www.sciencemag.org/content/351/6269/aad3876/suppl/DC1

Supplementary Materials for

Translation from the 5′ untranslated region shapes the integrated stress response

Shelley R. Starck,* Jordan C. Tsai, Keling Chen, Michael Shodiya, Lei Wang,

Kinnosuke Yahiro, Manuela Martins-Green, Nilabh Shastri,* Peter Walter*

* Corresponding author. E-mail: shelley@walterlab.ucsf.edu (S.R.S.); nshastri@berkeley.edu (N.S.); peter@walterlab.ucsf.edu (P.W.)

Published 29 January 2016 in Science 351, aad3876 (2016)

DOI: 10.1126/science.aad3876

This PDF file includes

Materials and Methods Figs. S1 to S15 References

2

Materials and Methods

Plasmid constructs

Human ATF4-luciferase reporter vector (pATF4.1) was prepared by PCR

amplifying human 5’ UTR ATF4-luciferase from pCAX-ATF4-FLuc (51) with forward

(5’-GTACGCGGATCCTTTCTACTTTGCCCGCCCAC-3’) and reverse (5’-

CACTAGGTCGACTTACACGGCGATCTTTCCGC-3’) primers and cloned into the

pcDNA1-based CCC[YL8] plasmid (58) using the BamHI and SalI sites. Tracer peptide

insertions (pATF4.2 – pATF4.5) and other mutations (pATF4.9) were carried out using

the QuikChange Site-Directed Mutagenesis Kit (Stratagene).

Human 5’ UTR BiP-FLAG vectors were prepared from cDNA of total HeLa-K

b

RNA (SuperScript III; Invitrogen), amplified by PCR with forward (5’-

GTACGCGGATCCGGGCTGGGGGAGGGTATATAAG-3’) and reverse (containing

FLAG sequence) (5’-

CACTAGCTCGAGCTACAACTCATCTTTTTCCTTGTCATCGTCATCCTTGTAATC

TGCTGTATCCTCTTCACCAGTTGG-3’) primers and cloned into pcDNA1 using the

BamHI and XhoI sites. Tracer peptide insertions and other mutations were carried out

using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) as described in detail

below.

Lentiviral reporter vectors (pATF4.6 and pATF4.7) were based on pSicoR (88).

Vectors contain a CMV promoter driving expression of ATF4-luciferase were sub-cloned

using an In-Fusion Cloning Kit (Clontech Laboratories). Human ATF4-luciferase

constructs were amplified by PCR, as described in detail below, and cloned into a

pMK1163 backbone linearized by PCR with forward (5’-

GAATTCCGCCCCTCTCCCTC-3’ ) and reverse (5’-

TGGGCGGGCAAAGTAGAAACTC-3’) primers.

Plasmid construct Sequences

ATF4-luciferase Plasmids; pATF4

pATF4.1: pcDNA1 ATF4-luciferase

Wild-type ATF4-luciferase (Human); parent plasmid - modified pcDNA1: CCC-

YL8 (58)

5’: BamHI and 3’: SalI (in red); uORF1 (blue); uORF2 (orange)

5’-

CCAAGCTTGGTACCGAGCTCGGATCCTTTCTACTTTGCCCGCCCACAGATGT

AGTTTTCTCTGCGCGTGTGCGTTTTCCCTCCTCCCCGCCCTCAGGGTCCACGG

CCACCATGGCGTATTAGGGGCAGCAGTGCCTGCGGCAGCATTGGCCTTTGC

AGCGGCGGCAGCAGCACCAGGCTCTGCAGCGGCAACCCCCAGCGGCTTAAG

CCATGGCGCTTCTCACGGCATTCAGCAGCAGCGTTGCTGTAACCGACAA

3

AGACACCTTCGAATTAAGCACATTCCTCGATTCCAGCAAAGCACCGCAA

CATGACCGAAATGAGCTTCCTGAGCAGCGAGGTGTTGGTGGGGGACTTG

ATGTCCCCCTTCGACCAGTCGGGTTTGGGGGCTGAAAGATCTGAAGACGC

CAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTGGAAGATGGAAC

CGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGA

ACAATTGCTTTTACAGATGCACATATCGAGGTGGACATCACTTACGCTGAGTA

CTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAAT

ACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCC

GGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTT

ATAATGAACGTGAATTGCTCAACAGTATGGGCATTTCGCAGCCTACCGTGGT

GTTCGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAGCTC

CCAATCATCCAAAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGAT

TTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAA

TACGATTTTGTGCCAGAGTCCTTCGATAGGGACAAGACAATTGCACTGATCAT

GAACTCCTCTGGATCTACTGGTCTGCCTAAAGGTGTCGCTCTGCCTCATAGAA

CTGCCTGCGTGAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATC

ATTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAAT

GTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTATA

GATTTGAAGAAGAGCTGTTTCTGAGGAGCCTTCAGGATTACAAGATTCAAAG

TGCGCTGCTGGTGCCAACCCTATTCTCCTTCTTCGCCAAAAGCACTCTGATTG

ACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGTGGCGCTCCCCTC

TCTAAGGAAGTCGGGGAAGCGGTTGCCAAGAGGTTCCATCTGCCAGGTATCA

GGCAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACACCCGA

GGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCG

AAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAAAGAGGCG

AACTGTGTGTGAGAGGTCCTATGATTATGTCCGGTTATGTAAACAATCCGGA

AGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATTCTGGAGACATA

GCTTACTGGGACGAAGACGAACACTTCTTCATCGTTGACCGCCTAAAGTCTCT

GATTAAGTACAAAGGCTATCAGGTGGCTCCCGCTGAATTGGAATCCATCTTG

CTCCAACACCCCCACATCTTCGACGCAGGTGTCGCAGGTCTTCCCGACGATG

ACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACGAT

GACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAA

AAAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACC

GGAAAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAG

GGCGGAAAGATCGCCGTGTAAGTCGACCCCCACCTTCAACTACCGGA-3’

pATF4.2: uORF1(MYL8)

Based on pATF4.1; tracer peptide MYL8 (underlined) inserted into uORF1

5’-CCACGGCCACCATGACCTTCAACTACCGGAATCTCTAGGGGCAGCA

pATF4.3: uORF2(KOVAK)

Based on pATF4.1; tracer peptide KOVAK (underlined) inserted into uORF2

5’-GCAGCAGCGTT---

AAATCGATAATCAACTTTGAACACCTCAAATTAAGCACATT

pATF4.4: uORF1(WI9)

4

Based on pATF4.1; tracer peptide WI9 (underlined) inserted into uORF1

5’-

CCACGGCCACCATGTGGATGCACCACAACATGGACCTCATATAGGGGCAG

CA

pATF4.5: uORF1(WI9)-uORF2(KOVAK)

Based on pATF4.1; contains the tracer peptides from both pATF4.4 and from pATF4.3

pATF4.6: WT ATF4-luciferase stable HeLa-Kb

Amplified insert from pATF4.1 using forward (5’- TTTCTACTTTGCCCGCCCACAG -

3’) and reverse (5’-AGAGGGGCGGAATTCTTACACGGCGATCTT-3’) primers and

inserted into pMK1163 backbone.

pATF4.7: uORF2(KOVAK) stable HeLa-Kb

Amplified insert from pATF4.3 using forward (5’- TTTCTACTTTGCCCGCCCACAG -

3’) and reverse (5’-AGAGGGGCGGAATTCTTACACGGCGATCTT-3’) primers and

inserted into pMK1163 backbone.

pATF4.8: Constitutively active ATF4-luciferase

Based on pATF4.1; T>A mutation and deleted C in uORF2 (49). This resulted in fusion

of uORF2 to the coding sequence of ATF4-luciferase.

5’-GACTTGATGACCCC-TTCGAC

BiP-FLAG Plasmids pBiP

pBiP.1: BiP-FLAG (Human); parent plasmid: pcDNA1 (Invitrogen)

5’: BamHI and 3’: XhoI (red); FLAG (cyan)

