Figure 1.
Schematic illustration of the workflow used to characterize the assembly states of (pre-)ribosomal particles.
In (A) and (B) an overview is given on the approach used to purify various (pre-)ribosomal particles from yeast cells and to characterize their protein composition by LC-MS/MS. (C) summarizes how the obtained data were further evaluated and visualized.
Figure 2.
Analysis of the (pre-) rRNAs contained in various affinity purified LSU (precursor) particles.
Yeast strains Y1878, Y1879, Y2398, Y2404, Y2410 and Y485 expressing the indicated TAP-tag fusion proteins (A) or carrying a C-terminally FLAG-tagged allele of RPS26 under control of the galactose-inducible GAL1/10 promoter (B) were cultivated to exponential growth phase in galactose-containing medium (YPG). The tagged proteins were affinity purified from total cellular extracts using rabbit IgG coupled to magnetic beads (A) or an anti FLAG M2 matrix (B) as described in the materials and methods. (pre-) rRNA species from total cellular extracts (Input) and affinity purified fractions (IP) were analyzed by northern blotting using oligonucleotide probes indicated on the right. Equal signal intensities of the Input and bead (IP) fractions indicate 3% (A) or 6% (B) co-purification efficiences of the respective (pre-) rRNA with bait proteins.
Figure 3.
Comparative analyses of the LSU biogenesis factor content in various affinity purified LSU (precursor) particles by semi-quantitative mass spectrometry.
(Pre-) ribosomal particles were affinity purified from total cellular extracts of yeast strains Y485, Y1878, Y1879, Y2398, Y2404 and Y2410 expressing FLAG-tagged rpS26 or TAP tagged ribosome biogenesis factors Nog2, Noc2, Rsa4, Nop53 or Arx1. Proteins contained in the affinity purified fractions were compared by semi-quantitative mass spectrometry in the pair wise combinations indicated in (A) using iTRAQ reagents as described in Materials and Methods. The resulting iTRAQ ratios of all LSU biogenesis factors that were identified in more than 50% of all pair wise comparisons were combined to one dataset which also included average ratios of housekeeping proteins, SSU biogenesis factors, LSU and SSU r-proteins. This dataset was further analyzed by statistical clustering methods as described in Materials and Methods. In the self-organizing tree shown in (A) most similar single comparisons are grouped in the same branches. In the self-organizing tree on the left side of (B) the similarity in behavior of the identified LSU biogenesis factors is analyzed. The normalized iTRAQ ratios for each of the proteins measured in the respective pair-wise particle comparison are visualized as a heat map in (B). Blue colors represent a low and yellow colors a high average iTRAQ ratio of the respective protein in each comparison (see color code on the right side). A grey color indicates that the respective protein was not identified in the individual experiment shown in this lane. Groups of proteins clustering in one branch which are discussed in more detail in the results part of the manuscript are labeled by bars on the right. In (C) the average iTRAQ ratios of a few LSU biogenesis factors are shown which were excluded from the statistical analyses since they were detected in less than 50% of all pair-wise comparisons.
Figure 4.
Comparative analyses of the LSU r-protein content in various affinity purified LSU (precursor) particles by semi-quantitative mass spectrometry.
The experimental dataset generated as described in Figure 3 was analyzed in regard to iTRAQ ratios of all LSU r-proteins that were identified in more than 50% of all pair wise comparisons. This dataset was further analyzed by statistical clustering methods as described in Materials and Methods. (A) shows again a self-organizing tree with most similar single comparisons grouped in the same branches. Accordingly, the similarity in behavior of individual identified LSU r-proteins is shown on the left side of (B). The normalized iTRAQ ratios for each protein measured in the respective pair wise particle comparison are visualized as a heat map in (B). Blue colors represent a low and yellow colors a high average iTRAQ of each protein in the respective comparison (see color code on the right side). A grey color indicates that the respective protein was not identified in the individual experiment shown in this lane. Groups of proteins clustering in one branch which are discussed in more detail in the results part of the manuscript are labeled on the right in (B). (C) shows average iTRAQ ratios of the remaining LSU r-proteins that were excluded from the statistical analyses since they were detected in less than 50% of all pair wise comparisons.
Figure 5.
Association of selected tagged LSU r-proteins with LSU precursor particles of different maturation states as indicated by co-purification of LSU (pre-) rRNAs.
