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

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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.