5’-

CCAAGCTTGGTACCGAGCTCGGATCCGGGCTGGGGGAGGGTATATAAGCCG

AGTAGGCGACGGTGAGGTCGACGCCGGCCAAGACAGCACAGACAGATTGAC

CTATTGGGGTGTTTCGCGAGTGTGAGAGGGAAGCGCCGCGGCCTGTATTTCT

AGACCTGCCCTTCGCCTGGTTCGTGGCGCCTTGTGACCCCGGGCCCCTGCCGC

CTGCAAGTCGGAAATTGCGCTGTGCTCCTGTGCTACGGCCTGTGGCTGGACTG

CCTGCTGCTGCCCAACTGGCTGGCAAGATGAAGCTCTCCCTGGTGGCCGCGA

TGCTGCTGCTGCTCAGCGCGGCGCGGGCCGAGGAGGAGGACAAGAAGGAGG

ACGTGGGCACGGTGGTCGGCATCGACCTGGGGACCACCTACTCCTGCGTCGG

CGTGTTCAAGAACGGCCGCGTGGAGATCATCGCCAACGATCAGGGCAACCGC

ATCACGCCGTCCTATGTCGCCTTCACTCCTGAAGGGGAACGTCTGATTGGCGA

TGCCGCCAAGAACCAGCTCACCTCCAACCCCGAGAACACGGTCTTTGACGCC

AAGCGGCTCATCGGCCGCACGTGGAATGACCCGTCTGTGCAGCAGGACATCA

AGTTCTTGCCGTTCAAGGTGGTTGAAAAGAAAACTAAACCATACATTCAAGT

TGATATTGGAGGTGGGCAAACAAAGACATTTGCTCCTGAAGAAATTTCTGCC

ATGGTTCTCACTAAAATGAAAGAAACCGCTGAGGCTTATTTGGGAAAGAAGG

TTACCCATGCAGTTGTTACTGTACCAGCCTATTTTAATGATGCCCAACGCCAA

GCAACCAAAGACGCTGGAACTATTGCTGGCCTAAATGTTATGAGGATCATCA

ACGAGCCTACGGCAGCTGCTATTGCTTATGGCCTGGATAAGAGGGAGGGGGA

GAAGAACATCCTGGTGTTTGACCTGGGTGGCGGAACCTTCGATGTGTCTCTTC

5

TCACCATTGACAATGGTGTCTTCGAAGTTGTGGCCACTAATGGAGATACTCAT

CTGGGTGGAGAAGACTTTGACCAGCGTGTCATGGAACACTTCATCAAACTGT

ACAAAAAGAAGACGGGCAAAGATGTCAGGAAAGACAATAGAGCTGTGCAGA

AACTCCGGCGCGAGGTAGAAAAGGCCAAACGGGCCCTGTCTTCTCAGCATCA

AGCAAGAATTGAAATTGAGTCCTTCTATGAAGGAGAAGACTTTTCTGAGACC

CTGACTCGGGCCAAATTTGAAGAGCTCAACATGGATCTGTTCCGGTCTACTAT

GAAGCCCGTCCAGAAAGTGTTGGAAGATTCTGATTTGAAGAAGTCTGATATT

GATGAAATTGTTCTTGTTGGTGGCTCGACTCGAATTCCAAAGATTCAGCAACT

GGTTAAAGAGTTCTTCAATGGCAAGGAACCATCCCGTGGCATAAACCCAGAT

GAAGCTGTAGCGTATGGTGCTGCTGTCCAGGCTGGTGTGCTCTCTGGTGATCA

AGATACAGGTGACCTGGTACTGCTTGATGTATGTCCCCTTACACTTGGTATTG

AAACTGTGGGAGGTGTCATGACCAAACTGATTCCAAGGAACACAGTGGTGCC

TACCAAGAAGTCTCAGATCTTTTCTACAGCTTCTGATAATCAACCAACTGTTA

CAATCAAGGTCTATGAAGGTGAAAGACCCCTGACAAAAGACAATCATCTTCT

GGGTACATTTGATCTGACTGGAATTCCTCCTGCTCCTCGTGGGGTCCCACAGA

TTGAAGTCACCTTTGAGATAGATGTGAATGGTATTCTTCGAGTGACAGCTGAA

GACAAGGGTACAGGGAACAAAAATAAGATCACAATCACCAATGACCAGAAT

CGCCTGACACCTGAAGAAATCGAAAGGATGGTTAATGATGCTGAGAAGTTTG

CTGAGGAAGACAAAAAGCTCAAGGAGCGCATTGATACTAGAAATGAGTTGG

AAAGCTATGCCTATTCTCTAAAGAATCAGATTGGAGATAAAGAAAAGCTGGG

AGGTAAACTTTCCTCTGAAGATAAGGAGACCATGGAAAAAGCTGTAGAAGA

AAAGATTGAATGGCTGGAAAGCCACCAAGATGCTGACATTGAAGACTTCAAA

GCTAAGAAGAAGGAACTGGAAGAAATTGTTCAACCAATTATCAGCAAACTCT

ATGGAAGTGCAGGCCCTCCCCCAACTGGTGAAGAGGATACAGCAGATTACA

AGGATGACGATGACAAGGAAAAAGATGAGTTGTAGCTCGAGCATGCATCT

AGAGGGCCCTA-3’

pBiP.2: BiP-FLAG(L416D)

Based on pBiP.1; Leu416Asp mutation

5’-CTGGTAGATCTTGAT

pBiP.3: -190 UUG(LYL8)

Based on pBiP.1; tracer peptide LYL8 (underlined) inserted into the -190 UUG uORF

5’-AGATTGACCTTCAACTACCGGAATCTC---TGA

pBiP.4: -190 UUG…UAG(LYL8)

Based on pBiP.3; codon mutated to a UAG stop codon

5’-AGATTGACCTTCAACTAGCGGAATCTC---TGA

pBiP.5: -61 CUG(KOVAK)

Based on pBiP.1; tracer peptide KOVAK (underlined) inserted into the -61 CUG uORF

5’-

CTGTGCTCCTGTGCTACGGCCTGTAAATCGATAATCAACTTTGAACACCTC

AAATGGCAA

pBiP.6: -61 CUG…UGA(KOVAK)

6

Based on pBiP.5; codon mutated to a UGA stop codon

5’-

CTGTGCTCCTGTGCTACGGCCTGAAAATCGATAATCAACTTTGAACACCTC

AAATGGCAA

pBiP.7: -61 CUG(KOVAK) fusion

Based on pBiP.1; tracer peptide KOVAK (underlined) inserted into the -61 CUG uORF,

removed a T to fuse -61 CUG uORF with BiP-FLAG CDS

5’-GCCTGTAAATCGATAATCAACTTTGAACACCTCAAA-GGCAAG

pBiP.8: -61 CUG. . .UGA(KOVAK) fusion

Based on pBiP.1; codon mutated to a UGA stop codon

5’-GCCTGAAAATCGATAATCAACTTTGAACACCTCAAA-GGCAAG

pBiP.9: uORF mutant

Based on pBiP.1; mutated -190 UUG, -182 UUG, -164 GUG, and -143 CUG to non-start

codons (33) and introduced additional mutations to prevent off-target uORF translation.

5’-

AGACCGACCTACCGGGGCGTTTCGCGAGTTTCAGAGGGAAGCGCCGCGGCC

CGCATTTCCAGACCTGCCC

pBiP.10: +1 ATG BiP-FLAG

Based on pBiP.1; mutated +1 ATG to GCC

pBiP.11: +24 ATG BiP-FLAG

Based on pBiP.1; mutated signal sequence +24 ATG to TTA

pBiP.12: +1 ATG;+24 ATG BiP-FLAG

Based on pBiP.1; mutated +1 ATG to GCC and +24 ATG to TTA

pBiP.13: -12 CTG BiP-FLAG

Based on pBiP.1; mutated -12 CTG to GCC

Integrated Stress Response Inducers

Subtilase cytotoxin (Mutant SubAB or SubAB) preparation: Escherichia coli

BL21(DE3) producing recombinant His-tagged subtilase cytotoxin (SubAB) or

catalytically inactivate mutant SubAS272AB (Mut SubAB) were used as the source of

toxins by purification according to the published procedure (89). Tunicamycin was

obtained from Calbiochem EMD Bioscience. Sodium Arsenite, thapsigargin,

cycloheximide ready-made solution in DMSO, anisomycin, and lipopolysaccharide from

Salmonella typhosa (LPS; L7895) were obtained from Sigma-Aldrich. ISRIB was

synthesized in house (51), polyinosinic:polycytidylic acid (Poly I:C) was purchased from

InvivoGen, and NSC119893 and bruceantin (NSC165563) were obtained from the

NCI/DTP Open Chemical Repository (http://dtp.nci.nih.gov).

7

Cell-based Expression Assays

HeLa-Kb, L-cell fibroblasts expressing H2-K

b (K

b-L cells; K89 cells) or L-cell

fibroblasts expressing H2-Db (D

b-L cells), and mouse bone-marrow-derived dendritic cell

assays (BM-dendritic cells; see preparation description below) were cultured in RPMI

containing 10% FBS (Life Technologies), 10 mM HEPES (Life Technologies), 1 mM

Sodium Pyruvate (Life Technologies), 2 mM L-glutamine (Sigma-Aldrich), 55 M beta-mercaptoethanol (Life Technologies), Penicillin-Streptomycin (Sigma-Aldrich), a human

retinal pigment epithelial (RPE-19) cell line (ARPE-19 originally from ATCC) was

cultured in DMEM:F12 (Life Technologies) containing 10% FBS (Life Technologies)

and Penicillin-Streptomycin (Sigma-Aldrich), and Human microvascular endothelial cell

line, HMVEC-1 (obtained as a gift from the Centers for Disease Control and Prevention;

Atlanta, GA) were cultured in DMEM with 10% FBS, L-glutamine, and Penicillin-

Streptomycin. Cells were usually plated in 6-well plates (60-80% confluence) 12–18 h

prior to transfection. Cells were transfected with 0.5–2 g plasmid DNA/well with

Lipofectamine 2000 (Invitrogen) for 12-18 h. For integrated stress response (ISR)

treatments, cells were either treated directly on 6-well plates or in suspension in 1.5 mL

tubes (typically 0.5–1 × 105 cells in 500 L). For [

35S]Met/Cys labeling, cells were

labeled at the end of treatment with 50 Ci of [35

S]Met/Cys/sample (EasyTag™

EXPRESS [35

S] Protein Labeling Mix; Perkin Elmer) for 5–10 min. The cells were

collected, rinsed with 1X PBS, and lysed in buffer containing 25 mM Tris-HCl (pH 8.0),

8 mM MgCl2, 1 mM DTT, 1% Triton™ X-100, 15% glycerol, 1X Halt™ Protease

Inhibitor Cocktail (Life Technologies), and 1X Halt™ Phosphatase Inhibitor Cocktail

