m6A Modification Prevents Formation of Endogenous Double-Stranded RNAs and Deleterious Innate Immune Responses during Hematopoietic Development.

N6-methyladenosine (m6A) is the most abundant RNA modification, but little is known about its role in mammalian hematopoietic development. Here, we show that conditional deletion of the m6A writer METTL3 in murine fetal liver resulted in hematopoietic failure and perinatal lethality. Loss of METTL3 and m6A activated an aberrant innate immune response, mediated by the formation of endogenous double-stranded RNAs (dsRNAs). The aberrantly formed dsRNAs were long, highly m6A modified in their native state, characterized by low folding energies, and predominantly protein coding. We identified coinciding activation of pattern recognition receptor pathways normally tasked with the detection of foreign dsRNAs. Disruption of the aberrant immune response via abrogation of downstream Mavs or Rnasel signaling partially rescued the observed hematopoietic defects in METTL3-deficient cells in vitro and in vivo. Our results suggest that m6A modification protects against endogenous dsRNA formation and a deleterious innate immune response during mammalian hematopoietic development.

Positive allosteric mechanisms of adenosine A1 receptor-mediated analgesia.

The adenosine A1 receptor (A1R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain1,2. However, development of analgesic orthosteric A1R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects3. Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A1R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A1R co-bound to adenosine, MIPS521 and a Gi2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts.

An α-ketoglutarate conformational switch controls iron accessibility, activation, and substrate selection of the human FTO protein.

The fat mass and obesity-associated fatso (FTO) protein is a member of the Alkb family of dioxygenases and catalyzes oxidative demethylation of N6-methyladenosine (m6A), N1-methyladenosine (m1A), 3-methylthymine (m3T), and 3-methyluracil (m3U) in single-stranded nucleic acids. It is well established that the catalytic activity of FTO proceeds via two coupled reactions. The first reaction involves decarboxylation of alpha-ketoglutarate (αKG) and formation of an oxyferryl species. In the second reaction, the oxyferryl intermediate oxidizes the methylated nucleic acid to reestablish Fe(II) and the canonical base. However, it remains unclear how binding of the nucleic acid activates the αKG decarboxylation reaction and why FTO demethylates different methyl modifications at different rates. Here, we investigate the interaction of FTO with 5-mer DNA oligos incorporating the m6A, m1A, or m3T modifications using solution NMR, molecular dynamics (MD) simulations, and enzymatic assays. We show that binding of the nucleic acid to FTO activates a two-state conformational equilibrium in the αKG cosubstrate that modulates the O2 accessibility of the Fe(II) catalyst. Notably, the substrates that provide better stabilization to the αKG conformation in which Fe(II) is exposed to O2 are demethylated more efficiently by FTO. These results indicate that i) binding of the methylated nucleic acid is required to expose the catalytic metal to O2 and activate the αKG decarboxylation reaction, and ii) the measured turnover of the demethylation reaction (which is an ensemble average over the entire sample) depends on the ability of the methylated base to favor the Fe(II) state accessible to O2.

Programmed RNA editing with an evolved bacterial adenosine deaminase.

Programmed RNA editing presents an attractive therapeutic strategy for genetic disease. In this study, we developed bacterial deaminase-enabled recoding of RNA (DECOR), which employs an evolved Escherichia coli transfer RNA adenosine deaminase, TadA8e, to deposit adenosine-to-inosine editing to CRISPR-specified sites in the human transcriptome. DECOR functions in a variety of cell types, including human lung fibroblasts, and delivers on-target activity similar to ADAR-overexpressing RNA-editing platforms with 88% lower off-target effects. High-fidelity DECOR further reduces off-target effects to basal level. We demonstrate the clinical potential of DECOR by targeting Van der Woude syndrome-causing interferon regulatory factor 6 (IRF6) insufficiency. DECOR-mediated RNA editing removes a pathogenic upstream open reading frame (uORF) from the 5' untranslated region of IRF6 and rescues primary ORF expression from 12.3% to 36.5%, relative to healthy transcripts. DECOR expands the current portfolio of effector proteins and opens new territory in programmed RNA editing.

Novel immunomodulatory properties of adenosine analogs promote their antiviral activity against SARS-CoV-2.

