Structural and Functional Studies of Wilms Tumour Protein - WT1; Novel Insights into Functionality of Zinc Finger Proteins

Sammanfattning: The molecular process under which message in DNA is relayed onto RNA is called transcription, and it accomplished through involvement of so-called transcription factors. Zinc finger proteins are one of the most abundant classes of regulatory proteins in eukaryotic cells where they play central roles in a variety of cellular activities. A classical zinc finger is a small protein motif with a simple beta-beta-alpha secondary structure stabilized by a zinc ion that is coordinated by two conserved cysteines and two histidines. Wilms tumour protein (WT1) is a transcription factor, which in normal cells regulates a vast network of genes during development of the kidney and the genitourinary system; its malfunction may lead to serious abnormalities such as WAGR syndrome, Denys Drash syndrome and Frasier syndrome. WT1 has been interesting for researchers as a zinc finger protein, and as a transcription factor having both oncogenic and tumor suppressor functions. WT1 contains four C2H2-type zinc fingers and it specifically binds to GC-rich sequences in the promoter regions of its target genes, which are either up- or down-regulated. It has a dual functionality: it can bind specifically to DNA and RNA. Alternative splicing of the immature WT1 mRNA at exon 5 and exon 9 produces 4 distinct isoforms with 17 and 3 amino acids (KTS) insertions, respectively. The KTS insertion into the linker between zinc fingers 3 and 4 abrogates binding to the DNA. On the other hand, it differentially affects affinity of WT1 for its single-stranded RNA targets ? this is not yet understood. Thus, two unique features of WT1 differentiate it from other zinc finger proteins of the same class: the KTS insertion and an unconventional amino acid composition of its zinc finger 1. We have undertaken an interdisciplinary approach in order to better understand the nucleic acid-binding features of WT1. In the Paper I we describe a study where we express, purify the WT1 protein and make its biochemical characterization. In Paper II a surface plasmon resonance-based study addresses the DNA-binding properties of WT1. Paper III describes an analogous study where RNA-binding properties of WT1 are investigated. Finally, Paper IV is a Bacterial 1-Hybrid System-based study that is directed towards elucidation of the DNA-binding preferences of zinc finger 1. Interesting conclusions about the functionality of the KTS insertion and the zinc finger 1 are reached.