Processing of the ribosomal ubiquitin-like fusion protein FUBI-eS30/FAU is required for 40S maturation and depends on USP36
- Institute of Biochemistry, Department of Biology, ETH Zurich, Switzerland
- Molecular Life Sciences Ph.D. Program, Switzerland
Figures
Figure 1
Mutant, non-cleavable FUBI-eS30 can be incorporated into (pre-)40S particles.
(A) Structures (PDB ID: 4UG0 Khatter et al., 2015) of the human 40S subunit shown from the subunit interface (top) and of the 80S ribosome viewed from the mRNA entry channel (bottom), highlighting …
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Figure 1—source data 1
Source data for Figure 1C and D with relevant bands labeled on the uncropped original blots.
- https://cdn.elifesciences.org/articles/70560/elife-70560-fig1-data1-v2.png.zip
Figure 2 with 1 supplement
40S ribosome biogenesis is defective upon expression of non-cleavable FUBI-eS30 mutants.
(A) Extracts from parental (P), wild-type (WT) or mutant (AA, GV) FUBI-eS30-StHA HEK293 cell lines were separated on a linear 15–45% sucrose gradient by centrifugation and analyzed by polysome …
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Figure 2—source data 1
Source data for Figure 2C with relevant areas labeled on the uncropped edited images.
- https://cdn.elifesciences.org/articles/70560/elife-70560-fig2-data1-v2.png.zip
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Figure 2—source data 2
Unedited image of the ITS1 blot shown in Figure 2C.
- https://cdn.elifesciences.org/articles/70560/elife-70560-fig2-data2-v2.tiff.zip
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Figure 2—source data 3
Unedited image of the ITS2 blot shown in Figure 2C.
- https://cdn.elifesciences.org/articles/70560/elife-70560-fig2-data3-v2.tiff.zip
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Figure 2—source data 4
Unedited image of the gel shown in Figure 2C.
- https://cdn.elifesciences.org/articles/70560/elife-70560-fig2-data4-v2.tif.zip
Figure 2—figure supplement 1
Quantification of pre-rRNAs based on the ITS2 signal.
Quantification of the indicated pre-rRNA species based on the ITS2 signal, normalized to mature 28S rRNA in three biological replicates as shown in Figure 2C, expressed as fold changes relative to …
Figure 3
eS30 joins pre-40S ribosomal subunits in the nucleus.
(A) Parental and tetracycline-inducible FUBI(WT)-eS30-StHA HeLa cell lines were treated with the indicated siRNAs (5 nM, 48 hr) and LMB (+; 20 nM, 90 min) before fixation. Cells were immunostained …
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Figure 3—source data 1
Source data for Figure 3B with relevant areas labeled on the uncropped edited image of the gel and with relevant bands labeled on the uncropped original blots.
- https://cdn.elifesciences.org/articles/70560/elife-70560-fig3-data1-v2.png.zip
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Figure 3—source data 2
Unedited image of the gel shown in Figure 3B.
- https://cdn.elifesciences.org/articles/70560/elife-70560-fig3-data2-v2.tif.zip
Figure 4 with 2 supplements
Non-cleavable FUBI-eS30 mutants have a dominant-negative effect on late cytoplasmic steps of 40S subunit biogenesis.
(A) Scheme of pre-40S maturation focusing on late nucleoplasmic to cytoplasmic maturation steps and highlighting the RBFs mentioned in the text. Nucleoplasmic pre-40S particles containing 18S-E …
Figure 4—figure supplement 1
Expression of non-cleavable FUBI-eS30 mutants affects nucleolar but not early cytoplasmic 40S or cytoplasmic 60S biogenesis.
(A) The localizations of the 40S RBFs LTV1, ENP1, and RRP12 and of the 60S RBF NMD3 were analyzed in parental, WT or mutant (AA, GV) FUBI-eS30-StHA HeLa cell lines by immunostaining. Where …
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Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1C with relevant bands labeled on the uncropped original blots.
- https://cdn.elifesciences.org/articles/70560/elife-70560-fig4-figsupp1-data1-v2.png.zip
Figure 4—figure supplement 2
Non-cleavable FUBI-eS30 mutants induce a persistent 40S ribosomal subunit biogenesis defect.
