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.

眼睛的透镜是一种透明的组织，它可以折射光线并将光线聚焦在视网膜上，从而获得清晰的视觉。如果透镜 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 全世界范围内的失明，常见于老年人（大约一半的80岁或以上的美国人 白内障），但也可以在出生时存在或在儿童早期发展-称为小儿白内障。遗传 据估计，异常占儿童白内障病例的约25-50%， 这些是未知的。重要的是，尽管信号和转录因子在透镜发育和发育中的作用是不明确的， 在过去的几十年里，白内障已经得到了很好的定义，即转录后基因中涉及的蛋白质 表达控制的研究严重不足，事实上，到目前为止， 透镜。我们对这些蛋白质的研究表明，转录后调节蛋白质的表达与转录后调节蛋白质的表达密切相关。 这些机制对于透镜的发育是至关重要的，它们的丧失导致早发性白内障和眼睛缺陷。 这些因子的鉴定是通过我们的新的生物信息学方法iSyTE（整合 眼基因发现系统工具），该工具可有效识别与透镜相关的高优先级靶基因 发育和白内障。我们现在已经使用iSyTE来鉴定透镜中的新的转录后调节因子， 即Elavl 1（胚胎致死异常视觉（ELAV）样RNA结合蛋白）。Elavl 1的功能包括： 未在透镜中检查。因此，我们开发了一种新的Elavl 1靶向晶状体特异性条件敲除方法， (KO)小鼠模型，并发现Elav 11 cKO小鼠表现出早期发作的眼缺陷，即白内障， 小眼症在这个提议中，我们将测试Elavl 1介导转录后的 基因表达控制透镜发育的关键调节因子，其破坏导致白内障， 小眼症具体而言，我们将实现以下目标。(Aim 1）表征透镜的发病机制 Elavl 1cKO小鼠中的缺陷，并通过以下方法深入了解这些缺陷的结构和分子基础： 透镜形态、转录组和蛋白质组的比较分析。(Aim 2）测试Elavl 1的机制- 介导控制透镜中的转录后基因表达。具体来说，我们将调查 Elavl 1调控透镜发育/分化关键转录因子的分子机制 Pax 6、c-Maf和Prox 1等靶标。用RNA免疫沉淀法鉴定Elavl 1的直接RNA靶点 然后进行RNA测序。(Aim 3）研究Elavl 1和Celf 1如何协同介导转录后 在透镜中控制并整合和分析以上生成的所有分子、基因组和功能数据， 推导出透镜中的Elavl 1和Celf 1调节网络。这些创新的预期总体影响 旨在揭示Elavl 1功能机制的调查方法是，它将继续推进 我们对透镜中转录后水平基因表达调控的了解， 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靶标。