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
- imunitní únik genetika imunologie MeSH
- lidé MeSH
- messenger RNA genetika imunologie MeSH
- MHC antigeny I. třídy 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
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
- introny MeSH
- lidé MeSH
- MHC antigeny I. třídy genetika imunologie 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
Yeast cells in general are known to be difficult to prepare for electron microscopy investigations particularly when the preservation of antigenicity is required. In this work, we compare various protocols for preparation of Saccharomyces cerevisiae cells for immunoelectron microscopy, ranging from classical chemical fixation to high-pressure freezing followed by freeze-substitution in different kinds of substitution media. Our aim was to establish a protocol giving optimal, routinely reproducible results for simultaneous retention of fine ultrastructural details and antigen immunoreactivity, with particular focus on the preservation of nuclear and nucleolar architecture. This was demonstrated by ultrastructural immunolocalization of various nucleolar (Nop1 and Nsr1), nuclear (Nsp1) and alpha-tubulin antigens. The protocol which we found to yield the best preserved Saccharomyces cerevisiae cells for both morphological and immunological studies included cryo-fixation by high-pressure freezing followed by freeze-substitution in acetone with 0.1% uranyl acetate and embedding in Lowicryl HM20. In addition, immunofluorescence detection of the antigens was performed and correlated with immunolabelling at the electron microscopy level.
- MeSH
- aceton chemie MeSH
- antigeny fungální chemie MeSH
- buněčné jadérko imunologie ultrastruktura MeSH
- buněčné jádro imunologie ultrastruktura MeSH
- financování organizované MeSH
- fluorescenční mikroskopie MeSH
- formaldehyd chemie MeSH
- glutaraldehyd chemie MeSH
- imunoelektronová mikroskopie metody MeSH
- mrazová substituce MeSH
- organokovové sloučeniny chemie MeSH
- Saccharomyces cerevisiae ultrastruktura MeSH
- transmisní elektronová mikroskopie MeSH
- MeSH
- akrodermatitida etiologie imunologie metabolismus MeSH
- buněčné jádro imunologie metabolismus patologie MeSH
- financování organizované MeSH
- gama-globuliny nedostatek MeSH
- imunologické testy metody využití MeSH
- kojenec MeSH
- lidé MeSH
- mateřské mléko chemie imunologie metabolismus MeSH
- nefelometrie a turbidimetrie metody využití MeSH
- nemoci imunitního systému diagnóza komplikace MeSH
- průjem kojenců etiologie MeSH
- průtoková cytometrie metody využití MeSH
- respirační vzplanutí účinky léků MeSH
- spektrofotometrie metody využití MeSH
- tvar buňky MeSH
- zinek metabolismus nedostatek terapeutické užití MeSH
- Check Tag
- kojenec MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
A protocol for high-pressure freezing and LR White embedding of mammalian cells suitable for fine ultrastructural studies in combination with immunogold labelling is presented. HeLa S3 cells enclosed in low-temperature gelling agarose were high-pressure frozen, freeze-substituted in acetone, and embedded in LR White at 0 degrees C. The morphology of such cells and the preservation of nuclear antigens were excellent in comparison with chemically fixed cells embedded in the same resin. The immunolabelling signal for different nuclear antigens was 4-to-13 times higher in high-pressure frozen than in chemically fixed cells. We conclude that one can successfully use high-pressure freezing/freeze-substitution and LR White embedding as an alternative of Lowicryl resins.
- MeSH
- akrylové pryskyřice MeSH
- antigeny jaderné analýza MeSH
- buněčné jádro imunologie ultrastruktura MeSH
- financování organizované MeSH
- HeLa buňky MeSH
- imunohistochemie MeSH
- kryoprezervace metody MeSH
- lidé MeSH
- mrazová substituce MeSH
- tlak MeSH
- transmisní elektronová mikroskopie MeSH
- zalévání tkání plastickou hmotou metody MeSH
- Check Tag
- lidé MeSH
- MeSH
- autoimunitní nemoci imunologie MeSH
- autoprotilátky analýza MeSH
- buněčné jádro imunologie MeSH
- dospělí MeSH
- lidé MeSH
- nadledviny imunologie MeSH
- ovarium imunologie MeSH
- štítná žláza etiologie imunologie komplikace MeSH
- vlasový folikul imunologie MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- MeSH
- antigeny imunologie izolace a purifikace MeSH
- buněčné jádro imunologie ultrastruktura MeSH
- elektroforéza využití MeSH
- hepatocelulární adenom imunologie MeSH
- imunochemie metody MeSH
- játra cytologie imunologie MeSH
- králíci MeSH
- křečci praví MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- křečci praví MeSH
- zvířata MeSH