The COVID-19 pandemic reminded us of the urgent need for new antivirals to control emerging infectious diseases and potential future pandemics. Immunotherapy has revolutionized oncology and could complement the use of antivirals, but its application to infectious diseases remains largely unexplored. Nucleoside analogs are a class of agents widely used as antiviral and anti-neoplastic drugs. Their antiviral activity is generally based on interference with viral nucleic acid replication or transcription. Based on our previous work and computer modeling, we hypothesize that antiviral adenosine analogs, like remdesivir, have previously unrecognized immunomodulatory properties which contribute to their therapeutic activity. In the case of remdesivir, we here show that these properties are due to its metabolite, GS-441524, acting as an Adenosine A2A Receptor antagonist. Our findings support a new rationale for the design of next-generation antiviral agents with dual - immunomodulatory and intrinsic - antiviral properties. These compounds could represent game-changing therapies to control emerging viral diseases and future pandemics.

Selective prebiotic formation of RNA pyrimidine and DNA purine nucleosides.

The nature of the first genetic polymer is the subject of major debate1. Although the 'RNA world' theory suggests that RNA was the first replicable information carrier of the prebiotic era-that is, prior to the dawn of life2,3-other evidence implies that life may have started with a heterogeneous nucleic acid genetic system that included both RNA and DNA4. Such a theory streamlines the eventual 'genetic takeover' of homogeneous DNA from RNA as the principal information-storage molecule, but requires a selective abiotic synthesis of both RNA and DNA building blocks in the same local primordial geochemical scenario. Here we demonstrate a high-yielding, completely stereo-, regio- and furanosyl-selective prebiotic synthesis of the purine deoxyribonucleosides: deoxyadenosine and deoxyinosine. Our synthesis uses key intermediates in the prebiotic synthesis of the canonical pyrimidine ribonucleosides (cytidine and uridine), and we show that, once generated, the pyrimidines persist throughout the synthesis of the purine deoxyribonucleosides, leading to a mixture of deoxyadenosine, deoxyinosine, cytidine and uridine. These results support the notion that purine deoxyribonucleosides and pyrimidine ribonucleosides may have coexisted before the emergence of life5.

N6-Methyladenosines Modulate A-to-I RNA Editing.

N6-methyladenosine (m6A) and adenosine-to-inosine (A-to-I) editing are two of the most abundant RNA modifications, both at adenosines. Yet, the interaction of these two types of adenosine modifications is largely unknown. Here we show a global A-to-I difference between m6A-positive and m6A-negative RNA populations. Both the presence and extent of A-to-I sites in m6A-negative RNA transcripts suggest a negative correlation between m6A and A-to-I. Suppression of m6A-catalyzing enzymes results in global A-to-I RNA editing changes. Further depletion of m6A modification increases the association of m6A-depleted transcripts with adenosine deaminase acting on RNA (ADAR) enzymes, resulting in upregulated A-to-I editing on the same m6A-depleted transcripts. Collectively, the effect of m6A on A-to-I suggests a previously underappreciated interplay between two distinct and abundant RNA modifications, highlighting a complex epitranscriptomic landscape.

Site-specific regulation of RNA editing with ribose-modified nucleoside analogs in ADAR guide strands.

Adenosine Deaminases Acting on RNA (ADARs) are enzymes that catalyze the conversion of adenosine to inosine in RNA duplexes. These enzymes can be harnessed to correct disease-causing G-to-A mutations in the transcriptome because inosine is translated as guanosine. Guide RNAs (gRNAs) can be used to direct the ADAR reaction to specific sites. Chemical modification of ADAR guide strands is required to facilitate delivery, increase metabolic stability, and increase the efficiency and selectivity of the editing reaction. Here, we show the ADAR reaction is highly sensitive to ribose modifications (e.g. 4'-C-methylation and Locked Nucleic Acid (LNA) substitution) at specific positions within the guide strand. Our studies were enabled by the synthesis of RNA containing a new, ribose-modified nucleoside analog (4'-C-methyladenosine). Importantly, the ADAR reaction is potently inhibited by LNA or 4'-C-methylation at different positions in the ADAR guide. While LNA at guide strand positions -1 and -2 block the ADAR reaction, 4'-C-methylation only inhibits at the -2 position. These effects are rationalized using high-resolution structures of ADAR-RNA complexes. This work sheds additional light on the mechanism of ADAR deamination and aids in the design of highly selective ADAR guide strands for therapeutic editing using chemically modified RNA.

