Characterization of an alternative splicing by a NAGNAG splice acceptor site in the porcine KIT gene

Abstract

The KIT gene has been shown to have multiple functions in hematopoiesis, melanogenesis, and gametogenesis. In addition, mutations of this gene cause pigmentation disorders in humans and mice and are responsible for coat color differences in pigs. While characterizing polymorphisms in the porcine KIT gene, we detected alternative splicing (AS) of the NAGNAG splice acceptor site at the boundary of intron 4 and exon 5. This AS event generated the E and I isoforms, characterized by insertion or deletion, respectively, of CAG at the borders of coding sequence. AS patterns measured in tissue samples from two randomly selected animals did not identified any tissue-specific outcomes. Analysis of AS patterns using three breeds demonstrated that Landrace and Large White pigs expressed both the E and I isoforms. In contrast, a subset of specimens from Korean Native Pigs (KNP) yielded a single I isoform. Alignment of the sequence from several species revealed that the region between the branch point sequence (BPS) and 3′ acceptor site is conserved. However, it is appeared that the selection of either the proximal or distal splice site varied between species. To test the breed specificity the NAGNAG splice acceptor site, we constructed two lineages of minigenes from KNP and Landrace pigs harboring breed-specific mutations. The minigene splicing assay demonstrated that both types of minigenes expressed both the E and I isoforms in two host cell lines, and no differences were detected in the AS pattern between the two breeds. We conclude that the AS at the NAGNAG splice acceptor site on intron 4/exon 5 in the porcine KIT gene is the result of noise selection at the splice site by the splicing machinery. Therefore, this AS event in the porcine KIT gene is unlikely to have any relationship with the coat color variations of Landrace and KNP breeds.

Divergent Evolutional Mode and Purifying Selection of theKITGene in European and Asian Domestic Pig Breeds

“…Up to now, 5 alleles have been identified in the Dominant White locus ( i , I , I P , I Be , and I Rn ) [ 7 , 11 , 33 , 34 ]. Furthermore, a large number of mutations were accumulated during the evolutional process either in coding regions or noncoding regions (many SNPs were found in intron4 and intron5, in the roan paper) [ 35 ]. None of these SNPs were identified as the causative mutation for the corresponding phenotype except the splice mutation in intron17 (G to A substitution), which contributed to the Dominant White phenotype with a normal copy of the KIT gene [ 5 ].…”

Cloning and association analysis of KIT and EDNRB polymorphisms with dominant white coat color in the Chinese raccoon dog (Nyctereutes procyonoides procyonoides)

“…In addition, an alternative splicing site, because of the NAGNAG splice acceptor sites at the boundary of exons 4 and 5 of the KIT gene, was identified and further confirmed by cloning and sequencing in both individuals (Figure 3). Two transcripts were designated as E and I, respectively, according to the description of Kim et al (2011). Because 3 SNPs were exclusively found in the dominant white individual, we obtained additional genotype information for these SNPs in the remaining 18 individuals.…”