Trinucleotide mRNA Cap Analogue N6-Benzylated at the Site of Posttranscriptional m6Am Mark Facilitates mRNA Purification and Confers Superior Translational Properties In Vitro and In Vivo.

Eukaryotic mRNAs undergo cotranscriptional 5'-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as m6Am─a common epitranscriptomic mark unique to the mRNA 5'-end. This modification is regulated by the Pcif1 methyltransferase and the FTO demethylase, but its biological function is still unknown. Here, we designed and synthesized a trinucleotide FTO-resistant N6-benzyl analogue of the m6Am-cap-m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped transcripts. The Bn6Am moiety was shown to act as a reversed-phase high-performance liquid chromatography (RP-HPLC) purification handle, allowing the separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, although the effect was cell-line-dependent. m7GpppBn6AmpG-capped mRNAs encoding reporter proteins administered intravenously to mice provided up to 6-fold higher protein outputs than reference mRNAs, while mRNAs encoding tumor antigens showed superior activity in therapeutic settings as anticancer vaccines. The biochemical characterization suggests several phenomena potentially underlying the biological properties of AvantCap: (i) reduced propensity for unspecific interactions, (ii) involvement in alternative translation initiation, and (iii) subtle differences in mRNA impurity profiles or a combination of these effects. AvantCapped-mRNAs bearing the Bn6Am may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of m6Am in mRNA.

The molecular language of RNA 5' ends: guardians of RNA identity and immunity.

RNA caps are deposited at the 5' end of RNA polymerase II transcripts. This modification regulates several steps of gene expression, in addition to marking transcripts as self to enable the innate immune system to distinguish them from uncapped foreign RNAs, including those derived from viruses. Specialized immune sensors, such as RIG-I and IFITs, trigger antiviral responses upon recognition of uncapped cytoplasmic transcripts. Interestingly, uncapped transcripts can also be produced by mammalian hosts. For instance, 5'-triphosphate RNAs are generated by RNA polymerase III transcription, including tRNAs, Alu RNAs, or vault RNAs. These RNAs have emerged as key players of innate immunity, as they can be recognized by the antiviral sensors. Mechanisms that regulate the presence of 5'-triphosphates, such as 5'-end dephosphorylation or RNA editing, prevent immune recognition of endogenous RNAs and excessive inflammation. Here, we provide a comprehensive overview of the complexity of RNA cap structures and 5'-triphosphate RNAs, highlighting their roles in transcript identity, immune surveillance, and disease.

Kinetic characterization of human mRNA guanine-N7 methyltransferase.

The 5'-mRNA-cap formation is a conserved process in protection of mRNA in eukaryotic cells, resulting in mRNA stability and efficient translation. In humans, two methyltransferases, RNA cap guanine-N7 methyltransferase (hRNMT) and cap-specific nucleoside-2'-O-methyltransferase 1 (hCMTr1) methylate the mRNA resulting in cap0 (N7mGpppN-RNA) and cap1 (N7mGpppN2'-Om-RNA) formation, respectively. Coronaviruses mimic this process by capping their RNA to evade human immune systems. The coronaviral nonstructural proteins, nsp14 and nsp10-nsp16, catalyze the same reactions as hRNMT and hCMTr1, respectively. These two viral enzymes are important targets for development of inhibitor-based antiviral therapeutics. However, assessing the selectivity of such inhibitors against human corresponding proteins is crucial. Human RNMTs have been implicated in proliferation of cancer cells and are also potential targets for development of anticancer therapeutics. Here, we report the development and optimization of a radiometric assay for hRNMT, full kinetic characterization of its activity, and optimization of the assay for high-throughput screening with a Z-factor of 0.79. This enables selectivity determination for a large number of hits from various screening of coronaviral methyltransferases, and also screening hRNMT for discovery of inhibitors and chemical probes that potentially could be used to further investigate the roles RNMTs play in cancers.

5'-cap RNA/SAM mimetic conjugates as bisubstrate inhibitors of viral RNA cap 2'-O-methyltransferases.

