Cap Analog Research Articles

Translation of human Δ133p53 mRNA and its targeting by antisense oligonucleotides complementary to the 5'-terminal region of this mRNA.

The p53 protein is expressed as at least twelve protein isoforms. Within intron 4 of the human TP53 gene, a P2 transcription initiation site is located and this transcript encodes two p53 isoforms: Δ133p53 and Δ160p53. Here, the secondary structure of the 5′-terminal region of P2-initiated mRNA was characterized by means of the SHAPE and Pb2+-induced cleavage methods and for the first time, a secondary structure model of this region was proposed. Surprisingly, only Δ133p53 isoform was synthetized in vitro from the P2-initiated p53 mRNA while translation from both initiation codons occurred after the transfection of vector-encoded model mRNA to HCT116 cells. Interestingly, translation performed in the presence of the cap analogue suggested that the cap-independent process contributes to the translation of P2-initiated p53 mRNA. Subsequently, several antisense oligonucleotides targeting the 5′-terminal region of P2-initiated p53 mRNA were designed. The selected oligomers were applied in in vitro translation assays as well as in cell lines and their impact on the Δ133p53 synthesis and on cell viability was investigated. The results show that these oligomers are attractive tools in the modulation of the translation of P2-initiated p53 mRNA through attacking the 5′ terminus of the transcript. Since cell proliferation is also reduced by antisense oligomers that lower the level of Δ133p53, this demonstrates an involvement of this isoform in tumorigenesis.

Nucleotide-decorated AuNPs as probes for nucleotide-binding proteins

Gold nanoparticles (AuNPs) decorated with biologically relevant molecules have variety of applications in optical sensing of bioanalytes. Coating AuNPs with small nucleotides produces particles with high stability in water, but functionality-compatible strategies are needed to uncover the full potential of this type of conjugates. Here, we demonstrate that lipoic acid-modified dinucleotides can be used to modify AuNPs surfaces in a controllable manner to produce conjugates that are stable in aqueous buffers and biological mixtures and capable of interacting with nucleotide-binding proteins. Using this strategy we obtained AuNPs decorated with 7-methylguanosine mRNA 5’ cap analogs and showed that they bind cap-specific protein, eIF4E. AuNPs decorated with non-functional dinucleotides also interacted with eIF4E, albeit with lower affinity, suggesting that eIF4E binding to cap-decorated AuNPs is partially mediated by unspecific ionic interactions. This issue was overcome by applying lipoic-acid-Tris conjugate as a charge-neutral diluting molecule. Tris-Lipo-diluted cap-AuNPs conjugates interacted with eIF4E in fully specific manner, enabling design of functional tools. To demonstrate the potential of these conjugates in protein sensing, we designed a two-component eIF4E sensing system consisting of cap-AuNP and 4E-BP1-AuNP conjugates, wherein 4E-BP1 is a short peptide derived from 4E-BP protein that specifically binds eIF4E at a site different to that of the 5’ cap. This system facilitated controlled aggregation, in which eIF4E plays the role of the agent that crosslinks two types of AuNP, thereby inducing a naked-eye visible absorbance redshift. The reported AuNPs-nucleotide conjugation method based on lipoic acid affinity for gold, can be harnessed to obtain other types of nucleotide-functionalized AuNPs, thereby paving the way to studying other nucleotide-binding proteins.

Efficient translation of Eggplant mottled dwarf nucleorhabdovirus N and X genes requires both 5\u2032 and 3\u2032 UTRs

