Pioneer Round Of Translation Research Articles

Regulation of DNA damage-induced HLA class I presentation

Immune checkpoint inhibitors (ICI) are cancer therapies that restore anti-tumor immunity; however, only a small percentage of patients have been completely cured by ICI alone. Multiple approaches in combination with other modalities have been used to improve the efficacy of ICI therapy. Among conventional cancer treatments, radiotherapy or DNA damage-based chemotherapy is a promising candidate as a partner of ICI because DNA damage signaling potentially stimulates immune activities turning the tumor’s immune environment into hot tumors. Programmed death-ligand 1 (PD-L1) and human leukocyte antigen class I (HLA-I), which are immune ligands, regulate the balance of anti-tumor immunity in the tumor microenvironment. PD-L1 functions as a brake to suppress cytotoxic T cell activity, whereas HLA-I is an immune accelerator that promotes the downstream of the T cell signaling. Accumulating evidence has demonstrated that DNA damage enhances the presentation of HLA-I on the surface of damaged cells. However, it is unclear how signal transduction in DNA-damaged cells upregulates the presentation of HLA-I with antigens. Our recent study uncovered the mechanism underlying DNA damage-induced HLA-I presentation, which requires polypeptide synthesis through a pioneer round of translation. In this review, we summarize the latest overview of how DNA damage stimulates antigen production presented by HLA-I.

Alternative sources of antigenic peptides for self and non-self recognition

MHC class I presentation pathway plays a crucial role in the immune system and allows to distinguish self from non-self. In the thymus, developing thymocytes undergo positive and negative selection once exposed to self-peptide:MHC complexes on cortical (cTECs) and medullar (mTECs) thymic epithelial cells as well as some antigen presenting cells (e.g. macrophages and dendritic cells). In the result of this process the immune system is capable of recognizing and responding to foreign peptides presented on self-MHC molecules. Discoveries of alternative sources of antigenic peptides (e.g. DRIPs and PTPs) challenged the notion about full-length proteins being the main supplier of material for the MHC class I pathway and shifted a focus of search for new sources to ribosomal scanning during pioneer round of translation. Works related to Epstein-Barr virus has shown that MHC class I immune surveillance is directly correlated with the mechanism that regulates protein synthesis. Together with other recent results it highlights the importance of pre-mRNA and mRNA processing in providing antigenic peptides for MHC class I surveillance. The lack of animal models to study the origin of alternative antigenic peptides hinders research in the field of neoantigens. I will describe results of the presentation of intron-derived antigenic peptides in mice model developed by our team. We will also revise some significant research related to the production of alternative antigenic peptides, their importance in cancer research, immunosurveillance and generation of tolerance.

DNA damage promotes HLA class I presentation by stimulating a pioneer round of translation-associated antigen production

Antigen presentation by the human leukocyte antigen (HLA) on the cell surface is critical for the transduction of the immune signal toward cytotoxic T lymphocytes. DNA damage upregulates HLA class I presentation; however, the mechanism is unclear. Here, we show that DNA-damage-induced HLA (di-HLA) presentation requires an immunoproteasome, PSMB8/9/10, and antigen-transporter, TAP1/2, demonstrating that antigen production is essential. Furthermore, we show that di-HLA presentation requires ATR, AKT, mTORC1, and p70-S6K signaling. Notably, the depletion of CBP20, a factor initiating the pioneer round of translation (PRT) that precedes nonsense-mediated mRNA decay (NMD), abolishes di-HLA presentation, suggesting that di-antigen production requires PRT. RNA-seq analysis demonstrates that DNA damage reduces NMD transcripts in an ATR-dependent manner, consistent with the requirement for ATR in the initiation of PRT/NMD. Finally, bioinformatics analysis identifies that PRT-derived 9-mer peptides bind to HLA and are potentially immunogenic. Therefore, DNA damage signaling produces immunogenic antigens by utilizing the machinery of PRT/NMD.

