RNA-binding proteins in early eye development.

早期眼睛发育中的 RNA 结合蛋白。

• 批准号: 10356066
• 负责人: Salil Lachke
• 金额: $ 33.67万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10356066
• 关键词: Address; Affect; Animals; Anophthalmos; Antibodies; Binding; Bioinformatics; Birth; Cataract; Cell Culture Techniques; Cell Differentiation process; Cell surface; Cellular Assay; Coloboma; Complement 1q; Complex; Congenital Abnormality; Data; Defect; Development; Disease; Ectoderm; Ectoderm Cell; Etiology; Event; Exhibits; Eye; Eye Development; Eye diseases; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Histology; Human; Immunoprecipitation; Knock-out; Knockout Mice; Knowledge; Lead; Lens Fiber; Lens Placodes; Lens development; Link; Mediating; Messenger RNA; MicroRNAs; Microphthalmos; Molecular; Morphogenesis; Morphology; Mus; Neurons; Online Systems; Optic vesicle; Organogenesis; Pathogenesis; Pathology; Pathway interactions; Patients; Post-Transcriptional Regulation; Protein Binding Domain; Protein Family; Proteins; Proteome; Proteomics; RNA; RNA Binding; RNA Splicing; RNA-Binding Proteins; Regulation; Regulator Genes; Research; Resources; Scientist; Signal Transduction; Specificity; System; Tertiary Protein Structure; Testing; Tissues; Translating; Translations; Visual impairment; base; conditional knockout; congenital cataract; crosslink; developmental disease; evidence base; eye formation; fiber cell; gene discovery; gene function; gene regulatory network; innovation; insight; interactive tool; lens; mRNA Precursor; mRNA Stability; mouse model; novel; tool; transcription factor; transcriptome; transcriptome sequencing; user-friendly

Project Summary The eye developmental disorders microphthalmia (small eye) and anophthalmia (no eye) result due to defects in early eye development and affect 2-6 in 50,000 live human births. Microphthalmia often presents with other ocular defects such as cataract and coloboma. The etiology of these genetically heterogeneous disorders is still not clearly understood; for example, thus far 24 genes are linked to anophthalmia in humans, and even here, the pathways involved in the regulation of these genes in eye development are not well established. We have developed a novel bioinformatics approach, iSyTE (integrated Systems Tool for Eye gene discovery, http://research.bioinformatics.udel.edu/iSyTE) to predict new genes that are associated with eye developmental defects. Based on iSyTE, our recent findings – namely, that deficiency of the RNA-binding proteins (RBPs) Celf1 and Tdrd7 cause congenital cataract – have demonstrated that RBP-based post-transcriptional regulatory mechanisms are essential for lens fiber cell differentiation. However, whether RBPs are involved in critically controlling other key aspects of ocular morphogenesis, especially in the early stages of optic vesicle and lens ectoderm/placode development, is unknown. Here, we used iSyTE to identify a new RBP, the RNA-binding motif protein Rbm24, that mediates distinct gene regulatory control in both optic vesicle and lens ectoderm in early eye development. We developed a new Rbm24-targeted conditional knockout (KO) mouse model and find that both constitutive and optic vesicle-specific Rbm24-KO mice exhibit fully penetrant eye defects namely, microphthalmia and/or anophthalmia. In this proposal, we will test the overarching hypothesis that Rbm24 mediates post-transcriptional control of key genes in optic vesicle and lens ectoderm /placode in early eye development. Specifically, we will address the following goals. (Aim 1) Characterize the pathogenesis of eye defects in Rbm24 KO mice (germline KO, and conditional KOs in optic vesicle and lens) and gain insights into the molecular underpinnings of the ocular defects by analysis of the Rbm24 cKO optic vesicle and lens placode transcriptome and proteome. (Aim 2) Elucidate the direct RNA targets of Rbm24 by RNA-immunoprecipitation (RIP) and cross-linked IP (CLIP) followed by RNA-Sequencing (RNA-seq). Further, test the mechanism of Rbm24-mediated control of key regulatory genes that function in early eye development and are linked to anophthalmia/microphthalmia. Specifically, we will investigate the molecular basis of Rbm24 function in: (1) control of Sox2 in the optic vesicle and the lens, (2) control of Lhx2 in the optic vesicle, and (3) control of Pax6 in the lens. (Aim 3) Integrate and analyze these Rbm24 data within the larger context of existing eye data to derive Rbm24 downstream gene regulatory networks (GRNs) in the optic vesicle and lens. The expected overall impact of this innovative proposal is that it will fundamentally advance our mechanistic understanding of post- transcriptional control of gene expression in optic vesicle and lens ectoderm/placode and lead to identification of new targets associated with the eye disorders microphthalmia and anophthalmia.

