Supporting Information

Loenarz et al. 10.1073/pnas.1311750111 SI Materials and Methods RPS23B for RPS23* variant replacements (including 100-bp 5′ Yeast Strains and Growth Media. Classical methods of yeast ge- and 97-bp 3′) was cloned into the pGEM-T Easy vector (using netics were used for plasmid selection and to construct strains 5′-TCAAGATCGAGATACTATTAGTGATGG-3′ and 5′-GCT- containing disruptions; transformations used the lithium TGATGTTTGATGATTTTGAA-3′). 2OG and iron-dependent acetate/PEG method. Saccharomyces cerevisiae was grown at oxygenase domain containing 1 (OGFOD1), Tpa1p, and Rps23p 30 °C with shaking in either YPD or synthetic-defined (S.D.) variants were obtained using PCR-based site-directed mutagenesis; medium for plasmid selection; Schizosaccharomyces pombe was domain deletions were obtained by PCR with overlapping primers. grown in YES medium. Genomic alterations were confirmed Full-lengthRps23pandN-terminally truncated human using colony PCR with subsequent sequencing. Cells were cured RPS2344–143 (full-length RPS23 was found to be insoluble when + off the [PSI ] factor (the prion state of the S. cerevisiae trans- expressed in Escherichia coli) were cloned into bacterial expres- lation termination factor eRF3) using growth in the presence of sion vectors providing N-terminal GST tags. Full-length S. pombe 5 mM guanidine hydrochloride followed by plating out. Ofd1 and S. cerevisiae Tpa1pΔ2–20 were cloned into the pNIC28 Unless specified otherwise, wild-type (WT) S. cerevisiae refers vector with N-terminal His6 tag from genomic DNA (gDNA). to the haploid strain BY4742 (MATα; his3Δ1; leu2Δ0; lys2Δ0; Full-length Ostreococcus tauri otOGFOD1 (National Center for ura3Δ0) (1) used in the systematic deletion project, which is Biotechnology Information GenInfo Identifier: 116061380) was derived from the strain used in the systematic sequencing cloned into the pET-28b vector with N-terminal His6 tag from project (S288c) (2). All strains for which PSI status is denoted gDNA kindly provided by Hervé Moreau (Observatoire Océan- + + are based on haploid YJW508 ([PSI ][PIN ]; MATα; leu2-3,-112; ologique de Banyuls-sur-Mer, Banyuls-sur-Mer, France). Full- his3-11,-15; trp1-1; ura3-1; ade1-4; can1-100) (3), which is de- length human OGFOD1 was cloned into the pET-28a vector with rived from W303. The YJW508 strain was a kind gift from N-terminal His6 tag. Used and plasmid backbones: Jonathan S. Weissman (University of California, San Francisco). + WT S. pombe refers to the MCW1221 strain (h ura4-D18 leu1-32 Tpa1p, Tpa1p H159A, Tpa1p D161A, Tpa1pΔ21–268 (ΔN) his3-D1 arg3-D4), a kind gift from Matthew C. Whitby (University and Tpa1pΔ2–21 (ΔNLS), Rps23bp: pCM189 of Oxford, Oxford). Tpa1p, OGFOD1 and Tpa1pΔ260–644 (ΔC): PGK1p-CYC1t- − tpa1 refers to tpa1:kanMX4 and tpa1::his3 replacement for YEplac195 − BY4742 and YJW508 strains, respectively. Rps23a refers to Rps23bp (WT, K62T, K62R, N65A): pGEM-T Easy rps23a::kanMX4 replacement; RPS23A and RPS23B encode the Rps23bp (WT, K62T, K62R, P64G, P64A, P64S, P64T, P64C, identical and essential ribosomal 40S subunit protein S23 N65A, N65D, N65C, N65P, N65Q, N65S, N65T, N65H, (Rps23p) in S. cerevisiae. We replaced RPS23B with LEU2 in S66A): PGK1p-pRS425 − rps23a rescued with RPS23B/pCM189 plasmid, the resulting GST-RPS2344–143: pGTvL1-SGC − rps23a rps23b::leu2 RPS23B/pCM189 strain was used to assess GST-Rps23bp: pGEX-6P-1 Δ – the viability conferred by transformation with Rps23p* variant His6-Ofd1, His6-Tpa1p 2 20: pNIC28 encoding RPS23B*/pRS425 plasmids by selection on doxycycline His6-OGFOD1, His6-otOGFOD1: pET-28 followed by removal of the WT RPS23B/pCM189 plasmid using repeated 5-fluoroorotic acid (5-FOA) selection. Genomic − Dual-Luciferase Reporter Construction and Inserts. A dual-luciferase Rps23p* variants were prepared from the rps23a rps23b::leu2 reporter vector iD3000 for use in mammalian cells was generated RPS23B/pCM189 strain using PCR-mediated gene deletion from by inserting a linker region with the sense/stop codon of interest RPS23B* templates on pGEM-T Easy plasmid backbones and an ORF encoding Firefly luciferase (Fluc) (from pAC99) (7) (Promega) followed by selection with doxycycline and 5-FOA. after a Renilla luciferase (Rluc) ORF (from pCI-Renilla-WT) (8). The pAC99 β-galactosidase/Fluc expression plasmid was Red/White Color Assays Investigating Readthrough of the ADE1 kindly provided by Jean-Pierre Rousset (University Paris-Sud, Nonsense Codon (4). Cells were grown in 25% YPD medium, Orsay, France). The pCI-Renilla-WT luciferase expression a growth condition at which endogenous adenine biosynthesis can plasmid was kindly provided by Matthias W. Hentze and An- be investigated. Development of the red pigment was similar in dreas E. Kulozik (Molecular Medicine Partnership Unit group, colonies on Agar plates and liquid cultures. Liquid cultures were European Molecular Biology Laboratory and Heidelberg Uni- photographed in front of a white background. Note that for- versity, Heidelberg). To generate the dual-luciferase reporter mation of the red pigment is a radical-based polymerization, and vector pRS425-iD2000 for use in yeast, the iD3000 linker se- can thus not be used to analyze the effects of hypoxia on ADE1 quence was adapted and the dual-luciferase reporter fragment readthrough. transferred into pAC99 (7). The dual-luciferase reporter cassette was then amplified, subcloned into pGEM-T Easy, and finally Plasmids. Genes and plasmid backbones are listed below. Yeast transferred into PGK1p-pRS425 (6). The resulting pRS425-iD2000 expression vectors included the URA3-selectable pCM189 (5) vector contains a high-copy-number 2-micron origin, a LEU2 se- (centromeric; doxycycline-repressible promoter under control lection marker, a strong, glycolytic PGK1 promoter, the dual-lucif- of tetO7; CYC1 translation terminator) plasmid, the LEU2- erase reporter cassette, and a downstream CYC1 transcription selectable episomal PGK1p-pRS425 (2-micron; PGK1 promoter; terminator for mRNA stabilization (Fig. S2A). Variants of pRS425- CYC1 translation terminator) plasmid (6), and the self-con- iD2000 contain different recoding sequences between the Rluc and structed URA3-selectable PGK1p-CYC1t-YEplac195 (2-micron; FLuc ORFs (Table S1). PGK1 promoter; CYC1 translation terminator) plasmid based on YEplac195 (American Tissue Culture Collection; 87589). The Description of Lentiviral shRNA Targeting Vectors. Sequence-verified PGK1p-pRS425 plasmid (6) was a kind gift from the Jamie Cate lentiviral plasmids containing shRNA hairpins targeting the CDS laboratory (University of California, Berkeley, CA). of human OGFOD1 (NM_018233.3) under control of a human