(A) Flowchart of the timeline for the tetracycline (tet)-mediated induction of FUBI-eS30-StHA expression in HeLa cell lines without (left panel) or with (right panel) a chase period in tet-free …
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Figure 4—figure supplement 2—source data 1
Source data for Figure 4—figure supplement 2C with relevant bands labeled on the uncropped original blots.
- https://cdn.elifesciences.org/articles/70560/elife-70560-fig4-figsupp2-data1-v2.png.zip
Figure 5
Identification of candidate proteases by differential affinity purification mass spectrometry of FUBI-eS30-StHA constructs.
(A) StrepTactin affinity purification of HASt-GFP (GFP), WT or mutant (AA, GV) FUBI-eS30-StHA from HEK293 cell lysates. Eluates were analyzed by SDS-PAGE followed by silver staining or mass …
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Figure 5—source data 1
Source data for Figure 5A and C with relevant areas and bands labeled on the uncropped gel and uncropped original blots, respectively.
- https://cdn.elifesciences.org/articles/70560/elife-70560-fig5-data1-v2.png.zip
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Figure 5—source data 2
Unedited image of the gel shown in Figure 5A.
- https://cdn.elifesciences.org/articles/70560/elife-70560-fig5-data2-v2.tif.zip
Figure 6
USP36 is required for FUBI-eS30 processing in vivo.
(A) Immunoblot analysis of HeLa K cells that were either left untreated (utr), treated with control (ctrl) siRNA or siRNAs against the indicated factors (48 hr, 10 nM, except for FAU/FUBI-eS30: 5 …
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Figure 6—source data 1
Source data for Figure 6 with relevant areas labeled on the uncropped original blots.
- https://cdn.elifesciences.org/articles/70560/elife-70560-fig6-data1-v2.png.zip
Figure 7 with 1 supplement
USP36 cleaves linear authentic and artificial UB(L) substrates in vitro.
In vitro processing assays for which 2.5 µM (A) His6-FUBI(WT)-eS30, (B) His6-FUBI(AA)-eS30, (C) His6-FUBI-EGFP, and (D) His6-Ub-EGFP were incubated with 0.5 µM His10-USP36-TEV-St WT or CA mutant at …
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Figure 7—source data 1
Source data for Figure 7 with relevant areas labeled on the uncropped images of the gels.
- https://cdn.elifesciences.org/articles/70560/elife-70560-fig7-data1-v2.png.zip
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Figure 7—source data 2
Unedited image of the gels shown in Figure 7.
- https://cdn.elifesciences.org/articles/70560/elife-70560-fig7-data2-v2.tif.zip
Figure 7—figure supplement 1
Analysis of purified His10-USP36-TEV-St.
Purified WT and CA His10-USP36-TEV-St (0.5 µM) were analyzed by Coomassie brilliant blue (CBB) staining of the gel or by immunoblotting using the indicated antibodies.
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Figure 7—figure supplement 1—source data 1
Source data for Figure 7—figure supplement 1 with relevant areas labeled on the uncropped gel and uncropped original blots.
- https://cdn.elifesciences.org/articles/70560/elife-70560-fig7-figsupp1-data1-v2.png.zip
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Figure 7—figure supplement 1—source data 2
Unedited image of the gel shown in Figure 7—figure supplement 1.
- https://cdn.elifesciences.org/articles/70560/elife-70560-fig7-figsupp1-data2-v2.tif.zip
Tables
Appendix 1—key resources table
.
