Analysis of two transcript isoforms of vacuolar ATPase subunit H in mouse and zebrafish.

ATP6V1H encodes the subunit H of vacuolar ATPase (V-ATPase) and has been recently proved to regulate osteoclast function. The alternative splicing of ATP6V1H gene results in two isoforms, and it is not clear whether and how the two isoforms function differently. In this report, we used bioinformatics methods to compare the differences of two isoforms in different species. The distributions and amounts of two isoforms were analyzed in eleven kinds of mouse tissues and mouse osteoclasts using RT-PCR, Q-PCR, western blot and immunohistochemical staining methods, respectively. In order to observe the in vivo biological differences of two isoforms during development, the zebrafish mRNA of two wild type atp6v1h transcripts as well as their mutant forms were also injected into zebrafish embryos, respectively. Bioinformatic analysis revealed that two isoforms were quite different in many ways, especially in protein size, internal space, phosphorylation state and H-bond binding. The amounts of two transcripts and the ratio of long and short transcript varied a lot from tissue to tissue or cell to cell, and osteoclasts were the cells only expressing long isoform among the tissues or cells we detected. The in vivo selective expression of two subunit H splice variants showed their different effects on the craniofacial development of zebrafish. The short isoform reduced the size of zebrafish head and did not play a complete function compared with the long isoform. We propose that long isoform of subunit H is necessary for the normal craniofacial bone development and the lack of short transcript might be necessary for the normal osteoclastic function.

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