BackgroundCircularization of RNA mediated by association of translation factors and RNA elements in 5′ and 3′ UTRs is a common feature for translation control in eukaryotes. There is no information about translation in plant rhabdoviruses and little information is known in animal rhabdoviruses.MethodsThe role of 5′ and 3′ UTRs in two genes of EMDV in translation were studied using luciferase constructs and RNA structures of these sequences were analyzed by SHAPE and Inline probing.ResultsWe have found that efficient translation of N and X mRNAs of nucleorhabdovirus Eggplant mottled dwarf virus (EMDV) requires elements present in both 5′ and 3′ UTRs. Luciferase reporter constructs containing precise 5′ and 3′ UTRs of the N and X genes had substantially higher translational activity compared with constructs containing only the 5′ or 3′ UTR. The 3′UTR of carmovirus Turnip crinkle virus, which contains a well-characterized cap-independent translation enhancer, was unable to complement the lack of EMDV 3′ UTR. Addition of cap analog to luciferase constructs containing the UTRs of the N gene did not restore translation, and translation of the reporter construct in the absence of the 5′ cap was higher than the capped construct. No RNA-RNA interactions between 5′ and 3′ UTRs were detected by EMSA or in-line cleavage structural assays. Deletion of 11 nucleotides from the 3′ terminus negated the synergistic activity of the 3′UTR.ConclusionsThe results with RNA-RNA interaction suggesting that translational synergy between the UTRs may utilize alternative means. Mutation analysis in 3′UTR suggesting that the polyadenylation signal sequence contained in this location may play a critical role in translation.

Trinucleotide Cap Analogue Bearing a Locked Nucleic Acid Moiety: Synthesis, mRNA Modification, and Translation for Therapeutic Applications.

The synthesis of a new trinucleotide cap analogue containing a locked nucleic acid (LNA) moiety such as m7(LNA)G(5')ppp(5')AmpG and its molecular biology applications are described. The most appealing feature is that this new cap analogue outperforms the standard trinucleotide cap m7G(5')ppp(5')AmpG and the anti-reverse cap analogue m27,3'-OG(5')ppp(5')G by a factor of 5 in terms of translational efficiency.

Construction and Immunogenicity of Modified mRNA-Vaccine Variants Encoding Influenza Virus Antigens.

Nucleic acid-based influenza vaccines are a promising platform that have recently and rapidly developed. We previously demonstrated the immunogenicity of DNA vaccines encoding artificial immunogens AgH1, AgH3, and AgM2, which contained conserved fragments of the hemagglutinin stem of two subtypes of influenza A—H1N1 and H3N2—and conserved protein M2. Thus, the aim of this study was to design and characterize modified mRNA obtained using the above plasmid DNA vaccines as a template. To select the most promising protocol for creating highly immunogenic mRNA vaccines, we performed a comparative analysis of mRNA modifications aimed at increasing its translational activity and decreasing toxicity. We used mRNA encoding a green fluorescent protein (GFP) as a model. Eight mRNA-GFP variants with different modifications (M0–M7) were obtained using the classic cap(1), its chemical analog ARCA (anti-reverse cap analog), pseudouridine (Ψ), N6-methyladenosine (m6A), and 5-methylcytosine (m5C) in different ratios. Modifications M2, M6, and M7, which provided the most intensive fluorescence of transfected HEK293FT cells were used for template synthesis when mRNA encoded influenza immunogens AgH1, AgH3, and AgM2. Virus specific antibodies were registered in groups of animals immunized with a mix of mRNAs encoding AgH1, AgH3, and AgM2, which contained either ARCA (with inclusions of 100% Ψ and 20% m6A (M6)) or a classic cap(1) (with 100% substitution of U with Ψ (M7)). M6 modification was the least toxic when compared with other mRNA variants. M6 and M7 RNA modifications can therefore be considered as promising protocols for designing mRNA vaccines.

Cap 1 Messenger RNA Synthesis with Co‐transcriptional CleanCap® Analog by In Vitro Transcription

Synthetic messenger RNA (mRNA)-based therapeutics are an increasingly popular approach to gene and cell therapies, genome engineering, enzyme replacement therapy, and now, during the global SARS-CoV-2 pandemic, vaccine development. mRNA for such purposes can be synthesized through an enzymatic in vitro transcription (IVT) reaction and formulated for in vivo delivery. Mature mRNA requires a 5'-cap for gene expression and mRNA stability. There are two methods to add a cap in vitro: via a two-step multi-enzymatic reaction or co-transcriptionally. Co-transcriptional methods minimize reaction steps and enzymes needed to make mRNA when compared to enzymatic capping. CleanCap® AG co-transcriptional capping results in 5 mg/ml of IVT with 94% 5'-cap 1 structure. This is highly efficient compared to first-generation cap analogs, such as mCap and ARCA, that incorporate cap 0 structures at lower efficiency and reaction yield. This article describes co-transcriptional capping using TriLink Biotechnology's CleanCap® AG in IVT. © 2021 Wiley Periodicals LLC. Basic Protocol 1: IVT with CleanCap Basic Protocol 2: mRNA purification and analysis.

