Function of SF3B4 in neural crest development

SF3B4 在神经嵴发育中的功能

• 批准号: 10450642
• 负责人: Casey Griffin
• 金额: $ 7.06万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 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/10450642
• 关键词: Affect; Animal Disease Models; Animal Model; Animals; Apoptosis; Automobile Driving; Binding; Bone structure; CRISPR/Cas technology; Cells; Cephalic; Cleft Palate; Collaborations; Complex; Craniofacial Abnormalities; Defect; Development; Disease; Elements; Embryo; Fissural; Genes; 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; Proteins; RNA; RNA Splicing; Radial; Resources; Skeleton; Specificity; Spliceosomes; Testing; Thumb structure; Time; Tissues; Transcript; Wood material; 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

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.

Nager综合征(OMIM#154400)是一种罕见的头面部和四肢疾病，以面中部后缩为特征， 小颌、拇指缺失和放射状发育不全。这种疾病是由SF3B4(剪接)突变引起的 因子3b，亚单位4)基因，它编码SAP49，这是剪接体的一个组成部分。这个 剪接体是一种RNA和蛋白质的复合体，它们共同作用于去除内含子和连接外显子。 转录的前信使核糖核酸。虽然剪接体存在并在身体的所有细胞中发挥作用，但许多 剪接体病--包括Nager综合征--在病理上通常是细胞或组织特异性的。在纳戈尔 综合征患者受影响的是神经脊(NC)衍生的头面部骨骼结构。这个 Nager综合征的病理机制及其组织特异性还知之甚少。在……里面 在此应用中，我们将使用最近培育的热带爪哇Sf3b4突变株和胚胎干细胞 (ESC)衍生的神经脊细胞(NCC)通过识别靶点和结合来梳理这些机制 SF3B4的合作伙伴。这种体内和体外方法的结合将为我们提供对 在Nager综合征的背景下，导致头面部缺陷的机制。拟议中的实验将检验 假设SF3B4具有NC特异性靶标和/或结合伙伴，并且在突变时这些相互作用 被破坏或丢失，导致与Nager综合征相关的头面部缺陷。我们精心设计了三个具体的 旨在测试这种可能性。具体目标1：与国家非洲爪哇资源(NXR；伍兹)合作 马萨诸塞州霍尔)我们已经使用CRISPR/Cas9技术产生了一个Sf3b4热带非洲爪哇突变系，以及 我们建议通过在不同的时间点评估NC来执行这些动物的表型特征 祖细胞的形成、增殖、迁移和随后的颅面软骨发育。目标2：我们 假设通过调节KEY的前mRNA加工，SF3B4是NC祖细胞形成所必需的 NC命运的调控者。为了确定这些监管机构，我们将分析SF3B4击倒对全球的影响 野生型和Sf3b4突变型热带爪哇胚胎转录本的比较研究 使用RNA-seq并专注于显示内含子保留的转录本。目标3：作为前信使核糖核酸的替代品 处理NC特异的调节剂，我们假设SF3B4的组织特异性功能可能取决于 与伙伴分子的相互作用优先在NC谱系中丰富。为此，我们将表达一个 在胚胎干细胞来源的神经干细胞中构建标志标记的人SF3B4通过免疫沉淀和质量鉴定 光谱学SF3B4 NC-特异性相互作用可能是SF3B4细胞类型特异性活性的元凶 纳格综合征。总之，这些研究将为研究NAGER的机制提供新的见解 综合征头面部缺陷，通过一种新的突变系的特征，和广泛的特征 SF3B4目标和互动伙伴的数量。