www.sciencemag.org/cgi/content/full/336/6089/1719/DC1

Supplementary Materials for

Leucine-tRNA Initiates at CUG Start Codons for Protein Synthesis and Presentation by MHC Class I

Shelley R. Starck, Vivian Jiang, Mariana Pavon-Eternod, Sharanya Prasad, Brian McCarthy, Tao Pan, Nilabh Shastri*

*To whom correspondence should be addressed. E-mail: nshastri@berkeley.edu

Published 29 June 2012, Science 336, 1719 (2012)

DOI: 10.1126/science.1220270

This PDF file includes:

Materials and Methods Figs. S1 to S10 Tables S1 References (39–42)

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Materials and Methods

Plasmid constructs

The ATG[YL8], CTG[YL8], CCC[YL8], and TAG[YL8] DNA plasmids used for

toeprinting and/or transfections were based upon those used earlier in functional antigen presentation and toeprinting assays (7, 16). The “Excellent Kozak” CTG[YL8] was in the pcDNA1 vector (Invitrogen): 5’-G TCG ACC CTG ACC TTC AAC TAC CGG AAT CTC TAG-3’. The ATG[YL8] and CCC[YL8] constructs were identical in length and sequence to CTG[YL8] and cloned using the SalI/XbaI restriction sites from Excellent Kozak CTG[YL8] above; ATG[YL8]: 5’-G TCG ACC ATG ACC TTC AAC TAC CGG AAT CTC TAG A-3’. The TAG[YL8] construct was cloned into the CCC[YL8] plasmid DNA using the BamHI/SalI restriction sites followed by addition of Luciferase downstream from the TAG[YL8] sequence using the PCR-amplified Luciferase gene from Promega's T7 Luciferase Control Plasmid (Promega, Madison, WI, U.S.A.) and the SalI/XbaI restriction sites. ATG[GFP] was cloned into the pcDNA1 plasmid using the GFP sequence from the pIRES2-EGFP plasmid (BD Bioscience) and the BamHI/HpaI restriction sites. The CTG start codon and surrounding Kozak context sequences were modified using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The GFP plasmid DNA sequences: ATG[GFP], 5’-GCC ACC ATG G and CTG[GFP], 5’-GCC ACC CTG G. The Myc sequence used for ribosome capture 5’-AG ACG CTG GAT TTT TTT CGG GTA GTG GAA AAC CAG CAG CCC CCG CGA CG was cloned into pcDNA1 using SalI/XbaI restriction sites from the CCC[YL8] construct above (start codons underlined). Synthesis of mRNAs

Plasmid DNA (pcDNA1) was linearized with HpaI or MslI and used as templates

for transcription by T7 RNA polymerase (mMessage mMachine T7; Ambion) to yield AUG[YL8], CUG[YL8], UAG[YL8] and AUG[GFP] and CUG[GFP] mRNAs. Transcription reactions contained m7GTP or ARCA m7GTP cap analog (Ambion) to yield naturally capped mRNAs. The Poly(A) Tailing Kit (Ambion) was used to add poly(A) tails onto mRNAs for cell transfections. Primer extension ‘toeprinting’ assay of initiation complexes

The DNA oligonucleotide 5′-GTC ACA CCA CAG AAG TAA GG-3′ was used as

the reverse primer and was labeled with T4 polynucleotide kinase and [γ-32P]ATP (3000 Ci/mmol; Perkin Elmer). For Odyssey measurements of toeprint gels, the DNA oligonucleotide 5’-(Alexa750)-GTC ACA CCA CAG AAG TAA GG-3’ (Invitrogen) was used. The DNA oligonucleotide 5’-(FAM)-GTC ACA CCA CAG AAG TAA GG-3’ (Applied Biosystems) was used for fragment analysis assays. These primers are complementary to the mRNA at position +76 from the AUG/CUG start codons in the pcDNA1 vector for AUG[YL8] and CUG[YL8]. The compounds Aurin Tricarboxylic acid, Suramine, Acriflavine, Emetine, Nogalamycin, Quinacrine Dihydrochloride, and

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Verrucarin A were obtained from Sigma and the remaining compounds obtained from the NCI/DTP Open Chemical Repository http://dtp.nci.nih.gov; see Table S1 for chemical identifier numbers except Bruceantin (NSC165563); Baccharinol (NSC269756); and Bouvardin (NSC227262).

The ribosome binding reactions utilized micrococcal nuclease-treated rabbit

reticulocyte lysate (RRL) (Flexi-rabbit from Promega). Reaction mixtures were assembled on ice in a total volume of 30 L containing 50% (v/v) reticulocyte lysate or active tRNA-depleted lysate fraction (see below), 500 g/mL cycloheximide (CHX), 200 M sparsomycin (SPR), 2 mM DTT, 100 mM KCl, 0.5 mM MgOAc2 (endogenous MgOAc2 concentrations varied between 2.7 and 3.2 mM; additional MgOAc2 was added if endogenous levels fell below 2.7 mM) and any additional test compounds, and pre-incubated at 30°C for 5 minutes to allow the drugs to interact with the translational machinery. Template mRNA (0.5 g/reaction) and 16.7 M amino acids were added to allow initiation complex assembly at 30°C for 10 minutes.

