BACKGROUND
secreted phosphoprotein  <dig>  is a matricellular glycoprotein mediating cell-adhesion, -migration, -survival and -signalling via integrin and cd <dig> receptors  <cit> .
in line with its versatility and widespread expression spp <dig> has been linked with various physiological and pathological events, amongst others development and regeneration of skeletal muscle  <cit> .
spp <dig> is regulated by the muscle regulatory factors myod and myf <dig>  <cit>  and has been shown to be expressed in vitro in myoblasts and myotubes  <cit> .
uaesoontrachoon et al.
<cit>  demonstrated that soluble spp <dig> stimulates proliferation of myoblasts whereas spp <dig> deposited in extracellular matrix promotes their differentiation.
recently we showed upregulation of spp <dig> mrna expression in prenatal musculus longissimus dorsi  at  <dig> and 63- <dig> days post conception  in pigs, i.e.
at the time points of the first and second myogenic wave respectively, thus providing additional, in vivo, evidence supporting involvement of spp <dig> in myogenesis.
in addition we showed consistent elevation of spp <dig> mrna level during myogenesis in the pig breed duroc compared to breed pietrain, the latter showing higher muscularity and higher proportion of fast twitch glycolytic fibers postnatally  <cit> .
moreover we mapped quantitative trait loci for the proportion of fast twitch glycolytic fibers and ph of m.l.d.
<dig> minutes post mortem on porcine chromosome  <dig> close to spp <dig> position  <cit> .
to detect polymorphisms that contribute to the breed-related differential mrna expression of spp <dig> and which are associated with differential microstructural and biophysical muscle properties we performed snp screening of the upstream regulatory region.
we identified and functionally characterized a snp located in the 3' terminal end of the first intron in an evolutionarily conserved transcriptional enhancer  <cit> .
RESULTS
identification and in silico characterization of snp g.3836a>g
we resequenced ~ <dig>  kb of the 5'-upstream region of the porcine spp <dig> including 5'-flanking region and promoter, exon <dig>  intron <dig>  as well as exon <dig> and identified two snp: a c>t snp in the 5'-flanking region  and an a>g snp in the first intron .
phylogenetic footprinting of the 5'-upstream region indicated that the g.1804c>t snp resides in an evolutionarily poorly conserved sequence of a repetitive element  and thus most likely possesses no function.
in contrast the g.3836a>g snp is located in an evolutionarily conserved region at the 3' terminal end of the first intron  that has been shown to harbor a strong transcriptional enhancer in the pig and in human  <cit> .
the snpinspector software predicted the elimination of binding sites of four transcription factors by this snp including ccaat/enhancer binding protein , adenovirus e <dig> promoter binding protein , thyrotrophic embryonic factor  and fork head homologous x .
interestingly, a ccaat/enhancer binding protein beta  binding site, overlapping the site corresponding to the g.3836a>g snp site in the human spp <dig>  has been shown to be important for the activity of the intronic enhancer  <cit> .
the c/ebpβ binding site at the 3' terminal end of the first intron of spp <dig> is evolutionarily conserved in the pig and is negatively affected by the snp g.3836a>g
in order to examine the effect of the snp g.3836a>g on the binding of nuclear proteins, specifically of c/ebpβ, we performed competitive electrophoretic mobility shift assay  and supershift assay respectively.
allelic probes centered around the g.3836a>g snp showed distinct differences in their ability to bind nuclear proteins from porcine fetal skeletal muscle .
the labelled probe containing the wild-type allele a formed a dna:protein complex  which was completely absent in all binding reactions containing the labelled mutant probe g .
complex "s" was specifically competed by the addition of excess of the corresponding unlabelled probe a  or unlabelled probe bearing consensus c/ebp binding motif  respectively, but was unaffected by the addition of a nonspecific competitor probe, in this case bearing consensus sp <dig> binding motif .
addition of c/ebpβ antibody to a binding reaction containing the labelled probe a caused disappearance of the complex "s" and caused an evident supershifted complex .
