Translation of pre-spliced RNAs in the nuclearcompartment generates peptides for the MHCclass I pathwaySébastien Apchera, Guy Millota, Chrysoula Daskalogiannia, Alexander Scherlb, Bénédicte Manouryc,and Robin Fåhraeusa,1

aCibles Thérapeutiques, Equipe Labellisé la Ligue Contre le Cancer, Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 940,Institut de Génétique Moléculaire, Hôpital St. Louis, Université Paris 7, F-75010 Paris, France; bBiomedical Proteomics Research Group, Centre MedicalUniversitaire, CH-1211 Geneva 4, Switzerland; and cInstitut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche S-1013, HôpitalNecker, Université Paris Descartes, 75743 Paris, France

Edited by Peter Cresswell, Yale University School of Medicine, New Haven, CT, and approved September 5, 2013 (received for review May 25, 2013)

The scanning of maturing mRNAs by ribosomes plays a key role inthe mRNA quality control process. When ribosomes first engagewith the newly synthesized mRNA, and if peptides are produced,is unclear, however. Here we show that ribosomal scanning ofprespliced mRNAs occurs in the nuclear compartment, and that thisevent produces peptide substrates for the MHC class I pathway.Inserting antigenic peptide sequences in introns that are splicedout before the mRNAs exit the nuclear compartment results in anequal amount of antigenic peptide products as when the peptidesare encoded from the main open reading frame (ORF). Taken to-gether with the detection of intron-encoded nascent peptides andRPS6/RPL7-carrying complexes in the perinucleolar compartment,these results show that peptides are produced by a translationevent occurring before mRNA splicing. This suggests that ribo-somes occupy and scan mRNAs early in the mRNA maturation pro-cess, and suggests a physiological role for nuclear mRNA translation,and also helps explain how the immune system tolerates peptidesderived from tissue-specific mRNA splice variants.

MHC class I restricted antigen presentation | mRNA maturation |nuclear translation

RNAs carrying premature termination codons or are recog-nized as defective in other aspects are prevented from fur-

ther translation and disposed of by the nonsense-mediated decay(NMD) pathway (1). The detection of premature stop codons ismediated by the scanning ribosome; however, when the ribo-somes first engage with the newly synthesized mRNA, and if thisevent results in the production of peptides, is unclear. Twoobservations from the field of immunology and the presentationof peptides on MHC class I molecules have highlighted someaspects relevant to the role of the ribosomes in the mRNAmaturation process. The first observation is related to the de-tection of antigenic peptides originating from intron sequences,and the second is related to the observation that the synthesis ofantigenic peptide substrates and full-length proteins are twospatiotemporarily distinct events (2–5).The presentation of antigenic peptides on MHC class I mol-

ecules allows CD8+ T cells to detect and eliminate cells in whichthe repertoire of peptide substrates has been altered owing to thepresence of viruses or to changes in the presentation of endog-enous antigens (6, 7). However, despite the key role of antigenpresentation on MHC class I molecules in immune surveillance,the source of peptides for the MHC class I pathway is not yetknown. Accumulating evidence indicates that the processing ofalternative peptide substrates plays a major role, and that deg-radation products derived from full-length proteins have limitedaccess to the MHC class I pathway (8–10). We recently dem-onstrated that pioneer translation products (PTPs) that formantigenic peptide substrates for the MHC class I pathway areproduced by a translation event that is distinct from the canonical

event giving rise to full-length proteins and does not require thecap-binding translation factor eIF4E, and also that mRNAs thathave stopped producing peptides substrates for the MHC class Ipathway still produce full-length proteins (8).Here we report that antigenic peptides for the MHC class I

pathway are synthesized during an early steps in the mRNAmaturation process by a noncanonical translation mechanism withinthe nuclear compartment and before introns are spliced out.

Results and DiscussionSubcellular Localization of Intron and Exon RNA Sequences. The de-tection of peptides on MHC class I molecules is highly sensitive,with only a few peptide–MHC class I complexes required toactivate specific T cells, and thus can serve to detect rare peptideproducts derived from noncanonical translation during the mRNAmaturation process (8). We aimed to take advantage of thisproperty to identify the early ribosomal scanning event that giverise to peptide products. To do so, we introduced two differentantigenic peptide sequences presented on the Kb [SIINFEKL(SL8) epitope from chicken ovalbumin] (11) and the Kk [MBP(79-87) epitope from myelin basic protein (MBP)] (12) MHCclass I molecules in the exon or intron sequences of mRNAsencoding the β-Globin gene (Fig. 1A and Fig. S1A). We alsointroduced premature termination codons (PTCs; Glob-intron/exon-PTCs) to provoke NMD and prevent the synthesis of full-length proteins from the mRNAs (1, 13).