Viral RNA cap 2'-O-methyltransferases are considered promising therapeutic targets for antiviral treatments, as they play a key role in the formation of viral RNA cap-1 structures to escape the host immune system. A better understanding of how they interact with their natural substrates (RNA and the methyl donor SAM) would enable the rational development of potent inhibitors. However, as few structures of 2'-O-MTases in complex with RNA have been described, little is known about substrate recognition by these MTases. For this, chemical tools mimicking the state in which the cap RNA substrate and SAM cofactor are bound in the enzyme's catalytic pocket may prove useful. In this work, we designed and synthesized over 30 RNA conjugates that contain a short oligoribonucleotide (ORN with 4 or 6 nucleotides) with the first nucleotide 2'-O-attached to an adenosine by linkers of different lengths and containing S or N-heteroatoms, or a 1,2,3-triazole ring. These ORN conjugates bearing or not a cap structure at 5'-extremity mimic the methylation transition state with RNA substrate/SAM complex as bisubstrates of 2'-O-MTases. The ORN conjugates were synthesized either by the incorporation of a dinucleoside phosphoramidite during RNA elongation or by click chemistry performed on solid-phase post-RNA elongation. Their ability to inhibit the activity of the nsp16/nsp10 complex of SARS-CoV-2 and the NS5 protein of dengue and Zika viruses was assessed. Significant submicromolar IC50 values and Kd values in the µM range were found, suggesting a possible interaction of some ORN conjugates with these viral 2'-O-MTases.

Diadenosine Tetraphosphate (Ap4 A) Serves as a 5' RNA Cap in Mammalian Cells.

The recent expansion of the field of RNA chemical modifications has changed our understanding of post-transcriptional gene regulation. Apart from internal nucleobase modifications, 7-methylguanosine was long thought to be the only eukaryotic RNA cap. However, the discovery of non-canonical RNA caps in eukaryotes revealed a new niche of previously undetected RNA chemical modifications. We are the first to report the existence of a new non-canonical RNA cap, diadenosine tetraphosphate (Ap4 A), in human and rat cell lines. Ap4 A is the most abundant dinucleoside polyphosphate in eukaryotic cells and can be incorporated into RNA by RNA polymerases as a non-canonical initiating nucleotide (NCIN). Using liquid chromatography-mass spectrometry (LC-MS), we show that the amount of capped Ap4 A-RNA is independent of the cellular concentration of Ap4 A. A decapping enzyme screen identifies two enzymes cleaving Ap4 A-RNA,NUDT2 and DXO, both of which also cleave other substrate RNAs in vitro. We further assess the translatability and immunogenicity of Ap4 A-RNA and show that although it is not translated, Ap4 A-RNA is recognized as self by the cell and does not elicit an immune response, making it a natural component of the transcriptome. Our findings open a previously unexplored area of eukaryotic RNA regulation.

DpCoA tagSeq: Barcoding dpCoA-Capped RNA for Direct Nanopore Sequencing via Maleimide-Thiol Reaction.

Recent discoveries of noncanonical RNA caps, such as nicotinamide adenine dinucleotide (NAD+) and 3'-dephospho-coenzyme A (dpCoA), have expanded our knowledge of RNA caps. Although dpCoA has been known to cap RNAs in various species, the identities of its capped RNAs (dpCoA-RNAs) remained unknown. To fill this gap, we developed a method called dpCoA tagSeq, which utilized a thiol-reactive maleimide group to label dpCoA cap with a tag RNA serving as the 5' barcode. The barcoded RNAs were isolated using a complementary DNA strand of the tag RNA prior to direct sequencing by nanopore technology. Our validation experiments with model RNAs showed that dpCoA-RNA was efficiently tagged and captured using this protocol. To confirm that the tagged RNAs are capped by dpCoA and no other thiol-containing molecules, we used a pyrophosphatase NudC to degrade the dpCoA cap to adenosine monophosphate (AMP) moiety before performing the tagSeq protocol. We identified 44 genes that transcribe dpCoA-RNAs in mouse liver, demonstrating the method's effectiveness in identifying and characterizing the capped RNAs. This strategy provides a viable approach to identifying dpCoA-RNAs that allows for further functional investigations of the cap.

Hepatitis C virus RNA is 5'-capped with flavin adenine dinucleotide.

