Nuclear export of the pre-60S ribosomal subunit through single nuclear pores observed in real time

Abstract

Ribosomal subunit biogenesis within mammalian cells initiates in the nucleolus with the assembly of a 90S precursor particle, which is subsequently split into the pre-40S and pre-60S subunits. During further processing steps, pre-ribosomal subunits are loaded with export receptors, which enables their passage through the pore complexes (NPCs) into the cytoplasm. Here export factors are released and both subunits can form a mature ribosome. Ribosomal biogenesis has been studied in great detail by biochemical, genetic and electron microscopic approaches, however, until now live cell data on the in vivo kinetics are still missing. We analysed export kinetics of the large ribosomal subunit ("pre-60S particle") through single NPCs in living human cells. To assess the in vivo dynamics of this process, we established a stable cell line co-expressing Halo-tagged eIF6 and GFP-fused NTF2 to simultaneously label ribosomal 60S subunits (eIF6) and NPCs (NTF2). By combining single molecule tracking and super resolution confocal microscopy in a highly customized microscopic setup, we visualized the dynamics of single pre-60S particles during the interaction with and export through single NPCs. In this way we obtained unprecedented insights into this key cellular process. Our results revealed that for export events, maximum particle accumulation is found in the centre of the pore, while unsuccessful export terminates within the nuclear basket. The export process takes place with a single rate limiting step and an export dwell time of ~24 milliseconds. Only about 1/3 of attempted export events were successful. Given the molecular mass of the pre-60S particles our results show that the mass flux through a single NPC can reach up to ~125 MDa{middle dot}s-1 in vivo.