Loenarz et al. www.pnas.org/cgi/content/short/1311750111 1of14 H6 promoter in a pLKO.1-puro backbone (9) were obtained as 10 mM MgCl2, 6 mM 2-mercaptoethanol in 20 mM Tris pH 7.5] glycerol stocks from the Sigma TRC-Hs 1.0 (human) MISSION in polyallomer tubes and centrifuged for 3.5 h at 100,000 × g library (shRNA-1: TRCN0000038904; shRNA-2: TRCN0000038905; using a Beckman Coulter Optima L-90 ultracentrifuge. The shRNA-3: TRCN0000038907; shRNA-4: TRCN0000038908). ribosomal pellet was resuspended in 0.7 mL resuspension buffer Doxycycline-inducible shRNA knockdown vectors were prepared (5 mM MgCl2, 25 mM KCl in 50 mM Tris pH 7.5) and purified for by cloning annealed oligos into a customized, modified pLKO.1- mass spectrometry (MS) as described (10). In outline, 5 μL1M puro backbone (pGINSENG, kindly provided by Bastiaan Evers, MgCl2 and0.7mLEtOHwereaddedtoprecipitatetheribosomes. Stockholm University, Stockholm). A scrambled sequence was The pellet was resuspended in 250 μL resuspension buffer; after μ μ used as a nontargeting control. Primers were diluted (2 μM of each adding 25 L1MMgCl2 followed by 550 L glacial acetic acid, primer, 10 mM Tris·HCl pH 8.5, 100 mM NaCl, 1 mM EDTA) and the solution was incubated on ice (45 min) with occasional mixing annealed (98 °C, 2 min, followed by cooling to 4 °C at a rate of by inversion. rRNA was pelleted by centrifugation (7,378 × g; 0.2 °C/s). Ligation and phosphorylation was performed using 10 min); ribosomal proteins were acetone precipitated (>2h;−20 °C), 50 ng predigested (XhoI/EcoRI) vector backbone, 1 ng an- pelleted (7,378 × g; 10 min), washed with acetone, and dried. nealed primers, 1 μL T4 polynucleotide kinase (Promega), 1 μL μ Polysome Analyses by Sucrose Gradient Centrifugation. Twelve- T4 DNA ligase (New England BioLabs) and 2 L T4 DNA ligase – buffer in a 20-μL reaction for 1 h at 37 °C. Ligation mixtures milliliter gradients [15 50% (wt/vol) sucrose in 50 mM ammonium were transformed into STBL-3 competent cells (Invitrogen) chloride, 12 mM MgCl2, 1 mM DTT, 0.1% diethylpyrocarbonate before selection on LB–carbenicillin. in 50 mM Tris acetate (pH 7.0 at 4 °C)] were prepared by layering Primer sequences used for generation of inducible OGFOD1 sucrose concentrations of decreasing density [50%, 41.25%, 32.5%, shRNA hairpins: 23.75% and 15% (wt/vol) sucrose] using intermittent freezing in liquid N2. Twenty-four hours before use, gradients were allowed to shRNA-1-Forward: 5′-TCGAGGCTGGTTTCATGGTCCAT- thaw for diffusion to establish a continuous gradient. CATTTCAAGAGAATGATGGACCATGAAACCAGCTTT- Cells were grown to an OD600 of 0.4–0.6 at 30 °C in 100 mL TTC YPD medium or an appropriate medium to maintain plasmid selection. All subsequent procedures were performed on ice or shRNA-1-Reverse: 5′-AATTGAAAAAGCTGGTTTCATG- in a cold room, using precooled equipment, unless otherwise GTCCATCATTCTCTTGAAATGATGGACCATGAAACC- stated. Cycloheximide (50 μg/mL) was added; the culture was AGCC swirled for 30 s and poured onto ∼100 mL ice. After centrifu- shRNA-2-Forward: 5′-TCGAGGCAGATTGTCAAGTCTC- gation (3,615 × g; 5 min), cells were resuspended in 20 mL lysis TTATTTCAAGAGAATAAGAGACTTGACAATCTGCTT- buffer [100 mM NaCl, 30 mM MgCl2,50μg/mL cycloheximide, TTTC 200 μg/mL heparin, 0.2% diethylpyrocarbonate in 10 mM Tris × ′ (pH 7.5 at 4 °C)] and centrifuged (3,615 g; 3 min). Cells were shRNA-2-Reverse: 5 -AATTGAAAAAGCAGATTGTCAA- resuspended in 0.2 mL lysis buffer, transferred to 1.5 mL Ep- GTCTCTTATTCTCTTGAAATAAGAGACTTGACAATC- pendorf tubes with ice-cold, baked acid-washed glass beads to TGCC ∼0.2 cm from the meniscus, and vortexed continuously (3 min). shRNA-3-Forward: 5′-TCGAGCCTTCCCAAACAGAACA- After centrifugation (295 × g; 2 min), the supernatant was GGTTTTCAAGAGAAACCTGTTCTGTTTGGGAAGGT- centrifuged (6,500 × g; 10 min), transferred to a fresh tube, and TTTTC its absorbance at 260 nm determined. Typically, solutions of 10 absorbance units (AU260) were loaded on the gradient, centrifuged shRNA-3-Reverse: 5′-AATTGAAAAACCTTCCCAAACAG- in a swinging bucket rotor (192,000 × g; 2.5 h), and analyzed using AACAGGTTTCTCTTGAAAACCTGTTCTGTTTGGGAA- a density gradient fractionator (ISCO) with UA-6 UV/Vis detector GGC (ISCO). shRNA-4-Forward: 5′-TCGAGCGGTCACTACACTTTAAT- General Cell Culture Procedures. HeLa and human embryonic kidney TCATTCAAGAGATGAATTAAAGTGTAGTGACCGTTT- 293T cell lines were cultured in DMEM, supplemented with 10% TTC (vol/vol) FBS, 1× GlutaMax, penicillin (50 U/mL), and strepto- shRNA-4-Reverse: 5′-AATTGAAAAACGGTCACTACAC- mycin (50 μg/mL). Incubations were performed at 37 °C under TTTAATTCATCTCTTGAATGAATTAAAGTGTAGTGA- a humidified, 5% CO2 atmosphere. CCGC Construction of shRNA Cell Lines. Pseudotyped replication- incompetent lentiviral transduction particles were generated Ribosomal Protein Purification. For yeast, growth medium (1 L; YPD using a third-generation lentiviral packaging mix (kindly provided or S.D. for plasmid selection) was inoculated with S. cerevisiae by Bastiaan Evers, Stockholm University, Stockholm). 293T cells – strains and grown at 30 °C to an OD600 of 2 4. Cells were har- were seeded at 50% confluence in six-well plates and left to attach vested (4,100 × g; 10 min), resuspended in lysis buffer (50 mM overnight. Cells were transfected with lentiviral transfer vector × potassium acetate, 1 mM 2-mercaptoethanol, 5 mM MgCl2,1 (4 μg), gag–pol plasmid (2 μg), rev plasmid (1 μg), and VSV-G EDTA-free cOmplete Protease Inhibitors (Roche Applied Sci- envelope plasmid (1.2 μg) using Lipofectamine 2000 (16 μL). ence) in 20 mM Tris·HCl pH 7.5), and lysed using a bead beater. Transfection complexes were replaced with fresh complete Crude lysate was centrifuged at 2,300 × g (5 min), then 20,000 × g growth medium 6 h posttransfection. Twenty-four hours after (15 min) and 37,000 × g (15 min). For human cell lines, cells transfection, the medium was replaced with high-BSA growth were washed in cold PBS before harvesting by scraping, resus- medium (500 mL DMEM, 200 mL FBS, 1× penicillin/strepto- pended in lysis buffer [100 mM NaCl, 20 mM KCl, 6 mM mycin, 1× GlutaMax). After another 24 h, the growth medium 2-mercaptoethanol, 10 mM MgCl2, 5% (wt/vol) sucrose, 1% was removed and filtered through a 0.45-μm syringe filter to af- Triton X-100, 1× EDTA-free Complete protease inhibitors in ford lentiviral stock solution. The filtrate containing active len- 20 mM Tris·HCl pH 7.5], lysed using a dounce homogenizer, tiviral particles was frozen at −80 °C in aliquots. Fresh high-FBS and centrifuged at 20,000 × g (20 min). medium was added to the producer cells allowing a second viral The cleared cell extract (10 mL) was layered over 29 mL su- harvest after another 24 h. Lentiviral work was conducted in crose buffer [25% (wt/vol) sucrose, 100 mM NaCl, 20 mM KCl, accordance with the Biosafety Considerations for Research with