RNA viruses have evolved elaborate strategies to protect their genomes, including 5' capping. However, until now no RNA 5' cap has been identified for hepatitis C virus1,2 (HCV), which causes chronic infection, liver cirrhosis and cancer3. Here we demonstrate that the cellular metabolite flavin adenine dinucleotide (FAD) is used as a non-canonical initiating nucleotide by the viral RNA-dependent RNA polymerase, resulting in a 5'-FAD cap on the HCV RNA. The HCV FAD-capping frequency is around 75%, which is the highest observed for any RNA metabolite cap across all kingdoms of life4-8. FAD capping is conserved among HCV isolates for the replication-intermediate negative strand and partially for the positive strand. It is also observed in vivo on HCV RNA isolated from patient samples and from the liver and serum of a human liver chimeric mouse model. Furthermore, we show that 5'-FAD capping protects RNA from RIG-I mediated innate immune recognition but does not stabilize the HCV RNA. These results establish capping with cellular metabolites as a novel viral RNA-capping strategy, which could be used by other viruses and affect anti-viral treatment outcomes and persistence of infection.

Blocking tri-methylguanosine synthase 1 (TGS1) stops anchorage-independent growth of canine sarcomas.

Tri methylguanosine synthase 1 (TGS1) is the enzyme that hyper methylates the hallmark 7-methyl-guanosine cap (m7G-cap) appended to the transcription start site of RNAs. The m7G-cap and the eIF4E-cap binding protein guide canonical cap-dependent translation of mRNAs, whereas hyper methylated cap, m2,2,7G-cap (TMG) lacks adequate eIF4E affinity and licenses entry into a different translation initiation pathway. The potential role for TGS1 and TMG-capped mRNA in neoplastic growth is unknown. Canine sarcoma has high translational value to the human disease. Cumulative downregulation of protein synthesis in osteosarcoma OSCA-40 was achieved cooperatively by siTGS1 and Torin-1. Torin-1 inhibited the proliferation of three canine sarcoma explants in a reversible manner that was eliminated by siRNA-downregulation of TGS1. TGS1 failure prevented the anchorage-independent growth of osteo- and hemangio-sarcomas and curtailed sarcoma recovery from mTOR inhibition. RNA immunoprecipitation studies identified TMG-capped mRNAs encoding TGS1, DHX9 and JUND. TMG-tgs1 transcripts were downregulated by leptomycin B and TGS1 failure was compensated by eIF4E mRNP-dependent tgs1 mRNA translation affected by mTOR. The evidence documents TMG-capped mRNAs are hallmarks of the investigated neoplasms and synergy between TGS1 specialized translation and canonical translation is involved in sarcoma recovery from mTOR inhibition. Therapeutic targeting of TGS1 activity in cancer is ripe for future exploration.

RIG-I recognizes metabolite-capped RNAs as signaling ligands.

The innate immune receptor RIG-I recognizes 5'-triphosphate double-stranded RNAs (5' PPP dsRNA) as pathogenic RNAs. Such RNA-ends are present in viral genomes and replication intermediates, and they activate the RIG-I signaling pathway to produce a potent interferon response essential for viral clearance. Endogenous mRNAs cap the 5' PPP-end with m7G and methylate the 2'-O-ribose to evade RIG-I, preventing aberrant immune responses deleterious to the cell. Recent studies have identified RNAs in cells capped with metabolites such as NAD+, FAD and dephosphoCoA. Whether RIG-I recognizes these metabolite-capped RNAs has not been investigated. Here, we describe a strategy to make metabolite-capped RNAs free from 5' PPP dsRNA contamination, using in vitro transcription initiated with metabolites. Mechanistic studies show that metabolite-capped RNAs have a high affinity for RIG-I, stimulating the ATPase activity at comparable levels to 5' PPP dsRNA. Cellular signaling assays show that the metabolite-capped RNAs potently stimulate the innate antiviral immune response. This demonstrates that RIG-I can tolerate diphosphate-linked, capped RNAs with bulky groups at the 5' RNA end. This novel class of RNAs that stimulate RIG-I signaling may have cellular roles in activating the interferon response and may be exploited with proper functionalities for RIG-I-related RNA therapeutics.

Thermodynamically Favorable Interactions between eIF4E Binding Domain of eIF4GI with Structured 5'-Untranslated Regions Drive Cap-Independent Translation of Selected mRNAs.

