Nager syndrome (OMIM#154400) is a rare craniofacial and limb disorder characterized by midface retrusion, micrognathia, absent thumbs, and radial hypoplasia. This disorder results from mutations in the SF3B4 (splicing factor 3b, subunit 4) gene, which encodes SAP49, a protein that is a component of the spliceosome. The spliceosome is a complex of RNA and proteins that function together to remove introns and join exons from transcribed pre-mRNA. While the spliceosome is present and functions in all cells of the body, many spliceosomopathies – including Nager syndrome – are often cell- or tissue-specific in their pathology. In Nager syndrome patients it is the neural crest (NC)-derived craniofacial skeletal structures that are affected. The mechanisms underlying Nager syndrome pathology, as well as its tissue-specificity are poorly understood. In this application, we will use a recently generated Xenopus tropicalis Sf3b4 mutant line and embryonic stem cell (ESC)-derived neural crest cells (NCCs) to tease apart these mechanisms by identifying the targets and binding partners of SF3B4. This combination of in vivo and in vitro approaches will provide novel insights into the mechanisms driving craniofacial defects in the context of Nager syndrome. The proposed experiments will test the hypothesis that SF3B4 has NC-specific targets and/or binding partners, and upon mutation these interactions are disrupted or lost, leading to Nager syndrome-associated craniofacial defects. We have crafted three specific aims to test this possibility. Specific Aim 1: In collaboration with the National Xenopus Resource (NXR; Woods Hole, MA) we have generated an Sf3b4 Xenopus tropicalis mutant line using the CRISPR/Cas9 technology, and we propose to perform the phenotypic characterization these animals by evaluating at different time points NC progenitor formation, proliferation, migration, and subsequent craniofacial cartilage development. Aim 2: We hypothesize that SF3B4 is required for NC progenitor formation by regulating the pre-mRNA processing of key regulators of NC fate. To identify these regulators, we will analyze the global impact of SF3B4 knockdown on pre-mRNA processing by comparing transcripts from wild-type and Sf3b4 mutant Xenopus tropicalis embryos using RNA-seq and focusing on transcripts showing intron retention. Aim 3: As an alternative to the pre-mRNA processing of NC-specific regulators, we hypothesize that the tissue-specific function of SF3B4 may depend on interactions with partner molecules preferentially enriched in the NC lineage. To this end, we will express a FLAG-tagged human SF3B4 construct in ESC-derived NCCs to identify by immunoprecipitation and mass spectrometry SF3B4 NC-specific interactors as the possible culprit for the cell-type specific activity of SF3B4 in Nager syndrome. Altogether these studies will provide novel insights into the mechanisms underlying Nager syndrome craniofacial defects, through the characterization of a new muta...