Imaging translation dynamics in live embryos reveals spatial heterogeneities

Abstract

The translation of individual mRNA molecules is a key biological process, yet this multi-step process has never been imaged in living multicellular organisms. Here we deploy the recently developed Suntag method to visualize and quantify translation dynamics of single mRNAs in living Drosophila embryos. By focusing on the translation of the conserved major epithelial-mesenchymal transition (EMT)-inducing transcription factor Twist, we identified spatial heterogeneity in mRNA translation efficiency and reveal the existence of translation factories, where clustered mRNAs are cotranslated preferentially at basal perinuclear regions. Simultaneous visualization of transcription and translation dynamics in a living multicellular organism opens exciting new avenues for understanding of gene regulation during development.

Dynamics ofhunchbacktranslation in real time and at single mRNA resolution in theDrosophilaembryo

“…The different MCP and scFv lines that we have generated will facilitate the quantitative study of translation of other Drosophila mRNAs, in live and/or fixed samples. Consistent with this, we note that another study, carried out in parallel to ours, has also recently described the application of the SunTag approach to the study of twi mRNA translation in the Drosophila embryo (Dufourt et al, 2020). Overall, the quantitative data from the fixed SunTag embryo images and dynamic information about translation sites gained from imaging live embryos represents a powerful combination for probing the spatiotemporal regulation of translation.…”

Tyramine induces dynamic RNP granule remodeling and translation activation in the Drosophila brain

“…As GFP-based reporters reflect translation status indirectly, and with poor temporal dynamics, we then aimed at monitoring translation activation with high spatiotemporal resolution, using the SunTag methodology ( Pichon et al, 2016 ; Wang et al, 2016 ; Wu et al, 2016 ; Yan et al, 2016 ), recently deployed in Drosophila ( Dufourt et al, 2021 ). SunTag-tagged profilin transcripts were co-expressed in MB γ neurons together with scFv-GFP-NLS fusions to detect translation sites and brains were imaged in real-time.…”

“…The UAS-SunTag- profilin plasmid was generated using the NEBuilder HiFi DNA Assembly Master Mix (NEB#E2621), through two successive Gibson assembly reactions. In the first one, UASt and profilin 5’UTR fragments were assembled into the twi_suntag_MS2 plasmid backbone ( Dufourt et al, 2021 ). The following primers were used for the PCR amplification of inserted fragments: UASt (1102_UASt_fwd 5’- tcgtcttcaagaattcgtttTGCTAGCGGATCCAAGCTTG -3’, 1103_UASt_rev 5’- ttactttcaaTTCCCTATTCAGAGTTCTCTTCTTG -3’); profilin 5’UTR (1104_5'UTR_prof_fwd 5’- gaatagggaaTTGAAAGTAAGTTACCCCAAG -3’, 1119_5'UTR_prof_rev 5’- gctgccgctaagcttggtCATacGGTGCTTTGTTTGTCGTG -3’).…”

“…With the notable exception of Stress Granules, most of these RNP granules are found constitutively and have been implicated in the regulation of various aspects of RNA expression, from decay to subcellular RNA localization and translation ( Besse and Ephrussi, 2008 ; Buchan, 2014 ; De Graeve and Besse, 2018 ; Formicola et al, 2019 ; Ivanov et al, 2019 ; Marnik and Updike, 2019 ; Trcek and Lehmann, 2019 ). Intriguingly, both repressor and activator functions have been assigned to RNA condensation: clustering of transcripts into translation factories, for example, appears to enhance translation ( Pichon et al, 2016 ; Pizzinga et al, 2019 ; Chouaib et al, 2020 ; Dufourt et al, 2021 ), while recruitment of mRNAs to P-bodies, germ granules, or neuronal granules is rather associated with translational repression ( Krichevsky and Kosik, 2001 ; Fritzsche et al, 2013 ; Hubstenberger et al, 2017 ; Ivanov et al, 2019 ; Kim et al, 2019 ; Tsang et al, 2019 ).…”

“…Functionally, we demonstrate that Tyramine signals through the TyrR receptor and through CamkII, a calcium-activated kinase associating with Imp, to induce RNP granule remodeling. Furthermore, we show that RNP granule remodeling is linked to the translation activation of granule-associated mRNAs, a process we monitor with unprecedented resolution via the SunTag amplification system ( Tanenbaum et al, 2014 ) recently implemented in Drosophila ( Dufourt et al, 2021 ). Together, our functional and cellular study sheds new light into the mechanisms underlying signal-induced disassembly of RNP granules.…”