60S ribosomal subunit assembly dynamics defined by semi-quantitative mass spectrometry of purified complexes

Institut Pasteur, Unité de Génétique des Interactions Macromoléculaires, CNRS-URA2171, Paris, France.
Nucleic Acids Research (Impact Factor: 9.11). 07/2008; 36(15):4988-99. DOI: 10.1093/nar/gkn469
Source: PubMed


During the highly conserved process of eukaryotic ribosome formation, RNA follows a maturation path with well-defined, successive intermediates that dynamically associate with many pre-ribosomal proteins. A comprehensive description of the assembly process is still lacking. To obtain data on the timing and order of association of the different pre-ribosomal factors, a strategy consists in the use of pre-ribsomal particles isolated from mutants that block ribosome formation at different steps. Immunoblots, inherently limited to only a few factors, have been applied to evaluate the accumulation or decrease of pre-ribosomal intermediates under mutant conditions. For a global protein-level description of different 60S ribosomal subunit maturation intermediates in yeast, we have adapted a method of in vivo isotopic labelling and mass spectrometry to study pre-60S complexes isolated from strains in which rRNA processing was affected by individual depletion of five factors: Ebp2, Nog1, Nsa2, Nog2 or Pop3. We obtained quantitative data for 45 distinct pre-60S proteins and detected coordinated changes for over 30 pre-60S factors in the analysed mutants. These results led to the characterisation of the composition of early, intermediate and late pre-ribosomal complexes, specific for crucial maturation steps during 60S assembly in eukaryotes.

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Available from: Abdelkader Namane, Dec 16, 2013
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    • "In agreement, affinity purifications of Ebp2-TAP and Rpf1-HA reveal, as observed for the Nsa1 pre-60S particle, a predominant association with 27SB pre-rRNAs [39,73,85]. Moreover, genetic depletion or mutational perturbation of Mak5, Ebp2 and Rpf1 elicit similar pre-rRNA processing phenotypes, i.e. delayed conversion of 27SA2 into 27SB pre-rRNAs and reduced formation of mature 25S rRNA from the 27SB pre-rRNAs [58,83–85,89], altogether indicating an instability of 27SB pre-rRNA containing pre-60S ribosomes. Moreover, mutations in ebp2 and mak5 likely entail similar structural alterations within pre-60S particles since both the ebp2.K287* and the mak5.R728* allele suppress the lethality of ∆nsa1 null mutant cells (Figures 1A and 7B). "
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    ABSTRACT: Ribosomes are the molecular machines that translate mRNAs into proteins. The synthesis of ribosomes is therefore a fundamental cellular process and consists in the ordered assembly of 79 ribosomal proteins (r-proteins) and four ribosomal RNAs (rRNAs) into a small 40S and a large 60S ribosomal subunit that form the translating 80S ribosomes. Most of our knowledge concerning this dynamic multi-step process comes from studies with the yeast Saccharomyces cerevisiae, which have shown that assembly and maturation of pre-ribosomal particles, as they travel from the nucleolus to the cytoplasm, relies on a multitude (>200) of biogenesis factors. Amongst these are many energy-consuming enzymes, including 19 ATP-dependent RNA helicases and three AAA-ATPases. We have previously shown that the AAA-ATPase Rix7 promotes the release of the essential biogenesis factor Nsa1 from late nucleolar pre-60S particles. Here we show that mutant alleles of genes encoding the DEAD-box RNA helicase Mak5, the C/D-box snoRNP component Nop1 and the rRNA-binding protein Nop4 bypass the requirement for Nsa1. Interestingly, dominant-negative alleles of RIX7 retain their phenotype in the absence of Nsa1, suggesting that Rix7 may have additional nuclear substrates besides Nsa1. Mak5 is associated with the Nsa1 pre-60S particle and synthetic lethal screens with mak5 alleles identified the r-protein Rpl14 and the 60S biogenesis factors Ebp2, Nop16 and Rpf1, which are genetically linked amongst each other. We propose that these 'Mak5 cluster' factors orchestrate the structural arrangement of a eukaryote-specific 60S subunit surface composed of Rpl6, Rpl14 and Rpl16 and rRNA expansion segments ES7L and ES39L. Finally, over-expression of Rix7 negatively affects growth of mak5 and ebp2 mutant cells both in the absence and presence of Nsa1, suggesting that Rix7, at least when excessively abundant, may act on structurally defective pre-60S subunits and may subject these to degradation.
    PLoS ONE 12/2013; 8(12):e82741. DOI:10.1371/journal.pone.0082741 · 3.23 Impact Factor
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    • "The precise mechanisms by which protein trans-acting factors operate are still largely unknown. The use of affinity purification combined with quantitative mass spectrometry techniques like isobaric Tag for Relative and Absolute Quantification or SILAC (Stable Isotope Labelling with Amino-acids in Cell culture) allow to measure the timing of binding to and dissociation from pre-ribosomal particles for many protein trans-acting factors [e.g., (5,6)]. To better understand the role of these factors in ribosome biogenesis, other experimental approaches have been developed, among them, in vivo cross-linking and cDNA analysis (CRAC). "
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    ABSTRACT: Ribosome biogenesis requires >300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C1 and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7(L)b within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.
    Nucleic Acids Research 08/2013; 41(20). DOI:10.1093/nar/gkt726 · 9.11 Impact Factor
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    • "Quantitative mass spectrometric approaches were previously used successfully to detect changes in the biogenesis factor compositions of various ribosomal precursors isolated from wildtype or mutant yeast strains [33,60–62]. Moreover, evidence for changes in the r-protein composition of early nuclear LSU intermediates in mutants of r-protein and biogenesis factor genes could be obtained by this approach [29,32]. "
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    ABSTRACT: During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed.
    PLoS ONE 07/2013; 8(7):e68412. DOI:10.1371/journal.pone.0068412 · 3.23 Impact Factor
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