Total cellular extracts were prepared of 16 yeast strains (indicated in (B) and (C)) each of which ectopically expressing a FLAG-tagged version of a LSU r-protein complementing the corresponding lethal gene deletion(s) (see Materials and Methods). Tagged proteins were affinity purified on a FLAG affinity matrix as described in Materials and Methods. (Pre-) rRNA species contained in cellular extracts (input fractions) and the corresponding purified fractions were analyzed by northern blotting (Figure S5A and B). In (A) the average co-purification efficiencies of the indicated RNAs with the group of analyzed LSU r-proteins were determined by relating the respective signal intensities detected in purified fractions to the ones detected in the extract of a reference yeast strain. In (B) is shown the ratio of the efficiency of 5.8S rRNA versus 7S pre-rRNA co-purification for each analyzed LSU r-protein. The ratios of the efficiencies of 7S pre-rRNA versus the 27S pre-rRNA co-purification are shown in (C) for each analyzed LSU r-protein. Values obtained when purifications were performed using buffers containing 200 mM or 300 mM potassium chloride (see Materials and Methods) are represented as dark grey or light grey bars, respectively, in (A) to (C). In (D) the green fluorescent protein (GFP) was immuno-detected on sections of chemically fixed yeast cells expressing fusions of GFP with the indicated r-proteins (see Materials and Methods for details). Yeast strains used for the analyses are indicated in brackets. GFP-fusion proteins were labeled with an anti GFP polycolonal antibody recognized by 10nm colloidal gold-conjugated secondary antibodies which are seen in the electron micrographs as black dots. Representative labeled yeast sections are shown. For better visualization the nucleolar regions were manually surrounded by a dashed black line.
Figure 6.
Analyses of the RNA and protein content of pre-60S particles purified via Nog1-TAP after in vivo depletion of selected ribosomal proteins.
The indicated yeast strains expressing a chromosomally-encoded TAP-tagged version of the LSU biogenesis factor Nog1 together with either RPL25, RPL2, RPL43, RPL21 or no LSU r-protein gene under control of the galactose-inducible GAL1/10 promoter were cultivated for four hours in glucose-containing medium to shut down expression of the respective LSU r-protein gene. Nog1-TAP and associated pre-ribosomal particles were then affinity purified from corresponding cellular extracts as described in Materials and Methods. The (pre-) rRNA content of total cellular extracts (Input lanes 1-5) or of parts of the affinity purified fractions (IP lanes 6-10) was analyzed by northern blotting and is shown in (A). Detected (pre-) rRNAs are indicated on the left and oligonucleotides used for (pre-) rRNA detection are indicated on the right. Equal signal intensities of the Input and IP fractions correspond to 1% (35S, 27S pre-rRNAs and 25S and 18S rRNAs) or 10% (7S pre-rRNA, 5.8S, 5S rRNAs and glutamyl-tRNA) co-purification efficiencies, respectively. Purification efficiencies of the bait protein Nog1-TAP were monitored by western blotting (see panel designated Nog1-TAP). Equal signal intensities in the western blot analyses indicate 25% purification efficiency of Nog1-TAP. Quantitation of the co-purification efficiencies (in %) of the 7S, 27S, 25S and 5.8S (pre-) rRNAs are shown in (B). Red bars in (B) designate internal background levels of the affinity purification procedure as measured by co-purification efficiencies of 20S pre-rRNA. One part of the affinity purified fractions was further processed for comparative protein analyses by semi-quantitative mass spectrometry using iTRAQ reagents as described in Materials and Methods. In each experiment particles purified from two strains expressing or not one of the r-proteins of interest were compared. The iTRAQ ratio for each LSU r-protein (C) or LSU biogenesis factor (D) which was identified by more than one peptide (except rpL43, marked by a (*)) in more than 70% of all comparisons are displayed as a heat map (see color code). A grey color indicates that the respective protein was not identified in the individual experiment shown in this lane. iTRAQ ratios in (C) were normalized to the median value of all LSU r-protein ratios. iTRAQ ratios in (D) were normalized to the iTRAQ ratio of the bait protein Nog1-TAP. Numbers in brackets behind protein names indicate the average number of peptides by which the respective protein was identified in a single experiment. In (C) the iTRAQ ratios of the LSU r-protein whose expression had been shut down are highlighted by red boxes. Several biological replicates of individual experiments are shown in (C) and (D). (E) shows a northern blot analysis of the amount of 7S pre-rRNA co-purifying with Noc2-TAP from extracts of a wild type strain or of conditional expression mutants of RPL2 or RPL43. Cultivation of the strains, affinity purification and northern blotting procedures were done as described above. Same signal intensities detected in the Input and IP fractions correspond to 2.2% purification efficiency of the 7S pre-rRNA.
Figure 7.
Relative amounts of selected proteins in Nog1-TAP associated LSU precursors depleted of rpL2, rpL43, rpL21 or rpL25 as analyzed by silver staining and western blotting.
Nog1-TAP and associated protein were affinity purified from cells of the indicated conditional expression mutants cultivated either in galactose- (left panels) or in glucose-containing (right panels) medium. Affinity purified fractions were analyzed by SDS-PAGE followed by silver staining (A) or western blotting (B) with appropriate antibodies for the presence of the proteins indicated on the left. The faster migrating of the two bands detected by the anti-Nog2 serum is a cross-contamination with the rabbit IgG.
Figure 8.
Analyses of the RNA and protein content of pre-60S particles purified via Nog2-TAP after in vivo depletion of selected ribosomal proteins.
