Internal Translation Initiation Generates Novel WT1 Protein Isoforms with Distinct Biological Properties

Abstract

The Wilms' tumor 1 gene, WT1, is homozygously mutated in a subset of Wilms' tumors. Heterozygous mutations in WT1 give rise to congenital anomalies. During embryogenesis, WT1 is expressed mainly in the kidneys, uterus, and testes.Alternative splicing of the WT1 mRNA results in synthesis of four main WT1 protein isoforms with molecular masses of 52-54 kDa. In addition, translation initiation at a CUG upstream of the initiator AUG generates four larger WT1 proteins of 60 -62 kDa.We describe here the existence of novel WT1 isoforms and demonstrate that they are derived from translation initiation at the second in-frame AUG of the WT1 mRNA. These N-terminally truncated WT1 proteins of 36 -38 kDa can be detected in several cell lines, mouse testes, and Wilms' tumor specimens. They can bind to DNA and direct transcription from reporter constructs. The shorter WT1 protein lacking the two splice inserts has a greater transcription activation potential than the corresponding main WT1 protein isoform but shows no transcription repression potential. Overexpression of full-length or N-terminally truncated WT1 efficiently induces apoptosis. These data show that additional WT1 isoforms with distinct transcription-regulatory properties exist, which further increases the complexity of WT1 expression and activity. Wilms' tumor (WT)1 is a pediatric kidney malignancy that affects 1 in 10,000 children and is thought to arise from pluripotent renal stem cells that fail to differentiate properly (1). Mutations in the WT1 gene are found in about 15% of all Wilms' tumors (2). Consequently, WT1 has been classified as a tumor suppressor gene. In addition to its involvement in Wilms' tumor, the WT1 gene is heterozygously mutated in several syndromes, all of which include malformations of the urogenital system (2, 3). An essential role for the WT1 gene product in urogenital development is further underscored by the finding that WT1 knockout mice fail to develop kidneys and gonads (4). In accordance with the phenotype of WT1-null mice, expression of WT1 is found mainly in kidneys, ovaries, and testes (5).The WT1 gene contains 10 exons and spans about 50 kilobases on chromosome 11p13. Exons 5 and 9 are differentially spliced, ultimately giving rise to four different protein isoforms with molecular masses ranging from 52 to 54 kDa. WT1(Ϫ/Ϫ) lacks both splice inserts, WT1(ϩ/ϩ) accommodates the 17-amino acid and the 3-amino acid KTS splice inserts, and WT1(ϩ/Ϫ) and WT1(Ϫ/ϩ) contain either the 17-amino acid or the KTS splice insert (Ref. 6; see Fig. 1). In addition to these WT1 isoforms, the existence of larger WT1 proteins, which result from translation initiation at an in-frame CUG upstream of the initiator AUG, has been reported (7). A further level of complexity is added by RNA editing at position 839 of the WT1 mRNA, which replaces leucine 280 in WT1 proteins by proline (8). The WT1 gene may thus produce 16 different protein isoforms.Exons 7-10 of the WT1 gene encode four zinc fingers of the Krü ppel type (9, 10), which can mediate binding ...

Wnt-4 regulation by the Wilms' tumour suppressor gene, WT1

“…The WT1 gene encodes multiple nuclear proteins by virtue of three di erent transcription start sites, RNA editing of a single nucleotide and two alternatively spliced regions; exon 5 (17 amino acids) and an alternate splice donor site after exon 9 Sharma et al, 1994;Bruening and Pelletier, 1996;Scharnhorst et al, 1999). The latter results in the addition of three amino acids (KTS) between exons 9 and 10.…”

Anlaysis of complementary expression profiles following WT1 induction versus repression reveals the cholesterol/fatty acid synthetic pathways as a possible major target of WT1

“…The WT1 gene actually encodes multiple protein isoforms as a result of alternate splicing and several transcriptional start sites Bruening and Pelletier, 1996;Scharnhorst et al, 1999). The most commonly described isoforms are four 52/54 kDa proteins that vary due to the presence of exon 5 (51 bp/17 amino acids) and the use of an alternative splice donor site in exon 9 resulting in the insertion of 9 bp that encodes the amino-acids lysinethreonine-serine (KTS).…”

Clinical Pictures and Novel Mutations ofWT1-Associated Denys–Drash Syndrome in Two Chinese Children

“…Thirty-two different transcripts of the gene have been predicted due to alternative translation start sites, alternative splicing, and RNA editing. 7 Notably, four major transcripts, due to alternative splicing of exon 5 and to the last nine nucleotides of exon 9, are expressed among various tissues in which WT1 is expressed. 7,8 The distinct polypeptides translated by alternative transcripts may have different targets and functions; hence, they may contribute to the role of WT1 as a regulator of cellular development and survival in a wide range of biological functions.…”