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Prefabrication of a ribosomal protein subcomplex essential for eukaryotic ribosome formation

  1. Cohue Peña
  2. Sabina Schütz
  3. Ute Fischer
  4. Yiming Chang
  5. Vikram G Panse  Is a corresponding author
  1. ETH Zurich, Switzerland
  2. University of Zurich, Switzerland
Research Article
Cite this article as: eLife 2016;5:e21755 doi: 10.7554/eLife.21755
9 figures and 1 additional file

Figures

Co-overexpression of Fap7 and the r-protein uS11 bypasses in vivo requirement for Tsr2.

(A) Co-overexpression of FAP7 and uS11 rescues slow growth of Tsr2-depleted cells. The PGAL1-TSR2 strain was transformed with indicated plasmids and spotted in 10-fold dilutions on selective and repressive glucose-containing plates and grown at indicated temperatures for 3–7 days. (B) Co-overexpression of FAP7 and uS11 rescues 20S pre-rRNA processing defect of Tsr2-depleted cells. Indicated strains were grown to mid-log phase at 25°C in selective glucose-containing medium. 20S pre-rRNA was localized by FISH using a Cy3-labeled oligonucleotide complementary to the 5’ portion of ITS1 (red). Nuclear and and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. (C) Indicated strains were grown as in above for extraction of total RNA and then analyzed by GelRed straining and Northern blotting using probes against 20S and 18S rRNAs. (D) Co-overexpression of FAP7 and uS11 restores protein levels of eS26 in Tsr2-depleted cells. Whole cell extracts (WCE) were prepared from indicated strains and subjected to Western analysis. (E) Co-overexpression of FAP7 and uS11 restores co-enrichment of eS26 with Enp1-TAP in Tsr2-depleted cells. Enp1-TAP was isolated from indicated strains and subjected to Western analysis. uS7 and CBP (TAP-tag) protein levels served as loading controls.

https://doi.org/10.7554/eLife.21755.002
uS11 and eS26 depend upon each other for their incorporation into pre-ribosomes.

(A) Fap7 is a nuclear localized protein. Fap7 and the nucleolar protein Gar1 were endogenously tagged with GFP and mCherry, respectively, and then strains expressing them were grown to mid-log phase at 25°C. Localization of Fap7-GFP and Gar1-mCherry was visualized by fluorescence microscopy. Scale bar = 5 µm. (B) uS11 and eS26, but not Fap7 or Tsr2 co-enrich with pre-ribosomal particles along the 40S maturation pathway. Pre-ribosomal particles in the 40S maturation pathway were purified using the indicated TAP-tagged baits. Calmodulin-eluates were analyzed by Silver staining, and Western analyses was performed using the indicated antibodies. The r-protein uS7 served as loading control for the TAPs. (C) Fap7-depletion does not impair pre-40S subunit nuclear export. The indicated strains expressing uS5-GFP were grown in repressive glucose-containing medium to mid-log phase at 25°C. Localization of uS5-GFP was monitored by fluorescence microscopy. Scale bar = 5 µm. (D) Fap7-depleted cells accumulate immature 20S pre-rRNA in the cytoplasm. Indicated strains were grown to mid-log phase at 25°C in selective glucose-containing medium. 20S pre-rRNA was localized by FISH using a Cy3-labeled oligonucleotide complementary to the 5’ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. (E) Slow growth of Fap7-depleted cells cannot be rescued by either (co-)overexpression of uS11, eS26 or TSR2. The PGAL1-FAP7 strain was transformed with indicated plasmids and spotted in 10-fold dilutions on selective and repressive glucose-containing plates and grown at indicated temperatures for 3–7 days. (F) Efficient recruitment of uS11 and eS26 to the 90S requires Fap7. Enp1-TAP was isolated from indicated strains and subjected to Western analysis. (G) Efficient recruitment of uS11 and eS26 to the 90S requires Tsr2. Enp1-TAP was isolated from indicated strains and subjected to Western analysis. Protein levels of uS7 and CBP (TAP-tag) served as loading control. (H) uS11, but not eS26 levels are strongly reduced in Fap7-depleted cells. (I) eS26, but not uS11 levels are strongly reduced in Tsr2-depleted cells. Whole cell extracts (WCE) were prepared from indicated strains and subjected to Western analysis. uS7 protein levels served as a loading control.

https://doi.org/10.7554/eLife.21755.003
Figure 3 with 1 supplement
The Fap7:uS11 complex recruits eS26.