Significance

The major histocompatibility complex (MHC) class I antigen pre-sentation pathway allows the immune system to distinguishbetween self and non-self. Despite extensive research on theprocessing of antigenic peptides, little is still known about theirorigin. We recently proposed that a unique class of peptides,termed pioneer translation products (PTPs), is produced duringthe pioneer rounds of mRNA translation and provides the majorsource of antigenic peptide substrates for direct presentation tothe MHC class I pathway. Here we show that a major portion ofthe substrates for the MHC class I pathway is synthesized duringthe early steps of mRNAmaturation via a noncanonical translationmechanism within the nucleus and before introns are spliced out.

Author contributions: S.A., C.D., G.M., A.S., and R.F. designed research; S.A., G.M., C.D.,and A.S. performed research; B.M. and R.F. contributed new reagents/analytic tools; S.A.,G.M., C.D., A.S., B.M., and R.F. analyzed data; and S.A. and R.F. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

See Commentary on page 17612.1To whom correspondence should be addressed. E-mail: robin.fahraeus@inserm.fr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1309956110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1309956110 PNAS | October 29, 2013 | vol. 110 | no. 44 | 17951–17956

IMMUNOLO

GY

SEECO

MMEN

TARY

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 20

, 202

1

RNA FISH using probes toward the intron sequences, theexon sequences, or both sequences demonstrated the presence ofexons from the Glob-exon-SL8 mRNA in the nuclear and cyto-plasmic compartments, whereas introns from the same constructwere detected in the nucleus only. Introduction of a PTC codon(Glob-exon-SL8-PTC) resulted in a sharp decrease in the amountof exons detected in the cytoplasm (Fig. 1B and Fig. S2). Frac-tionation of cellular compartments followed by RT-PCR con-firmed that intron sequences were removed before the RNA wasexported to the cytoplasm, and that the introduction of PTC codonstargeted the mRNAs for the NMD pathway (Fig. 1C). Westernblot analysis demonstrated that the presence of the PTC pre-vents synthesis of the full-length Globin protein from the Glob-exon-SL8-PTC and Glob-intron-SL8-PTC mRNAs (Fig. 1D).Thus, the positioning of antigenic peptide-encoding sequenceswithin introns and/or in the context of PTCs prevents the peptides

from being synthesized by the canonical translation machinerythat governs cap-dependent synthesis of full-length proteins inthe cytoplasm.

Ribosomal Scanning of Nonspliced mRNAs Gives Rise to Production ofAntigenic Peptides for the MHC Class I Pathway. We introduced theforegoing constructs in HEK293Kb cells that stably express theKb class I molecule, and then used the B3Z T-cell hybridoma(14) that specifically detects the SL8 epitope presented on Kbmolecules to determine the relative amount of antigenic peptidesderived from the different mRNA constructs. Increasing theamount of respective cDNA resulted in a gradual increase inantigen presentation from all constructs irrespective of whetheror not the SL8 epitope was located in the intron or the exonsequence or whether the mRNA was targeted for the NMDpathway (Fig. 2A).

Fig. 1. Detection of intron and exon RNA sequences in the nuclear andcytoplasmic compartments. (A) Cartoon illustrating the different positions ofthe SL8 antigenic epitope in the exon or introns of the Globin gene con-structs. The position of the PTC that triggers NMD in the Glob-exon-SL8-PTCand the Glob-intron1-proximal-SL8-PTC constructs and primer locations usedfor RT-PCR are indicated. A list of the constructs used is provided in Fig. S1A.(B) RNA FISH assays using a mix of exon and intron probes (Upper Left)showing the Glob-exon-SL8RNA in the nuclear and cytoplasmic compart-ments. Exon probes alone (Upper Right and Lower Right) detected thepresence of exon RNA sequences in the cytoplasm and nucleus. The in-troduction of a PTC resulted in loss of exon staining in the cytoplasm (LowerRight). Intron-specific probes (Lower Left) detected RNA in the nuclearcompartment only (Fig. S2). (C) Subcellular fractionation followed by RT-PCRusing exon-junction primers showing that the introduction of a PTC codontriggers NMD and depletes mRNA levels in both the nuclear and cytoplasmiccompartments (Fig. S3A). The graph shows the average of three experimentswith Glob-exon-SL8 mRNA levels set to 100%. (D) Western blot showing thatthe presence of the PTC prevents canonical mRNA translation and the syn-thesis of full-length proteins.

