RNA Binding by the KTS Splice Variants of Wilms' Tumor Suppressor Protein WT1

Area of Science:

• Molecular Biology
• Structural Biology
• Genetics

Background:

• Wilms' tumor suppressor protein WT1 (WT1) is a transcription factor that regulates gene expression.
• WT1 utilizes its Cys2His2 zinc finger domains for DNA binding and is also implicated in post-transcriptional regulation via RNA binding.
• Two major WT1 splice variants exist: -KTS and +KTS, differing by the presence of a Lys-Thr-Ser (KTS) peptide.

Purpose of the Study:

• To elucidate the structural mechanisms by which different WT1 splice variants recognize and bind to both DNA and RNA.
• To understand the role of specific zinc fingers and the KTS insertion in DNA and RNA binding specificity and affinity.

Main Methods:

• Solution structure determination of the WT1 (-KTS) zinc finger domain complexed with a stem-loop RNA.
• Nuclear magnetic resonance (NMR) chemical shift mapping and relaxation analysis to study protein-RNA interactions.

Main Results:

• WT1 zinc fingers 1-3 bind to a widened RNA major groove, with fingers 2 and 3 specifically contacting an AUGG sequence.
• NMR analysis indicates similar RNA complex formation by both WT1 (-KTS) and (+KTS) variants involving fingers 1-3.
• Finger 4 interacts differently: weakly with RNA loop in -KTS, but this is lost in +KTS; finger 4 is crucial for high-affinity DNA binding, and KTS insertion abolishes DNA binding.

Conclusions:

• WT1 splice variants exhibit distinct binding mechanisms for DNA and RNA.
• The KTS insertion significantly alters WT1's DNA binding capability while maintaining similar RNA binding affinity.
• Understanding these differential binding mechanisms is crucial for comprehending WT1's diverse regulatory roles.