(A) The Fap7:uS11 complex directly binds eS26 but not Tsr2:eS26 in vitro. Recombinant GST-tagged Fap7 and uS11 complexes were immobilized on Glutathione Sepharose beads before incubation with E. coli lysate containing recombinant eS26 or Tsr2:eS26. After washing away unbound proteins, beads were eluted and analyzed by SDS-PAGE followed by Coomassie Blue staining and Western blotting. L = 10% input. GST-baits are indicated with asterisks. (B) Conserved protein-protein interactions between uS11 and eS26 on the mature 40S subunit. uS11 (purple) binds to 18S rRNA helix 23 (grey) with its C-terminus embedded into the 18S rRNA 3’end. eS26 (blue) forms major contacts with uS11 via (1) a salt-bridge between uS11 R103 / R107 and eS26 D52 and (2) a hydrophobic side-chain interaction between uS11 R114 and eS26 Y59 / Y62. Yeast Fap7 which acts as a RNA mimic for helix 23 (light blue) was modeled into the structure by superposition of the Fap7:uS11 complex from Pyrococcus abyssi (PDB: 4CVN; Loc'h et al., 2014) onto yeast uS11 from the mature 40S ribosome (PDB: 4 V88; Ben-Shem et al., 2011). For the uS113R mutant, R103 / R107 / R114 were each mutated to aspartates. The sequences for the following organisms were aligned: Saccharomyces cerevisiae (Sc), Drosophila melanogaster (Dm), Caenorhabditis elegans (Ce), Mus musculus (Mm) and Homo sapiens (Hm) (C) The uS113R mutant is impaired in recruiting eS26 in vitro. Recombinant GST-Fap7:uS11 or GST-Fap7:uS113R was incubated with eS26 and analyzed by SDS-PAGE followed by Coomassie Blue staining and Western blotting. L = 100% input. GST-baits are indicated with asterisks. (D) Co-overexpression of FAP7 and uS113R does not rescue the slow growth of Tsr2-depleted cells. The PGAL1-TSR2 strain was transformed with indicated plasmids and spotted in 10-fold dilutions on selective and repressive glucose-containing plates and grown at indicated temperatures for 3–7 days. (E) Co-overexpression of FAP7 and uS113R does not rescue the 20S pre-rRNA processing defect of Tsr2-depleted cells. Indicated strains were grown to mid-log phase at 25°C in selective glucose-containing medium. 20S pre-rRNA was localized by FISH using a Cy3-labeled oligonucleotide complementary to the 5’ portion of ITS1 (red). Nuclear and and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. (F) Co-overexpression of FAP7 and uS113R does not restore protein levels of eS26 and neither allows co-enrichment with Enp1-TAP in Tsr2-depleted cells. Whole cell extracts (WCE, left panel) and Enp1-TAP (right panel) were prepared and isolated, respectively, from indicated strains and subjected to Western analysis. uS7 and CBP (TAP-tag) protein levels served as loading controls.

https://doi.org/10.7554/eLife.21755.004
Figure 3—figure supplement 1
r-proteins cluster on the eukaryotic ribosome via tertiary contacts.

Eukaryotic r-proteins interacting via tertiary protein-protein interactions are each highlighted in purple/red (universally conserved) or blue colors (archaea or eukaryote-specific) on the surface of ribosomal RNA (grey). Figures were constructed using the structure of the mature S. cerevisiae 80S ribosome (PDB: 4 V88; Ben-Shem et al., 2011).

https://doi.org/10.7554/eLife.21755.005
Figure 4 with 1 supplement
Fap7 ATPase activity is required to bypass Tsr2 requirement.