addition of the antibody to a binding reaction containing the labelled probe bearing the sp <dig> binding motif had no effect on the dna:protein complexes  and also produced no supershifted complex in the binding reaction containing the labelled mutant probe g , demonstrating specificity of the supershifted complex "ss".
our emsa experiment thus provides clear evidence that c/ebpβ participated on the formation of the complex "s" and consequently provides evidence that the c/ebpβ binding site present in human spp <dig> is evolutionarily conserved in porcine spp <dig>  the heterogeneity of the complex "s" is most likely due to the presence of multiple c/ebpβ isoforms or heterodimers with other members of the c/ebp family.
competitive inhibition of the complex "s" by addition of 100-fold molar excess of the unlabelled mutant probe g indicates that binding of c/ebpβ is not completely abolished by the snp g.3836a>g .
to characterize the effect of the g.3836a>g snp on the c/ebpβ binding site more precisely we performed competitive emsa using nuclear extracts from cells overexpressing n-terminally truncated bovine c/ebpβ .
the Δn-c/ebpβ spans the bovine c/ebpβ from amino acids  <dig> to  <dig>  i.e.
it retains the dna-binding domain and the bzip domain  <cit> .
the complex formed between the labelled probe a and Δn-c/ebpβ was competed away using 5- <dig> fold molar excess of unlabelled probe a and g respectively.
the decrease in relative intensity of the complex as a consequence of increasing unlabelled competitor concentration is an indication of the efficiency of displacement, thereby reflecting the relative affinity of the respective alleles to c/ebpβ.
the efficiency of displacement of the cold competitor probe g is more than twofold lower compared to the cold competitor probe a demonstrating that the mutation reduced the affinity of the binding site to c/ebpβ in excess of twofold .
snp g.3836a>g decreases mrna abundance of spp <dig> in fetal m.l.d
to examine whether the reduced affinity of the c/ebpβ binding site due to snp g.3836a>g leads to differential mrna expression of spp <dig> in vivo we genotyped  <dig> fetuses per each breed  and stage  with expression data from a previous study  <cit> .
the snp turned out to be fixed for the ancestral a allele in the pietrain fetuses, segregating only amongst the duroc fetuses with a frequency of 57% of the mutant g allele.
the analysis of the effect of the snp on spp <dig> mrna expression was therefore performed within the duroc breed.
the data were pooled across the four stages, which was accounted for in the statistical model.
the results revealed a decreasing trend in the spp <dig> mrna abundance associated with the mutant g allele .
quantification and separate analysis of spp <dig> mrna expression in m.l.d.
of additional seven  <dig> dpc fetuses per each genotype class revealed similar, although less pronounced, decreasing tendency .
the decreasing trend in mrna abundance of spp <dig> associated with the g allele is consistent with the evidence of an altered function of the mutated c/ebpβ binding site which normally acts as a transcriptional enhancer.
with regard to the breed-related differences in the mrna expression of spp <dig> gene during myogenesis the upregulation in duroc is apparently caused by factors different from snp g.3836a>g.
snp g.3836a>g leads to ubiquitous aberrant splicing of porcine spp <dig> in the 5' untranslated region
the mutant g allele generates a potential splice acceptor site  only  <dig> bp upstream of the naturally occurring intron1/exon <dig> junction.
to prove whether this potential de novo 3'ss is active in vivo we performed rt-pcr using the m.l.d.
cdna samples of  <dig> day old fetuses described in the preceding paragraph.
as shown in figure 5a carriers of the wild-type a allele expressed only one splice variant whereas multiple splice variants were detected in samples carrying the g allele.
cloning and sequencing revealed that the mutant g allele causes aberrant splicing of the first intron thus generating heterogeneity in the 5' untranslated region.
two aberrantly spliced transcript variants were identified: one variant with  <dig> bp longer leader sequence spliced at the de novo 3'ss generated by the g allele, and another with a  <dig> bp longer leader sequence spliced at a cryptic 3'ss located in the first intron  <dig> bp upstream of the g. <dig> snp site .
