Exploiting the SunTag system to study the developmental regulation of mRNA translation

Abstract

The ability to quantitatively study mRNA translation using SunTag imaging is transforming our understanding of the translation process. Here, we expand the SunTag method to study new aspects of translation regulation in Drosophila. Repression of the maternal hunchback (hb) mRNA in the posterior of the Drosophila embryo is a textbook example of translational control. Using SunTag imaging to quantify translation of maternal SunTag-hb mRNAs, we show that repression in the posterior is leaky, as ∼5% of SunTag-hb mRNAs are translated. In the anterior of the embryo, the maternal and zygotic SunTag-hb mRNAs show similar translation efficiency despite having different untranslated regions (UTRs). We demonstrate that the SunTag-hb mRNA can be used as a reporter to study ribosome pausing at single-mRNA resolution, by exploiting the conserved xbp1 mRNA and A60 pausing sequences. Finally, we adapt the detector component of the SunTag system to visualise and quantify translation of the short gastrulation (sog) mRNA, encoding an essential secreted extracellular BMP regulator, at the endoplasmic reticulum in fixed and live embryos. Together, these tools will facilitate the future dissection of translation regulatory mechanisms during development.

Keywords: Drosophila embryo; Hunchback; Ribosome pausing; Sog; SunTag; Translation.