(Life Technologies) on ice for 30 min. After removing cellular debris by centrifugation

at 14,000 x g for 10 min, 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 min, and resolved using ANY-KD SDS-PAGE

(Invitrogen) or directly combined with ONE-Glo™ Luciferase Assay Substrate

(Promega) for luminescence measurements on a luminometer. Protein was transferred to

Hybond ECL nitrocellulose membranes (Amersham) and probed with the following

antibodies in either 3% milk or 5% BSA (Sigma-Aldrich; #A7906) in PBS + 0.1%

Tween: 1:1000 mouse monoclonal anti-GFP antibody (Clones 7.1 and 13.1; Boehringer

Mannheim); 1:400 rabbit anti--actin (C-11) (Santa Cruz Biotechnology; #SC-1615);

1:1000 rabbit anti-GADPH (Abcam; #ab9485); 1:600 to 1:1000 rabbit anti-BiP

(Proteintech; #11587-1-AP); 1:1000 rabbit anti-eIF2A (Proteintech; #11233-1-AP);

1:1000 rabbit anti-PERK (Cell Signaling; #3192); 1:1000 rabbit anti-eIF2-P (S51) (Cell Signaling; #9721); 1:1000 rabbit anti-ATF4 (Santa Cruz Biotechnology; (anti-CREB)

#SC-200); and 1:1000 to 1:5000 mouse monoclonal anti-FLAG-M2 (Sigma-Aldrich;

#F1804). Secondary antibodies were horseradish peroxidase-conjugated anti-mouse or

anti-rabbit IgG, respectively (GE Healthcare Life Sciences or Promega) or horseradish

peroxidase-conjugated donkey anti-goat (Santa Cruz Biotechnology; #SC-2056). The

blots were developed with SuperSignal (Thermo Scientific) and imaged for

Chemluminescence detection using standard film or a ChemiDoc™ XRS+ Imaging

System (Bio-Rad). Immunoblots for endogenous BiP or BiP-FLAG expression were

primarily visualized using the ChemiDoc™ XRS+ Imaging System to prevent signal

saturation from the high levels of BiP present in the ER lumen of cells.

8

BiP Immunoprecipitation (IP)

HeLa-Kb cells (1×10

5–2×10

6 cells per IP) were transfected as described above.

Cells were either treated directly on the plate or aliquoted into 1.5 mL tubes for treatment

(typically 1×105 cells/500 L for suspension treatment). For radioactive IP experiments,

cells were treated for 1 h and then 50-100 Ci [35

S]Met/Cys was added directly to each

sample for an additional 1 h incubation. Cells were rinsed with 1X PBS and lysed in 150

L of RIPA buffer (25 mM HEPES-KOH (pH 7.4), 150 mM NaCl, 1% Triton™ X-100, 0.5% sodium deoxycholate (prepared fresh), 0.05% SDS, 1X Halt™ Protease Inhibitor

Cocktail, and 1X Halt™ Phosphatase Inhibitor Cocktail) alternating between incubation

on ice with 15 sec disruption by vortexing for a total of 15 min. After removing cellular

debris by centrifugation at 14,000 x g for 20 min, the cell lysate was combined with 5 g

of primary antibody (either rabbit anti-BiP; Proteintech or mouse monoclonal anti-

FLAG-M2; Sigma-Aldrich) and rotated for 1 h at 4ºC. To the cell lysate-primary antibody mixture, a 1:1 mixture of Dynabeads

® Protein-A and Dynabeads

® Protein-G was

added to each sample and rotated for 2 h at 4ºC. Beads were collected with a magnetic

and washed twice with RIPA buffer, and twice with RIPA buffer (without protease or

phosphastase inhibitors). To the samples was added SDS gel-loading buffer (50 mM

Tris-HCl (pH 6.8), 2% SDS, 0.1% bromophenol blue, 10% glycerol, 100 mM DTT) and

heated to 95°C for 5 min. Samples were resolved using ANY-KD SDS-PAGE

(Invitrogen). Gels were stained with Coomassie and dried [35

S]Met/Cys gels were

visualized by exposure to a PhosphoImager screen followed by analysis using a on a

Typhoon PhosphoImager (Molecular Dynamics) with ImageQuant software.

Tracing Translation by T cells (3T)

HeLa-Kb, D

b-L cells, or K

b-L cells were plated in 6-well plates (60-80%

confluence) 12–18 h prior to transfection. Cells were transfected with 0.5–1 g plasmid DNA (per well) using Lipofectamine 2000 (Invitrogen) for 12–18 h and titrated in 96-

well plates after treatment with ISR activators. Lac Z-inducible T cell hybridomas (1 ×

105/well) were added followed by overnight incubation at 37˚C for 16–20 h. The pMHC

I induced accumulation of intracellular β-galactosidase in the hybridoma and 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 (90).

T cell hybridomas: tracer peptide KSIINFEHLK (KOVAK) processed to

SIINFEHL (SHL8) is recognized by Kb-L cells by the B3Z hybridoma (46, 91); tracer

peptide MTFNYRNL (MYL8) or LTFNYRNL (LYL8) is recognized by Kb-L cells by

the BCZ103 hybridoma (92); tracer peptide WI9 translated from an AUG start codon to

yield MWMHHNMDLI (Met-WI9) processed to WMHHNMDLI (WI9) is recognized

by Db-L cells by the 11p9Z hybridoma (93).

For peptide extracts, cells were rinsed after treatments with 1X PBS, resuspended

in 10% acetic acid, and placed in boiling water for 10 min. Cell debris was removed by

centrifugation and filtered through a 10-kilodalton-cutoff filter (Millipore). The extract

9

was vacuum concentrated, resuspended in 1X PBS containing 25 g/mL phenol red or

RPMI (no additives), and the pH was adjusted to 7 with 0.1 N NaOH. The cell extract or

SHL8 synthetic peptide was titrated in 96-well plates and Kb-expressing cells (K

b-L cells

or HeLa-Kb) were added at 5×10

4 cells/per well together with 1×10

5 B3Z Lac Z-inducible

T cell hybridoma. The T cell response is proportional to the relative amount of peptide

translated from the uORF2(KOVAK)-ATF4-luciferase construct. Synthetic SHL8

peptide was synthesized by D. King (University of California, Berkeley) and the structure

was confirmed by mass spectrometry.

Stable Cell Generation

To produce lentivirus, HEK293T cells were transfected with standard packaging

vectors and pATF4.6 or pATF4.7 using TransIT-LT1 (Mirus). Viral supernatant was

harvested at 72 h and filtered through a 0.45 M PVDF syringe filter. HeLa-Kb were

transduced by centrifugation at 1000 x g for 2 h at 33°C in the presence of the viral

supernatant and 8 g/ml polybrene. Following centrifugation, the viral supernatant was

removed from HeLa-Kb, fresh RPMI media was added, and cells were allowed to recover

for 72 h before expansion and subsequent Luciferase and Tracer peptide assays.

siRNA knock-down experiments

In 6-well plates, 60% confluent HeLa-Kb cells were transfected with 200

pmol/well of either Control siRNA-A or human eIF2A siRNAs (Santa Cruz

Biotechnology) using Lipofectamine 2000 (Invitrogen) for 24–48 h. Cells were

transfected for 12–18 h with plasmid DNAs (0.5–1 g/well; increased signal to noise

observed with 1 g/well) containing BiP-FLAG and GFP Transfection controls: EBNA-

alpha-GFP or EBNA-delta-GFP (94). For transfection controls, cells were analyzed with

a BD LSR II flow cytometer and data were analyzed with BD FACSDiva software to calculate mean fluorescence intensity (MFI). To assess the levels of eIF2A knock-down,

cells were analyzed by immunoblot as described above with an anti-eIF2A antibody

(Proteintech). Expression from the BiP5’ UTR -190 uORF was assessed by titrating

siRNA/plasmid DNA-transfected cells with T cell hybridomas as described in the

Tracing Translation by T cells (3T) section. Full-length BiP levels were assessed by

immunoblot as described in the Cell-based Expression Assay section.

Animal work

Mouse bone-marrow-derived dendritic cells (BM-dendritic cells) were cultured

for 5–8 days with recombinant GM-CSF (10 ng/mL) (Peprotech, Inc) to yield BM-

dendritic cells. All mice were housed within the animal facilities at the University of

California at Berkeley according to IACUC guidelines.

HLA prediction

10

HLA binding prediction was assessed using three independent programs: 1)

SYFPEITHI: http://www.syfpeithi.de/ (61). This algorithm was used to assess the

known HLA peptides identified from (85) and compared to the peptides generated from

translation of the BiP 5’ UTR. 2) MHC I binding predictions were made using the IEDB

analysis resource consensus tool (87) which combines predictions from ANN, aka

NetMHC (3.4) (95, 96), SMM (97) and Comblib (98). 3) NetMHC 3.4 Server (95, 96).

mRNA transfection

pATF4.1: pcDNA1 ATF4-luciferase and pATF4.3: U2( KOVAK) plasmid DNA

was linearized with HpaI and used as templates for transcription by T7 RNA polymerase

(mMessage mMachine T7; Ambion) to yield wild-type- and uORF2(KOVAK)-ATF4-

luciferase mRNAs. Transcription reactions contained m7GTP or ARCA m

7GTP cap

analog (Ambion) to yield naturally capped mRNAs. The Poly(A) Tailing Kit (Ambion)

was used to add poly(A) tails onto mRNAs. Transcribed mRNA was purified using 1:1

phenol:chloroform extraction, followed by chloroform extraction and purification using illustra Microspin G-25 columns (GE Healthcare Life Sciences). Prior to mRNA

transfection (12–15 h), HeLa-Kb cells were plated (80% confluence) in 6-well plates.

