RNA Targets of the Wilm's Tumor Protein in the Kidney

描述(申请人提供)：肾母细胞瘤基因(WT1)的产物对于肾脏的发育以及维持正常的肾小球结构和功能都是必需的。在成人肾脏中，WT1的表达仅限于足细胞，WT1蛋白对正常足细胞功能的重要性因WT1蛋白的突变而突显，WT1蛋白突变导致两种肾小球疾病，Denys-Drash和Frasier综合征。由于编码足细胞特异性蛋白的其他基因的突变也会导致肾小球疾病，最近的工作主要集中在足细胞中维持裂隙隔膜正常形成和功能的分子途径。WT1的两个剪接变体导致含有或排除位于第三和第四锌指之间的三个氨基酸的蛋白质似乎在表达细胞的核中具有不同的功能。WT1(-KTS)蛋白以高亲和力结合DNA序列，具有转录调节功能，而WT1(-KTS)蛋白似乎是一种具有转录后功能的RNA结合蛋白。在小鼠中，由于突变或定向敲除导致的KTS变体的丢失分别会导致人类的Frasier综合征或小鼠的肾小球硬化。因此，KTS变异体是维持正常足细胞生理所必需的。这些研究针对的假设是，WT1(KTS)通过与足细胞基因在细胞核中的转录本直接相互作用，影响足细胞基因的转录后加工。然而，足细胞中WT1(KTS)结合的RNA仍未确定，因此该蛋白亚型影响足细胞功能的机制仍不清楚。我们打算通过以下特定目的来寻找这些特定RNA靶标的身份：1)通过体外选择鉴定与WT1(KTS)高亲和力结合的保守序列。为了鉴定与WT1(KTS)高亲和力结合的特定RNA共识序列，我们将使用系统进化配体系统富集法(SELEX)。2)用免疫共沉淀法鉴定分化足细胞中与WT1(KTS)相关的活体RNA靶点。我们将通过免疫沉淀(IP)特定的核糖核蛋白(RNP)复合体，直接在培养的足细胞中寻找相关的RNA转录。微阵列将用于识别与WTI(KTS)特定共IP的特定RNA。

DESCRIPTION (provided by applicant): Products of the Wilm's tumor gene (WT1) are required for both the development of the kidney as well as maintenance of normal glomerular structure and function. In the adult kidney, WT1 expression is confined to the podocyte and the importance of WT1 protein for normal podocyte function is highlighted by the mutations in the WT1 protein that result in two glomerular diseases, Denys-Drash and Frasier syndromes. Because mutations in other genes encoding podocyte specific proteins also result in glomerular diseases, much recent work has focused on molecular pathways in podocytes that maintain proper formation and function of the slit diaphragm. Two splice variants of WT1 that result in proteins that contain [WT1(+KTS)] or exclude [WT1(-KTS)] three amino acids between the third and fourth zinc fingers appear to have distinct functions in the nucleus of expressing cells. The WT1(-KTS) protein binds DNA sequences with high affinity and functions as a transcriptional regulator, whereas WT1 (+KTS) appears to function as an RNA binding protein with post-transcriptional functions. Loss of the +KTS variant due to mutation or by directed knockout in mice of this variant results in Frasier syndrome in humans or glomerular sclerosis in mice, respectively. Thus, the + KTS variant specifically is required for maintenance of normal podocyte physiology. These studies are directed towards the hypothesis that WT1 (+KTS) affects post-transcriptional processing of podocyte genes through direct interactions with their transcripts in the nucleus. However, RNAs that are bound by WT1 (+KTS) in podocytes remain undefined and thus the mechanism by which this protein isoform influences podocyte function remains unknown. We intend to pursue the identity of these specific RNA targets through the following specific aims: 1) Identify conserved sequences that are bound with high affinity by WT1(+ KTS) using in vitro selection. In order to identify specific RNA consensus sequences that bind to WT1(+KTS) with high affinity we will use the method of Systematic Evolution of Ligands by Systematic Enrichment (SELEX). 2) Identify in vivo RNA targets associated with WT1 (+ KTS) in differentiated podocytes using co-immunoprecipitation assays. We will pursue relevant RNA transcripts directly in cultured podocytes by immunoprecipitation (IP) of specific ribonucleoprotein (RNP) complexes. Microarrays will be used to identity of specific RNAs that specifically co-IP with WTI(+KTS).