The reverse transcriptase reaction was carried out in a total volume of 45 L

containing the entire ribosome binding reaction, 50 mM Tris-HCl (pH 8.3), 50 mM KCl, 10 mM MgCl2, 0.5 mM spermidine, 10 mM DTT, 500 M of each dNTPs, 3.1 pmol primer for either gel analysis or fragment analysis, and 10U Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT) (Promega). Reactions were incubated at 30°C for 35 minutes. Primer extension products were extracted by adding an equal volume of phenol:CHCl3 followed by precipitation with 1/10th the volume of 3 M NaOAc (pH 5.2) plus 2.5 volumes of EtOH. cDNAs were mixed with 40% formamide, 8 mM EDTA and heated to 95°C for 5 minutes before layering onto a 8% polyacrylamide sequencing gel. For reference, RNA sequencing ladders were generated by primer extension with dideoxynucleotides using AMV. Dried gels were visualized by either exposure on a PhosphoImager screen and analyzed using a Storm PhosphoImager (Molecular Dynamics) for 32P-primers or an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, Nebraska U.S.A) for Alexa750-primers. For fragment analysis, cDNAs were mixed with 10 μL of water, then 30 μL HiDi Formamide (Genetic Analysis Grade from Applied Biosystems Inc.). Additionally, RNA sequencing ladders were generated by primer extension with dideoxynucleotides using AMV. cDNA products were extracted by adding an equal volume of phenol:CHCl3, then an equal volume of CHCl3. Extracted products were then purified with G-25 Sephadex Microspin Columns (GE Healthcare) and combined with an equal volume of HiDi Formamide. All fragment analysis products and sequencing ladders were heated to 95°C for 3 minutes to denature, then flash cooled on wet ice for 2 minutes. Samples were then analyzed on a 96-capillary 3730xl DNA Analyzer system with a POP7 polymer and 50 cm array. Injection voltage: 1.5kV. Injection time: 15 seconds. Run temperature: 63°C (Applied Biosystems).

Cell-based translation assays

HeLa-H2-Kb cells (3 × 105 cells/well in a 6-well plate) were plated the night before

and the next day transfected with 2 g of AUG[GFP] or CUG[GFP] mRNA for 2 hours using TransMessenger Transfection Regent (Qiagen) prior to adding inhibitors for 2

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hours. GFP fluorescence was analyzed by flow cytometry with a Cytomics FC500 (Beckman Coulter) and data were analyzed with FlowJo software (TreeStar) to calculate mean fluorescence intensity (MFI). For acriflavine experiments, COS-7 cells (2 × 105/well in a 6-well plate) were plated the night before and transfected the next day with 1 g of AUG[GFP] or CUG[GFP] mRNA for 3 hours prior to adding acriflavine for 2 hours. For [35S]Met/Cys labeling, cells were pretreated with amino acid free media (containing inhibitor) for 15 minutes and then labeled with 50 Ci of [35S]Met/Cys for 15 to 30 minutes. The cells were trypsinized, lysed in buffer containing 25 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 1 mM DTT, 1% TritonX-100, 15% glycerol, 10 M leupeptin, 0.15 M aprotonin, and 0.1 mM PMSF on ice for 30 minutes. After removing cellular debris by centrifugation at 14,000 x g for 10 minutes, the cell lysate was combined with SDS gel-loading buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 0.1% bromophenol blue, 10% glycerol, 100 mM dithiotheitol), heated to 95°C for 5 minutes, and resolved using 4-10% SDS-PAGE. Protein was transferred to Hybond ECL nitrocellulose membranes (Amersham) and probed with a mouse monoclonal anti-GFP antibody (Boehringer Mannheim) and a polyclonal goat anti-actin antibody (Santa Cruz Biotechnology). Secondary antibodies were horseradish peroxidase-conjugated anti-mouse (GE Healthcare) or donkey anti-goat IgG, respectively (Santa Cruz Biotechnology). The blots were developed with the ECL system (GE Healthcare).

For HPLC experiments, COS-7 cells were plated the night before at 5 × 105

cells/well for 6-well plates. The next day, ATG[YL8] and CTG[YL8] along with H2-Kb plasmid DNAs were transfected using Fugene 6 (Roche) at 1 g per well for 6-well plates for 16 hours, according to the manufacturer’s recommendations. Cells were rinsed with 1X PBS and treated with 0.131 M citric acid, 0.066 M NaH2PO4 (pH 3.1) for 2 minutes, washed twice with 1X PBS, followed by addition of fresh media containing small molecule inhibitors with a 3 hours’ incubation. Transfected cells treated with NSC119893 were combined with untransfected cells to account for peptide supply losses as a result of significant protein synthesis inhibition. Extracts were prepared by resuspending cells in 10% acetic acid and placing in boiling water for 10 minutes. Cell debris was removed by centrifugation and filtered through a 10-kilodalton-cutoff filter (Millipore). The 10-kilodalton-cutoff filter filtrate (from cell transfections above) was then fractionated on a 2.1-mm × 250-mm C18 column (Vydac) over a gradient of 20–30% acetonitrile. Three drops per well over 48 fractions were collected in 96-well plates, dried, and analyzed with T cell hybridomas and L-cell fibroblasts expressing H2-Kb (L-Kb) as the APC. APCs were added at 5 × 104 cells/per well together with 1 × 105 BCZ103 Lac Z-inducible T cell hybridoma (39). The pMHC I induced accumulation of intracellular β-galactosidase in the hybridoma, was measured with the conversion of the substrate chlorophenol red-β-D-galactopyranoside with a 96-well plate reader at 595 nm and 655 nm as the reference wavelength (40). The T cell response is proportional to the relative amount of peptide translated from ATG[YL8] and CTG[YL8] plasmids in L-Kb cells. Synthetic LYL8 and MYL8 peptides were synthesized by D. King (University of California at Berkeley) and their structures were confirmed by mass spectrometry. Synthetic peptides and buffer alone were analyzed in identical conditions to establish their retention times and the absence of cross-contamination between samples (6).

