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

在高空间和时间分辨率下控制基因表达对早期胚胎发育至关重要，在早期胚胎发育中，负责细胞命运决定和细胞迁移的同时发生紧密协调的过程。斑马鱼是研究脊椎动物发育的极好的模式生物，因为它的胚胎是透明的，在母体外发育，这使它们更适合显微镜分析。然而，目前基因表达的实验操作方法并不适合某些应用，例如研究控制细胞迁移（如生殖细胞迁移）和快速亚细胞过程（如RNA定位和命运）的因素所需的时空控制。在相同的方向上，标记RNA分子以遵循其动力学将受益于标记程序，对核酸的化学和物理性质的影响最小。特别是，mrna中的某些序列元件，特别是位于3 ' -UTR的序列，通过控制RNA的稳定性、与RNA结合蛋白的相互作用以及RNA的定位来调节基因表达。由于这个原因，目前大多数基于向RNA附加标签的mRNA标记方法都可能影响这些特性。因此，我们开发了一种新的方法，在不改变mRNA的初级序列的情况下，我们在poly(a)尾部用多个荧光团标记mRNA。我们发现，当注入单细胞期受精卵时，这些mrna可以在发育中的胚胎中被跟踪，并表现出与其原生对应物相似的行为。在这个项目中，我们现在的目标是跟踪控制发育中的斑马鱼细胞命运的特定mrna，同时控制它们通过光的翻译。为此，我们开发了一种利用光笼基团对5΄-cap上的mrna进行酶修饰的方法，并证明这些修饰可以阻止翻译并且可以被光去除。基于这些结果，我们将合成各种光笼帽，并评估它们的相对性能。各自的光笼mrna将用于斑马鱼，以研究在特定位置和特定数量的光控引导线索（趋化因子）产生的细胞机制，从而指导我们用作体内模型的细胞（斑马鱼生殖细胞）的迁移。最后，我们的目标是结合cap- cage和poly(A)-labeling来剖析mrna及其编码蛋白的生物学作用。具体来说，我们将研究编码Dead end、Nanos和Vasa蛋白的mrna，通过研究它们的定位以及在突变胚胎发育的不同阶段光触发它们表达的影响。

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.