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Interrelationships between yeast ribosomal protein assembly events and transient ribosome biogenesis factors interactions in early pre-ribosomes.

Jakob S, Ohmayer U, Neueder A, Hierlmeier T, Perez-Fernandez J, Hochmuth E, Deutzmann R, Griesenbeck J, Tschochner H, Milkereit P - PLoS ONE (2012)

Bottom Line: One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain.Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes.They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Biochemie III, Universität Regensburg, Regensburg, Germany.

ABSTRACT
Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA). Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins) and ribosome precursors (pre-ribosomes) are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU) processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains.

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Analysis of ribosome biogenesis factors co-purifying with Noc4p after in vivo depletion of rpS5, rpS13, or rpS22.The yeast strain TY96 (wildtype) expressing chromosome encoded TAP tagged Noc4p and yeast conditional mutant strains TY1241, TY1897, and TY1902, expressing chromosomal encoded TAP tagged Noc4p and carrying in addition galactose inducible conditional alleles of RPS5 (TY1241), RPS13 (TY1897), or RPS22 (TY1902) were cultivated in medium containing galactose as carbon source and were subsequently transferred to glucose containing medium and cultivated for additional four hours. Noc4p-TAP was affinity purified from corresponding cellular extracts using IgG coupled magnetic bead matrix. Affinity purified proteins were digested using trypsin and the resulting peptides from each sample were labelled with different iTRAQ reagents. Labelled peptides of wildtype samples were combined with labelled peptides of samples derived either from the conditional mutant of RPS5 (A), RPS13 (B), or RPS22 (C) and were then further analyzed as described in material and methods. Average iTRAQ ratios of each SSU processome component identified by more than one peptide are indicated in (A)–(C). Numbers in brackets behind SSU processome component names indicate the number of peptides (confidence interval >95%) by which the respective protein was identified. (D) shows a heatmap representation of the three datasets. The factors are ordered according to a clustering analysis (see material and methods). Boxes in red colours represent relative enrichment and boxes in green colours relative deprivation of a protein in Noc4p-TAP fractions purified from mutant versus wildtype cells.
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pone-0032552-g005: Analysis of ribosome biogenesis factors co-purifying with Noc4p after in vivo depletion of rpS5, rpS13, or rpS22.The yeast strain TY96 (wildtype) expressing chromosome encoded TAP tagged Noc4p and yeast conditional mutant strains TY1241, TY1897, and TY1902, expressing chromosomal encoded TAP tagged Noc4p and carrying in addition galactose inducible conditional alleles of RPS5 (TY1241), RPS13 (TY1897), or RPS22 (TY1902) were cultivated in medium containing galactose as carbon source and were subsequently transferred to glucose containing medium and cultivated for additional four hours. Noc4p-TAP was affinity purified from corresponding cellular extracts using IgG coupled magnetic bead matrix. Affinity purified proteins were digested using trypsin and the resulting peptides from each sample were labelled with different iTRAQ reagents. Labelled peptides of wildtype samples were combined with labelled peptides of samples derived either from the conditional mutant of RPS5 (A), RPS13 (B), or RPS22 (C) and were then further analyzed as described in material and methods. Average iTRAQ ratios of each SSU processome component identified by more than one peptide are indicated in (A)–(C). Numbers in brackets behind SSU processome component names indicate the number of peptides (confidence interval >95%) by which the respective protein was identified. (D) shows a heatmap representation of the three datasets. The factors are ordered according to a clustering analysis (see material and methods). Boxes in red colours represent relative enrichment and boxes in green colours relative deprivation of a protein in Noc4p-TAP fractions purified from mutant versus wildtype cells.

