Most, if not all RNAs, are transcribed as precursors that require processing to gain functionality. Ribosomal RNAs (rRNA) from all organisms undergo both exo- and endonucleolytic processing. Also, in all organisms, rRNA processing occurs inside large preribosomal particles and is coupled to nucleotide modification, folding of the precursor rRNA (pre-rRNA), and assembly of the ribosomal proteins (r-proteins). In this review, we focus on the processing pathway of pre-rRNAs of cytoplasmic ribosomes in the yeast Saccharomyces cerevisiae, without doubt, the organism where this pathway is best characterized. We summarize the current understanding of the rRNA maturation process, particularly focusing on the pre-rRNA processing sites, the enzymes responsible for the cleavage or trimming reactions and the different mechanisms that monitor and regulate the pathway. Strikingly, the overall order of the various processing steps is reasonably well conserved in eukaryotes, perhaps reflecting common principles for orchestrating the concomitant events of pre-rRNA processing and ribosome assembly.

Ribosomal protein L3 is an evolutionarily conserved protein that participates in the assembly of early pre-60S particles. We report that the rpl3[W255C] allele, which affects the affinity and function of translation elongation factors, impairs cytoplasmic maturation of 20S pre-rRNA. This was not seen for other mutations in or depletion of L3 or other 60S ribosomal proteins. Surprisingly, pre-40S particles containing 20S pre-rRNA form translation-competent 80S ribosomes, and translation inhibition partially suppresses 20S pre-rRNA accumulation. The GTP-dependent translation initiation factor Fun12 (yeast eIF5B) shows similar in vivo binding to ribosomal particles from wild-type and rpl3[W255C] cells. However, the GTPase activity of eIF5B failed to stimulate processing of 20S pre-rRNA when assayed with ribosomal particles purified from rpl3[W255C] cells. We conclude that L3 plays an important role in the function of eIF5B in stimulating 3′ end processing of 18S rRNA in the context of 80S ribosomes that have not yet engaged in translation. These findings indicate that the correct conformation of the GTPase activation region is assessed in a quality control step during maturation of cytoplasmic pre-ribosomal particles.

Background:The contribution of ribosomal proteins to ribosome assembly and function is often not well understood. Results: L40 assembles within the cytoplasm into pre-60 S subunits and is required for Nmd3 and Rlp24 recycling. Conclusion: L40 contributes to formation of 60 S subunits competent for subunit joining and translation elongation. Significance: Our analysis of L40 function reveals an additional step during cytoplasmic pre-60 S maturation events.

Bud27, the yeast orthologue of human URI/RMP, is a member of the prefoldin-like family of ATP-independent molecular chaperones. It has recently been shown to mediate the assembly of the three RNA polymerases in an Rpb5-dependent manner. In this work, we present evidence of Bud27 modulating RNA pol II transcription elongation. We show that Bud27 associates with RNA pol II phosphorylated forms (CTD-Ser5P and CTD-Ser2P), and that its absence affects RNA pol II occupancy of transcribed genes. We also reveal that Bud27 associates in vivo with the Sth1 component of the chromatin remodeling complex RSC and mediates its association with RNA pol II. Our data suggest that Bud27, in addition of contributing to Rpb5 folding within the RNA polymerases, also participates in the correct assembly of other chromatin-associated protein complexes, such as RSC, thereby modulating their activity.

The archaea-/eukaryote-specific 40S-ribosomal-subunit protein S31 is expressed as an ubiquitin fusion protein in eukaryotes and consists of a conserved body and a eukaryote-specific N-terminal extension. In yeast, S31 is a practically essential protein, which is required for cytoplasmic 20S pre-rRNA maturation. Here, we have studied the role of the N-terminal extension of the yeast S31 protein. We show that deletion of this extension partially impairs cell growth and 40S subunit biogenesis and confers hypersensitivity to aminoglycoside antibiotics. Moreover, the extension harbours a nuclear localization signal that promotes active nuclear import of S31, which associates with pre-ribosomal particles in the nucleus. In the absence of the extension, truncated S31 inefficiently assembles into pre-40S particles and two subpopulations of mature small subunits, one lacking and another one containing truncated S31, can be identified. Plasmid-driven overexpression of truncated S31 partially suppresses the growth and ribosome biogenesis defects but, conversely, slightly enhances the hypersensitivity to aminoglycosides. Altogether, these results indicate that the N-terminal extension facilitates the assembly of S31 into pre-40S particles and contributes to the optimal translational activity of mature 40S subunits but has only a minor role in cytoplasmic cleavage of 20S pre-rRNA at site D.

