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. 2010 May 5;3:123.
doi: 10.1186/1756-0500-3-123.

A Study of Alternative Splicing in the Pig

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Free PMC article

A Study of Alternative Splicing in the Pig

Ann-Britt Nygard et al. BMC Res Notes. .
Free PMC article

Abstract

Background: Since at least half of the genes in mammalian genomes are subjected to alternative splicing, alternative pre-mRNA splicing plays an important contribution to the complexity of the mammalian proteome. Expressed sequence tags (ESTs) provide evidence of a great number of possible alternative isoforms. With the EST resource for the domestic pig now containing more than one million porcine ESTs, it is possible to identify alternative splice forms of the individual transcripts in this species from the EST data with some confidence.

Results: The pig EST data generated by the Sino-Danish Pig Genome project has been assembled with publicly available ESTs and made available in the PigEST database. Using the Distiller package 2,515 EST clusters with candidate alternative isoforms were identified in the EST data with high confidence. In agreement with general observations in human and mouse, we find putative splice variants in about 30% of the contigs with more than 50 ESTs. Based on the criteria that a minimum of two EST sequences confirmed each splice event, a list of 100 genes with the most distinct tissue-specific alternative splice events was generated from the list of candidates. To confirm the tissue specificity of the splice events, 10 genes with functional annotation were randomly selected from which 16 individual splice events were chosen for experimental verification by quantitative PCR (qPCR). Six genes were shown to have tissue specific alternatively spliced transcripts with expression patterns matching those of the EST data. The remaining four genes had tissue-restricted expression of alternative spliced transcripts. Five out of the 16 splice events that were experimentally verified were found to be putative pig specific.

Conclusions: In accordance with human and rodent studies we estimate that approximately 30% of the porcine genes undergo alternative splicing. We found a good correlation between EST predicted tissue-specificity and experimentally validated splice events in different porcine tissue. This study indicates that a cluster size of around 50 ESTs is optimal for in silico detection of alternative splicing. Although based on a limited number of splice events, the study supports the notion that alternative splicing could have an important impact on species differentiation since 31% of the splice events studied appears to be species specific.

Figures

Figure 1
Figure 1
Relationship between number of ESTs/cluster and percentage of clusters containing putative alternative splice events. The number of ESTs in clusters containing alternative splice events (with four as the minimum number of EST in a cluster) is shown at the x axis. The numbers at the y axis represents the percentages of clusters containing alternative splice events compared to the total number of clusters. The numbers at the data points refer to the bin of cluster size.
Figure 2
Figure 2
This figure illustrates how tissue-specific and tissue-restricted expression pattern of two splice events have been defined in the study. A) tissue-specific expression pattern is defined as one splice variant being dominant in some tissues whereas the other splice variant is dominant in other tissues. B) tissue-restricted expression is defined as expression pattern where the ratio between two splice variants is significantly different in different tissues, however always with the same splice variant as the dominant one.
Figure 3
Figure 3
Gene structure and QPCR data for genes with validated tissue-specific or tissue-restricted alternatively spliced variants. QPCR data presented as changes in fold ratios between expressions of two splice variants in different adult pig tissues for the genes: ANAPC11, ATP5S, AUH, CEP27, EIF4E2 and IGF2. Data are normalised with a reference gene index. Values are geometric means ± Geometric S.E.M.; N = 4 animals. The highest expressed splice variant in the majority of the tissues is used as the numerator and the less expressed splice variant is used as the denominator. The highest expressed splice variant is shown in the upper part of the "Gene" column and the less expressed splice variant is showed in the lower part of the "Gene" column. *sequence that have not been defined as exon in any other species.
Figure 4
Figure 4
Gene structure and QPCR data for genes that are neighed expressed in a tissue-specific manner nor in a tissue-restricted manner, however, with validated alternatively spliced variants. QPCR data presented as changes in fold ratios between expressions of two splice variants in different adult pig tissues for the genes: BSCL2, HNRPLL, PPP3CC and RBM4. Data are normalised with a reference gene index. Values are geometric means ± Geometric S.E.M.; N = 4 animals. The highest expressed splice variant in the majority of the tissues is used as the numerator and the less expressed splice variant is used as the denominator. The highest expressed splice variant is shown in the upper part of the "Gene" column and the less expressed splice variant is showed in the lower part of the "Gene" column. *sequence that have not been defined as exon in any other species.

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