Sébastien Apcher

Major source of antigenic peptides for the MHC class I pathway is produced during the pioneer round of mRNA translation

The MHC class I antigen presentation pathway allows the immune system to distinguish between self and nonself. Despite extensive research on the processing of antigenic peptides, little is known about their origin. Here, we show that mRNAs carrying premature stop codons that prevent the production of full-length proteins via the nonsense-mediated decay pathway still produce a majority of peptide substrates for the MHC class I pathway by a noncanonical mRNA translation process. Blocking the interaction of the translation initiation factor eIF4E with the cap structure suppresses the synthesis of full-length proteins but has only a limited effect on the production of antigenic peptides. These results reveal an essential cell biological function for a class of translation products derived during the pioneer round of mRNA translation and will have important implications for understanding how the immune system detects cells harboring pathogens and generates tolerance.

Translation of pre-spliced RNAs in the nuclear compartment generates peptides for the MHC class I pathway.

Significance The major histocompatibility complex (MHC) class I antigen presentation pathway allows the immune system to distinguish between self and non-self. Despite extensive research on the processing of antigenic peptides, little is still known about their origin. We recently proposed that a unique class of peptides, termed pioneer translation products (PTPs), is produced during the pioneer rounds of mRNA translation and provides the major source of antigenic peptide substrates for direct presentation to the MHC class I pathway. Here we show that a major portion of the substrates for the MHC class I pathway is synthesized during the early steps of mRNA maturation via a noncanonical translation mechanism within the nucleus and before introns are spliced out. The scanning of maturing mRNAs by ribosomes plays a key role in the mRNA quality control process. When ribosomes first engage with the newly synthesized mRNA, and if peptides are produced, is unclear, however. Here we show that ribosomal scanning of prespliced mRNAs occurs in the nuclear compartment, and that this event produces peptide substrates for the MHC class I pathway. Inserting antigenic peptide sequences in introns that are spliced out before the mRNAs exit the nuclear compartment results in an equal amount of antigenic peptide products as when the peptides are encoded from the main open reading frame (ORF). Taken together with the detection of intron-encoded nascent peptides and RPS6/RPL7-carrying complexes in the perinucleolar compartment, these results show that peptides are produced by a translation event occurring before mRNA splicing. This suggests that ribosomes occupy and scan mRNAs early in the mRNA maturation process, and suggests a physiological role for nuclear mRNA translation, and also helps explain how the immune system tolerates peptides derived from tissue-specific mRNA splice variants.

Melanoma miRNA trafficking controls tumour primary niche formation

Melanoma originates in the epidermis and becomes metastatic after invasion into the dermis. Prior interactions between melanoma cells and dermis are poorly studied. Here, we show that melanoma cells directly affect the formation of the dermal tumour niche by microRNA trafficking before invasion. Melanocytes, cells of melanoma origin, are specialized in releasing pigment vesicles, termed melanosomes. In melanoma in situ, we found melanosome markers in distal fibroblasts before melanoma invasion. The melanosomes carry microRNAs into primary fibroblasts triggering changes, including increased proliferation, migration and pro-inflammatory gene expression, all known features of cancer-associated fibroblasts (CAFs). Specifically, melanosomal microRNA-211 directly targets IGF2R and leads to MAPK signalling activation, which reciprocally encourages melanoma growth. Melanosome release inhibitor prevented CAF formation. Since the first interaction of melanoma cells with blood vessels occurs in the dermis, our data suggest an opportunity to block melanoma invasion by preventing the formation of the dermal tumour niche.

mRNA Translation Regulation by the Gly-Ala Repeat of Epstein-Barr Virus Nuclear Antigen 1

ABSTRACT The glycine-alanine repeat (GAr) sequence of the Epstein-Barr virus-encoded EBNA-1 prevents presentation of antigenic peptides to major histocompatibility complex class I molecules. This has been attributed to its capacity to suppress mRNA translation in cis. However, the underlying mechanism of this function remains largely unknown. Here, we have further investigated the effect of the GAr as a regulator of mRNA translation. Introduction of silent mutations in each codon of a 30-amino-acid GAr sequence does not significantly affect the translation-inhibitory capacity, whereas minimal alterations in the amino acid composition have strong effects, which underscores the observation that the amino acid sequence and not the mRNA sequence mediates GAr-dependent translation suppression. The capacity of the GAr to repress translation is dose and position dependent and leads to a relative accumulation of preinitiation complexes on the mRNA. Taken together with the surprising observation that fusion of the 5′ untranslated region (UTR) of the c-myc mRNA to the 5′ UTR of GAr-carrying mRNAs specifically inactivates the effect of the GAr, these results indicate that the GAr targets components of the translation initiation process. We propose a model in which the nascent GAr peptide delays the assembly of the initiation complex on its own mRNA.

Epstein Barr Virus-Encoded EBNA1 Interference with MHC Class I Antigen Presentation Reveals a Close Correlation between mRNA Translation Initiation and Antigen Presentation

Viruses are known to employ different strategies to manipulate the major histocompatibility (MHC) class I antigen presentation pathway to avoid recognition of the infected host cell by the immune system. However, viral control of antigen presentation via the processes that supply and select antigenic peptide precursors is yet relatively unknown. The Epstein-Barr virus (EBV)-encoded EBNA1 is expressed in all EBV-infected cells, but the immune system fails to detect and destroy EBV-carrying host cells. This immune evasion has been attributed to the capacity of a Gly-Ala repeat (GAr) within EBNA1 to inhibit MHC class I restricted antigen presentation. Here we demonstrate that suppression of mRNA translation initiation by the GAr in cis is sufficient and necessary to prevent presentation of antigenic peptides from mRNAs to which it is fused. Furthermore, we demonstrate a direct correlation between the rate of translation initiation and MHC class I antigen presentation from a certain mRNA. These results support the idea that mRNAs, and not the encoded full length proteins, are used for MHC class I restricted immune surveillance. This offers an additional view on the role of virus-mediated control of mRNA translation initiation and of the mechanisms that control MHC class I restricted antigen presentation in general.