the insertions do not lead to a change in the spp <dig> reading frame and also do not generate open reading frames upstream of the translation start site that could reduce translational efficiency.
inspection of the 3' terminal region of the first intron revealed two additional potential 3'ss between the authentic 3'ss and a putative branch site and one additional potential 3'ss between the cryptic 3'ss and a putative cryptic branch site.
to describe the splicing pattern in more detail we quantified relative amounts of the splice variants in m.l.d.
across different fetal stages and across various tissues/cell types of adult animals including tonsil, intestinal lymph node, liver, spleen, pituitary, adrenal gland, m.l.d., subcutaneous fat and leukocyte by fragment analysis on megabace capillary sequencer.
only heterozygous adult carriers were available.
we found aberrant splicing of spp <dig> mrna across all examined tissues of the carriers of the mutant g allele.
no additional splice variants were detected by the more sensitive analysis on the capillary sequencer.
the relative amounts of the three splice variants were allele dosage dependent; in homozygous gg animals the proportion of native: de novo: cryptic splice variants was about 56: 19:  <dig> and in heterozygous ag animals the proportion was about 78: 9:  <dig> .
we employed the maxentscore tool, implementing maximum entropy modelling , to examine the strength of the different identified active and potential 3'ss in silico.
vorechovsky  <cit>  showed that this method performs best in the prediction of the utilization of aberrant 3' ss.
maxentscore revealed that the mutation weakened the 3'ss, with the me score of the authentic 3'ss dropping from  <dig>  to  <dig>  for the mutated authentic 3'ss respectively.
the me score of the cryptic 3'ss  is higher than that of the de novo 3'ss .
the three potential 3'ss had low scores  in accord with the fact that they are not used in vivo.
discussion
the total number of myofibers and to some extent their metabolic and contractile properties are determined during the process of myogenesis.
insights into the regulation of this process will consequently bring about better knowledge of factors affecting postnatal growth and function of skeletal muscle.
we previously discovered breed-related differences in mrna expression of porcine spp <dig> during myogenesis  <cit>  and hypothesized that this variation might be caused by cis-regulatory dna-variation.
cis-regulatory dna variation has been shown to affect a large proportion  of genes  <cit>  and in fact has already been described in human spp <dig>  <cit> .
we identified an a>g snp at position g. <dig> at the 3' terminal end of the first intron of porcine spp <dig>  in silico analysis of the polymorphic sequence and knowledge about the regulation of spp <dig> in pigs and human suggested an effect of the snp on a c/ebpβ responsive transcriptional enhancer.
using competitive emsa and supershift assay we showed that the c/ebpβ binding site found in human is present also in the pig and is negatively affected by the mutation.
as a consequence of the reduced affinity of the c/ebpβ binding site the activity of the transcriptional enhancer is reduced, at least in fetal m.l.d., as indicated by the downregulation of spp <dig> mrna expression by fetuses carrying the g allele.
conservation of the c/ebpβ binding site at the 3' terminal end of the first intron of human and porcine spp <dig> points to an important function of this element in the regulation of spp <dig>  the relevance of c/ebpβ in positive regulation of mrna expression of spp <dig> is further emphasized by the identification of additional functional c/ebp transcriptional enhancer elements in the promoter of murine and human spp <dig>  <cit> .
c/ebps are a family of transcription factors involved in the regulation of proliferation and differentiation of diverse cell types, and play pivotal roles in a number of processes including adipogenesis  <cit> .
however, an involvement or a precise function of c/ebps in myogenesis has not been described so far.
we demonstrate that the snp g.3836a>g is functional also on the rna level and causes aberrant splicing of the first intron.
we found that this effect is ubiquitous with regard to temporo-spatial distribution which is in accord with the fact that it creates a general splicing signal.
naturally occurring dna-variation affecting splicing represents a valuable resource to identify sequence signals involved in the regulation of this process.
the g.3836a>g snp exhibits several features of de novo 3'ss activating aberrant splicing identified by kralovicova et al.
