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.

• Klíčová slova
• MHC-I epitope, Pioneer Translation Products, bi-directional transcripts, bi-directional translation, reverse strand antigenic peptides,
• MeSH
• aktivace lymfocytů imunologie   MeSH
• antisense RNA * genetika   imunologie   MeSH
• CD8-pozitivní T-lymfocyty * imunologie   MeSH
• leukocyty mononukleární imunologie   MeSH
• lidé MeSH
• MHC antigeny I. třídy * imunologie   genetika   MeSH
• peptidy * imunologie   genetika   MeSH
• prezentace antigenu MeSH
• proteosyntéza * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antisense RNA * MeSH
• MHC antigeny I. třídy * MeSH
• peptidy * 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
• imunitní systém metabolismus   MeSH
• infekce onkogenními viry * farmakoterapie   metabolismus   MeSH
• infekce virem Epsteina-Barrové * genetika   MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• onkogenní viry genetika   metabolismus   MeSH
• proteinarginin-N-methyltransferasy MeSH
• represorové proteiny MeSH
• virus Epsteinův-Barrové - jaderné antigeny genetika   metabolismus   MeSH
• virus Epsteinův-Barrové * genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• PRMT1 protein, human MeSH Prohlížeč
• PRMT2 protein, human MeSH Prohlížeč
• proteinarginin-N-methyltransferasy MeSH
• represorové proteiny MeSH
• virus Epsteinův-Barrové - jaderné antigeny 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
• alanin MeSH
• fosfoproteiny MeSH
• glycin MeSH
• infekce virem Epsteina-Barrové * MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• nukleolin MeSH
• peptidy genetika   MeSH
• proteinové agregáty MeSH
• proteiny vázající RNA metabolismus   MeSH
• virus Epsteinův-Barrové - jaderné antigeny metabolismus   MeSH
• virus Epsteinův-Barrové * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alanin MeSH
• fosfoproteiny MeSH
• glycin MeSH
• messenger RNA MeSH
• peptidy MeSH
• proteinové agregáty MeSH
• proteiny vázající RNA MeSH
• virus Epsteinův-Barrové - jaderné antigeny 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-kvadruplexy * MeSH
• intergenová DNA chemie   genetika   MeSH
• lidé MeSH
• regulace genové exprese MeSH
• RNA virová MeSH
• RNA chemie   genetika   MeSH
• transport RNA MeSH
• virus Epsteinův-Barrové - jaderné antigeny chemie   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• EBV-encoded nuclear antigen 1 MeSH Prohlížeč
• intergenová DNA MeSH
• RNA virová MeSH
• RNA MeSH
• virus Epsteinův-Barrové - jaderné antigeny 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
• buněčný cyklus genetika   MeSH
• fosforylace genetika   MeSH
• karcinogeneze genetika   MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• nádorový supresorový protein p14ARF genetika   MeSH
• nádorový supresorový protein p53 genetika   MeSH
• nádory genetika   virologie   MeSH
• onkogeny genetika   MeSH
• poškození DNA genetika   MeSH
• proliferace buněk genetika   MeSH
• proteinové domény genetika   MeSH
• protoonkogenní proteiny c-mdm2 genetika   MeSH
• RRM proteiny genetika   MeSH
• transkripční faktor E2F1 genetika   MeSH
• tumor supresorové geny MeSH
• virus Epsteinův-Barrové genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• E2F1 protein, human MeSH Prohlížeč
• MDM2 protein, human MeSH Prohlížeč
• messenger RNA MeSH
• nádorový supresorový protein p14ARF MeSH
• nádorový supresorový protein p53 MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• RRM proteiny MeSH
• TP53 protein, human MeSH Prohlížeč
• transkripční faktor E2F1 MeSH