UPF1 promotes rapid degradation of m6A-containing RNAs

N6-methyladenosine (m6A) is the most prevalent internal modification in eukaryotic mRNAs and affects RNA processing and metabolism. When YTHDF2, an m6A-recognizing protein, binds to m6A, it facilitates the destabilization of m6A-containing RNAs (m6A RNAs). Here, we demonstrate that upstream frameshift 1 (UPF1), a key factor for nonsense-mediated mRNA decay, interacts with YTHDF2, thereby triggering rapid degradation of m6A RNAs. The UPF1-mediated m6A RNA degradation depends on a specific interaction between UPF1 and N-terminal residues 101-168 of YTHDF2, UPF1 ATPase/helicase activities, and UPF1 interaction with proline-rich nuclear receptor coactivator 2 (PNRC2), a decapping-promoting factor preferentially involved in nonsense-mediated mRNA decay. Furthermore, transcriptome-wide analyses show that YTHDF2-bound mRNAs that are not substrates for HRSP12-RNase P/MRP-mediated endoribonucleolytic cleavage are destabilized with a higher dependency on UPF1. Collectively, our data indicate dynamic and multilayered regulation of the stability of m6A RNAs and highlight the multifaceted role of UPF1 in mRNA decay.

Widespread Exon Junction Complex Footprints in the RNA Degradome Mark mRNA Degradation before Steady State Translation.

Exon junction complexes (EJCs) are deposited on mRNAs during splicing and displaced by ribosomes during the pioneer round of translation. Nonsense-mediated mRNA decay (NMD) degrades EJC-bound mRNA, but the lack of suitable methodology has prevented the identification of other degradation pathways. Here, we show that the RNA degradomes of Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), worm (Caenorhabditis elegans), and human (Homo sapiens) cells exhibit an enrichment of 5' monophosphate (5'P) ends of degradation intermediates that map to the canonical EJC region. Inhibition of 5' to 3' exoribonuclease activity and overexpression of an EJC disassembly factor in Arabidopsis reduced the accumulation of these 5'P ends, supporting the notion that they are in vivo EJC footprints. Hundreds of Arabidopsis NMD targets possess evident EJC footprints, validating their degradation during the pioneer round of translation. In addition to premature termination codons, plant microRNAs can also direct the degradation of EJC-bound mRNAs. However, the production of EJC footprints from NMD but not microRNA targets requires the NMD factor SUPPRESSOR WITH MORPHOLOGICAL EFFECT ON GENITALIA PROTEIN7. Together, our results demonstrating in vivo EJC footprinting in Arabidopsis unravel the composition of the RNA degradome and provide a new avenue for studying NMD and other mechanisms targeting EJC-bound mRNAs for degradation before steady state translation.

MHC‐I presentation of peptides derived from intact protein products of the pioneer round of translation

Among the earliest protein products of most cellular genes are those synthesized during the pioneer round of translation (PRT), a key step in nonsense-mediated mRNA decay (NMD) that allows scanning of new transcripts for the presence of a premature termination codon (PTC). It has been demonstrated that at least some PRT degradation products can be targeted to major histocompatibility (MHC)-I presentation. To gain new insight into this putative PRT-to-MHC-I route, we have assembled 2 pairs of reporter genes so that the 2 genes in each pair encode an identical fusion protein between a model antigenic peptide and enhanced green fluorescent protein (EGFP), one of which harbors a PTC. We expressed these genes in different mouse and human cell lines and confirmed enhanced NMD activity for the PTC(+) gene in each pair by monitoring the effect of cycloheximide on the level of the respective mRNA. We then exploited several strategies for establishing the ratio between level of peptide presentation and total amount of protein product. We consistently observed significantly higher ratios for the PTC(+) mRNAs compared with the PTC(-) ones, pointing to correlation between the turnover of otherwise identical proteins and the fate of their template mRNA. Using confocal microscopy, we showed a clear link between NMD, the presence of misfolded EGFP polypeptides, and enhanced MHC-I peptide presentation. Altogether, these findings imply that identical full-length gene products differing only in 3' noncoding sequences can be differentially degraded and targeted to the MHC-I presentation pathway, suggesting a more general role for the PRT in establishing the MHC-I peptidome.-Weinstein-Marom, H., Hendel, L., Laron, E. A., Sharabi-Nov, A., Margalit, A., Gross, G. MHC-I presentation of peptides derived from intact protein products of the pioneer round of translation.

