Identification of the m6Am Methyltransferase PCIF1 Reveals the Location and Functions of m6Am in the Transcriptome.

mRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N6-methyladenosine (m6A), found within mRNAs, and N6,2'-O-dimethyladenosine (m6Am), which is found at the first transcribed nucleotide. Distinguishing these modifications in mapping studies has been difficult. Here, we identify and biochemically characterize PCIF1, the methyltransferase that generates m6Am. We find that PCIF1 binds and is dependent on the m7G cap. By depleting PCIF1, we generated transcriptome-wide maps that distinguish m6Am and m6A. We find that m6A and m6Am misannotations arise from mRNA isoforms with alternative transcription start sites (TSSs). These isoforms contain m6Am that maps to "internal" sites, increasing the likelihood of misannotation. We find that depleting PCIF1 does not substantially affect mRNA translation but is associated with reduced stability of a subset of m6Am-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize m6Am's function.

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.

A second type of N7-guanine RNA cap methyltransferase in an unusual locus of a large RNA virus genome.

The order Nidovirales is a diverse group of (+)RNA viruses, with a common genome organization and conserved set of replicative and editing enzymes. In particular, RNA methyltransferases play a central role in mRNA stability and immune escape. However, their presence and distribution in different Nidovirales families is not homogeneous. In Coronaviridae, the best characterized family, two distinct methytransferases perform methylation of the N7-guanine and 2'-OH of the RNA-cap to generate a cap-1 structure (m7GpppNm). The genes of both of these enzymes are located in the ORF1b genomic region. While 2'-O-MTases can be identified for most other families based on conservation of both sequence motifs and genetic loci, identification of the N7-guanine methyltransferase has proved more challenging. Recently, we identified a putative N7-MTase domain in the ORF1a region (N7-MT-1a) of certain members of the large genome Tobaniviridae family. Here, we demonstrate that this domain indeed harbors N7-specific methyltransferase activity. We present its structure as the first N7-specific Rossmann-fold (RF) MTase identified for (+)RNA viruses, making it remarkably different from that of the known Coronaviridae ORF1b N7-MTase gene. We discuss the evolutionary implications of such an appearance in this unexpected location in the genome, which introduces a split-off in the classification of Tobaniviridae.

Importance of RNA length for in vitro encapsidation by the nucleoprotein of human respiratory syncytial virus.

Respiratory syncytial virus has a negative-sense single-stranded RNA genome constitutively encapsidated by the viral nucleoprotein N, forming a helical nucleocapsid which is the template for viral transcription and replication by the viral polymerase L. Recruitment of L onto the nucleocapsid depends on the viral phosphoprotein P, which is an essential L cofactor. A prerequisite for genome and antigenome encapsidation is the presence of the monomeric, RNA-free, neosynthesized N protein, named N0. Stabilization of N0 depends on the binding of the N-terminal residues of P to its surface, which prevents N oligomerization. However, the mechanism involved in the transition from N0-P to nucleocapsid assembly, and thus in the specificity of viral genome encapsidation, is still unknown. Furthermore, the specific role of N oligomerization and RNA in the morphogenesis of viral factories, where viral transcription and replication occur, have not been elucidated although the interaction between P and N complexed to RNA has been shown to be responsible for this process. Here, using a chimeric protein comprising N and the first 40 N-terminal residues of P, we succeeded in purifying a recombinant N0-like protein competent for RNA encapsidation in vitro. Our results showed the importance of RNA length for stable encapsidation and revealed that the nature of the 5' end of RNA does not explain the specificity of encapsidation. Finally, we showed that RNA encapsidation is crucial for the in vitro reconstitution of pseudo-viral factories. Together, our findings provide insight into respiratory syncytial virus viral genome encapsidation specificity.

Access to High-Purity 7m G-cap RNA in Substantial Quantities by a Convenient All-Chemical Solid-Phase Method.

