Translation of pre-spliced RNAs in the nuclear
compartment generates peptides for the MHC
class I pathway
Sébastien 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 Medical
Universitaire, CH-1211 Geneva 4, Switzerland; andcInstitut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche S-1013, Hôpital
Necker, 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 in
the mRNA quality control process. When ribosomes first engage
with the newly synthesized mRNA, and if peptides are produced,
is unclear, however. Here we show that ribosomal scanning of
prespliced mRNAs occurs in the nuclear compartment, and that this
event produces peptide substrates for the MHC class I pathway.
Inserting antigenic peptide sequences in introns that are spliced
out before the mRNAs exit the nuclear compartment results in an
equal amount of antigenic peptide products as when the peptides
are encoded from the main open reading frame (ORF). Taken to-
gether with the detection of intron-encoded nascent peptides and
RPS6/RPL7-carrying complexes in the perinucleolar compartment,
these results show that peptides are produced by a translation
event 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 peptides
derived from tissue-specific mRNA splice variants.
MHC class I restricted antigen presentation|mRNA maturation|
ther translation and disposed of by the nonsense-mediated decay
(NMD) pathway (1). The detection of premature stop codons is
mediated by the scanning ribosome; however, when the ribo-
somes first engage with the newly synthesized mRNA, and if this
event results in the production of peptides, is unclear. Two
observations from the field of immunology and the presentation
of peptides on MHC class I molecules have highlighted some
aspects relevant to the role of the ribosomes in the mRNA
maturation 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 of
antigenic peptide substrates and full-length proteins are two
spatiotemporarily 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 which
the repertoire of peptide substrates has been altered owing to the
presence of viruses or to changes in the presentation of endog-
enous antigens (6, 7). However, despite the key role of antigen
presentation on MHC class I molecules in immune surveillance,
the source of peptides for the MHC class I pathway is not yet
known. Accumulating evidence indicates that the processing of
alternative peptide substrates plays a major role, and that deg-
radation products derived from full-length proteins have limited
access to the MHC class I pathway (8–10). We recently dem-
onstrated that pioneer translation products (PTPs) that form
antigenic peptide substrates for the MHC class I pathway are
produced by a translation event that is distinct from the canonical
NAs carrying premature termination codons or are recog-
nized as defective in other aspects are prevented from fur-
event giving rise to full-length proteins and does not require the
cap-binding translation factor eIF4E, and also that mRNAs that
have stopped producing peptides substrates for the MHC class I
pathway 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 mRNA
maturation process by a noncanonical translation mechanism within
the nuclear compartment and before introns are spliced out.
Results and Discussion
Subcellular 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 to
activate specific T cells, and thus can serve to detect rare peptide
products derived from noncanonical translation during the mRNA
maturation process (8). We aimed to take advantage of this
property to identify the early ribosomal scanning event that give
rise to peptide products. To do so, we introduced two different
antigenic 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) MHC
class I molecules in the exon or intron sequences of mRNAs
encoding the β-Globin gene (Fig. 1A and Fig. S1A). We also
introduced premature termination codons (PTCs; Glob-intron/
exon-PTCs) to provoke NMD and prevent the synthesis of full-
length proteins from the mRNAs (1, 13).
The major histocompatibility complex (MHC) class I antigen pre-
sentation pathway allows the immune system to distinguish
between self and non-self. Despite extensive research on the
processing of antigenic peptides, little is still known about their
origin. We recently proposed that a unique class of peptides,
termed pioneer translation products (PTPs), is produced during
the pioneer rounds of mRNA translation and provides the major
source of antigenic peptide substrates for direct presentation to
the MHC class I pathway. Here we show that a major portion of
the substrates for the MHC class I pathway is synthesized during
the early steps of mRNA maturation via a noncanonical translation
mechanism 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: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| October 29, 2013
| vol. 110
| no. 44
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 the
indicated constructs. At 48 h posttransfection, cells were washed with cold
PBS 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 for
5 min at 4 °C. The cytoplasmic fraction was collected and transferred to
a new microcentrifuge tube, and 120 μL of ProtK buffer [0.2 M Tris·HCl (pH
7.5), 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 of
samples at 37 °C for 20 min, cytoplasmic RNA was purified using the RNeasy
Mini Kit (Qiagen) following the manufacturer’s protocol and on-column
DNase treatment (RNeasy, Quiagen).
The nuclear RNA pellet was resuspended in 120 μL of LB buffer, after which
120 μL of ProtK buffer and 20 μL of proteinase K were added, and the nuclear
fractions were incubated at 37 °C for 20 min. Nuclear RNA was purified with
the 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 HEK293kbcells were lysed in 6 mL of 6 M guanidinium HCl, 0.1 M
Na2HPO4/NaH2PO4, 0.01 M Tris·HCl (pH 8.0), 5 mM imidazole, and 10 mM
β-mercaptoethanol, and then sonicated on ice for 20 s three times. Then
75 μL of Ni2+-NTA agarose beads (Qiagen) were added, and lysates were
rotated at room temperature for 6 h. Beads were then washed for 10 min
with 800 μL of 6 M guanidinium HCl, 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 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 plus
0.1% Triton X-100. After the last wash, His-6–tagged peptides were eluted
by incubating the beads in 80 μL of 200 mM imidazole, 0.15 M Tris·HCl (pH
6.7), 30% glycerol, and 0.72 M β-mercaptoethanol for 30 min at room
temperature. Peptides were then digested with trypsin before analysis by
LC-MS/MS as described previously (33).
