# Srsf3-mediated alternative RNA splicing in the facial mesenchyme

> **NIH NIH F31** · UNIVERSITY OF COLORADO DENVER · 2020 · $37,520

## Abstract

Project Summary
 Craniofacial development is a critical morphological event during embryogenesis, defects in which result
in highly prevalent human birth defects such as cleft lip and palate (CL/P). In both humans and mice this process
relies on signaling through the platelet-derived growth factor receptor alpha (PDGFRa). Mutations in human
PDGFRA are associated with CL/P and mouse models with mutations in this gene similarly display facial clefting
phenotypes. Identification of phosphorylation targets of PI3K/Akt downstream of PDGFRa signaling in E13.5
primary mouse embryonic palatal mesenchyme cells revealed an enrichment for proteins that regulate RNA
splicing, including the RNA-binding protein (RBP) Srsf3. While dysregulation of alternative RNA splicing has
been shown to cause numerous diseases in humans, the role of RBPs in regulating alternative splicing in neural
crest cells (NCCs) and their derivatives in the facial mesenchyme has been understudied. The aim of this
proposal is to examine the tissue-specific alternative RNA splicing mediated by Srsf3 in the facial mesenchyme
and the effect of PI3K/Akt-mediated PDGFRa signaling on Srsf3 activity during midface development. First, the
role of Srsf3 during craniofacial development in vivo will be characterized using a conditional Srsf3fl allele in
combination with the NCC-specific Wnt1-CreTg driver. The timing and extent of NCC migration will be analyzed
in this setting as well as the expression of markers specific to individual facial processes. Second, an RNA-
sequencing experiment will be performed to identify transcripts that are differentially alternatively spliced
between E11.5 Srsf3fl/fl;Wnt1-Cre+/+ and Srsf3fl/fl;Wnt1-Cre+/Tg maxillary processes. Validation of the differential
alternative splicing of select transcripts will be performed using semi-quantitative PCR. Finally, the effect of Akt
phosphorylation on Srsf3 subcellular localization, RNA binding and sequence specificity will be assessed through
the transfection of Srsf3 wild-type and phosphomutant GFP fusion constructs and enhanced crosslinking and
immunoprecipitation followed by sequencing, respectively, in the presence or absence of PDGF-AA ligand.
Further, genetic epistasis experiments will be performed between PdgfraPI3K and Srsf3fl mice and the resulting
craniofacial phenotypes analyzed to determine if the PdgfraPI3K/PI3K palatal clefting defect is rescued or
exacerbated upon loss of Srsf3 in the NCC lineage. This project will investigate a potentially novel role for Srsf3
as a splicing regulator in NCCs and their derivatives and explore how post-translational modification of this
protein downstream of PDGFRa signaling contributes to midface development. These studies will provide
significant insight into the mechanisms underlying mammalian craniofacial morphogenesis and new therapeutic
directions for the treatment of human craniofacial birth defects such as CL/P.

## Key facts

- **NIH application ID:** 9987250
- **Project number:** 1F31DE029364-01A1
- **Recipient organization:** UNIVERSITY OF COLORADO DENVER
- **Principal Investigator:** Brenna Jo Clay Dennison
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $37,520
- **Award type:** 1
- **Project period:** 2020-07-01 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9987250

## Citation

> US National Institutes of Health, RePORTER application 9987250, Srsf3-mediated alternative RNA splicing in the facial mesenchyme (1F31DE029364-01A1). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9987250. Licensed CC0.

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