Implication of nucleotides near the 3' end of 16S rRNA in guarding the translational reading frame.

Loss of the translational reading frame leads to misincorporation and premature termination, which can have lethal consequences. Based on structural evidence that A1503 of 16S rRNA intercalates between specific mRNA bases, we tested the possibility that it plays a role in maintenance of the reading frame by constructing ribosomes with an abasic nucleotide at position 1503. This was done by specific cleavage of 16S rRNA at position 1493 using the colicin E3 endonuclease and replacing the resulting 3'-terminal 49mer fragment with a synthetic oligonucleotide containing the abasic site using a novel splinted RNA ligation method. Ribosomes reconstituted from the abasic 1503 16S rRNA were highly active in protein synthesis but showed elevated levels of spontaneous frameshifting into the -1 reading frame. We then asked whether the residual frameshifting persisting in control ribosomes containing an intact A1503 is due to the absence of the N6-dimethyladenosine modifications at positions 1518 and 1519. Indeed, this frameshifting was rescued by site-specific methylation in vitro by the ksgA methylase. These findings thus implicate two different sites near the 3' end of 16S rRNA in maintenance of the translational reading frame, providing yet another example of a functional role for ribosomal RNA in protein synthesis.

Molecular basis of A. thaliana KEOPS complex in biosynthesizing tRNA t6A.

In archaea and eukaryotes, the evolutionarily conserved KEOPS is composed of four core subunits-Kae1, Bud32, Cgi121 and Pcc1, and a fifth Gon7/Pcc2 that is found in fungi and metazoa. KEOPS cooperates with Sua5/YRDC to catalyze the biosynthesis of tRNA N6-threonylcarbamoyladenosine (t6A), an essential modification needed for fitness of cellular organisms. Biochemical and structural characterizations of KEOPSs from archaea, yeast and humans have determined a t6A-catalytic role for Kae1 and auxiliary roles for other subunits. However, the precise molecular workings of KEOPSs still remain poorly understood. Here, we investigated the biochemical functions of A. thaliana KEOPS and determined a cryo-EM structure of A. thaliana KEOPS dimer. We show that A. thaliana KEOPS is composed of KAE1, BUD32, CGI121 and PCC1, which adopts a conserved overall arrangement. PCC1 dimerization leads to a KEOPS dimer that is needed for an active t6A-catalytic KEOPS-tRNA assembly. BUD32 participates in direct binding of tRNA to KEOPS and modulates the t6A-catalytic activity of KEOPS via its C-terminal tail and ATP to ADP hydrolysis. CGI121 promotes the binding of tRNA to KEOPS and potentiates the t6A-catalytic activity of KEOPS. These data and findings provide insights into mechanistic understanding of KEOPS machineries.

m6 A deposition is regulated by PRMT1-mediated arginine methylation of METTL14 in its disordered C-terminal region.

The N6-methyladenosine (m6 A) RNA modification serves crucial functions in RNA metabolism; however, the molecular mechanisms underlying the regulation of m6 A are not well understood. Here, we establish arginine methylation of METTL14, a component of the m6 A methyltransferase complex, as a novel pathway that controls m6 A deposition in mammalian cells. Specifically, protein arginine methyltransferase 1 (PRMT1) interacts with, and methylates the intrinsically disordered C terminus of METTL14, which promotes its interaction with RNA substrates, enhances its RNA methylation activity, and is crucial for its interaction with RNA polymerase II (RNAPII). Mouse embryonic stem cells (mESCs) expressing arginine methylation-deficient METTL14 exhibit significantly reduced global m6 A levels. Transcriptome-wide m6 A analysis identified 1,701 METTL14 arginine methylation-dependent m6 A sites located in 1,290 genes involved in various cellular processes, including stem cell maintenance and DNA repair. These arginine methylation-dependent m6 A sites are associated with enhanced translation of genes essential for the repair of DNA interstrand crosslinks; thus, METTL14 arginine methylation-deficient mESCs are hypersensitive to DNA crosslinking agents. Collectively, these findings reveal important aspects of m6 A regulation and new functions of arginine methylation in RNA metabolism.

Deep learning based method for predicting DNA N6-methyladenosine sites.

