Visualizing translocation of pre‐ribosomal particles through nuclear pore complexes by electron tomography

Abstract

Exchanges between the nucleus and the cytoplasm are ensured by the nuclear pore complexes (NPCs), massive multi‐molecular structures that form channels through the nuclear envelope. The molecular determinants that underlie the translocation of macromolecules through NPCs have been thoroughly studied, but less is known about the topology and the dynamics of the translocation events. The precursors to the ribosomal subunits, which ensure protein synthesis in the cells, are one of the biggest and most abundant nuclear export substrates. Most steps of ribosome synthesis take place in the nucleolus, but pre‐ribosomes (the precursors to the ribosomal subunits) leave the nucleolus and are exported into the cytoplasm at the end of the maturation process. In yeast Saccharomyces cerevisiae , the ribosome synthesis rate is estimated to reach 4000 subunits/minute in exponential growth phase. How cargoes of such large size and complex structure cross the barrier of the nuclear pore complex so efficiently remains poorly understood. In addition, the interactions of large cargoes with the NPC as well as their path in the central channel have been much debated and several models have been proposed, calling for experimental observations at high resolution. Using ultrafast high‐pressure freezing, cryo‐embedding and electron tomography, we could detect for the first time unlabelled large RNPs translocating through the NPC in yeast. Their size, abundance and morphological resemblance with both cytoplasmic ribosomes and nucleolar RNPs indicated that they were mainly pre‐ribosomes. In support to this hypothesis, the presence of these particles in NPCs was strongly diminished upon inhibition of ribosome synthesis in an RNA polymerase I mutant strain. Over 700 NPCs were observed in the tomograms: the occupancy rate of NPCs with RNP particles was only ~5‐6%, suggesting that pre‐ribosome nuclear export was not limited by the number of NPCs. Although pre‐ribosomes are synthesized in the nucleolus, they were detected with equal probability in NPCs located on the nucleolar and the nucleoplasmic sides of the nucleus, indicating no specialized function of either class of NPCs with respect to ribosomal export. To examine the position of the translocating particles relative to the NPC structure, we fitted a 3D reconstruction of the NPC from Dictyostellium discoideum into the tomograms (figure 1). On the nuclear side and within the core scaffold of the NPC, pre‐ribosomes follow the NPC central axis, but they deviate from this position when they reach the NPC cytoplasmic ring, which suggest that they interact with the cytoplasmic nucleoporins. Finally, to get access to the dynamics of this process, we established a model of pre‐ribosome nuclear export through NPCs according to a Jackson queueing network. Fitting of this model to the experimental data allowed us to approximate the translocation time of pre‐ribosomes through the NPC to 70‐150 ms. The rate limiting step appears to be the passage of the NPC core scaffold. These electron tomography data from ultrafast frozen samples not only deliver the first high‐resolution view of the trajectory of pre‐ribosomes through the NPC at nanometer scale, but also provide some dynamic parameters when combined with a probabilistic model of nuclear export.