Loenarz et al. www.pnas.org/cgi/content/short/1311750111 2of14 Lentiviral Vectors as set out by the Recombinant DNA Advisory machine (CS Bio CS336) using a Rink amide linker, PL-AMS Committee at the National Institutes of Health (11). resin (Polymer Laboratories), and the standard 9-fluorenylmeth- oxycarbonyl/N,N′-diisopropylcarbodiimide/N-hydroxybenzotriazole SDS/PAGE, Western Blotting, and Dot Blots. Cell lysates were strategy. Final cleavage [trifluoroacetic acid (CF3COOH):phenol: equalized for protein content before SDS/PAGE and transferred H2O:triisopropylsilane 88:5:5:2] gave peptides as C-terminal amides to nitrocellulose membranes (Hybond ECL; 0.2 μm). Membranes which were purified by preparative reversed-phase HPLC on a were blocked in 5% (wt/vol) milk/PBS/0.1% Tween-20 before Dionex Ultimate 3000 system using a Vydac 218TP C18 10- to analysis with antibodies. For dot blot analysis of ribosomes after 15-μm column (Grace Davison Discovery Sciences) and lyophilised. ultraperformance liquid chromatography (UPLC) separation, Alanine-scanning peptides were prepared using a Multipep 1 μL of individual UPLC-separated protein fractions, collected peptide synthesiser (Intavis AG Bioanalytical Instruments) on in 3-min intervals, was spotted onto nitrocellulose membranes. a Tentagel S-RAM resin, and deprotected by 2.5% triisopropylsilane/ After blocking in PBS with 0.05% Tween 20 containing 5% 97.5% CF3COOH for 3 h. HPLC-purified peptides were analyzed (wt/vol) nonfat dry milk (30 min), the membrane was incubated (90 using an Agilent 1200 series LC-MS system (6120 quadrupole MS) min) with monoclonal anti-RPS23 antibody (1:1,000; MCA3433Z; with a Waters Sunfire column. AbD Serotec) in blocking buffer. After washing and incubation Sequences of synthesized peptides were as follows (hs, Homo (20 min) with goat HRP–anti-mouse (1:1,000; A9917; Sigma- sapiens;sc,S. cerevisiae;sp,S. pombe; t3Hyp, trans-3-hydroxyprolyl): Aldrich) in blocking buffer, the washed membranes were de- veloped using ECL. hsRPS23: 51-VLEKVGVEAKQPNSAIRKCV-70 scRPS23: 53-VLEKLGIESKQPNSAIRKCV-72 LC–Tandem MS Protein Analysis. LC–tandem MS (LC-MS/MS) analysis of the digested material was initially performed on an spRPS23: 51-VVEKIGVEAKQPNSAIRKCV-70 Agilent 6520 quadrupole time-of-flight (Q-TOF) mass analyzer after separation on a 43 mm × 75 μm Zorbax 300SB-C18 5 μm hsRPS23 t3Hyp: 51-VLEKVGVEAKQxNSAIRKCV-70 chip column (Agilent) using a 23-min gradient of 5–40% solvent hsRPS23 K54A: 51-VLEAVGVEAKQPNSAIRKCV-70 B [solvent A: 2% acetonitrile, 0.1% formic acid (HCOOH); solvent B: 95% acetonitrile, 0.1% HCOOH]. Further analysis of hsRPS23 V55A: 51-VLEKAGVEAKQPNSAIRKCV-70 selected biological samples was carried out by nano-UPLC-MS/ hsRPS23 G56A: 51-VLEKVAVEAKQPNSAIRKCV-70 MS using a 75-μm inner diameter × 25 cm C18 nanoAcquity UPLC column (1.7-μm particle size; Waters Ltd.) with a 45-min hsRPS23 V57A: 51-VLEKVGAEAKQPNSAIRKCV-70 gradient of 2–40% solvent B [solvent A: 99.9% H2O, 0.1% HCOOH; solvent B: 99.9% MeCN, 0.1% HCOOH]. The Waters hsRPS23 E58A: 51-VLEKVGVAAKQPNSAIRKCV-70 nanoAcquity UPLC system (final flow rate, 250 nL/min; ∼7,000 hsRPS23 K60A: 51-VLEKVGVEAAQPNSAIRKCV-70 psi) was coupled to a Q-TOF Premier MS/MS (Waters Ltd.) run in positive-ion mode. MS analysis was performed in data- hsRPS23 Q61A: 51-VLEKVGVEAKAPNSAIRKCV-70 directed analysis mode with MS to MS/MS switching at precursor hsRPS23 P62A: 51-VLEKVGVEAKQANSAIRKCV-70 ion counts greater than 10 with a return from MS/MS to MS survey after 1 s (MS/MS collision energy is dependent on pre- hsRPS23 N63A: 51-VLEKVGVEAKQPASAIRKCV-70 cursor ion mass and charge state). All raw MS data were pro- hsRPS23 S64A: 51-VLEKVGVEAKQPNAAIRKCV-70 cessed using either the MassHunter Qualitative Analysis Version B.01.03 (Agilent) or ProteinLynx Global Server Version 2.3 hsRPS23 I66A: 51-VLEKVGVEAKQPNSAARKCV-70 (Waters Ltd.) software with deisotoping and deconvolution (converting masses with multiple charge states to m/z = 1). The hsRPS23 K68A: 51-VLEKVGVEAKQPNSAIRACV-70 mass accuracy of the raw data was corrected using Glu-fibrino- hsRPS23 C69A: 51-VLEKVGVEAKQPNSAIRKAV-70 peptide for the Waters Q-TOF and the background ion from dodecamethylcyclohexasiloxane at 445.12 Da for the Agilent Q-TOF. MS/MS spectra of the digested biological samples Protein Expression, Purification, and Coexpression Studies. All pro- (Agilent: Mascot generic format files; Waters Ltd.: Micromass teins were heterologously expressed in E. coli BL21-DE3 cells ProteinLynx files) were searched against the UniProt Knowl- and purified using Äkta FPLC systems (GE Healthcare Life edgebase/Swiss-Prot (Version 2010.08.13; 519,348 sequences) Sciences). Expression was induced by isopropyl-β-D-thio- database using Mascot Version 2.3.01 (Matrix Science) with the galactosidase (typically 0.5 mM, ∼14 h at 18 °C) before har- following parameters: peptide tolerance, 0.2 Da; 13C = 1; frag- vest. OGFOD1 and homologs were lysed (0.5 M NaCl, 10 mM · ment tolerance, 0.1 Da; missed cleavages, 2; instrument type, MgCl2 in 20 mM Tris HCl pH 7.9) by sonication and purified as electrospray ionization (ESI)-Q-TOF; fixed modification, car- N-terminally His-tagged proteins by nickel affinity and sub- bamidomethylation (C); and variable modifications, deamidation sequent size-exclusion chromatography. RPS23 and homologs (Asp, Glu), oxidation (Met, Asn, Pro), and dioxidation (Pro). All were lysed (200 mM NaCl in 50 mM Tris pH 7.5) by sonication database searches were performed on human or the corre- and purified as N-terminally GST-tagged proteins by immobi- sponding species’ entries. The interpretation and presentation of lized glutathione agarose affinity and subsequent size-exclusion MS/MS data were performed according to published guidelines chromatography. OGFOD1 and RPS23 homologs coexpressed (12). Assignments of hydroxylation on sites identified by Mascot in BL21-DE3 cells were purified by glutathione affinity chro- were verified by manual inspection. MS/MS spectra were processed matography, followed by in-solution proteolysis using sequenc- for documentation using the MassHunter Qualitative Analysis and ing-grade modified trypsin (Promega). MassLynx (Version 4.1) software for the Agilent and Waters data, respectively. Extracted ion chromatograms from the MS data were 1-[14C]-2-Oxoglutarate Decarboxylation Assays (13). OGFOD1 or generated using a mass window of ±0.1 Da. homologs were tested for their ability to stimulate decarboxylation of 1-[14C]–labeled 2-oxoglutarate (2OG) in the presence or oth- Solid-Phase Peptide Synthesis. Amino acids were from CS Bio, erwise of RPS23 peptides/proteins. Typical assay conditions com- Novabiochem, Sigma, TCI, Alfa Aesar, and AGTC Bioproducts. prised 4 mM ascorbate, 292 μM 2OG (1.25% 1-[14C]), 100 μM Peptides were prepared using a solid-phase peptide synthesis ammonium iron(II) sulfate·6H2O, 0.66 mg/mL catalase (H2O2