DNA N6 methyladenine (6mA) plays an important role in many biological processes, and accurately identifying its sites helps one to understand its biological effects more comprehensively. Previous traditional experimental methods are very labor-intensive and traditional machine learning methods also seem to be somewhat insufficient as the database of 6mA methylation groups becomes progressively larger, so we propose a deep learning-based method called multi-scale convolutional model based on global response normalization (CG6mA) to solve the prediction problem of 6mA site. This method is tested with other methods on three different kinds of benchmark datasets, and the results show that our model can get more excellent prediction results.

Deepm6A-MT: A deep learning-based method for identifying RNA N6-methyladenosine sites in multiple tissues.

N6-methyladenosine (m6A) is the most prevalent, abundant, and conserved internal modification in the eukaryotic messenger RNA (mRNAs) and plays a crucial role in the cellular process. Although more than ten methods were developed for m6A detection over the past decades, there were rooms left to improve the predictive accuracy and the efficiency. In this paper, we proposed an improved method for predicting m6A modification sites, which was based on bi-directional gated recurrent unit (Bi-GRU) and convolutional neural networks (CNN), called Deepm6A-MT. The Deepm6A-MT has two input channels. One is to use an embedding layer followed by the Bi-GRU and then by the CNN, and another is to use one-hot encoding, dinucleotide one-hot encoding, and nucleotide chemical property codes. We trained and evaluated the Deepm6A-MT both by the 5-fold cross-validation and the independent test. The empirical tests showed that the Deepm6A-MT achieved the state of the art performance. In addition, we also conducted the cross-species and the cross-tissues tests to further verify the Deepm6A-MT for effectiveness and efficiency. Finally, for the convenience of academic research, we deployed the Deepm6A-MT to the web server, which is accessed at the URL http://www.biolscience.cn/Deepm6A-MT/.

Copper catalyzed cycloaddition for the synthesis of non isomerisable 2' and 3'-regioisomers of arg-tRNAarg.

In this report, non-isomerisable analogs of arginine tRNA (Arg-triazole-tRNA) have been synthesized as tools to study tRNA-dependent aminoacyl-transferases. The synthesis involves the incorporation of 1,4 substituted-1,2,3 triazole ring to mimic the ester bond that connects the amino acid to the terminal adenosine in the natural substrate. The synthetic procedure includes (i) a coupling between 2'- or 3'-azido-adenosine derivatives and a cytidine phosphoramidite to access dinucleotide molecules, (ii) Cu-catalyzed cycloaddition reactions between 2'- or 3'-azido dinucleotide in the presence of an alkyne molecule mimicking the arginine, providing the corresponding Arg-triazole-dinucleotides, (iii) enzymatic phosphorylation of the 5'-end extremity of the Arg-triazole-dinucleotides with a polynucleotide kinase, and (iv) enzymatic ligation of the 5'-phosphorylated dinucleotides with a 23-nt RNA micro helix that mimics the acceptor arm of arg-tRNA or with a full tRNAarg. Characterization of nucleoside and nucleotide compounds involved MS spectrometry, 1H, 13C and 31P NMR analysis. This strategy allows to obtain the pair of the two stable regioisomers of arg-tRNA analogs (2' and 3') which are instrumental to explore the regiospecificity of arginyl transferases enzyme. In our study, a first binding assay of the arg-tRNA micro helix with the Arginyl-tRNA-protein transferase 1 (ATE1) was performed by gel shift assays.

Rolling circle extension-assisted loop-mediated isothermal amplification (Rol-LAMP) method for locus-specific and visible detection of RNA N6-methyladenosine.

N6-methyladenosine (m6A) is the most prevalent RNA modification in eukaryotic mRNAs. Currently available detection methods for locus-specific m6A marks rely on RT-qPCR, radioactive methods, or high-throughput sequencing. Here, we develop a non-qPCR, ultrasensitive, isothermal, and naked-eye visible method for m6A detection based on rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP), named m6A-Rol-LAMP, to verify putative m6A sites in transcripts obtained from the high-throughput data. When padlock probes hybridize to the potential m6A sites on targets, they are converted to circular form by DNA ligase in the absence of m6A modification, while m6A modification hinders the sealing of padlock probes. Subsequently, Bst DNA polymerase-mediated RCA and LAMP allow the amplification of the circular padlock probe to achieve the locus-specific detection of m6A. Following optimization and validation, m6A-Rol-LAMP can ultra-sensitively and quantitatively determine the existence of m6A modification on a specific target site as low as 100 amol under isothermal conditions. Detections of m6A can be performed on rRNA, mRNA, lincRNA, lncRNA and pre-miRNA from biological samples with naked-eye observations after dye incubation. Together, we provide a powerful tool for locus-specific detection of m6A, which can simply, quickly, sensitively, specifically, and visually determine putative m6A modification on RNA.