Mentions: Noc4p was identified above as a member of the Noc4p/Nop14p group of SSU processome components tending to be underrepresented in early pre-ribosomes purified from yeast cells depleted of rpS5, rpS13, or rpS14. We were interested to characterize in more detail the influence of r-protein assembly events on association of Noc4p with early pre-ribosomes. Conditional mutants of several SSU r-protein genes were constructed which express a chromosome encoded C-terminal TAP-fusion allele of Noc4p. The selected conditional r-protein gene mutants were the same as the ones studied in the experiments shown in Figure 2 and therefore included again the head domain binder rpS15 and the central domain/platform binder rpS14 together with the yeast homologues of five primary E. coli in vitro binders interacting with different regions of the SSU rRNA (see Fig. 1). Noc4p-TAP was affinity purified from extracts of these mutants either grown in permissive or restrictive conditions. As expected for a SSU processome component, Northern blot analyses indicated that Noc4p-TAP co-purified significant amounts of early SSU rRNA precursors (23S and 32/35S pre-rRNAs) from extracts of cells grown in permissive conditions (Fig. 4, compare 23S and 32/35S signals in lanes 1, 5, 9, 13, 17, 21 and 25 with 32/35S signals in lanes 2, 6, 10, 14, 18, 22 and 26). In vivo depletion of the various r-proteins led to the expected pre-rRNA processing phenotypes (Fig. 4, compare 32/35S signals in lanes 1, 5, 9, 13, 17, 21 and 25 with 32/35S signals in lanes 3,7,11,15,19,23 and 27, compare also with Fig. 2 and [9]). Interestingly, Noc4p-TAP efficiently co-purified large amounts of accumulating early 32/35S pre-rRNAs from extracts of a subset of the analyzed r-protein gene mutants shifted to restrictive conditions (RPS11, RPS9, RPS22, RPS15, see Fig. 4, compare 32/35S signals in lanes 3, 7, 11 and 15 with signals in lanes 4, 8, 12 and 16). By contrast, the efficiency of co-purification of early 32/35S pre-rRNA with Noc4p-TAP from extracts of strains depleted of another subset of r-proteins (rpS13, rpS14, rpS5) was reduced close to background levels, though co-precipitation was still detectable (Fig. 4, compare 35S/32S signals in lanes 15, 19 and 23 with signals in lanes 16, 20 and 24, quantification of the signals (see Materials and Methods) indicated a reduction of purification efficiency by a factor of 10). As stated above, E. coli homologues of rpS13 and rpS14 belong to the central domain assembly tree which is implicated in folding of the SSU platform. RpS5 is located adjacent to rpS14 in the head - platform cleft and, S7, the E. coli homologue of rpS5, initiates the establishment of the SSU head domain assembly tree [37] (See also Fig. 1). To further characterize the impact of specific r-protein assembly events on the association of Noc4p with early pre-ribosomes we performed a semi-quantitative comparative proteome analysis of ribosome biogenesis factors co-purifying with TAP-tagged Noc4p from extracts of wildtype cells and cells depleted of either rpS5, rpS13, or rpS22 (Fig. 5). Co-purification of SSU processome components other than Nop14p/Noc5p was clearly reduced in cells depleted of rpS5 or rpS13 (see Fig. 5A, Fig. 5B and Fig. 5D). This suggested that association of Noc4p and its interaction partner Nop14p/Noc5p [24], [25] with SSU processome complexes lacking rpS5 or rpS13 was destabilized. In addition, these analyses confirmed the results of the (pre-)rRNA precipitation experiments (Fig. 4) that Noc4p-TAP continued to be stably incorporated in SSU processomes formed in the absence of rpS22 (see Fig. 5C and heatmap representation in Fig. 5D).


Interrelationships between yeast ribosomal protein assembly events and transient ribosome biogenesis factors interactions in early pre-ribosomes.

Jakob S, Ohmayer U, Neueder A, Hierlmeier T, Perez-Fernandez J, Hochmuth E, Deutzmann R, Griesenbeck J, Tschochner H, Milkereit P - PLoS ONE (2012)

Analysis of ribosome biogenesis factors co-purifying with Noc4p after in vivo depletion of rpS5, rpS13, or rpS22.The yeast strain TY96 (wildtype) expressing chromosome encoded TAP tagged Noc4p and yeast conditional mutant strains TY1241, TY1897, and TY1902, expressing chromosomal encoded TAP tagged Noc4p and carrying in addition galactose inducible conditional alleles of RPS5 (TY1241), RPS13 (TY1897), or RPS22 (TY1902) were cultivated in medium containing galactose as carbon source and were subsequently transferred to glucose containing medium and cultivated for additional four hours. Noc4p-TAP was affinity purified from corresponding cellular extracts using IgG coupled magnetic bead matrix. Affinity purified proteins were digested using trypsin and the resulting peptides from each sample were labelled with different iTRAQ reagents. Labelled peptides of wildtype samples were combined with labelled peptides of samples derived either from the conditional mutant of RPS5 (A), RPS13 (B), or RPS22 (C) and were then further analyzed as described in material and methods. Average iTRAQ ratios of each SSU processome component identified by more than one peptide are indicated in (A)–(C). Numbers in brackets behind SSU processome component names indicate the number of peptides (confidence interval >95%) by which the respective protein was identified. (D) shows a heatmap representation of the three datasets. The factors are ordered according to a clustering analysis (see material and methods). Boxes in red colours represent relative enrichment and boxes in green colours relative deprivation of a protein in Noc4p-TAP fractions purified from mutant versus wildtype cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3303783&req=5

