1. Volume 28, Issue 3, Pages 446-457 (November 2007)
Analysis of the In Vivo Assembly Pathway of Eukaryotic 40S Ribosomal Proteins 
Sébastien Ferreira-Cerca, Gisela Pöll, Holger Kühn, Andreas Neueder, Steffen Jakob, Herbert Tschochner, Philipp Milkereit 
Molecular Cell 
Volume 28, Issue 3, Pages (November 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Simplified Scheme of Yeast SSU Pre-rRNA Processing Pathway
Simplified scheme of yeast SSU pre-rRNA processing pathway (adapted from Gallagher et al. [2004]).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Analysis of r-Protein Association with Mature and Precursor SSUs before and after In Vivo Depletion of rpS5
Yeast strain (ToY323) expressing RPS5 under the control of a galactose-inducible promoter was transformed with vectors supporting the constitutive expression of Flag-tagged SSU r-proteins. Positive transformants were selected, and logarithmically growing cells were diluted to an OD of 0.3–0.5 and incubated 4 hr in YP-galactose-containing media. Cultures were split; one half was further incubated 2 hr in YP-galactose-containing media (on) and the other half incubated 2 hr in YP-glucose-containing media to shut down the expression of RPS5 (off). Cell extracts were prepared, and association of SSU Flag-r-proteins with their rRNA targets was analyzed by RNA coimmunoprecipitation experiments (IP) followed by northern blotting (see the Experimental Procedures). rRNA species were determined using probes complementary to the ITS1 (D-A2) and the 18S region (see the Experimental Procedures). Signals in input lanes (In) correspond to 1% of loaded cell extracts onto the anti-Flag agarose beads (IP lanes). A representative rRNA analysis of supernatants is shown in Figure S1. In the upper left corner, a corresponding analysis of a Flag-Noc4 and a no-tag-carrying strain is depicted. Signals of coimmunoprecipitated 20S rRNA and 18S rRNA were quantified as described in the Experimental Procedures. Percentages of coimmunoprecipitated (pre-) rRNA were determined. Efficiency of 18S rRNA precipitation serves as an internal control of the IP experiments and of the general assembly characteristics of the Flag-rpS fusion proteins. The efficiency of coimmunoprecipitated 20S rRNA in permissive condition ([percent IP 20S rRNA/percent IP 18S rRNA] in galactose) versus the efficiency of coimmunoprecipitated 20S rRNA in nonpermissive condition ([percent IP 20S rRNA/percent IP 18S rRNA] in glucose) was compared and expressed as the following ratio: ([percent IP 20S rRNA/percent IP 18S rRNA] in galactose/[percent IP 20S rRNA/percent IP 18S rRNA] in glucose). A representative experiment from three independent experiments is shown.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Influence of Salt Concentrations on the Association of Flag-rpS13 with Mature and Precursor SSUs
(A) Yeast strain expressing an N-terminal Flag-tagged version of the primary binder-homolog rpS13 (ToY272) was grown in YP-galactose to an OD of 0.8–1. Association of Flag-rpS13 with (precursor)-rRNA was analyzed as described in Figure 2 except that the salt concentrations indicated (0.2, 0.4, 0.6, 0.8, and 1 M KCl) were present during cell breakage and coimmunoprecipitation procedures. Signal in input lanes (In) corresponds to 1% of loaded cell extract onto the anti-Flag agarose beads (IP lanes).
(B) Signals of coimmunoprecipitated rRNA species from blot shown in (A) were quantified as described in the Experimental Procedures. Percentages of coimmunoprecipitated rRNA species were normalized to the percentage of coimmunoprecipitated rRNA determined at 200 mM KCl.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Structural View of the Eukaryotic Small Ribosomal Subunit
Structural landmarks are indicated as follows: b, body; h, head; pt, platform; bk, beak. The thick dashed line indicates the “border” between the body and the head structure.
(A) Structure of the eukaryotic SSU. Eukaryotic SSU structure at 11.5 Å according to PDB code 1S1H (Spahn et al., 2004) shown from the solvent side. R-proteins analyzed in this study that were localized and modeled by Spahn et al. (2004) according to their homology with prokaryotic counterparts are colored as follows: red, r-proteins associated with the head structure; blue, r-proteins associated with the body structure of the SSU. The 18S rRNA and r-proteins modeled but not analyzed in this study are indicated in gray. The square indicates rpS0.
(B) Structure of the prokaryotic rpS0 homolog S2 and its rRNA/r-protein neighborhood in the T. thermophilus SSU at 5.5 Å according to PDB code 1J5E (Wimberly et al., 2000). S2 is colored in blue, and the central protein domain of 46 amino acids of S2 that is not conserved in the eukaryotic counterpart rpS0 is colored in green.
