Additional principles that govern the release of pre-ribosomes from the nucleolus into the nucleoplasm in yeast

Abstract

During eukaryotic ribosome biogenesis, pre-ribosomes travel from the nucleolus, where assembly is initiated, to the nucleoplasm and then are exported to the cytoplasm, where assembly concludes. Although nuclear export of pre-ribosomes has been extensively investigated, the release of pre-ribosomes from the nucleolus is an understudied phenomenon. Initial data indicate that unfolded rRNA interacts in trans with nucleolar components and that, when rRNA folds due to ribosomal protein (RP) binding, the number of trans interactions drops below the threshold necessary for nucleolar retention. To validate and expand on this idea, we performed a bioinformatic analysis of the protein components of the Saccharomyces cerevisiae ribosome assembly pathway. We found that ribosome biogenesis factors (RiBi factors) contain significantly more predicted trans interacting regions than RPs. We also analyzed cryo-EM structures of ribosome assembly intermediates to determine how nucleolar pre-ribosomes differ from post-nucleolar pre-ribosomes, specifically the capacity of RPs, RiBi factors, and rRNA components to interact in trans. We observed a significant decrease in the theoretical trans-interacting capability of pre-ribosomes between nucleolar and post-nucleolar stages of assembly due to the release of RiBi factors from particles and the folding of rRNA. Here, we provide a mechanism for the release of pre-ribosomes from the nucleolus.

Nucleolar dynamics are determined by the ordered assembly of the ribosome

“…However, our finding that NPM1 phase separation is more strongly impacted by interactions with rRNA as opposed to R-rich linkers suggests that additional factors may contribute to the observed phenotype. Our results also support that nucleolar release requires major architectural changes that occur with installation of the 5S RNP and formation of the CP, as previously preposed 7,11,18 . Indeed, although the exact function of the CP is still under investigation, it is thought to coordinate maturation of LSU functional centers 42,43 and mediate intersubunit bridges 44,54 , making its assembly a critical quality control checkpoint.…”

“…Increasing evidence suggests that the nucleolus forms through biological phase transitions, driven by heterotypic, multivalent interaction networks formed between nucleolar scaffolding proteins, RNA, and intrinsically disordered regions (IDRs) in nucleolar constituents 10 . Many of these interactions are thought to occur with the developing pre-ribosome itself, which is composed of a central rRNA scaffold that recruits ribosomal proteins (RPs) and assembly factors that are rich in IDRs 11 . It has therefore been hypothesized that pre-ribosomes are retained in the nucleolus through trans interactions with scaffolding proteins 11,12 , which suggests that ribosomal maturation is intimately tied to nucleolar assembly principles.…”

“…Many of these interactions are thought to occur with the developing pre-ribosome itself, which is composed of a central rRNA scaffold that recruits ribosomal proteins (RPs) and assembly factors that are rich in IDRs 11 . It has therefore been hypothesized that pre-ribosomes are retained in the nucleolus through trans interactions with scaffolding proteins 11,12 , which suggests that ribosomal maturation is intimately tied to nucleolar assembly principles. However, most understanding of nucleolar formation comes from in vitro reconstitution of a subset of nucleolar constituents, making it challenging to determine how the biophysical assembly features of the nucleolus relate to its molecular function in ribosome biogenesis.…”

Ribosome biogenesis factors—from names to functions

“…Along these lines, in vitro experiments showed a preferential partitioning of protein‐free bacterial rRNA but not mature ribosomal subunits into droplets formed by human NPM (Riback et al , 2020 ). Furthermore, a similar model has recently been proposed based on bioinformatic analyses of cryo‐EM structures, which revealed that earlier nucleolar ribosomal precursors contain more unstructured rRNA regions as well as RBFs with predicted intrinsically disordered regions compared to nucleoplasmic subunit assembly intermediates, and both these elements could contribute to nucleolar retention of subunits (LaPeruta et al , 2022 ).…”

Viscoelasticity and advective flow of RNA underlies nucleolar form and function

“… 24 , 25 Finally, the GC contains various proteins that assist in further rRNA processing and binding to ribosomal proteins (r-proteins), ultimately forming nearly complete RSUs, which then move into the nucleoplasm and the cytoplasm. 22 , 26 , 27 The size of the compartments suggests that rRNA processing and folding in the GC are the ratelimiting steps for RSU assembly.…”