Loenarz et al. www.pnas.org/cgi/content/short/1311750111 3of14 scavenger), 4 μM enzyme, and 50 μM RPS23 in 50 mM Tris·HCl quenched with an equal volume of MeCN (4 °C). Peptide (pH 7.5) at 100 μL total volume. Assays were set up in three modifications were analyzed using a MALDI-TOF microMX drops, one containing enzyme (10 μL), another containing RPS23 machine in positive-ion mode using an α-cyano-4-hydroxy-cinnamic (10 μL), and the other containing reagents. A 0.5-mL Eppendorf acid matrix (1:1). MS/MS analyses used a Bruker Daltonics vial containing 200 μL hyamine hydroxide was added and the vial Ultraflex MALDI TOF/TOF machine. was sealed with a rubber septum. The reaction mixtures were incubated with shaking at 37 °C for 15 min and then quenched Amino Acid Analyses. Amino acid analyses were performed by with acetonitrile (200 μL). Reaction tubes were then kept on ice hydrolysis using hydrochloric acid, followed by ion-exchange for 20 min, before the hyamine hydroxide was removed and chromatography with post-column derivatization with ninhydrin treated with scintillant liquid for radioactive count measurement using a Biochrom 30 amino acid analyzer. Proline and hydrox- (LS6500; Beckman). Assays were performed in triplicate. yproline isomers lack free α-amino groups and were thus moni- tored using the Biochrom 30 Proline Channel (440 nm). Sample MALDI MS-Coupled in Vitro Hydroxylation Assays. Assays with processing and analysis were performed by Peter Sharratt (Pro- recombinant enzymes/substrates used similar conditions to those tein and Nucleic Acid Chemistry Facility, Department of Bio- described (14), except without 1-[14C]-2OG; reactions were chemistry, University of Cambridge, Cambridge, UK).

