An adapted MS2-MCP system to visualize endogenous cytoplasmic mRNA with live imaging in < i > Caenorhabditis elegans < /i >

by Cristina Tocchini, Susan E. Mango

Live imaging of RNA molecules constitutes an invaluable means to track the dynamics of mRNAs, but live imaging inCaenorhabditis elegans has 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 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 accumulates in the cytoplasm, leading to loss-of-function phenotypes. In addition, during epithelial morphogenesis, MS2-tagged mRNAs fordlg-1 fail to associate with the adherens junction, as observed for untagged, endogenous mRNAs. These defects are reversed by inactivating the nonsense-mediated decay pathway. RNA accumulates in the cytoplasm, mutant phenotypes are rescued, anddlg-1 RNA associates with the adherens junction. These data suggest that MS2 repeats can induce the degradation of endogenous RNAs and alter their cytoplasmic distribution. Although our focus is RNAs expressed in epithelial cells during morphogenesis, we find that this method can be applied to other cell types and stages.

Source: PLoS Biology: Archived Table of Contents - March 1, 2024 Category: Biology Authors: Cristina Tocchini Source Type: research