60S ribosomal subunit assembly dynamics defined by semi-quantitative mass spectrometry of purified complexes.
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.
Lebreton
Alice
A
Institut Pasteur, Unité de Génétique des Interactions Macromoléculaires, CNRS-URA2171, Paris, France.
Rousselle
Jean-Claude
JC
Lenormand
Pascal
P
Namane
Abdelkader
A
Jacquier
Alain
A
Fromont-Racine
Micheline
M
Saveanu
Cosmin
C
D020558:GTP Phosphohydrolases; D007553:Isotope Labeling; D013058:Mass Spectrometry; D009154:Mutation; D012269:Ribosomal Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D054683:Ribosome Subunits, Large, Eukaryotic / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology
D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D020558:GTP Phosphohydrolases; C477781:NOG2 protein, S cerevisiae