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | HEK293 Flp-In T-REx | Thermo Fisher Scientific | Cat# R78007 RRID:CVCL_U427 | |
| Cell line (Homo sapiens) | HEK293 Flp-In T-REx FUBI-eS30-StHA | This paper | See Materials and methods, Cell culture, cell lines, and treatments | |
| Cell line (Homo sapiens) | HEK293 Flp-In T-REx FUBI(AA)-eS30-StHA | This paper | AA: G73,74A See Materials and methods, Cell culture, cell lines, and treatments | |
| Cell line (Homo sapiens) | HEK293 Flp-In T-REx FUBI(GV)-eS30-StHA | This paper | GV: G74V See Materials and methods, Cell culture, cell lines, and treatments | |
| Cell line (Homo sapiens) | HEK293 Flp-In T-REx HASt-C21orf70 | (Zemp et al., 2014) DOI: 10.1242/jcs.138719 | ||
| Cell line (Homo sapiens) | HEK293 Flp-In T-REx HASt-FUBI-eS30 | This paper | See Materials and methods, Cell culture, cell lines, and treatments | |
| Cell line (Homo sapiens) | HEK293 Flp-In T-REx HASt-GFP | (Wyler et al., 2011) DOI: 10.1261/rna.2325911 | ||
| Cell line (Homo sapiens) | HeLa Flp-In T-REx | (Häfner et al., 2014) DOI: 10.1038/ ncomms5397 | Obtained from T. Mayer (University of Konstanz) | |
| Cell line (Homo sapiens) | HeLa Flp-In T-REx FUBI-eS30-StHA | This paper | See Materials and methods, Cell culture, cell lines, and treatments | |
| Cell line (Homo sapiens) | HeLa Flp-In T-REx FUBI(AA)-eS30-StHA | This paper | AA: G73,74A See Materials and methods, Cell culture, cell lines, and treatments | |
| Cell line (Homo sapiens) | HeLa Flp-In T-REx FUBI(GV)-eS30-StHA | This paper | GV: G74V See Materials and methods, Cell culture, cell lines, and treatments | |
| Cell line (Homo sapiens) | HeLa FRT/TetR | Other | Obtained fromM. Beck (EMBL, Heidelberg) | |
| Cell line (Homo sapiens) | HeLa FRT/TetR EGFP-USP36 | This paper | See Materials and methods, Cell culture, cell lines, and treatments | |
| Cell line (Homo sapiens) | HeLa FRT/TetR EGFP-USP36(CA) | This paper | CA: C131A See Materials and methods, Cell culture, cell lines, and treatments | |
| Cell line (Homo sapiens) | HeLa Kyoto, HeLa K | Other | RRID:CVCL_1922 | Obtained from D. Gerlich (IMBA, Vienna) |
| Antibody | anti-AAMP (rabbit polyclonal) | This paper | WB (1:800) See Materials and methods, Antibodies | |
| Antibody | anti-β-actin (mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-47778 RRID:AB_626632 | WB (1:1000) |
| Antibody | anti-C21ORF70 (rabbit polyclonal) | (Montellese et al., 2020) DOI: 10.1093/nar/gkx253 | WB (1:500) | |
| Antibody | anti-EIF1AD | Proteintech | Cat# 20528-1-AP RRID:AB_10693533 | WB (1:1000) |
| Antibody | anti-ENP1 (rabbit polyclonal) | (Zemp et al., 2009) DOI: 10.1083/jcb.200904048 | IF (1:15,000) WB (1:1000) | |
| Antibody | anti-eS1 (RPS3A) (rabbit polyclonal) | (Wyler et al., 2011) DOI: 10.1261/rna.2325911 | WB (1:1000) | |
| Antibody | anti-eS26 (RPS26) (rabbit polyclonal) | Abcam | Cat# ab104050 RRID:AB_10710999 | WB (1:500) |
| Antibody | anti-FAU (FUBI-eS30) (rabbit polyclonal) | Abcam | Cat# ab135765 | WB (1:500) |
| Antibody | anti-FUBI (rabbit polyclonal) | This paper | WB (1:2000) See Materials and methods, Antibodies | |
| Antibody | anti-HA (mouse monoclonal) | Enzo Life Sciences | ENZ-ABS120-0200 | IF (1:2000) WB (1:1000) |