Abstract B187: Origins of neoantigens for the major histocompatibility complex class I pathway

Abstract Neoantigens are antigens generated by somatic mutations that can be recognized by the host immune system, firstl described as differentiating or tumor antigens back in 80s and 90s in research related to mice melanoma and breast cancers carried out by Houghton’s and Cheever’s teams, respectively. Now, during the era of potent immunotherapies, cancer vaccines and checkpoint inhibitors (CTLA-4, PD-1), neoantigens again attract much of scientists’ attention. With the use of cutting-edge technologies like next-generation sequencing, mass spectrometry, and predictive algorithms, more is known about antigen presentation and the links between occurrence of somatic mutations in cancer cells and antigen recognition by CD8+T-cells. Interestingly, discoveries of alternative sources of antigenic peptides (e.g., DRIPs and PTPs) challenged the notion about full-length proteins being the main supplier of material for MHC class I pathway and shifted focus of search for new sources to ribosomal scanning during pioneer round of translation. Despite the fact that pathways involved in processing and presentation of peptides have been thoroughly studied, there is still more to be learned about the sources of peptide material for the endogenous and exogenous MHC class I pathways. Based on works related to Epstein-Bar virus, it has been shown that MHC class I immune surveillance is directly correlated with the mechanism that regulates protein synthesis. Together with other results, it highlights the importance of pre-mRNA and mRNA processing in providing antigenic peptides for MHC class I surveillance. Here we revise some significant research related to the production of alternative antigenic peptides, their importance in cancer research, immunosurveillance and generation of tolerance. The lack of animal models to study the origin of alternative antigenic peptides hinders research in the field of neoantigens. I will describe results of the presentation of intron-derived antigenic peptides in mice model developed by our team. Citation Format: Ewa Maria Sroka, Rodrigo Prado Martins, Chrysoula Daskalogianni, Sebastien Apcher, Robin Fahraeus. Origins of neoantigens for the major histocompatibility complex class I pathway [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B187.

MRNAs containing NMD-competent premature termination codons are stabilized and translated under UPF1 depletion

mRNAs containing premature termination codons (PTCs) are rapidly degraded through nonsense-mediated mRNA decay (NMD). However, some PTC-containing mRNAs evade NMD, and might generate mutant proteins responsible for various diseases, including cancers. Using PTC-containing human genomic β-globin constructs, we show that a fraction (~30%) of PTC-containing mRNAs expressed from NMD-competent PTC-containing constructs were as stable as their PTC-free counterparts in a steady state. These PTC-containing mRNAs were monosome-enriched and rarely contributed to expression of mutant proteins. Expression of trace amounts of mutant proteins from NMD-competent PTC-containing constructs was not affected by inhibition of eIF4E-dependent translation and such expression was dependent on a continuous influx of newly synthesized PTC-containing mRNAs, indicating that truncated mutant proteins originated primarily in the pioneer round of translation. The generation of mutant proteins was promoted by UPF1 depletion, which induced polysome association of PTC-containing mRNAs, increased eIF4E-bound PTC-containing mRNA levels, and subsequent eIF4E-dependent translation. Our findings suggest that PTC-containing mRNAs are potent and regulatable sources of mutant protein generation.

RNA. A TRICK'n way to see the pioneer round of translation.

Watching where and when individual messenger RNAs direct protein synthesis in live cells [Also see Report by Halstead et al. ]

Two degrading decades for RNA.

Two degrading decades for RNA.

The EJC Binding and Dissociating Activity of PYM Is Regulated in Drosophila

In eukaryotes, RNA processing events in the nucleus influence the fate of transcripts in the cytoplasm. The multi-protein exon junction complex (EJC) associates with mRNAs concomitant with splicing in the nucleus and plays important roles in export, translation, surveillance and localization of mRNAs in the cytoplasm. In mammalian cells, the ribosome associated protein PYM (HsPYM) binds the Y14-Mago heterodimer moiety of the EJC core, and disassembles EJCs, presumably during the pioneer round of translation. However, the significance of the association of the EJC with mRNAs in a physiological context has not been tested and the function of PYM in vivo remains unknown. Here we address PYM function in Drosophila, where the EJC core proteins are genetically required for oskar mRNA localization during oogenesis. We provide evidence that the EJC binds oskar mRNA in vivo. Using an in vivo transgenic approach, we show that elevated amounts of the Drosophila PYM (DmPYM) N-terminus during oogenesis cause dissociation of EJCs from oskar RNA, resulting in its mislocalization and consequent female sterility. We find that, in contrast to HsPYM, DmPYM does not interact with the small ribosomal subunit and dismantles EJCs in a translation-independent manner upon over-expression. Biochemical analysis shows that formation of the PYM-Y14-Mago ternary complex is modulated by the PYM C-terminus revealing that DmPYM function is regulated in vivo. Furthermore, we find that whereas under normal conditions DmPYM is dispensable, its loss of function is lethal to flies with reduced y14 or mago gene dosage. Our analysis demonstrates that the amount of DmPYM relative to the EJC proteins is critical for viability and fertility. This, together with the fact that the EJC-disassembly activity of DmPYM is regulated, implicates PYM as an effector of EJC homeostasis in vivo.

