Control over mRNA translation by light-mediated uncaging of synthetic 5΄ caps in combination with fluorescent labeling of mRNAs for in vivo applications

通过合成 5μ 帽的光介导解禁结合体内应用的 mRNA 荧光标记来控制 mRNA 翻译

• 批准号: 426018296
• 负责人: Professor Dr. Erez Raz
• 金额: --
• 依托单位: Institut für Zellbiologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/426018296?language=en
• 关键词: Control; over; mRNA; translation; light

Controlling gene expression at high spatial and temporal resolution is critical for early embryonic development, where concurrent tightly coordinated processes responsible for cell fate decisions and cell migration take place. The zebrafish is an excellent model organism for studying vertebrate development, because the embryos are transparent and develop outside the mother’s body, making them more amenable to microscopic analysis. However, current methodology for experimental manipulation of gene expression is not optimal for certain applications, for example concerning the spatio-temporal control required for studying factors governing cell migration (e.g. germ cell migration) and rapid subcellular processes (e.g. RNA localization and fate). In the same direction, labeling of RNA molecules for following their dynamics would benefit from labeling procedures that have minimal effects on chemical and physical properties of the nucleic acid. In particular, certain sequence elements within mRNAs, in particular sequences located at the 3’-UTR contribute to regulation of gene expression by controlling RNA stability, interaction with RNA-binding proteins, and RNA localization. For this reason, most of the current mRNA labeling methods that are based on appending tags to the RNA can potentially affect these properties. We thus developed a new approach where we label mRNAs at the poly(A) tail with multiple fluorophores and without altering the primary sequence of mRNA. We found that when injected into one-cell stage fertilized zebrafish eggs, these mRNAs can be followed in the developing embryo and behave similarly to their native counterparts.In this project we now aim to follow specific mRNAs that control cell fate in developing zebrafish and at the same time to control their translation by light. To this end, we developed an approach to enzymatically modify mRNAs at the 5΄-cap with photo-caging groups and demonstrated that these modifications block translation and can be removed by light. Based on these results we will synthesize a variety of photo-caged caps and evaluate their relative performance. The respective photo-caged mRNAs will be used in zebrafish to investigate the cellular mechanisms by which light-controlled production of guidance cues (chemokines) at specific locations and at specific amounts direct the migration of the cells we use as an in vivo model (zebrafish germ cells). Finally, we aim to combine cap-caging and poly(A)-labeling to dissect the biological roles of mRNAs and the protein they encode for. Specifically, we will study the mRNAs encoding for the Dead end, Nanos and Vasa proteins by investigating both the localization as well as the effect of triggering their expression by light at different stages during development of mutant embryos.

在高空间和时间分辨率下控制基因表达对于早期胚胎发育至关重要，其中同时发生负责细胞命运决定和细胞迁移的紧密协调过程，斑马鱼是研究脊椎动物发育的优秀模型生物，因为胚胎是透明的且具有稳定性。然而，目前基因表达实验操作的方法对于某些应用来说并不是最佳的，例如研究控制细胞迁移的因素所需的时空控制。 （例如生殖细胞迁移）和快速亚细胞过程（例如RNA定位和命运）在同一方向上，对RNA分子进行标记以跟踪其动态将受益于对核酸的化学和物理性质影响最小的标记程序。特别是，mRNA 内的某些序列元件，特别是位于 3'-UTR 的序列，通过控制 RNA 稳定性、与 RNA 结合蛋白的相互作用以及 RNA 定位，有助于调节基因表达。标记方法因此，我们开发了一种新方法，用多个荧光团在 Poly(A) 尾部标记 mRNA，并且在注射到 mRNA 时不会改变 mRNA 的一级序列。在单细胞阶段受精斑马鱼卵中，这些 mRNA 可以在发育中的胚胎中追踪，并且其行为与其天然供体相似。在这个项目中，我们现在的目标是追踪控制发育中斑马鱼细胞命运的特定 mRNA，同时为此，我们开发了一种用光笼蔽基团在 5΄ 帽上酶促修饰 mRNA 的方法，并证明这些修饰会阻止翻译，并且可以通过光去除。合成各种光笼盖并评估它们的相对性能，将在斑马鱼中使用相应的光笼mRNA来研究光控制在特定位置和特定数量产生引导信号（趋化因子）的细胞机制。这最后，我们的目标是结合帽笼和聚腺苷酸标记来剖析 mRNA 及其编码的蛋白质的生物学作用。将通过研究突变胚胎发育过程中不同阶段的定位以及光触发表达的影响来研究编码 Dead end、Nanos 和 Vasa 蛋白的 mRNA。