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

Merl J, Jakob S, Ridinger K, Hierlmeier T, Deutzmann R, Milkereit P, Tschochner H - Nucleic Acids Res. (2010)

Bottom Line: We purified complexes of ribosome biogenesis factors from yeast cells in which de novo synthesis of rRNA precursors was down-regulated by genetic means.The experimental setup minimizes 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 candidate members of reported protein modules.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie, Genetik und Mikrobiologie, University of Regensburg, Universitätsstr. 31, 93053 Regensburg, Germany.

ABSTRACT
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 precursors 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 minimizes 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 candidate 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 particles can result from transient association of distinct building blocks.

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Schematic view of relative protein quantitation set up. A typical MS spectrum is shown, where each peak represents a mixture of sequence-identical but differentially iTRAQ-labelled peptides from affinity purifications of wild-type and rrn3-8 mutant strains. In the MSMS–mode peptides are selected for fragmentation, yielding in peptide fragments with sequence specific m/z ratios used for identification of the respective protein by database search. In addition the iTRAQ reporter ions of different masses are released and are used for relative quantitation and subsequent determination of pre-rRNA-dependent or pre-rRNA-independent co-purifications.
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Figure 5: Schematic view of relative protein quantitation set up. A typical MS spectrum is shown, where each peak represents a mixture of sequence-identical but differentially iTRAQ-labelled peptides from affinity purifications of wild-type and rrn3-8 mutant strains. In the MSMS–mode peptides are selected for fragmentation, yielding in peptide fragments with sequence specific m/z ratios used for identification of the respective protein by database search. In addition the iTRAQ reporter ions of different masses are released and are used for relative quantitation and subsequent determination of pre-rRNA-dependent or pre-rRNA-independent co-purifications.

Mentions: In the fragmentation chamber of the mass spectrometer different reporter groups (114, 115, 116, 117 Da) are released from the specific iTRAQ labels and can be used to determine how much of the analysed peptide was linked to the respective iTRAQ reagent (Figure 5). Furthermore, the peptide itself is fragmented and analysis of the resulting fragment masses allows identification of the protein from which it was derived (38).Figure 5.


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

Merl J, Jakob S, Ridinger K, Hierlmeier T, Deutzmann R, Milkereit P, Tschochner H - Nucleic Acids Res. (2010)

Schematic view of relative protein quantitation set up. A typical MS spectrum is shown, where each peak represents a mixture of sequence-identical but differentially iTRAQ-labelled peptides from affinity purifications of wild-type and rrn3-8 mutant strains. In the MSMS–mode peptides are selected for fragmentation, yielding in peptide fragments with sequence specific m/z ratios used for identification of the respective protein by database search. In addition the iTRAQ reporter ions of different masses are released and are used for relative quantitation and subsequent determination of pre-rRNA-dependent or pre-rRNA-independent co-purifications.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875017&req=5

Figure 5: Schematic view of relative protein quantitation set up. A typical MS spectrum is shown, where each peak represents a mixture of sequence-identical but differentially iTRAQ-labelled peptides from affinity purifications of wild-type and rrn3-8 mutant strains. In the MSMS–mode peptides are selected for fragmentation, yielding in peptide fragments with sequence specific m/z ratios used for identification of the respective protein by database search. In addition the iTRAQ reporter ions of different masses are released and are used for relative quantitation and subsequent determination of pre-rRNA-dependent or pre-rRNA-independent co-purifications.
Mentions: In the fragmentation chamber of the mass spectrometer different reporter groups (114, 115, 116, 117 Da) are released from the specific iTRAQ labels and can be used to determine how much of the analysed peptide was linked to the respective iTRAQ reagent (Figure 5). Furthermore, the peptide itself is fragmented and analysis of the resulting fragment masses allows identification of the protein from which it was derived (38).Figure 5.

Bottom Line: We purified complexes of ribosome biogenesis factors from yeast cells in which de novo synthesis of rRNA precursors was down-regulated by genetic means.The experimental setup minimizes 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 candidate members of reported protein modules.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie, Genetik und Mikrobiologie, University of Regensburg, Universitätsstr. 31, 93053 Regensburg, Germany.

ABSTRACT
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 precursors 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 minimizes 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 candidate 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 particles can result from transient association of distinct building blocks.

Show MeSH
Related in: MedlinePlus