项目概要 由于缺陷而导致的眼睛发育障碍小眼症（小眼睛）和无眼症（无眼） 影响早期眼睛发育，影响 50,000 名活产婴儿中的 2-6 名。小眼症常与其他疾病一起出现 眼部缺陷，例如白内障和缺损。这些遗传异质性疾病的病因仍不清楚 不清楚；例如，到目前为止，有 24 个基因与人类无眼症有关，甚至在这里， 眼睛发育过程中这些基因的调控途径尚未明确。我们有 开发了一种新颖的生物信息学方法 iSyTE（用于眼基因发现的集成系统工具， http://research.bioinformatics.udel.edu/iSyTE）预测与眼睛发育相关的新基因 缺陷。基于 iSyTE，我们最近的发现 – 即 RNA 结合蛋白 (RBP) Celf1 的缺陷 和 Tdrd7 导致先天性白内障 – 已证明基于 RBP 的转录后调节 机制对于晶状体纤维细胞分化至关重要。然而，RBP 是否参与关键的 控制眼形态发生的其他关键方面，特别是在视神经泡和晶状体的早期阶段 外胚层/基板发育尚不清楚。在这里，我们使用 iSyTE 鉴定了一个新的 RBP，即 RNA 结合基序 蛋白质 Rbm24，在早期介导视泡和晶状体外胚层中不同的基因调节控制 眼睛发育。我们开发了一种新的 Rbm24 靶向条件敲除 (KO) 小鼠模型，并发现 组成型和视神经囊泡特异性 Rbm24-KO 小鼠均表现出完全渗透性眼部缺陷，即 小眼症和/或无眼症。在本提案中，我们将测试 Rbm24 的总体假设 介导早期眼视囊和晶状体外胚层/基板关键基因的转录后控制 发展。具体来说，我们将实现以下目标。 （目标 1）表征眼病发病机制 Rbm24 KO 小鼠的缺陷（种系 KO 以及视神经泡和晶状体中的条件性 KO）并深入了解 通过分析 Rbm24 cKO 视泡和晶状体基板来了解眼部缺陷的分子基础 转录组和蛋白质组。 （目标 2）通过 RNA 免疫沉淀阐明 Rbm24 的直接 RNA 靶标 (RIP) 和交联 IP (CLIP)，然后进行 RNA 测序 (RNA-seq)。进一步测试其机制 Rbm24 介导的关键调控基因的控制在早期眼睛发育中发挥作用并与 无眼症/小眼症。具体来说，我们将研究 Rbm24 功能的分子基础：(1) 视泡和晶状体中 Sox2 的控制，(2) 视泡中 Lhx2 的控制，以及 (3) Pax6 的控制 镜头里。 （目标 3）在现有眼部数据的更大背景下整合和分析这些 Rbm24 数据，以 导出视神经泡和晶状体中的 Rbm24 下游基因调控网络 (GRN)。预期整体 这一创新提议的影响在于，它将从根本上增进我们对后人类的机械理解。 视泡和晶状体外胚层/基板中基因表达的转录控制，并导致鉴定 与小眼症和无眼症相关的眼部疾病的新目标。