During cellular stress conditions, particularly those seen in multiple cancers, canonical cap-dependent translation is suppressed and a subset of cellular mRNAs (e.g., those encoding FGF-9, HIF-1α, and p53, among others) is known to translate in a cap-independent manner. Human eIF4GI specifically binds to the highly structured 5'-untranslated regions (5'UTRs) of these mRNAs to promote cap-independent translation. The thermodynamics of these protein-RNA interactions have not been explored, and such information will aid in understanding the basic interactions and in potential design of therapeutic drugs. Using fluorescence quenching-based assays and site-directed mutagenesis, we determined the thermodynamic properties of three eIF4GI constructs binding to the 5'UTRs of FGF-9, HIF-1α, and p53 mRNA. These three constructs were designed to explore the importance of the eIF4E binding domain of eIF4GI, which has been shown to be important in binding and selectivity. eIF4GI557-1599, containing the eIF4E binding domain, had higher binding enthalpy (-21 to -14 kJ mol-1 higher), suggesting increased hydrogen bonding, whereas for eIF4GI682-1599 lacking the eIF4E binding domain, binding was entropically favored (TΔS/ΔG of 46-85%), suggesting hydrophobic forces and/or less specific binding. A third construct where a cluster of positively charged amino acids was changed to neutral amino acids showed intermediate properties. Circular dichroism spectra confirmed the significant role of eIF4E binding domain in stable bond formation between eIF4GI and mRNAs via conformational changes. Together, these data contribute to a better understanding of the molecular forces involved in eIF4GI-mRNA recognition and elucidate properties important for the design of small molecules to mediate these interactions.

Structural basis and dynamics of Chikungunya alphavirus RNA capping by nsP1 capping pores.

Alphaviruses are emerging positive-stranded RNA viruses which replicate and transcribe their genomes in membranous organelles formed in the cell cytoplasm. The nonstructural protein 1 (nsP1) is responsible for viral RNA capping and gates the replication organelles by assembling into monotopic membrane-associated dodecameric pores. The capping pathway is unique to Alphaviruses; beginning with the N7 methylation of a guanosine triphosphate (GTP) molecule, followed by the covalent linkage of an m7GMP group to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate RNA. Here, we provide structural snapshots of different stages of the reaction pathway showing how nsP1 pores recognize the substrates of the methyl-transfer reaction, GTP and S-adenosyl methionine (SAM), how the enzyme reaches a metastable postmethylation state with SAH and m7GTP in the active site, and the subsequent covalent transfer of m7GMP to nsP1 triggered by the presence of RNA and postdecapping reaction conformational changes inducing the opening of the pore. In addition, we biochemically characterize the capping reaction, demonstrating specificity for the RNA substrate and the reversibility of the cap transfer resulting in decapping activity and the release of reaction intermediates. Our data identify the molecular determinants allowing each pathway transition, providing an explanation for the need for the SAM methyl donor all along the pathway and clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together, our results set ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals.

eIF3d: A driver of noncanonical cap-dependent translation of specific mRNAs and a trigger of biological/pathological processes.

Eukaryotic initiation factor 3d (eIF3d), a known RNA-binding subunit of the eIF3 complex, is a 66 to 68-kDa protein with an RNA-binding motif and a cap-binding domain. Compared with other eIF3 subunits, eIF3d is relatively understudied. However, recent progress in studying eIF3d has revealed a number of intriguing findings on its role in maintaining eIF3 complex integrity, global protein synthesis, and in biological and pathological processes. It has also been reported that eIF3d has noncanonical functions in regulating translation of a subset of mRNAs by binding to 5'-UTRs or interacting with other proteins independent of the eIF3 complex and additional functions in regulating protein stability. The noncanonical regulation of mRNA translation or protein stability may contribute to the role of eIF3d in biological processes such as metabolic stress adaptation and in disease onset and progression including severe acute respiratory syndrome coronavirus 2 infection, tumorigenesis, and acquired immune deficiency syndrome. In this review, we critically evaluate the recent studies on these aspects of eIF3d and assess prospects in understanding the function of eIF3d in regulating protein synthesis and in biological and pathological processes.

Long noncoding RNAs are substrates for cytoplasmic capping enzyme.

Cytoplasmic capping returns a cap to specific mRNAs, thus protecting uncapped RNAs from decay. Prior to the identification of cytoplasmic capping, uncapped mRNAs were thought to be degraded. Here, we test whether long noncoding RNAs (lncRNAs) are substrates of the cytoplasmic capping enzyme (cCE). The subcellular localisation of 14 lncRNAs associated with sarcomas were examined in U2OS osteosarcoma cells. We used 5' rapid amplification of cDNA ends (RACE) to assay uncapped forms of these lncRNAs. Inhibiting cytoplasmic capping elevated uncapped forms of selected lncRNAs indicating a plausible role of cCE in targeting them. Analysis of published cap analysis of gene expression (CAGE) data shows increased prevalence of certain 5'-RACE cloned sequences, suggesting that these uncapped lncRNAs are targets of cytoplasmic capping.

