Early co-transcriptional events during eukaryotic ribosome assembly result in the formation of precursors of the small (40S) and large (60S) ribosomal subunits1. A multitude of transient assembly factors regulate and chaperone the systematic folding of preribosomal RNA subdomains. However, owing to a lack of structural information, the role of these factors during early nucleolar 60S assembly is not fully understood. Here we report cryo-electron microscopy (cryo-EM) reconstructions of the nucleolar pre-60S ribosomal subunit in different conformational states at resolutions of up to 3.4 Å. These reconstructions reveal how steric hindrance and molecular mimicry are used to prevent both premature folding states and binding of later factors. This is accomplished by the concerted activity of 21 ribosome assembly factors that stabilize and remodel pre-ribosomal RNA and ribosomal proteins. Among these factors, three Brix-domain proteins and their binding partners form a ring-like structure at ribosomal RNA (rRNA) domain boundaries to support the architecture of the maturing particle. The existence of mutually exclusive conformations of these pre-60S particles suggests that the formation of the polypeptide exit tunnel is achieved through different folding pathways during subsequent stages of ribosome assembly. These structures rationalize previous genetic and biochemical data and highlight the mechanisms that drive eukaryotic ribosome assembly in a unidirectional manner.

Making ribosomes in human cells The assembly of the human ribosome requires a vast number of assembly factors that first form a giant precursor of the ribosomal small subunit (SSU), the SSU processome. Singh et al . provide new insights into human ribosome assembly by using cryo–electron microscopy, x-ray crystallography, and functional studies to reveal the structures of the human SSU processome as it matures in the nucleolus. This study reveals the tightly controlled molecular choreography by which an ensemble of assembly factors controls early irreversible maturation steps that bring about the formation of the human SSU. —DJ

Early co-transcriptional events of eukaryotic ribosome assembly result in the formation of the small and large subunit processomes. We have determined cryo-EM reconstructions of the nucleolar large subunit processome in different conformational states at resolutions up to 3.4 Ångstroms. These structures reveal how steric hindrance and molecular mimicry are used to prevent premature folding states and binding of later factors. This is accomplished by the concerted activity of 21 ribosome assembly factors that stabilize and remodel pre-ribosomal RNA and ribosomal proteins. Mutually exclusive conformations of these particles suggest that the formation of the polypeptide exit tunnel is achieved through different folding pathways during subsequent stages of ribosome assembly.

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