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Trangenic mouse experiments Transgenic mice expressing the WI9*LYL9 transgene as previously described (6)

were used as the source of splenocytes and bone-marrow derived dendritic cells (BM-dendritic cells) and C57BL/6 mice were used as negative-transgenic controls. Bone-marrow cells were cultured for 5-8 days with GM-CSF (10 ng/mL) to yield BM-dendritic cells (30-40% CD11c+ cells). Splenocytes or BM-dendritic cells were incubated with 1 g/mL LPS (Sigma) for 3 hours in 96-well plates. After removing media, 1 × 105 11p9Z or BCZ103 Lac Z-inducible T cell hybridomas were added for 18 hours and analyzed as described above. For assays with NSC119893, cells were rinsed with 1X PBS and treated with 0.131 M citric acid, 0.066 M NaH2PO4 (pH 3.1) for 2 minutes, washed twice with 1X PBS, followed by addition of fresh media containing NSC119893 for 2 hours. Cells were then titrated in 96-well plates and 1 × 105 11p9Z or BCZ103 Lac Z-inducible T cell hybridomas were added. LPS injections were performed i.v. with 40 g per 200 L PBS or PBS alone. After 3 hours, mice were killed and their splenocytes harvested and subjected to red blood cell lysis (Qiagen). Peptide presentation from BM-dendritic cells or splenocytes was assessed with the T cell hybridomas as described above and normalized for cell number using an assay to measure metabolically active cells by conversion of the tetrazolium compound MTS into formazan (CellTiter 96® AQueous One Solution Cell Proliferation Assay; Promega). All mice were housed within the animal facilities at the University of California at Berkeley according to IACUC guidelines.

siRNA knock-down experiments

In 6-well plates, L-cell fibroblasts expressing H2-Kb (L-Kb) cells were transfected

with 200 pmol/well of either control, eIF2D, or eIF2A siRNAs (Santa Cruz Biotechnology) using Lipofectamine2000 (Invitrogen) for 24 hours and after a media change another 24 hours. Cells were transfected for 12-16 hours with plasmid DNAs containing the bicistronic ORF WI9*LYL8 and H2-Db to yield WI9/Db and LYL8/Kb, respectively. The siRNA/transfected cells were treated with 0.131 M citric acid, 0.066 M NaH2PO4 (pH 3.1) for 2 minutes, washed twice with 1X PBS and allowed to recover for 4 hours prior to adding the 11p9Z or BCZ103 Lac Z-inducible T cell hybridomas as described above. To confirm eIF2D or eIF2A siRNA knock-down, cells were subjected to Western blot analysis as described above with the polyclonal rabbit primary antibodies anti-eIF2D and anti-eIF2A (ProteinTech Group) and a goat anti-actin antibody (Santa Cruz Biotechnology). Secondary antibodies were horseradish peroxidase-conjugated anti-rabbit (GE Healthcare) or donkey anti-goat IgG, respectively (Santa Cruz Biotechnology). The blots were developed with the ECL system (GE Healthcare).

Ribosome initiation complex capture

Rabbit reticulocyte lysate (RRL) (Flexi-rabbit from Promega) used as the source of

ribosomes and initiation factors was pretreated with CHX and SPR as described above for the toeprinting assays, except each reaction was scaled up proportionally with 180-360 L RRL. mRNAs with long 3’-UTRs prepared from HpaI-linearized plasmid DNA

