Polypyrimidine tract binding protein regulates alternative splicing of an aberrant pseudoexon in NF1 Michela Raponi 1 , Emanuele Buratti 2 , Miriam Llorian 3 , Cristiana Stuani 2 , Christopher W. J. Smith 3 and Diana Baralle 1 1 Human Genetics Division, University of Southampton, UK 2 Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy 3 Department of Biochemistry, University of Cambridge, UK Pseudoexons are intronic sequences that are approxi- mately the same length as exons (200 bp) with appar- ently viable donor and acceptor splice sites but which are not normally spliced in the mature mRNA tran- script. Despite the known abundance of exon splicing silencer regulatory elements within introns [1], it is not possible to formulate general rules for pseudoexon repression without remembering that splicing is very much dependent upon local context [2,3]. Normal exons need to encode the information for both protein synthesis and RNA splicing, so one might expect dif- ferences in pseudoexons with regard to several fac- tors, including the distribution of splice consensus sequences, splicing enhancers, splicing silencers and secondary structures. In line with these considerations, bioinformatics studies have observed enrichment of exon splicing silencer elements within pseudoexons relative to exonic splicing enhancer elements [4,5]. Fur- thermore, it has been suggested that it is defective splice sites rather than the lack of enhancers that account for non-splicing of pseudoexon sequences, although even perfect consensus sequences are not always adequate for correct splicing [6]. To complicate matters further, it has been recently suggested that some pseudoexons are authentic exons whose inclusion leads to efficient nonsense-mediated decay, such as the pseudoexon located downstream of mutually exclusive exons 2 and 3 of the rat a-tropomyosin gene, which acts both as an alternative exon that leads to non- sense-mediated decay and as a zero-length exon [7]. Nonetheless, in a clinical setting, pathological pseudoexon sequences have been identified by simple activation of cryptic splice sites which reinforce their strength, de novo creation, the inactivation ⁄ activation Keywords intron; NF1; pseudoexon; PTB ⁄ nPTB; splicing Correspondence D. Baralle, Human Genetics Division, University of Southampton, Duthie Building (Mailpoint 808), Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK Fax: +44 2380794346 Tel: +44 2380796162 E-mail: D.Baralle@soton.ac.uk (Received 30 July 2008, revised 23 September 2008, accepted 9 October 2008) doi:10.1111/j.1742-4658.2008.06734.x In disease-associated genes, understanding the functional significance of deep intronic nucleotide variants represents a difficult challenge. We previ- ously reported that an NF1 intron 30 exonization event is triggered from a single correct nomenclature is ‘c.293-279 A>G’ mutation [Raponi M, Upadhyaya M & Baralle D (2006) Hum Mutat 27, 294–295]. In this paper, we investigate which characteristics play a role in regulating inclusion of the aberrant pseudoexon. Our investigation shows that pseudoexon inclu- sion levels are strongly downregulated by polypyrimidine tract binding pro- tein and its homologue neuronal polypyrimidine tract binding protein. In particular, we provide evidence that the functional effect of polypyrimidine tract binding protein is proportional to its concentration, and map the cis- acting elements that are principally responsible for this negative regulation. These results highlight the importance of evaluating local sequence context for diagnostic purposes, and the utility of developing therapies to turn off activated pseudoexons. Abbreviations NF1, neurofibromatosis type 1; nPTB, neuronal polypyrimidine tract binding protein; PTB, polypyrimidine tract binding protein. FEBS Journal 275 (2008) 6101–6108 ª 2008 The Authors Journal compilation ª 2008 FEBS 6101 of splicing regulatory elements, or manipulation of RNA structures [8–14]. Buratti et al. [2] provide an exhaustive table of pathological pseudoexon inclusion events. We previously reported a new intronic mutation c.31-279A>G in neurofibromatosis type 1 (NF1) intron 30 that results in creation of a new 3¢ splice site and activation of a cryptic 5¢ splice site, leading to par- tial inclusion of a pseudoexon [15]. The mutation caused aberrant splicing from the mutated allele with approximately 45% normal transcripts and 55% containing the cryptic exon. Intriguingly, the same c.31-279A>G variation has been found in the