<cit>  and vorechovsky  <cit> .
it creates an ag dinucleotide in intron in polypyrimidine tract of the authentic 3'ss by introducing a guanine residue, has an uridine in position + <dig> relative to the de novo 3'ss and activates a cryptic 3'ss.
vorechovsky  <cit>  has shown that authentic counterparts of de novo 3'ss are intrinsically weak.
in the case of the affected 3'ss of spp <dig> this is likely a consequence of the two concurrent functional constraints on the sequence of the 3' terminal end of the first intron; on one hand to provide a c/ebpβ responsive transcriptional enhancer and on the other to provide the canonical splicing acceptor signals.
the in silico predicted strength of the mutated authentic 3'ss compared to the cryptic 3'ss and their utilization in vivo are reversed.
an explanation for the lower utilization of the cryptic 3'ss compared to the mutated authentic 3'ss in vivo may be a suboptimal sequence of the putative cryptic branch site, in contrast to the predicted authentic branch site which perfectly matches with the consensus sequence ynyuray .
another explanation might be that the cryptic 3'ss is silenced by a splicing silencer or vice versa that the authentic 3'ss might be augmented by a splicing enhancer.
kralovicova and vorechovsky  <cit>  showed that in vivo selection of aberrant splice sites is extensively controlled by auxiliary splicing signals.
mutations that affect hnrna splicing account for up to 50% of disease-causing gene alterations in human and potentially represent the most frequent cause of hereditary disorders.
some  <dig> unique aberrant 3'ss, activated by disease-causing mutations in  <dig> genes, are presently known in human  <cit> .
our results show that about half of the spp <dig> messages expressed by the g allele comprise aberrantly spliced transcripts.
considering the versatile function of spp <dig> and the ubiquitous expression of the aberrant splice variants it could be speculated that the snp g.3836a>g might have pleiotropic effects on various traits including growth, reproduction and immune defence.
however the relatively high frequency of the g allele argues against a major negative phenotypic effect, because in that case the g allele would be quickly eliminated given the high selection pressure on commercial pigs.
furthermore, the aberrant splicing induced by the snp g.3836a>g does not change primary structure of the spp <dig> protein.
in fact, the aberrant splicing may counteract the negative effect of the g allele on mrna expression of spp <dig> by enhancing translational efficiency or rna stability.
aberrantly spliced mrna isoforms of the human insulin gene with longer 5'-leader sequence, induced by a snp in its first intron, were reported to generate more proinsulin in vitro compared to the native transcripts  <cit> .
CONCLUSIONS
functional characterization of the snp g.3836a>g revealed that it has two effects; it negatively affects a c/ebpβ binding site and activates aberrant splicing of the first intron.
however, although the snp g.3836a>g is functional it most likely does not represent the causative mutation responsible for the previously observed breed related differences in spp <dig> mrna expression.
nevertheless, the dual effect of the snp on spp <dig> function renders it as an interesting dna-marker for association studies concerning muscle-related, growth, reproduction, and immune defence traits.

