Post transcriptional control of gene expression in the lens

 描述(申请人提供)：白内障，人工晶状体混浊，是世界范围内导致失明的主要原因。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%。我们开发了一种新的生物信息学方法，iSyTE(眼睛基因发现集成系统工具，http://bioinformatics.udel.edu/Research/iSyTE))来预测与白内障相关的基因。最近，我们在人和动物模型中发现，RNA颗粒(RG)成分TDRD7的缺失会导致幼年性白内障，这表明转录后调控机制在晶状体透明度的发展和维持中具有重要意义。我们现在已经使用iSyTE鉴定了一种新的保守的RNA结合蛋白和RG成分CELF1，其在小鼠突变中的生殖系或晶状体特异性缺失会导致早发性白内障。在这项提案中，我们将检验CELF1介导转录后基因表达控制以调节晶状体发育的最重要的假设。具体地说，我们将解决以下目标。(目的1)通过对晶状体转录组和蛋白质组的分析，研究CELF1小鼠晶状体缺陷的发病机制，深入了解CELF1突变体晶状体缺陷的分子基础。(目的2)用RNA免疫沉淀-RNA测序技术(RNA-Seq)阐明CELF1的直接RNA靶点。此外，测试CELF1对高优先级候选基因的调节机制，这些候选基因在晶状体中起作用，并根据严格的筛选标准进行选择。具体来说，我们将 研究CELF1功能的分子机制：1)细胞周期蛋白激酶抑制物p27的翻译控制，2)控制晶状体上皮细胞中Prox1的表达，3)控制核降解所必需的纤维细胞分化因子Dnase2b的mRNA稳定性。(AI 3)最后，在现有晶状体数据的背景下，整合和分析CELF1上的这些数据，以得出晶状体中的自我调节网络。这些旨在阐明CELF1功能机制的研究将促进对转录后调控晶状体中基因表达的理解，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.