canine NF1 gene sequence, where the strength of the potential 3¢ splice site was estimated to be as high as the strength of the human pseudoexon 3¢ splice site. How- ever, no canine expressed sequence tag sequences have been reported that utilize this site. These data suggest that the sequence of the pseudoexon itself and the surrounding trans-acting factors may play a role in its definition. In this work, we have investigated which local context characteristics play a role in regulating exonization of this sequence. Results Deletion analysis of pseudoexon sequences In order to identify the splicing regulatory elements responsible for pseudoexon exonization, we performed a deletion analysis using a minigene carrying the )279A>G mutation. The del_a, del_b and del_c deletions are highlighted in Fig. 1A. The hybrid minig- enes carrying each deletion were transiently transfected into HeLa cells and the splicing outcome analysed (Fig. 1B). The results showed that the pseudoexon was only included in the minigene with the large central deletion, del_b. Transfection of hybrid minigenes with the del_a or del_c deletions resulted in complete pseudoexon exclusion. Pulldown analysis of deletion mutants del_a and del_c Affinity purification analysis was then performed in order to define putative trans-acting regulatory factors that might be lost ⁄ gained with the del_a and del_c deletions. Figure 1C shows the general protein binding profiles of wild-type (WT), del_a and del_c RNAs bound to adipic acid dehydrazide beads and incubated with total HeLa nuclear extract. Coomassie staining of the SDS–PAGE gel showed a very prominent change in the binding profile of protein bands migra- ting in the 60 kDa range (boxed area, Fig. 1C). In particular, the intensity of these bands was greatly increased in the del_a mutant but absent in del_c. Direct sequencing of these bands using MS identified them as polypyrimidine tract binding protein (PTB) hnRNP I. Analysis of the WT lane band yielded only the PTB protein (accession number AAH04383). On the other hand, the del_a mutant yielded three sequences: NP_114368 corresponding to PTB iso- form c, NP_114367 corresponding to PTB isoform b, and AAP35465 corresponding to PTB itself. No other clear differences in Coomassie intensities were seen between the profile binding patterns of WT, del_a and del_c RNAs in independent experiments (data not shown). In order to confirm this observation and to extend this analysis to specific splicing factors, western blot assays were with an array of specific antibodies. Figure 1D shows that the western blot experiments confirmed the variation in PTB binding ability to the WT, del_a and del_c RNAs. As in the Coomassie gel, PTB binding was increased for the del_a mutant, suggesting that its binding might be responsible for the increased exon skipping in the del_a mutant. In con- trast, PTB showed reduced binding to del_c. Binding of other well-known splicing factors, such as SRp55, ASF ⁄ SF2, SC35 and hnRNP A1, A2 and C showed no reproducible variations between the RNAs, even though dedicated bioinformatics tools such as ESEfinder (http://rulai.cshl.edu/tools/ESE/) and Splicing Rainbow (http://www.ebi.ac.uk/asd-srv/wb.cgi?method=8) had suggested their possible involvement. The only excep- tion was hnRNP H, which appeared to bind in the del_a mutant but not to the WT or del_c RNAs. The observation that this protein did not binding to the WT RNA (unlike PTB) suggested that it does not represent a natural regulator of pseudoexon inclusion under normal conditions. We therefore decided to concentrate on characterizing the functional effects of PTB alone. Functional effects of PTB/nPTB knockdown on pseudoexon inclusion RNA interference experiments were undertaken in order to determine whether PTB acts as a negative reg- ulator of pseudoexon splicing. As nPTB, the neuronal homologue of PTB, has been demonstrated to func- tionally compensate for PTB [16] and is upregulated when PTB is removed, we decided to investigate the effect of PTB and PTB + nPTB knockdown on pseudoexon inclusion. In this experiment, the WT minigene (pNF1, c.31-279A>G) was cotransfected into HeLa cells with siRNA against PTB and nPTB. As shown in Fig. 2A, PTB plays an important role as a PTB pseudoexon repression M. Raponi et al. 6102 FEBS Journal 275 (2008) 6101–6108 ª 2008 The Authors Journal compilation ª 2008 FEBS negative splicing regulator for the WT pseudoexon construct. WT pseudoexon inclusion levels increased moderately