In the last 2 years, we have been fighting against SARS-CoV-2 viral infection, which continues to claim victims all over the world. The entire scientific community has been mobilized in an attempt to stop and eradicate the infection. A well-known feature of RNA viruses is their high mutational rate, particularly in specific gene regions. The SARS-CoV-2 S protein is also affected by these changes, allowing viruses to adapt and spread more easily. The vaccines developed using mRNA coding protein S undoubtedly contributed to the “fight” against the COVID-19 pandemic even though the presence of new variants in the spike protein could result in protein conformational changes, which could affect vaccine immunogenicity and thus vaccine effectiveness.The study presents the findings of an in silico analysis using various bioinformatics tools finding conserved sequences inside SARS-CoV-2 S protein (encoding mRNA) same as in the vaccine RNA sequences that could be targeted by specific host RNA-binding proteins (RBPs). According to the results an interesting scenario emerges involving host RBPs competition and subtraction. The presence of viral RNA in cytoplasm could be a new tool in the virus’s armory, allowing it to improve its chances of survival by altering cell gene expression and thus interfering with host cell processes. In silico analysis was used also to evaluate the presence of similar human miRNA sequences within RBPs motifs that can modulate human RNA expression. Increased cytoplasmic availability of exogenous RNA fragments derived from RNA physiological degradation could potentially mimic the effect of host human miRNAs within the cell, causing modulation of the host cell network.Our in silico analysis could aid in shedding light on the potential effects of exogenous RNA (i.e. viruses and vaccines), thereby improving our understanding of the cellular interactions between virus and host biomolecules. Finally, using the computational approach, it is possible to obtain a safety assessment of RNA-based vaccines as well as indications for use in specific clinical conditions. SARS-CoV-2 has enlarged the family of coronaviruses of human interest by demonstrating increased virulence and the ability to cause serious disease forms . FollowiThe emergence of new strains has been linked to new pathogenic variants in the S protein, including missense, deletion, and insertion mutations .In January 2020, China reported the first SARS-CoV-2 variant in spike glycoprotein, which was an amino acid change at position 614. (p.D614G). In May 2020, the variant p.D614G was isolated in 20% of the Brazilian population, while it was found in almost all infected people in February 2021 .A new variant (p.N501Y) called B.1.1.7 was discovered in the UK in October 2020, and a new variant called B.1.351 was discovered in South Africa in December of the same year (501Y.V2). The other variant in position 484 (p.E484K) was also described in both new strains. Variants of the p.K417N, p.E484K, and p.N501Y were discovered in Japan, the USA, France, and Italy the following month [In December 2020, a new strain known as (P1) B.1.1.28 was discovered in Brazil, and 1 month later, a new strain known as P2 was discovered in Rio de Janeiro, but it was less aggressive than P1 . South ASome VOI variants have been linked to receptor-binding conformational changes, reduced neutralization by antibodies produced against previous infection or vaccination, and reduced treatment efficacy. The class of VOC, on the other hand, includes variants that increase transmissibility , decrease neutralization by antibodies generated during previous infection or vaccination, and prevent proper diagnostic classification .VOI strains include Eta (B.1.525), Iota (B.1.526), and Kappa , while VOC strains include Alpha (B.1.1.7), Beta , Delta , and Gamma . In this group, Omicron strain B.1.1.529 has also been introduced [These tests revealed a rapid decline in antibody titer, particularly in people who had an asymptomatic or paucisymptomatic infection. However, without accounting for the more complex mechanism in which memory cells play a role in their ability to reactivate, this decline has also been observed in those who have been vaccinated (B and T lymphocytes) , 11. TheCOVID-19 has the potential to become an endemic disease capable of spreading throughout the human population in a broad and recurrent manner, similar to how influenza viruses do . This isMiRNAs are non-coding, single-stranded RNA molecules with about 20–22 nucleotides that are found in plants, animals, and even some DNA viruses –16. TheyBy modulating parts of the machinery of host cells and inducing epigenetic and epitranscriptomic alterations of genes, evidence of the presence of viral mechanisms capable of directly interacting with genomic RNA has recently been reported in the literature . PositivA scenario of host transcriptional factors (TFs) competition and subtraction has also been reported . The simBased on these hypotheses, we used our pipeline to look for possible conserved RNA-binding domains sequences recognized specifically by host RBPs in all RNA sequences coding for the S protein, and then in the vaccine sequence. In addition, the in silico analysis was extended to focus on particular RNA sequences, capable to bind within the RBPs-binding motifs, than effects should be similar to role of human miRNAs. Namely, an incensement in the cytoplasmic availability of these small RNAs could mimic the effect of miRNAs within the cell by modulating the host cell’s network. This could lead to temporary delay of physiological degradation due to major “resistance” of RNA fragments bind to RBPs.p value [Examination of all SARS-CoV-2 Spike RNA-coding spike proteins derived from the most well-studied strains , as well as the full-length spike protein-encoding mRNA of Pfizer-BioNtech, Moderna, and CureVac vaccines containing proline substituted in positions 986 and 987 (p.K986P and p.V987P), were used to find conserved motifs on RNA sequences. MEME (multiple EM for motif elicitation) was used to analyze *.FASTA RNA sequences with default parameters. As shown in step 2 of Fig. p value .https://meme-suite.org/meme/tools/tomtom)), a MEME suite tool that compared the newly discovered motifs to RNA-binding motifs in Homo sapiens. Tomtom ranked the motifs in the database and used RNA-binding proteins to recognize the RNA motifs, which included 205 genes from 24 different eukaryotes. Because the sequence specificities of RNA-binding proteins are highly conserved throughout evolution, the recognition preferences of a large fraction of RNA-binding proteins can be inferred from the sequence of their RNA-binding domain. For all motif queries, a list of human RNA-binding proteins with the common conserved domain was obtained. Table All of the obtained motifs were used as queries in Tomtom ((Homo sapiens (hsa) database using the same conserved motifs as a query. The rna2meme utility was used to find mature microRNAs with RBPs-like motifs. Matches in the seeding region (positions 2 through 7) are heavily favored. Table Tomtom selected the miRBase v22 https://string-db.org/ /), a bioinformatics tool and biological database of known and predicted protein–protein interactions. The STRING database incorporates data from a variety of sources, including experimental data, computer prediction approaches, and publicly available text collections. Using a variety of functional classification methods such as GO, Pfam, and KEGG, the resource additionally highlights functional enrichments in user-provided protein lists. , an easy-to-use web tool that predicts miRNA-target interactions using data from TargetScan and miRTarBase, and GeneCodis4 ((https://genecodis.genyo.es/) ), which integrates multiple sources to provide functional enrichment analysis. From the input list of mature miRNAs, we combine the results of two different software to infer possible evidence of significant concurrent annotations  and evaluate those that are significantly enriched [The list of possible miRNA obtained through Tomtom analysis is used as a query for two different programs: MIENTURNET , in particular, binds to all three RBP motifs identified , ZNF638 (Zinc finger protein 638) and SNRNP70  proteins may also bind multiple spike mRNA motifs. The second and third RBP motifs are binded by MATR3, the first and third RBP motifs are binded by ZNF638, and the first and second RBP motifs are binded by SNRNP70 Fig. . Matrin Matrin 3 is also involved in the regulation of the transcriptional and translational networks, which control cell growth and proliferation, most likely through the formation of nuclear protein complexes that modulate pro and antiapoptotic signaling pathways. Reduced cell proliferation, which causes endothelial necrosis, is caused by matrin 3 downregulation .ZNF638 inhibition reduces adipocyte differentiation and expression of adipocyte-specific genes. ZNF638 promotes adipogenesis by acting as a transcriptional co-factor of CCAAT/enhancer-binding protein (C/EBP), which leads to the expression of the peroxisome proliferator-activated receptor (PPARG), which controls adipocyte differentiation .SNRNP70 is a nuclear ribonucleoprotein that binds to spliceosomal RNA U1 and is a key component of the spliceosome (U1snRNP). SNRNP70’s cytoplasmic pool plays an important role in nerve-dependent acetylcholine receptor (AChR) clustering and neuromuscular synaptogenesis. SNRNP70 can modulate the transcriptome composition independently of its nuclear activities, according to current evidence .The interaction of HNRNPA1 (heterogeneous nuclear ribonucleoprotein A1), HNRNPA1L2 (heterogeneous nuclear ribonucleoprotein A1 like 2), and HNRNPA2B1 (heterogeneous nuclear ribonucleoprotein A2/B1) is a key component of the network. The first motif can be binded by HNRNPA1, while the second motif can be binded by HNRNPH2 (Heterogeneous Nuclear Ribonucleoprotein H2). These proteins are part of a family of heterogeneous nuclear ribonucleoproteins (hnRNPs) that are involved in nucleoplasmic pre-mRNA processing, as well as mRNA maturation and transport .These proteins’ ability to recognize and bind other RNA molecules, such as hairpin-containing primary transcripts (pri-miRNA), appears to be very intriguing. By increasing the affinity of DGCR8 for primary miRNA transcripts, this specific bind process promotes pri-miRNA processing .HNRNPA1 is also an RNA-binding protein that plays a role in the life cycle of many DNA and RNA viruses. During viral infection and cellular stress, activation of the innate immune response causes a redistribution of the cytoplasmic HNRNPA1 process. HNRNPA1 is a trans-acting factor that binds to the 5′ untranslated RNA single strand of human rhinoviruses and influences virus translation .The RNA-binding proteins RBM5, RBM28, RBM42, and RBM6, which are all members of the same protein family, as well play a key role in the network. Despite the lack of a direct link between RBM28 and the core network, these proteins appear to play a role in a variety of cellular mechanisms, including cell cycle arrest and apoptosis induction via pre-mRNA splicing of multiple target genes, including TP53. They appear to be critical for cancer cell transformation and progression prevention in a variety of cancers, including lung cancer .In 75% of primary lung cancers, as well as prostate and breast cancer, a possible reduction in RBM5 availability, mostly localized in the cytoplasm, has already been described .Reduced cytoplasmic availability of the Strubbelig-receptor family protein group  can determine autophagy suppression regulated by SRSF1 and apoptosis of colon and lung cancer cells induced by SRSF7 and SRSF9 reduction –39.Other factors, such as LIN28A (Lin-28 homolog A), FXR2 , CELF6 (CUGBP Elav-like family member 6), and RBFOX1 (RNA-binding Fox-1 homolog 1), are also reported in the core network . Furthermore, CELF6 depletion promotes cell cycle progression, proliferation, and colony formation by modulating p21 gene expression . FinallyThe central network is not directly correlated with PABPC5 (polyadenylate-binding protein 5), YBX2 (Y-box-binding protein 2), SAMD4A .PABPC5 is involved in the regulation of mRNA metabolic processes in the cytoplasm, whereas YBX2 is a major constituent of messenger ribonucleoprotein particles and is involved in the regulation of the stability of germ cell mRNAs (mRNPs) , 45.Finally, SAMD4A is a conserved RBP found in a variety of species, and it regulates gene translation and stability. SAMD4A has been linked to angiogenesis and tumor progression in breast cancer, and its low expression in human breast tumor tissues/cells has been linked to a poor prognosis and survival .The in silico analysis was extended using the TomTom tool to analyze the presence of RNA fragment sequences generated by the degradation of RBP-binding motifs, taking into account a possible delay in physiological degradation of these motifs, such as a protective influence of RBPs-binding on motifs. With RBP target motif fragment sequences in common, we detected 39 candidate host miRNAs.  