Fig. 2. Antigenic peptides for the MHC class I pathway are produced fromnonspliced mRNAs. (A) Increasing amounts of DNAs expressing indicatedGlobin gene constructs in HEK293Kb cells followed by incubation with theSL8-specific CD8+ T-cell hybridoma (B3Z) revealed the production of the SL8epitope derived from PTPs irrespective of whether the epitope was in-troduced in the intron or exon sequence or whether the mRNAs carry a PTC.(B) Introducing the SL8 epitope in either the distal region of intron 1 orintron 2 led to an ∼40% reduction in antigen presentation compared withthat in the proximal region of intron 1 or in exon 1. (C) Changing the SL8epitope for the MBP epitope presented on Kk MHC class I molecules anddetected by specific CD8+ T cells showed a similar profile as the SL8 epitope.(D) A single nucleotide frame shift inserted before the SL8 epitope resultedin an ∼100-fold reduction in antigen presentation. The data show the av-erage of at least three independent experiments with SD minus the valuesfrom mock-transfected cells. Free SL8 peptide was added to cells to ensurethat T-cell assays were carried out at nonsaturated conditions and that theexpression of MHC class I molecules was not affected.

17952 | www.pnas.org/cgi/doi/10.1073/pnas.1309956110 Apcher et al.

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 20

, 202

1

To ensure that production of the SL8 peptide when inserted inthe first intron sequence was not related to missplicing that wouldretain the first intron in the spliced mRNA, we also introduced theSL8 epitope in the second intron (Glob-intron2-SL8). The pre-sentation of SL8 was observed from the second intron, but at an∼40% lower level compared with the first intron.The presentation of antigenic peptides depends on proteolytic

processing and hence on the amino acids surrounding the epi-tope; thus, moving an epitope within an RNA can alter its pro-cessing. In line with this notion, we observed a similar 40%reduction in antigen presentation when we moved the SL8 fromthe proximal region to the distal region of the first intron (Glob-intron1-distal-SL8) (Fig. 2B).The RNA sequence surrounding the epitope might possibly

play a role in governing noncanonical initiation of the antigenicpeptide substrate. This idea is consistent with the observationthat introducing a silent point mutation in codon 39 (CUC toCUG) upstream of the MBP(79-87) epitope in the MBP mRNAled to a 30% increase in antigen presentation (8). Exchangingthe SL8 peptide for the MBP peptide and using a T-cell hy-bridoma specific for this epitope (15) resulted in a similar pro-duction of antigenic peptide substrates from intron and exonsequences, demonstrating that presentation of epitopes derivedfrom introns is not restricted to the epitope or to a certain class Imolecule (Fig. 2C). Interestingly, the relative levels of SL8 vs.

MBP presentation differed depending on the context in whichthey were synthesized, further suggesting that the location andcontext of the antigenic peptide within the mRNA is importantto substrate production and peptide processing.Finally, we placed the SL8 epitope in a different ORF by in-

troducing a single nucleotide upstream of the antigenic peptide-encoding sequence (Glob-out-exon-SL8), and found an ∼100-fold decrease in antigen presentation (Fig. 2D and Fig. S1A).These results show that the synthesis of antigenic peptide sub-strates occurs on nonspliced mRNAs and depends on whether ornot the epitope is in frame with the initiation codon of the mainORF. This supports the idea that the scanning of presplicedmRNAs by a noncanonical translation event produces PTPs thatform an important source of antigenic peptide substrates for theMHC class I pathway.

Interfering with mRNA Nuclear Export Increases Production of MHCClass I Substrates. Introns were spliced out before the mRNAexited the nucleus, indicating that the production of peptidematerial occurs before the RNAs are exported out of the nu-cleus. To further test whether peptide epitopes inserted inintrons are indeed translated in the nucleus, we introducedthe Rev responsive element (RRE) of the HIV RNA in the 3′UTR of the Glob-exon/intron-SL8 constructs (16, 17) (Fig.S1A). This sequence targets nonspliced and spliced mRNAs

Fig. 3. Preventing mRNA nuclear export increases the production of MHC class I substrates. (A) Fusing the RRE sequence of HIV-1 to the 3′ UTR of the Globingene targeted nonspliced mRNAs for nuclear export via the CRM1 pathway in a Rev-dependent fashion, allowing the detection of intron sequences in thecytoplasm using RNA FISH (Left and Center) or by RT-PCR from nuclear and cytoplasmic cellular fractions (Right). NT, nontransfected control cells. (B) In-creasing levels of Rev resulted in a subsequent decrease in the relative amount of Glob-exon-SL8-RRE mRNA in the nucleus and a concomitant decrease in theamount of antigenic peptides produced. (C) CRM1-mediated export is blocked by the LMB compound and treating cells with increasing concentrations of LMBprevents Rev-mediated RNA export, with a subsequent increase in the relative amount of RNA in the nucleus and an increase in antigen presentation. (D) TheRev protein does not bind the IFN-α1 mRNA, but interferes with its nuclear export via CRM1. Increasing amounts of Rev led to accumulation of the IFN-α1-SL8mRNA in the nucleus and to an increase in antigen presentation. (E) Treatment of cells with the spliceosome inhibitor isoginkgetin resulted in an increase inantigen presentation when the SL8 epitope was inserted in Globin gene constructs, but not in the context of cDNA (Fig. S3D).