when just PTB was knocked down, but double knockdown of both PTB and nPTB caused a prominent increase in pseudoexon inclusion levels (from 41% to 81%). To confirm that PTB acts as a negative regulator of pseudoexon splicing, we then undertook cotransfection experiments of pNF1 c.31-279A>G with increasing amounts of a PTB expression minigene (Fig. 2B). As expected, PTB overexpression antagonized pseudoexon inclusion in this case and in also contexts where either PTB or nPTB were previously knocked down by siRNA treatment (Fig. 2C). Interestingly, in all cases, high con- centrations of PTB expression vector almost completely abolished the levels of pseudoexon inclusion, despite the presence of the c.31-279A>G mutation. The same result was also obtained by overexpressing nPTB*, a codon- optimized nPTB (data not shown). Splicing of the del_a and del_c mutants was un- affected by PTB ⁄ nPTB knockdown. In both cases, complete exon skipping was maintained upon PTB depletion, indicating that variations in PTB binding are not the main reason for the skipping observed with del_a and del_c, despite the strong increase in PTB binding to del_a (Fig. 1C,D). This observation sug- gests the presence of additional enhancer regulatory sequences or proteins that could not be detected using our affinity purification methods. Indeed, the presence of additional enhancer ⁄ silencer elements acting on pseudoexon inclusion in these regions is also evident from the results of single-nucleotide mutagenesis AB CD Fig. 1. Identification of pseudoexon splicing regulatory elements. (A) Pseudoexon sequence in upper case showing the del_a, del_b and del_c deletions (highlighted and underlined). Intronic sequences are shown in lower case. The acceptor and donor site sequences of the pseudoexon are in bold and underlined. (B) Transient transfection results for the hybrid minigenes carrying deletions. The + and ) signs indicate pseudoexon inclusion and exclusion, respectively. (C) nuclear extract protein binding profile of WT, del_a and del_c RNAs following Coomassie staining. The boxed area shows the protein bands that display the greatest change in binding profile. (D) Western blot probed for PTB, hnRNP C, hnRNP A1 ⁄ A2, hnRNP DAZAP, hnRNP H, ASF2 ⁄ SF2, SRp55 and SC35. M. Raponi et al. PTB pseudoexon repression FEBS Journal 275 (2008) 6101–6108 ª 2008 The Authors Journal compilation ª 2008 FEBS 6103 analysis based on human ⁄ dog intronic sequence com- parisons and ESEfinder in silico predictions. All dog ⁄ human and putative exonic splicing enhancer-inactivat- ing substitutions are capable of completely repressing pseudoexon inclusion (M. Raponi & D. Baralle, unpublished observations). Studies are now being performed to better characterize these additional elements and their cognate binding factors. Finally, it is interesting to note that PTB ⁄ nPTB knockdown had no effect on the pseudoexon sequence lacking the )279A>G mutation (Fig. 2E). This result suggests that PTB binding sites could be maintained in this pseudoexon sequence as a preventive measure against 3¢ splice site-activating mutations. Dissection of the PTB recognition elements The preferred RNA binding sites of PTB ⁄ nPTB are UCUU or CUCUCU in pyrimidine-rich contexts [17,18], and we therefore focused on the role played by such elements in the pseudoexon inclusion process. Three UCUU motifs were identified in the pseudoexon body itself and two were identified downstream of the pseudoexon cryptic 5¢ splice site (Fig. 3A, referred to as m1–m5), but no likely motifs for PTB binding were observed upstream of the pseudoexon (a common occurrence in several PTB-regulated exons). All these sites were modified by site-directed mutagenesis in order to inactivate putative PTB binding to each of B A C D E Fig. 2. PTB and nPTB regulate pseudoexon definition. (A) Transient transfection results for the WT minigene in the presence of siRNAs against PTB and nPTB. ‘Cont’, negative control siRNA; P1, siRNA against PTB; N1, siRNA against nPTB. The + and ) signs indicate pseudo- exon inclusion and exclusion, respectively. The top panel shows the effect of PTB and PTB ⁄ nPTB knockdown on pseudoexon inclusion. The middle and bottom panels show western blots probed for PTB, nPTB and ERK. The two bands observed in the PTB western blot corre- spond to the PTB-1 (lower) and PTB-4 (upper) isoforms. (B) Effect of PTB overexpression on pseudoexon inclusion. From left to right, increasing amounts (10, 100, 250 and 750 ng) of expression plasmid for