In-cell structures visualize human pre-ribosome assembly in the nucleolus.

In eukaryotes, ribosome biogenesis initiates within the nucleolus, a hallmark multilayered compartment of the nucleus. Structures of pre-ribosomes have been characterized ex situ, but their assembly pathways within human nucleoli have not been described. Here, we used cryogenic correlative light and electron tomography to visualize molecular landscapes within HeLa cell nucleoli and obtained in-cell structural snapshots of both ribosomal subunit precursors, the SSU processome and the pre-60S. These recapitulate major states resolved previously ex situ and reveal additional critical interaction partners, including the RNA exosome, rixosome, and nuclear export receptor CRM1-RanGTP. We further show how pre-ribosome assembly landscapes are altered upon RNA polymerase I inhibition. Our study combines molecular structures with cellular context to elucidate the spatiotemporal assembly pathway of the human ribosome.