Journal: eLife

Article Title: Prefabrication of a ribosomal protein subcomplex essential for eukaryotic ribosome formation

doi: 10.7554/eLife.21755

Figure Lengend Snippet: ( A ) Co-overexpression of FAP7 and uS11 rescues slow growth of Tsr2-depleted cells. The P GAL1 - 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. DOI: http://dx.doi.org/10.7554/eLife.21755.002

Article Snippet: The following primary antibodies were used in this study: α-Fap7/uS11 (1:2,000; this study), α-Tsr2/S26 (1:3,000; ), α-uS7 (yeast Rps5)(1:4,000; Proteintech Group Inc., Chicago, IL), α-uS3 (yeast Rps3)(1:3,000; M. Seedorf, University of Heidelberg, Heidelberg, Germany); α-TAP (CBP) (1:4,000; Thermo Scientific, Rockford, IL), α-Pno1 (1:10,000; K. Karbstein, Scripps Research Institute, Jupiter, FL), α-Dim1 (1:10,000; K. Karbstein, Scripps Research Institute, Jupiter, FL), α-Nob1 (1:500; Proteintech Group Inc., Chicago, IL), α-Tsr1 (1:10,000; K. Karbstein, Scripps Research Institute, Jupiter, FL).

Techniques: Over Expression, Transformation Assay, Labeling, Staining, Extraction, Northern Blot, Western Blot, Isolation

Journal: eLife

Article Title: Prefabrication of a ribosomal protein subcomplex essential for eukaryotic ribosome formation

doi: 10.7554/eLife.21755

Figure Lengend Snippet: ( 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 P GAL1 - 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. DOI: http://dx.doi.org/10.7554/eLife.21755.003

Article Snippet: The following primary antibodies were used in this study: α-Fap7/uS11 (1:2,000; this study), α-Tsr2/S26 (1:3,000; ), α-uS7 (yeast Rps5)(1:4,000; Proteintech Group Inc., Chicago, IL), α-uS3 (yeast Rps3)(1:3,000; M. Seedorf, University of Heidelberg, Heidelberg, Germany); α-TAP (CBP) (1:4,000; Thermo Scientific, Rockford, IL), α-Pno1 (1:10,000; K. Karbstein, Scripps Research Institute, Jupiter, FL), α-Dim1 (1:10,000; K. Karbstein, Scripps Research Institute, Jupiter, FL), α-Nob1 (1:500; Proteintech Group Inc., Chicago, IL), α-Tsr1 (1:10,000; K. Karbstein, Scripps Research Institute, Jupiter, FL).

Techniques: Expressing, Fluorescence, Microscopy, Purification, Silver Staining, Western Blot, Control, Labeling, Staining, Over Expression, Transformation Assay, Isolation

Journal: eLife

Article Title: Prefabrication of a ribosomal protein subcomplex essential for eukaryotic ribosome formation

doi: 10.7554/eLife.21755

Figure Lengend Snippet: ( 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; ) onto yeast uS11 from the mature 40S ribosome (PDB: 4 V88; ). For the uS11 3R 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 uS11 3R mutant is impaired in recruiting eS26 in vitro. Recombinant GST-Fap7:uS11 or GST-Fap7:uS11 3R 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 uS11 3R does not rescue the slow growth of Tsr2-depleted cells. The P GAL1 - 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 uS11 3R 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 uS11 3R 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. DOI: http://dx.doi.org/10.7554/eLife.21755.004

Article Snippet: The following primary antibodies were used in this study: α-Fap7/uS11 (1:2,000; this study), α-Tsr2/S26 (1:3,000; ), α-uS7 (yeast Rps5)(1:4,000; Proteintech Group Inc., Chicago, IL), α-uS3 (yeast Rps3)(1:3,000; M. Seedorf, University of Heidelberg, Heidelberg, Germany); α-TAP (CBP) (1:4,000; Thermo Scientific, Rockford, IL), α-Pno1 (1:10,000; K. Karbstein, Scripps Research Institute, Jupiter, FL), α-Dim1 (1:10,000; K. Karbstein, Scripps Research Institute, Jupiter, FL), α-Nob1 (1:500; Proteintech Group Inc., Chicago, IL), α-Tsr1 (1:10,000; K. Karbstein, Scripps Research Institute, Jupiter, FL).

Techniques: In Vitro, Recombinant, Incubation, SDS Page, Staining, Western Blot, Protein-Protein interactions, Mutagenesis, Over Expression, Transformation Assay, Labeling, Isolation

Journal: eLife

Article Title: Prefabrication of a ribosomal protein subcomplex essential for eukaryotic ribosome formation

doi: 10.7554/eLife.21755

Figure Lengend Snippet: ( 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 Mg 2+ (PDB: 4CW7; ). ( 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 P GAL1 - 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. DOI: http://dx.doi.org/10.7554/eLife.21755.006

Article Snippet: The following primary antibodies were used in this study: α-Fap7/uS11 (1:2,000; this study), α-Tsr2/S26 (1:3,000; ), α-uS7 (yeast Rps5)(1:4,000; Proteintech Group Inc., Chicago, IL), α-uS3 (yeast Rps3)(1:3,000; M. Seedorf, University of Heidelberg, Heidelberg, Germany); α-TAP (CBP) (1:4,000; Thermo Scientific, Rockford, IL), α-Pno1 (1:10,000; K. Karbstein, Scripps Research Institute, Jupiter, FL), α-Dim1 (1:10,000; K. Karbstein, Scripps Research Institute, Jupiter, FL), α-Nob1 (1:500; Proteintech Group Inc., Chicago, IL), α-Tsr1 (1:10,000; K. Karbstein, Scripps Research Institute, Jupiter, FL).

Techniques: Mutagenesis, In Vitro, Purification, Activity Assay, Enzymatic Assay, Binding Assay, Fluorescence, Over Expression, Transformation Assay, Labeling, Staining, Isolation, Western Blot