Cells were transfected with mRNA (2 g/well) for 3 h using TransMessenger

Transfection Regent (Qiagen). For mouse bone-marrow-derived dendritic cell assays

(BM-dendritic cells), bone-marrow cells (kind gift from Soo Jung Yang, University of

California, Berkeley) were cultured for 5–8 days with recombinant GM-CSF (10 ng/mL)

(Peprotech, Inc) to yield BM-dendritic cells. BM-dendritic cells (2–4 x106 cells) were

transfected with mRNA for 3 h as described above. Transfected HeLa-Kb or BM-

dendritic cells were titrated in 96-well plates and 1 × 105 B3Z Lac Z-inducible T cell

hybridoma were added and assayed as described in the Tracing Translation by T cells

section.

Fluorescence recovery after bleach (FRAP); TAP-FRAP Assay

Mel JuSo cells stably expressing TAP1-GFP (62) were maintained in Dulbecco's

modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS

(Invitrogen), 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin.

The cells were grown in 35mm glass-bottom cell culture dishes coated with collagen

(MatTek Corp., Ashland, MA, USA) and imaged as live cells. Images were obtained

with a Zeiss LSM 710 Axio Observer Inverted confocal microscope (Carl Zeiss Inc.,

Thornwood, N.Y.) equipped with an incubation chamber set to a condition of 37°C and

5% CO2. The 488 nm Argon laser was used to provide high-intensity pulses of light for

photobleaching of GFP in a circular spot (radius = 0.82 µm) in the endoplasmic reticulum

of the cell. Prior to and directly after the laser pulse, images were obtained using a time-

lapse imaging provided by Zeiss LSM software. Recovery of GFP fluorescence was

monitored every second for 20 sec. The fluorescence intensity of unbleached spot in the

endoplasmic reticulum within the same cell was used as baseline in analysis. To block

translation, cells were pre-incubated at 37°C in the presence of 50–100 g/mL cycloheximide for 30 min. To block standard translation, cells were incubated with 25

µM NSC119893 during imaging. Quantitative analyses of fluorescence intensities were

11

performed using EMBL Frap-analysis software with Soumpasis Diffusion model. The

obtained fluorescence recovery curves were normalized to the unbleached fluorescence.

Data was determined using 8 cells per experiment and depicted as mean ± SEM.

MTFNYRNLMAY-----MAY-----MAH-----MAL-----

ATF4 uORF1 Sequence Alignment

ATF4 uORF2 Sequence Alignment

Nested uORF2(KOVAK) Tracer PeptideHumanMouse

CowChicken

10MALLTAFSSSVKSIINFEHLKLSTFLD-SSKAPQHDRNELPEQRGVGGGLDVPLRPVGFGGMALLTAFSSSVA-VTDKDTFELSTFLDSS-KAPQHDRNELPEQRGVGGGLDVPLRPVGFGGMALFTKSSSSVA-VTDKDTFELSTFLESS-KAPQHDRDELPEQRSVGGGLDVPLRPVGFGGMALFTAFSSSLA-VTDKDIFELSTFLELSTKTSQHGRDELSEQRGVRGGLRVPLRPVGFGGMAFFKAPHSSTA-VVDKKTLSLSTSFDVK--ALKPDQDEPLEQRDAVGG-VLPLQPAVFGG

uORF1(MYL8) Tracer PeptideHumanMouse

CowChicken

20 30 40 50 60

Fig. S1: Conserved amino acid sequence alignment of uORF1 of human ATF4 with uORF1

tracer peptide MTFNYRNL (called MYL8) (top) and uORF2 of human ATF4 with nested

uORF2 tracer peptide SIINFEHL (called SHL8) flanked by K residues (called KOVAK)

(bottom).

12

BCZ1

03 T

Cel

l res

pons

e (A

595)

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8 uORF1(MYL8)Kb- L cells

uORF2(MYL8)uORF2(KOVAK)uORF1(WI9)

Cell Number102101 103 104 105

uORF1

ATF4 luciferase

uORF2

tracerpeptide

uORF1

ATF4 luciferase

uORF2

tracerpeptide

Fig. S2: Simultaneous detection of uORF peptides from ATF4 using 3T during normal growth

conditions. The tracer peptide constructs uORF1(MYL8)-ATF4-luciferase, uORF2(MYL8)-

ATF4-luciferase, uORF2(KOVAK)-ATF4-luciferase, or uORF1(WI9)-ATF4-luciferase were

transfected into Kb-L-cells. The tracer peptide MYL8 presented by K

b was detected by the

BCZ103 T cell hybridoma, while neither WI9 nor KOVAK tracer peptides were detected (data

are presented as mean ± SD of two biological replicates and are representative of n = 3).

13

WT ATF4

NS

uORF1(WI9)

uORF1(MYL8)

uORF1(WI9)-uORF2(KOVAK)

uORF2(KOVAK)

Relative ATF4-luciferase to WT (RLU)0 1 2 3 4 5 6 7 8 9 10 11 12

Constitutive ATF4

*

**

uORF1

ATF4 luciferase

uORF2

tracerpeptide

uORF1

ATF4 luciferase

uORF2

tracerpeptide

uORF1

ATF4 luciferase

uORF2

tracerpeptide

uORF1

ATF4 luciferase

uORF2

tracerpeptide

uORF1

ATF4 luciferase

uORF2

uORF1 uORF2 fusion

ATF4 luciferase

Fig. S3: Expression of ATF4-luciferase from constructs with uORF tracer peptides during

normal growth conditions. ATF4-luciferase was measured from constructs with various tracer

peptide insertions or a constitutively active variant and compared to wild-type-ATF4-luciferase

expression (from Fig. 2A) with additional tracer peptide insertion constructs (mean ± SEM; n =

3-4). Statistical significance was evaluated with the unpaired t test (NS, not significant; *P <

0.05; **P < 0.01).

14

A B C

0

0.5

1

1.5

2

2.5

Mut SubAB

SubAB SubAB+ ISRIB

1 2 3 4

[35S]

Met

/Cys

Wild

-typ

e AT

F4-lu

cife

rase

Mut SubAB

Mut SubAB

Mut SubAB + IS

RIBSubAB + IS

RIB

SubAB + ISRIB

SubAB

SubAB

eIF2α-P

eIF2α-P

BiPNS

β-actin

β-actin

PERK-PPERK

ATF4*

*

Fig. S4: Endogenous and transgene expression of ATF4 from cells used for 3T. (A) HeLa-Kb

cells treated for 1 h with Mutant SubAB (0.2 μg/mL) or SubAB (0.2 μg/mL), and ISRIB (200

nM) and labeled with [35

S]-Met/Cys prior to harvesting (n = 3). HeLa-Kb cells transfected with

wildt-ATF4-luciferase plasmid were treated as in (A) except for 3 h and analyzed by (B)

immunoblot (n = 3) or (C) for luciferase activity (mean ± SEM; n = 3). Statistical significance

was evaluated with the unpaired t test (NS, not significant; *P < 0.05).

15

UT ISRIBNaAsO 2

NaAsO 2 + IS

RIB

eIF2α-P

ATF4

1h Treatment

*

[35S]

Met

/Cys

Coom

assie

Fig. S5: Endogenous expression of ATF4 is induced with NaAsO2. (A) HeLa-Kb cells were treated for 1 h with NaAsO2 (10 μM) and/or ISRIB (200 nM) and analyzed by immunoblot (n = 3) (top) or pulsed with [35S]-Met/Cys prior to harvesting (bottom). * = non-specific anti-ATF4 antibody signal.

16

A B

0.2

0.4

0.6

0.8

1.0

Cell Equivalents

0.2

0.3

0.4

0.5

B3Z

Resp

onse

(A59

5)

B3Z

Resp

onse

(A59

5)

untreatedNaAsO2 (3h)no tracer peptide

Tracer Peptide Titrationtracer peptide (SHL8)no tracer peptide

104 105Tracer Peptide (SHL8, pM)

102 103 10410-2 10-1 100 101

Fig. S6: ATF4 uORF2 tracer peptide detection after peptide extraction. (A) Peptide extracts from uORF2(KOVAK)-ATF4-luciferase transfected HeLa-Kb cells after treatment for 3 h with NaAsO2 (10 μM) were prepared using 10% acetic acid with boiling and pH adjustment with NaOH, titrated onto either untransfected HeLa-Kb cells or Kb-L cells followed by addition of B3Z T cell hybridoma (mean ± SD from two biological replicates and are representative of n = 2). (B) Peptide titration of the processed uORF2(KOVAK) peptide (SHL8) used to estimate the number of peptides per cell presented from the uORF2(KOVAK)-ATF4-luciferase reporter: 7209 ± 3833.

