No abstract

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.

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.