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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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Analysis of cellular pre-rRNA and ribosome biogenesis factor levels after shut down of rRNA de novo synthesis. (A) Northern hybridization analysis of precursor and mature rRNA species of both ribosomal subunits was performed on RNA extracts from whole cells. Yeast strains with RRN3 and with rrn3-8 background were analysed at permissive (24°C) and restrictive (3 h 37°C) temperature. RNA from equal number of cells was loaded and different oligonucleotides (‘Materials and methods’ section) were used for detection of the different indicated (pre-) rRNA species (B) Protein levels of TAP–tagged ribosome biogenesis factors in RRN3 (rRNA synthesis +) and in rrn3-8 background (rRNA synthesis –) were analysed at restrictive (3h, 37°C) temperature by western blotting using PAP visualization reagent. Equal loading was controlled by determination of the protein level of rpS8.
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Figure 1: Analysis of cellular pre-rRNA and ribosome biogenesis factor levels after shut down of rRNA de novo synthesis. (A) Northern hybridization analysis of precursor and mature rRNA species of both ribosomal subunits was performed on RNA extracts from whole cells. Yeast strains with RRN3 and with rrn3-8 background were analysed at permissive (24°C) and restrictive (3 h 37°C) temperature. RNA from equal number of cells was loaded and different oligonucleotides (‘Materials and methods’ section) were used for detection of the different indicated (pre-) rRNA species (B) Protein levels of TAP–tagged ribosome biogenesis factors in RRN3 (rRNA synthesis +) and in rrn3-8 background (rRNA synthesis –) were analysed at restrictive (3h, 37°C) temperature by western blotting using PAP visualization reagent. Equal loading was controlled by determination of the protein level of rpS8.

Mentions: First, we analysed how the steady state levels of rRNA precursors were affected after specific shut down of the Pol-I machinery in a conditional temperature-sensitive mutant of the essential Pol-I transcription factor Rrn3p. Therefore, (pre-) rRNA levels in the rrn3-8 mutant strain YCC95 (31) and in the corresponding RRN3 wild-type strain CG379 were analysed at permissive (24°C) and restrictive temperature (37°C) by northern blot hybridization (Figure 1A). Pol-I transcribes a region of the rDNA-locus to yield the 35S rRNA precursor, which is processed in various endo- and exonucleolytic cleavage steps to finally result in the mature 18S, 5.8S and 25S rRNA. rRNA precursor levels of both large and small ribosomal subunit (respectively 27S and 20S pre-rRNA) were reduced after 3h incubation at 37°C to a residual amount of ∼5% in the rrn3-8 mutant when compared to the level in wild-type cells (Figure 1A, compare lane 2 with lane 4).Figure 1.


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)

Analysis of cellular pre-rRNA and ribosome biogenesis factor levels after shut down of rRNA de novo synthesis. (A) Northern hybridization analysis of precursor and mature rRNA species of both ribosomal subunits was performed on RNA extracts from whole cells. Yeast strains with RRN3 and with rrn3-8 background were analysed at permissive (24°C) and restrictive (3 h 37°C) temperature. RNA from equal number of cells was loaded and different oligonucleotides (‘Materials and methods’ section) were used for detection of the different indicated (pre-) rRNA species (B) Protein levels of TAP–tagged ribosome biogenesis factors in RRN3 (rRNA synthesis +) and in rrn3-8 background (rRNA synthesis –) were analysed at restrictive (3h, 37°C) temperature by western blotting using PAP visualization reagent. Equal loading was controlled by determination of the protein level of rpS8.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Analysis of cellular pre-rRNA and ribosome biogenesis factor levels after shut down of rRNA de novo synthesis. (A) Northern hybridization analysis of precursor and mature rRNA species of both ribosomal subunits was performed on RNA extracts from whole cells. Yeast strains with RRN3 and with rrn3-8 background were analysed at permissive (24°C) and restrictive (3 h 37°C) temperature. RNA from equal number of cells was loaded and different oligonucleotides (‘Materials and methods’ section) were used for detection of the different indicated (pre-) rRNA species (B) Protein levels of TAP–tagged ribosome biogenesis factors in RRN3 (rRNA synthesis +) and in rrn3-8 background (rRNA synthesis –) were analysed at restrictive (3h, 37°C) temperature by western blotting using PAP visualization reagent. Equal loading was controlled by determination of the protein level of rpS8.
Mentions: First, we analysed how the steady state levels of rRNA precursors were affected after specific shut down of the Pol-I machinery in a conditional temperature-sensitive mutant of the essential Pol-I transcription factor Rrn3p. Therefore, (pre-) rRNA levels in the rrn3-8 mutant strain YCC95 (31) and in the corresponding RRN3 wild-type strain CG379 were analysed at permissive (24°C) and restrictive temperature (37°C) by northern blot hybridization (Figure 1A). Pol-I transcribes a region of the rDNA-locus to yield the 35S rRNA precursor, which is processed in various endo- and exonucleolytic cleavage steps to finally result in the mature 18S, 5.8S and 25S rRNA. rRNA precursor levels of both large and small ribosomal subunit (respectively 27S and 20S pre-rRNA) were reduced after 3h incubation at 37°C to a residual amount of ∼5% in the rrn3-8 mutant when compared to the level in wild-type cells (Figure 1A, compare lane 2 with lane 4).Figure 1.

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