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Supporting Information 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 gene 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 genes 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-
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