17

WT∆ATG(+1)

∆ATG(+24)∆ATG(+1);∆ATG(+24)

∆CTG(-12)

5’-..CAACUGGCUGGCAAGAUGAAGCUCUCCCUGGUGGCCGCGAUGCUGCUGCUGCUC5’-..CAACUGGCUGGCAAGGCCAAGCUCUCCCUGGUGGCCGCGAUGCUGCUGCUGCUC5’-..CAACUGGCUGGCAAGAUGAAGCUCUCCCUGGUGGCCGCGUUACUGCUGCUGCUC5’-..CAACUGGCUGGCAAGGCCAAGCUCUCCCUGGUGGCCGCGUUACUGCUGCUGCUC5’-..CAAGCCGCUGGCAAGAUGAAGCUCUCCCUGGUGGCCGCGAUGCUGCUGCUGCUC

-12 +1 +24

WT

HeLa-Kb RPE-19 Kb-L Cells (K89)

BiP-FLAG

GAPDH

∆ATG(+1)

∆ATG(+1);∆ATG(+24)

∆CTG(-12)

WT ∆ATG(+1)

∆ATG(+1);∆ATG(+24)

∆CTG(-12)

WT ∆ATG(+1)

∆ATG(+24)

∆ATG(+1);∆ATG(+24)

∆CTG(-12)

BiP-FLAG constructs

Fig. S7: The annotated +1 AUG of BiP is required for full-length CDS expression. Immunoblot

analysis of BiP-FLAG expression from the +1 AUG start codon and nearby in-frame codon

mutants in HeLa-Kb and primary human retinal pigmented epithelium cells (RPE-19) or K

b-L

cells (n = 2). The +24 AUG is present in the BiP signal sequence.

18

B

A C

Mut SubAB

SubAB

BiP-Endo BiP-FLAG BiP(L416D)-FLAG

Mut SubAB

SubAB

1 2 3 4 5 6

PERK-PPERK

BiP-FLAG

C terminal BiP-FLAG

*

[35S]

Met

/Cys

Coom

assie

BiP(L416D)-FLAGMut SubAB

SubABPERK-PPERK

BiP-FLAG

eIF2α-P

β-actin

1 2

Fig. S8: SubAB treatment induces the UPR. (A) SubAB-treated HeLa-Kb cells (2 h) from

BiP(L416D)-FLAG-transfection and analyzed by immunoblot (n = 3). (B) Mutant SubAB (Mut

SubAB) or SubAB treatment (2 h) followed by [35

S]-Met/Cys labeling in RPE-19 cells. (C)

SubAB cleaves endogenous and BiP-FLAG, lanes 1 – 4, analyzed by immunoblot: is full-

length BiP or C-terminal SubAB cleavage product and * is a FLAG antibody non-specific band

used as a loading control. Persistent expression of BiP(L416D)-FLAG is observed with SubAB

treatment (C, lanes 5 and 6). BiP cleavage induces the UPR as shown by activation of PERK

(PERK-P) (n = 2).

19

A

B

BiP 5’ UTR uORF (-190 UUG)

BiP 5’ UTR uORF (-61 CUG)

BiP 5’ UTR uORF (-61 CUG)with nested tracer peptide

BiP 5’ UTR uORF (-190 UUG)with nested tracer peptide

-190

L T Y W G V S R V

L T F N Y R N L

L C S C A T A C G W T A C C C P T G W N

L C S C A T A C K S I I N F E H L K W N

5’-GGG...GACAGAUUGACCUAUUGGGGUGUUUCGCGAGUGUGA

5’-...CUGUGCUCCUGUGCUACGGCCUGUGGCUGGACUGCCUGCUGCUGCCCAACUGGCUGGCAAG...

AUGAAG...

BiP CDS

AUGAAG...

5’-...CUGUGCUCCUGUGCUACGGCCUGUAAAUCGAUAAUCAACUUUGAACACCUCAAAUGGCAAG...

5’-GGG...GACAGAUUGACCUUCAACUACCGGAAUCUC---UGA

UAG

UGA

-61 +1

Fig. S9: Human BiP uORFs present in the 5’ UTR with tracer peptide insertions. (A) The LTFNYRNL tracer peptide (called LYL8) was nested in the -190 UUG uORF. An UAG stop codon (in blue) was inserted in the middle of the -190 UUG uORF and (B) the uORF2 tracer peptide SIINFEHL (SHL8) flanked by K residues (KOVAK) was nested in the -61 CUG uORF. An UGA stop codon (in blue) was inserted after the -61 CUG uORF start codon but before the tracer peptide sequence.

20

A

B

PERK-PPERK

BiP-FLAG

no plasmidDNA BiP-FLAG

-61 CUG N-extended(KOVAK)-BiP-FLAG fusion

5’-...CUGUGCUCCUGUGCUACGGCCUGUAAAUCGAUAAUCAACUUUGAACACCUCAAAGGCAAGAUGAAG...

-190 UUG (MYL8)BiP-FLAG

-61 CUG (KOVAK)BiP-FLAG fusion

β-actin

ATF4*

eIF2α-P

Mut SubABSubAB

UGA

-61 +10.5

0.4

0.3

0.2

102 103 104 105

B3Z

resp

onse

(A59

5)

Cell Number

-61 CUG N-extended(KOVAK) BiP-FLAG fusion-61 CUG...UGAno tracer peptide

Fig. S10: Tracer peptide insertion in the BiP 5’ UTR does not alter expression of the BiP CDS during steady-state and stress conditions. (A) HeLa-Kb cells transfected with BiP-FLAG tracer peptide constructs and treated with Mut SubAB/SubAB (0.2 μg/mL) for 2 h were analyzed by immunoblot (n = 2). (B) Translation of the -61 CUG uORF from -61 CUG uORF(KOVAK)-BiP-FLAG fusion transfected HeLa-Kb cells was assayed with the B3Z T cell hybridoma and compared to cells transfected with a no tracer peptide (BiP-FLAG) construct or a construct with in-frame UGA stop codon (in blue) inserted after the -61 CUG uORF start codon but before the tracer peptide (data are presented as mean ± SD of two biological replicates and are representative of n = 3).

21

A B

[35S]

Met

/Cys

cntrl siRNAMut SubABSubABISRIB

Coom

assie

[35S]

Met

/Cys

Coom

assie

eIF2A siRNA cntrlsiRNA

eIF2AsiRNA

DMSOThapsigargin

Fig. S11: eIF2A knock-down does not alter global protein synthesis. eIF2A siRNA knock-

down (48 h) in (A) mouse Kb-L cells followed by treatment with Mut SubAB/SubAB (0.2

μg/mL), and ISRIB (200 nM) and labeled with [35

S]-Met/Cys prior to harvesting (n = 2) or in (B)

HeLa-Kb cells followed by treatment with thapsigargin (1 μM) (3 h) and labeled with [

35S]-

Met/Cys prior to harvesting (n = 2).

22

Rela

tive

GFP

Pos

itive

Cel

ls

cntrlsiRNA

eIF2AsiRNA

0

20

40

60

80

100

120

Fig. S12: Transfection efficiency after control versus eIF2A siRNA knock-down is equivalent.

Transfection efficiency was determined by flow cytometry analysis of GFP-positive cells after

48h siRNA knock-down in HeLa-Kb cells followed by 16-20 h GFP plasmid (EBNA-GFP and

) transfection (mean ± SD; n = 4).

23

A

B

0

20

40

60

80

100

120

140

160

*

NS

Thapsigargin

cntrl siRNA eIF2A siRNA

cntrlsiRNA

eIF2AsiRNA

ThapsigarginPERK-PPERK

BiP-endogenous

BiP-

endo

geno

us le

vels

β-actin

ATF4*

Fig. S13: eIF2A knock-down deregulates BiP expression during normal growth condition and

ER stress. (A) Immunoblot analysis of endogenous BiP expression after 48 h eIF2A siRNA

knock-down in HeLa-Kb cells followed by treatment with thapsigargin (1 μM) (3 h). (B)

Endogenous BiP expression is enhanced after thapsigargin treatment, while eIF2A siRNA

knock-down depresses basal BiP expression and compromises enhanced expression during

thapsigargin treatment (mean ± SD; n = 3). Statistical significance was evaluated with the

unpaired t test (NS, not significant; *P < 0.05).

24

Human BiP 5’ UTR uORF (-190 UUG)

MH

C I e

pito

pe sc

ore

HLAB*27:05

Known HLAepitopes

-61 CUG peptide 27-35G R D A A A A Q R

otherHLA

HLAB*27:05

LTYWGVSRVA

D

0

5

10

15

20

25

30

******

B

E

0

5

10

15

20

25

Known HLAEpitopes

MH

C I e

pito

pe sc

ore

HLAB*15:16

otherHLA

HLA A*02:01 HLA B*15:16

-190 UUG peptideL T Y W G V S R V

HLAA*02:01

Human BiP 5’ UTR uORF (-61 CUG)

LCSCATACGWTACCCPTGWQDEA

LPGGRDAAAAQRGAGRGGGQEGGRGHGGRHRPGDHLLLRRRVQERP

RGDHRQRSGQPHHAVLCRLHS

C

Peptide

LTYWGVSRV 0.567 108320.678

HLA-A*02:01HLA-A*02:01

WBSBMTYWGVSRV

logscore Affinity (nM) Bind Level Allele

Fig. S14: Translation of the BiP 5’-UTR is predicted to generate human MHC I peptides (HLA epitopes). (A and B) HLA epitope prediction from the BiP 5’ UTR -190 UUG uORF was determined using SYFPEITHI. The predicted -190 UUG peptide epitope (LTYWGVSRV) score was compared to the score for all HLA (except HLA A*02:01 and HLA B*15:16) and to scores for known HLA A*02:01 and HLA B*15:16 epitopes identified from Kowalewski et al. (C and D) HLA epitope prediction from the BiP 5’ UTR -61 CUG uORF was determined using SYFPEITHI. The predicted -61 CUG peptide epitope (GRDAAAAQR, underlined) score was compared to the score for all HLA (except HLA B*27:05) and to scores for known HLA B*27:05 epitopes identified from Kowalewski et al. (E) HLA epitope prediction from the BiP 5’ UTR -190 UUG uORF (LTYWGVSRV or MTYWGVSRV) was determined using the NetMHC 3.4 Server.

