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.

项目摘要 眼睛发育障碍会导致缺陷引起的微观恐惧症（小眼）和性质的疾病（无眼） 在早期的眼睛发育中，有50,000名活人分娩中的2-6。微观情绪通常与其他 眼部缺陷，例如白内障和古罗巴马。这些遗传异质性疾病的病因仍然是 尚未清楚地理解；例如，迄今 这些基因在眼睛发育中涉及的途径尚未确定。我们有 开发了一种新型的生物信息学方法，即Isyte（Eye Gene Discovery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的缺乏 和TDRD7导致先天性白内障 - 证明基于RBP的转录后调节 机制对于透镜纤维细胞分化至关重要。但是，RBP是否涉及批判性 控制眼形形态发生的其他关键方面，尤其是在视力囊泡和镜头的早期 外胚层/placode开发未知。在这里，我们使用ISYTE识别新的RBP，即RNA结合基序 蛋白RBM24，介导了早期视力囊泡和镜头外胚层的不同基因调节对照 眼睛发育。我们开发了一种新的RBM24靶向条件敲除（KO）鼠标模型，发现 本构和视囊特异性RBM24-KO小鼠都暴露了完全渗透的眼缺损，即 微观恐惧症和/或性病。在此提案中，我们将测试RBM24的总体假设 在早期眼睛中介导关键基因和镜头外胚层 /placode的关键基因的转录后控制 发展。具体来说，我们将解决以下目标。 （AIM 1）表征眼睛的发病机理 RBM24 KO小鼠的缺陷（种系KO和光囊泡和镜头中的有条件KO），并洞悉 通过分析RBM24 CKO视囊和透镜置换镜头的分子缺损分子基础 转录组和蛋白质组。 （AIM 2）通过RNA免疫沉淀阐明RBM24的直接RNA靶标 （RIP）和交联的IP（夹子），然后进行RNA序列（RNA-Seq）。此外，测试机制 RBM24介导的关键调节基因的控制，该基因在早期眼发发育中起作用，并与 Anophthalmia/Microphthalmia。特别是，我们将研究RBM24功能的分子基础：（1） 控制视囊和镜头中Sox2的控制，（2）控制视力囊泡中的LHX2，（3）控制PAX6 在镜头。 （目标3）在现有眼睛数据的较大上下文中集成和分析这些RBM24数据 推导RBM24下游基因调节网络（GRNS）在视囊和镜头中。总体预期 这项创新提议的影响是，它将从根本上提高我们对后的机械理解 基因表达在视囊和镜头外胚层/置换术中的转录控制，并导致鉴定 与眼睛疾病有关的新靶标微粒症和性心血症。