| Antibody | anti-His (mouse monoclonal) | Sigma-Aldrich | Cat# H1029 RRID:AB_260015 | WB (1:2000) |
| Antibody | anti-LSG1 (rabbit polyclonal) | (Wyler et al., 2014) DOI: 10.1016/j.febslet.2014.08.013 | WB (1:3000) | |
| Antibody | anti-LTV1 (rabbit polyclonal) | (Zemp et al., 2009) DOI: 10.1083/jcb.200904048 | IF (1:4000) WB (1:2000) | |
| Antibody | anti-NMD3 (rabbit polyclonal) | (Zemp et al., 2009) DOI: 10.1083/jcb.200904048 | IF (1:1000) WB (1:10,000) | |
| Antibody | anti-NOB1 (rabbit polyclonal) | (Zemp et al., 2009) DOI: 10.1083/jcb.200904048 | IF (1:5000) WB (1:2000) | |
| Antibody | anti-NOC4L (rabbit polyclonal) | (Wyler et al., 2011) DOI: 10.1261/rna.2325911 | WB (1:5000) | |
| Antibody | anti-PNO1 (DIM2) (rabbit polyclonal) | (Zemp et al., 2009) DOI: 10.1083/jcb.200904048 | IF (1:2000) WB (1:2000) | |
| Antibody | anti-RIOK1 (rabbit polyclonal) | (Widmann et al., 2012) DOI: 10.1091/mbc.E11-07-0639 | IF (1:8000) WB (1:1000) | |
| Antibody | anti-RIOK2 (rabbit polyclonal) | (Zemp et al., 2009) DOI: 10.1083/jcb.200904048 | IF (1:5000) WB (1:5000) | |
| Antibody | anti-RRP12 (rabbit polyclonal) | (Wyler et al., 2011) DOI: 10.1261/rna.2325911 | IF (1:2000) WB (1:1000) | |
| Antibody | anti-Strep (mouse monoclonal) | IBA GmbH | Cat# 2-1507-001 RRID:AB_513133 | WB (1:1000) |
| Antibody | anti-TRIP4 (rabbit polyclonal) | This paper | WB (1:50,000) See Materials and methods, Antibodies | |
| Antibody | anti-TSR1 (rabbit polyclonal) | ##(Zemp et al., 2014)# DOI: 10.1242/jcs.138719 | WB (1:10,000) | |
| Antibody | anti-uL23 (RPL23A) (rabbit polyclonal) | (Wyler et al., 2011) DOI: 10.1261/rna.2325911 | WB (1:200) | |
| Antibody | anti-uS3 (RPS3) (rabbit polyclonal) | (Zemp et al., 2009) DOI: 10.1083/jcb.200904048 | WB (1:1000) | |
| Antibody | anti-USP10 (rabbit polyclonal) | Sigma-Aldrich | Cat# HPA006731 RRID:AB_1080495 | WB (1:1000) |
| Antibody | anti-USP16 (rabbit polyclonal) | Bethyl Laboratories | Cat# A301-615A RRID:AB_1211387 | WB (1:500) |
| Antibody | anti-USP36 (rabbit polyclonal) | Sigma-Aldrich | Cat# HPA012082 RRID:AB_1858682 | IF (1:1000) WB (1:250) |
| Antibody | goat anti-mouse Alexa Fluor 594 | Thermo Fisher Scientific | A-11005, RRID:AB_2534073 | IF (1:250) |
| Antibody | goat anti-rabbit Alexa Fluor 488 | Thermo Fisher Scientific | A-11008, RRID:AB_143165 | IF (1:250) |
| Antibody | goat anti-mouse Alexa Fluor Plus 680 | Thermo Fisher Scientific | Cat# A32729 RRID:AB_2633278 | WB (1:10,000) |
| Antibody | goat anti-rabbit Alexa Fluor Plus 800 | Thermo Fisher Scientific | Cat# A32735, RRID:AB_2633284 | WB (1:10,000) |
| Recombinant DNA reagent | pC2Pi/control | (Boneberg et al., 2019) DOI: 10.1261/rna.069609.118 | See Materials and methods, Molecular cloning | |
| Recombinant DNA reagent | pC2Pi/USP36i-g1 | This paper | USP36 guide 1 See Materials and methods, Molecular cloning | |
| Recombinant DNA reagent | pC2Pi/USP36i-g2 | This paper | USP36 guide 2 See Materials and methods, Molecular cloning | |
| Recombinant DNA reagent | pCDNA5/FRT/TO/FUBI-eS30-StHA | This paper | See Materials and methods, Molecular cloning | |
| Recombinant DNA reagent | pCDNA5/FRT/TO/FUBI(AA)-eS30-StHA | This paper | AA: G73,74A See Materials and methods, Molecular cloning | |
