RNA-binding proteins in early eye development.

早期眼睛发育中的 RNA 结合蛋白。

• 批准号: 10589082
• 负责人: Salil Lachke
• 金额: $ 34.7万
• 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 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10589082
• 关键词: Acceleration; Affect; Animal Model; 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; Fertilization; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Histology; Human; Immunoprecipitation; Knock-out; Knockout Mice; Knowledge; Lens Fiber; Lens Placodes; Lens development; Link; LoxP-flanked allele; 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 analysis; RNA-Binding Proteins; Regulation; Regulator Genes; Research; Resources; Scientist; Signal Transduction; Specificity; System; Tertiary Protein Structure; Testing; Tissues; Translating; Translations; Visual impairment; 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; mRNA Translation; mouse model; novel; posttranscriptional; 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.

项目摘要 眼睛发育障碍小眼球(小眼)和无眼症(无眼)是由缺陷引起的 在早期眼睛发育和影响2-6在50,000个活的人类出生。小眼球通常表现为其他 眼部缺陷，如白内障和缺损。这些遗传异质性疾病的病因学仍然是 不清楚；例如，到目前为止，有24个基因与人类无眼症有关，即使在这里， 这些基因在眼睛发育过程中的调控途径还不是很清楚。我们有 开发了一种新的生物信息学方法，iSyTE(眼睛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(RBPs)CELF1的缺陷 和Tdrd7导致先天性白内障-已经证明基于RBP的转录后调控 晶状体纤维细胞分化的机制是必不可少的。然而，限制性商业惯例是否参与了关键的 控制眼睛形态发生的其他关键方面，特别是在视泡和晶状体的早期阶段 外胚层/胎盘的发育，是未知的。在这里，我们使用iSyTE来鉴定一个新的RBP，RNA结合基序 Rbm24蛋白，在早期视泡和晶状体外胚层中介导不同的基因调控 眼睛发育。我们开发了一种新的Rbm24靶向条件性基因敲除(KO)小鼠模型，并发现 结构性和视泡特异性的Rbm24-KO小鼠都表现出完全穿透性的眼睛缺陷，即， 小眼球和/或无眼症。在这个提案中，我们将测试Rbm24的总体假设 介导早期眼的视泡和晶状体外胚层/胎盘关键基因转录后调控 发展。具体地说，我们将解决以下目标。(目标1)描述眼睛的发病机制 Rbm24 KO小鼠的缺陷(生殖系KO，以及视泡和晶状体中的条件性KO)，并获得对 Rbm24 CKO视泡和晶状体胎盘的分子基础研究 转录组和蛋白质组。(目的2)用RNA免疫沉淀法阐明Rbm24的直接RNA靶标 (RIP)和交联型IP(CLIP)，然后进行RNA测序(RNA-SEQ)。此外，还测试了 Rbm24介导的对关键调控基因的控制，这些基因在眼睛早期发育中发挥作用，并与 无眼球/小眼球。具体地说，我们将在以下方面研究Rbm24功能的分子基础：(1) 视泡和晶状体中Sox2的调控，(2)视泡中LHX2的调控，以及(3)Pax6的调控 在镜头里。(目标3)在现有眼睛数据的更大背景下整合和分析这些Rbm24数据，以 在视泡和晶状体中衍生Rbm24下游基因调控网络(GRN)。预期的总体情况 这一创新建议的影响是，它将从根本上推进我们对后发展的机械性理解 视泡和晶状体外胚层/胎盘基因表达的转录调控 与眼病、小眼球和无眼症相关的新靶点。