Structural Insights into the Interaction of Clinically Relevant Phosphorothioate mRNA Cap Analogs with Translation Initiation Factor 4E Reveal Stabilization via Electrostatic Thio-Effect.

mRNA-based therapies and vaccines constitute a disruptive technology with the potential to revolutionize modern medicine. Chemically modified 5′ cap structures have provided access to mRNAs with superior translational properties that could benefit the currently flourishing mRNA field. Prime examples of compounds that enhance mRNA properties are antireverse cap analog diastereomers that contain an O-to-S substitution within the β-phosphate (β-S-ARCA D1 and D2), where D1 is used in clinically investigated mRNA vaccines. The compounds were previously found to have high affinity for eukaryotic translation initiation factor 4E (eIF4E) and augment translation in vitro and in vivo. However, the molecular basis for the beneficial “thio-effect” remains unclear. Here, we employed multiple biophysical techniques and captured 11 cap analog-eIF4E crystallographic structures to investigate the consequences of the β-O-to-S or -Se substitution on the interaction with eIF4E. We determined the SP/RP configurations of β-S-ARCA and related compounds and obtained structural insights into the binding. Unexpectedly, in both stereoisomers, the β-S/Se atom occupies the same binding cavity between Lys162 and Arg157, indicating that the key driving force for complex stabilization is the interaction of negatively charged S/Se with positively charged amino acids. This was observed for all structural variants of the cap and required significantly different conformations of the triphosphate for each diastereomer. This finding explains why both β-S-ARCA diastereomers have higher affinity for eIF4E than unmodified caps. Binding affinities determined for di-, tri-, and oligonucleotide cap analogs suggested that the “thio-effect” was preserved in longer RNAs. Our observations broaden the understanding of thiophosphate biochemistry and enable the rational design of translationally active mRNAs and eIF4E-targeting drugs.

Chemical Synthesis of a Locked Nucleic Acid-Substituted Dinucleotide Cap Analog.

This article describes a reliable and efficient method for synthesis of the dinucleotide cap analog m7(LNA) G[5']ppp[5']G containing a locked nucleic acid moiety. The required LNA intermediate for the final coupling reaction, m7(LNA) GDP, is prepared in six steps starting from 5'-DMTr-N-DMF LNA guanosine. The overall reaction involves removal of DMTr and DMF groups, 5' monophosphorylation, imidazolide formation, diphosphorylation, and regioselective m7 methylation. The final coupling reaction of m7(LNA) GDP with ImGMP in the presence of zinc chloride as a catalyst affords m7(LNA) G[5']ppp[5']G in 59% yield. © 2021 Wiley Periodicals LLC. Basic Protocol: Synthesis of an LNA-substituted dinucleotide cap analog Support Protocol: Preparation of the tris(tributylammonium) phosphate linker.

Enzymatic characterization of three human RNA adenosine methyltransferases reveals diverse substrate affinities and reaction optima

RNA methylations of varied RNA species (mRNA, tRNA, rRNA, non-coding RNA) generate a range of modified nucleotides, including N6-methyladenosine. Here we study the enzymology of three human RNA methyltransferases that methylate the adenosine amino group in diverse contexts, when it is: the first transcribed nucleotide after the mRNA cap (PCIF1), at position 1832 of 18S rRNA (MettL5-Trm112 complex), and within a hairpin in the 3′ UTR of the S-adenosyl-l-methionine synthetase (MettL16). Among these three enzymes, the catalytic efficiency ranges from PCIF1, with the fastest turnover rate of >230 h−1 μM−1 on mRNA cap analog, down to MettL16, which has the lowest rate of ∼3 h−1 μM−1 acting on an RNA hairpin. Both PCIF1 and MettL5 have a binding affinity (Km) of ∼1 μM or less for both substrates of SAM and RNA, whereas MettL16 has significantly lower binding affinities for both (Km >0.4 mM for SAM and ∼10 μM for RNA). The three enzymes are active over a wide pH range (∼5.4–9.4) and have different preferences for ionic strength. Sodium chloride at 200 mM markedly diminished methylation activity of MettL5-Trm112 complex, whereas MettL16 had higher activity in the range of 200 to 500 mM NaCl. Zinc ion inhibited activities of all three enzymes. Together, these results illustrate the diversity of RNA adenosine methyltransferases in their enzymatic mechanisms and substrate specificities and underline the need for assay optimization in their study.