Reversible Control of Gene Expression by Guest-Modified Adenosines in a Cell-Free System via Host-Guest Interaction.

Gene expression technology has become an indispensable tool for elucidating biological processes and developing biotechnology. Cell-free gene expression (CFE) systems offer a fundamental platform for gene expression-based technology, in which the reversible and programmable control of transcription can expand its use in synthetic biology and medicine. This study shows that CFE can be controlled via the host-guest interaction of cucurbit[7]uril (CB[7]) with N6-guest-modified adenosines. These adenosine derivatives were conveniently incorporated into the DNA strand using a post-synthetic approach and formed a selective and stable base pair with complementary thymidine in DNA. Meanwhile, alternate addition of CB[7] and the exchanging guest molecule induced the reversible formation of a duplex structure through the formation and dissociation of a bulky complex on DNA. The kinetics of the reversibility was fine-tuned by changing the size of the modified guest moieties. When incorporated into a specific region of the T7 promoter sequence, the guest-modified adenosines enabled tight and reversible control of in vitro transcription and protein expression in the CFE system. This study marks the first utility of the host-guest interaction for gene expression control in the CFE system, opening new avenues for developing DNA-based technology, particularly for precise gene therapy and DNA nanotechnology.

Regioselective Homolytic C2-H Borylation of Unprotected Adenosine and Adenine Derivatives via Minisci Reaction.

A Minisci-type borylation of unprotected adenosine, adenine nucleotide, and adenosine analogues was successfully achieved through photocatalysis or thermal activation. Despite the challenges posed by the presence of two potential reactive sites (C2 and C8) in the purine motif, the unique nucleophilic amine-ligated boryl radicals effortlessly achieved excellent C2 site selectivity and simultaneously avoided the formation of multifunctionalized products. This protocol proved effective for the late-stage borylation of some important biomolecules as well as a few antiviral and antitumor drug molecules, such as AMP, cAMP, Vidarabine, Cordycepin, Tenofovir, Adefovir, GS-441524, etc. Theoretical calculations shed light on the site selectivity, revealing that the free energy barriers for the C2-Minisci addition are further lowered through the chelation of additive Mg2+ to N3 and furyl oxygen. This phenomenon has been confirmed by an IGMH analysis. Preliminary antitumor evaluation, derivation of the C2-borylated adenosine to other analogues with high-value functionalities, along with the CuAAC click reactions, suggest the potential application of this methodology in drug molecular optimization studies and chemical biology.

AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA.

RNA molecules undergo various chemical modifications that play critical roles in a wide range of biological processes. N6,N6-Dimethyladenosine (m6,6A) is a conserved RNA modification and is essential for the processing of rRNA. To gain a deeper understanding of the functions of m6,6A, site-specific and accurate quantification of this modification in RNA is indispensable. In this study, we developed an AlkB-facilitated demethylation (AD-m6,6A) method for the site-specific detection and quantification of m6,6A in RNA. The N6,N6-dimethyl groups in m6,6A can cause reverse transcription to stall at the m6,6A site, resulting in truncated cDNA. However, we found that Escherichia coli AlkB demethylase can effectively demethylate m6,6A in RNA, generating full-length cDNA from AlkB-treated RNA. By quantifying the amount of full-length cDNA produced using quantitative real-time PCR, we were able to achieve site-specific detection and quantification of m6,6A in RNA. Using the AD-m6,6A method, we successfully detected and quantified m6,6A at position 1851 of 18S rRNA and position 937 of mitochondrial 12S rRNA in human cells. Additionally, we found that the level of m6,6A at position 1007 of mitochondrial 12S rRNA was significantly reduced in lung tissues from sleep-deprived mice compared with control mice. Overall, the AD-m6,6A method provides a valuable tool for easy, accurate, quantitative, and site-specific detection of m6,6A in RNA, which can aid in uncovering the functions of m6,6A in human diseases.

Chemoproteomic Approaches to Studying RNA Modification-Associated Proteins.