| Recombinant DNA reagent | pCDNA5/FRT/TO/FUBI(GV)-eS30-StHA | This paper | GV: G74V See Materials and methods, Molecular cloning | |
| Recombinant DNA reagent | pCDNA5/FRT/TO/EGFP-USP36 | This paper | See Materials and methods, Molecular cloning | |
| Recombinant DNA reagent | pCDNA5/FRT/TO/EGFP-USP36(CA) | This paper | CA: C131A See Materials and methods, Molecular cloning | |
| Recombinant DNA reagent | pCDNA5/FRT/TO/HASt-FUBI-eS30 | This paper | See Materials and methods, Molecular cloning | |
| Recombinant DNA reagent | pDEST/LTR/Flag-HA-USP36 | Addgene (Sowa et al., 2009) DOI: 10.1016/j.cell.2009.04.042 | Cat# 22579 RRID:Addgene_22579 | See Materials and methods, Molecular cloning |
| Recombinant DNA reagent | pET-28b(+)/His6-FUBI-EGFP | This paper | See Materials and methods, Molecular cloning | |
| Recombinant DNA reagent | pET-28b(+)/His6-Ub-EGFP | This paper | See Materials and methods, Molecular cloning | |
| Recombinant DNA reagent | pFBD/EGFP/His10-USP36-TEV-St | This paper | pFBD: pFastBac Dual See Materials and methods, Molecular cloning | |
| Recombinant DNA reagent | pFBD/EGFP/His10-USP36(CA)-TEV-St | This paper | pFBD: pFastBac Dual, CA: C131A See Materials and methods, Molecular cloning | |
| Recombinant DNA reagent | pQE30/His6-FUBI-eS30 | This paper | See Materials and methods, Molecular cloning | |
| Recombinant DNA reagent | pQE30/His6-FUBI(AA)-eS30 | This paper | AA: G73,74A See Materials and methods, Molecular cloning | |
| Sequence-based reagent | QuikChange primers for FUBI(AA)-eS30 | Sigma-Aldrich | 5'-GCAGGCCGCATGCTTGcAGcTAAAGTTCATGGTTCC-3', 5'-GGAACCATGAACTTTAgCTgCAAGCATGCGGCCTGC-3' See Materials and methods, Molecular cloning | |
| Sequence-based reagent | QuikChange primers for FUBI(GV)-eS30 | Sigma-Aldrich | 5'-GGCCGCATGCTTGGAGtTAAAGTTCATGGTTCC-3', 5'-GGAACCATGAACTTTAaCTCCAAGCATGCGGCC-3' See Materials and methods, Molecular cloning | |
| Sequence-based reagent | QuikChange primers for USP36(CA) | Sigma-Aldrich | 5'-CCACAACCTaGGCAACACCgcCTTTCTCAATGCCACC-3', 5'-GGTGGCATTGAGAAAGgcGGTGTTGCCtAGGTTGTGG-3' See Materials and methods, Molecular cloning | |
| Sequence-based reagent | QuikChange primers for USP36(NdeI) | Sigma-Aldrich | 5’- CCGTGTGCAAGAGCGTCagcGAtACaTAtGACCCCTACTTGGAC-3’, 5'-GTCCAAGTAGGGGTCaTAtGTaTCgctGACGCTCTTGCACACGG-3' See Materials and methods, Molecular cloning | |
| Sequence-based reagent | 5'ITS1 | Microsynth (Rouquette et al., 2005) DOI: 10.1038/sj.emboj.7600752 | 5'-CCTCGCCCTCCGGGCTCCGTTAATGATC-3' See Materials and methods, Northern blot analysis | |
| Sequence-based reagent | ITS2 | Microsynth (Rouquette et al., 2005) DOI: 10.1038/sj.emboj.7600752 | 5'-GCGCGACGGCGGACGACACCGCGGCGTC-3' See Materials and methods, Northern blot analysis | |
| Sequence-based reagent | Cy3-5'ITS1 | Microsynth (Rouquette et al., 2005) DOI: 10.1038/sj.emboj.7600752 | 5'-CCTCGCCCTCCGGGCTCCGTTAATGATC-3' See Materials and methods, Fluorescence in situ hybridization | |
| Sequence-based reagent | si-AAMP | Microsynth | 5’-CAGGAUGGCAGCUUGAUCCUA-3 See Materials and methods, RNA interference | |
| Sequence-based reagent | si-control | Qiagen | Cat# 1027281 | Allstars Negative Control siRNA See Materials and methods, RNA interference |
