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

Ribosome biogenesis is an essential multi-step process. In eukaryotes, it is orchestrated within the nucleolus, a hallmark multi-layered compartment of the nucleus. Structures of pre-ribosomes have been characterized in vitro, but their assembly pathways within human nucleoli have not been described. Here, we used cryogenic correlative light and electron tomography to visualize the 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 in vitro while additionally revealing critical interaction partners, including the RNA exosome, rixosome, and nuclear export receptor CRM1-RanGTP. We further show how pre-ribosome assembly is altered upon RNA polymerase I inhibition. Our study combines molecular structures with cellular context to elucidate the spatiotemporal assembly pathways and regulation of human ribosome biogenesis.