Given the importance of mRNA with 5'-cap, easy access to RNA substrates with different 7m G caps, of high quality and in large quantities is essential to elucidate the roles of RNA and the regulation of underlying processes. In addition to existing synthetic routes to 5'-cap RNA based on enzymatic, chemical or chemo-enzymatic methods, we present here an all-chemical method for synthetic RNA capping. The novelty of this study lies in the fact that the capping reaction is performed on solid-support after automated RNA assembly using commercial 2'-O-propionyloxymethyl ribonucleoside phosphoramidites, which enable final RNA deprotection under mild conditions while preserving both 7m G-cap and RNA integrity. The capping reaction is efficiently carried out between a 5'-phosphoroimidazolide RNA anchored on the support and 7m GDP in DMF in the presence of zinc chloride. Substantial amounts of 7m G-cap RNA (from 1 to 28 nucleotides in length and of any sequence with or without internal methylations) containing various cap structures (7m GpppA, 7m GpppAm , 7m Gpppm6 A, 7m Gpppm6 Am , 7m GpppG, 7m GpppGm ) were obtained with high purity after IEX-HPLC purification. This capping method using solid-phase chemistry is convenient to perform and provides access to valuable RNA substrates as useful research tools to unravel specific issues regarding cap-related processes.

The methyltransferase domain of the Respiratory Syncytial Virus L protein catalyzes cap N7 and 2'-O-methylation.

Respiratory syncytial virus (RSV) is a negative sense single-stranded RNA virus and one of the main causes of severe lower respiratory tract infections in infants and young children. RSV RNA replication/transcription and capping are ensured by the viral Large (L) protein. The L protein contains a polymerase domain associated with a polyribonucleotidyl transferase domain in its N-terminus, and a methyltransferase (MTase) domain followed by the C-terminal domain (CTD) enriched in basic amino acids at its C-terminus. The MTase-CTD of Mononegavirales forms a clamp to accommodate RNA that is subsequently methylated on the cap structure and depending on the virus, on internal positions. These enzymatic activities are essential for efficient viral mRNA translation into proteins, and to prevent the recognition of uncapped viral RNA by innate immunity sensors. In this work, we demonstrated that the MTase-CTD of RSV, as well as the full-length L protein in complex with phosphoprotein (P), catalyzes the N7- and 2'-O-methylation of the cap structure of a short RNA sequence that corresponds to the 5' end of viral mRNA. Using different experimental systems, we showed that the RSV MTase-CTD methylates the cap structure with a preference for N7-methylation as first reaction. However, we did not observe cap-independent internal methylation, as recently evidenced for the Ebola virus MTase. We also found that at µM concentrations, sinefungin, a S-adenosylmethionine analogue, inhibits the MTase activity of the RSV L protein and of the MTase-CTD domain. Altogether, these results suggest that the RSV MTase domain specifically recognizes viral RNA decorated by a cap structure and catalyzes its methylation, which is required for translation and innate immune system subversion.

Supramolecular Recognition of Phosphodiester-Based Donor and Acceptor Oligomers Forming Gels in Water.

Inspired by automated DNA synthesis, electron-rich dialkoxynaphthalene (DAN) donor and electron-deficient naphthalene-tetracarboxylic diimide (NDI) acceptor phosphodiester-linked homohexamers were synthesized by the phosphoramidite method. Two types of hexamers were prepared, one with only one phosphodiester between the aromatics (i.e., DAN or NDI) and a second with two phosphodiesters around a propanediol between the aromatics, leading to the latter more flexible and more hydrophilic hexamers. The folding properties of these homohexamers alone or mixed together, in water only, were studied by UV-visible absorption spectroscopy and atomic force microscopy (AFM). AFM imaging revealed that a 1:1 mixture of hexaDAN and hexaNDI formed fibers by charge transfer donor-acceptor recognition leading to a hydrogel after drying. The organization of the resulting structures is strongly dependent on the nature of the complementary partner, leading to the formation of mono- or multilayer hydrogel networks with different compactness.

