Srsf3-mediated alternative RNA splicing in craniofacial development

Srsf3介导的颅面发育中的选择性RNA剪接

基本信息

批准号：
10650417
负责人：
Katherine Ann Fantauzzo
金额：
$ 48.61万
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：
2022-07-01 至 2028-02-29
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10650417
关键词：
Ablation Affect Alleles Alternative Splicing Automobile Driving Binding Biological Process Cell Nucleus Cell Proliferation Cell Survival Cell physiology Cell surface Cells Cephalic Complex Congenital Abnormality Consensus Craniofacial Abnormalities Cytoplasm Development Dilated Cardiomyopathy Disease Ectoderm Embryo Face Gene Expression Gene Expression Regulation Genetic Epistasis Goals Histologic Human Immunoprecipitation Knock-in Ligands Malignant Neoplasms Mass Spectrum Analysis Mediating Mesenchyme Modeling Molecular Morphogenesis Morphology Mus Muscular Dystrophies Mutation Neural Crest Neural Crest Cell Nuclear Proteins PDGF receptor tyrosine kinase PDGFRA gene PIK3CG gene Pathway interactions Phenotype Phosphorylation Phosphotransferases Platelet-Derived Growth Factor Platelet-Derived Growth Factor alpha Receptor Play Post-Translational Protein Processing Process Proliferating Protein Isoforms Protein Kinase Protein-Serine-Threonine Kinases Proteins RNA RNA Binding RNA Sequences RNA Splicing RNA-Binding Proteins Receptor Protein-Tyrosine Kinases Research Retinitis Pigmentosa Role Sampling Serine Signal Pathway Signal Transduction Site Skeletal Development Specificity Spliceosomes Structure Transcript cleft lip and palate conditional knockout craniofacial craniofacial development crosslink experimental study in vivo insight mutant mutant mouse model nervous system disorder novel novel therapeutics phosphoproteomics programs protein protein interaction response transcriptome sequencing

项目摘要

Project Summary Craniofacial development is a complex morphogenetic process, disruptions in which result in highly prevalent human birth defects. Signaling through the platelet-derived growth factor receptor alpha (PDGFRa) plays a critical role in this process in humans and mice. Pdgfra mutant mouse models display midline facial clefting phenotypes. Phosphatidylinositol 3-kinase (PI3K) is the primary effector of PDGFRa signaling during skeletal development in the mouse, leading to activation of the kinase Akt. A previous phosphoproteomic screen identified Akt phosphorylation targets downstream of PI3K-mediated PDGFRa signaling in primary mouse embryonic palatal mesenchyme (MEPM) cells, revealing a significant enrichment for proteins that regulate alternative RNA splicing (AS), such as the RNA-binding protein (RBP) Srsf3. Ablation of Srsf3 in the mouse neural crest lineage leads to facial clefting due to defective cranial neural crest cell proliferation and survival. Further, Srsf3 regulates the AS of transcripts encoding protein kinases in the mouse facial process mesenchyme to regulate PDGFRa-dependent intracellular signaling. These findings have shifted the paradigm on how RTKs regulate gene expression and have identified post-translational modification of RBPs involved in AS downstream of PDGFRa signaling as a critical mechanism contributing to craniofacial development. The goal of this proposal is to determine the molecular mechanisms by which Srsf3 activity is regulated to generate protein isoforms necessary for midface development. First, to identify proteins that differentially interact with Srsf3 depending on its phosphorylation in response to PDGFRa signaling, Srsf3-interacting proteins will be immunoprecipitated from MEPM cells with or without PDGF-AA treatment and identified by mass spectrometry. Separately, Srsf3-RNA interactions will be purified and sequenced in response to PDGF-AA ligand stimulation through enhanced crosslinking and immunoprecipitation analysis to identify which transcripts are directly bound by Srsf3 and to determine if the extent of RNA binding and/or sequence specificity of these interactions changes upon Srsf3 phosphorylation. Second, craniofacial phenotypes will be assessed in Srsf3 phosphomutant knock-in embryos and RNA-seq analysis will be performed to identify AS targets of Srsf3 that depend on Srsf3 phosphorylation. The relationship between PDGFRa and Srsf3 will be dissected using this allele in genetic epistasis experiments. Finally, craniofacial phenotypes will be assessed in Srsf3 ectoderm- specific conditional knock-out embryos. The AS targets of Srsf3 will be identified in the facial mesenchyme and overlying ectoderm at the onset of craniofacial phenotypes through RNA-seq analysis. The splicing programs regulated by Srsf3 in each compartment will be correlated with biological processes active during craniofacial development. This project will delineate a complete pathway from PDGFRa at the cell surface to Srsf3 and its target transcripts in the nucleus. These studies will provide significant insight into what are likely broadly- applicable mechanisms underlying gene expression regulation during mammalian craniofacial development.

项目摘要颅面发育是一个复杂的形态发生过程，干扰导致高度普遍的人类先天缺陷。通过血小板衍生的生长因子受体α（PDGFRA）信号传导在人类和小鼠中在这一过程中起关键作用。 PDGFRA突变鼠标模型显示中线面部裂作表型。磷脂酰肌醇3-激酶（PI3K）是PDGFRA信号传导的主要效应因子小鼠的骨骼发育，导致激酶Akt的激活。先前的磷酸化蛋白质组学屏幕鉴定出Akt磷酸化目标是PI3K介导的pDGFRA信号的下游原始目标小鼠胚胎palatal间充质（MEPM）细胞，揭示了蛋白质的显着富集调节替代RNA剪接（AS），例如RNA结合蛋白（RBP）SRSF3。在小鼠神经rest谱系导致面部裂口由于缺陷颅神经rest细胞增殖和生存。此外，SRSF3调节在小鼠面部过程中编码蛋白激酶的转录本间充质调节PDGFRA依赖性细胞内信号传导。这些发现改变了范式关于RTK如何调节基因表达并确定了参与RBP的翻译后修饰作为PDGFRA信号的下游，是导致颅面发育的关键机制。这该提案的目标是确定SRSF3活性被调节以生成的分子机制中心发育所需的蛋白质同工型。首先，确定与差异相互作用的蛋白质 SRSF3取决于其响应PDGFRA信号传导的磷酸化，SRSF3相互作用蛋白将是从MEPM细胞进行免疫沉淀，经过或不接受PDGF-AA处理，并通过质谱法鉴定。另外，SRSF3-RNA相互作用将被纯化和测序，以响应PDGF-AA配体刺激通过增强的交联和免疫沉淀分析，以确定哪些转录本直接由SRSF3结合，并确定这些相互作用的RNA结合和/或序列特异性的程度是否存在 SRSF3磷酸化的变化。其次，将在SRSF3中评估颅面表型将进行磷化剂敲入胚胎和RNA-seq分析，以确定为SRSF3的靶标取决于SRSF3磷酸化。 PDGFRA和SRSF3之间的关系将使用此阐述遗传性遗传实验中的等位基因。最后，将在SRSF3外胚层中评估颅面表型特定的条件敲除胚胎。 SRSF3的AS目标将在面部间充质和通过RNA-Seq分析在颅面表型开始时外胚层上覆盖。剪接程序每个隔室中由SRSF3调节将与颅面期间活跃的生物过程相关发展。该项目将描绘从细胞表面的PDGFRA到SRSF3及其的完整途径核中的目标转录本。这些研究将为可能广泛的洞察力提供重大洞察力 - 哺乳动物颅面发育过程中基因表达调节的适用机制。