 secreted phosphoprotein is a matricellular glycoprotein mediating cell-adhesion, -migration, -survival and -signalling via integrin and cd receptors. in line with its versatility and widespread expression spp has been linked with various physiological and pathological events, amongst others development and regeneration of skeletal muscle. spp is regulated by the muscle regulatory factors myod and myf and has been shown to be expressed in vitro in myoblasts and myotubes. uaesoontrachoon et al. demonstrated that soluble spp stimulates proliferation of myoblasts whereas spp deposited in extracellular matrix promotes their differentiation. recently we showed upregulation of spp mrna expression in prenatal musculus longissimus dorsi at and 63- days post conception in pigs, i.e. at the time points of the first and second myogenic wave respectively, thus providing additional, in vivo, evidence supporting involvement of spp in myogenesis. in addition we showed consistent elevation of spp mrna level during myogenesis in the pig breed duroc compared to breed pietrain, the latter showing higher muscularity and higher proportion of fast twitch glycolytic fibers postnatally. moreover we mapped quantitative trait loci for the proportion of fast twitch glycolytic fibers and ph of m.l.d. minutes post mortem on porcine chromosome close to spp position. to detect polymorphisms that contribute to the breed-related differential mrna expression of spp and which are associated with differential microstructural and biophysical muscle properties we performed snp screening of the upstream regulatory region. we identified and functionally characterized a snp located in the 3' terminal end of the first intron in an evolutionarily conserved transcriptional enhancer.  identification and in silico characterization of snp g.3836a>g we resequenced ~ kb of the 5'-upstream region of the porcine spp including 5'-flanking region and promoter, exon intron as well as exon and identified two snp: a c>t snp in the 5'-flanking region and an a>g snp in the first intron. phylogenetic footprinting of the 5'-upstream region indicated that the g.1804c>t snp resides in an evolutionarily poorly conserved sequence of a repetitive element and thus most likely possesses no function. in contrast the g.3836a>g snp is located in an evolutionarily conserved region at the 3' terminal end of the first intron that has been shown to harbor a strong transcriptional enhancer in the pig and in human. the snpinspector software predicted the elimination of binding sites of four transcription factors by this snp including ccaat/enhancer binding protein, adenovirus e promoter binding protein, thyrotrophic embryonic factor and fork head homologous x. interestingly, a ccaat/enhancer binding protein beta binding site, overlapping the site corresponding to the g.3836a>g snp site in the human spp has been shown to be important for the activity of the intronic enhancer. the c/ebpβ binding site at the 3' terminal end of the first intron of spp is evolutionarily conserved in the pig and is negatively affected by the snp g.3836a>g in order to examine the effect of the snp g.3836a>g on the binding of nuclear proteins, specifically of c/ebpβ, we performed competitive electrophoretic mobility shift assay and supershift assay respectively. allelic probes centered around the g.3836a>g snp showed distinct differences in their ability to bind nuclear proteins from porcine fetal skeletal muscle. the labelled probe containing the wild-type allele a formed a dna:protein complex which was completely absent in all binding reactions containing the labelled mutant probe g. complex "s" was specifically competed by the addition of excess of the corresponding unlabelled probe a or unlabelled probe bearing consensus c/ebp binding motif respectively, but was unaffected by the addition of a nonspecific competitor probe, in this case bearing consensus sp binding motif. addition of c/ebpβ antibody to a binding reaction containing the labelled probe a caused disappearance of the complex "s" and caused an evident supershifted complex. addition of the antibody to a binding reaction containing the labelled probe bearing the sp binding motif had no effect on the dna:protein complexes and also produced no supershifted complex in the binding reaction containing the labelled mutant probe g, demonstrating specificity of the supershifted complex "ss". our emsa experiment thus provides clear evidence that c/ebpβ participated on the formation of the complex "s" and consequently provides evidence that the c/ebpβ binding site present in human spp is evolutionarily conserved in porcine spp the heterogeneity of the complex "s" is most likely due to the presence of multiple c/ebpβ isoforms or heterodimers with other members of the c/ebp family. competitive inhibition of the complex "s" by addition of 100-fold molar excess of the unlabelled mutant probe g indicates that binding of c/ebpβ is not completely abolished by the snp g.3836a>g. to characterize the effect of the g.3836a>g snp on the c/ebpβ binding site more precisely we performed competitive emsa using nuclear extracts from cells overexpressing n-terminally truncated bovine c/ebpβ. the Δn-c/ebpβ spans the bovine c/ebpβ from amino acids to i.e. it retains the dna-binding domain and the bzip domain. the complex formed between the labelled probe a and