Human Molecular Genetics Advance Access originally published online on July 6, 2005
Human Molecular Genetics 2005 14(16):2289-2303; doi:10.1093/hmg/ddi233
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Up-regulation of the ubiquitous alternative splicing factor Tra2ß causes inclusion of a germ cell-specific exon
1Institute of Human Genetics, University of Newcastle upon Tyne, International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK and 2Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP, 67404 Illkirch, C.U. Strasbourg, France
* To whom correspondence should be addressed. Tel: +44 1912418636; Fax: +44 1912418666; Email: j.venables{at}ncl.ac.uk
Received March 22, 2005; Accepted June 24, 2005
We have discovered a new exon of the homeodomain-interacting kinase HipK3 that incorporates a premature stop codon and is included only in the human testis. To investigate this, we tested the effects of transfecting cells with green fluorescent protein fusions of RNA-binding proteins implicated in spermatogenesis using a novel assay based on multi-fraction fluorescence-activated cell sorting (MF-FACS). This allows the effect of a controlled titration of any splicing factor on the splicing of endogenous genes to be studied in vivo. We found that Tra2ß recapitulates testis-specific splicing of endogenous HipK3 in a concentration-dependent manner and binds specifically to a long purine-rich sequence in the novel exon. This sequence was also specifically bound by hnRNP A1, hnRNP H, ASF/SF2 and SRp40, but not by 9G8. Consistent with these observations, in vitro studies showed that this sequence shifts splicing to a downstream 5' splice site within a heterologous pre-mRNA substrate in the presence of Tra2ß, ASF/SF2 and SRp40, whereas hnRNP A1 specifically inhibits this choice. By mutating the purine-rich sequence in the context of the HipK3 gene, we also show that it is the major determinant of Tra2ß- and hnRNP A1-mediated regulation. Tra2 is essential for sex determination and spermatogenesis in flies, and Tra2ß protein was most highly expressed in testis out of six mouse tissues, whereas hnRNP A1 is down-regulated during germ cell development. Therefore, our data imply an evolutionarily conserved role for Tra2 proteins in spermatogenesis and suggest that an elevated concentration of Tra2ß may convert it into a tissue-specific splicing factor.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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