Deciphering the underlying mechanisms of craniofacial spliceosomopathies

PROJECT ABSTRACT 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/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. Interestingly, other craniofacial spliceosomopathies, such as craniofacial microsomia (SF3B2), mandibulofacial

dysostosis Guion-Almeida type (EFTUD2), Burn-McKeown syndrome (TXNL4A), and Verheij syndrome (PUF60) share similar clinical features with Nager syndrome, however it is unclear if they are caused by the same underlying mechanisms. In this application, I will combine use of a Xenopus tropicalis sf3b4 mutant line

and Nager syndrome patient-derived induced pluripotent stem cells (iPSCs) to tease apart the mechanisms underlying Nager syndrome. 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, which can then be translated to

other craniofacial spliceosomopathies to determine if they share a common root cause. 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 abnormal NC development and subsequent Nager

syndrome-associated craniofacial defects. I have crafted three specific aims to test this possibility. Specific Aim 1: Using preliminary data from RNA-seq analyses performed on a Xenopus tropicalis sf3b4 mutant line, I propose to identify the biological pathways disrupted in Nager syndrome by testing candidate genes via gain-

and loss-of-function experiments in Xenopus tropicalis. Specific Aim 2: In collaboration with the Columbia Stem Cell Core, I propose to generate and characterize a Nager syndrome patient-derived iPSC line, and use this line to characterize NC in the context of Nager syndrome. Specific Aim 3: In the R00 phase of the application, I

plan to use the knowledge and experience from modeling Nager syndrome and develop in vivo and in vitro models of other craniofacial spliceosomopathies in order to determine their root cause. Altogether these studies will provide novel insights into the mechanisms underlying Nager syndrome craniofacial defects, which

can be used to inform work on other craniofacial spliceosomopathies, establishing a unique view on understanding craniofacial development and disorders.