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Nucleic Acids Research Advance Access published online on July 25, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn469
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

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

Alice Lebreton1, Jean-Claude Rousselle2, Pascal Lenormand2, Abdelkader Namane2, Alain Jacquier1, Micheline Fromont-Racine1 and Cosmin Saveanu1,*

1Institut Pasteur, Unité de Génétique des Interactions Macromoléculaires, CNRS-URA2171 and 2Institut Pasteur, Plate-Forme Protéomique, 75724 Paris Cedex 15, France

*To whom correspondence should be addressed. Tel: +1 33 140613431; Fax: +1 33 140613456; Email: cosmin.saveanu{at}pasteur.fr

Received April 30, 2008. Revised July 2, 2008. Accepted July 5, 2008.

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.

Present address: Alice Lebreton, Équipe Labellisée La ligue, Centre de Génétique Moléculaire, CNRS UPR2167 associée à l'Université P. et M. Curie, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France


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