Nucleolin‐enhanced deadenylation and bulk translation (566.1)

Rate of translation is one of the critical factors, determining protein abundance in a cell. After a pioneer round of translation in which 80S ribosome scans the linear mRNA, a cap‐dependent mRNP closed‐loop is formed for bulk translation.Nucleolin interacts with the PABPC and works together for deadenylation and mRNP closed‐loop formation. Nucleolin interacts to the Pan2/Pan3 and CCR4‐NOT deadenylation complex, indicating nucleolin’s involvement in the mRNA deadenylation. Nucleolin also interacts with the eIF4F translation initiation complex, suggesting another role of nucleolin in translation initiation. We found that an increased level of deadenylated mRNAs is associated with heavy polyribosomes in nucleolin overexpressed cell lines. Together these results indicate that nucleolin‐mediated deadenylation is not followed by mRNA decay, but can lead to the bulk translation by forming poly(A)n‐less mRNP closed‐loop.We propose that a non‐canonical mRNP closed‐loop lacking poly(A)n tail can be formed by nucleolin for bulk translation. In this model, nucleolin enhances deadenylation of the mRNA and induces the formation of poly(A)n‐less mRNP closed‐loop by binding dsRNA formed between the mRNA 5’‐ and 3’‐UTR complementary sequences. Nucleolin is proposed as a mediator for the transition from the canonical to non‐canonical poly(A)n‐less mRNP closed‐loop to enhance the bulk translation.

EIF4E-bound mRNPs are substrates for nonsense-mediated mRNA decay in mammalian cells

Eukaryotic mRNAs with premature translation termination codons (PTCs) are recognized and degraded through a process termed nonsense-mediated mRNA decay (NMD). The evolutionary conservation of the core NMD factors UPF1, UPF2 and UPF3 implies a similar basic mechanism of PTC recognition in all eukaryotes. However, while PTC-containing mRNAs in yeast seem to be available to NMD at each round of translation, mammalian NMD has been reported to be restricted to cap-binding complex (CBC)-bound mRNAs during the pioneer round of translation. Here, we compared decay kinetics of two NMD reporter genes in mRNA fractions bound to either CBC or the eukaryotic initiation factor 4E (eIF4E) in human cells and demonstrate that NMD destabilizes eIF4E-bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event.

Equal Immune Rights For All Polypeptides (106.38)

Abstract Effective immune surveillance requires that MHC class I molecules display a peptide repertoire on the surface representing all cellular proteins. How the peptide repertoire can be comprehensive despite large differences in abundance and stability of individual proteins is not known. We show here that peptide presentation by MHC I molecules is strongly influenced by the pioneer round of translation (PRT) associated with nonsense mediated decay (NMD) of mRNAs. The PRT allows cells to detect and eliminate aberrant mRNAs containing premature stop codons. Inhibition of PRT by knock-down of CBP80, a unique cap-binding protein reduced peptide presentation by MHC class I molecules on the cell surface without affecting global protein synthesis. On the other hand, introduction of premature stop codons changed mRNA stability without affecting peptide presentation by MHC I on the cell surface. Thus, the pioneer round of translation could be used to derive peptides presentation by MHC I molecules to generate a comprehensive display of virtually all endogenous proteins.

MRNA for N-Bak, a neuron-specific BH3-only splice isoform of Bak, escapes nonsense-mediated decay and is translationally repressed in the neurons

mRNA for neuronal Bak (N-Bak), a splice variant of pro-apoptotic Bcl-2 family member Bak is expressed in the neurons. Surprisingly the endogeneous N-Bak protein cannot be demonstrated in the neurons, although the antibodies recognize N-Bak protein from in vitro translation or transiently transfected cells. As N-Bak mRNA contains premature termination codon (PTC) at 89 nucleotides upstream from the last exon–exon junction, it could be degraded by nonsense-mediated decay (NMD) during the pioneer round of translation thus explaining the absence of the protein. We show here that the endogeneous neuronal N-Bak mRNA is not the NMD substrate, as it is not accumulating by cycloheximide treatment, it has a long lifetime, and even prevention of PTC by interfering with the alternative splicing did not lead to translation of the Bak mRNA. N-Bak protein is also not revealed by proteasome inhibitors. Our data suggest strong translational arrest of N-Bak mRNA in the neurons. We show that this arrest is partially mediated by 5′-untranslated region of Bak mRNA and it is not released during mitochondrial apoptosis.

