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The calcium channel beta2 (CACNB2) subunit repertoire in teleosts.

Ebert AM, McAnelly CA, Srinivasan A, Mueller RL, Garrity DB, Garrity DM - BMC Mol. Biol. (2008)

Bottom Line: Moreover, phenotypes may be obscured by secondary effects of hypoxia.Moreover, a different subset of spliced beta2 transcript variants is detected in the embryonic heart compared to the adult.These studies refine our understanding of beta2 subunit diversity arising from alternative splicing, and provide the groundwork for functional analysis of beta2 subunit diversity in the embryonic heart.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Colorado State University, Fort Collins, CO 80523, USA. amebert@lamar.colostate.edu

ABSTRACT

Background: Cardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary beta subunits to chaperone the pore-forming alpha subunit to the plasma membrane, and to modulate channel electrophysiology 1. Several beta subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse beta2, but not in the other three beta family members, are embryonic lethal at E10.5 due to defects in cardiac contractility 2. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of beta subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of beta2 subunits in zebrafish and other teleosts.

Results: Cloning of two zebrafish beta2 subunit genes (beta2.1 and beta2.2) indicated they are membrane-associated guanylate kinase (MAGUK)-family genes. Zebrafish beta2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the "core" of MAGUK proteins, but beta2.2 is much more divergent in sequence than beta2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both beta2 genes, alternative short ATG-containing first exons are separated by some of the largest introns in the genome, suggesting that individual transcript variants could be subject to independent cis-regulatory control. In the Tetraodon nigrovidis and Fugu rubripes genomes, we identified single beta2 subunit gene loci. Comparative analysis of the teleost and human beta2 loci indicates that the short 5' exon sequences are highly conserved. A subset of 5' exons appear to be unique to teleost genomes, while others are shared with mammals. Alternative splicing is temporally and spatially regulated in embryo and adult. Moreover, a different subset of spliced beta2 transcript variants is detected in the embryonic heart compared to the adult.

Conclusion: These studies refine our understanding of beta2 subunit diversity arising from alternative splicing, and provide the groundwork for functional analysis of beta2 subunit diversity in the embryonic heart.

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Large intron sizes in β2 genomic loci. Boxes denote the location of exons that encode the 5' region of β2 subunit genes. The intervening regions denote the relative sizes of introns, labeled in bp. The scale is expanded for the teleost gene diagrams relative to human. The various N-terminal (grey) exons splice to the solid black exon, which begins with amino acids GSAD...The following genomic contigs were used for the analysis: Human β2 (NT_008705.15), Danio β2.2 (NC_007113), Danio β2.1 (NC_007133), Tetraodon β2 (CAAE01015017.1), Fugu β2 (CAAB01000004.1), Gasterosteus β2 (AANH01005391.1, using the EST DW608729, which resembles zebrafish β2.2).
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Figure 5: Large intron sizes in β2 genomic loci. Boxes denote the location of exons that encode the 5' region of β2 subunit genes. The intervening regions denote the relative sizes of introns, labeled in bp. The scale is expanded for the teleost gene diagrams relative to human. The various N-terminal (grey) exons splice to the solid black exon, which begins with amino acids GSAD...The following genomic contigs were used for the analysis: Human β2 (NT_008705.15), Danio β2.2 (NC_007113), Danio β2.1 (NC_007133), Tetraodon β2 (CAAE01015017.1), Fugu β2 (CAAB01000004.1), Gasterosteus β2 (AANH01005391.1, using the EST DW608729, which resembles zebrafish β2.2).

Mentions: Remarkably, the introns that separate the first few 5' exons of the β2 loci are among the largest introns known in the zebrafish, pufferfish or human genomes. Each Tetraodon and Fugu β2 locus contains one intron over 10 kb, a size that ranks within the top 5% of the largest introns in pufferfish [51]. In addition, the two pufferfish genes, the stickleback β2.2-like gene, and both zebrafish β2.2 and β2.1 each contain introns of > 5000 bp that separate 5'-most exons (Fig. 5). In pufferfish, the modal value for intron size is 79 bp, with 75% of introns < 425 bp in length [51]. Given the compact nature of the pufferfish genomes [52], it was not surprising that introns of the human β2 locus exceeded the size of those in fish. Nevertheless, the trend of megasized introns in the N-terminus of β2 loci extends to the human genome. Three giant introns ranging in size from 60–100 kb each separate the human β2 exons in the N-terminus. Thus, when human exon 3 is spliced to exon 7 (beginning GSAD...), the splicing machinery must exclude over 250 kb of intronic sequence. In contrast, introns in the remaining part of the human β2 gene averaged 3261 bp in size. In humans, the mean size of introns adjoining coding sequences is 3749 bp, but 75% of introns are smaller than 2609 bp [51,53,54]. A recent analysis of the genomes of Arabidopsis thaliana, Drosophila, mouse and human indicated that the median sizes of introns separating 5' UTR (non-coding) sequence are significantly larger than introns separating coding sequence [54]. Even so, the median size of human 5' UTR introns (8223 bp, [54]) is still much smaller than the human 5' β2 introns, which separate coding sequences.