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were used in experiments described in Fig. 3, A to D. mRNAs with minimal 3’-UTRs (and no additional AUG or non-AUG start codons) prepared from MslI-linearized plasmid DNA were used in experiments described in Fig. 3, E to G and fig. S9. After adding the primer 5’-biotin-TEG-GTC ACA CCA CAG AAG TAA GG-3’ (IDT) for mRNA prepared from HpaI-linearized plasmid or 5’-biotin-TEG-CAC TAT AGA ATA GGG CCC TC-3’ (IDT) for mRNA prepared from MslI-linearized plasmid and incubation at 30˚C for 30 minutes, initiation complexes were captured on streptavidin-agarose (Novagen) in the presence of an equal volume of binding buffer (20 mM Tris-Cl (pH 8.0), 5 mM MgCl2, 100 mM NaCl, 2 mM DTT, and 8% sucrose) at RT with rotation for 1 hour. Non-specifically bound tRNA was removed by consecutive washing in binding buffer. Initiation complexes were eluted by adding 10-fold excess of an unlabeled primer and heating the reactions to 65˚C for 10 minutes. The RNA fraction was extracted by adding equal molar extraction buffer (0.4 M guanidine thiocyanate, 25 mM sodium citrate, 5 mM EDTA (pH 8.0), 0.5% N-lauryl sarcosine, 0.1 M -mercaptoethanol) and saturated phenol/CHCl3 and precipitated with 1/10th the volume of 3 M NaOAc (pH 5.5) + 2 volumes of EtOH. The RNA fraction was washed with 70% EtOH and stored at -80˚C until characterization. The RNA fraction was 3’-end-labeled using 5’-[32P]-pCp and T4 RNA ligase (Ambion). The 3’-end-labeled RNA was loaded onto a 10% UREA-polyacrylamide gel to resolve the various tRNA species. Dried gels were visualized by exposure to a PhosphoImager screen and analyzed using a Storm PhosphoImager (Molecular Dynamics). The RNA fraction was also resolved on a 1% formaldehyde gel (Ambion) and transferred to a Hybond-XL membrane (GE Healthcare). The blot was probed with full-length tRNA probes labeled with T4 polynucleotide kinase and [γ-32P]ATP (3000 Ci/mmol; Perkin Elmer) at 56˚C in hybridization buffer containing 6X SSC, 0.1% SDS, and 4X Denhardt’s solution. Blots were washed successively with 3X SSC, 0.1% SDS and 1.5X SSC, 0.1% SDS and exposed for 30-60 minutes to a PhosphoImager screen as described above. Prior to re-probing, blots were stripped with a boiling solution of 5 mM EDTA (pH 8.0), 0.1% SDS and re-exposed to a PhosphoImager screen to confirm probe removal.

Preparation of T7 transcribed tRNAs

Two partially overlapping oligos Leu-tRNA-CAG: Top – 5’-GCT AAC TAG AGA

ACC CAC TGC TTA CTG GCT TAT CGA AAT TAA TAC GAC TCA CTA TAG TCA GGA TGG CCG AGC GGT CTA AGG CGC and Bottom – 5’- TGG TGT CAG GAG TGG GAT TCG AAC CCA CGC CTC CAG GGG AGA CTG CGA CCT GAA CGC AGC GCC TTA GAC CGC TCG GCC ATC CTG AC; Leu-tRNA-UAA: Top – 5’-GCT AAC TAG AGA ACC CAC TGC TTA CTG GCT TAT GAA ATT AAT ACG ACT CAC TAT AAC CGG GAT GGC TGA GTG GTT AAG GCG and Bottom – 5’-TGG TAC CGG GAG TGG GGC TCG AAC CCA CGC GGA CAC CTG TCC ATT GGA TCT TAA GTC CAA CGC CTT AAC CAC TCA GCC ATC CCG G; Leu-tRNA-UAG: Top – 5’-GCT AAC TAG AGA ACC CAC TGC TTA CTG GCT TAT CGA AAT TAA TAC GAC TCA CTA TAG GTA GCG TGG CCG AGC GGT CTA AGG CGC and Bottom – 5’-TGG TGG CAG CGG TGG GAT TCG AAC CCA CGC CCC CGA AGA GAC TGG AGC CTA AAT CCA GCG CCT TAG ACC GCT CGG CCA CGC TAC (Top oligo contained a T7 promoter site) were annealed and subjected to a Klenow

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fill-in reaction using DNA polymerase I (Klenow fragment). The double stranded DNA was a template in a T7 transcription reaction containing (1X T7 transcription buffer (Promega), 10 mM DTT, 1U/L RNasin (Promega), 0.5 mM of each NTP, either 1.875 mM AMP or GMP, and 0.5 U/L T7 polymerase (Promega) to generate tRNA. The tRNA product was 1:1 phenol/CHCl3 extracted and purified using a 10% UREA-polyacrylamide gel. Bands corresponding to full-length tRNA were excised, crushed and tRNA was purified using Performa DTR gel filtration cartridges (Edge Biosystems) to remove salts and UREA.

tRNA-depleted RRL

Flexi-rabbit reticulocyte lysate from Promega was run through an ethanolamine

column to remove unbound tRNA exactly as described previously in (30). The resulting fractions were tested for translational activity of a Luciferase mRNA reporter (Promega) with either total calf liver or RRL tRNA according the manufacturer’s protocol (Promega). Fractions were stored at -80˚C prior to use.

tRNA Aminoacylation

To verify that T7-transcribed tRNA was aminoacylated, active tRNA-depleted

fractions (10 L) were combined with 0.5 g of tRNA and 7.5 Ci [3H]-leucine in a total volume of 15 L and incubated at 30˚C for 1 hour. To preserve the [3H]-leucine attachment to tRNA, 3 M NaOAc (pH 5.5) was added to each reaction followed by extraction with 1:1 phenol:CHCl3. To remove unincorporated [3H]-leucine, the aqueous fraction was subjected to a G-25 Sephadex spin column. The level of aminoacylation was determined by combining the RNA fraction with scintillation fluid and measuring CPM of [3H]-leucine in each reaction with a liquid scintillation counter.

tRNA Microarray

The basic features of the tRNA microarray have been described previously to

determine tRNA expression in human tissues and breast tumors (25, 41). The tRNA microarray used for this study includes 48 human nuclear tRNA probes, 22 human mitochondrial tRNA probes, and 26 other probes as specificity and background controls. Each probe is replicated 8 times on the array, for a total of 768 spots.

