Project Summary
Craniofacial development involves complex signaling to coordinate tissue organization to form the head
and face, and disruptions in this process result in common congenital malformations. A key question in this
field is how external stimuli lead to gene expression changes required to form fully developed craniofacial
structures. Signaling through the Platelet-derived growth factor receptor alpha (PDGFRa) plays a critical role in
this process, as mutations in PDGFR¿ are associated with cleft lip/palate in humans. Relatedly, Pdgfra mutant
mouse models develop a range of phenotypes from cleft palate to complete facial clefting. Phosphatidylinositol
3-kinase (PI3K) is the primary effector of PDGFRa signaling during skeletal development in the mouse, leading
to the activation of the kinase Akt. A previous phosphoproteomic screen demonstrated that Akt phosphorylates
the RNA-binding protein (RBP) Serine/arginine-rich splicing factor 3 (Srsf3) downstream of PI3K-mediated
PDGFRa signaling in mouse embryonic palatal mesenchyme (MEPM) cells, leading to translocation of
phosphorylated Srsf3 into the nucleus. Srsf3 is ubiquitously expressed with enhanced expression in the neural
crest-derived mesenchyme and overlying ectoderm of mouse facial processes at mid-gestation. Additionally,
ablation of Srsf3 in the murine neural crest cell lineage (cKO) results in a severe midline facial clefting
phenotype due to defects in proliferation and survival of cranial neural crest cells. Further, RNA-sequencing of
Srsf3 cKO facial process mesenchyme identified alternative RNA splicing events that were enriched for
transcripts encoding protein serine/threonine kinases, suggesting that alternative splicing may serve as a novel
feedback mechanism for intracellular kinase signaling. The goal of this proposal is to test the hypothesis that
PI3K/Akt-mediated PDGFRa signaling regulates Srsf3 protein and RNA interactions to affect the alternative
RNA splicing of transcripts necessary for craniofacial development. First, Srsf3 will be immunoprecipitated from
MEPM cells in the absence or presence of PDGF-AA ligand and analyzed by mass spectrometry to
comprehensively map phosphorylation changes in response to PDGFRa signaling. Further, craniofacial
phenotypes will be analyzed in a Srsf3 phosphomutant knock-in mouse model to determine the role of Akt-
mediated phosphorylation of Srsf3 in craniofacial development. Next, BioID2 proximity labeling and mass
spectrometry will be used to identify Srsf3 protein interacting partners in response to PDGFRa signaling in
MEPM cells. Finally, Srsf3-RNA interactions will be purified and sequenced in response to PDGFRa signaling
in MEPM cells through enhanced crosslinking and immunoprecipitation analysis to identify direct targets of
Srsf3 and determine if RNA binding and/or sequence specificity changes upon Srsf3 phosphorylation. This
project will determine the molecular mechanisms by which Srsf3 activity is controlled in response to PDGFRa
signaling in the facial mesenchyme, thus providing considerable insight into mechanisms underlying gene
expression regulation during mammalian craniofacial development.