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). ? ?
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