(A) To generate a catalytically inactive ATPase mutant for in vitro assays, the conserved D82 and H84 residues in the Walker B motif of Fap7 were each mutated to alanine (left panel), and expressed and purified from E. coli (right panel). The structure shows the Fap7:uS11 complex from P. abyssi in complex with ATP and Mg2+ (PDB: 4CW7; Loc'h et al., 2014). (B) Fap7-2 is deficient in ATP hydrolysis. The ATPase activity of indicated Fap7 and uS11 complexes was monitored by an indirect enzyme assay that detects formation of ADP, which is then coupled to β-NADH oxidation via the action of pyruvate kinase (PK) and lactate dehydrogenase (LDH). The decrease in β-NADH absorbance over time was measured at 340 nm in arbitrary units (AU). (C) Fap7-2 is deficient in ATP binding. Fluorescent mant-ATP was mixed with purified Fap7 or Fap7-2 and the change in emission spectra upon nucleotide binding was measured in relative fluorescence units (RFUs). (D) Co-overexpression of fap7-2 and uS11 does not rescue the slow growth of Tsr2-depleted cells. The PGAL1-TSR2 strain was transformed with indicated plasmids and spotted in 10-fold dilutions on selective and repressive glucose-containing plates and grown at indicated temperatures for 3–7 days. (E) Co-overexpression of fap7-2 and uS11 does not rescue the 20S pre-rRNA processing defect of Tsr2-depleted cells. Indicated strains were grown to mid-log phase at 25°C in selective glucose-containing medium. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5’ portion of ITS1 (red). Nuclear and and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. (F) Co-overexpression of fap7-2 and uS11 does not restore protein levels of eS26 and neither allows co-enrichment with Enp1-TAP in Tsr2-depleted cells. Whole cell extracts (WCE, left panel) and Enp1-TAP (right panel) were prepared and isolated, respectively, from indicated strains and subjected to Western analysis. uS7 and CBP (TAP-tag) protein levels served as loading controls.

https://doi.org/10.7554/eLife.21755.006
Figure 4—figure supplement 1
Co-overexpression of FAP7 and uS113R or fap7-2 and uS11 does not rescue the 20S pre-rRNA processing defect of Tsr2-depleted cells.

Indicated strains were grown to mid-log phase at 25°C in selective glucose-containing medium for extraction of total RNA and then analyzed by GelRed straining and Northern blotting using probes against 20S and 18S rRNAs.

https://doi.org/10.7554/eLife.21755.007
Fap7 ATPase activity organizes and recruits the uS11:eS26 subcomplex to helix 23 of 18S rRNA.

(A) Schematic for ATP-dependent loading of uS11:eS26 onto helix 23 of 18S rRNA by Fap7. Asterisk indicates a potential intermediate Fap7-ATP:uS11:eS26 complex. (B) Recombinant GST-uS11 was immobilized on Glutathione Sepharose beads before incubation with Fap7 or Fap7-2, eS26, ATP or non-hydrolysable ATP analog AMP-PNP and 18S helix 23 rRNA. (C) Recombinant GST-eS26 was immobilized on Glutathione Sepharose beads before incubation with 18S helix 23 rRNA. After washing away unbound proteins and RNA, beads were eluted and analyzed by SDS-PAGE followed by Coomassie Blue staining and Western blotting. For analysis of RNA, samples were Phenol-extracted and separated by denaturing PAGE followed by GelRed staining. RNA was quantified with respect to the input. L = 10% input. GST-baits and pulled-down proteins are indicated with asterisks.

https://doi.org/10.7554/eLife.21755.008
Figure 6 with 1 supplement
RanGTP-dependent nuclear import of uS11 and Fap7.

(A) Nuclear uptake of uS11-GFP is impaired in pse1-1 and pse1-1 kap104∆ mutants. Strains expressing uS11-GFP were grown in synthetic media at 25°C to mid-log phase. Ts-mutant strains were then shifted to 37°C for 4 hr and localization of uS11-GFP was analyzed by fluorescence microscopy. Scale bar = 5 µm. (B) Fap7:uS11, but not Fap7, interacts with Pse1. Recombinant GST-Pse1 or GST alone were immobilized on Glutathione Sepharose beads and incubated with buffer, purified Fap7 or Fap7:uS11 for 1 hr. After washing away unbound proteins, beads were eluted and analyzed by SDS-PAGE followed by Coomassie Blue staining and Western blotting. L = 10% input. GST-baits are indicated with asterisks. RanGTP (His6-Gsp1Q71L-GTP) efficiently releases Fap7:uS11 from Pse1. GST-Pse1:Fap7:uS11 complexes immobilized on Glutathione Sepharose were incubated with either buffer alone or with RanGTP for 10 min and then analyzed as described above. (D) Nuclear uptake of uS11-GFP and GFP-Fap7 is impaired in prp20-1 and rna1-1 mutants. Strains expressing uS11-GFP or GFP-Fap7 were grown in synthetic media at 25°C to mid-log phase. Ts-mutant strains were then shifted to 37°C for 4 hr and localization of uS11-GFP and GFP-Fap7 was analyzed by fluorescence microscopy. Scale bar = 5 µm.

https://doi.org/10.7554/eLife.21755.009
Figure 6—figure supplement 1
Nuclear import of uS11 and Fap7.