Yeast strains expressing a chromosomally encoded TAP-tagged version of the LSU biogenesis factor Nog2 together with either RPL25, RPL2, RPL43, RPL21 or no LSU r-protein gene under control of the galactose-inducible GAL1/10 promoter were cultivated for four hours in glucose containing medium to shut down expression of the respective LSU r-protein gene. Nog2-TAP and associated pre-ribosomal particles were then affinity purified from corresponding cellular extracts as described in Materials and Methods. The (pre-) rRNA content of total cellular extracts (Input lanes 1-5) or of the affinity purified fractions (IP lanes 6-10) are shown in (A) as analyzed by northern blotting (see Materials and Methods). Detected (pre-) rRNAs are indicated on the left and oligonucleotides used for (pre-) rRNA detection are indicated on the right. Equal signal intensities of the Input and IP fractions correspond to 1% (35S, 27S pre-rRNAs and 25S and 18S rRNAs) or 10% (7S pre-rRNA, 5.8S, 5S rRNAs and glutamyl-tRNA) co-purification efficiencies, respectively. Purification efficiencies of the bait protein Nog2-TAP were monitored by western blotting (see panel designated Nog2-TAP). Equal signal intensities in the western blot analyses indicate 25% purification efficiency of Nog2-TAP. Quantitation of the co-purification efficiencies (in %) of the 7S, 27S, 25S and 5.8S (pre-) rRNAs are shown in (B). Red bars in (B) designate internal background levels of the affinity purification procedure as measured by co-purification efficiencies of 20S pre-rRNA. One part of the affinity purified fractions was further processed for comparative protein analyses by semi-quantitative mass spectrometry using iTRAQ reagents as described in Materials and Methods. In each experiment particles purified from two strains expressing or not one of the r-proteins of interest were compared. The iTRAQ ratio for each LSU r-protein (C) or LSU biogenesis factor (D) which was identified by more than one peptide (except rpL43, rpL29 and rpL40, marked by a (*)) in more than 70% of all comparisons are displayed as a heat map (see color code). A grey color indicates that the respective protein was not identified in the individual experiment shown in this lane. iTRAQ ratios in (C) and (D) were normalized to the iTRAQ ratio of the bait protein Nog2-TAP. Numbers in brackets behind protein names indicate the average number of peptides by which the respective protein was identified in a single experiment. In (C) the iTRAQ ratios of the LSU r-protein whose expression had been shut down are highlighted by red boxes. Several biological replicates of individual experiments are shown in (C) and (D).
Figure 9.
Analyses of the RNA and protein content of pre-60S particles purified via Rsa4-TAP after in vivo depletion of selected ribosomal proteins.
Yeast strains expressing a chromosomally-encoded TAP-tagged versions of the LSU biogenesis factor Rsa4 together with either RPL25, RPL2, RPL43, RPL21 or no LSU r-protein gene under control of the galactose-inducible GAL1/10 promoter were cultivated for four hours in glucose-containing medium to shut down expression of the respective LSU r-protein gene. Rsa4-TAP and associated pre-ribosomal particles were then affinity purified from corresponding cellular extracts as described in Materials and Methods. The (pre-) rRNA content of total cellular extracts (Input lanes 1-5) or of parts of the affinity purified fractions (IP lanes 6-10) are shown in (A) as analyzed by total RNA extraction and northern blotting (see Materials and Methods). Detected (pre-) rRNAs are indicated on the left and oligonucleotides used for (pre-) rRNA detection are indicated on the right. Equal signal intensities of the Input and IP fractions correspond to 1% (35S, 27S pre-rRNAs and 25S and 18S rRNAs) or 10% (7S pre-rRNA, 5.8S, 5S rRNAs and glutamyl-tRNA) co-purification efficiences, respectively. Purification efficiencies of the bait protein Rsa4-TAP were monitored by western blotting (see panel designated Rsa4-TAP). Equal signal intensities in the western blot analyses indicate 25% purification efficiency of Rsa4-TAP. Quantitation of the co-purification efficiencies (in %) of the 7S, 27S, 25S and 5.8S (pre-) rRNAs are shown in (B). Red bars in (B) designate internal background levels of the affinity purification procedure as measured by co-purification efficiencies of 20S pre-rRNA. One part of the affinity purified fractions was further processed for comparative protein analyses by semi-quantitative mass spectrometry using iTRAQ reagents as described in Materials and Methods. In each experiment particles purified from two strains expressing or not one of the r-proteins of interest were compared. The iTRAQ ratio for each LSU r-protein (C) or LSU biogenesis factor (D) that was identified with more than one peptide (except rpL40, marked by a (*)) in more than 70% of all comparisons are displayed as a heat map (see color code). A grey color indicates that the respective protein was not identified in the individual experiment shown in this lane. iTRAQ ratios in (C) and (D) were normalized to the iTRAQ ratio of the bait protein Rsa4-TAP. Numbers in brackets behind protein names indicate the average number of peptides by which the respective protein was identified in a single experiment. In (C) the iTRAQ ratios of the LSU r-protein whose expression had been shut down are highlighted by red boxes. Several biological replicates of individual experiments are shown in (C) and (D).