Abstract
Visualizing mRNA in real time invivo at high resolution is critical for a full understanding of the spatiotemporal dynamics of gene regulation and function. Here, using a PP7/PCP-based mRNA-tagging approach, we construct a collection of tissue-specific and differentially expressed toolkit strains for visualizing mRNAs encoding apical, basolateral, and junctional proteins in Caenorhabditis elegans epithelia. We precisely delineate the spatiotemporal organization and dynamics of these transcripts across multiple subcellular compartments and tissues. Remarkably, all the transcripts exhibit an asymmetric, membrane-associated localization during epithelial polarization and maturation, which suggests that mRNA localization is a prerequisite for epithelial polarization and function. Single-particle tracking reveals striking features of the transport dynamics of the mRNAs in a gene-specific, compartment-linked, and time-resolved manner. The toolkit can be used to identify the cis-regulatory elements and trans-acting factors for mRNA localization. This study provides a valuable resource to investigate complex RNA dynamics in epithelial polarity and morphogenesis.
Highlights
MRNA localization is a highly evolutionarily conserved mechanism in a variety of cells and organisms
We generated a protein reporter consisting of a fusion of tandem dimers of PCP and Tomato fluorescent protein (FP), preceded by a nuclear localization signal (NLS) to restrict PCP-Tomato to the nucleus if it is not complexed to RNA
The localization of erm-1 in spermatheca was confirmed by single-molecule FISH (smFISH) (Figures 2E and S1D, bottom panels). These results reveal a robust correlation between asymmetric erm-1 localization and epithelial polarization in multiple tissues. erm-1 mRNAs appear to be enriched on the membranes at early stages when the epithelia start to polarize, which suggests that mRNA targeting may be important in immature epithelia when polarity is still labile
Summary
MRNA localization is a highly evolutionarily conserved mechanism in a variety of cells and organisms. Global fluorescent in situ hybridization (FISH) analyses in Drosophila illustrated that most mRNAs are post-transcriptionally localized in distinct temporal and spatial patterns (Wilk et al, 2016; Jambor et al, 2015; Lecuyer et al, 2007). The differentially localized maternal transcripts in Xenopus and Drosophila function to establish morphogen gradients that determine cell fates and body patterning (Berleth et al, 1988; Rebagliati et al, 1985). Over 200 mRNA transcripts have been identified to be enriched asymmetrically in Caenorhabditis elegans 2-cell stage embryos, revealing an important mechanism for cell fate specification (Osborne Nishimura et al, 2015)
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