Apcher et al. PNAS | October 29, 2013 | vol. 110 | no. 44 | 17953

IMMUNOLO

GY

SEECO

MMEN

TARY

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 20

, 202

1

for Rev-dependent nuclear export through the CRM1 pathway (18).Using either RNA FISH on fixed cells or RT-PCR on subcellularfractions, we detected the presence of introns in the cytoplasm afteroverexpression of Rev (Fig. 3A and Fig. S3A). Introduction of in-creasing amounts of Rev in cells expressing a fixed amount of theGlob-exon/intron-SL8-RRE resulted in a dose-dependent decreasein mRNA levels in the nucleus, accompanied by decreased pre-sentation of the SL8 epitope on the Kb molecules (Fig. 3B and Fig.S3B). In contrast, prevention of Rev-dependent nuclear export ofthe Glob-exon/intron-SL8-RRE mRNAs using the CRM1 inhibitorleptomycin B (LMB) (19) resulted in increased antigen presentation(Fig. 3C and Fig. S3C).

We further tested the correlation between mRNA export andantigen presentation by inserting the SL8 epitope in the IFN-α1mRNA, which is exported through the CRM1 but does not bindthe Rev protein (20). In this situation, Rev instead blocks IFN-α1mRNA export by interfering with the CRM1, resulting in in-creased SL8 peptide production (Fig. 3D). In further support ofprespliced mRNAs as a source for antigenic peptide substrates,we observed an increase in antigen presentation when the splicingmachinery was inhibited using the isoginkgetin compound (21,22) (Fig. 3E and Fig. S3D). These results may shed further lighton the finding that PTC codons can be detected while the na-scent RNA is still linked to the RNA polymerase (23).

Fig. 4. PTPs are produced in the nuclear compartment. (A) The antigenic peptide epitope was exchanged for the HA epitope in indicated β-Globin geneconstructs and used to visualize PTPs by immunohistochemistry analysis. Treatment of cells with the proteasome inhibitor epoxomycin for 15 or 30 min in-creased the amount of HA epitopes detected in the cytoplasmic compartment. (B) The compound puromycin is incorporated in the nascent peptide and,together with the elongation inhibitor emetine, can be used to visualize nascent peptides attached to the ribosome using anti-puromycin antibodies (27).Expression of the HA epitope from within an intron (Glob-intron-HA-PTC), followed by fixation and anti-puromycin (mouse mAb) and anti-HA (rabbitpolyclonal sera) shows colocalization in the nucleus but not in the cytoplasm. (C) The PLA allows identification of two primary antibodies in close proximity.Secondary PLA-labeled antibodies revealed a specific signal toward anti-puromycin and anti-HA antibodies in the nuclear and cytoplasmic compartmentsfrom the Glob-exon-HA construct (Upper) and in the nuclear compartment using Glob-intron-HA-PTC (Lower). (D) (Left) Immunohistochemistry detected S6 ofthe small ribosomal subunit in the cytoplasm, nucleoli, and perinucleoli. (Center) The combination of anti-HA and anti-S6 showed a nuclear PLA reaction only.(Right) Anti-S6 (mouse Ab) and anti-L7 of the large subunit (polyclonal Ab) showed PLA complexes in the cytoplasmic and nuclear compartments. The S6 andL7 PLA complexes were not detected in nucleoli despite the large amount of respective proteins in this compartment (Fig. S4D).

17954 | www.pnas.org/cgi/doi/10.1073/pnas.1309956110 Apcher et al.

