Abstract
Live imaging of mRNA in cells and organisms is important for understanding the dynamic aspects underlying its function. Ideally, labeling of mRNA should not alter its structure or function, nor affect the biological system. However, most methods applied in vivo make use of genetically encoded tags and reporters that significantly enhance the size of the mRNA of interest. Alternately, we utilize the 3' poly(A) tail as a non-coding repetitive hallmark to covalently label mRNAs via bioorthogonal chemistry with different fluorophores from a wide range of spectra without significantly changing the size. We demonstrate that the labeled mRNAs can be visualized in cells and zebrafish embryos, and that they are efficiently translated. Importantly, the labeled mRNAs acquired the proper subcellular localization in developing zebrafish embryos and their dynamics could be tracked in vivo.
Highlights
Harnessing the potential of this labeling strategy, in the current study we investigated if different types of uorophores could be used to visualize mRNA in living systems
We found that the non-labeled STOP mcherry-nos mRNA (Fig. 4A upper le panel, with the mcherry sequence targeted by an RNAscope probe) was localized to granules within which the endogenous nanos mRNA resides (Fig. 4A upper third panel, with the nanos open reading frame targeted by an RNAscope probe)
We demonstrate a generally applicable bioorthogonal approach to label mRNAs in a way that maintains their translational activity in adherent cells and zebra sh embryos, and does not affect their subcellular localization in the latter
Summary
The de ned localization of different mRNAs to speci c subcellular domains provides a mechanism for regulating gene expression temporally and spatially, in particular during dynamic processes such as embryonic development.[1,2,3,4,5,6,7] Classical examples of localized mRNAs include the ASH1 in yeast,[8] the bicoid, oskar and nanos in Drosophila embryos,[9] the vg[1] in Xenopus oocytes,[10] as well as beta actin in mammalian neurons.[11,12,13] The development of the germline is a good model for studying subcellular mRNA localization, as it heavily relies on tight posttranscriptional regulation, with many key components being highly conserved across species. To test whether poly(A) tail-labeling of mRNAs containing a nos 30 UTR would be suitable for visualization in zebra sh embryos, we injected the in vitro-labeled mRNA into 1-cell stage embryos and assessed the dye signal in PGCs at 10 hours post fertilization (hpf) (Fig. 3A).
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