In Vivo RNA Labeling Using MS2

Abstract

The traffi cking and asymmetric distribution of cytoplasmic RNA is a fundamental process during development and signaling across phyla. Plants support the intercellular traffi cking of RNA molecules such as gene transcripts, small RNAs, and viral RNA genomes by targeting these RNA molecules to plasmodesmata (PD). Intercellular transport of RNA molecules through PD has fundamental implications in the cell-to- cell and systemic signaling during plant development and in the systemic spread of viral disease. Recent advances in time-lapse microscopy allow researchers to approach dynamic biological processes at the molecular level in living cells and tissues. These advances include the ability to label RNA molecules in vivo and thus to monitor their distribution and traffi cking. In a broadly used RNA labeling approach, the MS2 method, the RNA of interest is tagged with a specifi c stem-loop (SL) RNA sequence derived from the origin of assembly region of the bacteriophage MS2 genome that binds to the bacteriophage coat protein (CP) and which, if fused to a fl uorescent protein, allows the visualization of the tagged RNA by fl uorescence microscopy. Here we describe a protocol for the in vivo visualization of transiently expressed SL-tagged RNA and discuss key aspects to study RNA localization and traffi cking to and through plasmodesmata in Nicotiana benthamiana plants.