Srsf3-mediated alternative RNA splicing in craniofacial development

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Project Summary Craniofacial development is a complex morphogenetic process, disruptions in which result in highly prevalent human birth defects. Signaling through the platelet-derived growth factor receptor alpha (PDGFRa) plays a critical role in this process in humans and mice. Pdgfra mutant mouse models display midline facial clefting phenotypes. Phosphatidylinositol 3-kinase (PI3K) is the primary effector of PDGFRa signaling during skeletal development in the mouse, leading to activation of the kinase Akt. A previous phosphoproteomic screen identified Akt phosphorylation targets downstream of PI3K-mediated PDGFRa signaling in primary mouse embryonic palatal mesenchyme (MEPM) cells, revealing a significant enrichment for proteins that regulate alternative RNA splicing (AS), such as the RNA-binding protein (RBP) Srsf3. Ablation of Srsf3 in the mouse neural crest lineage leads to facial clefting due to defective cranial neural crest cell proliferation and survival. Further, Srsf3 regulates the AS of transcripts encoding protein kinases in the mouse facial process mesenchyme to regulate PDGFRa-dependent intracellular signaling. These findings have shifted the paradigm on how RTKs regulate gene expression and have identified post-translational modification of RBPs involved in AS downstream of PDGFRa signaling as a critical mechanism contributing to craniofacial development. The goal of this proposal is to determine the molecular mechanisms by which Srsf3 activity is regulated to generate protein isoforms necessary for midface development. First, to identify proteins that differentially interact with Srsf3 depending on its phosphorylation in response to PDGFRa signaling, Srsf3-interacting proteins will be immunoprecipitated from MEPM cells with or without PDGF-AA treatment and identified by mass spectrometry. Separately, Srsf3-RNA interactions will be purified and sequenced in response to PDGF-AA ligand stimulation through enhanced crosslinking and immunoprecipitation analysis to identify which transcripts are directly bound by Srsf3 and to determine if the extent of RNA binding and/or sequence specificity of these interactions changes upon Srsf3 phosphorylation. Second, craniofacial phenotypes will be assessed in Srsf3 phosphomutant knock-in embryos and RNA-seq analysis will be performed to identify AS targets of Srsf3 that depend on Srsf3 phosphorylation. The relationship between PDGFRa and Srsf3 will be dissected using this allele in genetic epistasis experiments. Finally, craniofacial phenotypes will be assessed in Srsf3 ectoderm- specific conditional knock-out embryos. The AS targets of Srsf3 will be identified in the facial mesenchyme and overlying ectoderm at the onset of craniofacial phenotypes through RNA-seq analysis. The splicing programs regulated by Srsf3 in each compartment will be correlated with biological processes active during craniofacial development. This project will delineate a complete pathway from PDGFRa at the cell surface to Srsf3 and its target transcripts in the nucleus. These studies will provide significant insight into what are likely broadly- applicable mechanisms underlying gene expression regulation during mammalian craniofacial development.