Function of SF3B4 in neural crest development

Nager综合征（OMIM#154400）是一种罕见的颅面和肢体疾病，其特征是面中部凹陷， 小颌畸形拇指缺失桡骨发育不全这种疾病是由SF 3B 4（剪接）突变引起的。 因子3b，亚基4）基因，其编码作为剪接体组分的蛋白质SAP 49。的 剪接体是RNA和蛋白质的复合物，其共同起作用以从DNA中去除内含子并连接外显子。 转录前mRNA。虽然剪接体存在于身体的所有细胞中并发挥作用，但许多细胞都有剪接体。 剪接体病--包括Nager综合征--在其病理学上通常是细胞或组织特异性。因纳格尔 综合征患者的神经嵴（NC）衍生的颅面骨骼结构受到影响。的 Nager综合征病理学的潜在机制以及其组织特异性知之甚少。在 在本申请中，我们将使用最近产生的热带爪蟾Sf 3b 4突变系和胚胎干细胞 （ESC）衍生的神经嵴细胞（NCC）通过识别靶点和结合这些机制来梳理这些机制。 SF 3B 4的合作伙伴。这种体内和体外方法的组合将提供新的见解， 在Nager综合征背景下驱动颅面缺陷的机制。拟议的实验将测试 假设SF 3B 4具有NC特异性靶标和/或结合配偶体，并且在突变后这些相互作用 被破坏或丢失，导致Nager综合征相关的颅面缺陷。我们精心制作了三个 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），我们已经使用CRISPR/Cas9技术产生了Sf 3b 4热带爪蟾突变株系，并且 我们建议通过在不同时间点评价NC来进行这些动物的表型表征， 祖细胞形成、增殖、迁移和随后的颅面软骨发育。目标2：我们 假设SF 3B 4是通过调节关键蛋白的前mRNA加工而形成NC祖细胞所必需的， NC的命运。为了确定这些监管机构，我们将分析SF 3B 4击倒对全球的影响。 通过比较野生型和Sf 3b 4突变体热带爪蟾胚胎的转录本进行前mRNA加工 使用RNA-seq并专注于显示内含子保留的转录本。目的3：作为前mRNA的替代品 我们推测SF 3B 4的组织特异性功能可能取决于 与优先富集在NC谱系中的伴侣分子的相互作用。为此，我们将表达一个 通过免疫沉淀和质谱鉴定ESC衍生的NCC中的FLAG标记的人SF 3B 4构建体 光谱SF 3B 4 NC-特异性相互作用物作为SF 3B 4细胞类型比活性的可能罪魁祸首， 纳格综合征。总之，这些研究将为Nager的潜在机制提供新的见解 综合症颅面缺陷，通过新突变系的表征和广泛的表征 SF 3B 4靶点和相互作用的伙伴。

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.