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Loenarz et al. www.pnas.org/cgi/content/short/1311750111 4of14 Fig. S1. Polysome gradient analysis of ribosomes from WT and modified S. cerevisiae grown in atmospheres with different oxygen concentration, and yeast genes predicted to encode for 2OG oxygenases. S. cerevisiae was grown in YPD medium at different O2 atmospheres: 21% (vol/vol) O2 (A), 3% (vol/vol) O2,(B), and 0.1% O2 (C). (D) Overlay of WT S. cerevisiae (control; in black) and a TPA1 deletion strain (in red) grown in YPD medium. (E) Overlay of WT S. cerevisiae at 0.1% O2 (in red) and a LIA1 deletion strain at 21% (vol/vol) O2 (in green) grown in YPD medium. (F) Genes predicted to likely encode for 2OG oxygenases in S. cerevisiae, with predicted functions as derived from www.yeastgenome.org.

Loenarz et al. www.pnas.org/cgi/content/short/1311750111 5of14 Fig. S2. Dual-luciferase assays investigating the effects of TPA1 deletion on aspects of translation. (A) For the analysis of translational accuracy, the sequences of interest, including those containing a stop codon, were cloned into the recoding window and the FLuc:RLuc activity ratio was detected, analogous to previously used accuracy reporters (1–3). To account for effects of the insert sequence on luciferase stability/activity or potential differences in ribosomal processivity, a reporter with sense codon control (CGA for UGA; CAA for UAA; CAG for UAG) was used. The percentage readthrough was defined as the relative FLuc/RLuc ratio of the stop codon reporter over the FLuc/RLuc ratio of the sense codon control. (B) Codon frequency (10 consecutive rare arginine codons) (4). n = 3; mean ± SD. (C) Programmed ribosomal frameshifting (Ty1 −1 site) (5). n = 3; mean ± SD. (D) Programmed ribosomal frameshifting (HIV +1 site) (6). n = 3; mean ± SD. For the sequences inserted between the Fluc and Rluc genes, see Table S1. (E) TPA1 deletion significantly alters the readthrough of BSC4 stop codons UGA, UAA, and UAG (P = 0.0027, 0.026, and 0.0068, respectively). n = 3; mean ± SD; *P < 0.05; **P < 0.01. (F) TPA1 deletion significantly alters readthrough of ADE1 stop codons UGA, UAA and UAG (P = 0.00015, 0.00006, and 0.029, respectively). n = 3; mean ± SD; *P < 0.05; ***P < 0.001. (G) Relative stop codon readthrough of tpa1− compared with WT cells; note the apparent correlation of the +4 stop codon nucleotide with the directional effect of TPA1 deletion on accuracy. For full dual-luciferase reporter insert sequences, see Table S1. Relative ratios refer to BY4742 tpa1− strains over WT controls (n = 3; mean ± SD; logarithmic y axis).

