558 - Proteomic Profiling Identifies a Role for WT1 in RNA Splicing and DNA Double-Strand Repair

Publication Number: 4546.558

Leah Bolsius, Boston Children's Hospital, Cambridge, MA, United States; Xin Wang, nationwide childrens hospital, Columbus, OH, United States; Lilian R Hiltebeitel, Icahn School of Medicine at Mount Sinai, New York, NY, United States; F. Melisa Bilgin, Nationwide Children's Hospital, Columbus, OH, United States; Wafaa Albalawy, Nationwide Children's Hospital, Columbus, OH, United States; Friedhelm Hildebrandt, Boston Children's Hospital / Harvard Medical School, Boston, MA, United States; Nina Mann, Nationwide Children's Hospital, Columbus, OH, United States; Rebecca H. Nelson, Boston Children's Hospital, Granite Bay, CA, United States

Background: The transcription factor Wilms’ Tumor 1 (WT1) is essential for nephrogenesis and podocyte maintenance. Pathogenic WT1 variants cause severe podocytopathies characterized by heavy proteinuria and progression to end-stage kidney disease. Over 36 WT1 isoforms have been described, with the two predominant forms, WT1(-)KTS and WT1(+)KTS, differing by three amino acids between the third and fourth zinc fingers. However, the distinct and overlapping functions of these isoforms, as well as the molecular effects of podocytopathy-causing patient variants, remain incompletely understood.

Objective: This study aims to define the interactome of the WT1 (-)KTS and (+)KTS isoforms using proximity-dependent labeling and to elucidate how the podocytopathy-causing N-terminal p.Val167Asp and C-terminal p.Arg467Trp variants alter these interactions.

Design/Methods: We generated stable hsPodocyte cell lines expressing wild-type and mutant WT1 proteins fused to a promiscuous biotin ligase (miniTurbo). Following biotin addition, proteins were harvested, and streptavidin-based pull-downs were performed. Biotinylated proteins from each cell line were analyzed separately by label-free mass spectrometry to define condition-specific WT1 interactomes.

Results: The WT1 (-)KTS wild type and (-)KTS p.Val167Asp mutant proteins fused to miniTurbo localized diffusely within the podocyte nucleus, whereas the WT1 (+)KTS wild type and WT1 (-)KTS p.Arg467Trp mutant proteins fused to miniTurbo localized to punctate nuclear domains. Our analyses showed that RNA splicing and DNA double-strand break repair proteins are enriched in proximity to both WT1(-)KTS and WT1 (+)KTS wild type proteins, suggesting a role for WT1 in these processes. No significant differences were observed between WT1(-)KTS and WT1(+)KTS interactomes. The WT1 p.Arg467Trp mutant protein showed disrupted interactions with putative transcriptional co-activators and repressors as well as DNA double-strand break repair proteins, while the WT1 p.Val67Asp mutant protein lost interactions with RNA splicing machinery.

Conclusion(s): Our proximity-dependent labeling studies indicate that, in addition to its role in transcriptional regulation, WT1 contributes to RNA splicing and DNA double-strand break repair. These functions appear essential for nephrogenesis and podocyte maintenance, as the N-terminal p.Val167Asp and C-terminal p.Arg467Trp variants each disrupt interactions within one of these pathways, leading to podocytopathy.

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