Independent association of Pwp2p- and Rrp7p-containing complexes with the 35S pre-rRNA. (A and C) Cellular extracts from the indicated yeast strains (top) grown in either galactose (left panels)- or glucose (right panels)-containing medium were subjected to sucrose gradient sedimentation analysis. After the ultracentrifugation step, fractions were automatically collected and polysome profiles recorded by continuous reading of the A₂₅₄ (upper panels). The distribution of Rrp7p-MYC (A) or Pwp2p-MYC (C) in aliquots of the collected fractions was analyzed by anti-MYC immunoblotting (second panels from the top). In parallel, total RNAs were obtained from aliquots of the collected fractions and subjected to Northern blot analysis with ³²P-labeled oligonucleotide probes to the 35S pre-rRNA EC₂ (third panels from top) and A₂-A₃ (fourth panels from top) regions or to the U3 snoRNA (bottom panels) (see Materials and Methods and Fig. S1 in the supplemental material). The number of each gradient fraction is indicated at the bottom of the first and fifth panels from top. Gal, galactose; Glu, glucose; WB, Western blot; NB, Northern blot. (B and D) Total cellular lysates (lanes 1 to 4) and anti-MYC immunoprecipitates (lanes 5 to 8) obtained for the indicated yeast strains and growth conditions (top) were analyzed by anti-MYC immunoblotting (upper panels), anti-HA immunoblotting (second panels from top), and Northern blot analyses using ³²P-labeled oligonucleotide probes to the 35S pre-rRNA DA₂ region (third panels from the top; see Fig. S1 in the supplemental material) and the U3 snoRNA (bottom panel). TCL, total cellular lysates; IP, immunoprecipitation.

Asymmetrical dependency of Nan1p- and Rrp7p-containing complexes for their assembly onto the 35S pre-rRNA. (A and C) Cellular extracts from cultures of the indicated yeast strains (top) grown in either galactose (left panels)- or glucose (right panels)-containing medium were subjected to sucrose gradient sedimentation analysis. The distribution of Nan1p-MYC (A) and Rrp7p-MYC (C) in aliquots of the collected fractions was analyzed by anti-MYC immunoblotting (second panels from the top). In parallel, the sedimentation profiles of ribosomal subunits (top panels), pre-rRNA precursor molecules (third and fourth panels from top), and the U3 snoRNA (bottom panels) were analyzed in aliquots of the same gradient fractions, as indicated in Fig. 2. WB, Western blot; NB, Northern blot. (B and D) RNAs and proteins from either total cellular lysates (lanes 1 to 4) or anti-MYC immunoprecipitates (lanes 5 to 8) obtained for the indicated yeast strains and growth conditions (top) were probed with anti-MYC (upper panels) and anti-HA (second panels from the top) antibodies or ³²P-labeled oligonucleotides hybridizing to sequences within the 35S pre-RNA DA₂ region (third panels from the top; see Fig. S1 in the supplemental material) and the U3 snoRNA (bottom panel). TCL, total cellular lysates; IP, immunoprecipitation.

Rrp5p is required for the association of Rrp7p, but not of Pwp2p-, Nan1p-, or U3 snoRNP-containing complexes, with the 35S pre-rRNA precursor. (A) Cellular extracts from the indicated yeast strain (top) grown in either galactose (left panels)- or glucose (right panels)-containing medium were subjected to sucrose gradient sedimentation analysis. The distribution of Rrp7p-MYC in the gradient fractions was analyzed using anti-MYC immunoblots (second panels from the top). In parallel, the sedimentation profiles of ribosomal subunits (top panels), pre-rRNA precursor molecules (third and fourth panels from top), and the U3 snoRNA (bottom panels) were analyzed in aliquots of the gradient fractions, as indicated in Fig. 2. The distinctive accumulation of an aberrant 12S′ pre-rRNA species is observed in Rrp5-depleted cells, as previously described (35). WB, Western blot; NB, Northern blot. (B to D) Identification by mass spectrometry of molecules associated with the indicated MYC-tagged bait proteins (top) purified from yeast cells lacking Rrp5p. Large-scale extracts from yeast cells that had been depleted of Rrp5p were incubated with anti-MYC antibodies covalently bound to a solid matrix. After incubation and washes, the proteins bound to the MYC-tagged bait were released from the matrix by boiling in SDS-polyacrylamide gel electrophoresis sample buffer, fractionated electrophoretically, stained with Sypro Ruby, and identified by mass spectrometry. The identified proteins are indicated on the left of the original gel used for their purification. The proteins belonging to the bait's subunit are indicated in blue. The migration of molecular mass markers is indicated on the right.

