An in vivo translation-reporter system for the study of protein synthesis in zebrafish embryos

Control of gene expression at the translation level is increasingly regarded as a key feature in many biological processes. Simple, inexpensive and reliable procedures to visualize sites of protein production are required to allow observation of the spatiotemporal patterns of mRNA translation at sub...

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Bibliographic Details
Published in:Biology open 2018-12, Vol.7 (12)
Main Authors: Garcez Palha, Inês, Anselme, Isabelle, Schneider-Maunoury, Sylvie, Giudicelli, François
Format: Article
Language:English
Subjects:
Biological activity
Danio rerio
Development Biology
Embryos
Fluorescence
Gene expression
In vivo methods and tests
Life Sciences
Local translation
Methods and Techniques
Neuron
Protease
Protease inhibitors
Protein biosynthesis
Protein synthesis
Proteinase inhibitors
Proteins
SPoT
TimeStamp
Translation
Zebrafish
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Summary:Control of gene expression at the translation level is increasingly regarded as a key feature in many biological processes. Simple, inexpensive and reliable procedures to visualize sites of protein production are required to allow observation of the spatiotemporal patterns of mRNA translation at subcellular resolution. We present a method, named SPoT (for Subcellular Patterns of Translation), developed upon the original TimeStamp technique ( Lin et al., 2008), consisting in the expression of a fluorescent protein fused to a tagged, self-cleavable protease domain. The addition of a cell-permeable protease inhibitor instantly stabilizes newly produced tagged protein allowing us to distinguish recently synthesized proteins from pre-existing ones. After a brief protease inhibitor treatment, the ratio of tagged versus non-tagged forms is highest at sites where proteins are the most recent, i.e. sites of synthesis. Therefore, by comparing tagged and non-tagged proteins it is possible to spotlight sites of translation. By specifically expressing the SPoT cassette in neurons of transgenic zebrafish embryos, we reveal sites of neuronal protein synthesis in diverse cellular compartments during early development.
ISSN:2046-6390
2046-6390
DOI:10.1242/bio.039362