Contribution of individual zinc fingers of WT1 in RNA aptamer binding




Foster, Julie Lynne

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The WTI gene encodes for a transcription factor which is mutated in approximately 15% of sporadic Wilms' tumors (Orkin et al., 1984; Fearon et al., 1990). Mutations in WTI are also associated with a number of clinical disorders such as Denys-Drash and Frasier syndromes. which are distinguished by genitourinary malformation and kidney disease (McTaggart et al., 2001). The nucleic acid binding domain of WTI is comprised of four tandemly arranged C2H2 type zinc fingers (Haber et al., 1990; Rauscher et al.. 1990; Morris et al., 1991). The WT1 transcript is regulated by two different alternative splicing events. The first alternative splice introduces 17 amino acids between the proline-rich amino terminus and the zinc finger domain (Haber et al.. 1991). The second alternative splice inserts the amino acids KTS between zinc fingers 3 and 4 (Haber et al., 1991). The +KTS and -KTS isoforms of WT1 have different nucleic acid binding specificities. The .-KTS isoform readily binds to specific sequences in both DNA and RNA. while the +KTS isoform only binds to specific sequences in RNA (Zhai et al., 2001). The RNA binding capabilities of the +KTS variant combined with its presence in spliceosomes (Davies et al.. 1998) and nuclear poly(A)+ ribonucleoprotein (Ladomery et at., 1999), suggests that it is involved in RNA metabolism. WT1 has been shown to interact with RNA through the zinc finger domain (Caricasole et al, 1996), but as of yet. there is not a purified crystal structure of WT l interacting with RNA. There have been many experiments that have attempted to decipher the mechanism for WTI RNA binding (Caricasole et al, 1996; Bardeesy and Pelletier, 1998). One zinc finger knock-out experiment has proposed that it is zinc finger number 1 which plays the most important role in this RNA interaction (Caricasole et at, 1996) while another suggests that it is finger 4 (Bardeesy and Pelletier, 1998). In order to determine which zinc finger of WT 1 is the most crucial for RNA binding, WT 1 swap mutants and deletion mutants were created by PCR and a filter binding assay was employed to determine dissociation binding constants of the mutant proteins with the WT1-specific RNA aptamer Pe122. The mutant W12P8W4, which has finger 3 of WTI replaced with finger 8 of the zinc finger protein p43, demonstrated a relative affinity for Pe122 of less than 0.29. The deletion mutant WTlAF4 which had finger 4 of WTI deleted showed no affinity for Pe122 RNA, while the mutant Wp4 which had finger 4 of WTI replaced with finger 9 of p43 demonstrated a 10 times greater affinity for Pe122 than wild type WT1. These results suggest that finger 3 makes important residue-base contacts with the RNA and that perhaps finger 4 confers some sort of stability to the WT1-RNA complex.



Zinc-finger proteins, Nephroblastoma