A model for the stepwise assembly of the 90S preribosomal particle. The 90S preribosome is formed by independent building blocks that follow hierarchical rules of assembly onto the 35S pre-rRNA. The binding of the t-UTP subunit to the pre-rRNA precursor (step 1) is required for the subsequent assembly of other 90S preribosome components. Experimental evidence suggests that the assembly of t-UTP might occur at an early and independent step. However, it cannot be excluded that the recruitment of this subunit is simultaneous with the assembly events shown as immediately subsequent in this model (step 2 and/or step3) (see Discussion). The other building modules associate with the 35S pre-rRNA through two mutually independent assembly branches. In one of the branches (step 2), the Pwp2p/UTP-B subunit, the U3 snoRNP, the Mpp10 complex, and seven other proteins stably associate with the pre-rRNA. Previously published evidence suggests that the Pwp2p/UTP-B subunit and the U3 snoRNP coassemble with the 35S pre-rRNA precursor in one cooperative event (6). Whether the Mpp10p complex and the other proteins of the same assembly branch (Utp20, Bms1p, Kre33p, Nop14p, Enp2p, Noc4p, and Krr1p) also incorporate into the particle in a cooperative manner remains to be determined. In the second assembly branch (step 3), Rrp5 binds to the 35S pre-rRNA prior to the UTP-C subunit. Further details about this model are given in the text.

Schematic depiction of the experimental strategy used in this work. The experimental basis of this study relied on the analysis of how depletion of specific markers for putative 90S preribosome subunits affected the assembly of other 90S preribosomal particle proteins onto the 35S pre-rRNA. A description of the steps outlined in this figure, including the techniques employed and the aims pursued, is given in the text. The results corresponding to the experiments of each step are shown in Fig. 2 to 7 as follows. (i) Polysome profiles (step 3A) are shown in the upper panels of Fig. 2A and C, 3A and C, 4A and C, and 6A and Fig. S2, S3, and S5 in the supplemental material. (ii) The sedimentation patterns of MYC-tagged proteins (step 3B) are shown in the second panels (from the top) of Fig. 2A and C, 3A and C, 4A and C, and 6A and Fig. S2, S3, and S5 in the supplemental material. (iii) The sedimentation of pre-rRNA precursors and the U3 snoRNA (step 3C) are shown in the third (in the cases of the 35S and 27S pre-rRNA precursors), fourth (in the cases of the 35S, 27SA₂, and 23S pre-rRNA precursors), and fifth (in the cases of the U3 snoRNA) panels (from the top) of Fig. 2A and C, 3A and C, 4A and C, and 6A and Fig. S2, S3, and S5 in the supplemental material. (iv) The results of coimmunoprecipitation experiments (step 4A) are shown in Fig. 2B and D, 3B and D, and 4B and D. (v) Proteins associated with MYC-tagged proteins identified by mass spectrometry (step 4B) are shown in Fig. 5 and 6B to D and Fig. S4 in the supplemental material. vi) The network of pairwise interactions and the hierarchical clustering of 90S preribosome components generated by bioinformatic analysis are shown in Fig. 7A and B. GAL, GAL1 promoter; Pr, endogenous promoter; MS, mass spectrometry.

A Nan1p-containing complex is required for the interaction of Pwp2p with the 35S pre-rRNA but not vice versa. (A and C) Cellular extracts from cultures of the indicated yeast strains (top) grown in either galactose (left panels)- or glucose (right panels)-containing medium were subjected to sucrose gradient sedimentation analysis. The distribution of Nan1p-MYC (A) and Pwp2p-MYC (C) in aliquots of the collected fractions was visualized by anti-MYC immunoblotting (second panels from the top). In parallel, the sedimentation profiles of ribosomal subunits (top panels), pre-rRNA precursor molecules (third and fourth panels from top), and the U3 snoRNA (bottom panels) were analyzed in aliquots of each of the gradient fractions, as indicated in Fig. 2. WB, Western blot; NB, Northern blot. (B and D) RNAs and proteins from either total cellular lysates (lanes 1 to 4) or anti-MYC immunoprecipitates (lanes 5 to 8) obtained for the indicated yeast strains and growth conditions (top) were analyzed by Western blotting using anti-MYC (upper panels) and anti-HA (second panels from top) antibodies or by Northern blot analyses using ³²P-labeled oligonucleotide probes to the 35S pre-rRNA DA₂ region (third panels from the top; see Fig. S1 in the supplemental material) and the U3 snoRNA (bottom panel). TCL, total cellular lysates; IP, immunoprecipitation.

Proteomic analysis of partially assembled subunits of the 90S preribosomal particle. The MYC-tagged bait proteins used for the immunopurifications are indicated in the first row from the top. The proteins that have been depleted in the lysates used for the immunopurifications are indicated in the second row from top. The prey proteins identified in these analyses, their open reading frame names, and the type of UTP subcomplex they belong to are indicated in the second, third, and first columns on the left, respectively. Shaded areas indicate a positive association between the appropriate bait and 90S particle proteins.

A bioinformatic-based model depicting the hierarchical interactions established among 90S preribosome particle components. (A) Network of pairwise interactions established by components of t-UTP (labeled in yellow), Pwp2p/UTP-B (labeled in green), UTP-C (labeled in purple), the Mpp10p complex (labeled in blue), and nine additional 90S preribosomal proteins (labeled in gray). Blue lines indicate interactions between protein pairs that have been detected in at least two independent proteomic experiments. First-order interactors are underlined. (B) Hierarchical clustering analysis of the interaction data available for the 32 proteins shown in panel A. Eight specific branching points of the dendrogram that gave bootstrap values of ≥745 (optimal score = 1,000) are depicted as closed circles and identified by arabic numerals. The specific stability value of each branching point is indicated in parentheses.