Function of SF3B4 in neural crest development

SF3B4 在神经嵴发育中的功能

基本信息

批准号：
10669171
负责人：
Casey Griffin
金额：
$ 7.46万
依托单位：
NEW YORK UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10669171
关键词：
Affect Animal Disease Models Animal Model Animals Apoptosis Automobile Driving Binding Bone structure CRISPR/Cas technology Cells Cephalic Classification Cleft Palate Collaborations Complex Craniofacial Abnormalities Defect Development Disease Elements Embryo Genes Genetic Transcription Heterozygote Human Immunoprecipitation In Vitro Introns Joining Exons Limb structure Mass Spectrum Analysis Mediating Mesoderm Cell Micrognathism Mutation Nager syndrome Neural Crest Neural Crest Cell Online Mendelian Inheritance In Man Pathology Patients Phenotype Proliferating Proteins RNA RNA Splicing Radial Resources Skeleton Specificity Spliceosomes Testing Thumb structure Time Tissues Transcript Xenopus Xenopus laevis cartilage development cell type craniofacial craniofacial complex developmental disease embryonic stem cell experimental study face bone structure in vivo insight knock-down mRNA Precursor malformation migration mutant novel progenitor protein function skeletal transcriptome sequencing

项目摘要

Nager syndrome (OMIM#154400) is a rare craniofacial and limb disorder characterized by midface retrusion, micrognathia, absent thumbs, and radial hypoplasia. This disorder results from mutations in the SF3B4 (splicing factor 3b, subunit 4) gene, which encodes SAP49, a protein that is a component of the spliceosome. The spliceosome is a complex of RNA and proteins that function together to remove introns and join exons from transcribed pre-mRNA. While the spliceosome is present and functions in all cells of the body, many spliceosomopathies – including Nager syndrome – are often cell- or tissue-specific in their pathology. In Nager syndrome patients it is the neural crest (NC)-derived craniofacial skeletal structures that are affected. The mechanisms underlying Nager syndrome pathology, as well as its tissue-specificity are poorly understood. In this application, we will use a recently generated Xenopus tropicalis Sf3b4 mutant line and embryonic stem cell (ESC)-derived neural crest cells (NCCs) to tease apart these mechanisms by identifying the targets and binding partners of SF3B4. This combination of in vivo and in vitro approaches will provide novel insights into the mechanisms driving craniofacial defects in the context of Nager syndrome. The proposed experiments will test the hypothesis that SF3B4 has NC-specific targets and/or binding partners, and upon mutation these interactions are disrupted or lost, leading to Nager syndrome-associated craniofacial defects. We have crafted three specific aims to test this possibility. Specific Aim 1: In collaboration with the National Xenopus Resource (NXR; Woods Hole, MA) we have generated an Sf3b4 Xenopus tropicalis mutant line using the CRISPR/Cas9 technology, and we propose to perform the phenotypic characterization these animals by evaluating at different time points NC progenitor formation, proliferation, migration, and subsequent craniofacial cartilage development. Aim 2: We hypothesize that SF3B4 is required for NC progenitor formation by regulating the pre-mRNA processing of key regulators of NC fate. To identify these regulators, we will analyze the global impact of SF3B4 knockdown on pre-mRNA processing by comparing transcripts from wild-type and Sf3b4 mutant Xenopus tropicalis embryos using RNA-seq and focusing on transcripts showing intron retention. Aim 3: As an alternative to the pre-mRNA processing of NC-specific regulators, we hypothesize that the tissue-specific function of SF3B4 may depend on interactions with partner molecules preferentially enriched in the NC lineage. To this end, we will express a FLAG-tagged human SF3B4 construct in ESC-derived NCCs to identify by immunoprecipitation and mass spectrometry SF3B4 NC-specific interactors as the possible culprit for the cell-type specific activity of SF3B4 in Nager syndrome. Altogether these studies will provide novel insights into the mechanisms underlying Nager syndrome craniofacial defects, through the characterization of a new mutant line, and the broad characterization of SF3B4 targets and interacting partners.

纳格综合征 (OMIM#154400) 是一种罕见的颅面和肢体疾病，其特征是中面部后缩，小颌畸形、拇指缺失和桡骨发育不全这种疾病是由 SF3B4（剪接）突变引起的。因子 3b，亚基 4) 基因，编码 SAP49，一种剪接体成分的蛋白质。剪接体是RNA和蛋白质的复合体，它们共同作用以去除内含子并连接外显子虽然剪接体存在于身体的所有细胞中并发挥作用，但许多细胞中都存在转录的前 mRNA。剪接体病（包括 Nager 综合征）的病理学通常具有细胞或组织特异性。综合征患者的神经嵴 (NC) 衍生的颅面骨骼结构受到影响。纳格病理综合征的机制及其组织特异性尚不清楚。在此应用中，我们将使用最近生成的热带爪蟾 Sf3b4 突变系和胚胎干细胞 (ESC) 衍生的神经嵴细胞 (NCC) 通过识别目标和结合来梳理这些机制 SF3B4 的合作伙伴将体内和体外方法相结合，将为我们提供新的见解。拟议的实验将测试纳格综合征背景下导致颅面缺陷的机制。假设 SF3B4 具有 NC 特异性靶标和/或结合伴侣，并且在突变时这些相互作用被破坏或丢失，导致与纳格综合征相关的颅面缺陷。我们精心设计了三种特定的颅面缺陷。旨在测试这种可能性。具体目标 1：与国家非洲爪蟾资源中心（NXR；Woods）合作。 Hole，MA）我们使用 CRISPR/Cas9 技术生成了 Sf3b4 热带爪蟾突变体系，并且我们建议通过在不同时间点进行评估来进行这些动物的表型表征 NC 目标2：我们 SF3B4 通过调节关键基因的前体 mRNA 加工，是 NC 祖细胞形成所必需的为了识别这些调节因子，我们将分析 SF3B4 敲除对全球的影响。通过比较野生型和 Sf3b4 突变体热带爪蟾胚胎的转录本进行前 mRNA 处理使用 RNA-seq 并关注显示内含子保留的转录本目标 3：作为前 mRNA 的替代方案。通过对 NC 特异性调节因子的处理，我们发现 SF3B4 的组织特异性功能可能取决于与NC谱系中优先富集的伙伴分子的相互作用为此，我们将表达一个 ESC 衍生的 NCC 中带有 FLAG 标签的人 SF3B4 构建体，可通过免疫沉淀和质量进行鉴定光谱测定 SF3B4 NC 特异性相互作用子可能是 SF3B4 细胞类型特异性活性的罪魁祸首总的来说，这些研究将为纳格综合征的潜在机制提供新的见解。颅面缺陷综合征，通过新突变系的表征以及广泛的表征 SF3B4 目标和交互伙伴。