Gly-Ala repeats induce position- and substrate-specific regulation of 26 S proteasome-dependent partial processing.

Partial degradation or regulated ubiquitin proteasome-dependent processing by the 26 S proteasome has been demonstrated, but the underlying molecular mechanisms and the prevalence of this phenomenon remain obscure. Here we show that the Gly-Ala repeat (GAr) sequence of EBNA1 affects processing of substrates via the ubiquitin-dependent degradation pathway in a substrate- and position-specific fashion. GAr-mediated increase in stability of proteins targeted for degradation via the 26 S proteasome was associated with a fraction of the substrates being partially processed and the release of the free GAr. The GAr did not cause a problem for the proteolytic activity of the proteasome, and its fusion to the N terminus of p53 resulted in an increase in the rate of degradation of the entire chimera. Interestingly the GAr had little effect on the stability of EBNA1 protein itself, and targeting EBNA1 for 26 S proteasome-dependent degradation led to its complete degradation. Taken together, our data suggest a model in which the GAr prevents degradation or promotes endoproteolytic processing of substrates targeted for the 26 S proteasome by interfering with the initiation step of substrate unfolding. These results will help to further understand the underlying mechanisms for partial proteasome-dependent degradation.

The cell-penetrating peptide octa-arginine is a potent inhibitor of proteasome activities.

Oligo-arginines are cell-penetrating peptides and find use as carriers for transportation of various membrane-impermeable biopharmaceuticals into target cells. We have found that oligo-arginines of a length of 4-10 amino acids, but especially (Arg)(8), are able to inhibit the major intracellular proteolytic system, the proteasome, with mixed-type inhibition characteristics. The IC(50) values of (Arg)(8) for the proteasomal chymotrypsin-like and caspase-like activities are approximately 100 and 200 nM, respectively. The inhibition of the trypsin-like activity never exceeds 50% even at micromolar concentrations. (Arg)(8) also inhibits 20S proteasome/PA28 complexes as well as 26S proteasomes, although with a decreased efficiency. Due to its cell membrane-penetrating capability, incubation of HeLa cells in the presence of (Arg)(8) resulted in an impaired activity of proteasomes going along with an accumulation of high-molecular mass ubiquitin-conjugated proteins, the preferred substrates of 26S proteasomes. The in vivo susceptibility of the three proteasome activities resembles that found in vitro with chymotrypsin-like>caspase-like>trypsin-like activities. Since inhibition of the proteasome system might affect fundamental basic cellular processes but on the other side might also prevent the degradation of a proteinacous cargo, we suggest that this proteasome inhibitory activity should be taken into account when oligo-arginines are being considered for use as vectors for the intracellular delivery of pharmaceuticals.

The role of mRNA translation in direct MHC class I antigen presentation

It has been over 40 years since it was discovered that the major histocompatibility (MHC) class I molecules present peptides for the CD8 T cell-mediated immune response. The pathways for delivering and processing peptides for the MHC class I complexes have since been thoroughly studied but it is only until more recently that we start to understand the mechanisms that provide peptide material for the endogenous MHC class I pathway. Interestingly, the main source of antigenic peptide substrates does not come from the same mRNA translation mechanism that produces full length proteins but from ribosomes which products sole purpose might be to produce antigenic peptide substrates. We will discuss the latest development in this field and its implications for a better understanding of one of the corner stones for how the immune system distinguishes between self and non-self.

Beyond the classical: Influenza virus and the elucidation of alternative MHC class II-restricted antigen processing pathways

CD4+ T cells (TCD4+) are activated by peptides, generally 13–17 amino acids in length, presented at the cell surface in combination with highly polymorphic MHC class II molecules. According to the classical model, these peptides are generated by endosomal digestion of internalized antigen and loaded onto MHC class II molecules in the late endosome. Historically, this “exogenous” pathway has been defined through the extensive use of purified proteins. However, the relatively recent use of clinically relevant antigens, those of influenza virus in our case, has revealed several additional pathways of peptide production, including some that are truly “endogenous”, entailing synthesis of the protein within the infected cell. Indeed, some peptides appear to be created only via endogenous processing. The cell biology that underlies these alternative pathways remains poorly understood as do their relative contributions to defence against infectious agents and cancer, and the triggering of autoimmune diseases.

The source of MHC class I presented peptides and its implications.

The source of peptides that enter the major histocompatibility class I (MHCI) pathway has been intensively debated over the last two decades. The initial assumption that peptides are derived from degradation of full length proteins was challenged by a model in which alternative translation products are a source of peptides. This model has been tested and supported by scientific data. We now need new hypotheses on the physiological implications of different sources of peptides for the MHCI pathway. The aim of this overview is to give an up-to-date account of the source of antigenic peptide material for the MHCI pathway and to incorporate the more recent observations of alternative mRNA translation products into existing models of the direct and cross-presentation pathways.