Δn-c/ebpβ was competed away using 5- fold molar excess of unlabelled probe a and g respectively. the decrease in relative intensity of the complex as a consequence of increasing unlabelled competitor concentration is an indication of the efficiency of displacement, thereby reflecting the relative affinity of the respective alleles to c/ebpβ. the efficiency of displacement of the cold competitor probe g is more than twofold lower compared to the cold competitor probe a demonstrating that the mutation reduced the affinity of the binding site to c/ebpβ in excess of twofold. snp g.3836a>g decreases mrna abundance of spp in fetal m.l.d to examine whether the reduced affinity of the c/ebpβ binding site due to snp g.3836a>g leads to differential mrna expression of spp in vivo we genotyped fetuses per each breed and stage with expression data from a previous study. the snp turned out to be fixed for the ancestral a allele in the pietrain fetuses, segregating only amongst the duroc fetuses with a frequency of 57% of the mutant g allele. the analysis of the effect of the snp on spp mrna expression was therefore performed within the duroc breed. the data were pooled across the four stages, which was accounted for in the statistical model. the results revealed a decreasing trend in the spp mrna abundance associated with the mutant g allele. quantification and separate analysis of spp mrna expression in m.l.d. of additional seven dpc fetuses per each genotype class revealed similar, although less pronounced, decreasing tendency. the decreasing trend in mrna abundance of spp associated with the g allele is consistent with the evidence of an altered function of the mutated c/ebpβ binding site which normally acts as a transcriptional enhancer. with regard to the breed-related differences in the mrna expression of spp gene during myogenesis the upregulation in duroc is apparently caused by factors different from snp g.3836a>g. snp g.3836a>g leads to ubiquitous aberrant splicing of porcine spp in the 5' untranslated region the mutant g allele generates a potential splice acceptor site only bp upstream of the naturally occurring intron1/exon junction. to prove whether this potential de novo 3'ss is active in vivo we performed rt-pcr using the m.l.d. cdna samples of day old fetuses described in the preceding paragraph. as shown in figure 5a carriers of the wild-type a allele expressed only one splice variant whereas multiple splice variants were detected in samples carrying the g allele. cloning and sequencing revealed that the mutant g allele causes aberrant splicing of the first intron thus generating heterogeneity in the 5' untranslated region. two aberrantly spliced transcript variants were identified: one variant with bp longer leader sequence spliced at the de novo 3'ss generated by the g allele, and another with a bp longer leader sequence spliced at a cryptic 3'ss located in the first intron bp upstream of the g. snp site. the insertions do not lead to a change in the spp reading frame and also do not generate open reading frames upstream of the translation start site that could reduce translational efficiency. inspection of the 3' terminal region of the first intron revealed two additional potential 3'ss between the authentic 3'ss and a putative branch site and one additional potential 3'ss between the cryptic 3'ss and a putative cryptic branch site. to describe the splicing pattern in more detail we quantified relative amounts of the splice variants in m.l.d. across different fetal stages and across various tissues/cell types of adult animals including tonsil, intestinal lymph node, liver, spleen, pituitary, adrenal gland, m.l.d., subcutaneous fat and leukocyte by fragment analysis on megabace capillary sequencer. only heterozygous adult carriers were available. we found aberrant splicing of spp mrna across all examined tissues of the carriers of the mutant g allele. no additional splice variants were detected by the more sensitive analysis on the capillary sequencer. the relative amounts of the three splice variants were allele dosage dependent; in homozygous gg animals the proportion of native: de novo: cryptic splice variants was about 56: 19: and in heterozygous ag animals the proportion was about 78: 9:. we employed the maxentscore tool, implementing maximum entropy modelling, to examine the strength of the different identified active and potential 3'ss in silico. vorechovsky showed that this method performs best in the prediction of the utilization of aberrant 3' ss. maxentscore revealed that the mutation weakened the 3'ss, with the me score of the authentic 3'ss dropping from to for the mutated authentic 3'ss respectively. the me score of the cryptic 3'ss is higher than that of the de novo 3'ss. the three potential 3'ss had low scores in accord with the fact that they are not used in vivo. discussion the total number of myofibers and to some extent their metabolic and contractile properties are determined during the process of myogenesis. insights into the regulation of this process will consequently bring about better knowledge of factors affecting postnatal growth and function of skeletal muscle. we previously discovered breed-related differences in mrna expression of porcine spp during myogenesis and hypothesized that this variation might be caused by cis-regulatory dna-variation. cis-regulatory