1. Discovering Nuclear Vs. Cytoplasmic Roles of ESRP1 and ESRP2 RNA Splicing Regulators in Craniofacial Development

PURPOSE: Disruption of epithelial splicing regulators ESRP1 and ESRP2 contributes to cleft lip and palate. ESRP1/2 are crucial in the periderm, a transient epithelial layer facilitating lip and palate fusion during development. ESRP1 harbors a nuclear localization signal (NLS) essential for its function. The roles of nuclear and cytoplasmic localization in craniofacial morphogenesis remain unclear. METHODS: Zebrafish esrp1/2 vectors with GFP tags were created. The NLS was altered to target Esrp1 to the nucleus and Esrp2 to the cytoplasm. These vectors were introduced into Py2T cells. Immunofluorescence visualized Esrp1/2 in Py2T and HEK293 cells. CRISPR/Cas9 introduced Esrp1/2 mutations. Wildtype and knockout cells were transfected with Esrp1/2 for RT-PCR analysis. In esrp1/2 mutant zebrafish, Esrp1/2 isoforms were injected. RESULTS: Esrp1 is cytoplasmic in zebrafish and mouse, while in mouse and human, it also localizes to the nucleus. Esrp2 is nuclear in zebrafish, mouse, and human. RT-PCR analysis for splicing targets (e.g., Arhgef11) showed distinct splicing patterns in Esrp1/2 knockouts, indicating different roles through subcellular localization. In vivo rescue showed NLS is required for esrp2 function, while cytoplasmic esrp1 still achieved rescue, possibly via tp63 regulation. CONCLUSION: Cytoplasmic Esrp1 and nuclear Esrp2 are crucial for periderm function, while cytoplasmic Esrp2 is dispensable. Correct isoform localization is essential, with an unexpected role for cytoplasmic Esrp1 involving tp63 regulation. Understanding these mechanisms may lead to therapies for preventing orofacial clefts.