Loenarz et al. www.pnas.org/cgi/content/short/1311750111 6of14 1. Manuvakhova M, Keeling K, Bedwell DM (2000) Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system. RNA 6(7):1044–1055. 2. Bidou L, et al. (2000) Nonsense-mediated decay mutants do not affect programmed -1 frameshifting. RNA 6(7):952–961. 3. Keeling KM, et al. (2004) Leaky termination at premature stop codons antagonizes nonsense-mediated mRNA decay in S. cerevisiae. RNA 10(4):691–703. 4. Letzring DP, Dean KM, Grayhack EJ (2010) Control of translation efficiency in yeast by codon-anticodon interactions. RNA 16(12):2516–2528. 5. Belcourt MF, Farabaugh PJ (1990) Ribosomal frameshifting in the yeast retrotransposon Ty: tRNAs induce slippage on a 7 nucleotide minimal site. Cell 62(2):339–352. 6. Jacks T, et al. (1988) Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature 331(6153):280–283.

Fig. S3. Experiments showing shRNA-mediated reduction of OGFOD1 levels in human cells, and sequence alignments illustrating the extent of evolutionary conservation of OGFOD1 and its substrate RPS23. (A) Exon structure of the canonical OGFOD1 (gi: 94536835) and sequences targeted by the selected shRNA sequences. Numbering is relative to the transcriptional start site. TGA denotes the location of an upstream, in-frame stop codon forming an ORF (uORF). CDS, coding sequence; CTD, OGFOD1 C-terminal domain. (B) Western blots showing the efficiency of tested shRNA constructs targeting OGFOD1 in HeLa cells. NTC, nontargeting control (Sigma; SHC002). (C) View from a crystal structure of Tpa1p [ (PDB) ID code 3KT7] (1) into which Rps23p (yellow) from the structure of the eukaryotic ribosome (PDB ID code 3U5C) (2) was manually docked. The N- and C-terminal domains of Tpa1p are highlighted in cyan and red, respectively; protein N and C termini are indicated. Rps23p Pro-64 is located at the apex of a loop protruding from a β-strand, modeled into the active site of Tpa1p. (D) Sequence alignment of RPS23 homologs; the residue corresponding to human Pro-62 is highlighted in red. HUMAN, H. sapiens; DROME, Drosophila melanogaster; YEAST, S. cerevisiae; POMBE, S. pombe; OSTTA, O. tauri.(E) Sequence alignment of the N-terminal double-stranded beta-helix domain of OGFOD1 homologs. Residues coordinating Fe(II) (corresponding to Tpa1p His-159, Asp-161 and His-227) and 2OG (corresponding to Arg-238) are highlighted in red.

1. Kim HS, et al. (2010) Crystal structure of Tpa1 from Saccharomyces cerevisiae, a component of the messenger ribonucleoprotein complex. Nucleic Acids Res 38(6):2099–2110. 2. Ben-Shem A, et al. (2011) The structure of the eukaryotic ribosome at 3.0 Å resolution. Science 334(6062):1524–1529.

Loenarz et al. www.pnas.org/cgi/content/short/1311750111 7of14 Fig. S4. MALDI MS hydroxylation assays and 2OG turnover assays demonstrate catalytic activity of OGFOD1 and homologs; human OGFOD1 mono- hydroxylates (+16 Da) RPS23 fragments from different organisms. (A) An RPS23 peptide (51-VLEKVGVEAKQPNSAIRKCV-70; calculated 2168.5 Da) was in- cubated with the indicated reagents. OGFOD1 apparently copurifies with iron ions. OGFOD1 activity (hydroxylation of RPS23 as evidenced by +16 Da mass + shift) requires 2OG, is stimulated by Fe(II), independent of ascorbate, and inhibited by Co2 and 2,4-pyridine dicarboxylate (2,4-PDCA). (B) Whereas human OGFOD1 modified peptides with sequences corresponding to human RPS23 (51-VLEKVGVEAKQPNSAIRKCV-70, calculated 2168.5 Da; the underlined P is the site of OGFOD1-mediated hydroxylation), S. cerevisiae Rps23p (53-VLEKLGIESKQPNSAIRKCV-72, calculated 2212.6 Da), and S. pombe Rps23 (51-VVE- KIGVEAKQPNSAIRKCV-70; calculated 2168.5 Da), S. cerevisiae Tpa1p and S. pombe Ofd1 did not modify any of these peptides. Incubations contained 300 μM 2OG, 100 μM RPS23 peptide from the indicated species, and 25 μM enzyme in 50 mM Tris pH 7.5 buffer containing 100 mM NaCl, 1 mM DTT, and 4 mM ascorbate. Reactions were conducted for 1 h at 37 °C. (C) OGFOD1 hydroxylation of RPS23 peptides with systematic alanine substitutions. The percentage hydroxylation was determined using MALDI MS analysis (a representative of two repeats is shown). All variants, except RPS23 P62A, were modified by OG- FOD1; however, the N63A and S64A variants were weaker substrates. Numbers on the x axis indicate the residue position relative to Pro-62. (D) Human

OGFOD1 activity is stimulated by the addition of the RPS2351–70 peptide. OGFOD1 activity does not require ascorbate, is stimulated by Fe(II) addition, and 14 14 inhibited by the broad-spectrum 2OG oxygenase inhibitor 2,4-PDCA. Turnover of 1-[ C]-2OG to CO2 and succinate was determined (mean ± SD from three technical replicates). Assay conditions: 8 μM His-OGFOD1, 300 μM 2OG, 15 min, 37 °C. (E) Both Tpa1p and otOGFOD1 stimulate turnover of 2OG in the presence 14 14 of GST-RPS2344–143 protein, but not RPS2351–70 peptide. Turnover of 1-[ C]-2OG to CO2 and succinate was determined (mean ± SD from three technical replicates).

