An adapted MS2-MCP system to visualize endogenous cytoplasmic mRNA with live imaging inCaenorhabditis elegans

Abstract

AbstractLive imaging of RNA molecules constitutes an invaluable means to track the dynamics of mRNAs, but live imaging inCaenorhabditis eleganshas been difficult to achieve. Endogenous transcripts have been observed in nuclei, but endogenous mRNAs have not been detected in the cytoplasm, and functional mRNAs have not been generated. Here, we have adapted live imaging methods to visualize mRNA in embryonic epithelial cells. We have tagged endogenous transcripts with MS2 hairpins in the 3’ Untranslated Region (UTR) and visualized them after adjusting MS2 Coat Protein (MCP) expression. A reduced number of these transcripts accumulate in the cytoplasm, leading to loss-of-function phenotypes. In addition, mRNAs fordlg-1fail to associate with the adherens junction, as observed for the endogenous mRNA. These defects are reversed by inactivating the nonsense-mediated decay pathway. RNA accumulates in the cytoplasm,dlg-1associates with the adherens junction, and mutant phenotypes are rescued. These data suggest that MS2 repeats can induce the degradation of endogenous targets and alter the cytoplasmic distribution. Although our focus is RNAs expressed in epithelial cells during morphogenesis, this method can likely be applied to other cell types and stages.Summary statementAn adapted MS2-MCP method to tag endogenous transcripts inC. elegansembryos for live imaging without affecting mRNA stability.