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.

在高空间和临时分辨率下控制基因表达对于早期胚胎发育至关重要，在这些胚胎发育中，同时紧密协调的过程导致细胞命运决策和细胞迁移。斑马鱼是研究脊椎动物发育的出色模型生物，因为胚胎是透明的，并且在母亲的身体外发育，使它们更适合微观分析。但是，当前对基因表达操纵的方法对于某些应用并不是最佳的，例如研究研究细胞迁移（例如生殖细胞迁移）和快速亚细胞过程所需的空间对照（例如，RNA定位和命运）。在同一方向上，RNA分子遵循其动力学的标记将受益于对核酸的化学和物理特性最小影响的标记程序。特别是，MRNA中的某些序列元素，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和RNA定位来促进基因表达的调节。因此，基于将标签附加到RNA的大多数当前mRNA标记方法可能会影响这些特性。因此，我们开发了一种新方法，在该方法中，我们将mRNA标记为具有多个荧光团的聚（A）尾巴，而不会改变mRNA的主要序列。我们发现，当注射到一细胞阶段受精的斑马鱼卵中时，可以在发育中的胚胎中遵循这些mRNA，并且与本地对应物相似。在这个项目中，我们的目标是遵循特定的mRNA，以控制斑马鱼中的细胞命运，并同时通过光照来控制它们的翻译。为此，我们开发了一种方法，可以用光扣组在5΄-CAP上修改mRNA，并证明这些修饰可以阻止翻译，并且可以通过光线去除。基于这些结果，我们将合成各种光扣帽并评估其相对性能。斑马鱼将使用相对光夹的mRNA来研究在特定位置的指导提示（趋化因子）的光照机制，并以特定量指导我们用作体内模型（Zebrafish生殖细胞）的细胞迁移。最后，我们旨在结合帽胶和聚（A）标签，以剖析mRNA的生物学作用及其编码的蛋白质。具体而言，我们将研究为死端，纳米和VASA蛋白编码的mRNA编码，通过研究定位以及在突变胚胎发展过程中不同阶段通过光触发其表达的效果。