interact with each other with a cytoplasmic localization, in particular as part of cytoplasmic ribonucleoprotein granules in processing bodies (P-bodies) primarily composed of translationally repressed mRNAs . Surprisingly, an increase in small RNA that mimics the effect of has-mir-455-3p may have the opposite and protective effect .Furthermore, increased levels of hsa-miR-21-3p in hepatocellular carcinoma were found to be significantly elevated, indicating poor overall survival .Finally, hsa-miR-15b-5p has been shown to either promote or inhibit tumor progression in a variety of tumor types. In breast cancer, hsa-miR-15b-5p expression was upregulated compared to normal breast tissue, and it was associated with poor overall survival in patients. In breast cancer-promoting breast cancer cell proliferation, migration, and invasion, hsa-miR-15b-5p availability significantly decreased HPSE2 expression at both the mRNA and protein levels .In recent years, new evidence has been reported about the possible role of exogenous RNA in causing variations in cytoplasmic protein distribution, resulting in a wide range of structural, functional, and biochemical changes within host cells, furthering our understanding of the various “strategies” that RNA viruses can adapt to evade and suppress the innate immune system . Even thOver 250 new viral miRNAs were discovered at the same time, allowing researchers to investigate the function and biogenesis of virus-encoded miRNAs. Several studies have found that miRNAs encoded by RNA viruses have specific biological functions in gene regulation and the alteration of cellular pathways . Based oFurthermore, in silico analysis has been extended to assess the presence of specific sequences similar to human miRNAs in the same RBPs-binding motifs.In the first hypothesis, we found conserved motifs targeted by host RBPs within the mRNA coding S protein and vaccine sequence, which could lead to abnormal protein recruitment. In this way, the impact of various cellular networks has been assessed.In the second hypothesis, researchers looked into the possibility of RNA sequences similar to human mature miRNA being found within the same targeted RBPs motifs. Following the physiological degradation of exogenous RNA, the persistence of these RNA fragments  may be able to modulate various pathways.The discovery of close interconnections between 17 of the 22 RBPs identified has led to the suggestion of an intricate network of RBPs  have been linked to the progression of 75% of primary lung cancers, as well as prostate and some breast cancers. Low expression of SAMD4A in human breast tumor tissues and cells was also linked to a poor prognosis.The function of the HNRNPA1 protein is also intriguing because it has been described as an IRES trans-acting factor that binds the 5′ untranslated region of ssRNA human rhinovirus-2 and regulates its translation. The HNRNPA1 protein has been shown to have a high affinity for the SARS-CoV nucleocapsid protein (N) . BlockinOther intricate connections capable of regulating different networks inside the host cell are highlighted by the in-depth in silico analysis. RBPs target motifs were analyzed, and 39 miRNAs with similar sequences to RBPs target motif were observed. We found that increased levels of miR-455-3p were associated with protective effects in pancreatic cancer cells by reducing cell migration and invasion, that hsa-miR-21-3p was significantly elevated in hepatocellular carcinoma and associated with poor overall survival, and that hsa-miR-15b-5p was associated with poor overall survival in breast cancer patients.The use of our pipeline allowed for the identification of highly conserved motifs capable of binding RBPs, reducing their cytoplasmic concentration, and inducing cellular system modifications in all known SARS-Cov-2 spike protein RNA sequences and then vaccine sequences.The use of inexpensive and fast in silico analysis allowed for the provision of useful information that helped to focus the work in the wet lab. This approach is critical, especially in emergency situations like a pandemic, because it allows for rapid re-evaluation of new evidence (reverse genomics).The results of this analysis provide fascinating insights into the possible variations in infected cellular functions, opening up new opportunities for understanding concurrent symptomatology and possible side effects during infections and vaccinations at the molecular level.


RNAs provide considerable opportunities as therapeutic agent to expand the plethora of classical therapeutic targets, from extracellular and surface proteins to intracellular nucleic acids and its regulators, in a wide range of diseases. RNA versatility can be exploited to recognize cell types, perform cell therapy, and develop new vaccine classes. Therapeutic RNAs  can modulate or induce protein expression, inhibit molecular interactions, achieve genome editing as well as exon-skipping. A common RNA thread, which makes it very promising for therapeutic applications, is its structure, flexibility, and binding specificity. Moreover, RNA displays peculiar structural plasticity compared to proteins as well as to DNA. Here we summarize the recent advances and applications of therapeutic RNAs, and the experimental and computational methods to analyze their structure, by biophysical techniques , with a focus on dynamic and flexibility aspects and to binding analysis. This will provide insights on the currently available RNA therapeutic applications and on the best techniques to evaluate its dynamics and reactivity. Modern molecular biology research redefined the central dogma that explains the flow of genetic information, from DNA to RNA and from RNA to protein  and shedad hoc the RNA construct sequence or to adapt to a new therapeutic request, such as pathogen variants.Therapeutic RNAs can modulate transcript level, inhibit RNA modulator activity, encode therapeutic protein, or induce alternative splicing, and also bind target proteins . AdvantaDespite the advantages of RNA-based therapies, the development of RNA therapeutics meets challenges. RNAs are rapidly catabolized by ubiquitous RNases  and exogRNA molecules fold, based on the Watson–Crick base pairing, into secondary structures and specific sub-structures, such as characteristic RNA loops, like hairpin loops, internal and external loops, base-pair stackings, multi-branch loops, bulge loops, junctions, pseudoknots, kissing hairpins, and so forth. Functions of ncRNAs as well as their effectiveness as therapeutic agents are deeply related to their folding because these structures are recognized by proteins, other RNAs and other parts of the same RNA. Their thermodynamic stability is crucial, and it is determined by conditions that occur and change when interacting with proteins or other ligands . The preThe implementation of therapeutics drug design and bioinformatics platforms, structural modeling and machine learning play a key role in this new RNA therapeutics field. Small molecule drug development pipelines that target enzymes, and protein–protein interactions cannot be applied to nucleic acids. The progresses in experimental resolution and computational modelling of RNA structures have enabled clinical translation of RNA-based therapy. In this review we will discuss the characteristic of RNA therapeutics and recent advances in this field, summarizing from the structural viewpoint, the available strategies, and the computational and experimental analysis methods.RNA-based therapies rely on both coding and non-coding RNAs, and mainly targets nucleic acids (ether DNA or RNA) and proteins. These therapies can control gene function by silencing or activation, splice modulation, transcript degradation, translational activation, or antigen synthesis, up to protein encoding and function, as also decrease or block protein production . AptamerAptamers are short (20–100 nt) single strand nucleic acids that fold into specific tertiary structures . AptamerIn 1990 two different