Post transcriptional control of gene expression in the lens

 描述（由申请人提供）：白内障，即眼透镜的混浊，是全球失明的主要原因。大多数散发性儿童白内障的病因尚不清楚，估计遗传异常占这些病例的25-50%。我们已经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：//bioinformatics.udel.edu/Research/iSyTE）来预测与白内障相关的基因。在最近的过去，我们的发现，RNA颗粒（RG）的组成部分TDRD 7的缺陷，导致青少年白内障在人类和动物模型表明转录后调控机制的发展和维持透镜透明度的重要性。我们现在已经使用iSyTE来鉴定一种新的保守的RNA结合蛋白和RG组分Celf 1，其在小鼠突变体中的种系或晶状体特异性缺失导致早发性白内障。在本提案中，我们将测试Celf 1介导基因表达的转录后控制以调节透镜发育的总体假设。具体而言，我们将实现以下目标。(Aim 1）通过分析透镜转录组和蛋白质组，表征Celf 1小鼠突变体中透镜缺陷的发病机制，并深入了解Celf 1突变体中透镜缺陷的分子基础。(Aim 2）通过RNA免疫沉淀，然后进行RNA测序（RNA-Seq）来阐明Celf 1的直接RNA靶标。此外，测试Celf 1介导的调节机制，用于在透镜中起作用的高优先级候选基因，并基于严格的筛选标准进行选择。具体来说，我们将 研究Celf 1功能的分子机制：1）细胞周期激酶抑制剂p27的翻译控制，2）透镜上皮中Prox 1表达的控制，和3）核降解所必需的纤维细胞分化因子Dnase 2b的mRNA稳定性的控制。(Ai 3）最后，在现有透镜数据的背景下整合和分析Celf 1上的这些数据，以导出透镜中的Celf调控网络。这些旨在阐明Celf 1功能机制的研究将促进对透镜中基因表达的转录后控制的理解，并导致识别与白内障相关的新靶点。

 DESCRIPTION (provided by applicant): Cataract, the opacification of the ocular lens, is the leading cause of blindness worldwide. The etiology of most cases of sporadic pediatric cataract is still unknown, and genetic anomalies are estimated to account for 25-50% of these cases. We have developed a novel bioinformatics approach, iSyTE (integrated Systems Tool for Eye gene discovery, http://bioinformatics.udel.edu/Research/iSyTE) to predict genes that are associated with cataract. In the recent past, our finding that deficiency of the RNA granule (RG) component TDRD7 causes juvenile cataracts in human and animal models have indicated the significance of post-transcriptional regulatory mechanisms in the development and maintenance of lens transparency. We have now used iSyTE to identify a new conserved RNA binding protein and RG component Celf1 whose germline or lens-specific deletion in mouse mutants causes early onset cataracts. In this proposal, we will test the overarching hypothesis that Celf1 mediates post-transcriptional control of gene expression to regulate lens development. Specifically, we will address the following goals. (Aim 1) Characterize the pathogenesis of lens defects in Celf1 mouse mutants and gain insights into the molecular underpinning of lens defects in Celf1 mutants by analysis of the lens transcriptome and proteome. (Aim 2) Elucidate the direct RNA targets of Celf1 by RNA-immunoprecipitation followed by RNA-Sequencing (RNA- Seq). Further, test the mechanism of Celf1-mediated regulation for high-priority candidate genes that function in the lens, and are selected based on stringent filtering criteria. Specifically, we will investigate the molecular mechanism of Celf1 function in: 1) translational control of the cell cycl kinase inhibitor p27, 2) control of Prox1 expression in lens epithelium, and 3) control of mRNA stability for the fiber cell differentiation factor essential for nuclear degradation, Dnase2b. (Ai 3) Finally, integrate and analyze these data on Celf1 within the context of existing lens data to derive Celf-regulatory networks in the lens. These investigations aimed at elucidating the mechanism of Celf1 function will advance the understanding of post-transcriptional control of gene expression in the lens and lead to identification of new targets associated with cataracts.