Antisense transcripts play an important role in generating regulatory non-coding RNAs but whether these transcripts are also translated to generate functional peptides remains poorly understood. In this study, RNA sequencing and six-frame database generation were combined with mass spectrometry analysis of peptides isolated from polysomes to identify Nascent Pioneer Translation Products (Na-PTPs) originating from alternative reading frames of bi-directional transcripts. Two Na-PTP originating peptides derived from antisense strands stimulated CD8+ T cell proliferation when presented to peripheral blood mononuclear cells (PBMCs) from nine healthy donors. Importantly, an antigenic peptide derived from the reverse strand of two cDNA constructs was presented on MHC-I molecules and induced CD8+ T cell activation. The results demonstrate that three-frame translation of bi-directional transcripts generates antigenic peptide substrates for the immune system. This discovery holds significance for understanding the origin of self-discriminating peptide substrates for the major histocompatibility class I (MHC-I) pathway and for enhancing immune-based therapies against infected or transformed cells.

Department of Medical Biosciences Umeå University Umeå Sweden

Inserm UMRS1131 Institut de Génétique Moléculaire Université Paris Cité Hôpital St Louis Paris France

Laboratory of Growth Regulators Institute of Experimental Botany The Czech Academy of Sciences Olomouc Czechia

RECAMO Masaryk Memorial Cancer Institute Brno Czechia

UMR 1015 Immunologie des tumeurs et immunothérapie contre le cancer B2M Gustave Roussy Université Paris Sud Villejuif France

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Blum JS, Wearsch PA, Cresswell P. Pathways of antigen processing. Annu Rev Immunol. (2013) 31:443–73. doi: 10.1146/annurev-immunol-032712-095910 PubMed DOI PMC

van de Weijer ML, Luteijn RD, Wiertz EJ. Viral immune evasion: Lessons in MHC class I antigen presentation. Semin Immunol. (2015) 27:125–37. doi: 10.1016/j.smim.2015.03.010 PubMed DOI

Dersh D, Holly J, Yewdell JW. A few good peptides: MHC class I-based cancer immunosurveillance and immunoevasion. Nat Rev Immunol. (2021) 21:116–28. doi: 10.1038/s41577-020-0390-6 PubMed DOI

Zhang Y, Zhang Z. The history and advances in cancer immunotherapy: understanding the characteristics of tumor-infiltrating immune cells and their therapeutic implications. Cell Mol Immunol. (2020) 17:807–21. doi: 10.1038/s41423-020-0488-6 PubMed DOI PMC

Chen J, Brunner AD, Cogan JZ, Nunez JK, Fields AP, Adamson B, et al. . Pervasive functional translation of noncanonical human open reading frames. Science. (2020) 367:1140–6. doi: 10.1126/science.aay0262 PubMed DOI PMC

Ingolia NT, Brar GA, Stern-Ginossar N, Harris MS, Talhouarne GJ, Jackson SE, et al. . Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes. Cell Rep. (2014) 8:1365–79. doi: 10.1016/j.celrep.2014.07.045 PubMed DOI PMC

Laumont CM, Vincent K, Hesnard L, Audemard E, Bonneil E, Laverdure JP, et al. . Noncoding regions are the main source of targetable tumor-specific antigens. Sci Transl Med. (2018) 10(470). doi: 10.1126/scitranslmed.aau5516 PubMed DOI

Starck SR, Shastri N. Nowhere to hide: unconventional translation yields cryptic peptides for immune surveillance. Immunol Rev. (2016) 272:8–16. doi: 10.1111/imr.12434 PubMed DOI PMC

Weingarten-Gabbay S, Klaeger S, Sarkizova S, Pearlman LR, Chen DY, Gallagher KME, et al. . Profiling SARS-CoV-2 HLA-I peptidome reveals T cell epitopes from out-of-frame ORFs. Cell. (2021) 184:3962–3980 e3917. doi: 10.1016/j.cell.2021.05.046 PubMed DOI PMC

Camarena ME, Theunissen P, Ruiz M, Ruiz-Orera J, Calvo-Serra B, Castelo R, et al. . Microproteins encoded by noncanonical ORFs are a major source of tumor-specific antigens in a liver cancer patient meta-cohort. Sci Adv. (2024) 10:eadn3628. doi: 10.1126/sciadv.adn3628 PubMed DOI PMC