Sample preparation and analysis: RNA from AUG or CUG initiation complexes was

3’end-labeled with 5’-[32P]-pCp. Briefly, 5’[32P]-pCp was synthesized from cytidine-3’-monophosphate (Sigma) and [32P]-ATP (Perkin-Elmer) using T4 polynucleotide kinase (USB), then ligated to the RNA using T4 RNA ligase (USB). The 3’-end-labeled RNA was loaded onto a 10% UREA-polyacrylamide gel and the tRNA bands were gel purified. The 3’-end-labeled tRNAs thus obtained were analyzed on the tRNA microarrays.

Arrray hybridization was performed in PerfectHyb Plus Hybridization Buffer

(Sigma) for 16 hours at 60˚C on a Genomic Solutions Hyb4 station. After hybridization, the arrays were washed successively with 2X SSC, 0.1% SDS, then 0.1X SSC, and

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finally 0.1% SDS and 0.1X SSC. The arrays were spun dry, wrapped in plastic film, and exposed to phosphorimager plates (Fuji Medicals) for 10 to 30 minutes. Spot intensity was quantified using Fuji Imager software.

. tRNA suppression assays

HeLa-H2-Kb cells were plated 12 hours prior to transfection in 24-well plates.

UAG-YL8 mRNA and Leu-tRNA suppressor were transfected using Lipofecatime 2000 (Invitrogen) for 16-18 hours according to the manufacturer’s recommendations and following the previously reported technique (32). The Leu-tRNA suppressor is the same sequence as endogenous Leu-tRNA-CAG except the anticodon is mutated to 5’-CUA-3’ to recognize the UAG stop codon. The pMHC I induced accumulation of intracellular β-galactosidase in the hybridoma, was measured with the conversion of the substrate chlorophenol red-β-D-galactopyranoside with a 96-well plate reader at 595 nm and 655 nm as the reference wavelength (40).

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5’-UCGACCAUGACCUUCAACUACCGGAAUCUCUAG

5’-UCGACCCUGACCUUCAACUACCGGAAUCUCUAG

AUG-YL8

CUG-YL8

+1-3 +4

+1-3 +4

Leu Thr Phe Asn Tyr Arg Asn Leu ***

Met MYL8

LYL8

Presented byKb MHC I

anddetectedby the

BCZ103 T cell

hybridoma

mRNA

peptides

Fig. S1.

Antigenic peptide constructs, AUG-YL8 and CUG-YL8, encoding MYL8 and LYL8 peptide antigens were used for toeprinting and antigen presentation assays. MYL8 and LYL8 peptides are presented by H2-Kb MHC class I molecules and stimulate the BCZ103 T cell hybridoma.

10

0

20

40

60

80

100

120

UT NSC119893

Rela

tive G

FP

exp

ressio

n (

MF

I)

A

**NS

*AUG-GFPCUG-GFP

C

GFP

0 10 50AUG-GFP

0 10 50CUG-GFP

25kDa Acriflavine (μM)

β-actin

UT

Propidium iodide

100 101 102 103

Cell

nu

mb

er

(x10

2)

AUG-GFP

CUG-GFP

8.0 7.4

3.9 2.7

B NSC119893

% dead cells

10

5

15

0

10

5

15

0

10

5

15

020

10

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15

0100 101 102 103

Fig. S2. CUG- versus AUG-initiated translation of GFP is differentially affected by protein synthesis inhibitors. (A) HeLa-H2-Kb cells were transfected with AUG-GFP or CUG-GFP mRNA and treated with NSC119893 (50 M) for 2 hours. GFP expression was determined by flow cytometry. Mean fluorescence intensity (MFI) of GFP+ cells relative to untreated samples (mean ± S.E.M.; n = 3). Statistical significance was evaluated with the unpaired t test (NS, not significant; *P < 0.05; **P < 0.01). (B) Relative cell viability as determined by propidium iodide staining (stains dead cells only) from (A) is unchanged with NSC119893 treatment in AUG vs. CUG-GFP mRNA transfected HeLa-H2-Kb cells. (C) AUG vs. CUG-GFP mRNA translation in COS-7 cells is differentially inhibited by acriflavine treatment for 2 hours. Western analysis of COS-7 cell lysates in the presence of acriflavine is representative of three independent experiments. A 0.25:1 AUG- versus CUG-GFP sample ratio is analyzed to prevent signal saturation from AUG-GFP.

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0

20

40

60

80

100

120

untreated

NSC119893

50 μM

Acriflavine

10 μM

COS-7

(x106)

[35S]Met/

Cys label

Coomassie stain

A

75100

5037

25

150250

20

15

kDa

10

un

treate

d

NS

C119893 (5

0 μ

M)

Ac

riflavin

e (1

0 μ

M)

****

B

% P

rote

in S

yn

thesis

(of

un

treate

d)

1.2 0.94 0.62 1.2 0.94 0.62 1.2 0.94 0.62

Fig. S3.