Structural basis for substrate selection by the SARS-CoV-2 replicase.

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC)1. Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogues, including remdesivir2. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogues must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogues compete, has not been discerned in detail. Here, we use cryogenic-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart adenosine triphosphate3,4. Our results explain the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase), an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds guanosine triphosphate, strengthening proposals for the role of this domain in the formation of the 5' RNA cap6.

mRNA ageing shapes the Cap2 methylome in mammalian mRNA.

The mRNA cap structure is a major site of dynamic mRNA methylation. mRNA caps exist in either the Cap1 or Cap2 form, depending on the presence of 2'-O-methylation on the first transcribed nucleotide or both the first and second transcribed nucleotides, respectively1,2. However, the identity of Cap2-containing mRNAs and the function of Cap2 are unclear. Here we describe CLAM-Cap-seq, a method for transcriptome-wide mapping and quantification of Cap2. We find that unlike other epitranscriptomic modifications, Cap2 can occur on all mRNAs. Cap2 is formed through a slow continuous conversion of mRNAs from Cap1 to Cap2 as mRNAs age in the cytosol. As a result, Cap2 is enriched on long-lived mRNAs. Large increases in the abundance of Cap1 leads to activation of RIG-I, especially in conditions in which expression of RIG-I is increased. The methylation of Cap1 to Cap2 markedly reduces the ability of RNAs to bind to and activate RIG-I. The slow methylation rate of Cap2 allows Cap2 to accumulate on host mRNAs, yet ensures that low levels of Cap2 occur on newly expressed viral RNAs. Overall, these results reveal an immunostimulatory role for Cap1, and that Cap2 functions to reduce activation of the innate immune response.

Inhibition of cellular RNA methyltransferase abrogates influenza virus capping and replication.

Orthomyxo- and bunyaviruses steal the 5' cap portion of host RNAs to prime their own transcription in a process called "cap snatching." We report that RNA modification of the cap portion by host 2'-O-ribose methyltransferase 1 (MTr1) is essential for the initiation of influenza A and B virus replication, but not for other cap-snatching viruses. We identified with in silico compound screening and functional analysis a derivative of a natural product from Streptomyces, called trifluoromethyl-tubercidin (TFMT), that inhibits MTr1 through interaction at its S-adenosyl-l-methionine binding pocket to restrict influenza virus replication. Mechanistically, TFMT impairs the association of host cap RNAs with the viral polymerase basic protein 2 subunit in human lung explants and in vivo in mice. TFMT acts synergistically with approved anti-influenza drugs.

Click-iT trinucleotide cap analog: Synthesis, mRNA translation, and detection.

The first example of the synthesis of a new trinucleotide cap analog containing propargyl group such as m7,3'-O-propargylG(5')PPP(5')AmpG is reported. The effect of the propargyl group in trinucleotide analog with a standard trinucleotide cap analog (GAG), m7G(5')ppp(5')AmpG was evaluated with respect to their capping efficiency, in vitro T7 RNA polymerase transcription efficiency, and translation activity using cultured A549 lung carcinoma epithelial cells. The new propargyl cap analog is a substrate for T7 RNA polymerase. Notably, the mRNA capped with the propargyl cap is translated âˆ¼ 1.3 times more efficiently than the mRNA capped with the GAG cap. The most characteristic feature of the new propargyl cap analog is that the presence of the propargyl group allows further modification of the mRNA by chemical ligation of an azide-containing fluorescent-labeled substrate to the mRNA via click chemistry.

A gel electrophoresis-based assay for measuring enzymatic RNA decapping activity.