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 20

, 202

1

Detection of PTPs on the Nascent Ribosome in the Nuclear Compartment.We next inserted the HA tag next to the antigenic epitope tovisualize PTPs derived from introns or from mRNAs targeted forthe NMD pathway using immunohistochemistry. We observeda specific signal in the nucleus of cells expressing the Glob-intron-HA-PTC constructs using anti-HA antibodies (Fig. 4A).Application of 15- and 30-min pulses with the proteasomeinhibitor epoxomycin resulted in increased detection of HA-carrying peptides in the nucleus accompanied by detection inthe cytoplasm as well. This indicates a short half-life of PTPs,which is in line with the first model to predict alternative trans-lation products, or defective ribosomal products (DRiPs) as thesource of peptides for the MHC class I pathway (10, 24).The presentation of PTPs for the class I pathway requires

proteasomal activity, and the rapid degradation of these sub-strates ensures their prompt availability for the immune system,and also minimizes the potential risk of PTPs interfering withother cellular processes. Translational profiling and mass spec-trometry (MS) studies have recently identified a large pool ofnoncanonical small translation products in mammalian cellsinitiated from non-AUG codons and from intron sequences (25,26). After insertion of the 6xHis-tag after the SL8 epitope in theGlob-exon-SL8-PTC construct (Glob-exon/intron-SL8-His-PTC)(Fig. S1A) and enrichment of His-tagged PTP products throughnickel agarose affinity chromatography, MS analysis detecteda series of peptides of various lengths carrying the SL8 epitopederived from both intron and exon sequences (Table 1 andTable S1).The puromycin compound is incorporated into the nascent

peptide by the ribosome and, together with the elongation inhibitoremetine and an anti-puromycin antibody, can be used to detectnascent peptides on the polysome (27, 28). Incubation of cellsexpressing the Glob-intron-HA-PTC construct with these twocompounds, followed by fixation and anti-HA-tag (rabbit Ab)and anti-puromcyin (mouse Ab) antibodies, revealed colocali-zation between HA and puromycin in the nuclear compartment(Fig. 4B). We did not observe such colocalization in the cytoplasm,presumably because the majority of HA-carrying epitopes in thiscompartment are not linked to polysomes (Fig. S4A).To determine whether the HA tag is present on nascent PTPs,

we used proximal ligation assay (PLA)-labeled secondary anti-bodies, which allows detection of two primary antibodies in closeproximity (29). The combination of PLA-labeled anti-HA and

anti-puromycin antibodies and the use of saponin to permeabilizecells produced a specific signal in the nuclear and cytoplasmiccompartments of cells expressing the Glob-exon-HA construct.Expression of the Glob-intron-HA-PTC gave rise to a nuclearPLA signal, however, demonstrating the presence of the nascentHA epitope derived from an intron sequence in the nuclearcompartment (Fig. 4C). Estimating the amount of nascent PTPsusing this approach is difficult, given the potential destruction ofsome HA epitopes by puromycin and relatively short lengths ofthe PTPs. However, anti-HA alone under these conditions pro-duced no PLA reaction, and no positive signals were detected innontransfected cells (Fig. S4B).The majority of the S6 ribosomal protein is detected in the

cytoplasm and nucleoli, with a small fraction detected in the peri-nucleoli (Fig. 4D, Left and Fig. S4C). We carried out PLA-basedcolocalization using anti-HA and anti-RPS6 antibodies and, in-terestingly, detected colocalization in the nucleus but not in thenucleoli (Fig. 4D, Center). Similar to RPS6, we detected the ma-jority of L7 of the large subunit in the cytoplasm and in thenucleoli, as expected, but also a small but significant amount inthe perinucleoli (Fig. S4D). Using a combination of anti-RPS6 andanti-RPL7 antibodies, we detected PLA complexes in the cyto-plasm and the nucleus. Importantly, we did not detect the nu-clear RPS6-RPL7 PLA complexes in the nucleoli, even thoughthe majority of RPS6 and RPL7 were present in this compart-ment (Fig. 4D, Right). Taken together, these results suggest thatthe production of PTPs occurs in the nucleus, but outsidethe nucleoli.

Concluding RemarksNuclear translation has been a controversial issue. Previousreports have suggested that nuclear translation indeed can occur(27, 30–32), but the physiological context has remained obscure.Our data presented here indicate that a major portion of thesubstrates for the MHC class I pathway are synthesized in theearly steps of mRNA maturation by a noncanonical translationmechanism within the nucleus and before introns are spliced out.Thus, the ribosomes engage with the newly synthesized mRNAearlier than previously thought. This translation event ensuresthat the first peptides derived from an mRNA are presented tothe MHC class I pathway, allowing early immune detection ofinfected cells. CD8+ T cells toward epitopes derived from intronshave been identified previously (2, 5), and our results provide anexplanation for these observations. The production of PTPs fromnonspliced RNAs in thymic cells would guarantee that peptidesderived from all possible splice variants of hnRNAs are pre-sented during T-cell selection, and thereby safeguard against thepossibility that alternative tissue-specific splicing events in pe-ripheral tissues could give rise to autoimmune reactions. In fact, theproduction of antigenic peptides from prespliced mRNAs could bea prerequisite for allowing tissue-dependent splicing to occur.Our results open up new possibilities for investigating the pro-

duction of antigenic peptides that will have implications for gener-ating vaccines, understanding dysfunctional antigen presentationin disease, and developing novel immune therapies, includingthose against cancer cells. It also sheds light on a unique mRNAtranslation event and raises the possibility that different ribo-somes produce peptide products with different functions, similarto how different RNA polymerases produce different classes ofRNAs, each with a defined function.