Endogenous protein synthesis is differentially affected by protein synthesis inhibitors. (A) COS-7 cells were treated with NSC119893 or acriflavine for 3 hours during which amino acid free medium was added after 2 hours, 15 minutes followed by [35S]-Met/Cys labeling for 15 minutes. Cell lysate was prepared and resolved using 4-12% SDS-PAGE. Prior to drying, gels were stained with Coomassie dye. (B) Levels of total protein synthesis with each treatment from (A) (relative to untreated samples) were determined from three independent experiments (mean ± S.E.M). Statistical significance was evaluated with the unpaired t test (*P < 0.05; ***P < 0.001).

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10 20 30 40 50 600.00.10.20.30.40.50.6

Kb: LYL8 – BCZ103Db: WI9 – 11p9Z

Spleen

Db: WI9

Conventional

Met-initiation

Kb: LYL8

Cryptic

Leu-initiation

. . .UAGAUGUGGAUGCAUCAUAAUAUGGAUCUAAUUUAGCUGACUUUCAACUAUCGUAAUCUCUAG. . .

* M W M H H N M D L I * L T F N Y R N L *

MHC I promoter ββ-globin splice + poly(A) signalWI9 LYL8*

Conventional ORF Cryptic RF

A

B

WI9 LYL8

MHC I:

peptide

T cell

hybridoma

T c

ell

re

sp

on

se (

A595)

HPLC fraction

Fig. S4.

(A) The bicistronic transgene regulated by the H2-Kb MHC class I promoter directs expression of a conventional AUG-initiated peptide (WI9) and a cryptic CUG-initiated peptide (LYL8) in the 3’-UTR. (B) Peptides from spleen cells from transgenic mice were extracted and analyzed by reverse-phase HPLC. Each fraction was assayed with antigen presenting cells (APC) expressing Db and the T cell hybridoma 11p9Z or APCs expressing Kb and the T cell hybridoma BCZ103.

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C

020406080

100120

un

treate

d

NS

C119893 (4

0 μ μ

M)

% P

rote

in S

yn

thesis

(of

un

treate

d) ***

104 105

0.40.50.60.70.80.9

MT

S A

49

0 (

ce

ll v

iab

ilit

y)

untreatedNSC119893 20 μMNSC119893 40 μMCHX

A

BM-dendritic cells

untreated

BM-dendritic

cells (x105)

[35S]

Met/Cys

label

Coomassie stain

75100

5037

25

150250

20

15

kDa

10

B

8 4 4 8 4 4

NSC119893

40 μM

Fig. S5. Bone marrow-derived dendritic cells (BM-dendritic cells) are sensitive to protein synthesis inhibitors NSC119893 or cycloheximide (CHX). (A) Cells were exposed to mild acid (see materials and methods) and allowed to recover in the presence of NSC119893 or CHX (100 g/mL) for 2 hours. A portion of the cells were titrated and cell viability was determined by using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) with absorbance reading at A490 (Promega). (B) BM-dendritic cells are sensitive to the protein synthesis inhibitor NSC119893. Cells were exposed to mild acid (see materials and methods) and allowed to recover in the presence of NSC119893 for 2 hours with [35S]-Met/Cys added for labeling for 15 minutes. Cell lysate was prepared and resolved using 4-12% SDS-PAGE. Prior to drying, gels were stained with Coomassie. (C) Levels of total protein synthesis with each treatment from (B) (relative to untreated samples) were determined from two independent experiments (mean ± S.E.M.) Statistical significance was evaluated with the unpaired t test (***P < 0.001).

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103 104 105 106

0.10.20.30.40.50.6

103 104 105

0.1

0.2

0.3

0.4

103 104 105 1060.0

0.1

0.2

0.3

103 104 1050.0

0.1

0.2

0.3

0.4

0.5

T c

ell r

esp

on

se (

A595)

Splenocytes

UTNSC119893(10 μM)

NSC119893(20 μM)

B6:UT

UT

NSC119893 (10 μM)

NSC119893 (20 μM)

B6:UT

AT

cell

re

sp

on

se (

A595)

B

UT

NSC119893(20 μM)

B6:UT,NSC119893

BM-dendritic cells

B6:UT,NSC119893

UT

NSC119893 (20 μM)

11p9Z

AUG/methionine

WI9

BCZ103

CUG/leucine

LYL8

11p9Z

AUG/methionine

WI9

BCZ103

CUG/leucine

LYL8

Fig. S6.

CUG/leucine-initiated presentation is upregulated with limiting Met-tRNAiMet activity in

splenocytes and bone marrow-derived dendritic cells (BM-dendritic cells). Primary cells from WI9*LYL8 transgenic mice were treated with mild acid and allowed to recover for 3 hours (splenocytes) and 2 hours (BM-dendritic cells) in the presence of NSC119893. T cell hybridoma responses to either AUG/Met- (11p9Z) or CUG/Leu- (BCZ103) initiated peptides are representative of at least three independent experiments from three mice. C57BL/6 (B6) mice were used as transgene-negative controls.