UPF1 Association with the Cap-Binding Protein, CBP80, Promotes Nonsense-Mediated mRNA Decay at Two Distinct Steps

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance mechanism that in mammals generally occurs upon recognition of a premature termination codon (PTC) during a pioneer round of translation. This round involves newly synthesized mRNA that is bound at its 5' end by the cap-binding protein (CBP) heterodimer CBP80-CBP20. Here we show that precluding the binding of the NMD factor UPF1 to CBP80 inhibits NMD at two steps: the association of SMG1 and UPF1 with the two eukaryotic release factors (eRFs) during SURF complex formation at a PTC, and the subsequent association of SMG1 and UPF1 with an exon-junction complex. We also demonstrate that UPF1 binds PTC-containing mRNA more efficiently than the corresponding PTC-free mRNA in a way that is promoted by the UPF1-CBP80 interaction. A unifying model proposes a choreographed series of protein-protein interactions occurring on an NMD target.

UGA hopping: a sport for nephrologists too?

The genetic information encoded by DNA is ultimately ex-pressed into specific polypeptides by way of transcriptionand translation. Transcription is the process whereby thecoding gene sequence is transferred into a messengerRNA (mRNA). Through translation, polypeptides aresynthesized at the ribosomes, collinearly with the sequenceof codons in mRNA. Codons are sets of three ribonucleo-tides in mRNA that signal the initiation, elongation and ter-mination stages of translation. With the exception of thenonsense codons (UAG, UAA and UGA), all codons havecorrespondingcomplementaryanti-codonsintransferRNA(tRNA) molecules. Each tRNA carries a specific aminoacid, which is incorporated into the elongating polypeptideaccording to the codon sequence in mRNA. The correctmRNA reading frame is defined at the start of translationbythestartcodon(AUG),whichcodesfor methionine.Ter-mination of translation is normally signalled by the occur-rence of any of the three codons for which no tRNA isavailable, hence named stop codons. Protein release factorsspecificallybindtostopcodons,mediatingthereleaseofthenewly synthesized polypeptide from the ribosome. How-ever, the stop codons are not equally effective in determin-ing the efficiency of translation termination, with therelative rank order of efficiency being UAA>UAG>UGA[1].Whentranslationalreadingthroughstopcodonsoccurs,UAAandUAGusuallydirecttheadditionofglutamineintothenascentpolypeptide,whereastryptophanisincorporatedin response to UGA in bacteria [2].Nonsense mutations are single-nucleotide alterations inthe DNA that directly change sense to nonsense codons,causing a premature stop of the mRNA translation process.Stop codons may also be generated by mutations that alterthe reading frame of mRNA, either as a result of insertionand/or deletion of a number of nucleotides not evenlydivisiblebythree,orofanabnormallysplicedRNA.Prema-tureterminationcodons(PTC)leadtotheformationoftrun-cated proteins that do not function properly. The frequencyof PTCs is rather variable, ranging from 5% to 70% ofreported mutations for different diseases [3–5]. Cellulardefence mechanisms against the potential deleteriousconsequences of PTCs involve the degradation of thetruncated proteins, and the detection and degradationof mRNAs containing these codons through nonsense-mediated mRNA decay (NMD) (Figure 1). NMD is aribosomal function that scans newly synthesizedmRNAs for the presence of PTCs in a pioneer roundof translation [6,7].Normal stop codons are surrounded by upstream anddownstream sequences, which enhance the efficiency oftranslation termination [8,9]. As PTCs lack these surround-ing complementary signals, pharmacological approachesmay be devised that allow translational read-through ofin-frame PTCs, whilekeepingthe physiological stop codonactive, thus potentially yielding a full-length normal orfunctional protein (Figure 1) [10].

