The ratio of ±KTS splice variants of the Wilms’ tumour suppressor protein WT1 mRNA is determined by an intronic enhancer

Abstract

Alternative splicing of the primary transcript of the Wilms’ tumour suppressor gene WT1 involving 2 overlapping 5′ splice sites at the end of exon 9 results in the insertion of 2 amino acids (KTS) between zinc fingers 3 and 4 of the protein. The presence or absence of these 3 amino acids has consequences for DNA binding affinity, protein–protein interactions, and subnuclear localization. Disruption of the characteristic +KTS to –KTS ratio of mRNA isoforms as a result of mutations in the +KTS splice site results in Frasier syndrome. Mutational analysis of a WT1 minigene construct was carried out to search for sequences that regulate the ±KTS alternative splicing event. A strong pyrimidine-rich intronic enhancer that increases the use of the +KTS splice site was identified. Cross-linking experiments with nuclear extracts demonstrated that this enhancer specifically binds a protein with a molecular mass of 42 ± 2 kDa. One candidate for this trans-factor is the splicing regulator TIA-1, which binds to the pyrimidine-rich enhancer in the primary transcript from the minigene construct and increases the ±KTS splicing ratio. The observation that TIA-1 and WT1 are both involved in apoptosis supports our proposal for a functional link between these proteins.