Taking a re-look at cap-binding signatures of the mRNA cap-binding protein eIF4E orthologues in trypanosomatids.

Protein translation leading to polypeptide synthesis involves three distinct events, namely, initiation, elongation, and termination. Translation initiation is a multi-step process that is carried out by ribosomes on the mRNA with the assistance of a large number of proteins called translation initiation factors. Trypanosomatids are kinetoplastidas (flagellated protozoans), some of which cause acute disease syndromes in humans. Vector-borne transmission of protozoan parasites like Leishmania and Trypanosoma causes diseases that affect a large section of the world population and lead to significant morbidity and mortality. The mechanisms of translation initiation in higher eukaryotes are relatively well understood. However, structural and functional conservation of initiation factors in trypanosomatids are only beginning to be understood. Studies carried out so far suggests that at least in Leishmania and Trypanosoma eIF4E function may not be restricted to canonical translation initiation and some of the homologues may have alternate/non-canonical functions. Nonetheless, all of them bind the cap analogs, albeit with different efficiencies, indicating that this property may play an important role in the functionality of eIF4Es. Here, I give a brief background of trypanosomatid eIF4Es and revisit the cap-binding signatures of eIF4E orthologues in trypanosomatids, whose genome sequences are available, in detail, in comparison to human eIF4E1 and Trypanosoma cruzi eIF4E5, with an expanded list of members of this group in light of newer findings. The group 1 and 2 eIF4Es may use either a variation of heIF4E1 or T. cruzi eIF4E5 cap-4-binding signatures, while eIF4E5 and eIF4E6 use distinct amino acid contacts.

Interaction of ferritin iron responsive element (IRE) mRNA with translation initiation factor eIF4F.

The interaction of ferritin iron responsive element (IRE) mRNA with eIF4F was examined by fluorescence and circular dichroism spectroscopy. Fluorescence quenching data indicated that eIF4F contains one high affinity binding site for ferritin IRE RNA. The Scatchard analysis revealed strong binding affinity (Ka=11.1×107M-1) and binding capacity (n=1.0) between IRE RNA and eIF4F. The binding affinity of IRE RNA for eIF4F decreased (~4-fold) as temperature increased (from 5°C to 30°C). The van't Hoff analysis revealed that IRE RNA binding to eIF4F is enthalpy-driven (ΔH=-47.1±3.4kJ/mol) and entropy-opposed (ΔS=-30.1±1.5J/mol/K). The addition of iron increased the enthalpic, while decreasing the entropic contribution towards the eIF4F•IRE RNA complex, resulting in favorable free energy (ΔG=-49.8±2.8kJ/mol). Thermodynamic values and ionic strength data suggest that the presence of iron increases hydrogen bonding and decreases hydrophobic interactions, leading to formation of a more stable complex. The interaction of IRE RNA with eIF4F at higher concentrations produced significant changes in the secondary structure of the protein, as revealed from the far-UV CD results, clearly illustrating the structural alterations resulted from formation of the eIF4F•IRE RNA complex. A Lineweaver-Burk plot showed an uncompetitive binding behavior between IRE RNA and m7G cap for the eIF4F, indicating that there are different binding sites on the eIF4F for the IRE RNA and the cap analog; molecular docking analysis further supports this notion. Our findings suggest that the eIF4F•IRE RNA complex formation is accompanied by an elevated hydrogen bonding and weakened hydrophobic interactions, leading to an overall conformational change, favored in terms of its free energy. The conformational change in the eIF4F structure, caused by the IRE RNA binding, provides a more stable platform for effective IRE translation in iron homeostasis.