(A) A C-terminally GFP-tagged uS11 cannot complement the lethality of a rps14a∆rps14b∆ double knockout strain. The rps14a∆rps14b∆ pURA-uS11B shuffle strain was transformed with a plasmid encoding C-terminally tagged uS11-GFP and spotted in 10-fold dilutions on selective Leucine-deficient media plates with or without FOA to select against pURA-uS11B and grown at 30°C for 3–4 days. (B) Nuclear uptake of uS11-GFP is not impaired in kap123∆, msn5∆, kap114∆ sxm1∆ and kap120∆ sxm1∆ nmd5∆ mutants. Strains expressing uS11-GFP were grown in synthetic media at 25°C to mid-log phase. Ts-mutant strains were then shifted to 37°C for 4 hr and localization of uS11-GFP was analyzed by fluorescence microscopy. Scale bar = 5 µm. (C) Fap7:uS11, but not Fap7, interacts with Kap104. uS11, but not Fap7 interacts with Kap95, Kap114, Pdr6. Fap7 and uS11 alone, but not the Fap7:uS11 dimer interact with Kap123. Only Fap7 interacts with Msn5. Recombinant GST-tagged importins were immobilized on Glutathione Sepharose beads and incubated with buffer, purified Fap7 or Fap7:uS11 for 1 hr. After washing away unbound proteins, beads were eluted and analyzed by SDS-PAGE followed by Coomassie Blue staining and Western blotting. L = 10% input. GST-baits are indicated with asterisks. (D) Nuclear uptake of GFP-Fap7 is not impaired in several importin mutants and upon depletion of uS11. Strains expressing GFP-Fap7 were grown in synthetic media at 25°C to mid-log phase. Ts-mutant strains were then shifted to 37°C for 4 hr while PGal1-RPS14A rps14b∆ was grown overnight at 25°C in repressive glucose medium. Localization of GFP-Fap7 was analyzed by fluorescence microscopy. Scale bar = 5 µm.

https://doi.org/10.7554/eLife.21755.010
Fap7:uS11 facilitates RanGTP-dependent release of eS26 from Pse1.

(A) eS26 and Fap7:uS11 interact with Pse1. Recombinant GST-Pse1 was immobilized on Glutathione Sepharose beads and incubated with buffer or eS26 alone. After washing away unbound proteins, beads were further incubated in presence of buffer or Fap7:uS11 for 1 hr and washed again. Beads were then eluted and analyzed by SDS-PAGE followed by Coomassie Blue staining and Western blotting. L = 10% input. GST-baits are indicated with asterisks. (B) Fap7:uS11 facilitates RanGTP-dependent release of eS26. Immobilized GST-Pse1 was incubated first with eS26 alone (left panel). After washing away unbound proteins, beads were further incubated with RanGTP alone or RanGTP together with either Fap7:uS11 or Fap7:uS113R for indicated time points and washed again. Beads were then eluted and analyzed as described above. Right panel shows a schematic for how Fap7:uS11 facilitates RanGTP-dependent release of eS26 from Pse1.

https://doi.org/10.7554/eLife.21755.011
Author response image 1

(A) Whole cell extracts (WCE) were prepared from indicated strains and subjected to Western analysis using antibodies directed against Pse1 and Kap123. uS7 served as loading control. (B) eS26-FLAG was isolated from indicated strains. Equal levels of eS26-FLAG were loaded and separated on a SDS-gel and subjected to Western analysis using the indicated antibodies.

https://doi.org/10.7554/eLife.21755.013
Author response image 2

(A) Wild-type and pse1-1 strains expressing uS11-GFP were grown in synthetic media at 25°C to mid-log phase and then shifted to 37°C for 4 h. uS11-GFP was then immuno-precipitated and subjected to Western analysis.(B) Wild-type and prp20-1 strains expressing Fap7-TAP were grown in synthetic media at 25°C to mid-log phase and then shifted to 37°C for 4 h. Fap7-TAP was then purified and subjected to Western analysis.

https://doi.org/10.7554/eLife.21755.014

Additional files

Supplementary file 1

Yeast strains and plasmids used in this study.

(A) Yeast strains used in this study. (B) Plasmids used in this study.

https://doi.org/10.7554/eLife.21755.012

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