Wilms' Tumor 1
Created by Emily Lee
Wilms' Tumor 1 (WT1) gene encodes a protein that contains four zinc finger (zf) motifs at the C-terminus. It has an essential role in urogenital development. Mutation or deletion of the WT1 gene leads to Wilms tumor, Aniridia, Genitourinary anomalies, mental Retardation syndrome (WAGR), Isolated Diffuse Mesangial Sclerosis (IDMS), Denys-Drash syndrome (DDS), Fraser Syndrome (FS), and cancer (Barbaux et al., 1997; Davies et al., 1998).
Alternative splicing of the WT1 transcripts results in the production of proteins that differ by the presence or absence of three amino acids (lysine, threonine, and serine; KTS). The WT1 variant lacking these amino acids, WT1(-KTS), is well-characterized as a transcription factor and has a stronger affinity for DNA and the transcription factor SF1 as compared to the splicing factor U2AF65 (Davies, Bratt, & Hastie, 2000). In contrast, WT1(+KTS) functions mainly at the post-transcriptional level, as evidenced by its affinity for RNA and splicing factor U2AF65 (Davies et al., 1998; Davies et al., 2000). Substituting different amino acids in for lysine, threonine, and serine or increasing/decreasing the number of amino acids inserted does not affect the U2AF65-binding capacity (Davie et al., 2000). Despite these facts, the nucleotide sequence at the KTS insertion site is highly conserved suggesting something significant about WT1's sequence (Davies et al., 2000).
The ratio between the two isoforms is crucial, since mutations that alter the ratio lead to disease. Mutation after the KTS residues leads to a change in alternative splicing efficiency and loss of the +KTS isoform phenotype causing FS (Barbaux et al., 1997; Davies et al., 1998). Alternatively, a mutated -KTS isoform displays a +KTS phenotype, as evidenced by its affinity for U2AF65 (Davies et al., 1998). This excess of +KTS phenotype causes isoform imbalance and leads to DDS (Davies et al., 1998). Several years ago, Drs Hammarskjöld's and Rekosh's laboratories at UVa identified a novel RNA element, called constitutive transport element (CTE), that is essential for the export of unspliced mRNA in the genome of Mason-Pfizer Monkey Virus (MPMV). They were the first to show that WT1(+KTS) promotes the function of CTE at the post-transcriptional level (Bor et al., 2006). Its affinity for the splicing factor U2AF65 suggests its involvement with splicing machinery which would explain why it is able to export intron-retaining mRNA in the case of this discovery.
The structural features of the WT1 protein are essential to its function as a transcription factor and therefore a DNA-binding protein. It was discovered bound to DNA; however, the WT1 protein itself is composed of one main chain that contains 119 residues. This main chain is 35% helical, with 4 helices, and 15% beta sheet, with 8 strands.
WT1 protein associates with zinc ions to form the fingers that facilitate binding to DNA. Zinc Finger 1 is the product of the residues C-325, C-330, H-343, and H-347 interacting with zinc. Zinc Finger 2 is the product of the residues C-355, C-360, H-373, and H-377 interacting with zinc. Zinc Finger 3 is the product of the residues C-385, C-388, H-401, and H-405 interacting with zinc. And Zinc Finger 4 is the product of the residues C413, C418, H431, and H435 interacting with zinc.
In addition to the zinc fingers, there are key residues on the WT1 protein that hydrogen bond intramolecularly or with DNA. On zinc finger 2, S-367 and K-371 and H-373 and R-376 hydrogen bond intramolecularly; R-366 hydrogen bonds with DNA. On zinc finger 3, R394 and D396 and H397 and H401 form intramolecular hydrogen bonds. Lastly, R-424 on zinc finger 4 hydrogen bonds with DNA.
NCBI Blast showed the protein Krueppel-like Factor 4 (KL4) zinc finger domain as having 96% homology to Wilms Tumor Suppressor protein (WT1) zinc finger domain. In terms of primary structure, WT1 had a pI at ph: 10.18 and a molecular weight of 14377.4 g/mol (Gasteiger et al., 2005). KL4 had similar properties with a pI at pH: 9.66 and a molecular weight of 10528.8 g/mol (Gasteiger et al., 2005).
In terms of tertiary structure, TopMatch superimposed the two tertiary structures and found two alignment stretches of significant similarity. One of them, a 57-residue stretch, had 56% relative similarity and showed KL4 overlapping with 50% of WT1 (Sippl, 2008; Sippl & Wiederstein, 2008). Another, 53-residue stretch, had 52% relative similarity and showed KL4 overlapping with 46% of WT1 (Sippl, 2008; Sippl & Wiederstein, 2008). It is not surprising to see such similarity when one considers that both are found bound to DNA and both have a zinc finger domain.