Enzymatic Protein Biopolymers as a Tool to Synthetize Eukaryotic Messenger Ribonucleic Acid (mRNA) with Uses in Vaccination, Immunotherapy and Nanotechnology

Multi-subunit enzymes are protein biopolymers that are involved in many cellular processes. The enzyme that carries out the process of transcription of mRNAs is RNA polymerase II (RNAPII), which is a multi-subunit enzyme in eukaryotes. This protein biopolymer starts the transcription from specific sites and is positioned by transcription factors, which form a preinitiation complex (PIC) on gene promoters. To recognize and position the RNAPII and the transcription factors on the gene promoters are needed specific DNA sequences in the gene promoters, which are named promoter elements. Those gene promoter elements can vary and therefore several kinds of promoters exist, however, it appears that all promoters can use a similar pathway for PIC formation. Those pathways are discussed in this review. The in vitro transcribed mRNA can be used as vaccines to fight infectious diseases, e.g., in immunotherapy against cancer and in nanotechnology to deliver mRNA for a missing protein into the cell. We have outlined a procedure to produce an mRNA vaccine against the SARS-CoV-2 virus, which is the causing agent of the big pandemic, COVID-19, affecting human beings all over the world. The potential advantages of using eukaryotic RNAPII to synthetize large transcripts are outlined and discussed. In addition, we suggest a method to cap the mRNA at the 5′ terminus by using enzymes, which might be more effective than cap analogs. Finally, we suggest the construction of a future multi-talented RNAPII, which would be able to synthetize large mRNA and cap them in the test tube.

1H, 13C, and 15N backbone chemical shift assignments of m7GTP cap-bound Leishmania initiation factor 4E-1

Most of the translational control of gene expression in higher eukaryotes occurs during the initiation step of protein synthesis. While this process is well characterized in mammalian cells, it is less defined in parasites, including the ones that cause human Leishmaniasis. The Leishmania cap-binding isoform 1 (LeishIF4E-1) is the only isoform that binds the specific trypanosomatids-specific hypermethylated 5′ cap, called cap-4, in the human stage of the parasite life cycle. We report here the extensive NMR resonance assignment of LeishIF4E-1 bound to a cap analog, m7GTP. The chemical shift data constitute a prerequisite to understanding specific translation initiation mechanisms used in Leishmania parasites and to developing antiparasitic drugs targeting their translation initiation factors.

Effect of the His-Tag Location on Decapping Scavenger Enzymes and Their Hydrolytic Activity toward Cap Analogs.

Decapping scavenger enzymes (DcpSs) are important players in mRNA degradation machinery and conserved in eukaryotes. Importantly, human DcpS is the recognized target for spinal muscular atrophy (SMA) and acute myeloid leukemia (AML) therapy, and has recently been connected to development of intellectual disability. Most recombinant DcpSs used in biochemical and biophysical studies are prepared as tagged proteins, with polyhistidine (His-tag) at the N-terminus or C-terminus. Our work is the first report on the parallel characterization of three versions of DcpSs (native and N- or C-terminally tagged) of three species (humans, Caenorhabditis elegans, and Ascaris suum). The native forms of all three enzymes were prepared by N-(His)10 tag cleavage. Protein thermal stability, measured by differential scanning fluorimetry (DSF), was unaffected in the case of native and tagged versions of human and A. suum DcpS; however, the melting temperature (Tm) of C. elagans DcpS of was significantly influenced by the presence of the additional N- or C-tag. To investigate the impact of the tag positioning on the catalytic properties of DcpS, we tested the hydrolytic activity of native DcpS and their His-tagged counterparts toward cap dinucleotides (m7GpppG and m32,2,7GpppG) and m7GDP. The kinetic data indicate that dinucleotide substrates are hydrolyzed with comparable efficiency by native human and A. suum DcpS and their His-tagged forms. In contrast, both His-tagged C. elegans DcpSs exhibited higher activity toward m7GpppG than the native enzyme. m7GDP is resistant to enzymatic cleavage by all three forms of human and nematode DcpS.

