditions. Mol. Cell. Neurosci. 37,622–633.

Wahle, E., 1991. A novel poly(A) binding protein acts as a specificity factor in the secondphase of messenger RNA polyadenylation. Cell 66, 759–768.