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Lookup NU author(s): Dr Julian Venables, Caroline Dalgliesh, Professor David Elliott
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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 Tra2β 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 Tra2β, 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 Tra2β- and hnRNP A1-mediated regulation. Tra2 is essential for sex determination and spermatogenesis in flies, and Tra2β 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 Tra2β may convert it into a tissue-specific splicing factor. © The Author 2005. Published by Oxford University Press. All rights reserved.
Author(s): Venables JP, Bourgeois CF, Dalgliesh C, Kister L, Stevenin J, Elliott DJ
Publication type: Article
Publication status: Published
Journal: Human Molecular Genetics
Year: 2005
Volume: 14
Issue: 16
Pages: 2289-2303
ISSN (print): 0964-6906
ISSN (electronic): 1460-2083
Publisher: Oxford University Press
URL: http://dx.doi.org/10.1093/hmg/ddi233
DOI: 10.1093/hmg/ddi233
PubMed id: 16000324
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