Role of the yeast ribosomal protein L16 in ribosome biogenesis

Abstract

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.

Ribosomal protein L14 contributes to the early assembly of 60S ribosomal subunits in Saccharomyces cerevisiae

“…The specificity of the purification was evaluated by assessing the presence of the selected trans -acting factors Nop1, Erb1, Has1 and Mrt4 by western blot using specific antibodies (Figure 1 ). Western blot analyses also showed that L16 significantly co-enriched with practically all tested pre-60S particles (Figure 1 ; see also ( 81 )), as did L1, which was used as a control of an early assembling r-protein ( 19 , 32 ). In contrast, L10, which has been reported to assemble in the cytoplasm ( 19 , 93 ), only co-enriched with late and cytoplasmic pre-60S r-particles and P0-GFP-containing particles that correspond mainly to mature ribosomes ( 33 ).…”

“…Total yeast protein extracts were prepared as described previously ( 81 ). Total protein extracts and samples from purified particles were analysed by western blotting according to standard procedures.…”

Pol5 is an essential ribosome biogenesis factor required for 60S ribosomal subunit maturation in Saccharomyces cerevisiae

“…This is consistent with a delayed processing at the early A 0 , A 1 , and A 2 cleavage sites upon Pol5 depletion that, however, affects only minorly the overall synthesis of 18S rRNA and 40S r-subunit production. Delayed processing of 35S pre-rRNA, which is common for practically all loss-of-function mutations in genes involved in 60S r-subunit biogenesis including 60S r-proteins, has previously been well discussed (e.g., Axt et al 2014;Espinar-Marchena et al 2016;Talkish et al 2016) and arises as the consequence of a functional limitation of early trans-acting factors that are sequestered on aberrant pre-60S intermediates and/or the specific switch of pre-rRNA processing to the so-called A 3 pathway (Kos-Braun et al 2017). Recently, Gallagher has reported comparable results after assessing the steady-state levels of pre-rRNAs from an equivalent GAL::POL5 strain shifted to glucose medium for 24 h (Gallagher 2019).…”

“…Role of Pol5 in yeast ribosome synthesis www.rnajournal.org 1571 Affinity purification of Pol5-GFP Complexes containing GFP-tagged Pol5 protein were precipitated following the one-step GFP-Trap A procedure (Chromotek) with GFP-Trap A beads, as exactly described in Babiano and de la Cruz (2010). The proteins from the purified complexes were extracted by boiling the beads with Laemmli buffer and analyzed by western blotting (Espinar-Marchena et al 2016). Pre-and mature rRNAs were recovered from the beads by phenol-chloroform extraction and assayed by northern hybridization as indicated above.…”

Comparative Analysis of the 5’ Flanking Region in <i>Arabidopsis</i> and <i>O. sative</i> RP Genes Revealed Conserved and Divergent Regulating Mechanisms

“…In contrast to the relatively well-defined regulatory mechanisms for yeast and animal RP genes, little is known about the regulation of expression of plant RP genes. Recent studies in Arabidopsis have identified two cis elements presented in most of the RP genes promoters, suggesting that it could be involved in the coordinated expression of this class of genes at transcriptional level [7]. One of these cis elements is the proliferating cell nuclear antigen (PCNA) site II motif (5'-GCCCR-3') [8].…”