Regulation of mRNA decay in retinal development and maintenance

项目概要/摘要 基因调控在转录水平和转录后水平都发挥着重要作用 在视网膜发育和功能方面。到目前为止，视网膜中基因调控的研究主要集中在 转录水平。尽管转录后机制也在各种生物学过程中起重要作用， 虽然视网膜的发育过程中，很少有人知道如何转录后调控影响视网膜的发育和功能。 在这个项目中，我们建议通过研究TTP（tristetraprolin）mRNA的两个成员来解决这个问题。 结合蛋白家族，Zfp 36 l1和Zfp 36 l2（统称为Zfp 36 l1/2）。TTP蛋白是CCCH锌 指蛋白在进化过程中高度保守，参与多种生物过程。他们携带 通过与靶mRNA的3' UTR中的富含AU的元件（战神）结合以促进它们的功能， 腐烂我们发现Zfp 36 l1/2在视网膜前体细胞（RPCs）中高表达， 成熟视网膜中的Müller神经胶质细胞和感光细胞。我们还创造了视网膜- 这两个基因的特定敲除小鼠。我们对突变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）的视网膜在发育上有缺陷 并在出生后退化尽管RPC产生所有视网膜细胞类型，但Müller神经胶质细胞被认为是 哺乳动物视网膜中的静止RPC。因此，Zfp 36 l1/2可能在这两种细胞类型中发挥共同的作用。我们 Zfp 36 l1/2也在光感受器中表达，这一发现表明这两种蛋白质也可能直接作用于光感受器。 参与感光细胞的维护。基于这些考虑，我们假设mRNA的调节 Zfp 36 l1/2是mRNA衰变功能的两个关键调节因子 在视网膜的发育和维持中都是多余的。为了验证这一假设，我们建议研究 通过研究Zfp 36 l1和Zfp 36 l2的缺失如何影响这两种蛋白在视网膜中的功能， 视网膜的发育和维持，并通过解剖Zfp 36 l1/2控制的基因和途径， 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 免疫沉淀）-Seq和生物信息学。这些实验的结果将使我们能够 揭示Zfp 36 l1/2如何参与视网膜发育和维持，以确定mRNA靶点， 相关的途径调节，并揭示这两个共同的和独特的机制， 蛋白质在不同的发育阶段发挥作用。因此，该项目提供了一个独特的机会， 推进我们对mRNA衰变在正常和疾病条件下所起作用的理解， 我们的发现将为我们对视网膜基因调控的认识增加一个新的层面。

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.