Reversible methylation of m6Am in the 5' cap controls mRNA stability.

Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5' end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N6,2'-O-dimethyladenosine (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5' cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability.

First insights into the structural features of Ebola virus methyltransferase activities.

The Ebola virus is a deadly human pathogen responsible for several outbreaks in Africa. Its genome encodes the 'large' L protein, an essential enzyme that has polymerase, capping and methyltransferase activities. The methyltransferase activity leads to RNA co-transcriptional modifications at the N7 position of the cap structure and at the 2'-O position of the first transcribed nucleotide. Unlike other Mononegavirales viruses, the Ebola virus methyltransferase also catalyses 2'-O-methylation of adenosines located within the RNA sequences. Herein, we report the crystal structure at 1.8 Å resolution of the Ebola virus methyltransferase domain bound to a fragment of a camelid single-chain antibody. We identified structural determinants and key amino acids specifically involved in the internal adenosine-2'-O-methylation from cap-related methylations. These results provide the first high resolution structure of an ebolavirus L protein domain, and the framework to investigate the effects of epitranscriptomic modifications and to design possible antiviral drugs against the Filoviridae family.

Applications of the Reversible Boronic Acids/Boronate Switch to Nucleic Acids.

Over the last decades, boron and nucleic acids chemistries have gained a lot of attention for biological, medicinal and analytical applications. Our laboratory has a long-standing interest in both chemistries and owing to the ability of boronic acids to react with cis-diol function in aqueous media we developed over the years a variety of applications ranging from molecular recognition and sensing to the development of reversible dynamic systems in which the natural phosphodiester linkage was replaced by a boronate. In this account, we summarize research results from our group from our preliminary studies on molecular recognition of ribonucleosides to the dynamic assembly of functional DNAzymes. In particular, the various parameters influencing the dynamic nature of these reversible covalent bonds able to respond to external stimuli are discussed. Finally, current challenges and opportunities for boron-based nucleic acids are also addressed.

Design and NMR characterization of reversible head-to-tail boronate-linked macrocyclic nucleic acids.

Inspired by the ability of boronic acids to bind with compounds containing diol moieties, we envisioned the formation in solution of boronate ester-based macrocycles by the head-to-tail assembly of a nucleosidic precursor that contains both a boronic acid and the natural 2',3'-diol of ribose. DOSY NMR spectroscopy experiments in water and anhydrous DMF revealed the dynamic assembly of this precursor into dimeric and trimeric macrocycles in a concentration-dependent fashion as well as the reversibility of the self-assembly process. NMR experimental values and quantum mechanics calculations provided further insight into the sugar pucker conformation profile of these macrocycles.

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.

The C-Terminal Domain of the Sudan Ebolavirus L Protein Is Essential for RNA Binding and Methylation.

The large (L) protein of Ebola virus is a key protein for virus replication. Its N-terminal region harbors the RNA-dependent RNA polymerase activity, and its C terminus contains a cap assembling line composed of a capping domain and a methyltransferase domain (MTase) followed by a C-terminal domain (CTD) of unknown function. The L protein MTase catalyzes methylation at the 2'-O and N-7 positions of the cap structures. In addition, the MTase of Ebola virus can induce cap-independent internal adenosine 2'-O-methylation. In this work, we investigated the CTD role in the regulation of the cap-dependent and cap-independent MTase activities of the L protein. We found that the CTD, which is enriched in basic amino acids, plays a key role in RNA binding and in turn regulates the different MTase activities. We demonstrated that the mutation of CTD residues modulates specifically the different MTase activities. Altogether, our results highlight the pivotal role of the L protein CTD in the control of viral RNA methylation, which is critical for Ebola virus replication and escape from the innate response in infected cells.IMPORTANCE Ebola virus infects human and nonhuman primates, causing severe infections that are often fatal. The epidemics, in West and Central Africa, emphasize the urgent need to develop antiviral therapies. The Ebola virus large protein (L), which is the central protein for viral RNA replication/transcription, harbors a methyltransferase domain followed by a C-terminal domain of unknown function. We show that the C-terminal domain regulates the L protein methyltransferase activities and consequently participates in viral replication and escape of the host innate immunity.