Functional interactions between mRNA turnover and surveillance and the ubiquitin proteasome system

The proteasome is a critical regulator of protein levels within the cell and is essential for maintaining homeostasis. A functional proteasome is required for effective mRNA surveillance and turnover. During transcription, the proteasome localizes to sites of DNA breaks, degrading RNA polymerase II and terminating transcription. For fully transcribed and processed messages, cytoplasmic surveillance is initiated with the pioneer round of translation. The proteasome is recruited to messages bearing premature termination codons, which trigger nonsense-mediated decay (NMD), as well as messages lacking a termination codon, which trigger nonstop decay, to degrade the aberrant protein produced from these messages. A number of proteins involved in mRNA translation are regulated in part by proteasome-mediated decay, including the initiation factors eIF4G, eIF4E, and eIF3a, and the poly(A)-binding protein (PABP) interacting protein, Paip2. eIF4E-BP (4E-BP) is differentially regulated by the proteasome: truncated to generate a protein with higher eIF4B binding or completely degraded, depending on its phosphorylation status. Finally, a functional proteasome is required for AU-rich-element (ARE)-mediated decay but the specific role the proteasome plays is unclear. There is data indicating the proteasome can bind to AREs, act as an endonuclease, and degrade ARE-binding proteins. How these events interact with the 5'-to-3' and 3'-to-5' decay pathways is unclear at this time; however, data is provided indicating that proteasomes colocalize with Xrn1 and the exosome RNases Rrp44 and Rrp6 in untreated HeLa cells.

Remodeling of the pioneer translation initiation complex involves translation and the karyopherin importin β

Mammalian mRNAs lose and acquire proteins throughout their life span while undergoing processing, transport, translation, and decay. How translation affects messenger RNA (mRNA)-protein interactions is largely unknown. The pioneer round of translation uses newly synthesized mRNA that is bound by cap-binding protein 80 (CBP80)-CBP20 (also known as the cap-binding complex [CBC]) at the cap, poly(A)-binding protein N1 (PABPN1) and PABPC1 at the poly(A) tail, and, provided biogenesis involves pre-mRNA splicing, exon junction complexes (EJCs) at exon-exon junctions. Subsequent rounds of translation engage mRNA that is bound by eukaryotic translation initiation factor 4E (eIF4E) at the cap and PABPC1 at the poly(A) tail, but that lacks detectable EJCs and PABPN1. Using the level of intracellular iron to regulate the translation of specific mRNAs, we show that translation promotes not only removal of EJC constituents, including the eIF4AIII anchor, but also replacement of PABPN1 by PABPC1. Remarkably, translation does not affect replacement of CBC by eIF4E. Instead, replacement of CBC by eIF4E is promoted by importin beta (IMPbeta): Inhibiting the binding of IMPbeta to the complex of CBC-IMPalpha at an mRNA cap using the IMPalpha IBB (IMPbeta-binding) domain or a RAN variant increases the amount of CBC-bound mRNA and decreases the amount of eIF4E-bound mRNA. Our studies uncover a previously unappreciated role for IMPbeta and a novel paradigm for how newly synthesized messenger ribonucleoproteins (mRNPs) are matured.

Diverse Role of Three Tyrosines in Binding of the RNA 5′ Cap to the Human Nuclear Cap Binding Complex

The heterodimeric nuclear cap-binding complex (CBC) specifically recognizes the monomethylguanosine 5′ cap structure of the eukaryotic RNA polymerase II transcripts such as mRNA and U snRNA. The binding is essential for nuclear maturation of mRNA, for nuclear export of U snRNA in metazoans, and for nonsense-mediated decay of mRNA and the pioneer round of translation. We analysed the recognition of the cap by native human CBC and mutants in which each tyrosine that stacks with the 7-methylguanosine moiety was replaced by phenylalanine or alanine and both tyrosines were replaced by phenylalanines. The equilibrium association constants ( K as) for two selected cap analogues, P 1-7-methylguanosine-5′ P 3-guanosine-5′ triphosphate and 7-methylguanosine triphosphate, were determined by two independent methods, fluorescence titration and surface plasmon resonance. We could distinguish two tyrosines, Y43 and Y20, in stabilization of the cap inside the CBC-binding pocket. In particular, lack of Y20 in CBC leads to a greater affinity of the mono- than the dinucleotide cap analogue, in contrast to the wild-type protein. A crucial role of cation–π stacking in the mechanism of the specific cap recognition by CBC was postulated from the comparison of the experimentally derived Gibbs free binding energy (Δ G°) with the stacking energy (Δ E) of the 7-methylguanosine/Y binary and ternary complexes calculated by the Møller–Plesset second-order perturbation method. The resulting kinetic model of the association between the capped RNA and CBC, based on the experimental data and quantum calculations, is discussed with respect to the “CBC-to-eukaryotic initiation factor 4E handoff” of mRNA.