Barczak W, Carr SM, Liu G, Munro S, Nicastri A, Lee LN, et al. . Long non-coding RNA-derived peptides are immunogenic and drive a potent anti-tumour response. Nat Commun. (2023) 14:1078. doi: 10.1038/s41467-023-36826-0 PubMed DOI PMC

Lv D, Chang Z, Cai Y, Li J, Wang L, Jiang Q, et al. . TransLnc: a comprehensive resource for translatable lncRNAs extends immunopeptidome. Nucleic Acids Res. (2022) 50:D413–20. doi: 10.1093/nar/gkab847 PubMed DOI PMC

Apcher S, Daskalogianni C, Lejeune F, Manoury B, Imhoos G, Heslop L, et al. . Major source of antigenic peptides for the MHC class I pathway is produced during the pioneer round of mRNA translation. Proc Natl Acad Sci U.S.A. (2011) 108:11572–7. doi: 10.1073/pnas.1104104108 PubMed DOI PMC

Schwab SR, Li KC, Kang C, Shastri N. Constitutive display of cryptic translation products by MHC class I molecules. Science. (2003) 301:1367–71. doi: 10.1126/science.1085650 PubMed DOI

Apcher S, Millot G, Daskalogianni C, Scherl A, Manoury B, Fahraeus R. Translation of pre-spliced RNAs in the nuclear compartment generates peptides for the MHC class I pathway. Proc Natl Acad Sci United States America. (2013) 110:17951–6. doi: 10.1073/pnas.1309956110 PubMed DOI PMC

Martins RP, Malbert-Colas L, Lista MJ, Daskalogianni C, Apcher S, Pla M, et al. . Nuclear processing of nascent transcripts determines synthesis of full-length proteins and antigenic peptides. Nucleic Acids Res. (2019) 47:3086–100. doi: 10.1093/nar/gky1296 PubMed DOI PMC

Fahraeus R. Has translation in the nucleus found its purpose? Nat Rev Mol Cell Biol. (2024) 25:1–2. doi: 10.1038/s41580-023-00651-6 PubMed DOI

Core LJ, Waterfall JJ, Lis JT. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science. (2008) 322:1845–8. doi: 10.1126/science.1162228 PubMed DOI PMC

Preker P, Nielsen J, Schierup MH, Jensen TH. RNA polymerase plays both sides: vivid and bidirectional transcription around and upstream of active promoters. Cell Cycle. (2009) 8:1106–7. doi: 10.4161/cc.8.8.8236 PubMed DOI

He Y, Vogelstein B, Velculescu VE, Papadopoulos N, Kinzler KW. The antisense transcriptomes of human cells. Science. (2008) 322:1855–7. doi: 10.1126/science.1163853 PubMed DOI PMC

Seila AC, Core LJ, Lis JT, Sharp PA. Divergent transcription: a new feature of active promoters. Cell Cycle. (2009) 8:2557–64. doi: 10.4161/cc.8.16.9305 PubMed DOI

Scruggs BS, Gilchrist DA, Nechaev S, Muse GW, Burkholder A, Fargo DC, et al. . Bidirectional transcription arises from two distinct hubs of transcription factor binding and active chromatin. Mol Cell. (2015) 58:1101–12. doi: 10.1016/j.molcel.2015.04.006 PubMed DOI PMC

Sroka EM, Lavigne M, Pla M, Daskalogianni C, Tovar-Fernandez MC, Prado Martins R, et al. . Major histocompatibility class I antigenic peptides derived from translation of pre-mRNAs generate immune tolerance. Proc Natl Acad Sci U.S.A. (2023) 120:e2208509120. doi: 10.1073/pnas.2208509120 PubMed DOI PMC

Reynisson B, Alvarez B, Paul S, Peters B, Nielsen M. NetMHCpan-4.1 and NetMHCIIpan-4.0: improved predictions of MHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data. Nucleic Acids Res. (2020) 48:W449–54. doi: 10.1093/nar/gkaa379 PubMed DOI PMC

Zaks TZ, Rosenberg SA. Immunization with a peptide epitope (p369-377) from HER-2/neu leads to peptide-specific cytotoxic T lymphocytes that fail to recognize HER-2/neu+ tumors. Cancer Res. (1998) 58:4902–8. PubMed

Clarke SR, Barnden M, Kurts C, Carbone FR, Miller JF, Heath WR. Characterization of the ovalbumin-specific TCR transgenic line OT-I: MHC elements for positive and negative selection. Immunol Cell Biol. (2000) 78:110–7. doi: 10.1046/j.1440-1711.2000.00889.x PubMed DOI