RNA 5' ends are remarkably heterogeneous. In addition to the eukaryotic 5' methyl-7-Guanosine (m7G) cap, a number of primarily metabolite-based cap structures have been identified both in prokaryotic and eukaryotic systems. These metabolite caps include Nicotinamide Adenine Dinucleotide (NAD+/NADH), dephosphoCoenzyme A (dpCoA), Flavin Adenine Dinucleotide (FAD), dinucleotide polyphosphates and Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) (Chen et al., 2009; Kowtoniuk et al., 2009; Wang et al., 2019). The most highly studied of these new cap structures, 5' NAD, has significant effects on RNA stability (Bird et al., 2016; Jiao et al., 2017). Both prokaryotes and eukaryotes have decapping enzymes specific to these metabolite caps and decapping is an integral step in the control of RNA stability (Cahová et al., 2015; Jiao et al., 2017; Sharma et al., 2020; Zhang et al., 2020). To better study how these 5' metabolite RNAs are decapped, we present a method to (1) generate radiolabeled dinucleotide and "full length" 5' capped RNA substrates for use in decapping assays, (2) a simple decapping assay to test the activity of various enzymes on different 5' capped transcripts and (3) a gel electrophoresis-based method for the visualization and differentiation of 5' capped transcripts.

Ferritin Iron Responsive Elements (IREs) mRNA Interacts with eIF4G and Activates In Vitro Translation.

BACKGROUND: Eukaryotic initiation factor (eIF) 4G plays an important role in assembling the initiation complex required for ribosome binding to mRNA and promote translation. Translation of ferritin IRE mRNAs is regulated by iron through iron responsive elements (IREs) and iron regulatory protein (IRP). The noncoding IRE stem-loop (30-nt) structure control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. High cellular iron concentrations promote IRE RNA binding to ribosome and initiation factors, and allow synthesis of ferritin. METHODS: In vitro translation assay was performed in depleted wheat germ lysate with supplementation of initiation factors. Fluorescence spectroscopy was used to characterize eIF4F/IRE binding. RESULTS: Eukaryotic initiation factor eIF4G increases the translation of ferritin through binding to stem loop structure of iron responsive elements mRNA in the 5'-untranslated region. Our translation experiment demonstrated that exogenous addition of eIF4G selectively enhanced the translation of ferritin IRE RNA in depleted WG lysate. However, eIF4G facilitates capped IRE RNA translation significantly higher than uncapped IRE RNA translation. Addition of iron with eIF4G to depleted WG lysate significantly enhanced translation for both IRE mRNA (capped and uncapped), confirming the contribution of eIF4G and iron as a potent enhancer of ferritin IRE mRNA translation. Fluorescence data revealed that ferritin IRE strongly interacts to eIF4G (Kd = 63 nM), but not eIF4E. Further equilibrium studies showed that iron enhanced (~4-fold) the ferritin IRE binding to eIF4G. The equilibrium binding effects of iron on ferritin IRE RNA/eIFs interaction and the temperature dependence of this reaction were measured and compared. The Kd values for the IRE binding to eIF4G ranging from 18.2 nM to 63.0 nM as temperature elevated from 5 °C to 25 °C, while the presence of iron showed much stronger affinity over the same range of temperatures. Thermodynamic parameter revealed that IRE RNA binds to eIF4G with ΔH = -42.6 ± 3.3 kJ. mole-1, ΔS = -11.5 ± 0.4 J. mole-1K-1, and ΔG = -39.2 ± 2.7 kJ. mole-1, respectively. Furthermore, addition of iron significantly changed the values of thermodynamic parameters, favoring stable complex formation, thus favoring efficient protein synthesis. This study first time demonstrate the participation of eIF4G in ferritin IRE mRNA translation. CONCLUSIONS: eIF4G specifically interacts with ferritin IRE RNA and promotes eIF4G-dependent translation.

Facile access to 4'-(N-acylsulfonamide) modified nucleosides and evaluation of their inhibitory activity against SARS-CoV-2 RNA cap N7-guanine-methyltransferase nsp14.

N-Acylsulfonamides possess an additional carbonyl function compared to their sulfonamide analogues. Due to their unique physico-chemical properties, interest in molecules containing the N-acylsulfonamide moiety and especially nucleoside derivatives is growing in the field of medicinal chemistry. The recent renewal of interest in antiviral drugs derived from nucleosides containing a sulfonamide function has led us to evaluate the therapeutic potential of N-acylsulfonamide analogues. While these compounds are usually obtained by a difficult acylation of sulfonamides, we report here the easy and efficient synthesis of 20 4'-(N-acylsulfonamide) adenosine derivatives via the sulfo-click reaction. The target compounds were obtained from thioacid and sulfonyl azide synthons in excellent yields and were evaluated as potential inhibitors of the SARS-CoV-2 RNA cap N7-guanine-methyltransferase nsp14.