Exploring tryptamine conjugates as pronucleotides of phosphate-modified 7-methylguanine nucleotides targeting cap-dependent translation

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed in many cancers deregulating translational control of the cell cycle. mRNA 5′ cap analogs targeting eIF4E are small molecules with the potential to counteract elevated levels of eIF4E in cancer cells. However, the practical utility of typical cap analogs is limited because of their reduced cell membrane permeability. Transforming the active analogs into their pronucleotide derivatives is a promising approach to overcome this obstacle. 7-Benzylguanosine monophosphate (bn7GMP) is a cap analog that has been successfully transformed into a cell-penetrating pronucleotide by conjugation of the phosphate moiety with tryptamine. In this work, we explored whether a similar strategy is applicable to other cap analogs, particularly phosphate-modified 7-methylguanine nucleotides. We report the synthesis of six new tryptamine conjugates containing N7-methylguanosine mono- and diphosphate and their analogs modified with thiophosphate moiety. These new potential pronucleotides and the expected products of their activation were characterized by biophysical and biochemical methods to determine their affinity towards eIF4E, their ability to inhibit translation in vitro, their susceptibility to enzymatic degradation and their turnover in cell extract. The results suggest that compounds containing the thiophosphate moiety may act as pronucleotides that release low but sustainable concentrations of 7-methylguanosine 5′-phosphorothioate (m7GMPS), which is a translation inhibitor with in vitro potency higher than bn7GMP.

Effect of HIV-1 TAT Peptide Fusion on 5' mRNA Cap Analogs Cell Membrane Permeability and Translation Inhibition.

The development of targeted anticancer drugs has been one of the most challenging goals of current research. Eukaryotic translation initiation factor 4E (eIF4E) is an oncogene that stimulates mRNA translation via binding to the 5' endcap structure. It is well documented that eIF4E is overexpressed in many cancers including breast, prostate, head and neck, and stomach malignancies and leads to oncogenic transformation and metastasis. One approach to block eIF4E function in cancer cells is based on the disruption of the interaction between eIF4E and the 5' mRNA cap structure using cap analog inhibitors. Since analogs are cell-impermeable due to their anionic nature, we used a cell penetrating peptide (CPP) for delivery of model cap analogs into cancer cells. The human immunodeficiency virus I (HIV-1) transactivator of transcription derived peptide (TAT) was conjugated with the analogs m7GMP and m7GpppG using click chemistry methodology. We observed that both conjugates (m7GMP-TAT and m7GpppG-TAT), contrary to TAT alone, did not translocate through the artificial phospholipid membrane of giant unilamellar vesicles. This suggests that passive transport is not the mechanism by which translocation of cap analogs occurs. In contrast, synthesized fluorescently labeled m7GpppG-TAT translocated into the human breast adenocarcinoma cancer cell line MCF-7. Furthermore, we demonstrated that m7GMP-TAT and m7GpppG-TAT inhibited cap-dependent translation up to 30% both in vivo and in vitro while simultaneously not affecting cell growth and viability. These results demonstrate the usefulness of cell penetration peptides as carriers for the internalization of cap analogs.

Functional Cyclization of Eukaryotic mRNAs.

The closed-loop model of eukaryotic translation states that mRNA is circularized by a chain of the cap-eIF4E-eIF4G-poly(A)-binding protein (PABP)-poly(A) interactions that brings 5′ and 3′ ends together. This circularization is thought to promote the engagement of terminating ribosomes to a new round of translation at the same mRNA molecule, thus enhancing protein synthesis. Despite the general acceptance and the elegance of the hypothesis, it has never been proved experimentally. Using continuous in situ monitoring of luciferase synthesis in a mammalian in vitro system, we show here that the rate of translation initiation at capped and polyadenylated reporter mRNAs increases after the time required for the first ribosomes to complete mRNA translation. Such acceleration strictly requires the presence of a poly(A)-tail and is abrogated by the addition of poly(A) RNA fragments or m7GpppG cap analog to the translation reaction. The optimal functional interaction of mRNA termini requires 5′ untranslated region (UTR) and 3′ UTR of moderate lengths and provides stronger acceleration, thus a longer poly(A)-tail. Besides, we revealed that the inhibitory effect of the dominant negative R362Q mutant of initiation factor eIF4A diminishes in the course of translation reaction, suggesting a relaxed requirement for ATP. Taken together, our results imply that, upon the functional looping of an mRNA, the recycled ribosomes can be recruited to the start codon of the same mRNA molecule in an eIF4A-independent fashion. This non-canonical closed-loop assisted reinitiation (CLAR) mode provides efficient translation of the functionally circularized mRNAs.