dna variation has been shown to affect a large proportion of genes and in fact has already been described in human spp. we identified an a>g snp at position g. at the 3' terminal end of the first intron of porcine spp in silico analysis of the polymorphic sequence and knowledge about the regulation of spp in pigs and human suggested an effect of the snp on a c/ebpβ responsive transcriptional enhancer. using competitive emsa and supershift assay we showed that the c/ebpβ binding site found in human is present also in the pig and is negatively affected by the mutation. as a consequence of the reduced affinity of the c/ebpβ binding site the activity of the transcriptional enhancer is reduced, at least in fetal m.l.d., as indicated by the downregulation of spp mrna expression by fetuses carrying the g allele. conservation of the c/ebpβ binding site at the 3' terminal end of the first intron of human and porcine spp points to an important function of this element in the regulation of spp the relevance of c/ebpβ in positive regulation of mrna expression of spp is further emphasized by the identification of additional functional c/ebp transcriptional enhancer elements in the promoter of murine and human spp. c/ebps are a family of transcription factors involved in the regulation of proliferation and differentiation of diverse cell types, and play pivotal roles in a number of processes including adipogenesis. however, an involvement or a precise function of c/ebps in myogenesis has not been described so far. we demonstrate that the snp g.3836a>g is functional also on the rna level and causes aberrant splicing of the first intron. we found that this effect is ubiquitous with regard to temporo-spatial distribution which is in accord with the fact that it creates a general splicing signal. naturally occurring dna-variation affecting splicing represents a valuable resource to identify sequence signals involved in the regulation of this process. the g.3836a>g snp exhibits several features of de novo 3'ss activating aberrant splicing identified by kralovicova et al. and vorechovsky. it creates an ag dinucleotide in intron in polypyrimidine tract of the authentic 3'ss by introducing a guanine residue, has an uridine in position + relative to the de novo 3'ss and activates a cryptic 3'ss. vorechovsky has shown that authentic counterparts of de novo 3'ss are intrinsically weak. in the case of the affected 3'ss of spp this is likely a consequence of the two concurrent functional constraints on the sequence of the 3' terminal end of the first intron; on one hand to provide a c/ebpβ responsive transcriptional enhancer and on the other to provide the canonical splicing acceptor signals. the in silico predicted strength of the mutated authentic 3'ss compared to the cryptic 3'ss and their utilization in vivo are reversed. an explanation for the lower utilization of the cryptic 3'ss compared to the mutated authentic 3'ss in vivo may be a suboptimal sequence of the putative cryptic branch site, in contrast to the predicted authentic branch site which perfectly matches with the consensus sequence ynyuray. another explanation might be that the cryptic 3'ss is silenced by a splicing silencer or vice versa that the authentic 3'ss might be augmented by a splicing enhancer. kralovicova and vorechovsky showed that in vivo selection of aberrant splice sites is extensively controlled by auxiliary splicing signals. mutations that affect hnrna splicing account for up to 50% of disease-causing gene alterations in human and potentially represent the most frequent cause of hereditary disorders. some unique aberrant 3'ss, activated by disease-causing mutations in genes, are presently known in human. our results show that about half of the spp messages expressed by the g allele comprise aberrantly spliced transcripts. considering the versatile function of spp and the ubiquitous expression of the aberrant splice variants it could be speculated that the snp g.3836a>g might have pleiotropic effects on various traits including growth, reproduction and immune defence. however the relatively high frequency of the g allele argues against a major negative phenotypic effect, because in that case the g allele would be quickly eliminated given the high selection pressure on commercial pigs. furthermore, the aberrant splicing induced by the snp g.3836a>g does not change primary structure of the spp protein. in fact, the aberrant splicing may counteract the negative effect of the g allele on mrna expression of spp by enhancing translational efficiency or rna stability. aberrantly spliced mrna isoforms of the human insulin gene with longer 5'-leader sequence, induced by a snp in its first intron, were reported to generate more proinsulin in vitro compared to the native transcripts.  functional characterization of the snp g.3836a>g revealed that it has two effects; it negatively affects a c/ebpβ binding site and activates aberrant splicing of the first intron. however, although the snp g.3836a>g is functional it most likely does not represent the causative mutation responsible for the previously observed breed related differences in spp mrna expression. nevertheless, the dual effect of the snp on spp function renders it as an interesting dna-marker for association studies concerning muscle-related, growth, reproduction, and immune defence traits. 
