The nascent polypeptide exit tunnel (NPET) is a major functional center of 60S ribosomal subunits. However, little is known about how the NPET is constructed during ribosome assembly. We utilized molecular genetics, biochemistry, and cryo-electron microscopy (cryo-EM) to investigate the functions of two NPET-associated proteins, ribosomal protein uL4 and assembly factor Nog1, in NPET assembly. Structures of mutant pre-ribosomes lacking the tunnel domain of uL4 reveal a misassembled NPET, including an aberrantly flexible ribosomal RNA helix 74, resulting in at least three different blocks in 60S assembly. Structures of pre-ribosomes lacking the C-terminal extension of Nog1 demonstrate that this extension scaffolds the tunnel domain of uL4 in the NPET to help maintain stability in the core of pre-60S subunits. Our data reveal that uL4 and Nog1 work together in the maturation of ribosomal RNA helix 74, which is required to ensure proper construction of the NPET and 60S ribosomal subunits.

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.

Ribosomes are responsible for translating the genome, in the form of mRNA, into the proteome in all organisms. Biogenesis of ribosomes in eukaryotes is a complex process involving numerous remodeling events driven in part by the concerted actions of hundreds of protein assembly factors. A major challenge in studying eukaryotic ribosome assembly has, until recently, been a lack of structural data to facilitate understanding of the conformational and compositional changes the pre-ribosome undergoes during its construction. Cryo-electron microscopy (cryo-EM) has begun filling these gaps; recent advances in cryo-EM have enabled the determination of several high resolution pre-ribosome structures. This review focuses mainly on lessons learned from the study of pre-60S particles purified from yeast using the assembly factor Nog2 as bait. These Nog2 particles provide insight into many aspects of nuclear stages of 60S subunit assembly, including construction of major 60S subunit functional centers and processing of the ITS2 spacer RNA.

The final product of ribosome assembly consists of two ribosomal subunits capable of functioning with high fidelity. In eukaryotes, this process is facilitated by protein assembly factors that can promote proper formation of functional centers through structural probing, functional test‐driving, acting as a scaffold, or protecting the site from premature activity. The polypeptide exit tunnel (PET) of the ribosomal large subunit (LSU) is the conduit through which nascent polypeptide chains are passed as they are synthesized. Protein and RNA components of the PET lumen actively participate in translation by interacting with the nascent chain in order to regulate ribosome stalling events, thus regulating translation speed and gene expression. Recent cryo‐EM structures of immature LSU particles from different stages of assembly spanning the nucleoplasm to the cytoplasm have shown that the PET is sequentially probed by the C‐termini of three assembly factors: first, Nog1, then Rei1, and finally Reh1. The C‐termini of these three assembly factors mimic nascent chains, plugging the PET for almost the entirety of LSU assembly. No biological function, however, has been assigned to this redundant occupation of the PET. Here, we show that truncating PET‐inclusive residues from the C‐terminus of Nog1 causes LSU assembly defects that are exacerbated by analogous truncations to one or both of the Rei1 and Reh1 C‐termini. These defects manifest in processing of late immature ribosomal RNA intermediates. Interestingly, these assembly defects are further exacerbated at lower temperatures. Despite these mutants displaying slow growth at permissive temperatures, however, mature ribosomes are produced that may be defective in translation. Our data supports the model that Nog1 is necessary for efficient construction of the PET during early stages of assembly while Rei1 and Reh1 carry out checkpoints during later stages.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.