The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5' cap modulates protein expression in living cells.

7-Methylguanosine 5′ cap on mRNA is necessary for efficient protein expression in vitro and in vivo. Recent studies revealed structural diversity of endogenous mRNA caps, which carry different 5′-terminal nucleotides and additional methylations (2′-O-methylation and m6A). Currently available 5′-capping methods do not address this diversity. We report trinucleotide 5′ cap analogs (m7GpppN(m)pG), which are utilized by RNA polymerase T7 to initiate transcription from templates carrying Φ6.5 promoter and enable production of mRNAs differing in the identity of the first transcribed nucleotide (N = A, m6A, G, C, U) and its methylation status (±2′-O-methylation). HPLC-purified mRNAs carrying these 5′ caps were used to study protein expression in three mammalian cell lines (3T3-L1, HeLa and JAWS II). The highest expression was observed for mRNAs carrying 5′-terminal A/Am and m6Am, whereas the lowest was observed for G and Gm. The mRNAs carrying 2′-O-methyl at the first transcribed nucleotide (cap 1) had significantly higher expression than unmethylated counterparts (cap 0) only in JAWS II dendritic cells. Further experiments indicated that the mRNA expression characteristic does not correlate with affinity for translation initiation factor 4E or in vitro susceptibility to decapping, but instead depends on mRNA purity and the immune state of the cells.

Isoxazole-containing 5' mRNA cap analogues as inhibitors of the translation initiation process.

Herein we describe a synthesis of new isoxazole-containing 5' mRNA cap analogues via a cycloaddition reaction. The obtained analogues show a capability to inhibit cap-dependent translation in vitro and are characterized by a new binding mode in which an isoxazolic ring, instead of guanine, is involved in the stacking effect. Our study provides valuable information toward designing new compounds that can be potentially used as anticancer therapeutics.

Structural studies of the eIF4E-VPg complex reveal a direct competition for capped RNA: Implications for translation.

Viruses have transformed our understanding of mammalian RNA processing, including facilitating the discovery of the methyl-7-guanosine (m7G) cap on the 5' end of RNAs. The m7G cap is required for RNAs to bind the eukaryotic translation initiation factor eIF4E and associate with the translation machinery across plant and animal kingdoms. The potyvirus-derived viral genome-linked protein (VPg) is covalently bound to the 5' end of viral genomic RNA (gRNA) and associates with host eIF4E for successful infection. Divergent models to explain these observations proposed either an unknown mode of eIF4E engagement or a competition of VPg for the m7G cap-binding site. To dissect these possibilities, we resolved the structure of VPg, revealing a previously unknown 3-dimensional (3D) fold, and characterized the VPg-eIF4E complex using NMR and biophysical techniques. VPg directly bound the cap-binding site of eIF4E and competed for m7G cap analog binding. In human cells, VPg inhibited eIF4E-dependent RNA export, translation, and oncogenic transformation. Moreover, VPg formed trimeric complexes with eIF4E-eIF4G, eIF4E bound VPg-luciferase RNA conjugates, and these VPg-RNA conjugates were templates for translation. Informatic analyses revealed structural similarities between VPg and the human kinesin EG5. Consistently, EG5 directly bound eIF4E in a similar manner to VPg, demonstrating that this form of engagement is relevant beyond potyviruses. In all, we revealed an unprecedented modality for control and engagement of eIF4E and show that VPg-RNA conjugates functionally engage eIF4E. As such, potyvirus VPg provides a unique model system to interrogate eIF4E.