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: 10589140
• 负责人: Salil Lachke
• 金额: $ 39.18万
• 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/10589140
• 关键词: 3' Untranslated Regions; Acceleration; Address; American; Animal Model; Antibodies; Binding; Binding Sites; Bioinformatics; Biological Assay; Biology; Birth; Blindness; CISH gene; Cataract; Cellular Assay; Complex; Crystalline Lens; Data; Defect; Development; Disease; E-Cadherin; Embryo; Ensure; Etiology; Exhibits; Eye; Fertilization; Funding; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genomics; Goals; Histology; Homeostasis; Immunoprecipitation; Impairment; Individual; Investigation; Knock-out; Knockout Mice; Knowledge; Lens development; Light; Link; LoxP-flanked allele; Maintenance; Mediating; Messenger RNA; Microphthalmos; Molecular; Morphology; Mus; Nature; Online Systems; Organogenesis; Pathogenesis; Pathology; Pathway interactions; Post-Transcriptional Regulation; 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; bioinformatics resource; childhood cataract; combinatorial; comparative; conditional knockout; crosslink; dosage; early childhood; early onset; gene discovery; gene regulatory network; human model; innovation; insight; interactive tool; lens; lens transparency; mRNA Precursor; mRNA Stability; mRNA Translation; mouse model; novel; online resource; posttranscriptional; 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.

眼晶状体是一种透明组织，它将光线折射并聚焦到视网膜上，以实现清晰的视力。如果镜头 失去透明度，视力受损，这种疾病被称为“白内障”。白内障是导致白内障的主要原因。 全世界范围内的失明，普遍存在于老年人(大约一半80岁或以上的美国人患上 儿童白内障)，但也可在出生时出现或在儿童早期发展--称为儿童白内障。遗传 据估计，异常约占儿童白内障病例的25%-50%，而大多数儿童白内障的病因 这些都是未知的。重要的是，虽然信号和转录因子在晶状体发育和 在过去的几十年里，白内障已经被很好地定义为参与转录后基因的蛋白质 对表达调控的研究严重不足--事实上，到目前为止，只有四种这样的蛋白质被表征在 镜头。我们对这些蛋白质的研究表明，转录后调控 机制对晶状体的发育至关重要，它们的缺失会导致早发性白内障和眼睛缺陷。 通过我们新的生物信息学方法iSyTE(集成)，可以识别这些因素 眼睛基因发现的系统工具)，它有效地识别与晶状体相关的高优先级靶基因 发育与白内障。我们现在已经使用iSyTE在晶状体中识别了一个新的转录后调控因子， 即Eavl1(胚胎致死性视觉异常(ELAV)样RNA结合蛋白)。Elavl1的功能有 没有在镜片中检查。因此，我们开发了一种新的针对Elevl1的晶状体特异性条件基因敲除 (KO)小鼠模型，发现Eavl1cKO小鼠表现出早发性眼缺陷，即白内障和 小眼炎。在这项提议中，我们将测试最重要的假设，即Elavl1介导转录后 基因表达控制晶状体发育的关键调控因素，其干扰会导致白内障和 小眼炎。具体地说，我们将解决以下目标。(目标1)描述晶状体的发病机制 并通过以下方法深入了解这些缺陷的结构和分子基础 晶状体形态、转录组和蛋白质组的比较分析。(目标2)测试Elevl1的作用机制- 调节晶状体中转录后基因的表达。具体来说，我们将调查 Eavl1调控晶状体发育分化关键转录因子的分子机制 Pax6、c-Maf和Prox1等靶标。用RNA免疫沉淀法鉴定Elawl1的直接RNA靶点 然后进行RNA测序。(目标3)研究Eavl1和CELF1如何协调调节转录后 控制在镜头中，并整合和分析上述产生的所有分子、基因组和功能数据 在晶状体中衍生Eavl1和CELF1-调节网络。这些创新产品的预期总体影响 旨在揭示Eavl1功能机制的研究途径是，它将继续推进 我们在转录后水平上对晶状体中基因表达调控的了解，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有关的新目标。