25

[35S]

Met

/Cys

Coom

assie

DMSOBruce

antin

NSC119893

CHX

BiP

A B

GAPDH

Thapsigargin

DMSOAniso

mycin

NSC119893

Fig. S15: BiP expression is unaltered during limiting levels of eIF2•GTP•Met•tRNAi

Met. (A)

Total protein synthesis in HeLa-Kb cells were treated for 8h with the protein synthesis inhibitors

bruceantin (100 nM), NSC119893 (50 μM; replenished every 2h), and cycloheximide (CHX) (100 μg/mL) and pulse labeled with [35S]-Met/Cys prior to harvesting (n = 3). (B) Confluent endothelial human microvascular endothelial cells (HMVEC-1) were treated for 4 h with thapsigargin (4 μM), anisomycin (50 μM), NSC119893 (50 μM; replenished after 2h), or DMSO and analyzed by immunoblot (n = 3).

26

27

REFERENCES AND NOTES

1. P. Walter, D. Ron, The unfolded protein response: From stress pathway to homeostatic regulation. Science 334, 1081–1086 (2011). Medline doi:10.1126/science.1209038

2. Y. Shi, K. M. Vattem, R. Sood, J. An, J. Liang, L. Stramm, R. C. Wek, Identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in translational control. Mol. Cell. Biol. 18, 7499–7509 (1998). Medline doi:10.1128/MCB.18.12.7499

3. H. P. Harding, Y. Zhang, D. Ron, Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397, 271–274 (1999). Medline doi:10.1038/16729

4. J. Galabru, M. G. Katze, N. Robert, A. G. Hovanessian, The binding of double-stranded RNA and adenovirus VAI RNA to the interferon-induced protein kinase. Eur. J. Biochem. 178, 581–589 (1989). Medline doi:10.1111/j.1432-1033.1989.tb14485.x

5. E. Meurs, K. Chong, J. Galabru, N. S. Thomas, I. M. Kerr, B. R. Williams, A. G. Hovanessian, Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon. Cell 62, 379–390 (1990). Medline doi:10.1016/0092-8674(90)90374-N

6. J. J. Berlanga, J. Santoyo, C. De Haro, Characterization of a mammalian homolog of the GCN2 eukaryotic initiation factor 2alpha kinase. Eur. J. Biochem. 265, 754–762 (1999). Medline doi:10.1046/j.1432-1327.1999.00780.x

7. L. Lu, A. P. Han, J. J. Chen, Translation initiation control by heme-regulated eukaryotic initiation factor 2alpha kinase in erythroid cells under cytoplasmic stresses. Mol. Cell. Biol. 21, 7971–7980 (2001). Medline doi:10.1128/MCB.21.23.7971-7980.2001

8. R. J. Jackson, C. U. Hellen, T. V. Pestova, The mechanism of eukaryotic translation initiation and principles of its regulation. Nat. Rev. Mol. Cell Biol. 11, 113–127 (2010). Medline doi:10.1038/nrm2838

9. T. W. Hai, F. Liu, W. J. Coukos, M. R. Green, Transcription factor ATF cDNA clones: An extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers. Genes Dev. 3 (12B), 2083–2090 (1989). Medline doi:10.1101/gad.3.12b.2083

10. P. D. Lu, H. P. Harding, D. Ron, Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response. J. Cell Biol. 167, 27–33 (2004). Medline doi:10.1083/jcb.200408003

11. K. M. Vattem, R. C. Wek, Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 101, 11269–11274 (2004). Medline doi:10.1073/pnas.0400541101

12. A. J. Fornace Jr., I. Alamo Jr., M. C. Hollander, DNA damage-inducible transcripts in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 85, 8800–8804 (1988). Medline doi:10.1073/pnas.85.23.8800

28

13. D. Ron, J. F. Habener, CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription. Genes Dev. 6, 439–453 (1992). Medline doi:10.1101/gad.6.3.439

14. J. Han, S. H. Back, J. Hur, Y. H. Lin, R. Gildersleeve, J. Shan, C. L. Yuan, D. Krokowski, S. Wang, M. Hatzoglou, M. S. Kilberg, M. A. Sartor, R. J. Kaufman, ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death. Nat. Cell Biol. 15, 481–490 (2013). Medline doi:10.1038/ncb2738

15. A. G. Hinnebusch, The scanning mechanism of eukaryotic translation initiation. Annu. Rev. Biochem. 83, 779–812 (2014). Medline doi:10.1146/annurev-biochem-060713-035802

16. G. Joslin, W. Hafeez, D. H. Perlmutter, Expression of stress proteins in human mononuclear phagocytes. J. Immunol. 147, 1614–1620 (1991). Medline

17. R. Panniers, Translational control during heat shock. Biochimie 76, 737–747 (1994). Medline doi:10.1016/0300-9084(94)90078-7

18. K. Richter, M. Haslbeck, J. Buchner, The heat shock response: Life on the verge of death. Mol. Cell 40, 253–266 (2010). Medline doi:10.1016/j.molcel.2010.10.006

19. S. D. Der, A. S. Lau, Involvement of the double-stranded-RNA-dependent kinase PKR in interferon expression and interferon-mediated antiviral activity. Proc. Natl. Acad. Sci. U.S.A. 92, 8841–8845 (1995). Medline doi:10.1073/pnas.92.19.8841

20. F. D. Gilfoy, P. W. Mason, West Nile virus-induced interferon production is mediated by the double-stranded RNA-dependent protein kinase PKR. J. Virol. 81, 11148–11158 (2007). Medline doi:10.1128/JVI.00446-07

21. L.-C. Hsu, J. M. Park, K. Zhang, J.-L. Luo, S. Maeda, R. J. Kaufman, L. Eckmann, D. G. Guiney, M. Karin, The protein kinase PKR is required for macrophage apoptosis after activation of Toll-like receptor 4. Nature 428, 341–345 (2004). Medline doi:10.1038/nature02405

22. R. P. Shiu, J. Pouyssegur, I. Pastan, Glucose depletion accounts for the induction of two transformation-sensitive membrane proteins[ ]in Rous sarcoma virus-transformed chick embryo fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 74, 3840–3844 (1977). Medline doi:10.1073/pnas.74.9.3840

23. I. G. Haas, M. Wabl, Immunoglobulin heavy chain binding protein. Nature 306, 387–389 (1983). Medline doi:10.1038/306387a0

24. S. Munro, H. R. Pelham, An Hsp70-like protein in the ER: Identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell 46, 291–300 (1986). Medline doi:10.1016/0092-8674(86)90746-4

25. J. Li, A. S. Lee, Stress induction of GRP78/BiP and its role in cancer. Curr. Mol. Med. 6, 45–54 (2006). Medline doi:10.2174/156652406775574523

26. M. S. Gorbatyuk, O. S. Gorbatyuk, The molecular chaperone GRP78/BiP as a therapeutic target for neurodegenerative disorders: A mini review. J. Genet. Syndr. Gene Ther. 4, 128 (2013). Medline doi:10.4172/2157-7412.1000128

29

27. L. Booth, J. L. Roberts, D. R. Cash, S. Tavallai, S. Jean, A. Fidanza, T. Cruz-Luna, P. Siembiba, K. A. Cycon, C. N. Cornelissen, P. Dent, GRP78/BiP/HSPA5/Dna K is a universal therapeutic target for human disease. J. Cell. Physiol. 230, 1661–1676 (2015). Medline doi:10.1002/jcp.24919

28. C. U. Hellen, P. Sarnow, Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev. 15, 1593–1612 (2001). Medline doi:10.1101/gad.891101

29. J. Zhou, J. Wan, X. Gao, X. Zhang, S. R. Jaffrey, S. B. Qian, Dynamic m(6)A mRNA methylation directs translational control of heat shock response. Nature 526, 591–594 (2015). Medline doi:10.1038/nature15377

30. S. E. Calvo, D. J. Pagliarini, V. K. Mootha, Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans. Proc. Natl. Acad. Sci. U.S.A. 106, 7507–7512 (2009). Medline doi:10.1073/pnas.0810916106

31. A. M. Resch, A. Y. Ogurtsov, I. B. Rogozin, S. A. Shabalina, E. V. Koonin, Evolution of alternative and constitutive regions of mammalian 5′UTRs. BMC Genomics 10, 162 (2009). Medline doi:10.1186/1471-2164-10-162

32. N. T. Ingolia, L. F. Lareau, J. S. Weissman, Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147, 789–802 (2011). Medline doi:10.1016/j.cell.2011.10.002

33. S. Lee, B. Liu, S. Lee, S. X. Huang, B. Shen, S. B. Qian, Global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution. Proc. Natl. Acad. Sci. U.S.A. 109, E2424–E2432 (2012). Medline doi:10.1073/pnas.1207846109

34. N. T. Ingolia, G. A. Brar, N. Stern-Ginossar, M. S. Harris, G. J. Talhouarne, S. E. Jackson, M. R. Wills, J. S. Weissman, Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes. Cell Reports 8, 1365–1379 (2014). Medline doi:10.1016/j.celrep.2014.07.045

35. M. Cazzola, R. C. Skoda, Translational pathophysiology: A novel molecular mechanism of human disease. Blood 95, 3280–3288 (2000). Medline