15

Kb (AF6-88.5)

C

0

25

50

75

100

125

**

******

AUG

Met

CUG

Leu

LPSUT

Rela

tive

sp

len

ocyte

s f

or

½-m

ax T

cell

re

sp

on

se

D LPS

PBS

B6: PBS

104 105 1060.00.10.20.30.4

102 103 1040.00.10.20.30.40.50.6

Splenocytes

T c

ell

re

sp

on

se (

A595)

11p9Z

AUG/methionine

WI9

BCZ103

CUG/leucine

LYL8

LPS

PBS

B6: PBS

E

0

25

50

75

100

125NS

***

Rela

tive

sp

len

ocyte

s

for

½-m

ax T

cell

re

sp

on

se

AUG

Met

CUG

Leu

UT

0

10

20

30

40A

Db (28-14-8)

020

406080

100%

of

Ma

x

100 101 102 103 100 101 102 1030

2040

6080

100 unstaineduntreatedLPS (1 μg/mL)

Rela

tive

MH

C c

lass I

wit

h L

PS

(M

FI)

Db Kb

NSB

LPS

Fig. S7.

Global MHC class I expression and T cell response to LPS-activated bone marrow- derived dendritic cells (BM-dendritic cells) and splenocytes from WI9*LYL8 transgenic mice. (A) Cell surface expression of H2-Db and H2-Kb on BM-dendritic cells with LPS treatment (1 g/mL) for 3 hours was determined using flow cytometry and is representative of four independent experiments. (B) The percent mean fluorescent intensity (MFI) of MHC class I-positive cells relative to untreated samples was determined (mean ± S.E.M; n = 4). CUG/leucine-initiated LYL8 presentation detected with the BCZ103 T cell hybridoma and AUG/methionine-initiated WI9 presentation with the 11p9Z T cell hybridoma from splenocytes of WI9*LYL8 transgenic mice exposed to LPS (1 g/mL) (C) in culture or (D and E) from i.v. LPS administration (3 hours’ stimulation). (D) An individual experiment with each mouse represented by one symbol/line: two PBS- and three LPS-treated WI9*LYL8 transgenic mice is shown and is representative of two independent experiments. C57BL/6 (B6) mice were used as transgene-negative controls. (C and E) T cell hybridoma responses to either AUG/methionine- or CUG/leucine-initiated peptides are presented as number of splenocytes required for ½-max T cell hybridoma response in the presence of LPS (mean ± S.E.M.; n = 3 in (C) and n = 2 independent experiments with a sum total of 5 PBS- or 6 LPS-treated mice in (E)). Statistical significance was evaluated with the unpaired t test (NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001).

16

Am7G

Initiator

tRNA

UG

Capture on streptavidin-agarose beads and

Wash to remove non-specifically bound tRNAs

Analyze tRNA pool from initiation complexes

CHX

SPR

Elute with competitor oligo (no biotin)

CUG

Assemble ribosomes at start codons and

hybridize a biotin-linked complementary oligo

mRNA

1) [32P]-end label tRNA

2) tRNA microarray

3) Northern analysis

Fig. S8.

Ribosome capture strategy to isolate initiator tRNAs from AUG and CUG initiation complexes. Rabbit reticulocyte lysate (RRL) was used to prepare initiation complexes on in vitro transcribed mRNA (see materials and methods). A biotinylated primer complementary to sites downstream from the initiation complex was used to selectively purify ribosome-associated tRNAs which were analyzed by various methods described in the text and materials and methods.

17

A

Full-

length

ATGC

sequencing

Myc

mRNA+ _

ribosomes

CUG toeprint (+17)

Myc CUG ORF: Met/Leu-Asp-Phe . . .

BtRNA probe

(codon)

mRNA

_Myc

CUGTotal

tRNA

Meti (AUG)

Asp

(GAC/GAU)

Phe-tRNA

(UUC/UUU)

Pro (CCU)

Leu (CUG)

Leu

(CUA/CUC/CUU)

tRNA probe

(codon)

mRNA

_Myc

CUGTotal

tRNA

(+20)*

*

*

Fig. S9.

Leu-tRNA is present in translation initiation complexes isolated from the endogenous Myc CUG start codon. (A) Toeprinting on the CUG-initiated Myc isoform mRNA (only one CUG codon is present in this mRNA prepared by MslI-linearized plasmid) using rabbit reticulocyte lyate (RRL) as the source of ribosomes (see arrow, CUG toeprint). Additional larger and 1 nucleotide shorter bands representing reverse transcriptase stops (see *) with higher intensity are observed in the ribosome-containing lane (RRL) compared to the ribosome-minus lane (no RRL). These may be trapped scanning small ribosomal subunits due to mRNA secondary structure or enhanced RNA secondary structure in the presence of RRL since these stops are observed in the same location in the ribosome-minus lane. These stops do not likely reflect initiation complexes since this mRNA does not contain any other CUG (or AUG) start codons, other than the annotated Myc CUG. The identified Myc CUG toeprint at +17 nt and another toeprint at +19 nt, which would correspond to one round of elongation, suggest that initiation complexes do assemble on the annotated CUG start codon of Myc. Sequencing reactions using the same mRNA are also shown (data are representative of three independent experiments). (B) Northern blot analysis of tRNAs isolated from Myc CUG-initiation complexes from (A). tRNAs resulting from A-site occupancy and elongation are also shown (Asp and Phe). The same Northern blot was stripped/re-probed with the indicated full-length tRNA probes and each blot shown is from an equivalent PhosphoImager exposure. Data are representative of three independent experiments.

18

Fig. S10.