groups  developein vitro, with a very low variability among different batches, and lower production cost  single strand DNA or RNA molecules . OverallSteric blocking ASOs are RNase H-independent and can inhibit or increase protein translation by binding to different targets. Inhibition is obtained by binding directly to mRNA and pre-mRNA or to trans-acting factors, like small nuclear RNAs, miRNA, long non-coding RNAs or RNA binding proteins . This inExon-skipping ASOs are single strand splice-switching oligonucleotides (SSOs) that recognize pre-mRNA altering the splicing process . SSOs arin silico prescreening using statistical modelling  are short (20–25 nt) double stranded non-coding RNAs , obtaine(19)_TT was suggested to be advantageous  are short (22 nt) double strand non-coding RNAs , that reMiRNA-based therapeutic can mimic endogenous miRNAs or inhibit them (antimiRs): mimic miRNAs, administered as pre-miRNA, are double-stranded, match the endogenous miRNA and display the same activity restoring the lost miRNA expression in disease ; miRNA iMiRNAs are dysregulated into a wide variety of disease, therefore there is a clear interest for the use of miRNAs as therapeutic agents or targets . Moreovevia non-homologous end joining (NHEJ) or homology-directed repair (HDR), thereby resulting in gene disruptions and inactivation of the targeted gene , a DNA endonuclease, and a single guide RNA (sgRNA) . In sgRNted gene . NHEJ isted gene   must be present at sufficient concentrations to form intracellular ribonucleoproteins ; and difThe fine-knowledge of this large complex, obtained by biochemical and structural studies on Cas9 at different stages of DNA target surveillance, provides key hints for its customization and application in therapies . Recentlex vivo to guide differentiation or alter cell phenotype, and then these cells are injected back to the patient  .A strategic role in the development of RNA-based therapies is played by physical-chemistry, structural biology, and computational modelling of biomacromolecules. These disciplines ensure the fine tuning and/or prediction of the intramolecular interactions that drive molecular recognition. Hence, mastering the RNA chemistry is critical in this respect. Despite the apparent chemical similarity between DNA and RNA nucleotide monomers sidechains, the subtle difference between the two backbone chemical moieties enormously influences both the reactivity and the dynamics of the two polymers. Each nucleotide consists of a planar aromatic base attached to a furanose ring with a 5′-phosphate group. Phosphodiester linkages between successive sugar residues groups (linking the 3′-carbon of each sugar to the 5′-carbon of the next one) drives the polymerization of RNA chains, leaving a free 5′-position at one end of the chain and a free 3′-position at the other. If this process is basically the same for DNA, the 2′-OH group of the RNA ribose (2′-H in DNA) moiety makes RNA less chemically stable than DNA by facilitating self-cleavage reactions, thus making RNA more flexible and prone to adaptation in cells. Moreover, 2′-OH group is a versatile hydrogen bond donor and acceptor, allowing the formation of unique macromolecular spatial arrangements with a functional role. This is reflected by the necessity of describing RNA molecules hierarchically by analyzing both their secondary structure, i.e., the interaction between nucleic acids regardless their three-dimensional orientation and the overall folding, and their tertiary structures, i.e., the spatial organization of atoms belonging to the RNA polymer. Moreover, post-translational modification of RNA has an impact on topological and spatial arrangement of RNA molecules .Secondary (2D) structure is defined by the pattern of Watson−Crick canonical base pairs . Canonical double helices alternate with unpaired regions, namely that do not form canonical base pairs. RNA topology is determined by the backbone, which depends on the position and direction of the backbone segments attached to the bases. This links a local property (base pair geometry) with the global topology, which determines the molecule’s biological function. Building on the basic architecture defined by the 2D structure, RNA 3D structure is determined by some canonical and many noncanonical base pair interactions that often involve H-bonding through the 2′-OH group. RNA folding kinetics is hierarchical and sequential secondary (2D) structure motifs display an overall higher stability compared to 3D structures, they fold autonomously, and usually  before aA single RNA molecule, with a well-defined sequence, often has multiple accessible 2D and 3D structures that lie within a narrow range of folding free energies. These are sampled because of thermal fluctuations or proteins and other cofactors interactions induce or select specific RNA conformations. The time scales over which these alternate structures form and disappear can range from microseconds for simple base pairing changes to seconds (or even longer) for complex 2D refolding events . BecauseAfter at least three decades of investigations led by physico-chemical techniques, the structure-function paradigm is currently integrated in almost all the studies that address how biomacromolecules mechanistically exert their function. More recently the role of the internal motions has been elucidated and has revealed to be crucial in molecular recognition between partners. Macromolecular structures obtained by X-ray crystallography, high-resolution NMR spectroscopy and from cryoelectron microscopy constitute an essential technique for characterizing the lowest  energy state of a molecular system and understanding molecular recognition. Nowadays, structural plasticity has become a stable asset to understand macromolecular functionality. The combination of complementary physico-chemical techniques  allows to extensive investigate structural equilibrium of molecules, also to obtain truthful models. It is of key importance also the experimental characterization of the less populated states , that aIn this section we will discuss biophysical methods that are now routinely used for the characterization of the interaction between biomolecules and their partners, either other macromolecules or small molecules, with a focus on their application to RNA. In this respect, due also to the relatively recent role of RNA molecules as drugs, these techniques will be reviewed regardless the oligonucleotides function as ligand or as target, with the aim of highlighting the general principles behind the characterization of their interaction with any partner. Moreover, in this respect a purpose of this review is the suggestion of the application of these techniques, once they have not been intended in this way, to the world of RNA-based drug discovery.Nuclear magnetic resonance (NMR) spectroscopy is the most advanced experimental technique that can efficiently provide details ranging from atomic positions to inner or global dynamics . The innThis technique monitors RNA dynamics