Materials and MethodsT-Cell Hybridoma, Cell Culture, and Transfection. The SIINFEKL:Kb-specific (B3Z)and theMBP(79-87):Kk-specific T-cell reporter hybridomas have been describedpreviously (14). Human cell lines were cultivated under standard conditions inRPMI medium 1640. Human HEK-293 T Kb stable cell lines were a gift from L.Eisenlohr (Thomas Jefferson University, Philadelphia). Plasmid constructionsand quantitative RT-PCR (qRT-PCR) are described in SI Materials and Methods.

Table 1. Purified peptides uniquely expressed from theβ-Globin gene in HEK-293 cells expressing the YFP-Glob-exon-SL8-HIS-PTC construct identified by LC-MS/MS

Peptide sequence Peptide length, aa

TAAGITLGMDELYK 14AGITLGMDELYK 12GITLGMDELYK 11YQTSLYKK 8KAGYTMVHLTPEEK 14AGYTMVHLTPEEK 13YTMVHLTPEEK 11TMVHLTPEEK 10SAVTSIINFEK 11AVTSIINFEK 10VTSIINFEK 9VNVDEVGGEALGR 13VDEVGGEALGR 11NWACGDREDSWVSDRH 16LLVVTPWT 10

Amino acids in bold type are encoded by the first intron sequence of theβ-Globin gene. A complete list of peptides is provided in Table S1.

Apcher et al. PNAS | October 29, 2013 | vol. 110 | no. 44 | 17955

IMMUNOLO

GY

SEECO

MMEN

TARY

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 20

, 202

1

RNA Preparation, RT-PCR, and qRT-PCR. For nuclear/cytoplasmic RNA extrac-tion, HEK-293 cells were plated in six-well plates and transfected with theindicated constructs. At 48 h posttransfection, cells were washed with coldPBS and then resuspended in 120 μL of LB buffer [10 mM NaCl, 2 mM MgCl2,10 mM Tris·HCl (pH 7.8), 5 mM DTT, 0.5% Nonidet P-40, and 3 μL/mL RNaseOUT).Samples were placed on ice for 5 min and then centrifuged at 6000 × g for5 min at 4 °C. The cytoplasmic fraction was collected and transferred toa new microcentrifuge tube, and 120 μL of ProtK buffer [0.2 M Tris·HCl (pH7.5), 25 mM EDTA, 0.3 M NaCl, 2% SDS, and 3 μL/mL RNaseOUT] was added.After the addition of 20 μL of Proteinase K (10 mg/mL) and incubation ofsamples at 37 °C for 20 min, cytoplasmic RNA was purified using the RNeasyMini Kit (Qiagen) following the manufacturer’s protocol and on-columnDNase treatment (RNeasy, Quiagen).

The nuclear RNA pellet was resuspended in 120 μL of LB buffer, after which120 μL of ProtK buffer and 20 μL of proteinase K were added, and the nuclearfractions were incubated at 37 °C for 20 min. Nuclear RNA was purified withthe RNeasy Mini Kit (Qiagen). Reverse transcription was carried out as de-scribed previously (8). Primers are listed in SI Materials and Methods.

Peptide Purification, Peptide Digestion, MS Analysis, and Peptide Identification.Transfected HEK293kb cells were lysed in 6 mL of 6 M guanidinium HCl, 0.1 MNa2HPO4/NaH2PO4, 0.01 M Tris·HCl (pH 8.0), 5 mM imidazole, and 10 mMβ-mercaptoethanol, and then sonicated on ice for 20 s three times. Then75 μL of Ni2+-NTA agarose beads (Qiagen) were added, and lysates wererotated at room temperature for 6 h. Beads were then washed for 10 minwith 800 μL of 6 M guanidinium HCl, 0.1 M Na2HPO4/NaH2PO4, 0.01 MTris·HCl (pH 8.0), and 10 mM β-mercaptoethanol; 6 M urea, 0.1 M Na2HPO4/NaH2PO4, 0.01 M Tris·HCl (pH 8.0) and 10 mM β-mercaptoethanol; 6 M urea,0.1 M Na2HPO4/NaH2PO4, 0.01 M Tris·HCl (pH 6.8), 10 mM β-mercaptoetha-nol (buffer A) plus 0.2% Triton X-100; buffer A again; and then buffer A plus0.1% Triton X-100. After the last wash, His-6–tagged peptides were elutedby incubating the beads in 80 μL of 200 mM imidazole, 0.15 M Tris·HCl (pH6.7), 30% glycerol, and 0.72 M β-mercaptoethanol for 30 min at roomtemperature. Peptides were then digested with trypsin before analysis byLC-MS/MS as described previously (33).

