Analysis of ribosome biogenesis factor-modules in yeast cells depleted from pre-ribosomes

Formation of eukaryotic ribosomes requires more than 150 biogenesis factors which transiently interact with the nascent ribosomal subunits. Previously, many pre-ribosomal intermediates could be distinguished by their protein composition and rRNA precursor (pre-rRNA) content. We purified complexes of ribosome biogenesis factors from yeast cells in which de novo synthesis of rRNA pre-cursors was down-regulated by genetic means. We compared the protein composition of these largely pre-rRNA free assemblies with the one of analogous pre-ribosomal preparations by semi-quantitative mass spectrometry. The experimental setup mini-mizes the possibility that the analysed pre-rRNA free protein modules were derived from (partially) disrupted pre-ribosomal particles and provides thereby strong evidence for their pre-ribosome independent existence. In support of the validity of this approach (i) the predicted composition of the analysed protein modules was in agreement with previously described rRNA-free complexes and (ii) in most of the cases we could identify new candi-date members of reported protein modules. An unexpected outcome of these analyses was that free large ribosomal subunits are associated with a specific set of ribosome biogenesis factors in cells where neo-production of nascent ribosomes was blocked. The data presented strengthen the idea that assembly of eukaryotic pre-ribosomal par-ticles can result from transient association of distinct building blocks