The calcium channel beta2 (CACNB2) subunit repertoire in teleosts.

Ebert AM, McAnelly CA, Srinivasan A, Mueller RL, Garrity DB, Garrity DM - BMC Mol. Biol. (2008)

Large intron sizes in β2 genomic loci. Boxes denote the location of exons that encode the 5' region of β2 subunit genes. The intervening regions denote the relative sizes of introns, labeled in bp. The scale is expanded for the teleost gene diagrams relative to human. The various N-terminal (grey) exons splice to the solid black exon, which begins with amino acids GSAD...The following genomic contigs were used for the analysis: Human β2 (NT_008705.15), Danio β2.2 (NC_007113), Danio β2.1 (NC_007133), Tetraodon β2 (CAAE01015017.1), Fugu β2 (CAAB01000004.1), Gasterosteus β2 (AANH01005391.1, using the EST DW608729, which resembles zebrafish β2.2).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2365960&req=5

Figure 5: Large intron sizes in β2 genomic loci. Boxes denote the location of exons that encode the 5' region of β2 subunit genes. The intervening regions denote the relative sizes of introns, labeled in bp. The scale is expanded for the teleost gene diagrams relative to human. The various N-terminal (grey) exons splice to the solid black exon, which begins with amino acids GSAD...The following genomic contigs were used for the analysis: Human β2 (NT_008705.15), Danio β2.2 (NC_007113), Danio β2.1 (NC_007133), Tetraodon β2 (CAAE01015017.1), Fugu β2 (CAAB01000004.1), Gasterosteus β2 (AANH01005391.1, using the EST DW608729, which resembles zebrafish β2.2).
Mentions: Remarkably, the introns that separate the first few 5' exons of the β2 loci are among the largest introns known in the zebrafish, pufferfish or human genomes. Each Tetraodon and Fugu β2 locus contains one intron over 10 kb, a size that ranks within the top 5% of the largest introns in pufferfish [51]. In addition, the two pufferfish genes, the stickleback β2.2-like gene, and both zebrafish β2.2 and β2.1 each contain introns of > 5000 bp that separate 5'-most exons (Fig. 5). In pufferfish, the modal value for intron size is 79 bp, with 75% of introns < 425 bp in length [51]. Given the compact nature of the pufferfish genomes [52], it was not surprising that introns of the human β2 locus exceeded the size of those in fish. Nevertheless, the trend of megasized introns in the N-terminus of β2 loci extends to the human genome. Three giant introns ranging in size from 60–100 kb each separate the human β2 exons in the N-terminus. Thus, when human exon 3 is spliced to exon 7 (beginning GSAD...), the splicing machinery must exclude over 250 kb of intronic sequence. In contrast, introns in the remaining part of the human β2 gene averaged 3261 bp in size. In humans, the mean size of introns adjoining coding sequences is 3749 bp, but 75% of introns are smaller than 2609 bp [51,53,54]. A recent analysis of the genomes of Arabidopsis thaliana, Drosophila, mouse and human indicated that the median sizes of introns separating 5' UTR (non-coding) sequence are significantly larger than introns separating coding sequence [54]. Even so, the median size of human 5' UTR introns (8223 bp, [54]) is still much smaller than the human 5' β2 introns, which separate coding sequences.

Bottom Line: Moreover, phenotypes may be obscured by secondary effects of hypoxia.Moreover, a different subset of spliced beta2 transcript variants is detected in the embryonic heart compared to the adult.These studies refine our understanding of beta2 subunit diversity arising from alternative splicing, and provide the groundwork for functional analysis of beta2 subunit diversity in the embryonic heart.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Colorado State University, Fort Collins, CO 80523, USA. amebert@lamar.colostate.edu

ABSTRACT

Background: Cardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary beta subunits to chaperone the pore-forming alpha subunit to the plasma membrane, and to modulate channel electrophysiology 1. Several beta subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse beta2, but not in the other three beta family members, are embryonic lethal at E10.5 due to defects in cardiac contractility 2. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of beta subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of beta2 subunits in zebrafish and other teleosts.

Results: Cloning of two zebrafish beta2 subunit genes (beta2.1 and beta2.2) indicated they are membrane-associated guanylate kinase (MAGUK)-family genes. Zebrafish beta2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the "core" of MAGUK proteins, but beta2.2 is much more divergent in sequence than beta2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both beta2 genes, alternative short ATG-containing first exons are separated by some of the largest introns in the genome, suggesting that individual transcript variants could be subject to independent cis-regulatory control. In the Tetraodon nigrovidis and Fugu rubripes genomes, we identified single beta2 subunit gene loci. Comparative analysis of the teleost and human beta2 loci indicates that the short 5' exon sequences are highly conserved. A subset of 5' exons appear to be unique to teleost genomes, while others are shared with mammals. Alternative splicing is temporally and spatially regulated in embryo and adult. Moreover, a different subset of spliced beta2 transcript variants is detected in the embryonic heart compared to the adult.

Conclusion: These studies refine our understanding of beta2 subunit diversity arising from alternative splicing, and provide the groundwork for functional analysis of beta2 subunit diversity in the embryonic heart.

Show MeSH
Related in: MedlinePlus