Boronic Acid-Mediated Activity Control of Split 10-23 DNAzymes.

The 10-23 DNAzyme is an artificially developed Mg2+ -dependent catalytic oligonucleotide that can cleave an RNA substrate in a sequence-specific fashion. In this study, new split 10-23 DNAzymes made of two nonfunctional fragments, one of which carries a boronic acid group at its 5' end, while the other has a ribonucleotide at its 3' end, were designed. Herein it is demonstrated that the addition of Mg2+ ions leads to assembly of the fragments, which in turn induces the formation of a new boronate internucleoside linkage that restores the DNAzyme activity. A systematic evaluation identified the best-performing system. The results highlight key features for efficient control of DNAzyme activity through the formation of boronate linkages.

FTO controls reversible m6Am RNA methylation during snRNA biogenesis.

Small nuclear RNAs (snRNAs) are core spliceosome components and mediate pre-mRNA splicing. Here we show that snRNAs contain a regulated and reversible nucleotide modification causing them to exist as two different methyl isoforms, m1 and m2, reflecting the methylation state of the adenosine adjacent to the snRNA cap. We find that snRNA biogenesis involves the formation of an initial m1 isoform with a single-methylated adenosine (2'-O-methyladenosine, Am), which is then converted to a dimethylated m2 isoform (N6,2'-O-dimethyladenosine, m6Am). The relative m1 and m2 isoform levels are determined by the RNA demethylase FTO, which selectively demethylates the m2 isoform. We show FTO is inhibited by the oncometabolite D-2-hydroxyglutarate, resulting in increased m2-snRNA levels. Furthermore, cells that exhibit high m2-snRNA levels show altered patterns of alternative splicing. Together, these data reveal that FTO controls a previously unknown central step of snRNA processing involving reversible methylation, and suggest that epitranscriptomic information in snRNA may influence mRNA splicing.

Charge-Transfer Interactions Stabilize G-Quadruplex-Forming Thrombin Binding Aptamers and Can Improve Their Anticoagulant Activity.

In the search for optimized thrombin binding aptamers (TBAs), we herein describe the synthesis of a library of TBA analogues obtained by end-functionalization with the electron-rich 1,5-dialkoxy naphthalene (DAN) and the electron-deficient 1,8,4,5-naphthalenetetra-carboxylic diimide (NDI) moieties. Indeed, when these G-rich oligonucleotides were folded into the peculiar TBA G-quadruplex (G4) structure, effective donor-acceptor charge transfer interactions between the DAN and NDI residues attached to the extremities of the sequence were induced, providing pseudo-cyclic structures. Alternatively, insertion of NDI groups at both extremities produced TBA analogues stabilized by π-π stacking interactions. All the doubly-modified TBAs were characterized by different biophysical techniques and compared with the analogues carrying only the DAN or NDI residue and unmodified TBA. These modified TBAs exhibited higher nuclease resistance, and their G4 structures were markedly stabilized, as evidenced by increased Tm values compared to TBA. These favorable properties were also associated with improved anticoagulant activity for one DAN/NDI-modified TBA, and for one NDI/NDI-modified TBA. Our results indicated that TBA pseudo-cyclic structuring by ad hoc designed end-functionalization represents an efficient approach to improve the aptamer features, while pre-organizing and stabilizing the G4 structure but allowing sufficient flexibility to the aptamer folding, which is necessary for optimal thrombin recognition.

Modified Galacto- or Fuco-Clusters Exploiting the Siderophore Pathway to Inhibit the LecA- or LecB-Associated Virulence of Pseudomonas aeruginosa.