in real-time , and is the most intuitive NMR-based method, as the changes that occur in a biological system can be directly associated with disappearance and appearance of signals. In a classical drug discovery context  kinetic properties of non-equilibrium ligand-dependent RNA can be followed using time-resolved NMR . Real tiThe proper understanding of biomolecular recognition mechanisms that take place in drug targeting is of paramount importance to improve the efficiency of drug discovery and development. The flexible nature of RNA influences the molecular recognition mechanisms and its characterization plays a key role in a drug discovery context, i.e., allowing the identification of multiple conformers involved in the process. NMR spectroscopy offers a wide range of techniques that can be informative in this context also including time resolution of structural interconversion.13C, 15N) (in vitro transcription using the T7 RNA polymerase (RNAP) enzyme  . As exte) enzyme . Recentl) enzyme , leading) enzyme , as wellSpin relaxation, that can be seen as a measurement of the decay of the excited magnetization perpendicular to an externally applied magnetic field, has been widely used in the last two decades for the characterization of internal protein motions. The theoretical framework for its interpretation correlates the macromolecule overall shape, reference structure and hydrodynamics to its relaxation properties. While this provides a proper description of the relaxation-related observables for globular proteins, its application to flexible objects like intrinsically disordered proteins  or RNA hChemical bonds and structural motifs orientation have an influence on dipolar couplings (DCs), i.e., the spectroscopic observable that reflects the interaction between individual nuclear spins energy levels as mediated by their distance, electronic environment, and relative orientation. Residual dipolar couplings (RDCs) are variations of complete motional averaging of DCs due to a partial molecular alignment to the external magnetic field. RDCs can also provide information about dynamics on micro-milliseconds timescale. An application of RDCs for the determination of RNA binding modes to proteins has been reported in Small Angle X-rays scattering (SAXS) provides an accurate and diverse set of parameters that describe biomolecules, including global information about macromolecular size and shape, intermolecular association, domain motion and linker flexibility . Recentlvia readily available computational tools to model structures from SAXS data (2O with D2O (deuterated water). Contrast variation methods for neutron scattering are perhaps more effective to implement as they involve replacing H2O with D2O and enable “blanking” of either the protein or nucleic acid components of the sample. Contrast variation SAXS presents unique opportunities for studying the dynamics of RNA–protein complexes, for example adding sucrose to the buffer to increase its electron density to equal that of the protein, ensuring significant scattering of the RNA even above this electron dense background. The contrast variation method has recently been applied to study DNA–protein complexes, where the use of high intensity X-ray sources enables time resolved studies  and AIR-3A were investigated by different methods including RNA structure probing, SAXS and microscale thermophoresis. SAXS experiments allowed the determination of the overall shape  of self-recognition during AIR-3/A assembly’s formation, providing an indication of its dimeric state in the apo- and -holo- forms with the help of MD simulations.SAXS can be used to investigate dynamics of nucleic acids, except for the experimental characterization of complex formation and hydrodynamics. Dynamics of biological relevant nucleic acids has been investigated  buildingChemical reactivity of flexible molecules is influenced by inner dynamics, and it is also altered by binding. In the last decade, RNA profiling is employed for characterizing the inner dynamics and topology of RNA, both as single molecule and during molecular recognition. This is challenging, but a promising development in the field of RNA-based drug discovery, offers the possibility to comparatively monitor the effect of RNA binders, flexibility, and selectivity, including several quantitative interpretations of reactivity, in a relatively fast and direct way.RNA chemical probing is a versatile technique that can be used to elucidate RNA secondary and tertiary structures as well as RNA-ligand interactions at single-nucleotide resolution in the solution state  Table 2Table 2. Escherichia coli thiamine pyrophosphate (TPP) riboswitch aptamer domain in the presence and absence of saturating concentrations of the TPP ligand: SHAPE-MaP confirmed the known reactivity pattern for the folded, ligand-bound RNA and accurately reported nucleotide-resolution reactivity differences that occur upon ligand binding. The latter has been performed on the HIV-1 RNA genome, thus updating, and completing its high-resolution topological model. An extension of this technology has been proposed for its performance in live cells , MD simulations may be complemented by quantum mechanical (QM) calculations, leaving to molecular mechanical (MM) the treatment of distal layers of the RNA that are not directly involved in forming or breaking covalent bonds, to provide context and to capture the impact of conformational dynamics on the reaction . DespiteIn the protein and protein-protein interactions field, standard MD simulations have been considered a gold standard methodology for the inspection of biomolecules plasticity and their role in molecular recognition, for many years. Concerning RNAs, the high heterogeneity of the transitions timescales disallows the straightforward application of the aforementioned methodologies. Thus, enhanced sampling methods are of strong interest to overcome complicated energy barriers and to efficiently explore a rough free energy surface, simulating events that occur in the range of biologically and thermodynamically relevant RNA motions timescale. Indeed, MD simulation has been recently employed to overcome the high costs and time-consuming SELEX process, as well as to study the interaction mechanism between the aptamer and the target molecule on the atomic scale .In general, the standard MD simulations, for the study of recognition dynamics, are relatively often used for RNA, but always coupled to other techniques like free energy perturbation or MMPBSA calculations  or steerThe huge increase in the computational power available at relatively low expense allowed in the recent years the performance of extensive MD simulations that can be analyzed in terms of states and their relative population. A transition probability matrix can be constructed from MD simulations providing the probability of observing transitions between pairs of population clusters, by means of discrete jumps . The sysBeing already successfully applied to the study of many proteins systems , they arVia additional docking calculations, the theophylline binding site is found only in one ensemble of conformers accounting for one quarter of the overall population, whereas most of them are binding-incompetent, lacking a binding pocket that can accommodate theophylline. Moreover, the complete theophylline binding pathway has been simulated, supporting prior experimental observations of slow theophylline binding kinetics by showing that the binding site must undergo a large conformational rearrangement after the aptamer and theophylline forms an initial complex, namely, the rearrangement of a single base from a buried to a solvent-exposed orientation.Few examples can be found in the literature on the application of the MSM to RNA-ligand complexes. A notable exception is constituted by the study of the structural diversity and dynamics of a theophylline-binding RNA aptamer in its unbound state . MSM havAnother example of the application of MSM applied to RNA complex, to characterizing the RNA fraying kinetics, is reported in In replica exchange schemes (RE) numerous replicas of the system are simultaneously simulated using different parallelization parameters  and, periodically, exchanges are attempted between them according to a Metropolis criterion . The mosHigh temperature simulations alone have sometimes been used qualitatively to enhance sampling of RNA systems; however, they cannot be used to directly estimate the values of experimental observables at physiological temperatures. H-REMD sampling technique has been used by In metadynamics , a bias A recent interesting application of MetaMD has been illustrated for the characterization of the RNA targeting by Peptide Nucleic Acids (PNAs), one of the most established and efficient artificial systems for targeting DNA or RNA so far described . PNAs coAlthough the feasible time scales of biomolecules all-atom MD simulations have impressively increased in recent decades, and enhanced sampling methods allow deep exploration of the free energy surfaces of large molecules, there are many phenomena that still cannot be efficiently studied by atomistic MD. Biologically interesting processes, such as the folding of RNA structures or the domain motions of ribosomal subunits during translocation, can occur time scales of the order of milliseconds or longer, and might also involve millions of atoms or more. One alternative approach to all-atoms simulations is the use of simplified coarse-grained (CG) descriptions, i.e., the reduction of the degrees of freedom of the original atomistic system by grouping selected sets of atoms and representing them as a smaller set of CG particles (beads) that interact through effective energy functions. This reduction reduces the complexity of the calculation, and, in general, defines a smoother energy surface, which can be explored more quickly, allowing larger systems and longer simulation timescales. Because of the used approximations, CG models will generally only faithfully reproduce a specific set of observables in a limited region around the conditions (thermodynamic and non-thermodynamic) assumed in their parameterization. Therefore, these approaches must be used with care, and may sometimes require a redefinition of the observables of interest based on the model resolution. However, CG simulations can provide important insights that complement atomistic MD simulations or contribute to interpret experimental observations.Many codes are currently available for performing simulations of RNA applying the coarse-grained scheme [OxDNA , SimRNA 2+ ions binding sites on the surface and in the RNA cavities. This topic has been recently covered by a similar study in the field . Despite having lower throughput relative to HTS, NMR spectroscopy is also a powerful tool for identifying and validating small molecules that interact with biomolecules and has played a significant role in protein-targeted drug discovery. Excellent reviews have been published in recent years  NMR spectroscopy is one of the most widely used NMR methods in drug discovery, such as fragment-based drug discovery (FBDD) screening for protein targets. STD experiments build upon magnetization transfer between biomolecules, such as proteins or RNAs, and small ligands . If a livia gradient spectroscopy) is a ligand-observed NMR technique that can be used in target-directed drug screening or ligand validation to assess the binding of molecules to macromolecules  and low to moderate affinity are identified that bind a target of interest, and these fragments are then either elaborated or linked to create more effective ligands . Fragmenbuilding . This mevia multiple subsites, which are regions of a binding pocket that contact a ligand in an independent or cooperative manner (Zeller and coworkers have recently shown that many RNAs bind their ligands e manner . Besides12–1015) of ∼20–100 nucleotides containing flanking constant sequences required for PCR amplification. For RNA SELEX (in vitro transcription of dsDNA templates using T7 RNA polymerases. Modified nucleotide triphosphates (NTPs) are typically incorporated during the selection process to reduce the possibility of post-selection modifications impairing aptamer function  of chemical immobilization that may compromise the target molecule before even starting the selection process. The difficulties in the selection of small molecule binding aptamers, can be avoided by a target-immobilization free protocol called Capture-SELEX , where tSAXS-based RNA screening (SAXScreen) procedure has been used to categorizes ligand titrations by computing pairwise agreement between scattering curves and by estimating affinities through the quantification of complex formation as deviation from the linear combination properties of solution SAXS . The resvia flexible-RNA and flexible-ligand docking  one example of induced-fit binding  docking . Besides docking , until r docking , all of ab initio a ssRNA (ad hoc scoring function are under development (Actually, just some examples of RNA-protein docking are available; Chauvot de Beauchene group used a fragment-based approach and docking to design  a ssRNA , and Gui a ssRNA ; ad hoc elopment . Besideselopment , could b