(C) Structure of rpS0 and its rRNA neighborhood in S. cerevisiae according to PDB code 1S1H (Spahn et al., 2004). rpS0 is colored in blue, while 18S rRNA and other r-proteins are in gray.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Analysis of r-Protein Association with Mature and Precursor SSUs before and after In Vivo Depletion of rpS15
Yeast strain (ToY89) expressing RPS15 under the control of a galactose inducible promoter was transformed with vectors supporting the constitutive expression of Flag-tagged SSU r-proteins. RNA coimmunoprecipitation experiments were performed as described in Figure 2. Signals in input lanes (In) correspond to 1% of loaded cell extracts onto the anti-Flag agarose beads (IP lanes). For rpS5∗ and rpS10∗, signals in input lanes (In) correspond to 3% of loaded cell extracts onto the anti-Flag agarose beads (IP lanes). Signals of coimmunoprecipitated 20S rRNA and 18S rRNA were quantified as described in the Experimental Procedures. Quantifications were performed using different loading as shown as representative results for rpS5/rpS5∗ and rpS10/rpS10∗. Percentages of coimmunoprecipitated rRNA were determined. Efficiency of 18S rRNA precipitation serves as an internal control of the IP experiments and of the general assembly characteristics of the Flag-rpS fusion proteins. The efficiency of coimmunoprecipitated 20S rRNA in permissive condition ([percent IP 20S rRNA/percent IP 18S rRNA] in galactose) versus the efficiency of coimmunoprecipitated 20S rRNA in nonpermissive condition ([percent IP 20S rRNA/percent IP 18S rRNA] in glucose) was compared and expressed as the following ratio: ([percent IP 20S rRNA/percent IP 18S rRNA] in galactose/[percent IP 20S rRNA/percent IP 18S rRNA] in glucose). The result of these ratios is indicated as an average of the value determined from two independent experiments and two different loading for each experiment.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Association of SSU r-Proteins with Mature and Precursor SSUs after Inactivation of Exportin Crm1
(A) Northern analysis of SSU (precursor) rRNA of nuclear/cytoplasmic fractions of cells treated with (+) or without (−) 100 nM leptomycin B (LMB) (see the Experimental Procedures). To control the localization of 20S rRNA before and after inhibition of the exportin Crm1 with LMB, nuclear and cytoplasmic fractions of a yeast strain (ToY376) carrying a leptomycin B-sensitive allele of exportin Crm1 were prepared after treatment with (+) or without (−) 100 nM leptomycin B (LMB) for 45 min as described in Leger-Silvestre et al. (2004) and in the Experimental Procedures. A representative experiment is shown.
(B) Association of Flag-tagged SSU r-proteins with (precursor-) SSUs after inactivation of exportin Crm1. Yeast strain (ToY376) carrying a leptomycin B-sensitive allele of exportin Crm1 (Neville and Rosbash, 1999) was transformed with vectors supporting constitutive expression of Flag-tagged SSU r-proteins. Logarithmically growing transformants were diluted to an OD of 0.3–0.5 and incubated 2 hr in YP-glucose. Cultures were split and further incubated for 45 min with (+) or without (−) 100 nM of leptomycin B (LMB). Cell extracts were prepared and association of SSU Flag-r-proteins with their rRNA targets was analyzed by RNA coimmunoprecipitation experiments followed by northern blotting (see the Experimental Procedures). rRNA species were determined using probes complementary to the ITS1 (D-A2) and the 18S region (see the Experimental Procedures). Signals in input lanes (In) correspond to 1% of loaded cell extracts onto the anti-Flag agarose beads (IP lanes).
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Summary of In Vivo Analysis of r-Protein Association with Precursor SSUs in Conditional Mutants in Permissive and Nonpermissive Conditions
(A) Schematic representation of relative coimmunoprecipitation efficiency of 20S rRNA versus 35S rRNA (left panel) and 20S rRNA versus 23S rRNA (right panel) with Flag-tagged rpS. The percentages of coimmunoprecipitated rRNA species were determined from strains carrying each one of 21 Flag-tagged rpS as the only source for this r-protein. Ratio between percentage of coimmunoprecipitated 20S rRNA versus 35S rRNA (left panel) and ratio between percentage of coimmunoprecipitated 20S rRNA versus 23S rRNA (right panel) were determined. The y axis indicates how many times more efficient 20S rRNA was coprecipitated than 35S rRNA and 23S rRNA, respectively. The minimum and maximum values for these ratios (parallel bar) were plotted on a graphic representation and the respective ratios average indicated with a white lane in a ball. Average (percent IP 20S rRNA/percent IP 35S rRNA) = 18.3 ± 8.3 and average (percent IP 20S rRNA/percent IP 23S rRNA) = 7.7 ± 3.3.
(B) Schematic representation of relative coimmunoprecipitation efficiency of 20S rRNA with Flag-tagged r-proteins in permissive versus nonpermissive condition in mutants for rpS5 (left panel), rpS15 (center panel), and crm1 (right panel) are shown. Quantified results from Figures 2, 5, and 6 were plotted on a graphic representation according to the following formula: ([percent IP 20S rRNA/percent IP 18S rRNA] in permissive conditions)/([percent IP 20S rRNA/percent IP 18S rRNA] in nonpermissive conditions). The y axis indicates how many times more efficient 20S rRNA was coprecipitated in permissive versus nonpermissive conditions. R-proteins supposed to assemble in the body-like part of the SSU are indicated in blue and in the head-like part in red.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions