Srsf3-mediated alternative RNA splicing in craniofacial development

项目摘要 颅面发育是一个复杂的形态发生过程，其中的中断导致高度的 普遍存在的人类出生缺陷。通过血小板衍生生长因子受体α（PDGFRa）的信号传导 在人类和小鼠的这一过程中起着关键作用。PDGFRA突变小鼠模型显示中线面部 分裂表型磷脂酰肌醇3-激酶（PI 3 K）是PDGFRa信号转导过程中的主要效应子。 小鼠骨骼发育，导致激酶Akt的激活。以前的磷酸化蛋白质组学 筛选鉴定了PI 3 K介导的PDGFRa信号传导下游的Akt磷酸化靶点， 小鼠胚胎腭间充质（MEPM）细胞，揭示了蛋白质的显着富集， 调节可变RNA剪接（AS），如RNA结合蛋白（RBP）Srsf 3。消融Srsf 3 小鼠神经嵴谱系由于有缺陷的颅神经嵴细胞增殖而导致面部裂开， 生存此外，Srsf 3调节小鼠面部过程中编码蛋白激酶的转录物的AS 间充质调节PDGFRa依赖性细胞内信号传导。这些发现改变了 RTKs如何调节基因表达，并确定了参与基因表达的RBP的翻译后修饰。 AS下游的PDGFRa信号传导作为一个关键机制，有助于颅面发育。的 该提案的目标是确定Srsf 3活性被调节以产生 面中部发育所必需的蛋白质异构体。首先，为了识别与蛋白质相互作用的差异， Srsf 3依赖于其响应于PDGFRa信号传导的磷酸化，Srsf 3相互作用蛋白将被激活。 从有或没有PDGF-AA处理的MEPM细胞免疫沉淀，并通过质谱鉴定。 单独地，响应于PDGF-AA配体刺激，Srsf 3-RNA相互作用将被纯化和测序 通过增强的交联和免疫沉淀分析，以确定哪些转录本直接 并确定RNA结合的程度和/或这些相互作用的序列特异性 Srsf 3磷酸化的变化。其次，将在Srsf 3中评估颅面表型 将进行磷酸突变体敲入胚胎和RNA-seq分析以鉴定Srsf 3的AS靶， 依赖于Srsf 3磷酸化。PDGFRa和Srsf 3之间的关系将使用该方法进行剖析。 等位基因的遗传上位性实验。最后，将在Srsf 3外胚层中评估颅面表型。 特定条件敲除胚胎。Srsf 3的AS靶标将在面部间充质中鉴定， 通过RNA-seq分析，在颅面表型开始时覆盖外胚层。拼接程序 由Srsf 3调节的每个隔室将与颅面过程中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Srsf 3的完整途径， 靶向细胞核中的转录物。这些研究将提供重要的见解，什么是可能广泛- 哺乳动物颅面发育过程中基因表达调控的可能机制。

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.