The function of cellular RNA is modulated by a host of post-transcriptional chemical modifications installed by dedicated RNA-modifying enzymes. RNA modifications are widespread in biology, occurring in all kingdoms of life and in all classes of RNA molecules. They regulate RNA structure, folding, and protein-RNA interactions, and have important roles in fundamental gene expression processes involving mRNA, tRNA, rRNA, and other types of RNA species. Our understanding of RNA modifications has advanced considerably; however, there are still many outstanding questions regarding the distribution of modifications across all RNA transcripts and their biological function. One of the major challenges in the study of RNA modifications is the lack of sequencing methods for the transcriptome-wide mapping of different RNA-modification structures. Furthermore, we lack general strategies to characterize RNA-modifying enzymes and RNA-modification reader proteins. Therefore, there is a need for new approaches to enable integrated studies of RNA-modification chemistry and biology.In this Account, we describe our development and application of chemoproteomic strategies for the study of RNA-modification-associated proteins. We present two orthogonal methods based on nucleoside and oligonucleotide chemical probes: 1) RNA-mediated activity-based protein profiling (RNABPP), a metabolic labeling strategy based on reactive modified nucleoside probes to profile RNA-modifying enzymes in cells and 2) photo-cross-linkable diazirine-containing synthetic oligonucleotide probes for identifying RNA-modification reader proteins.We use RNABPP with C5-modified cytidine and uridine nucleosides to capture diverse RNA-pyrimidine-modifying enzymes including methyltransferases, dihydrouridine synthases, and RNA dioxygenase enzymes. Metabolic labeling facilitates the mechanism-based cross-linking of RNA-modifying enzymes with their native RNA substrates in cells. Covalent RNA-protein complexes are then isolated by denaturing oligo(dT) pulldown, and cross-linked proteins are identified by quantitative proteomics. Once suitable modified nucleosides have been identified as mechanism-based proteomic probes, they can be further deployed in transcriptome-wide sequencing experiments to profile the substrates of RNA-modifying enzymes at nucleotide resolution. Using 5-fluorouridine-mediated RNA-protein cross-linking and sequencing, we analyzed the substrates of human dihydrouridine synthase DUS3L. 5-Ethynylcytidine-mediated cross-linking enabled the investigation of ALKBH1 substrates. We also characterized the functions of these RNA-modifying enzymes in human cells by using genetic knockouts and protein translation reporters.We profiled RNA readers for N6-methyladenosine (m6A) and N1-methyladenosine (m1A) using a comparative proteomic workflow based on diazirine-containing modified oligonucleotide probes. Our approach enables quantitative proteome-wide analysis of the preference of RNA-binding proteins for modified nucleotides across a range of affinities. Interestingly, we found that YTH-domain proteins YTHDF1/2 can bind to both m6A and m1A to mediate transcript destabilization. Furthermore, m6A also inhibits stress granule proteins from binding to RNA.Taken together, we demonstrate the application of chemical probing strategies, together with proteomic and transcriptomic workflows, to reveal new insights into the biological roles of RNA modifications and their associated proteins.

m6A Methylation of Transcription Leader Sequence of SARS-CoV-2 Impacts Discontinuous Transcription of Subgenomic mRNAs.

The SARS-CoV-2 genome has been shown to be m6A methylated at several positions in vivo. Strikingly, a DRACH motif, the recognition motif for adenosine methylation, resides in the core of the transcriptional regulatory leader sequence (TRS-L) at position A74, which is highly conserved and essential for viral discontinuous transcription. Methylation at position A74 correlates with viral pathogenicity. Discontinuous transcription produces a set of subgenomic mRNAs that function as templates for translation of all structural and accessory proteins. A74 is base-paired in the short stem-loop structure 5'SL3 that opens during discontinuous transcription to form long-range RNA-RNA interactions with nascent (-)-strand transcripts at complementary TRS-body sequences. A74 can be methylated by the human METTL3/METTL14 complex in vitro. Here, we investigate its impact on the structural stability of 5'SL3 and the long-range TRS-leader:TRS-body duplex formation necessary for synthesis of subgenomic mRNAs of all four viral structural proteins. Methylation uniformly destabilizes 5'SL3 and long-range duplexes and alters their relative equilibrium populations, suggesting that the m6A74 modification acts as a regulator for the abundance of viral structural proteins due to this destabilization.