Single-Molecule Imaging of RNA in Live Cells

Abstract

Expression of genetic information in eukaryotes involves a series of interconnected processes that ultimately determine the quality and amount of proteins in the cell. Many individual steps in gene expression are kinetically coupled, but tools are lacking to determine how temporal relationships between chemical reactions contribute to the output of the final gene product. Previous studies have imaged RNAs in living cells by genetically inserting the binding sites for bacteriophage coat proteins in the RNA of interest. However, multiple nascent RNAs were simultaneously detected at the site of transcription, necessitating a modelling approach to infer kinetic information. To circumvent these significant limitations and potential problems in data interpretation, we developed a strategy that permits direct tracking of single nascent pre-mRNA molecules in live cells. We are using this approach to study how kinetic mechanisms impact on RNA biogenesis.