Anomalies of the urogenital system: role of WT1 isoforms and partners – Anaform

The Wilms' tumour suppressor WT1 is a key regulator of development and mutations in this gene have been identified in a wide range of genetic diseases, including disorders of sex development (DSD), abnormalities in kidney formation, pathologies affecting the diaphragm, and cancers. Paradoxically, the molecular and developmental role of WT1 in many of these processes is still poorly understood. This is due to the complexity of this gene, which can produce a variety of protein isoforms. The two major alternatively spliced isoforms of WT1 differ in the presence or absence of the three amino acids KTS, giving rise to variants known as +KTS or -KTS. The importance of these isoforms is highlighted by point mutations that disrupt the normal ratio of +KTS and -KTS variants, leading to Frasier syndrome, a congenital disease characterized by XY sex reversal and glomerulopathy. The aim of the current project (Anaform) is to elucidate the molecular roles of these two isoforms in urogenital development and related disorders.

Through genetic analysis, we have recently shown that -KTS acts as the elusive ovarian determinant in mammals. However, the molecular functions of -KTS in gonadal differentiation and the broader roles of +KTS variants during urogenital development remain enigmatic. Here, we aim to leverage our expertise in sex determination, kidney development and WT1 function to elucidate the mechanism of action of these two isoforms under normal and pathological conditions. We will use genetic models expressing only one isoform in combination with next-generation ChIP and RIP sequencing techniques to identify their specific functions and their DNA and RNA targets during gonadal development and nephrogenesis.

Previous work has identified WTAP (Wilms’ Tumor Associated Protein) as a direct interaction partner of WT1. WTAP plays a crucial role in RNA methylation, as it has been shown to stabilize the methylation complex and promote the addition of a methyl group to the targeted adenosine at position N6 in mRNAs. RNA methylation has emerged as an important regulatory mechanism that impacts post-transcriptional processes such as alternative splicing, transcript stability and translation efficiency. Our previous results have shown that WT1 is involved in regulating the stability of some RNAs and we hypothesize cooperation between WTAP and WT1 in jointly regulating the methylation of specific RNA targets. To test this hypothesis, we conduct genetic experiments to assess the role of WTAP in urogenital development comparing the observed phenotype with that of WT1 mutants. Subsequently, we will analyze the methylation profiles of the different mutants, delineate the interactions between WT1 isoforms and m6A RNA methylation, and integrate these factors into the genetic network governing organ differentiation.

Taken together, our project will elucidate the molecular processes governing urogenital development that are regulated by WT1 and m6A RNA methylation, and uncover novel pathogenic mechanisms associated with WT1 in human patients. Therefore, our findings hold significance not only for fundamental scientific research but also for clinical counseling. Ultimately, they may contribute to the development of innovative therapeutic strategies for WT1-related diseases.