Ubiquitin is generated by proteolytic cleavage of precursor proteins in which it is fused either to itself, constituting a linear polyubiquitin protein of head‐to‐tail monomers, or as a single N‐terminal moiety to one of two ribosomal proteins, eL40 (Ubi1/2 precursors) and eS31 (Ubi3 precursor). It has been proposed that the ubiquitin moiety fused to these ribosomal proteins could act as a chaperone by facilitating their efficient production, folding and ribosome assembly in Saccharomyces cerevisiae. We have previously shown that ubiquitin release from eS31 is required for yeast viability and that noncleaved Ubi3 can get incorporated into translation‐competent 40S subunits. In this study, we have analysed the effects of mutations that partially or totally impair cleavage of the ubiquitin‐eL40A fusion protein. While noncleaved Ubi1 is not able to support growth when it is the sole cellular source of eL40, it can assemble into nascent pre‐60S particles. However, Ubi1‐containing 60S ribosomal subunits are not competent for translation. This is likely due to a steric interference of the unprocessed ubiquitin with the binding and function of factors that interact with the ribosome's GTPase‐associated centre. In agreement with this suggestion, Ubi1‐containing ribosomes affect the efficient recycling of the anti‐association factor Tif6 and have a reduced presence of translation elongation factors. We conclude that the removal of the ubiquitin moiety from ribosomal protein eL40 is an essential prerequisite for both the cytoplasmic maturation and the functionality of 60S ribosomal subunits.

Ubiquitin is a highly conserved small eukaryotic protein. It is generated by proteolytic cleavage of precursor proteins in which it is fused either to itself, constituting a polyubiquitin precursor of head-to-tail monomers, or as a single N-terminal moiety to ribosomal proteins. Understanding the role of the ubiquitin fused to ribosomal proteins becomes relevant, as these proteins are practically invariably eS31 and eL40 in the different eukaryotes. Herein, we used the amenable yeast Saccharomyces cerevisiae to study whether ubiquitin facilitates the expression of the fused eL40 (Ubi1 and Ubi2 precursors) and eS31 (Ubi3 precursor) ribosomal proteins. We have analyzed the phenotypic effects of a genomic ubi1∆ub-HA ubi2∆ mutant, which expresses a ubiquitin-free HA-tagged eL40A protein as the sole source of cellular eL40. This mutant shows a severe slow-growth phenotype, which could be fully suppressed by increased dosage of the ubi1∆ub-HA allele, or partially by the replacement of ubiquitin by the ubiquitin-like Smt3 protein. While expression levels of eL40A-HA from ubi1∆ub-HA are low, eL40A is produced practically at normal levels from the Smt3-S-eL40A-HA precursor. Finally, we observed enhanced aggregation of eS31-HA when derived from a Ubi3∆ub-HA precursor and reduced aggregation of eL40A-HA when expressed from a Smt3-S-eL40A-HA precursor. We conclude that ubiquitin might serve as a cis-acting molecular chaperone that assists in the folding and synthesis of the fused eL40 and eS31 ribosomal proteins.

Most ribosomal proteins play essential roles in ribosome synthesis and function. In this study, we have analysed the contribution of yeast ribosomal protein L16 to ribosome biogenesis. We show that in vivo depletion of the essential L16 protein results in a deficit in 60S subunits and the appearance of half-mer polysomes. This phenotype is likely due to the instability and rapid turnover of early and intermediate pre-60S particles, as evidenced by the reduced steady-state levels of 27SB S and 7S L/S pre-rRNA, and the low amounts of de novo synthesized 27S pre-rRNA and 25S rRNA. Additionally, depletion of L16 blocks nucleocytoplasmic export of pre-60S particles. Moreover, we show that L16 assembles in the nucleolus and binds to early 90S preribosomal particles. Many evolutionarily conserved ribosomal proteins possess extra eukaryote-specific amino-or carboxy-terminal extensions and/or internal loops. Here, we have also investigated the role of the eukaryote-specific carboxy-terminal extension of L16. Progressive truncation of this extension recapitulates, albeit to a lesser extent, the growth and ribosome biogenesis defects of the L16 depletion. We conclude that L16 assembly is a prerequisite to properly stabilize rRNA structures within early pre-60S particles, thereby favouring efficient 27S pre-rRNA processing within the internal transcribed spacer 1 at sites A 3 and B 1 . Upon depletion of L16, the lack of this stabilization aborts early pre-60S particle assembly and subjects these intermediates to turnover.