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

• 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
• 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

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.

项目摘要 颅面发育涉及复杂的信号，以协调组织形成头部 和脸部，在这个过程中的破坏会导致常见的先天性畸形。这其中的一个关键问题 研究领域是外部刺激如何导致形成完全发育的颅面所需的基因表达变化 结构。通过血小板衍生生长因子受体α(PDGFRα)传递的信号在 这一过程，因为PDGFRΑ的突变与人类的唇腭裂有关。与此相关，PDGFRA突变体 小鼠模型发育出一系列的表型，从腭裂到完全性面部裂隙。磷脂酰肌醇 3-激酶(PI3K)是小鼠骨骼发育过程中PDGFRα信号的主要效应者，主要 与Akt的激活有关。先前的磷酸蛋白质组筛查显示Akt磷酸化 PI3K介导的RNA结合蛋白(RBP)丝氨酸/精氨酸富含剪接因子3下游 小鼠胚胎腭间充质细胞中PDGFRα信号转位的研究 将Srsf3磷酸化进入细胞核。Srsf3在神经中普遍表达，表达增强。 孕中期小鼠面部突起的冠源性间充质和覆盖的外胚层。另外， 切除小鼠神经脊细胞系(CKO)中的Srsf3可导致严重的正中线面裂 脑神经脊细胞增殖和存活缺陷所致的表型。进一步，RNA测序 Srsf3 CKO面部突起间充质细胞鉴定了富含 编码蛋白丝氨酸/苏氨酸激酶的转录本，表明选择性剪接可能是一种新的 细胞内激酶信号的反馈机制。这项提议的目标是检验这一假设 PI3K/Akt介导的PDGFRRNA信号调节α蛋白和RNA的相互作用以影响替代 头面部发育所必需的转录本的RNA剪接。首先，srsf3将从 MEPM细胞在没有或存在PDGF-AA配体的情况下，用质谱仪分析 全面定位PDGFRα信号反应中的磷酸化变化。此外，颅面 我们将分析Srsf3基因突变的敲入小鼠模型中的表型，以确定Akt- 在颅面发育中介导的Srsf3的磷酸化。接下来，BioID2邻近标记和质量 光谱分析将被用来确定响应PDGFRα信号的sRf3蛋白相互作用伙伴 MEPM细胞。最后，将对srsf3-rna相互作用进行纯化和测序，以响应pdgfrα信号。 在MEPM细胞中通过增强交联和免疫沉淀分析确定其直接靶点 并确定当Srsf3磷酸化时，RNA结合和/或序列特异性是否发生变化。这 该项目将确定控制srsf3活性以响应PDGFRA的分子机制 面部间充质中的信号，从而为基因潜在的机制提供了相当大的洞察力 哺乳动物头面部发育过程中的表达调控。