| Sequence-based reagent | si-eS4X (RPS4X) | Qiagen | 5’-CUGGAGGUGCUAACCUAGGAA-3’ See Materials and methods, RNA interference | |
| Sequence-based reagent | si-FAU (FUBI-eS30) | Qiagen | 5’-CCGGCGCUUUGUCAACGUUGU-3’ See Materials and methods, RNA interference | |
| Sequence-based reagent | si-uS19 (RPS15) | Microsynth (Rouquette et al., 2005) DOI: 10.1038/sj.emboj.7600752 | 5’-UCACCUACAAGCCCGUAAA-3’ See Materials and methods, RNA interference | |
| Sequence-based reagent | si-USP10-1 | Qiagen | 5'-UCGCUUUGGAUGGAAGUUCUA-3' See Materials and methods, RNA interference | |
| Sequence-based reagent | si-USP10-2 | Qiagen | 5’-UACGUCAACACCCAUGAUAGA-3’ See Materials and methods, RNA interference | |
| Sequence-based reagent | si-USP10-3 | Qiagen | 5’-AACACAGCUUCUGUUGACUCU-3’ See Materials and methods, RNA interference | |
| Sequence-based reagent | si-USP10-4 | Qiagen | 5’-AAGAACUAGUUCUUACUUCAA-3’ See Materials and methods, RNA interference | |
| Sequence-based reagent | si-USP36-1 | Qiagen, Microsynth | 5’-CAAGAGCGUCUCGGACACCUA-3’ See Materials and methods, RNA interference | |
| Sequence-based reagent | si-USP36-2 | Qiagen | 5’-UCCGUAUAUGUCCCAGAAUAA-3’ See Materials and methods, RNA interference | |
| Sequence-based reagent | si-USP36-3 | Qiagen | 5’-CCGCAUCGAGAUGCCAUGCAU-3’ See Materials and methods, RNA interference | |
| Sequence-based reagent | si-USP36-4 | Qiagen | 5’-UUCCUUGUGAGUAGCUCUCAA-3’ See Materials and methods, RNA interference | |
| Sequence-based reagent | si-XPO1 (CRM1) | Microsynth (Zemp et al., 2009) DOI: 10.1083/jcb.200904048 | 5’-UGUGGUGAAUUGCUUAUAC-3’ See Materials and methods, RNA interference | |
| Chemical compound, drug | cycloheximide, CHX | Sigma-Aldrich | Cat# C7698 | |
| Chemical compound, drug | Leptomycin B | LC Laboratories | Cat# L-6100 | |
| Chemical compound, drug | tetracycline, tet | Invitrogen | Cat# 550205 |
Additional files
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Supplementary file 1
Proteomic analysis of the interactome of WT and non-cleavable FUBI-eS30-StHA.
(S2-1) Spectral counts and SAINT Bayesian false discovery rates (BFDRs) of proteins identified in affinity purification mass spectrometry experiments of HASt-GFP (GFP), FUBI-eS30-StHA wild-type (WT), G73,74A (AA) or G74V (GV) performed in three independent biological replicates. (S2-2) Spectral counts of the confidently identified interactors with a BFDR < 0.01 for at least one bait were normalized to protein length (per 1000 amino acids, UniProt database) after addition of a pseudocount of 0.1. The average spectral counts identified on the individual (AA, GV) and combined (mut) non-cleavable mutants compared to WT FUBI-eS30-StHA were determined as a log2 fold change (log2FC). The enrichment of interactors on mut vs. WT was tested by ANOVA (p value and adjusted p values using FDR correction). Significantly enriched interactors (|log2FC| > one with an adjusted p value < 0.05) were categorized as deubiquitinase (DUB), 40S ribosome biogenesis factor (RBF), 60S RBF, ribosome-associated, or other enriched interactor.
- https://cdn.elifesciences.org/articles/70560/elife-70560-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/70560/elife-70560-transrepform-v2.docx