PTB1 were cotransfected with the WT plasmid. Percentages of pseudoexon inclusion are reported below the gel. (C) PTB overexpression can overcome the PTB ⁄ nPTB knockdown effect on pseudoexon inclusion in the WT plasmid. (D) Knockdown of PTB and PTB ⁄ nPTB does not affect splicing in the artificial mutants del_a and del_c. (E) Knockdown of PTB ⁄ nPTB does not affect splicing in the pseudoexon sequence lacking the )279a>g activating mutation (WT-279a). PTB pseudoexon repression M. Raponi et al. 6104 FEBS Journal 275 (2008) 6101–6108 ª 2008 The Authors Journal compilation ª 2008 FEBS them, and the effects of the mutations were then tested by transient transfection. As shown in Fig. 3B, the m1 and m5 mutations had negligible effects upon pseudoexon splicing. However, the pseudoexon inclusion efficiency (48%) was moder- ately increased to 62% and 72%, respectively, follow- ing introduction of the m2 and m3 mutations. Strikingly, mutating the UCUU motif m4 immediately downstream of the pseudoexon 5¢ splice site induced almost complete pseudoexon exclusion. However, given that the m4 motif is immediately adjacent to the 5¢ splice site, it is very likely that this mutation is involved in recognition of this sequence either directly or through interaction with a positive trans-acting factor. Taken together, these results suggest that the m2 and m3 are the sequences that are principally responsi- ble for mediating the repressive activity of PTB. Consistent with this, pulldown and western blot anal- ysis showed decreased PTB binding to pseudoexon sequences carrying mutations m2 and ⁄ or m3 compared with the WT pseudoexon sequence [Fig. 3C, normal- ized using the uniformly binding deleted in Azoosper- mia associated protein (DAZAP) protein]. Moreoever, although PTB overexpression in HeLa cells induced a fourfold decrease in WT pseudoexon inclusion, this effect was reduced to threefold for the individual m2 and m3 mutants (Fig. 3D) and to 1.7-fold for the double mutant m2 + m3. In conclusion, these results suggest that the effects of PTB on pseudoexon exclusion are mediated by the two central UCUUCUU (m2) and UCUU (m3) sequences. Discussion Introns frequently embed potential exonic sequences [19], and their activation through the creation or acti- vation of a cryptic splice site is a common cause of genetic disease. We recently reported the example of a deep intronic mutation c.31-279A>G in the NF1 gene of a patient with a severe form of neurofibromatosis type 1, in whom this mutation was associated with a 3¢ A BD C Fig. 3. Role played by UCUU-type motifs in pseudoexon splicing. (A) Pseudoexon (upper case) and partial downstream intron (lower case) nucleotide composition. The acceptor and donor site sequences of the pseudoexon are in bold and underlined. m1–m5 indicate the nucleo- tide substitutions analysed. (B) RT-PCR products from transfection experiments using minigenes carrying each substitution. The + and ) signs indicate pseudoexon inclusion and exclusion, respectively. The percentages of pseudoexon inclusion are also shown. The nucleotide substitutions analysed are indicated above each lane and labelled m1–m5. (C) Western blot probed for PTB and DAZAP. Comparison between the PTB binding capacity of the WT pseudoexon and mutant pseudoexons m2, m3 and m2 + m3. (D) Effect of PTB overexpression on mutants with various m2 and m3 combinations; 750 ng of expression plasmid for PTB1 was cotransfected in each case. M. Raponi et al. PTB pseudoexon repression FEBS Journal 275 (2008) 6101–6108 ª 2008 The Authors Journal compilation ª 2008 FEBS 6105 splice site activation event [15]. In this work, we have focused on identifying the internal pseudoexon sequences and trans-acting factors capable of affecting the levels of inclusion of the pseudoexon. The fact that such sequences ⁄ factors are likely to exist is based on a previous comparison [15] with the dog genome, where the presence of a naturally occurring )279A>G substi- tution does not lead to any exonization event. In line with this hypothesis, we provide experimental evidence that altering the human pseudoexon sequence can heavily affect its recognition. In particular, our findings demonstrate that PTB and nPTB are major repressors of pseudoexon splicing, with a role in regu- lating inclusion of the pseudoexon. These functional effects are in line with the view that PTB ⁄ nPTB might act as general repressors of weak exons, including pseudoexons [20], although PTB may also act as a positive splicing regulator [21]. We were able to detect increased or decreased binding of PTB to the pseudo- exon when the 5¢ (del_a) or 3¢ (del_c) thirds of the pseudoexon, respectively, were deleted. In line with this, we show that mutation of putative PTB binding motif m1 (which is deleted in del_a) had no influence on pseudoexon inclusion rates but mutation of m3 (which is deleted in del_c) had a strong effect on pseudoexon exclusion. The two deletions leading to strong repression of pseudoexon inclusion may have also damaged splice site recognition as well as altered binding sites for exonic splicing enhancer regulatory elements. In parti- cular, changes in secondary structure could explain why PTB binds more strongly to the deletion mutant del_a. Furthermore, involvement of positive regulatory elements is suggested by the strong repression of pseudoexon inclusion in mutant m4. This effect could be due to disruption of a T-cell intracellular antigen 1 (TIA-1) binding site immediately downstream of the 5¢ splice site, as this splicing factor has been shown to bind at pyrimidine tracts, competing with PTB [22]. In general, however, PTB cannot be considered the sole determinant of pseudoexon splicing, which is evidently controlled by more complex processes that are currently under investigation. This complexity may well be important in explaining the severe phenotype observed in the patient, where the requirement and balance of antagonistic splicing factors involved in pseudoexon definition defines the degree of aberrant NF1 intron 30 exonization in various tissues. The functional effect of PTB on intervening sequence 30 (IVS30) pseudoexon inclusion appears to be mediated by cooperative binding sites within the pseudoexon. In particular, the central m2 and m3 elements have the strongest effect on pseudoexon inclusion and can function as an exon splicing silencer. The requirement for multiple PTB binding sites for optimal repression has already been highlighted in other model pre-mRNAs. Recent research on other genes such as c-src and Fas has shown that PTB can repress exon inclusion by interfering with several steps of intron ⁄ exon definition (reviewed in [23]). Most importantly, we have established the physio- logical importance of these results by using an expression vector to increase PTB and nPTB protein levels in living cells, and shown that this results in efficient repression of pseudoexon aberrant splicing both with and without PTB ⁄ nPTB knockdown. Our observation that different PTB ⁄ nPTB expression levels can successfully alter pseudoexon inclusion suggests that quantitative differences in PTB ⁄ nPTB expression may be responsible for cell-type-specific restrictions (upregulation) in pseudoexon splicing. This has considerable importance when considering potential methods for the control of aberrant splicing (see below). Indeed, PTB ⁄ nPTB expression levels in differ- ent tissues may be the cause of the patient’s particu- larly severe spinal NF1 phenotype described previously [15]. It is important to note that the effect of nPTB in regulating aberrant pseudoexon exclusion in neurons may be weak due to translational repres- sion. It has been recently shown that nPTB is expressed in vivo at a lower level than PTB or codon- optimized nPTB* [24]. Finally, our findings open the way to development of novel therapeutic strategies aimed at rescuing splic- ing inhibition in patient cells. In general, pseudoexon inclusion in pathological situations can be targeted through use of antisense oligonucleotides or modified U7 snRNA molecules against the cryptic 5¢ and 3¢ splice sites, as recently described for PCCA, PCCB, PTCH1 and BRCA1 [25,26]. Although these strategies may represent viable therapeutic approaches for repression of the NF1 pseudoexon, the results pre- sented in this work have expanded the list of poten- tial options. The use of bifunctional oligonucleotides {targeted oligonucleotide enhancer of splicing (TOES)/ targeted oligonucleotide silencing of splicing (TOSS) methodology reviewed by Garcia-Blanco et al. [27]} that carry a binding domain and an effector domain with binding sites for known splicing factors has been recently described for the successful splicing recovery of spinal muscular atrophy disease gene (SMN) exon 7. In our case, we hypothesize that the use of such reagents will increase recruitment of additional PTB molecules and in this way achieve downregula- tion of pseudoexon inclusion. This type of strategy would also have the advantage that