A single molecule view on Dbp5 and mRNA at the nuclear pore
Nucleus-Austin, 2013, 4(1): 8-13.
Numerous molecular details of intracellular mRNA processing have been revealed in recent years. However, the export process of single native mRNA molecules, the actual translocation through the nuclear pore complex (NPC), could not yet be examined in vivo. The problem is observing mRNA molecules without interfering with their native behavior. We used a protein-based labeling approach to visualize single native mRNPs in live salivary gland cells of Chironomus tentans, an iconic system used for decades to study the mRNA life cycle. Recombinant hrp36, the C. tentans homolog of mammalian hnRNP A1, was fluorescence labeled and microinjected into living cells, where it was integrated into nascent mRNPs. Intranuclear trajectories of single mRNPs, including their NPC passage, were observed with high space and time resolution employing a custom-built light sheet fluorescence microscope. We analyzed the kinetics and dynamics of mRNP export and started to study its mechanism and regulation by measuring the turnover-kinetics of single Dbp5 at the NPC.
mRNA-export; mRNA labelling; light sheet microscope; nuclear pore complex nuclear retention; Dbp5; nuclear probing; cytoplasmic probing; nucleocytoplasmic trafficking