Ted in a further decrease of the wild-type residual transcript, from about 5 to about 1 of the total FGG transcripts, thus confirming the role of this factor 
in promoting pseudoexon inclusion (194423-15-9 web Mirin web Figure 2B). In this case, to have a more accurate measure of the relative amount of the two splicing variants, a fluorescent RT-PCR approach was used (see Materials and Methods).A 25-bp Region Binds hnRNP F and is Important for Pseudoexon InclusionBesides G-runs, additional regulatory elements modulating pseudoexon inclusion were predicted using the ESEfinder software [21,22]. The program predicted multiple binding sites for SF2/ ASF, SC35, SRp40, and SRp55 proteins. The higher density of high-score motifs (score .3) was found in a 25-bp region comprised between nucleotides 7 and 27 of the pseudoexon (Figure 3A), this region also contains one of the three G-run motifs that may bind hnRNP F. To identify which trans-acting factors can bind this ESE-enriched 25-bp region, an affinity pulldown protocol was used. Both the wild-type and a scrambled (negative control) 25-bp sequence (Figure 3B), were covalently coupled to adipic acid dehydrazide beads and incubated with HeLa nuclear extracts. As shown in Figure 3B (left panel), Western blotting withResultsA previous work from our group demonstrated that a single nucleotide substitution within intron 6 of the FGG gene (IVS6320A.T) results in the inclusion of a disease-causing pseudoexon in nearly the totality of mature transcripts (Figure 1) [17]. This nucleotide substitution produces an extended complementarity to U1snRNA at a cryptic donor splice site. However, the exiguity of residual wild-type splicing, as well as the existence in other FGG exons (i.e. exons 3 and 9) of physiologic donor splice sites withG-runs Regulating FGG Pseudoexon InclusionFigure 1. Schematic representation of the 75-bp FGG pseudoexon activated by the IVS6-320A.T mutation. (top) The fibrinogen cluster; boxes and lines represent exons and intronic/intergenic regions, respectively (only exons are drawn to scale); the two parallel slanted lines indicate breaks in the scale. (middle) The FGG minigene (M) cloned in pTargeT vector; the star marks the IVS6-320A.T mutation. (bottom) The complete 75-bp-long pseudoexon sequence and flaking splice sites; nucleotides belonging to the pseudoexon are in capital letters; the strength of pseudoexon splice sites, calculated by using the NNSPLICE 0.9 (http://www.fruitfly.org/seq_tools/splice.html) and the Netgene2 (http://www.cbs.dtu. dk/services/NetGene2/) software is reported below the corresponding sequence; G-stretches are shaded in gray. doi:10.1371/journal.pone.0059333.gantibodies against the principal hnRNPs evidenced signals for all four tested proteins (hnRNP H, F, A1, and A2). However, comparison with results 1527786 obtained using a scrambled or an unrelated oligoribonucleotide revealed that the only protein exclusively binding the 25-bp target sequence was hnRNP F. Moreover, the binding efficiency of hnRNP H to the 25-bp probe was much higher than that of the permutated sequence. The same 16574785 experiment performed immunodecorating with antibodies against SR proteins evidenced a weak binding of SRp40 to the 25-bp sequence (Figure 3B, right panel), supporting the ESEfinder prediction for this protein. Finally, probing with additional antibodies (i.e. anti PTB, hnRNP C) ruled out the binding of additional common hnRNP factors to this sequence (data not shown). The relevance of the 25-bp region in p.Ted in a further decrease of the wild-type residual transcript, from about 5 to about 1 of the total FGG transcripts, thus confirming the role of this factor in promoting pseudoexon inclusion (Figure 2B). In this case, to have a more accurate measure of the relative amount of the two splicing variants, a fluorescent RT-PCR approach was used (see Materials and Methods).A 25-bp Region Binds hnRNP F and is Important for Pseudoexon InclusionBesides G-runs, additional regulatory elements modulating pseudoexon inclusion were predicted using the ESEfinder software [21,22]. The program predicted multiple binding sites for SF2/ ASF, SC35, SRp40, and SRp55 proteins. The higher density of high-score motifs (score .3) was found in a 25-bp region comprised between nucleotides 7 and 27 of the pseudoexon (Figure 3A), this region also contains one of the three G-run motifs that may bind hnRNP F. To identify which trans-acting factors can bind this ESE-enriched 25-bp region, an affinity pulldown protocol was used. Both the wild-type and a scrambled (negative control) 25-bp sequence (Figure 3B), were covalently coupled to adipic acid dehydrazide beads and incubated with HeLa nuclear extracts. As shown in Figure 3B (left panel), Western blotting withResultsA previous work from our group demonstrated that a single nucleotide substitution within intron 6 of the FGG gene (IVS6320A.T) results in the inclusion of a disease-causing pseudoexon in nearly the totality of mature transcripts (Figure 1) [17]. This nucleotide substitution produces an extended complementarity to U1snRNA at a cryptic donor splice site. However, the exiguity of residual wild-type splicing, as well as the existence in other FGG exons (i.e. exons 3 and 9) of physiologic donor splice sites withG-runs Regulating FGG Pseudoexon InclusionFigure 1. Schematic representation of the 75-bp FGG pseudoexon activated by the IVS6-320A.T mutation. (top) The fibrinogen cluster; boxes and lines represent exons and intronic/intergenic regions, respectively (only exons are drawn to scale); the two parallel slanted lines indicate breaks in the scale. (middle) The FGG minigene (M) cloned in pTargeT vector; the star marks the IVS6-320A.T mutation. (bottom) The complete 75-bp-long pseudoexon sequence and flaking splice sites; nucleotides belonging to the pseudoexon are in capital letters; the strength of pseudoexon splice sites, calculated by using the NNSPLICE 0.9 (http://www.fruitfly.org/seq_tools/splice.html) and the Netgene2 (http://www.cbs.dtu. dk/services/NetGene2/) software is reported below the corresponding sequence; G-stretches are shaded in gray. doi:10.1371/journal.pone.0059333.gantibodies against the principal hnRNPs evidenced signals for all four tested proteins (hnRNP H, F, A1, and A2). However, comparison with results 1527786 obtained using a scrambled or an unrelated oligoribonucleotide revealed that the only protein exclusively binding the 25-bp target sequence was hnRNP 
F. Moreover, the binding efficiency of hnRNP H to the 25-bp probe was much higher than that of the permutated sequence. The same 16574785 experiment performed immunodecorating with antibodies against SR proteins evidenced a weak binding of SRp40 to the 25-bp sequence (Figure 3B, right panel), supporting the ESEfinder prediction for this protein. Finally, probing with additional antibodies (i.e. anti PTB, hnRNP C) ruled out the binding of additional common hnRNP factors to this sequence (data not shown). The relevance of the 25-bp region in p.