Leu-tRNA stimulates ribosome initiation at CUG but not AUG codons. (A) Initiation complex assembly analysis (toeprinting) using rabbit reticulocyte lysate (RRL) on AUG-YL8 and CUG-YL8 mRNAs with total RRL tRNA and in vitro transcribed Leu-tRNAs (5’-anticodon-3’ shown; 500 ng tRNA/reaction). AUG-YL8 sequencing and CUG-YL8 lanes are from a higher exposure relative to AUG-YL8 to better assess weak signals particularly for the CUG toeprints, which are 10-25% the intensity of AUG toeprints (16). Data are representative of at least four experiments. (B) % Toeprint intensity from AUG-YL8 vs. CUG-YL8 mRNAs in the presence of added tRNA (500 ng/reaction) in RRL is measured relative to the no tRNA sample after normalizing the toeprint signal relative to the full-length reverse transcriptase cDNA (mean ± S.E.M, n = 4). (C) tRNA-depleted RRL was prepared by fractionating RRL through an ethanolamine column to specifically remove unbound tRNA (see materials and methods) to test the effect of added Leu-tRNA during translation initiation. To verify the activity of the fractions prior to use in toeprinting assays, Luciferase mRNA was added to each fraction with or without added RRL tRNA (20 ng/L) and the relative Luciferase activity (RLU) was measured. Data reflect at least three independent measurements. (D) Toeprinting on AUG-YL8 and CUG-YL8 mRNAs in tRNA-depleted RRL (from C) in the presence of total RRL tRNA or in vitro transcribed Leu-tRNA-CAG (500 ng tRNA/reaction). Additional tRNA can inhibit the reverse transcriptase reaction (42) particularly in tRNA-depleted RRL. Therefore, to obtain accurate toeprint intensity the % toeprint presented is the toeprint intensity relative to full-length and relative to the no tRNA reaction (mean ± S.E.M; n = 3). Statistical significance was evaluated with the unpaired t test (*P < 0.05; **P < 0.01; ***P < 0.001).

C ****

0

50

100

150

200

250 ***AUG

no

tRNA

total

tRNA

Leu-tRNA

(CAG)

0

50

100

150

200

250

no

tRNA

total

tRNA

****

Leu-tRNA

(CAG)

CUGD

% T

oe

pri

nt

Leu-tRNA_ +_ +U

AG

CA

G

+UA

A

_ +_ +UA

G

CA

G

+0

50

100

150**

NS NS*

total tRNA+_ _ _ _0

50

100

150

*** ***

*** NS

AUG CUG

+_ _ _ _

UA

A

B

0123456789

10

3 4 5 6 7 8 9 10 11 12tRNA

tRNA-depleted

RRL fractions

Lu

cif

era

se A

cti

vit

y

(RL

U x

10

3)

+ + + +++++++fraction

AUG CUG

A T G C

sequencing

total

tRNA

Leu-tRNA

_

UA

GC

AG

+_ _

_ _ _

UA

G

CA

G

+_ _

_ _

Full-

length

toeprint

% T

oe

pri

nt

A

19

Table S1. AUG vs. CUG ribosome initiation complex sensitivity to various compounds. Toeprinting on AUG-YL8 or CUG-YL8 mRNAs using either fragment analysis or traditional toeprinting gel analysis in the presence of each compound (20 M). Results are presented as toeprint intensity relative to the untreated sample (mean ± S.E.M; n = 3). NSC compounds and Baccharinol and Bouvardin are from the NCI/DTP Open Chemical Repository (http://dtp.nci.nih.gov).

Fragment Analysis Toeprint Gel Analysis

Response Compound (20 M) AUG

Toeprint CUG

Toeprint AUG

Toeprint CUG Toeprint

AUG inhibited, CUG unaffected

Bruceantin (from 16)

60% (+19%) 100% (+20%) 54% (+1.2%) 100% (+37%)

NSC-119889 8.6% (+0.11%) 69% (+13%) 15% (+0.2%) 56% (+7.6%)

NSC-119893 55% (+18%) 82% (+5.7%) - -

NSC-119911 12% (+1.1%) 76% (+5.0%) - -

NSC-119913 11% (+1.3%) 81% (+19%) - -

NSC-119915 19% (+1.7%) 89% (+14%) - -

AUG inhibited, CUG enhanced

Aurin Tricarboxylic Acid 36% (+4.2%) 177% (+57%) 27% (+7.2%) 220% (+57%)

Suramine 30% (+5.0%) 193% (+75%) 42% (+9.8%) 130% (+17%)

CUG inhibited more than AUG

Acriflavine 51% (+9.2%) 21% (+5.2%) 67% (+3.5%) 34% (+5.5%)

Nonspecific, elongation inhibitors

Baccharinol 130% (+50%) 112% (+15%) - -

Bouvardin 79% (+15%) 91% (+3.9%) - -

Emetine (from 16)

66% (+24%) 150% (+58%) 110% (+12%) 110% (+17%)

Nogalamycin - - 80% (+25%) 100% (+0.62%)

Quinacrine

Dihydrochloride - - 100% (+45%) 100% (+35%)

Verrucarin A - - 92% (+12%) 150% (+35%)

NSC-111041 86% (+8.3%) 64% (+2.1%) 91% (+10%) 120% (+8.6%)

NSC-115183 84% (+27%) 110% (+29%) - -

NSC-119910 83% (+23%) 110% (+42%) - -

NSC-378139 72% (+3.8%) 92% (+3.0%) - -

20

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