Overlapping RNA and DNA binding domains of the wt1 tumor suppressor gene product.

The Wilms' tumour suppressor gene (wt1) is mutated in a subset of patients with Wilms' tumour and has a critical role in urogenital development. wt1 encodes a zinc finger transcription factor which regulates expression of several genes involved in cellular proliferation and differentiation. Although a number of studies have characterized the DNA binding properties of the WT1 protein, recent evidence has suggested that WT1 may also have a role in RNA metabolism. We have used an RNA selection method to identify WT1 binding ligands from a random RNA pool. Three groups of RNA ligands specifically recognized by WT1 were identified. Mutational analysis pinpointed ribonucleotide sequences critical for binding. Analysis of truncated WT1 proteins demonstrated that three of four zinc fingers were necessary for RNA-protein interaction. The naturally occurring WT1 isoforms with insertion of lysine, threonine and serine between zinc fingers three and four were unable to bind the selected RNAs. The selected RNA ligands competed with the cognate WT1 DNA binding site for complex formation with WT1. Our findings suggest potential cellular RNA target sequences for WT1 and provide tools for studying the structural and functional properties of this tumour suppressor protein.肾母细胞瘤（Wilms' tumour）抑癌基因wt1在部分肾母细胞瘤患者中发生突变，且在泌尿生殖系统发育过程中发挥关键调控作用。wt1编码一种锌指转录因子，可调控多种参与细胞增殖与分化的基因的表达。尽管已有多项研究对WT1蛋白的DNA结合特性开展了表征工作，但近期研究证据表明，WT1或许还在RNA代谢过程中扮演重要角色。本研究采用RNA筛选法，从随机RNA库中筛选可与WT1结合的配体，最终鉴定出三类可被WT1特异性识别的RNA配体。通过突变分析精准确定了WT1结合所必需的核糖核苷酸序列；对截短型WT1蛋白的分析显示，四个锌指结构中的三个是RNA-蛋白质相互作用所必需的。在锌指3与锌指4之间插入赖氨酸、苏氨酸和丝氨酸的天然WT1同工型，无法结合本次筛选得到的RNA配体。本次筛选得到的RNA配体可与同源WT1 DNA结合位点竞争结合WT1复合物。本研究结果为WT1潜在的细胞内RNA靶序列提供了理论线索，同时也为研究该抑癌蛋白的结构与功能特性提供了有效的研究工具。