Regulation of mRNA decay in retinal development and maintenance

视网膜发育和维护中 mRNA 衰减的调节

• 批准号: 10580686
• 负责人: Xiuqian Mu
• 金额: $ 49.62万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10580686
• 关键词: 3' Untranslated Regions; Address; Affect; Alleles; Apoptosis; Binding; Binding Proteins; Bioinformatics; Biological Process; Biology; Defect; Development; Dimensions; Disease; Electrophysiology (science); Electroretinography; Elements; Embryo; Epitopes; Equilibrium; Evolution; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Histology; Immunofluorescence Immunologic; Immunoprecipitation; Knock-out; Knockout Mice; LoxP-flanked allele; Maintenance; Messenger RNA; Muller' s cell; Mutate; Neuroglia; Pathway interactions; Phenotype; Photoreceptors; Play; Post-Transcriptional Regulation; Process; Proliferating; Protein Family; Proteins; RNA Decay; Regulation; Research; Retina; Retinal Degeneration; Rod; Role; Stains; TIS11 protein; Testing; Transgenic Organisms; Zinc Fingers; cell type; crosslink; experimental study; insight; mRNA Decay; member; mouse genetics; mutant; notch protein; novel; postnatal; posttranscriptional; protein function; retinal progenitor cell; single-cell RNA sequencing; transcriptome sequencing

Project Summary/Abstract Gene regulation, which takes place at both transcriptional and post-transcriptional levels, plays important roles in retinal development and function. So far, studies of gene regulation in the retina have largely focused on the transcriptional level. Although post-transcriptional mechanisms also are critically involved in various biological processes, little is known about how post-transcriptional regulation impacts retinal development and function. In this project, we propose to address this issue by studying two members of the TTP (tristetraprolin) mRNA binding protein family, Zfp36l1 and Zfp36l2 (collectively referred to as Zfp36l1/2). TTP proteins are CCCH zinc finger proteins highly conserved through evolution, and are involved in diverse biological processes. They carry out their functions by binding to the AU-rich elements (AREs) in the 3’ UTR of target mRNAs to promote their decay. We discovered that Zfp36l1/2 were highly expressed in retinal progenitor cells (RPCs) during development and Müller glial cells and photoreceptors in the mature retina. Further, we have created retina- specific knockout mice of the two genes. Our preliminary analysis of the mutant retinas revealed that single knockout retinas appeared largely normal, but the double knockout (DKO) retina had defects in development and degenerated postnatally. Whereas RPCs give rise to all retinal cell types, Müller glial cells are considered quiescent RPCs in the mammalian retina. Thus Zfp36l1/2 likely play shared roles in these two cell types. Our finding that Zfp36l1/2 were also expressed in photoreceptors indicated that the two proteins may also be directly involved in photoreceptor maintenance. Based on these considerations, we hypothesize that regulation of mRNA decay plays essential roles in the retina, and that Zfp36l1/2 are two critical regulators of mRNA decay functioning redundantly in both retinal development and maintenance. To test this hypothesis, we propose to study the function of these two proteins in the retina by investigating how deletion of Zfp36l1 and Zfp36l2 affects both the development and maintenance of the retina, and by dissecting the genes and pathways controlled by Zfp36l1/2 at different developmental stages and in different cell types using a combined approach of mouse genetics, histology, immunofluorescence, electrophysiology, RNA-seq and single cell RNA-seq, CLIP (cross-linking immunoprecipitation)-seq, and bioinformatics. The results from these experiments collectively will allow us to uncover how Zfp36l1/2 are involved in retinal development and maintenance, to identify mRNA targets and relevant pathways regulated by them, and to reveal the shared and unique mechanisms by which these two proteins function at different developmental stages. Therefore, this project affords a unique opportunity to advance our understanding of the roles mRNA decay plays in both normal and disease conditions, and the discoveries we make will add a new dimension to our knowledge of gene regulation in the retina.

项目摘要/摘要 基因调控在转录水平和转录后水平上都发挥着重要作用。 在视网膜的发育和功能方面。到目前为止，对视网膜基因调控的研究主要集中在 转录水平。尽管转录后机制也关键地涉及到各种生物 对于转录后调控如何影响视网膜的发育和功能，人们知之甚少。 在这个项目中，我们建议通过研究TTP(Tristetraprolin)的两个成员来解决这个问题 结合蛋白家族Zfp36l1和Zfp36l2(统称为Zfp36l1/2)。TTP蛋白是CCCH锌 Finger蛋白在进化过程中高度保守，并参与多种生物学过程。他们携带着 通过与靶mRNA3‘端非编码区中的富含AU元件(ARE)结合来促进其功能 腐烂。我们发现Zfp36l1/2在视网膜祖细胞(RPC)中高表达 成熟视网膜内Müler神经胶质细胞和光感受器的发育。此外，我们还创造了视网膜- 这两种基因的特异性敲除小鼠。我们对突变型视网膜的初步分析显示， 基因敲除的视网膜看起来基本正常，但双基因敲除(DKO)的视网膜在发育中存在缺陷 并在出生后退化。尽管RPC产生所有类型的视网膜细胞，但Müler神经胶质细胞被认为是 哺乳动物视网膜中静止的RPC。因此，Zfp36l1/2可能在这两种细胞类型中扮演共同的角色。我们的 发现Zfp36l1/2也在光感受器中表达，表明这两种蛋白也可能是直接 参与光感受器的维护。基于这些考虑，我们假设，对mRNA的调控 衰变在视网膜中起着至关重要的作用，而Zfp36l1/2是mRNA衰变功能的两个关键调节因子 在视网膜发育和维护方面都是多余的。为了检验这一假设，我们建议研究 通过研究Zfp36l1和Zfp36l2的缺失如何影响这两种蛋白质在视网膜中的功能 视网膜的发育和维持，并通过解剖Zfp36l1/2控制的基因和通路 在不同的发育阶段和不同的细胞类型中使用小鼠遗传学的组合方法， 组织学、免疫荧光、电生理学、RNA-SEQ和单细胞RNA-SEQ、CLIP(交联体 免疫沉淀)-序列和生物信息学。这些实验的总体结果将使我们能够 揭示Zfp36l1/2如何参与视网膜的发育和维持，以确定mRNA靶点和 相关途径，并揭示这两者共同和独特的机制 蛋白质在不同的发育阶段起作用。因此，这个项目提供了一个独特的机会 进一步加深我们对正常和疾病条件下mRNA衰退所起作用的理解，以及 我们的发现将为我们对视网膜基因调控的知识增加一个新的维度。