36. T. Kondo, M. Okabe, M. Sanada, M. Kurosawa, S. Suzuki, M. Kobayashi, M. Hosokawa, M. Asaka, Familial essential thrombocythemia associated with one-base deletion in the 5′-untranslated region of the thrombopoietin gene. Blood 92, 1091–1096 (1998). Medline

37. A. Wiestner, R. J. Schlemper, A. P. van der Maas, R. C. Skoda, An activating splice donor mutation in the thrombopoietin gene causes hereditary thrombocythaemia. Nat. Genet. 18, 49–52 (1998). Medline doi:10.1038/ng0198-49

38. N. Ghilardi, R. C. Skoda, A single-base deletion in the thrombopoietin (TPO) gene causes familial essential thrombocythemia through a mechanism of more efficient translation of TPO mRNA. Blood 94, 1480–1482 (1999). Medline

39. S. A. Slavoff, A. J. Mitchell, A. G. Schwaid, M. N. Cabili, J. Ma, J. Z. Levin, A. D. Karger, B. A. Budnik, J. L. Rinn, A. Saghatelian, Peptidomic discovery of short open reading frame-encoded peptides in human cells. Nat. Chem. Biol. 9, 59–64 (2013). Medline doi:10.1038/nchembio.1120

30

40. S. J. Andrews, J. A. Rothnagel, Emerging evidence for functional peptides encoded by short open reading frames. Nat. Rev. Genet. 15, 193–204 (2014). Medline doi:10.1038/nrg3520

41. V. Olexiouk, J. Crappé, S. Verbruggen, K. Verhegen, L. Martens, G. Menschaert, sORFs.org: A repository of small ORFs identified by ribosome profiling. Nucleic Acids Res. gkv1175 (2015). Medline doi:10.1093/nar/gkv1175

42. A. Pauli, M. L. Norris, E. Valen, G. L. Chew, J. A. Gagnon, S. Zimmerman, A. Mitchell, J. Ma, J. Dubrulle, D. Reyon, S. Q. Tsai, J. K. Joung, A. Saghatelian, A. F. Schier, Toddler: An embryonic signal that promotes cell movement via Apelin receptors. Science 343, 1248636 (2014). Medline doi:10.1126/science.1248636

43. D. E. Andreev, P. B. O’Connor, C. Fahey, E. M. Kenny, I. M. Terenin, S. E. Dmitriev, P. Cormican, D. W. Morris, I. N. Shatsky, P. V. Baranov, Translation of 5′ leaders is pervasive in genes resistant to eIF2 repression. eLife 4, e03971 (2015). Medline doi:10.7554/eLife.03971

44. C. Sidrauski, A. M. McGeachy, N. T. Ingolia, P. Walter, The small molecule ISRIB reverses the effects of eIF2α phosphorylation on translation and stress granule assembly. eLife 4, (2015). Medline doi:10.7554/eLife.05033

45. J. Somers, T. Pöyry, A. E. Willis, A perspective on mammalian upstream open reading frame function. Int. J. Biochem. Cell Biol. 45, 1690–1700 (2013). Medline doi:10.1016/j.biocel.2013.04.020

46. J. Karttunen, S. Sanderson, N. Shastri, Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens. Proc. Natl. Acad. Sci. U.S.A. 89, 6020–6024 (1992). Medline doi:10.1073/pnas.89.13.6020

47. Materials and methods are available as supplementary materials on Science Online.

48. H. P. Harding, Y. Zhang, H. Zeng, I. Novoa, P. D. Lu, M. Calfon, N. Sadri, C. Yun, B. Popko, R. Paules, D. F. Stojdl, J. C. Bell, T. Hettmann, J. M. Leiden, D. Ron, An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol. Cell 11, 619–633 (2003). Medline doi:10.1016/S1097-2765(03)00105-9

49. L. M. Mielnicki, R. G. Hughes, P. M. Chevray, S. C. Pruitt, Mutated Atf4 suppresses c-Ha-ras oncogene transcript levels and cellular transformation in NIH3T3 fibroblasts. Biochem. Biophys. Res. Commun. 228, 586–595 (1996). Medline doi:10.1006/bbrc.1996.1702

50. A. W. Paton, T. Beddoe, C. M. Thorpe, J. C. Whisstock, M. C. Wilce, J. Rossjohn, U. M. Talbot, J. C. Paton, AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP. Nature 443, 548–552 (2006). Medline doi:10.1038/nature05124

51. C. Sidrauski, D. Acosta-Alvear, A. Khoutorsky, P. Vedantham, B. R. Hearn, H. Li, K. Gamache, C. M. Gallagher, K. K. Ang, C. Wilson, V. Okreglak, A. Ashkenazi, B. Hann, K. Nader, M. R. Arkin, A. R. Renslo, N. Sonenberg, P. Walter, Pharmacological brake-release of mRNA translation enhances cognitive memory. eLife 2, e00498 (2013). Medline doi:10.7554/eLife.00498

52. C. Sidrauski, J. C. Tsai, M. Kampmann, B. R. Hearn, P. Vedantham, P. Jaishankar, M. Sokabe, A. S. Mendez, B. W. Newton, E. L. Tang, E. Verschueren, J. R. Johnson, N. J.

31

Krogan, C. S. Fraser, J. S. Weissman, A. R. Renslo, P. Walter, Pharmacological dimerization and activation of the exchange factor eIF2B antagonizes the integrated stress response. eLife 4, e07314 (2015). Medline doi:10.7554/eLife.07314

53. Y. Sekine, A. Zyryanova, A. Crespillo-Casado, P. M. Fischer, H. P. Harding, D. Ron, Mutations in a translation initiation factor identify the target of a memory-enhancing compound. Science 348, 1027–1030 (2015). Medline doi:10.1126/science.aaa6986

54. K. Zhan, K. M. Vattem, B. N. Bauer, T. E. Dever, J. J. Chen, R. C. Wek, Phosphorylation of eukaryotic initiation factor 2 by heme-regulated inhibitor kinase-related protein kinases in Schizosaccharomyces pombe is important for resistance to environmental stresses. Mol. Cell. Biol. 22, 7134–7146 (2002). Medline doi:10.1128/MCB.22.20.7134-7146.2002

55. J. P. Abastado, P. F. Miller, B. M. Jackson, A. G. Hinnebusch, Suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis for GCN4 translational control. Mol. Cell. Biol. 11, 486–496 (1991). Medline

56. K. Gülow, D. Bienert, I. G. Haas, BiP is feed-back regulated by control of protein translation efficiency. J. Cell Sci. 115, 2443–2452 (2002). Medline

57. S. R. Schwab, J. A. Shugart, T. Horng, S. Malarkannan, N. Shastri, Unanticipated antigens: Translation initiation at CUG with leucine. PLOS Biol. 2, e366 (2004). Medline doi:10.1371/journal.pbio.0020366

58. S. R. Starck, V. Jiang, M. Pavon-Eternod, S. Prasad, B. McCarthy, T. Pan, N. Shastri, Leucine-tRNA initiates at CUG start codons for protein synthesis and presentation by MHC class I. Science 336, 1719–1723 (2012). Medline doi:10.1126/science.1220270

59. H. Liang, S. He, J. Yang, X. Jia, P. Wang, X. Chen, Z. Zhang, X. Zou, M. A. McNutt, W. H. Shen, Y. Yin, PTENα, a PTEN isoform translated through alternative initiation, regulates mitochondrial function and energy metabolism. Cell Metab. 19, 836–848 (2014). Medline

60. F. Robert, L. D. Kapp, S. N. Khan, M. G. Acker, S. Kolitz, S. Kazemi, R. J. Kaufman, W. C. Merrick, A. E. Koromilas, J. R. Lorsch, J. Pelletier, Initiation of protein synthesis by hepatitis C virus is refractory to reduced eIF2·GTP·Met-tRNAi

Met ternary complex availability. Mol. Biol. Cell 17, 4632–4644 (2006). Medline doi:10.1091/mbc.E06-06-0478

61. H. Rammensee, J. Bachmann, N. P. Emmerich, O. A. Bachor, S. Stevanović, SYFPEITHI: Database for MHC ligands and peptide motifs. Immunogenetics 50, 213–219 (1999). Medline doi:10.1007/s002510050595

62. E. A. Reits, J. C. Vos, M. Grommé, J. Neefjes, The major substrates for TAP in vivo are derived from newly synthesized proteins. Nature 404, 774–778 (2000). Medline doi:10.1038/35008103

63. M. J. Androlewicz, P. Cresswell, How selective is the transporter associated with antigen processing? Immunity 5, 1–5 (1996). Medline doi:10.1016/S1074-7613(00)80304-0

64. G. E. Hammer, T. Kanaseki, N. Shastri, The final touches make perfect the peptide-MHC class I repertoire. Immunity 26, 397–406 (2007). Medline doi:10.1016/j.immuni.2007.04.003

32

65. K. R. Brandvold, R. I. Morimoto, The chemical biology of molecular chaperones—Implications for modulation of proteostasis. J. Mol. Biol. 427, 2931–2947 (2015). Medline doi:10.1016/j.jmb.2015.05.010

66. B. Wu, C. Hunt, R. Morimoto, Structure and expression of the human gene encoding major heat shock protein HSP70. Mol. Cell. Biol. 5, 330–341 (1985). Medline doi:10.1128/MCB.5.2.330