we focused our investigation on the snp g.3836a>g, because in silico analysis and knowledge about the regulation of spp suggested an effect of this snp on a ccaat/enhancer binding protein beta responsive transcriptional enhancer. this snp affects the function of the spp gene at the dna level by affecting a c/ebpβ binding site and at the rna level by activating aberrant splicing of the first intron, and thus represents an interesting dna-marker to study phenotypic effects of spp dna-variation. with the aim to identify putative contributing cis-regulatory dna variation we resequenced the 5' upstream region of the gene in the respective breeds pietrain and duroc. using electrophoretic mobility shift assay we demonstrated that, similar to human spp the 3' terminal end of the first intron of porcine spp harbors a c/ebpβ binding site and showed that this binding site is negatively affected by the mutant g allele. at the 3' terminal end of the first intron of the porcine spp we identified a unique, dually functional snp g.3836a>g. using real-time quantitative pcr we showed that, consistent with its negative effect on a transcriptional enhancer element, the g allele tends to decrease mrna abundance of spp in the fetal musculus longissimus dorsi. secreted phosphoprotein is a multifunctional matricellular glycoprotein involved in development and regeneration of skeletal muscle. moreover, we showed that the snp g.3836a>g leads to ubiquitous aberrant splicing of the first intron by generating a de novo and activating a cryptic splice acceptor site. previously, we have demonstrated that porcine spp shows breed-related differential mrna expression during myogenesis. genotyping of fetuses per breed revealed that the g allele segregated exclusively in duroc fetuses with a frequency of percent. aberrantly spliced transcripts comprise about half of the spp messages expressed by the g allele.
 secreted phosphoprotein is a multifunctional matricellular glycoprotein involved in development and regeneration of skeletal muscle. previously, we have demonstrated that porcine spp shows breed-related differential mrna expression during myogenesis. with the aim to identify putative contributing cis-regulatory dna variation we resequenced the 5' upstream region of the gene in the respective breeds pietrain and duroc. we found two single nucleotide polymorphisms. we focused our investigation on the snp g.3836a>g, because in silico analysis and knowledge about the regulation of spp suggested an effect of this snp on a ccaat/enhancer binding protein beta responsive transcriptional enhancer.  using electrophoretic mobility shift assay we demonstrated that, similar to human spp the 3' terminal end of the first intron of porcine spp harbors a c/ebpβ binding site and showed that this binding site is negatively affected by the mutant g allele. genotyping of fetuses per breed revealed that the g allele segregated exclusively in duroc fetuses with a frequency of percent. using real-time quantitative pcr we showed that, consistent with its negative effect on a transcriptional enhancer element, the g allele tends to decrease mrna abundance of spp in the fetal musculus longissimus dorsi. moreover, we showed that the snp g.3836a>g leads to ubiquitous aberrant splicing of the first intron by generating a de novo and activating a cryptic splice acceptor site. aberrantly spliced transcripts comprise about half of the spp messages expressed by the g allele. both aberrant splice variants differ from the native transcript by insertions in the leader sequences which do not change the reading frame of spp  at the 3' terminal end of the first intron of the porcine spp we identified a unique, dually functional snp g.3836a>g. this snp affects the function of the spp gene at the dna level by affecting a c/ebpβ binding site and at the rna level by activating aberrant splicing of the first intron, and thus represents an interesting dna-marker to study phenotypic effects of spp dna-variation. 