LEVER, which mapped 236 kb upstream of the β-globin cluster as confirmed by Nanopore sequencing. LEVER RNA interacts with PRC2 in its nascent form, and this prevents the accumulation of the H3K27 repressive histone marks within LEVER locus. Interestingly, the accessible LEVER chromatin, in turn, suppresses the chromatin interactions between the ε-globin locus and β-globin locus control region (LCR), resulting in a repressive effect on ε-globin gene expression. Our findings validate that the nascent RNA-PRC2 interaction inhibits local PRC2 function in situ. More importantly, we demonstrate that such a local process can in turn regulate the expression of neighboring genes.Polycomb Repressive Complex 2 (PRC2) is an epigenetic regulator required for gene silencing during development. Although PRC2 is a well-established RNA-binding complex, the biological function of PRC2-RNA interaction has been controversial. Here, we study the gene-regulatory role of the inhibitory PRC2-RNA interactions. We report a nuclear long non-coding RNA,  Identification of a long non-coding RNA LEVER, that inhibits the Polycomb Repressive Complex 2 (PRC2) and controls nearby embryonic form of beta-globin gene, provides additional evidence for PRC2-RNA functional interaction. These biological functions are achieved mainly through establishing and maintaining the global methylation landscape of lysine 27 on histone H3. In particular, the final H3K27 tri-methylated product (H3K27me3) generally marks transcriptionally inactive regions and is considered as the major epigenetic output through which PRC2 supervises the repressive genomeXIST2 and HOTAIR4, were proposed to serve as co-factors to direct PRC2 to its specific targets in the mammalian genome. Importantly, this model highlights an axis of a specific lncRNA-PRC2-specific target gene loci.However, in mammals, how PRC2 selectively represses target genes remains elusive. Many long non-coding RNAs (lncRNAs), such as 18. First, the promiscuous RNA-binding pattern of PRC2 in vitro and in cells challenged the selectivity of PRC2 for its specific lncRNA partners12. Second, RNA can inhibit PRC2 enzymatic activity10 by competing with histone for PRC2-binding in vitro14 and in cells12. Furthermore, extensive interaction between PRC2 and nascent RNAs was observed in mouse embryonic stem cells (ESCs) at transcriptionally active gene loci12, which brings into question the transcription repressive effect by local PRC2-RNA interaction at these loci. In line with these findings, PRC2 occupancy and H3K27me3 levels at previously active gene loci were increased upon global RNA degradation12 or transcription blockage9.This lncRNA-directed PRC2-mediated gene repression model has been questioned by several follow-up studies on PRC2-RNA interactiongene autonomous’. The nascent RNAs sequester and inhibit local PRC2, thereby protect their own gene loci from being accidentally repressed18. Meanwhile, these nascent RNAs also locally reserve PRC2, preparing the complex to occupy and function quickly once chromatin repressive signals are detected18. These two functions theoretically maintain the current cellular state while renders adaptiveness to environmental changes, and phenotypically this has been supported by one recent study showing that global disruption of PRC2-RNA interaction led to differentiation defects in induced pluripotent stem cells (iPSC)17. Notably, while this RNA inhibiting PRC2 model is well supported mechanistically, the histone modification was the major readout in most studies and its direct effect on autonomous gene transcription was less evidenced due to technical limitations caused by the autonomous regulation.While an increasing amount of evidence suggested that PRC2 interacts with nascent RNAs and such interaction inhibits PRC2 function, the consequential effects of such PRC2-inhibitory interaction on gene expression have been mostly proposed as ‘LEVER, short for the regulation of ε-globin through inactivating EZH2 by an upstream non-coding RNA), and we further characterized LEVER by direct-RNA Nanopore sequencing. LEVER RNA represses the expression of the neighboring ε-globin gene. Mechanistically, LEVER RNA inhibits PRC2 chromatin occupancy and thereby regulates H3K27me2/me3 levels at the LEVER locus. The epigenetic status at the LEVER locus in turn influences chromatin interactions involving the β-globin Locus Control Region (LCR) that further regulates ε-globin transcription. In summary, our study reveals a model that nascent RNA sequesters PRC2 from its genome locus and blocks PRC2 activity in cis, which remodels local chromatin interaction and regulates neighboring gene expression. This provides an example of a robust non-autonomous gene-regulatory role for nascent RNAs in suppressing PRC2.Here, we report a non-autonomous gene-regulatory role of the inhibitory PRC2-RNA interaction. We identified a PRC2-interacting non-coding RNA  and adenosine (A) was significantly enriched from our motif analysis  in K562 cells for endogenous EZH2, which is the enzymatic subunit and the subunit with the highest RNA-binding affinity in PRC212. Collectively, these results support previous findings that RNA antagonizes PRC2 chromatin binding and thereby inhibits its methyltransferase activity in K562 cells.To further study how RNA affects PRC2 chromatin occupancy biochemically in K562, we inhibited global transcription by treating the cells with the RNA Polymerase II (Pol II) inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) in culture and performed co-immunoprecipitation against PRC2 subunits EZH2 or SUZ12  whose transcription start site locates 236 kb upstream of the β-globin gene cluster on chromosome 11  do not overlap with the spliced exons identified by our Nanopore polyA+ RNA sequencing. Therefore, we confirmed that EZH2 binds multiple intronic rather than exonic RNAs of the long LEVER nascent transcript  spanning the LEVER TSS site  and the embryonic form globin (ε-globin) genes were dominantly expressed, while the adult globins (β-and δ-globins) are undetectable by RT-qPCR. Interestingly, the mRNA level of ε-globin gene, which locates closest to the LEVER locus among the β-globin cluster genes  system with a dead Cas9 . Notably, the dCas9 we used in our CRISPRi21 experiments is not fused with any epigenetic silencer, e.g., KRAB22. Similar to the LEVER knock-out cells, ε-globin expression increased after LEVER knock-down -derived erythroblasts 25 in the knock-out cells restored ε-globin expression, while the truncated EZH2 mutant without methyltransferase motif (EZH2ΔSET) failed to do so  as a negative control. Indeed, we found that re-expression of the EZH2-interacting LEVER fragments can reverse the ε-globin activating effect caused by the loss of the whole LEVER transcript. Changes in ε-globin expression were demonstrated at both ε-globin mRNA  or CCDC26 (negative control) regions  at the LEVER locus was correspondingly reduced, further supporting the above observations. These ChIP-seq results were further validated at selected regions by qPCR 26. Therefore, we asked whether the epigenetic changes within the LEVER locus could remodel local chromatin interactions and thereby influence ε-globin expression. To test this hypothesis, we performed 4C-seq analysis before and after LEVER promoter knock-out to compare local chromatin interaction changes upon LEVER RNA loss. We positioned two viewpoints at the ε-globin promoter and another two viewpoints at HS3 and HS4 regions within the β-globin LCR, which are well-established activating elements of the β-globin cluster27  nascent transcript without disrupting its transcription through currently available RNA targeting systems, we tried but failed to reduce LEVER nascent RNA abundance using shRNA or Cas13a32. Therefore, the independent effects of LEVER RNA and transcription remain