Galacto- and fuco-clusters conjugated with one to three catechol or hydroxamate motifs were synthesised to target LecA and LecB lectins of Pseudomonas aeruginosa (PA) localised in the outer membrane and inside the bacterium. The resulting glycocluster-pseudosiderophore conjugates were evaluated as Trojan horses to cross the outer membrane of PA by iron transport. The data suggest that glycoclusters with catechol moieties are able to hijack the iron transport, whereas those with hydroxamates showed strong nonspecific interactions. Mono- and tricatechol galactoclusters (G1C and G3C) were evaluated as inhibitors of infection by PA in comparison with the free galactocluster (G0). All of them exhibited an inhibitory effect between 46 to 75 % at 100 µM, with a higher potency than G0. This result shows that LecA localised in the outer membrane of PA is involved in the infection mechanism.

DNA-Based Asymmetric Inverse Electron-Demand Hetero-Diels-Alder.

While artificial cyclases hold great promise in chemical synthesis, this work presents the first example of a DNA-catalyzed inverse electron-demand hetero-Diels-Alder (IEDHDA) between dihydrofuran and various α,ß-unsaturated acyl imidazoles. The resulting fused bicyclic O,O-acetals containing three contiguous stereogenic centers are obtained in high yields (up to 99 %) and excellent diastereo- (up to >99:1 dr) and enantioselectivities (up to 95 % ee) using a low catalyst loading. Most importantly, these results show that the concept of DNA-based asymmetric catalysis can be expanded to new synthetic transformations offering an efficient, sustainable, and highly selective tool for the construction of chiral building blocks.

Fine-tuning the properties of the thrombin binding aptamer through cyclization: Effect of the 5'-3' connecting linker on the aptamer stability and anticoagulant activity.

A small library of cyclic TBA analogues (named cycTBA I-IV), obtained by covalently connecting its 5'- and 3'-ends with flexible linkers, has been synthesized with the aim of improving its chemical and enzymatic stability, as well as its anticoagulant properties. Two chemical procedures have been exploited to achieve the desired cyclization, based on the oxime ligation method (providing cycTBA I and II) or on Cu(I)-assisted azide-alkyne cycloaddition (CuAAC) protocols (for cycTBA III and IV), leading to analogues containing circularizing linkers with different chemical nature and length, overall spanning from 22 to 48 atoms. The resulting cyclic TBAs have been characterized using a variety of biophysical methods (UV, CD, gel electrophoresis, SE-HPLC analyses) and then tested for their serum resistance and anticoagulant activity under in vitro experiments. A fine-tuning of the length and flexibility of the linker allowed identifying a cyclic analogue, cycTBA II, with improved anticoagulant activity, associated with a dramatically stabilized G-quadruplex structure (ΔTm = +17 °C) and a 6.6-fold higher enzymatic resistance in serum compared to unmodified TBA.

La-related protein 1 (LARP1) repression of TOP mRNA translation is mediated through its cap-binding domain and controlled by an adjacent regulatory region.

Cell growth is a complex process shaped by extensive and coordinated changes in gene expression. Among these is the tightly regulated translation of a family of growth-related mRNAs defined by a 5' terminal oligopyrimidine (TOP) motif. TOP mRNA translation is partly controlled via the eukaryotic initiation factor 4F (eIF4F), a translation factor that recognizes the mRNA 5' cap structure. Recent studies have also implicated La-related protein 1 (LARP1), which competes with eIF4F for binding to mRNA 5' ends. However, it has remained controversial whether LARP1 represses TOP mRNA translation directly and, if so, what features define its mRNA targets. Here, we show that the C-terminal half of LARP1 is necessary and sufficient to control TOP mRNA translation in cells. This fragment contains the DM15 cap-binding domain as well as an adjacent regulatory region that we identified. We further demonstrate that purified LARP1 represses TOP mRNA translation in vitro through the combined recognition of both the TOP sequence and cap structure, and that its intrinsic repressive activity and affinity for these features are subject to regulation. These results support a model whereby the translation of TOP mRNAs is controlled by a growth-regulated competition between eIF4F and LARP1 for their 5' ends.