pone-0032552-g005: Analysis of ribosome biogenesis factors co-purifying with Noc4p after in vivo depletion of rpS5, rpS13, or rpS22.The yeast strain TY96 (wildtype) expressing chromosome encoded TAP tagged Noc4p and yeast conditional mutant strains TY1241, TY1897, and TY1902, expressing chromosomal encoded TAP tagged Noc4p and carrying in addition galactose inducible conditional alleles of RPS5 (TY1241), RPS13 (TY1897), or RPS22 (TY1902) were cultivated in medium containing galactose as carbon source and were subsequently transferred to glucose containing medium and cultivated for additional four hours. Noc4p-TAP was affinity purified from corresponding cellular extracts using IgG coupled magnetic bead matrix. Affinity purified proteins were digested using trypsin and the resulting peptides from each sample were labelled with different iTRAQ reagents. Labelled peptides of wildtype samples were combined with labelled peptides of samples derived either from the conditional mutant of RPS5 (A), RPS13 (B), or RPS22 (C) and were then further analyzed as described in material and methods. Average iTRAQ ratios of each SSU processome component identified by more than one peptide are indicated in (A)–(C). Numbers in brackets behind SSU processome component names indicate the number of peptides (confidence interval >95%) by which the respective protein was identified. (D) shows a heatmap representation of the three datasets. The factors are ordered according to a clustering analysis (see material and methods). Boxes in red colours represent relative enrichment and boxes in green colours relative deprivation of a protein in Noc4p-TAP fractions purified from mutant versus wildtype cells.
Mentions: Noc4p was identified above as a member of the Noc4p/Nop14p group of SSU processome components tending to be underrepresented in early pre-ribosomes purified from yeast cells depleted of rpS5, rpS13, or rpS14. We were interested to characterize in more detail the influence of r-protein assembly events on association of Noc4p with early pre-ribosomes. Conditional mutants of several SSU r-protein genes were constructed which express a chromosome encoded C-terminal TAP-fusion allele of Noc4p. The selected conditional r-protein gene mutants were the same as the ones studied in the experiments shown in Figure 2 and therefore included again the head domain binder rpS15 and the central domain/platform binder rpS14 together with the yeast homologues of five primary E. coli in vitro binders interacting with different regions of the SSU rRNA (see Fig. 1). Noc4p-TAP was affinity purified from extracts of these mutants either grown in permissive or restrictive conditions. As expected for a SSU processome component, Northern blot analyses indicated that Noc4p-TAP co-purified significant amounts of early SSU rRNA precursors (23S and 32/35S pre-rRNAs) from extracts of cells grown in permissive conditions (Fig. 4, compare 23S and 32/35S signals in lanes 1, 5, 9, 13, 17, 21 and 25 with 32/35S signals in lanes 2, 6, 10, 14, 18, 22 and 26). In vivo depletion of the various r-proteins led to the expected pre-rRNA processing phenotypes (Fig. 4, compare 32/35S signals in lanes 1, 5, 9, 13, 17, 21 and 25 with 32/35S signals in lanes 3,7,11,15,19,23 and 27, compare also with Fig. 2 and [9]). Interestingly, Noc4p-TAP efficiently co-purified large amounts of accumulating early 32/35S pre-rRNAs from extracts of a subset of the analyzed r-protein gene mutants shifted to restrictive conditions (RPS11, RPS9, RPS22, RPS15, see Fig. 4, compare 32/35S signals in lanes 3, 7, 11 and 15 with signals in lanes 4, 8, 12 and 16). By contrast, the efficiency of co-purification of early 32/35S pre-rRNA with Noc4p-TAP from extracts of strains depleted of another subset of r-proteins (rpS13, rpS14, rpS5) was reduced close to background levels, though co-precipitation was still detectable (Fig. 4, compare 35S/32S signals in lanes 15, 19 and 23 with signals in lanes 16, 20 and 24, quantification of the signals (see Materials and Methods) indicated a reduction of purification efficiency by a factor of 10). As stated above, E. coli homologues of rpS13 and rpS14 belong to the central domain assembly tree which is implicated in folding of the SSU platform. RpS5 is located adjacent to rpS14 in the head - platform cleft and, S7, the E. coli homologue of rpS5, initiates the establishment of the SSU head domain assembly tree [37] (See also Fig. 1). To further characterize the impact of specific r-protein assembly events on the association of Noc4p with early pre-ribosomes we performed a semi-quantitative comparative proteome analysis of ribosome biogenesis factors co-purifying with TAP-tagged Noc4p from extracts of wildtype cells and cells depleted of either rpS5, rpS13, or rpS22 (Fig. 5). Co-purification of SSU processome components other than Nop14p/Noc5p was clearly reduced in cells depleted of rpS5 or rpS13 (see Fig. 5A, Fig. 5B and Fig. 5D). This suggested that association of Noc4p and its interaction partner Nop14p/Noc5p [24], [25] with SSU processome complexes lacking rpS5 or rpS13 was destabilized. In addition, these analyses confirmed the results of the (pre-)rRNA precipitation experiments (Fig. 4) that Noc4p-TAP continued to be stably incorporated in SSU processomes formed in the absence of rpS22 (see Fig. 5C and heatmap representation in Fig. 5D).

Bottom Line: One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain.Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes.They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Biochemie III, Universität Regensburg, Regensburg, Germany.

ABSTRACT
Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA). Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins) and ribosome precursors (pre-ribosomes) are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU) processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains.

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Related in: MedlinePlus