67. X. Zhang, X. Gao, R. A. Coots, C. S. Conn, B. Liu, S. B. Qian, Translational control of the cytosolic stress response by mitochondrial ribosomal protein L18. Nat. Struct. Mol. Biol. 22, 404–410 (2015). Medline

68. W. L. Zoll, L. E. Horton, A. A. Komar, J. O. Hensold, W. C. Merrick, Characterization of mammalian eIF2A and identification of the yeast homolog. J. Biol. Chem. 277, 37079–37087 (2002). Medline doi:10.1074/jbc.M207109200

69. J. H. Kim, S. M. Park, J. H. Park, S. J. Keum, S. K. Jang, eIF2A mediates translation of hepatitis C viral mRNA under stress conditions. EMBO J. 30, 2454–2464 (2011). Medline doi:10.1038/emboj.2011.146

70. I. Ventoso, M. A. Sanz, S. Molina, J. J. Berlanga, L. Carrasco, M. Esteban, Translational resistance of late alphavirus mRNA to eIF2alpha phosphorylation: A strategy to overcome the antiviral effect of protein kinase PKR. Genes Dev. 20, 87–100 (2006). Medline doi:10.1101/gad.357006

71. D. G. Macejak, P. Sarnow, Internal initiation of translation mediated by the 5′ leader of a cellular mRNA. Nature 353, 90–94 (1991). Medline doi:10.1038/353090a0

72. Y. K. Kim, S. K. Jang, Continuous heat shock enhances translational initiation directed by internal ribosomal entry site. Biochem. Biophys. Res. Commun. 297, 224–231 (2002). Medline doi:10.1016/S0006-291X(02)02154-X

73. M. C. Lai, Y. H. Lee, W. Y. Tarn, The DEAD-box RNA helicase DDX3 associates with export messenger ribonucleoproteins as well as tip-associated protein and participates in translational control. Mol. Biol. Cell 19, 3847–3858 (2008). Medline doi:10.1091/mbc.E07-12-1264

74. V. P. Pisareva, A. V. Pisarev, A. A. Komar, C. U. Hellen, T. V. Pestova, Translation initiation on mammalian mRNAs with structured 5′UTRs requires DExH-box protein DHX29. Cell 135, 1237–1250 (2008). Medline doi:10.1016/j.cell.2008.10.037

75. M. A. Skabkin, O. V. Skabkina, V. Dhote, A. A. Komar, C. U. Hellen, T. V. Pestova, Activities of Ligatin and MCT-1/DENR in eukaryotic translation initiation and ribosomal recycling. Genes Dev. 24, 1787–1801 (2010). Medline doi:10.1101/gad.1957510

76. M. Oyama, C. Itagaki, H. Hata, Y. Suzuki, T. Izumi, T. Natsume, T. Isobe, S. Sugano, Analysis of small human proteins reveals the translation of upstream open reading frames of mRNAs. Genome Res. 14 (10B), 2048–2052 (2004). Medline doi:10.1101/gr.2384604

77. M. A. Purbhoo, D. J. Irvine, J. B. Huppa, M. M. Davis, T cell killing does not require the formation of a stable mature immunological synapse. Nat. Immunol. 5, 524–530 (2004). Medline doi:10.1038/ni1058

33

78. D. F. Hunt, R. A. Henderson, J. Shabanowitz, K. Sakaguchi, H. Michel, N. Sevilir, A. L. Cox, E. Appella, V. H. Engelhard, Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry. Science 255, 1261–1263 (1992). Medline doi:10.1126/science.1546328

79. H. Escobar, D. K. Crockett, E. Reyes-Vargas, A. Baena, A. L. Rockwood, P. E. Jensen, J. C. Delgado, Large scale mass spectrometric profiling of peptides eluted from HLA molecules reveals N-terminal-extended peptide motifs. J. Immunol. 181, 4874–4882 (2008). Medline doi:10.4049/jimmunol.181.7.4874

80. V. H. Engelhard, The contributions of mass spectrometry to understanding of immune recognition by T lymphocytes. Int. J. Mass Spectrom. 259, 32–39 (2007). Medline doi:10.1016/j.ijms.2006.08.009

81. B. Vanderperre, J. F. Lucier, C. Bissonnette, J. Motard, G. Tremblay, S. Vanderperre, M. Wisztorski, M. Salzet, F. M. Boisvert, X. Roucou, Direct detection of alternative open reading frames translation products in human significantly expands the proteome. PLOS ONE 8, e70698 (2013). Medline doi:10.1371/journal.pone.0070698

82. V. L. Crotzer, R. E. Christian, J. M. Brooks, J. Shabanowitz, R. E. Settlage, J. A. Marto, F. M. White, A. B. Rickinson, D. F. Hunt, V. H. Engelhard, Immunodominance among EBV-derived epitopes restricted by HLA-B27 does not correlate with epitope abundance in EBV-transformed B-lymphoblastoid cell lines. J. Immunol. 164, 6120–6129 (2000). Medline doi:10.4049/jimmunol.164.12.6120

83. G. Lubec, L. Afjehi-Sadat, Limitations and pitfalls in protein identification by mass spectrometry. Chem. Rev. 107, 3568–3584 (2007). Medline doi:10.1021/cr068213f

84. S. R. Starck, N. Shastri, Non-conventional sources of peptides presented by MHC class I. Cell. Mol. Life Sci. 68, 1471–1479 (2011). Medline doi:10.1007/s00018-011-0655-0

85. D. J. Kowalewski, H. Schuster, L. Backert, C. Berlin, S. Kahn, L. Kanz, H. R. Salih, H. G. Rammensee, S. Stevanovic, J. S. Stickel, HLA ligandome analysis identifies the underlying specificities of spontaneous antileukemia immune responses in chronic lymphocytic leukemia (CLL). Proc. Natl. Acad. Sci. U.S.A. 112, E166–E175 (2015). Medline

86. G. L. Law, A. Raney, C. Heusner, D. R. Morris, Polyamine regulation of ribosome pausing at the upstream open reading frame of S-adenosylmethionine decarboxylase. J. Biol. Chem. 276, 38036–38043 (2001). Medline

87. A. Ventura, A. Meissner, C. P. Dillon, M. McManus, P. A. Sharp, L. Van Parijs, R. Jaenisch, T. Jacks, Cre-lox-regulated conditional RNA interference from transgenes. Proc. Natl. Acad. Sci. U.S.A. 101, 10380–10385 (2004). Medline doi:10.1073/pnas.0403954101

88. N. Morinaga, K. Yahiro, G. Matsuura, M. Watanabe, F. Nomura, J. Moss, M. Noda, Two distinct cytotoxic activities of subtilase cytotoxin produced by shiga-toxigenic Escherichia coli. Infect. Immun. 75, 488–496 (2007). Medline doi:10.1128/IAI.01336-06

89. S. Sanderson, N. Shastri, LacZ inducible, antigen/MHC-specific T cell hybrids. Int. Immunol. 6, 369–376 (1994). Medline doi:10.1093/intimm/6.3.369

34

90. J. Kunisawa, N. Shastri, The group II chaperonin TRiC protects proteolytic intermediates from degradation in the MHC class I antigen processing pathway. Mol. Cell 12, 565–576 (2003). Medline doi:10.1016/j.molcel.2003.08.009

91. S. Malarkannan, P. P. Shih, P. A. Eden, T. Horng, A. R. Zuberi, G. Christianson, D. Roopenian, N. Shastri, The molecular and functional characterization of a dominant minor H antigen, H60. J. Immunol. 161, 3501–3509 (1998). Medline

92. S. Malarkannan, L. M. Mendoza, N. Shastri, Generation of antigen-specific, lacZ-inducible T-cell hybrids. Methods Mol. Biol. 156, 265–272 (2001). Medline

93. S. Cardinaud, S. R. Starck, P. Chandra, N. Shastri, The synthesis of truncated polypeptides for immune surveillance and viral evasion. PLOS ONE 5, e8692 (2010). Medline doi:10.1371/journal.pone.0008692

94. Y. Kim, J. Ponomarenko, Z. Zhu, D. Tamang, P. Wang, J. Greenbaum, C. Lundegaard, A. Sette, O. Lund, P. E. Bourne, M. Nielsen, B. Peters, Immune epitope database analysis resource. Nucleic Acids Res. 40 (W1), W525–W530 (2012). Medline doi:10.1093/nar/gks438

95. M. Nielsen, C. Lundegaard, P. Worning, S. L. Lauemøller, K. Lamberth, S. Buus, S. Brunak, O. Lund, Reliable prediction of T-cell epitopes using neural networks with novel sequence representations. Protein Sci. 12, 1007–1017 (2003). Medline doi:10.1110/ps.0239403

96. C. Lundegaard, K. Lamberth, M. Harndahl, S. Buus, O. Lund, M. Nielsen, NetMHC-3.0: Accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8-11. Nucleic Acids Res. 36 (Web Server), W509–W512 (2008). Medline doi:10.1093/nar/gkn202

97. B. Peters, A. Sette, Generating quantitative models describing the sequence specificity of biological processes with the stabilized matrix method. BMC Bioinformatics 6, 132 (2005). Medline doi:10.1186/1471-2105-6-132

98. J. Sidney, E. Assarsson, C. Moore, S. Ngo, C. Pinilla, A. Sette, B. Peters, Quantitative peptide binding motifs for 19 human and mouse MHC class I molecules derived using positional scanning combinatorial peptide libraries. Immunome Res. 4, 2 (2008). Medline doi:10.1186/1745-7580-4-2