Loenarz et al. www.pnas.org/cgi/content/short/1311750111 8of14 Fig. S5. Amino acid analysis of the RPS2351–70 peptide incubated with human OGFOD1 identifies the catalytic product as a trans-3-hydroxyprolyl residue. (A) Chromatograms of synthetic standards of four hydroxyproline isomers. Note, whereas t3H [retention time (Rt) 7.091 min] and t4H (Rt 7.715 min) are distin- guishable, c3H (Rt 9.869 min) and c4H (Rt 9.826 min) coelute (Rt of standards can vary by ±0.05 min between analyses). (B–E) Chromatograms of hydrolysates of in vitro hydroxylation assays containing cofactors after precipitation of enzyme using 50% (vol/vol) MeCN: RPS2351–70 only (B); RPS2351–70 after treatment with OGFOD1, hydroxylation was ∼65% complete as assessed by MALDI MS (C); sample as in C, but spiked with ∼0.5 eq trans-3-hydroxyproline (D); and OGFOD1 only (E). c3H, cis-3-hydroxyproline; c4H, cis-4-hydroxyproline; t3H, trans-3-hydroxyproline; t4H, trans-4-hydroxyproline.

Loenarz et al. www.pnas.org/cgi/content/short/1311750111 9of14 Fig. S6. LC-MS/MS analyses of trypsinized GST-RPS23 coexpressed with Tpa1p/OGFOD1 homologs. (A) MS/MS spectrum demonstrating that RPS23 Pro-62 is unmodified in the absence of Tpa1p/OGFOD1 homologs (control for B and C). (B and C) MS/MS spectra indicating that RPS23 Pro-62 was either mono- or dihydroxylated (+32 Da) when coexpressed with otOGFOD1 or Ofd1 (only the dihydroxylated form is shown). (D–F) RPS23 Pro-62 was observed in both mono- and dihydroxylated forms after coexpression with Tpa1p in E. coli (only the dihydroxylated form is shown). The hydroxylation pattern was independent of which species’ RPS23 was used, as shown for S. cerevisiae Tpa1p and S. cerevisiae Rps23p (D). (G) The tryptic fragment 55-VGVEAKQPNSAIRK-68 as MH2+ ion

(predicted monoisotopic mass: 748.93 Da) is shown: (i) unmodified, (ii) monohydroxylated, and (iii) dihydroxylated. Note that Rt decreases with the increasing number of hydroxylations at Pro-62, consistent with the higher Rt for anoxic Rps23p (Fig. 1A). Also note that the RPS23 dihydroxyprolyl modification catalyzed by Tpa1p is relatively stable, including to the proteolysis procedure, providing support for dihydroxylation at prolyl C-3/C-4 positions.

Loenarz et al. www.pnas.org/cgi/content/short/1311750111 10 of 14 Fig. S7. Deconvoluted UPLC intact protein ESI-MS analysis of ribosomal proteins from different yeast strains. (A) Proteins coeluting with S. cerevisiae Rps23p after ribosomal UPLC separation (Fig. 1A) were identified as Rpl21ap (observed 18,111 Da, calculated 18,111 Da) and Rpl21bp (observed 18,145 Da, calculated − + 18,143 Da). (B) The S. cerevisiae tpa1 BY4742 strain rescued with different TPA1 or OGFOD1 plasmids and WT YJW508 [PSI ] strain. Masses correspond to Rps23p unmodified (calculated 15,906 Da) and with one and two additional oxygen atoms (15,922 and 15,938 Da, respectively). TPA1 domain deletion variants were as follows: ΔNLS, Tpa1pΔ2–21; ΔN, Tpa1pΔ21–268; and ΔC, Tpa1pΔ260–644. doxy, 100 ng/mL doxycycline. (C) Ribosomal Rps23 from S. pombe is dihydroxylated (observed 15,557.5 ± 1.2 Da; calculated 15,524 Da). All protein masses were calculated with the N-terminal methionine cleaved.

Loenarz et al. www.pnas.org/cgi/content/short/1311750111 11 of 14 − − Fig. S8. Effect of Rps23p variants on growth. (A) The viability of Rps23p* variants was assessed by RPS23*/pCM189 plasmid rescue of an rps23a rps23b strain; variants highlighted in red were found to be lethal. (B and C) Rps23a− rps23b− strains harboring plasmid-based RPS23*/pCM189 variants were grown in suspension (S.D.–URA medium) in triplicates with OD600 measurements every 10 min. The ratio of curve-fitted EC50 values (sigmoidal dose–response with variable slope, using GraphPad Prism) compared with a WT RPS23/pCM189 plasmid is expressed as percentages; values are pooled from three technical rep- licates. (D) Readthrough of the BSC4 stop codon context is significantly different for genomic Rps23p K62T and K62R variants (P = 0.0083): The Rps23p K62T and K62R variants are approximately fivefold hyperaccurate and approximately ninefold less accurate than a WT strain, respectively (P = 0.0031 and 0.0099, respectively). n = 3; mean ± SD; **P < 0.01. (E) Full photograph of suspension cultures, views of which are presented in Fig. 3C and the description thereof in the legend.

Loenarz et al. www.pnas.org/cgi/content/short/1311750111 12 of 14 Table S1. pRS425-iD2000 insert DNA sequences Name Sequence (5′ to 3′) Protein translation

