Post transcriptional control of gene expression in the lens

晶状体中基因表达的转录后控制

• 批准号: 10338126
• 负责人: Salil Lachke
• 金额: $ 38万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10338126
• 关键词: 3' Untranslated Regions; Address; American; Animals; Antibodies; Binding; Binding Sites; Bioinformatics; Biological Assay; Biology; Birth; Blindness; Cataract; Cellular Assay; Complex; Crystalline Lens; Data; Defect; Development; Disease; E-Cadherin; Embryo; Ensure; Etiology; Exhibits; Eye; Funding; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genetic Translation; Genomics; Goals; Histology; Homeostasis; Human; Immunoprecipitation; Impairment; Individual; Investigation; Knock-out; Knockout Mice; Knowledge; Lens development; Light; Link; Maintenance; Mediating; Messenger RNA; Microphthalmos; Molecular; Morphology; Mus; Nature; Online Systems; Organogenesis; Pathogenesis; Pathology; Pathway interactions; Post-Transcriptional Regulation; Precipitation; Process; Proteins; Proteome; Proteomics; RNA; RNA Splicing; RNA-Binding Proteins; Regulation; Research; Retina; Role; Scanning Electron Microscopy; Scientist; Signal Transduction; System; Testing; Tissues; Translations; Vision; aged; base; bioinformatics resource; childhood cataract; combinatorial; comparative; conditional knockout; crosslink; dosage; early childhood; early onset; gene discovery; gene regulatory network; innovation; insight; interactive tool; lens; lens transparency; mRNA Precursor; mRNA Stability; mouse model; novel; online resource; tool; transcription factor; transcriptome; transcriptome sequencing

The eye lens is a transparent tissue that refracts and focuses light on the retina to allow clear vision. If the lens loses its transparency, vision is impaired, and the disease is termed “cataract”. Cataract is the major cause of blindness worldwide, commonly found in aged individuals (about half of Americans aged 80 or older develop cataract), but can also be present at birth or develop in early childhood – termed pediatric cataract. Genetic anomalies are estimated to account for about 25-50% of pediatric cataract cases, and the etiology of majority of these are unknown. Importantly, while the role of signaling and transcription factors in lens development and cataract have been well defined over the last few decades, that of proteins involved in post-transcriptional gene expression control is grossly understudied – indeed, only four such proteins have been characterized so far in the lens. Our efforts in characterizing these proteins have demonstrated that post-transcriptional regulatory mechanisms are critical for lens development, and their loss results in early onset cataract and eye defects. Identification of these factors was made possible by our novel bioinformatics approach iSyTE (integrated Systems Tool for Eye gene discovery), which is effective in identifying high-priority target genes linked to lens development and cataract. We have now used iSyTE to identify a new post-transcriptional regulator in the lens, namely, Elavl1 (Embryonic lethal abnormal vision (ELAV) like RNA-binding protein). The function of Elavl1 has not been examined in the lens. Therefore, we developed a new Elavl1-targeted lens-specific conditional knockout (KO) mouse model and find that Elavl1cKO mice exhibit early onset eye defects namely, cataract and microphthalmia. In this proposal, we will test the overarching hypothesis that Elavl1 mediates post-transcriptional gene expression control over key regulators of lens development, disruption of which causes cataract and microphthalmia. Specifically, we will address the following goals. (Aim 1) Characterize the pathogenesis of lens defects in Elavl1cKO mice and gain insights into the structural and molecular underpinning of these defects by comparative analysis of lens morphology, transcriptome and proteome. (Aim 2) Test the mechanism of Elavl1- mediated control of post-transcriptional gene expression in the lens. Specifically, we will investigate the molecular mechanism of Elavl1 function in control of the key lens development/differentiation transcription factors Pax6, c-Maf and Prox1 among other targets. Identify direct RNA targets of Elavl1 by RNA-immunoprecipitation followed by RNA-Sequencing. (Aim 3) Examine how Elavl1 and Celf1 coordinately mediate post-transcriptional control in the lens and integrate and analyze all the molecular, genomic and functional data generated above to derive Elavl1 and Celf1-regulatory networks in the lens. The expected overall impact of these innovative investigative approaches aimed at uncovering the mechanism of Elavl1 function is that it will continue to advance our knowledge of gene expression regulation in the lens at the post-transcriptional level while informing on the nature of RBP-based combinatorial control, in turn leading to identification of new targets linked to cataract.

眼镜是一种透明的组织，可折射并集中在视网膜上以允许清晰的视力。如果镜头 失去其透明度，视力受损，该疾病被称为“白内障”。白内障是 全球盲目，通常在老年人中发现（大约一半的80岁以上的美国人发展 白内障），但也可以在出生时出现或在幼儿生长 - 称为小儿白内障。遗传 估计异常占小儿白内障病例的25-50％，大多数病因 这些是未知的。重要的是，信号传导和转录因子在晶状体发展中的作用以及 在过去的几十年中，白内障的定义很好，涉及转录后基因的蛋白质 表达控制已被严格理解 - 实际上，到目前为止，只有四种此类蛋白质被表征 镜头。我们在表征这些蛋白质方面的努力表明，转录后调节性 机制对于晶状体的发展至关重要，它们的损失导致早期发作性白内障和眼睛缺陷。 通过我们的新型生物信息学方法Isyte（综合）使这些因素的识别成为可能 眼基因发现的系统工具），该工具有效地识别与镜头相关的高优先级靶基因 发育和白内障。现在，我们已经使用Isyte识别镜头中的新的转录后调节器， 也就是说，elavl1（胚胎致死异常（ELAV）如RNA结合蛋白）。 elavl1的功能 没有在镜头中检查。因此，我们开发了一个新的Elavl1靶向镜头特异性条件敲除 （KO）鼠标模型，发现Elavl1cko小鼠暴露于早期发作眼缺陷，即白内障和 微观心血症。在此提案中，我们将测试Elavl1介导后转录的总体假设 基因表达控制晶状体发育的关键调节剂，导致白内障和 微观心血症。具体来说，我们将解决以下目标。 （AIM 1）表征镜头的发病机理 elavl1cko小鼠的缺陷，并获得对这些缺陷的结构和分子基础的见解 晶状体形态，转录组和蛋白质组的比较分析。 （AIM 2）测试Elavl1-的机制 介导的晶状体转录后基因表达的控制。具体来说，我们将调查 Elavl1的分子机制在控制关键透镜发育/分化转录因子方面的功能 PAX6，C-MAF和PROX1等目标。通过RNA免疫沉淀确定ELAVL1的直接RNA靶标 然后进行RNA序列。 （AIM 3）检查elavl1和celf1如何协调转录后介导 控制镜头，并整合并分析上述生成的所有分子，基因组和功能数据 在镜头中推导Elavl1和CELF1调节网络。这些创新的预期总体影响 旨在发现Elavl1功能机制的调查方法是，它将继续前进 我们对转录后镜头中基因表达调节的了解，同时告知 基于RBP的组合控制的性质，进而导致识别与白内障相关的新目标。