Tandem MS data were searched against the UniProt database and the
translated 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 spectra
matches with a false-discovery rate <1% were retained.
Cell Fixation and Immunostaining. Cells were plated on 24 × 24 mm coverslips
in six-well plates or on 12-mm-diameter coverslips in 24-well plates. At 24 h
posttransfection, the cells were washed briefly with cold PBS and then fixed
in 4% PFA for 10 min at room temperature, rinsed twice with PBS 1×, and
saturated 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 or
overnight at 4 °C, and secondary antibodies were incubated for 30 min at
room 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 transfected
with indicated constructs for 48 h and treated with 500 nM epoxomicin
(Peptanova) for 15 min, 208 μM emetine (Sigma-Aldrich) for 5 min, and then
91 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 cells
were 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 mounting
medium (OLink Bioscience) with DAPI. Slides were analyzed by fluorescence
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 Regional
Development Fund, and the State Budget of the Czech Republic (Regional
Centre 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 on
HLA-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 gene
transcript encodes an epitope recognized by melanoma-reactive tumor-infiltrating
lymphocytes. J Immunol 159(1):303–308.
4. Starck SR, Shastri N (2011) Non-conventional sources of peptides presented by MHC
class I. Cell Mol Life Sci 68(9):1471–1479.
5. Coulie PG, et al. (1995) A mutated intron sequence codes for an antigenic peptide
recognized by cytolytic T lymphocytes on a human melanoma. Proc Natl Acad Sci USA
6. Blum JS, Wearsch PA, Cresswell P (2013) Pathways of antigen processing. Annu Rev
7. Shastri N, Schwab S, Serwold T (2002) Producing nature’s gene-chips: The generation
of 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 pathway
is produced during the pioneer round of mRNA translation. Proc Natl Acad Sci USA
9. Khan S, et al. (2001) Cutting edge: Neosynthesis is required for the presentation of a T
cell 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): A
major source of antigenic peptides for MHC class I molecules? J Immunol 157(5):
11. Rötzschke O, et al. (1991) Exact prediction of a natural T cell epitope. Eur J Immunol
12. Huseby ES, Ohlén C, Goverman J (1999) Cutting edge: Myelin basic protein-specific
cytotoxic T cell tolerance is maintained in vivo by a single dominant epitope in H-2k
mice. J Immunol 163(3):1115–1118.
13. Lejeune F, Ranganathan AC, Maquat LE (2004) eIF4G is required for the pioneer
round 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 tolerance
to 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-1
recombinant Rev protein to the Rev-responsive element in vitro. Nature 342(6251):
17. Zapp ML, Green MR (1989) Sequence-specific RNA binding by the HIV-1 Rev protein.
18. Daugherty MD, Liu B, Frankel AD (2010) Structural basis for cooperative RNA binding
and 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 the
CRM1-Rev nuclear export signal interaction is mediated by RanGTP. J Biol Chem
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 is
a 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):
24. Schubert U, et al. (2000) Rapid degradation of a large fraction of newly synthesized
proteins by proteasomes. Nature 404(6779):770–774.
25. Ingolia NT, Lareau LF, Weissman JS (2011) Ribosome profiling of mouse embryonic
stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147(4):
26. Slavoff SA, et al. (2013) Peptidomic discovery of short open reading frame-encoded
peptides in human cells. Nat Chem Biol 9(1):59–64.
27. David A, et al. (2012) Nuclear translation visualized by ribosome-bound nascent chain
puromycylation. J Cell Biol 197(1):45–57.
28. David A, Bennink JR, Yewdell JW (2013) Emetine optimally facilitates nascent chain
puromycylation and potentiates the ribopuromycylation method (RPM) applied to
inert cells. Histochem Cell Biol 139(3):501–504.
29. Söderberg O, et al. (2006) Direct observation of individual endogenous protein
complexes 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):
31. Iborra FJ, Jackson DA, Cook PR (2001) Coupled transcription and translation within
nuclei of mammalian cells. Science 293(5532):1139–1142.
32. Dolan BP, Knowlton JJ, David A, Bennink JR, Yewdell JW (2010) RNA polymerase II
inhibitors dissociate antigenic peptide generation from normal viral protein synthesis:
A role for nuclear translation in defective ribosomal product synthesis? J Immunol
33. Gluck F, et al. (2013) EasyProt—an easy-to-use graphical platform for proteomics
data analysis. J Proteomics 79:146–160.
| www.pnas.org/cgi/doi/10.1073/pnas.1309956110Apcher et al.