Visualization of Nonengineered Single mRNAs in Living Cells Using Genetically Encoded Fluorescent Probes

Single mRNA imaging in live cells is a useful technique to elucidate its precise localization and dynamics. We developed a method for visualizing endogenous mRNAs in living cells with single molecule sensitivity using genetically encoded probes. An RNA-binding protein of human PUMILIO1 (PUM-HD) was used for recognizing base sequences of a target mRNA, beta-actin mRNA. Two PUM-HDs were modified by amino acid mutations to bind specifically to tandem 8-base sequences of the target mRNA. Because each PUM-HD was connected with amino- and carboxyl-terminal fragments of enhanced green fluorescent protein (EGFP), the probes emit fluorescence by reconstitution of EGFP fragments upon binding to beta-actin mRNAs. The EGFP reconstituted on the mRNAs was monitored with a total internal reflection fluorescence microscope. Results show that each fluorescent spot in live cells represented a single beta-actin mRNA and that distinct spatial and temporal movement of the individual beta-actin mRNAs was visualized. We also estimated the average velocity of the movement of the single mRNAs along microtubules in live cells. This method is widely applicable to tracking various mRNAs of interest in the native state of living cells with single-mRNA sensitivity. :