RNA-binding proteins in early eye development.

项目摘要 眼睛发育障碍小眼症（小眼）和无眼症（无眼）是由于缺陷造成的 在眼睛早期发育中，每50，000个活产婴儿中就有2-6个受影响。小眼症通常与其他 眼睛缺陷，如白内障和缺损。这些遗传异质性疾病的病因仍然是 还不清楚;例如，到目前为止，有24个基因与人类的无眼症有关，即使在这里， 在眼睛发育中涉及这些基因的调节的途径还没有很好地建立。我们有 开发了一种新的生物信息学方法，iSyTE（用于眼睛基因发现的集成系统工具， http：//research.bioinformatics.udel.edu/iSyTE）来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）Celf 1的缺乏 和Tdrd 7导致先天性白内障-已经证明，基于RBP的转录后调节 机制对于透镜纤维细胞分化是必不可少的。然而，限制性商业惯例是否涉及关键的 控制眼形态发生的其他关键方面，特别是在视泡和透镜的早期阶段 外胚层/基板发育，未知。在这里，我们使用iSyTE来识别一个新的RBP，RNA结合基序 Rbm 24蛋白，在早期视泡和透镜外胚层中介导不同的基因调节控制 眼睛发育我们开发了一种新的Rbm 24靶向条件性敲除（KO）小鼠模型，并发现 组成型和视囊特异性Rbm 24-KO小鼠均表现出完全穿透性眼缺陷即， 小眼症和/或无眼症。在本提案中，我们将测试总体假设，即Rbm 24 介导早期眼视泡和透镜外胚层/基板关键基因的转录后调控 发展具体而言，我们将实现以下目标。(Aim 1）表征眼睛的发病机制 Rbm 24 KO小鼠的缺陷（种系KO和视泡和透镜中的条件性科斯），并深入了解 通过对Rbm 24 cKO视泡和透镜基板的分析来探讨眼部缺陷的分子基础 转录组和蛋白质组。(Aim 2）通过RNA免疫沉淀法阐明Rbm 24的直接RNA靶点 (RIP)和交联IP（CLIP），随后进行RNA测序（RNA-seq）。此外，测试 rbm 24介导的关键调控基因的控制，这些基因在早期眼睛发育中起作用，并与 无眼/小眼。具体而言，我们将研究Rbm 24功能的分子基础：（1） 视泡和透镜中Sox 2的控制，（2）视泡中Lhx 2的控制，和（3）Pax 6的控制 在透镜中。(Aim 3）在现有眼睛数据的更大背景下整合和分析这些Rbm 24数据， 推导出视泡和透镜中的Rbm 24下游基因调控网络（GRNs）。预期总体 这一创新性建议的影响是，它将从根本上推进我们对后社会机制的理解， 视泡和透镜外胚层/基板中基因表达的转录控制，并导致鉴定 与眼部疾病小眼症和无眼症相关的新目标。

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.