Srsf3-mediated alternative RNA splicing in the facial mesenchyme

Srsf3介导的面部间质中的替代RNA剪接

基本信息

批准号：
10190759
负责人：
Brenna Jo Clay Dennison
金额：
$ 1.8万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2021-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10190759
关键词：
1-Phosphatidylinositol 3-Kinase Ablation Address Alleles Alternative Splicing Antibodies Arginine Binding Binding Sites Biochemical Biological Process Biology Cell Lineage Cell membrane Cells Cellular biology Cephalic Cleft Lip Cleft Palate Congenital Abnormality Craniofacial Abnormalities Defect Development Dilated Cardiomyopathy Disease Embryo Embryonic Development Event Exhibits Face Genes Genetic Genetic Epistasis Goals Human Immunoprecipitation Incidence Individual Knock-in Length Ligands Live Birth Malignant Neoplasms Mammals Mass Spectrum Analysis Maxilla Maxillary Prominence Mediating Mesenchyme Messenger RNA Molecular Morphogenesis Morphology Mus Muscular Dystrophies Mutation Nasal cavity Neural Crest Cell Ontology Oral PDGFRA gene Phenotype Phosphorylation Phosphotransferases Platelet-Derived Growth Factor Platelet-Derived Growth Factor alpha Receptor Play Post-Translational Protein Processing Process Protein Array Proteins RNA Binding RNA Processing RNA Sequences RNA Splicing RNA-Binding Proteins Regulation Reporter Retinitis Pigmentosa Role Serine Signal Transduction Specificity Techniques Time Tissues Transcript Transfection United States Validation Xenopus base cell motility cleft lip and palate cohesion craniofacial craniofacial development crosslink experimental study in vivo insight mouse model mutant mutant mouse model nervous system disorder novel novel therapeutics palatal shelves palatogenesis phosphoproteomics programs protein function recruit transcriptome sequencing

项目摘要

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.

项目概要颅面发育是胚胎发生过程中的一个关键形态事件，其缺陷会导致高度普遍的人类出生缺陷，例如唇裂和腭裂（CL/P）。在人类和小鼠中都有这个过程依赖于通过血小板衍生生长因子受体α（PDGFRa）的信号传导。人类的突变 PDGFRA 与 CL/P 相关，并且该基因突变的小鼠模型同样表现出面部裂痕表型。 E13.5 中 PDGFRa 信号传导下游 PI3K/Akt 磷酸化靶点的鉴定原代小鼠胚胎腭间充质细胞显示调节 RNA 的蛋白质富集剪接，包括 RNA 结合蛋白 (RBP) Srsf3。虽然RNA选择性剪接的失调已已被证明会导致人类多种疾病，RBP 在调节神经选择性剪接中的作用面部间充质中的嵴细胞（NCC）及其衍生物尚未得到充分研究。此举的目的提议是检查面部间充质中 Srsf3 介导的组织特异性替代 RNA 剪接以及中面部发育过程中 PI3K/Akt 介导的 PDGFRa 信号对 Srsf3 活性的影响。首先， Srsf3 在体内颅面发育过程中的作用将使用条件 Srsf3fl 等位基因来表征与 NCC 特定的 Wnt1-CreTg 驱动程序相结合。将分析 NCC 迁移的时间和范围在这种情况下，以及特定于个人面部过程的标记的表达。其次，RNA- 将进行测序实验以鉴定差异选择性剪接的转录本 E11.5 Srsf3fl/fl;Wnt1-Cre+/+ 和 Srsf3fl/fl;Wnt1-Cre+/Tg 上颌突之间。差异验证将使用半定量 PCR 进行选定转录本的选择性剪接。最后是Akt的效果 Srsf3 亚细胞定位、RNA 结合和序列特异性的磷酸化将通过以下方式进行评估 Srsf3 野生型和磷酸突变型 GFP 融合构建体的转染以及增强的交联和分别在存在或不存在 PDGF-AA 配体的情况下进行免疫沉淀，然后进行测序。此外，将在 PdgfraPI3K 和 Srsf3fl 小鼠之间进行遗传上位实验，所得结果分析颅面表型以确定 PdgfraPI3K/PI3K 腭裂缺陷是否得到挽救或 NCC 谱系中 Srsf3 丢失后加剧。该项目将研究 Srsf3 的潜在新角色作为 NCC 及其衍生物的剪接调节因子，并探讨如何对其进行翻译后修饰 PDGFRa 信号下游的蛋白质有助于中面部发育。这些研究将提供对哺乳动物颅面形态发生和新治疗机制的重要见解治疗人类颅面出生缺陷（例如 CL/P）的指导。