Multicolor Live-Cell mRNA Imaging Using RNA-Regulated Destabilization Domains

mRNA molecules are critical for relaying genetic information from the genome into functional proteins for maintaining cellular function. Real-time imaging of mRNA in living cells reveals the spatiotemporal dynamics of RNA and offers insights into RNA-mediated regulation of gene expression and cellular function. Here, we describe a method of using RNA-regulated destabilization domains to simultaneously image multiple mRNAs in living cells. In this method, three mRNAs of interest are engineered to contain three distinct RNA imaging tags. Each tag specifically binds to and stabilizes its cognate RNA-regulated destabilization domain fused to fluorescent proteins, resulting in selective activation of fluorescence on the corresponding mRNA for imaging.