Tandem MS data were searched against the UniProt database and thetranslated sequence from the Glob-SL8-PTC-His construct, without enzyme

specificity. Database searches and false-discovery rate estimation were per-formed using the EasyProt software platform (33). Only peptide spectramatches with a false-discovery rate <1% were retained.

Cell Fixation and Immunostaining. Cells were plated on 24 × 24 mm coverslipsin six-well plates or on 12-mm-diameter coverslips in 24-well plates. At 24 hposttransfection, the cells were washed briefly with cold PBS and then fixedin 4% PFA for 10 min at room temperature, rinsed twice with PBS 1×, andsaturated with PBS, 3% BSA, and 0.1% saponin (saturation buffer) for 30 min.Primary antibodies (see below) were incubated for 2 h at room temperature orovernight at 4 °C, and secondary antibodies were incubated for 30 min atroom temperature, both in saturation buffer. The anti-nucleolin antibody(Abcam) and anti-Globin antibody (Sigma-Aldrich) were mouse Abs.

Proximal Ligation Assay. HEK cells were grown on coverslips and transfectedwith indicated constructs for 48 h and treated with 500 nM epoxomicin(Peptanova) for 15 min, 208 μM emetine (Sigma-Aldrich) for 5 min, and then91 μM puromycin (Sigma-Aldrich) for 3 min. The cells were fixed in 4%paraformaldehyde for 15 min before being permeabilized in PBS and 3%BSA containing 0.1% saponin. Primary antibodies—mouse anti-puromycin(a kind gift from Alexandre David, Institut de Génomique Fonctionnelle,Montpellier, France), mouse anti-S6 (Cell Signaling), rabbit polyclonal anti-L7(Cell Signaling), rabbit polyclonal anti-HA (Sigma-Aldrich), or mouse anti-HA(a kind gift from Borek Vojtesek, Masaryk Memorial Cancer Institute, Brno,Czech Republic)—were incubated in the same buffer for 2 h. After the cellswere washed, PLA probes were added, followed by hybridization, ligation,and amplification according to the manufacturer’s protocol (Olink Bio-science). Coverslips were mounted on slides using Duolink in situ mountingmedium (OLink Bioscience) with DAPI. Slides were analyzed by fluorescencemicroscopy.

ACKNOWLEDGMENTS. We thank Alexander David for providing the anti-puromycin antibody. This work was supported by la Ligue Contre le Cancer(Grant 5933), Agence National de Recherche (ANR), the European RegionalDevelopment Fund, and the State Budget of the Czech Republic (RegionalCentre for Applied Molecular Oncology, CZ.1.05/2.1.00/03.0101).

1. Chang YF, Imam JS, Wilkinson MF (2007) The nonsense-mediated decay RNA sur-veillance pathway. Annu Rev Biochem 76:51–74.

2. Guilloux Y, et al. (1996) A peptide recognized by human cytolytic T lymphocytes onHLA-A2 melanomas is encoded by an intron sequence of the N-acetylglucosaminyl-transferase V gene. J Exp Med 183(3):1173–1183.

3. Robbins PF, et al. (1997) The intronic region of an incompletely spliced gp100 genetranscript encodes an epitope recognized by melanoma-reactive tumor-infiltratinglymphocytes. J Immunol 159(1):303–308.

4. Starck SR, Shastri N (2011) Non-conventional sources of peptides presented by MHCclass I. Cell Mol Life Sci 68(9):1471–1479.

5. Coulie PG, et al. (1995) A mutated intron sequence codes for an antigenic peptiderecognized by cytolytic T lymphocytes on a human melanoma. Proc Natl Acad Sci USA92(17):7976–7980.

6. Blum JS, Wearsch PA, Cresswell P (2013) Pathways of antigen processing. Annu RevImmunol 31:443–473.

7. Shastri N, Schwab S, Serwold T (2002) Producing nature’s gene-chips: The generationof peptides for display by MHC class I molecules. Annu Rev Immunol 20:463–493.

8. Apcher S, et al. (2011) Major source of antigenic peptides for the MHC class I pathwayis produced during the pioneer round of mRNA translation. Proc Natl Acad Sci USA108(28):11572–11577.

9. Khan S, et al. (2001) Cutting edge: Neosynthesis is required for the presentation of a Tcell epitope from a long-lived viral protein. J Immunol 167(9):4801–4804.

