In the past 25 years, genetic and biochemical analyses of ribosome assembly in yeast have identified most of the factors that participate in this complex pathway and have generated models for the mechanisms driving the assembly. More recently, the publication of numerous cryo-electron microscopy structures of yeast ribosome assembly intermediates has provided near-atomic resolution snapshots of ribosome precursor particles. Satisfyingly, these structural data support the genetic and biochemical models and provide additional mechanistic insight into ribosome assembly. In this Review, we discuss the mechanisms of assembly of the yeast small ribosomal subunit and large ribosomal subunit in the nucleolus, nucleus and cytoplasm. Particular emphasis is placed on concepts such as the mechanisms of RNA compaction, the functions of molecular switches and molecular mimicry, the irreversibility of assembly checkpoints and the roles of structural and functional proofreading of pre-ribosomal particles.

Ribosome assembly coming into focus.

Eukaryotic ribosome synthesis is a complex, energy-consuming process that takes place across the nucleolus, nucleoplasm and cytoplasm and requires more than 200 conserved assembly factors. Here, we discuss mechanisms by which the ribosome assembly and nucleocytoplasmic transport machineries collaborate to produce functional ribosomes. We also highlight recent cryo-EM studies that provided unprecedented snapshots of ribosomes during assembly and quality control.

Eukaryotic ribosome assembly, transport and quality control.

https://doc.rero.ch/record/288894/files/kre_pfc.pdf

A Puzzle of Life: Crafting Ribosomal Subunits

Visualizing the Assembly Pathway of Nucleolar Pre-60S Ribosomes.

Post-transcriptional Regulatory Functions of Mammalian Pumilio Proteins.

Eukaryotic small ribosomal subunits are first assembled into 90S pre-ribosomes. The complete 90S is a gigantic complex with a molecular mass of approximately five megadaltons. Here, we report the nearly complete architecture of Saccharomyces cerevisiae 90S determined from three cryo-electron microscopy single particle reconstructions at 4.5 to 8.7 angstrom resolution. The majority of the density maps were modeled and assigned to specific RNA and protein components. The nascent ribosome is assembled into isolated native-like substructures that are stabilized by abundant assembly factors. The 5' external transcribed spacer and U3 snoRNA nucleate a large subcomplex that scaffolds the nascent ribosome. U3 binds four sites of pre-rRNA, including a novel site on helix 27 but not the 3' side of the central pseudoknot, and crucially organizes the 90S structure. The 90S model provides significant insight into the principle of small subunit assembly and the function of assembly factors.

Molecular architecture of the 90S small subunit pre-ribosome

The assembly of eukaryotic ribosomes requires numerous factors that transiently associate with evolving pre-ribosomal particles. The Pumilio repeat-containing protein Nop9 briefly associates with the 90S pre-ribosome during its co-transcriptional assembly. Here, we show that Nop9 specifically binds an 11-nucleotide sequence of 18S rRNA that forms the 3΄ side of the central pseudoknot and helix 28 in the mature subunit. Crystal structures of Nop9 in the free and RNA-bound states reveal a new type of Pumilio repeat protein with a distinct structure, target sequence and RNA-binding mode. Nop9 contains 10 Pumilio repeats arranged into a U-shaped scaffold. The target RNA is recognized by two stretches of repeats in a bipartite manner, and three central bases are unrecognized as a result of the degeneracy of repeats 6 and 7. Our data suggest that Nop9 regulates the folding of 18S rRNA at early assembly stages of 90S.

Bing Wang

Papers

Molecular architecture of the 90S small subunit pre-ribosome

Nop9 binds the central pseudoknot region of 18S rRNA.

Correction: Molecular architecture of the 90S small subunit pre-ribosome.

Author response: Molecular architecture of the 90S small subunit pre-ribosome