权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Investigating Srsf3-mediated alternative RNA splicing in craniofacial development

研究颅面发育中 Srsf3 介导的替代 RNA 剪接

基本信息

批准号：
10663828
负责人：
Thomas E Forman
金额：
$ 3.97万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-08 至 2025-07-07
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10663828
关键词：
Ablation Affect Alleles Alternative Splicing Apoptosis Arginine Biotinylation Cell Lineage Cell Nucleus Cell membrane Cells Cephalic Cleft Palate Complex Congenital Abnormality Consensus Consensus Sequence Craniofacial Abnormalities Cytoplasm Data Defect Disease Dissociation Ectoderm Embryo Event Face Feedback Gene Expression Gene Expression Regulation Genes Goals Head Histologic Human Immunoprecipitation In Vitro Knock-in Knock-in Mouse Label Lead Ligands Maps Mass Spectrum Analysis Mediating Membrane Mesenchyme Molecular Morphology Mus Mutation Neural Crest Neural Crest Cell Nuclear Proteins Ontology PDGFRA gene PIK3CG gene Phenotype Phosphopeptides Phosphorylation Phosphotransferases Physiological Platelet-Derived Growth Factor Platelet-Derived Growth Factor alpha Receptor Play Post-Translational Protein Processing Pregnancy Process Proliferating Protein-Serine-Threonine Kinases Proteins RNA RNA Binding RNA Sequences RNA Splicing RNA-Binding Proteins Research Role Serine Signal Transduction Site Skeletal Development Small Interfering RNA Specificity Stimulus Structure Techniques Testing Tissues Transcript Western Blotting cleft lip and palate craniofacial craniofacial development craniofacial structure crosslink experimental study in vivo innovation insight interest knock-down mouse model mutant mouse model novel novel therapeutics nucleocytoplasmic transport orofacial cleft phosphoproteomics recruit response stem transcriptome sequencing

项目摘要

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 (PDGFRα) plays a critical role in this process, as mutations in PDGFRΑ 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 PDGFRα 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 PDGFRα 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 PDGFRα 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 PDGFRα 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 PDGFRα signaling in MEPM cells. Finally, Srsf3-RNA interactions will be purified and sequenced in response to PDGFRα 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.

项目总结