10. Yewdell JW, Antón LC, Bennink JR (1996) Defective ribosomal products (DRiPs): Amajor source of antigenic peptides for MHC class I molecules? J Immunol 157(5):1823–1826.

11. Rötzschke O, et al. (1991) Exact prediction of a natural T cell epitope. Eur J Immunol21(11):2891–2894.

12. Huseby ES, Ohlén C, Goverman J (1999) Cutting edge: Myelin basic protein-specificcytotoxic T cell tolerance is maintained in vivo by a single dominant epitope in H-2kmice. J Immunol 163(3):1115–1118.

13. Lejeune F, Ranganathan AC, Maquat LE (2004) eIF4G is required for the pioneerround of translation in mammalian cells. Nat Struct Mol Biol 11(10):992–1000.

14. Shastri N, Gonzalez F (1993) Endogenous generation and presentation of the oval-bumin peptide/Kb complex to T cells. J Immunol 150(7):2724–2736.

15. Perchellet A, Stromnes I, Pang JM, Goverman J (2004) CD8+ T cells maintain toleranceto myelin basic protein by “epitope theft.” Nat Immunol 5(6):606–614.

16. Daly TJ, Cook KS, Gray GS, Maione TE, Rusche JR (1989) Specific binding of HIV-1recombinant Rev protein to the Rev-responsive element in vitro. Nature 342(6251):816–819.

17. Zapp ML, Green MR (1989) Sequence-specific RNA binding by the HIV-1 Rev protein.Nature 342(6250):714–716.

18. Daugherty MD, Liu B, Frankel AD (2010) Structural basis for cooperative RNA bindingand export complex assembly by HIV Rev. Nat Struct Mol Biol 17(11):1337–1342.

19. Askjaer P, Jensen TH, Nilsson J, Englmeier L, Kjems J (1998) The specificity of theCRM1-Rev nuclear export signal interaction is mediated by RanGTP. J Biol Chem273(50):33414–33422.

20. Kimura T, Hashimoto I, Nagase T, Fujisawa J (2004) CRM1-dependent, but not ARE-mediated, nuclear export of IFN-alpha1 mRNA. J Cell Sci 117(Pt 11):2259–2270.

21. O’Brien K, Matlin AJ, Lowell AM, Moore MJ (2008) The biflavonoid isoginkgetin isa general inhibitor of pre-mRNA splicing. J Biol Chem 283(48):33147–33154.

22. Disney MD (2008) Short-circuiting RNA splicing. Nat Chem Biol 4(12):723–724.23. de Turris V, Nicholson P, Orozco RZ, Singer RH, Mühlemann O (2011) Cotranscriptional

effect of a premature termination codon revealed by live-cell imaging. RNA 17(12):2094–2107.

24. Schubert U, et al. (2000) Rapid degradation of a large fraction of newly synthesizedproteins by proteasomes. Nature 404(6779):770–774.

25. Ingolia NT, Lareau LF, Weissman JS (2011) Ribosome profiling of mouse embryonicstem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147(4):789–802.

26. Slavoff SA, et al. (2013) Peptidomic discovery of short open reading frame-encodedpeptides in human cells. Nat Chem Biol 9(1):59–64.

27. David A, et al. (2012) Nuclear translation visualized by ribosome-bound nascent chainpuromycylation. J Cell Biol 197(1):45–57.

28. David A, Bennink JR, Yewdell JW (2013) Emetine optimally facilitates nascent chainpuromycylation and potentiates the ribopuromycylation method (RPM) applied toinert cells. Histochem Cell Biol 139(3):501–504.

29. Söderberg O, et al. (2006) Direct observation of individual endogenous proteincomplexes in situ by proximity ligation. Nat Methods 3(12):995–1000.

30. Allfrey VG, Mirsky AE (1955) Protein synthesis in isolated cell nuclei. Nature 176(4492):1042–1049.

31. Iborra FJ, Jackson DA, Cook PR (2001) Coupled transcription and translation withinnuclei of mammalian cells. Science 293(5532):1139–1142.

32. Dolan BP, Knowlton JJ, David A, Bennink JR, Yewdell JW (2010) RNA polymerase IIinhibitors dissociate antigenic peptide generation from normal viral protein synthesis:A role for nuclear translation in defective ribosomal product synthesis? J Immunol185(11):6728–6733.

33. Gluck F, et al. (2013) EasyProt—an easy-to-use graphical platform for proteomicsdata analysis. J Proteomics 79:146–160.

17956 | www.pnas.org/cgi/doi/10.1073/pnas.1309956110 Apcher et al.

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 20

, 202

1