BSC4 (UGA) AAAATAGTCATTATATACGTAGTGCGCTTTCATTGACAACTACATTTTT KIVIIYVVRFH*QLHFCATFGTPLH GTGCAACTTTTGGTACACCGCTACACGTATTGAAAGCGCAAA VLKAQKTIAPPAGKTH AGACTATAGCTCCTCCAGCAGGGAAAACTCAC BSC4 (CGA) AAAATAGTCATTATATACGTAGTGCGCTTTCATCGACAAC KIVIIYVVRFHRQLHFCATFGTPLHVLKA TACATTTTTGTGCAACTTTTGGTACACCGCTACACGTATTG QKTIAPPAGKTH AAAGCGCAAAAGACTATAGCTCCTCCAGCAGGGAAAACTCAC BSC4 (UAA) AAAATAGTCATTATATACGTAGTGCGCTTTCATTAACAACTA KIVIIYVVRFH*QLHFCATFGTPLHVLKA CATTTTTGTGCAACTTTTGGTACACCGCTACACGTATTG QKTIAPPAGKTH AAAGCGCAAAAGACTATAGCTCCTCCAGCAGGGAA AACTCAC BSC4 (CAA) AAAATAGTCATTATATACGT KIVIIYVVRFHQQLHFCATFGTPLHVLKAQ AGTGCGCTTTCATCAACAACTACATTTTTGTGCAA KTIAPPAGKTH CTTTTGGTACACCGCTACACGTAT TGAAAGCGCAAAAGACTATAGCTC CTCCAGCAGGGAAAACTCAC BSC4 (UAG) AAAATAGTCATTATATACG KIVIIYVVRFH*QLHFCATFGTPLHVLK TAGTGCGCTTTCATTAGCAACTACATTTTTGTGCA AQKTIAPPAGKTH ACTTTTGGTACACCGCTACACGTATT GAAAGCGCAAAAGACTATAGCTCCTC CAGCAGGGAAAACTCAC BSC4 (CAG) AAAATAGTCATTATATACGTAGTGCG KIVIIYVVRFHQQLHFCATFGTPLHVLK CTTTCATCAGCAACTACATTTTTGTGC AQKTIAPPAGKTH AACTTTTGGTACACCGCTACACGTAT TGAAAGCGCAAAAGACTATAGCT CCTCCAGCAGGGAAAACTCAC ADE1 (UGAA) CCAGACTCCTCTAGATTCTGAAACGGTGCCTCTTAT PDSSRF*NGASY ADE1 (CGAA) CCAGACTCCTCTAGATTCCGAAACGGTGCCTCTTAT PDSSRFRNGASY ADE1 (UAAA) CCAGACTCCTCTAGATTCTAAAACGGTGCCTCTTAT PDSSRF*NGASY ADE1 (CAAA) CCAGACTCCTCTAGATTCCAAAAC PDSSRFQNGASY GGTGCCTCTTAT ADE1 (UAGA) CCAGACTCCTCTAGATTCTAGAACGGTGCCTCTTAT PDSSRF*NGASY ADE1 (CAGA) CCAGACTCCTCTAGATTCCAGAACGGTGCCTCTTAT PDSSRFQNGASY ADE1 (UGAC) CCAGACTCCTCTAGATTCTGACACGGTGCCTCTTAT PDSSRF*HGASY ADE1 (CGAC) CCAGACTCCTCTAGATTCCGACACGGTGCCTCTTAT PDSSRFQHGASY Mdx TCTTTGAAAGAGCAATAAAATGGCTTCAACTAT SLKEQ*NGFNY Mdx ctrl TCTTTGAAAGAGCAACAAAATGGCTTCAACTAT SLKEQQNGFNY TMV (UAGC) ACACAATAGCAATTACAG TQ*QLQ TMV (CAGC) ACACAACAGCAATTACAG TQQQLQ TMV (UAGA) ACACAATAGAAATTACAG TQ*KLQ TMV (CAGA) ACACAACAGAAATTACAG TQQKLQ IDUA GTCCTCAGCTGGGACTAGCAGCTCAACCTCGCC VLSWD*QLNLA IDUA ctrl GTCCTCAGCTGGGACCAGCAGCTCAACCTCGCC VLSWDQQLNLA PDE2 CCACAATAGCAAGAATATCAAAAT PQ*QEYQN PDE2 ctrl CCACAACAGCAAGAATATCAAAAT PQQQEYQN 2014 ACGTGCGATTGACCGTTCGGA TCD*PFG 2014 ctrl ACGTGCGATCGACCGTTCGGA TCDRPFG rare (Arg)10 CGACGACGACGACGACGACGACGACGACGA RRRRRRRRRR

(Arg)10 ctrl AGAAGAAGAAGAAGAAGAAGAAGAAGAAGA RRRRRRRRRR HIV −1 AAGGGACAGGCTAATTTTTTAGGGAAGATCTGGCCTTCCT KGQANFLGKIWPSYKGRPGNFL*T ACAAGGGAAGGCCAGGGAA TTTTCTTTAAACGCC HIV −1 ctrl AAGGGACAGGCTAATTTCCTAAGGGAAGATCT KGQANFLREDLAFLQGKAREFSLN GGCCTTCCTACAAGGGAAGG CCAGGGAATTTTCTTTAAACGCC Ty1 +1 AATGTATCCACATCTAA NVSTSNNSPSTDNDSISKSTT TAACTCTCCCAGCACGGACAACGATTC EPIQLNNKHDLHLRPETY*IYSKSY*SF***TPW CATCAGTAAATCAACTACTGAACCGAT TPPSRFRSITNPYK TCAATTGAACAATAAGCACGACCTT CATCTTAGGCCAGAAACTTACTGAATCTACAGTAAATCATACTA ATCATTCTGATGATGAACTCCCTG GACACCTCCTTCTCGATTCAGGAGC ATCACGAACCCTTATAAGA

Loenarz et al. www.pnas.org/cgi/content/short/1311750111 13 of 14 Table S1. Cont. Name Sequence (5′ to 3′) Protein translation

Ty1 +1 ctrl AATGTATCCACATCTAATAACTCTCCCAGCAC NVSTSNNSPSTDNDSI GGACAACGATTCCATCAGTAAATCAACTAC SKSTTEPIQLNNKHDLHLRP TGAACCGATTCAATTGAACAATAAGC ETYESTVNHTNHSDDE ACGACCTTCATCTTAGGCCAGAAACTTACGAA LPGHLLLDSGASRTLIR TCTACAGTAAATCATACTAATCATTCTGATGATG AACTCCCTGGACACCTCCTTCTCGATTCA GGAGCATCACGAACCCTTATAAGA

The underlined sequences correspond to the stop codon and its respective sense codon control. Asterisks in the protein translation column symbolize the position of translation termination. ADE1, ADEnine-requiring; BSC4, S. cerevisiae bypass of stop codon 4 gene; IDUA, human alpha-L-iduronidase gene from Hurler syndrome patient; Mdx, dystrophin gene of the mdx mouse strain; PDE2, S. cerevisiae PhosphoDiEsterase 2 gene; TMV, Tobacco mosaic virus.

Loenarz et al. www.pnas.org/cgi/content/short/1311750111 14 of 14