Dual effect of a single nucleotide polymorphism in the first intron of the porcine Secreted phosphoprotein 1 gene: allele-specific binding of C/EBP beta and activation of aberrant splicing

BACKGROUND: Secreted phosphoprotein 1 (SPP1 or Osteopontin, OPN) is a multifunctional matricellular glycoprotein involved in development and regeneration of skeletal muscle. Previously, we have demonstrated that porcine SPP1 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 (SNP; [GenBank:M84121]: g.1804C>T and g.3836A>G). We focused our investigation on the SNP g.3836A>G, because in silico analysis and knowledge about the regulation of SPP1 suggested an effect of this SNP on a CCAAT/enhancer binding protein beta (C/EBPβ) responsive transcriptional enhancer. RESULTS: Using electrophoretic mobility shift assay we demonstrated that, similar to human SPP1, the 3' terminal end of the first intron of porcine SPP1 harbors a C/EBPβ binding site and showed that this binding site is negatively affected by the mutant G allele. Genotyping of 48 fetuses per breed revealed that the G allele segregated exclusively in Duroc fetuses with a frequency of 57 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 SPP1 in the fetal musculus longissimus dorsi (~1.3 fold; P ≥ 0.1). 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 SPP1 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 SPP1. CONCLUSION: At the 3' terminal end of the first intron of the porcine SPP1 we identified a unique, dually functional SNP g.3836A>G. This SNP affects the function of the SPP1 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 SPP1 DNA-variation.