successful skip- PTB pseudoexon repression M. Raponi et al. 6106 FEBS Journal 275 (2008) 6101–6108 ª 2008 The Authors Journal compilation ª 2008 FEBS ping of the pseudoexon would remove the bifunctional oligonucleotide from the rescued mRNA and would not eventually interfere with subsequent steps of the mRNA life cycle such as transport to the cytoplasm and ⁄ or its translation. Experimental procedures Site-directed mutagenesis and deletion Site-directed mutagenesis was performed by the overlap extension method [28] using previously described pNF1c.31-279A>G as a template and NF31-F and NF31-R as flanking primers [15]. Deletions were introduced using the same method with overlapping primers designed according to the portion to be deleted as follows: DELa reverse 5¢-TCCTCCACTATAAAAGGAAATG-3¢ and DELa forward 5¢-TTATAGTGGAGGAAAATAAGAC-3¢; DELb reverse 5¢-AACAGTCCATTTTAGTCCTT-3¢ and DELb forward 5¢-AAAATGGACTGTTCTTTCTT-3¢; DELc reverse 5¢-TACCTAGAAGAAAGAACAGT-3¢ and DELc forward 5¢-TCTTTCTTCTAGGTAATAGT-3¢. Transient transfection assay and pre-mRNA splicing analysis HeLa cells were grown in Dulbecco’s modified Eagle’s medium (supplemented with 10% fetal calf serum, 450 mgÆL )1 glucose, 110 mgÆL )1 sodium pyruvate, 2 mm l-glutamine and 50 mgÆmL )1 penicillin ⁄ streptomycin) on 35 mm plates. Each minigene plasmid (0.8 lg) was trans- fected into 3 · 10 5 Hela cells in serum-free medium with 8 mL Lipofectamine reagent (Invitrogen, Carlsbad, CA, USA). Cells were grown overnight, washed with NaCl ⁄ P i , and fresh medium with 10% fetal calf serum was added. Cells were grown for an additional 24 h followed by RNA extraction. siRNA transfection of HeLa cells was carried out using 10 pmol of P1 and N1 siRNA accord- ing to a 7-day, two-hit protocol as described previously [16,20], where the target genes for knockdown were PTB and nPTB, respectively. For the add-back experiments, increasing amounts of PTB1 expression plasmid (10, 100, 250 and 750 ng) were cotransfected on the 5th day of the knockdown protocol together with 250 ng of reporter plasmid using 4 lL of Lipofectamine (Invitrogen). Total RNA was extracted using an RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Total RNA (3 lg) was reverse-transcribed using random hexamer primers, and cDNA was then amplified by PCR in a total volume of 50 lL using prim- ers specifically designed to amplify processed transcripts derived from the minigene. Each transfection experiment was perfomed at least three times, and representative gels are shown in each case. Pulldown assay Pulldown assays were performed essentially as described previously [29]. Briefly, 500 pmol of the target RNA (approximately 15 lg of a 100-mer RNA) were placed in a 400 lL reaction mixture containing 100 mm NaOAC pH 5.0 and 5 mm sodium m-periodate (Sigma, St Louis, MO, USA), incubated for 1 h in the dark at room temperature, ethanol-precipitated, and resuspended in 100 lL of 0.1 m NaOAC, pH 5.0. To this RNA, 300 lL of an adipic acid dehydrazide agarose bead 50% slurry (Sigma) equilibrated in 100 mm NaOAC pH 5.0 were added, and the mix was incubated for 12 h at 4 °C on a rotator. The beads with the bound RNA were then pelleted, washed (5 min) three times with 1 mL of 2 m NaCl, and equilibrated in washing buffer (5 mm HEPES pH 7.9, 1 mm MgCl 2 , 0.8 mm magnesium acetate). They were then incubated on a rotator with approximately 1 mg of HeLa cell nuclear extract for 30 min at room temperature in 1 mL final volume. Heparin was added to a final concentration of 5 mgÆmL )1 . The beads were then pelleted by centrifugation at 3000 g for 3 min and washed for 5 min, four times with 1.5 mL of washing buffer, before addition of SDS sample buffer and loading onto a 10% SDS–PAGE gel. Western blot Pulldown samples were electroblotted onto a Hybond-C Extra membrane (Amersham, Chalfont St Giles, UK), and antibody recognition was then performed using several in-house antibodies against PTB, hnRNP A1 ⁄ A2 ⁄ C and H proteins and commercial antibodies against ASF ⁄ SF2 (Zymed, Carlsbad, CA, USA), SC35 (Sigma) and SRp55 (1H4 antibody, Zymed). Protein bands were detected using an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Acknowledgements E.B. and C.S. are supported by the Telethon Onlus Foundation (GGP06147), Fondo per l’Investimento sulla Ricerca di Base (FIRB) (RBNE01W9PM), and by EC grant EURASNET-LSHG-CT-2005-518238. R.M. and B.D. are supported by Action Medical Research (grant SP4175) and EURASNET. 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