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.

• Keywords
• MHC-I epitope, Pioneer Translation Products, bi-directional transcripts, bi-directional translation, reverse strand antigenic peptides,
• MeSH
• Lymphocyte Activation immunology   MeSH
• RNA, Antisense * genetics   immunology   MeSH
• CD8-Positive T-Lymphocytes * immunology   MeSH
• Leukocytes, Mononuclear immunology   MeSH
• Humans MeSH
• Histocompatibility Antigens Class I * immunology   genetics   MeSH
• Peptides * immunology   genetics   MeSH
• Antigen Presentation MeSH
• Protein Biosynthesis * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• RNA, Antisense * MeSH
• Histocompatibility Antigens Class I * MeSH
• Peptides * MeSH

The oncogenic Epstein-Barr virus (EBV) evades the immune system but has an Achilles heel: its genome maintenance protein EBNA1. Indeed, EBNA1 is essential for viral genome maintenance but is also highly antigenic. Hence, EBV seemingly evolved a system in which the glycine-alanine repeat (GAr) of EBNA1 limits the translation of its own mRNA to the minimal level to ensure its essential function, thereby, at the same time, minimizing immune recognition. Therefore, defining intervention points at which to interfere with GAr-based inhibition of translation is an important step to trigger an immune response against EBV-carrying cancers. The host protein nucleolin (NCL) plays a critical role in this process via a direct interaction with G-quadruplexes (G4) formed in the GAr-encoding sequence of the viral EBNA1 mRNA. Here we show that the C-terminal arginine-glycine-rich (RGG) motif of NCL is crucial for its role in GAr-based inhibition of translation by mediating interaction of NCL with G4 of EBNA1 mRNA. We also show that this interaction depends on the type I arginine methyltransferase family, notably PRMT1 and PRMT3: drugs or small interfering RNA that target these enzymes prevent efficient binding of NCL on G4 of EBNA1 mRNA and relieve GAr-based inhibition of translation and of antigen presentation. Hence, this work defines type I arginine methyltransferases as therapeutic targets to interfere with EBNA1 and EBV immune evasion.

• MeSH
• Immune System metabolism   MeSH
• Tumor Virus Infections * drug therapy   metabolism   MeSH
• Epstein-Barr Virus Infections * genetics   MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• Oncogenic Viruses genetics   metabolism   MeSH
• Protein-Arginine N-Methyltransferases MeSH
• Repressor Proteins MeSH
• Epstein-Barr Virus Nuclear Antigens genetics   metabolism   MeSH
• Herpesvirus 4, Human * genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Messenger MeSH
• PRMT1 protein, human MeSH Browser
• PRMT2 protein, human MeSH Browser
• Protein-Arginine N-Methyltransferases MeSH
• Repressor Proteins MeSH
• Epstein-Barr Virus Nuclear Antigens MeSH

Protein aggregates and abnormal proteins are toxic and associated with neurodegenerative diseases. There are several mechanisms to help cells get rid of aggregates but little is known on how cells prevent aggregate-prone proteins from being synthesised. The EBNA1 of the Epstein-Barr virus (EBV) evades the immune system by suppressing its own mRNA translation initiation in order to minimize the production of antigenic peptides for the major histocompatibility (MHC) class I pathway. Here we show that the emerging peptide of the disordered glycine-alanine repeat (GAr) within EBNA1 dislodges the nascent polypeptide-associated complex (NAC) from the ribosome. This results in the recruitment of nucleolin to the GAr-encoding mRNA and suppression of mRNA translation initiation in cis. Suppressing NAC alpha (NACA) expression prevents nucleolin from binding to the GAr mRNA and overcomes GAr-mediated translation inhibition. Taken together, these observations suggest that EBNA1 exploits a nascent protein quality control pathway to regulate its own rate of synthesis that is based on sensing the nascent GAr peptide by NAC followed by the recruitment of nucleolin to the GAr-encoding RNA sequence.

