Work

41. Discovering Nuclear Vs. Cytoplasmic Roles of Esrp1 and Esrp2 RNA Splicing Regulators in Craniofacial Development.

Year: 2025

Type: article

Abstract: PURPOSE: ESRP1 and ESRP2 are epithelial-specific regulators of RNA splicing function, disruption of which result in cleft of the lip and palate in humans, mouse and zebrafish. Esrp1 and Esrp2 function in the transient embryonic epithelial cells termed the periderm, a crucial epithelial layer involved in fusion of the embryonic lip and palate structures. Previous research has shown that mouse Esrp1 contains a nuclear localization signal (NLS), facilitating the mRNA splicing activity. While cross-species analyses have identified NLS motifs in Esrp1 and Esrp2 among vertebrates, the functional role of the nuclear versus cytoplasmic localization signals on Esrp1/2 function and morphogenesis remain unclear. METHODS: To study Esrp protein localization, vectors were created to express zebrafish esrp1 (cytoplasmic) and esrp2 (nuclear) with green fluorescent protein tags. The nuclear localization signal was altered to ectopically target Esrp1 to nuclear and Esrp2 cytoplasmic fractions, respectively. These vectors were also introduced into murine Py2T epithelial cells. Immunofluorescence assays visualized Esrp1/2 in Py2T and HEK293 cells. CRISPR/Cas9 introduced targeted mutations in Esrp1/2 genes in Py2T cells. These cells, including wildtype and knockouts, were transfected with different Esrp1/2 isoforms for RT-PCR analysis of splicing targets. In esrp1/2 mutant zebrafish, different esrp1/2 isoforms (with and without the NLS) were injected at the one-cell stage to study gene overexpression effects. RESULTS: The Esrp1 isoform constitutively locates to the cytoplasm in zebrafish and mouse. In mouse and human, Esrp1 locates to the cytoplasm and to the nucleus. In contrast, Esrp2 exclusively locates to the nucleus in zebrafish, mouse, and human. RT-PCR analysis for Esrp1/2 splicing targets (i.e., Arhgef11, Enah, and Atp6v1c2) showed different splicing patterns for Esrp1 and Esrp2 single and double knockouts, suggesting different roles through different subcellular protein localizations. In vivo rescue assay demonstrates that the NLS is required for esrp2 function. Unexpectedly, esrp1 targeted to the cytoplasm still achieved functional rescue, likely through post-transcriptional regulation of tp63 mRNA, a transcription factor that mediates epithelial-mesenchymal transition (EMT). CONCLUSION: This study demonstrates that cytoplasmic Esrp1 and nuclear Esrp2 are crucial for normal periderm function, and that the cytoplasmic Esrp2 is dispensable. The findings emphasize that the correct isoforms of Esrp1 and Esrp2 must localize to their appropriate subcellular domains, highlighting an as-yet-unidentified role for cytoplasmic Esrp1. Notably, the unexpected function of cytoplasmic Esrp1 may involve post-transcriptional regulation of tp63 mRNA, a transcription factor that mediates EMT. Unraveling the specific mechanisms by which cytoplasmic Esrp isoforms, such as Esrp1, influence gene regulation through factors like tp63 could lead to targeted molecular therapies aimed at preventing orofacial clefts.

Source: Plastic & Reconstructive Surgery Global Open

DOI: 10.1097/01.gox.0001112120.54537.98