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Alternative splicing of exons encoding the calmodulin-binding domains and C termini of plasma membrane Ca(2+)-ATPase
T P Keeton1, S E Burk, G E Shull
1Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Ohio 45267-0524.
Abstract:
Rat plasma membrane Ca(2+)-ATPase (PMCA) mRNAs were examined by S1 nuclease protection (isoform 1) and polymerase chain reaction (isoforms 1, 2, 3, and 4) and the corresponding genes were analyzed to determine the tissue-specific splicing patterns involving exons encoding the calmodulin-binding domains and C termini. Splicing of PMCA1 involves a single 154-nucleotide exon that can be either included or excluded; when the exon is included four different splice donor sites, at positions 87, 114, 152, and 154, can be utilized. PMCA2 mRNAs are generated either by the inclusion of a 172-nucleotide exon, by the inclusion of both the 172-nucleotide exon and a 55-nucleotide exon, or by the exclusion of both exons. Four PMCA3 mRNAs arise by alternative splicing of a 154-nucleotide exon, in patterns that are analogous to those of PMCA1, and additional mRNAs are generated by the inclusion of a 68-nucleotide exon immediately before the 154-nucleotide exon. The simplest splicing pattern occurs in PMCA4, where a single 175-nucleotide exon is either included or excluded. The alternative mRNAs for each of the four genes are expressed in a tissue-specific manner and encode enzyme variants with different combinations of calmodulin-binding domains and C termini.
Insights
Rat plasma membrane Ca(2+)-ATPase (PMCA) gene expression shows tissue-specific alternative splicing. Different mRNA variants encode distinct enzyme structures, impacting cellular calcium regulation.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Plasma membrane Ca(2+)-ATPase (PMCA) pumps are crucial for maintaining calcium homeostasis.
- Alternative splicing of PMCA genes generates diverse protein isoforms with potentially varied functions.
Purpose of the Study:
- To investigate the tissue-specific alternative splicing patterns of rat PMCA isoforms (PMCA1-4).
- To understand how alternative splicing affects the calmodulin-binding domains and C termini of PMCA enzymes.
Main Methods:
- S1 nuclease protection assays for PMCA1 mRNA.
- Polymerase chain reaction (PCR) for PMCA isoforms 1-4.
- Analysis of gene sequences to identify splicing variations.
Main Results:
- PMCA1 and PMCA3 exhibit complex alternative splicing involving multiple donor sites and exon inclusion/exclusion.
- PMCA2 utilizes inclusion or exclusion of two distinct exons.
- PMCA4 shows a simpler pattern of including or excluding a single exon.
- All four PMCA isoforms demonstrate tissue-specific expression of alternative mRNA variants.
Conclusions:
- Rat PMCA genes undergo extensive tissue-specific alternative splicing.
- These splicing events generate distinct enzyme variants with unique calmodulin-binding domains and C termini.
- The diversity in PMCA isoforms likely contributes to specialized calcium handling in different tissues.