Apcher S, Tovar-Fernadez M, Ducellier S, Thermou A, Nascimento M, Sroka E, et al. . mRNA translation from an antigen presentation perspective: A tribute to the works of Nilabh Shastri. Mol Immunol. (2022) 141:305–8. doi: 10.1016/j.molimm.2021.12.010 PubMed DOI

Najafi S, Tan SC, Raee P, Rahmati Y, Asemani Y, Lee EHC, et al. . Gene regulation by antisense transcription: A focus on neurological and cancer diseases. BioMed Pharmacother. (2022) 145:112265. doi: 10.1016/j.biopha.2021.112265 PubMed DOI

Pelechano V, Steinmetz LM. Gene regulation by antisense transcription. Nat Rev Genet. (2013) 14:880–93. doi: 10.1038/nrg3594 PubMed DOI

Katsikis PD, Ishii KJ, Schliehe C. Challenges in developing personalized neoantigen cancer vaccines. Nat Rev Immunol. (2024) 24:213–27. doi: 10.1038/s41577-023-00937-y PubMed DOI

Darrigrand R, Pierson A, Rouillon M, Renko D, Boulpicante M, Bouyssie D, et al. . Isoginkgetin derivative IP2 enhances the adaptive immune response against tumor antigens. Commun Biol. (2021) 4:269. doi: 10.1038/s42003-021-01801-2 PubMed DOI PMC

Lu SX, De Neef E, Thomas JD, Sabio E, Rousseau B, Gigoux M, et al. . Pharmacologic modulation of RNA splicing enhances anti-tumor immunity. Cell. (2021) 184:4032–4047 e4031. doi: 10.1016/j.cell.2021.05.038 PubMed DOI PMC

Xie N, Shen G, Gao W, Huang Z, Huang C, Fu L. Neoantigens: promising targets for cancer therapy. Signal Transduct Target Ther. (2023) 8:9. doi: 10.1038/s41392-022-01270-x PubMed DOI PMC

Cai Y, Chen R, Gao S, Li W, Liu Y, Su G, et al. . Artificial intelligence applied in neoantigen identification facilitates personalized cancer immunotherapy. Front Oncol. (2022) 12:1054231. doi: 10.3389/fonc.2022.1054231 PubMed DOI PMC

Charneau J, Suzuki T, Shimomura M, Fujinami N, Mishima Y, Hiranuka K, et al. . Development of antigen-prediction algorithm for personalized neoantigen vaccine using human leukocyte antigen transgenic mouse. Cancer Sci. (2022) 113:1113–24. doi: 10.1111/cas.15291 PubMed DOI PMC

Gnanasundram SV, Pyndiah S, Daskalogianni C, Armfield K, Nylander K, Wilson JB, et al. . PI3Kdelta activates E2F1 synthesis in response to mRNA translation stress. Nat Commun. (2017) 8:2103. doi: 10.1038/s41467-017-02282-w PubMed DOI PMC

Deutsch EW, Bandeira N, Perez-Riverol Y, Sharma V, Carver JJ, Mendoza L, et al. . The ProteomeXchange consortium at 10 years: 2023 update. Nucleic Acids Res. (2023) 51:D1539–48. doi: 10.1093/nar/gkac1040 PubMed DOI PMC

Perez-Riverol Y, Bai J, Bandla C, Garcia-Seisdedos D, Hewapathirana S, Kamatchinathan S, et al. . The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. (2022) 50:D543–52. doi: 10.1093/nar/gkab1038 PubMed DOI PMC

Cunningham F, Allen JE, Allen J, Alvarez-Jarreta J, Amode MR, Armean IM, et al. . Ensembl 2022. Nucleic Acids Res. (2022) 50:D988–95. doi: 10.1093/nar/gkab1049 PubMed DOI PMC

Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. (2013) 14:R36. doi: 10.1186/gb-2013-14-4-r36 PubMed DOI PMC

Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, et al. . Twelve years of SAMtools and BCFtools. Gigascience. (2021) 10(2). doi: 10.1093/gigascience/giab008 PubMed DOI PMC

Koboldt DC, Zhang Q, Larson DE, Shen D, McLellan MD, Lin L, et al. . VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res. (2012) 22(3):568–76. doi: 10.1101/gr.129684.111 PubMed DOI PMC