• MeSH
• Alanine MeSH
• Phosphoproteins MeSH
• Glycine MeSH
• Epstein-Barr Virus Infections * MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Nucleolin MeSH
• Peptides genetics   MeSH
• Protein Aggregates MeSH
• RNA-Binding Proteins metabolism   MeSH
• Epstein-Barr Virus Nuclear Antigens metabolism   MeSH
• Herpesvirus 4, Human * genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Alanine MeSH
• Phosphoproteins MeSH
• Glycine MeSH
• RNA, Messenger MeSH
• Peptides MeSH
• Protein Aggregates MeSH
• RNA-Binding Proteins MeSH
• Epstein-Barr Virus Nuclear Antigens MeSH

The role of G-quadruplex (G4) RNA structures is multifaceted and controversial. Here, we have used as a model the EBV-encoded EBNA1 and the Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded LANA1 mRNAs. We have compared the G4s in these two messages in terms of nucleolin binding, nuclear mRNA retention, and mRNA translation inhibition and their effects on immune evasion. The G4s in the EBNA1 message are clustered in one repeat sequence and the G4 ligand PhenDH2 prevents all G4-associated activities. The RNA G4s in the LANA1 message take part in similar multiple mRNA functions but are spread throughout the message. The different G4 activities depend on flanking coding and non-coding sequences and, interestingly, can be separated individually. Together, the results illustrate the multifunctional, dynamic and context-dependent nature of G4 RNAs and highlight the possibility to develop ligands targeting specific RNA G4 functions. The data also suggest a common multifunctional repertoire of viral G4 RNA activities for immune evasion.

• MeSH
• G-Quadruplexes * MeSH
• DNA, Intergenic chemistry   genetics   MeSH
• Humans MeSH
• Gene Expression Regulation MeSH
• RNA, Viral MeSH
• RNA chemistry   genetics   MeSH
• RNA Transport MeSH
• Epstein-Barr Virus Nuclear Antigens chemistry   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• EBV-encoded nuclear antigen 1 MeSH Browser
• DNA, Intergenic MeSH
• RNA, Viral MeSH
• RNA MeSH
• Epstein-Barr Virus Nuclear Antigens MeSH

Cell growth requires a high level of protein synthesis and oncogenic pathways stimulate cell proliferation and ribosome biogenesis. Less is known about how cells respond to dysfunctional mRNA translation and how this feeds back into growth regulatory pathways. The Epstein-Barr virus (EBV)-encoded EBNA1 causes mRNA translation stress in cis that activates PI3Kδ. This leads to the stabilization of MDM2, induces MDM2's binding to the E2F1 mRNA and promotes E2F1 translation. The MDM2 serine 166 regulates the interaction with the E2F1 mRNA and deletion of MDM2 C-terminal RING domain results in a constitutive E2F1 mRNA binding. Phosphorylation on serine 395 following DNA damage instead regulates p53 mRNA binding to its RING domain and prevents the E2F1 mRNA interaction. The p14Arf tumour suppressor binds MDM2 and in addition to preventing degradation of the p53 protein it also prevents the E2F1 mRNA interaction. The data illustrate how two MDM2 domains selectively bind specific mRNAs in response to cellular conditions to promote, or suppress, cell growth and how p14Arf coordinates MDM2's activity towards p53 and E2F1. The data also show how EBV via EBNA1-induced mRNA translation stress targets the E2F1 and the MDM2 - p53 pathway.

• MeSH
• Cell Cycle genetics   MeSH
• Phosphorylation genetics   MeSH
• Carcinogenesis genetics   MeSH
• Humans MeSH
• RNA, Messenger genetics   MeSH
• Tumor Suppressor Protein p14ARF genetics   MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• Neoplasms genetics   virology   MeSH
• Oncogenes genetics   MeSH
• DNA Damage genetics   MeSH
• Cell Proliferation genetics   MeSH
• Protein Domains genetics   MeSH
• Proto-Oncogene Proteins c-mdm2 genetics   MeSH
• RNA Recognition Motif Proteins genetics   MeSH
• E2F1 Transcription Factor genetics   MeSH
• Genes, Tumor Suppressor MeSH
• Herpesvirus 4, Human genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• E2F1 protein, human MeSH Browser
• MDM2 protein, human MeSH Browser
• RNA, Messenger MeSH
• Tumor Suppressor Protein p14ARF MeSH
• Tumor Suppressor Protein p53 MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH
• RNA Recognition Motif Proteins MeSH
• TP53 protein, human MeSH Browser
• E2F1 Transcription Factor MeSH