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
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
- 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
The accumulation of protein aggregates is toxic and linked to different diseases such as neurodegenerative disorders, but the role of the immune system to target and destroy aggregate-carrying cells is still relatively unknown. Here we show a substrate-specific presentation of antigenic peptides to the direct MHC class I pathway via autophagy. We observed no difference in presentation of peptides derived from the viral EBNA1 protein following suppression of autophagy by knocking down Atg5 and Atg12. However, the same knock down treatment suppressed the presentation from ovalbumin. Fusing the aggregate-prone poly-glutamine (PolyQ) to the ovalbumin had no effect on antigen presentation via autophagy. Interestingly, fusing the EBNA1-derived gly-ala repeat (GAr) sequence to ovalbumin rendered the presentation Atg5/12 independent. We also demonstrate that the relative levels of protein expression did not affect autophagy-mediated antigen presentation. These data suggest a substrate-dependent presentation of antigenic peptides for the MHC class I pathway via autophagy and indicate that the GAr of the EBNA1 illustrates a novel virus-mediated mechanism for immune evasion of autophagy-dependent antigen presentation.
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
Peptides presented on major histocompatibility (MHC) class I molecules form an essential part of the immune system's capacity to detect virus-infected or transformed cells. Earlier works have shown that pioneer translation peptides (PTPs) for the MHC class I pathway are as efficiently produced from introns as from exons, or from mRNAs targeted for the nonsense-mediated decay pathway. The production of PTPs is a target for viral immune evasion but the underlying molecular mechanisms that govern this non-canonical translation are unknown. Here, we have used different approaches to show how events taking place on the nascent transcript control the synthesis of PTPs and full-length proteins. By controlling the subcellular interaction between the G-quadruplex structure (G4) of a gly-ala encoding mRNA and nucleolin (NCL) and by interfering with mRNA maturation using multiple approaches, we demonstrate that antigenic peptides derive from a nuclear non-canonical translation event that is independently regulated from the synthesis of full-length proteins. Moreover, we show that G4 are exploited to control mRNA localization and translation by distinguishable mechanisms that are targets for viral immune evasion.
- MeSH
- antigeny genetika imunologie MeSH
- buněčné jádro genetika imunologie MeSH
- G-kvadruplexy MeSH
- histokompatibilita - antigeny třídy I genetika imunologie MeSH
- imunitní únik genetika imunologie MeSH
- lidé MeSH
- messenger RNA genetika imunologie MeSH
- nonsense mediated mRNA decay genetika imunologie MeSH
- peptidy genetika imunologie MeSH
- proteosyntéza genetika imunologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Protein-RNA interactions (PRIs) control pivotal steps in RNA biogenesis, regulate multiple physiological and pathological cellular networks, and are emerging as important drug targets. However, targeting of specific protein-RNA interactions for therapeutic developments is still poorly advanced. Studies and manipulation of these interactions are technically challenging and in vitro drug screening assays are often hampered due to the complexity of RNA structures. The binding of nucleolin (NCL) to a G-quadruplex (G4) structure in the messenger RNA (mRNA) of the Epstein-Barr virus (EBV)-encoded EBNA1 has emerged as an interesting therapeutic target to interfere with immune evasion of EBV-associated cancers. Using the NCL-EBNA1 mRNA interaction as a model, we describe a quantitative proximity ligation assay (PLA)-based in cellulo approach to determine the structure activity relationship of small chemical G4 ligands. Our results show how different G4 ligands have different effects on NCL binding to G4 of the EBNA1 mRNA and highlight the importance of in-cellulo screening assays for targeting RNA structure-dependent interactions.
- MeSH
- aminochinoliny chemie MeSH
- biotest metody MeSH
- fosfoproteiny metabolismus MeSH
- G-kvadruplexy * MeSH
- kyseliny pikolinové chemie MeSH
- lidé MeSH
- messenger RNA metabolismus MeSH
- nádorové buněčné linie MeSH
- proteiny vázající RNA metabolismus MeSH
- virus Epsteinův-Barrové - jaderné antigeny genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The c-myc oncogene stimulates ribosomal biogenesis and protein synthesis to promote cellular growth. However, the pathway by which cells sense and restore dysfunctional mRNA translation and how this is linked to cell proliferation and growth is not known. We here show that mRNA translation stress in cis triggered by the gly-ala repeat sequence of Epstein-Barr virus (EBV)-encoded EBNA1, results in PI3Kδ-dependent induction of E2F1 mRNA translation with the consequent activation of c-Myc and cell proliferation. Treatment with a specific PI3Kδ inhibitor Idelalisib (CAL-101) suppresses E2F1 and c-Myc levels and causes cell death in EBNA1-induced B cell lymphomas. Suppression of PI3Kδ prevents E2F1 activation also in non-EBV-infected cells. These data illustrate an mRNA translation stress-response pathway for E2F1 activation that is exploited by EBV to promote cell growth and proliferation, offering new strategies to treat EBV-carrying cancers.
- MeSH
- buněčné linie MeSH
- fosfatidylinositol-3-kinasy třídy I genetika metabolismus MeSH
- interakce hostitele a patogenu genetika MeSH
- lidé MeSH
- messenger RNA genetika MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory genetika patologie virologie MeSH
- proliferace buněk genetika MeSH
- proteosyntéza * MeSH
- regulace genové exprese u nádorů MeSH
- transkripční faktor E2F1 genetika metabolismus MeSH
- virus Epsteinův-Barrové - jaderné antigeny genetika metabolismus MeSH
- virus Epsteinův-Barrové genetika fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The notion that alternative peptide substrates can be processed and presented to the MHC class I pathway has opened for new aspects on how the immune system detects infected or damaged cells. Recent works show that antigenic peptides are derived from intron sequences in pre-mRNAs target for the nonsense-mediated degradation pathway. Introns are spliced out co-transcriptionally suggesting that such pioneer translation products (PTPs) are synthesized on the nascent RNAs in the nuclear compartment to ensure that the first peptides to emerge from an mRNA are destined for the class I pathway. This illustrates an independent translation event during mRNA maturation that give rise to specific peptide products with a specific function in the immune system. The characterization of the translation apparatus responsible for PTP synthesis will pave the way for understanding how PTP production is regulated in different tissues under different conditions and will help designing new vaccine strategies.
- MeSH
- buněčné jádro genetika imunologie MeSH
- CD8-pozitivní T-lymfocyty cytologie imunologie MeSH
- cytosol imunologie metabolismus MeSH
- dendritické buňky cytologie imunologie metabolismus MeSH
- fagozomy genetika imunologie MeSH
- histokompatibilita - antigeny třídy I genetika imunologie MeSH
- introny MeSH
- lidé MeSH
- peptidy genetika imunologie MeSH
- prekurzory RNA genetika imunologie MeSH
- prezentace antigenu genetika MeSH
- proteasomový endopeptidasový komplex genetika imunologie MeSH
- proteosyntéza imunologie MeSH
- sestřih RNA imunologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