to be resolved in future studies. This question could be addressed by a localized overexpression experiment with techniques enabling localized enrichment of LEVER fragments at the LEVER locus, similarly as designed by Beltran et al.16. Such a method would further demonstrate the causal relationships between PRC2-regulated epigenetics, chromatin interaction, and ε-globin expression proposed in our model  and were used in experiments at their early passages. K562 cells were cultured in RPMI (Biowest) supplemented with 10% fetal bovine serum (FBS) (Biowest). HEK293T cells were cultured in DMEM supplemented with 4500 mg/L glucose (Biowest), 2 mM L-glutamine (Thermo Scientific), 1X MEM non-essential amino acids (Thermo Scientific), 1 mM sodium pyruvate, and 10% fetal bovine serum (FBS) (Biowest). All cell lines were tested for mycoplasma every 6 months.pCMV-dR8.91, pCMV-VSV-G, and lentivectors were transfected into 10 million 293T cells using Lipofectamine 2000 following the manufacturer’s instructions. Supernatants were harvested at 48 and 72 h after transfection. Viruses were concentrated 100–300 times through centrifugation after filtering through 0.45 µM syringe filters and stored at −80 °C. The titers of viruses were determined on HeLa cells using flow cytometry. The plasmids used in this study are listed in Supplementary Data LEVER KO) or western blot (for EZH2 KO) were expanded and analyzed by genotyping. The list of sgRNAs used can be found in Supplementary Data K562 cells were transduced with the FUCas9Cherry or FUdCas9Cherry lentiviruses at 0.5 MOI with 5 µg/mL polybrene. The transduced cells were sorted for mCherry positive as K562-Cas9/dCas9 stable lines. FgH1tUTG-sgRNA viruses were further transduced into these K562-Cas9/dCas9 stable lines and sorted for GFP positives. To generate EZH2 KO, in vitro synthesized sgRNA (Synthego) was transiently transfected into K562-Cas9 cells with DharmaFECT 1 (DHARMACON) following the manufacturer’s instructions. Of note, the DNA editing efficiency of the sgRNAs at their specific targeting regions was determined in K562-Cas9-sgRNA cells by the T7 endonuclease I (NEB) assay (as described in the manufacturer’s protocol) after sgRNA induction by 1 µg/mL doxycycline for 3 days. EZH2-sgRNA transfected (48 h) K562-Cas9 cells or Induced K562-Cas9-sgRNA cells were seeded into single-cell clones in 96-well plates. Successful KO clones screened by RT-qPCR  was cloned from K562 cDNA into a modified pLX304 plasmid in which the CMV promoter was replaced with the CAG promoter. The EZH2Protein lysates were prepared in RIPA buffer , 0.1% SDS), and the lysates were mildly sonicated into clear supernatants before subjected to protein quantification by the Pierce BCA assay (Thermo Scientific). 10–20 µg of protein lysate was loaded into each well of SDS-PAGE mini gel. The electrophoresis was performed in Tris-glycine buffer supplemented with 0.1% SDS. Proteins were then transferred onto nitrocellulose membranes (Bio-Rad) in Tris-glycine-methanol transfer buffer. The membrane was blocked by 5% skim milk/TBST and incubated with primary antibodies followed by HRP-conjugated secondary antibodies diluted in 5% skim milk/TBST. Chemiluminescence was detected by exposing X-ray film to the blot pre-incubated with the Luminata Crescendo Western HRP substrate (Millipore). The list of antibodies used can be found in Supplementary Table One microgram of RNA was reverse transcribed using Qscript cDNA Supermix (QuantaBio). qPCR on cDNA or ChIP DNA was performed with GoTaq qPCR Master Mix (Promega) under the manufacturer’s instruction on a QS5 system (Thermo Scientific). The copy numbers were measured by predetermined standard curves. The relative expression or enrichment to control samples was calculated based on the ΔΔCt method. Quantitative digital droplet PCR was performed on a QX200 Droplet Digital PCR (ddPCR) System (Bio-Rad) with EvaGreen Digital PCR Supermix (Bio-Rad) under the manufacturer’s instructions. Primers used in this study are listed in Supplementary Data 35. Briefly, 12 million cells were harvested and washed first with 20 mL of cold PBS supplemented with 5 mM ribonucleoside Vanadyl complex (NEB) and 1 mM phenylmethylsulphonyl fluoride (PMSF), and then with 20 mL of cold PBS supplemented with 1 mM PMSF. The cell pellet was lysed in 1.7 mL of cold hypotonic buffer  supplemented with 1 mM PMSF, EDTA-free protease inhibitor cocktail (Roche) and 20 U/mL SUPERase-In (Invitrogen). The cells were homogenized with a 15 mL Dounce homogenizer (by 10 strokes with Pestle A and 45 strokes with Pestle B). The homogenized cells were diluted in 7.8 mL of NP-40 free hypotonic buffer containing 2 M sucrose. 3.2 mL of cushion buffer (BSA and NP-40 free hypotonic buffer containing 2 M sucrose) was added to the bottom of a polypropylene tube , and the cell lysate was overlaid on the cushion buffer without disrupting the bottom phase. Nuclei were harvested after ultra-centrifugation at 100,000 × g for 1 h at 4 °C with a SW41Ti rotor and Beckman Coulter TH641 ultra-centrifuge. The supernatant was removed, and the nuclear RNA was extracted by Trizol according to the manufacturer’s protocol. Nuclear polyA+ RNA was extracted with a NEBNext Poly(A) mRNA Magnetic Isolation Module according to the manufacturer’s protocol, and the nuclear polyA− fraction was extracted from the unbound supernatant by ethanol precipitation. Cytoplasmic RNA was isolated with a Qiagen RNeasy kit according to the manufacturer’s protocol. The fractionated RNA was subjected to DNase I digestion and then re-purified using Trizol. The RNA was quantified with the Qubit RNA HS assay kit (Thermo Scientific), and rRNA was removed by the Ribo-Zero Gold rRNA Removal Kit (Illumina). The stranded RNA-seq libraries were constructed with the Script-seq V2 RNA-seq library preparation kit (Illumina). The 250–500 bp size-selected libraries were sequenced on the Illumina MiniSeq platform with MiniSeq High Output 2 × 150 paired-end kit.Nuclear RNA purification was performed as described previously2, 0.1% Triton X-100) supplemented with EDTA-free protease inhibitor cocktail (Roche), phosphatase inhibitor (Roche), 1 mM DTT, and 1 mM PMSF and then centrifuged at 800 RPM for 10 min at 4 °C. The nuclei pellets were washed once with the cell lysis buffer without Triton X-100 and harvested by centrifugation at 1300 × g for 4 min at 4 °C. Nuclei were resuspended in resuspension buffer  supplemented with EDTA-free protease inhibitor cocktail (Roche), phosphatase inhibitor (Roche), 80 U/mL RNaseOUT (ThermoScietific), and 1 mM PMSF. The nuclei were sonicated using EpiShear Probe Sonicator (Active Motif) for 15 s at 30% amplitude. 500 µl of the lysate was incubated with EZH2 antibody (1:250), SUZ12 antibody (2 µg), or rabbit IgG isotype (2 µg) in the cold room for 12 h. The lysate was incubated with 50 µl pre-blocked dynabeads protein A (Invitrogen) for 2 h at 4 °C. The beads were washed with washing buffer  for three times before eluted with 30 µl of RIPA buffer and 15 µl of 6X Laemmli buffer at 95 °C for 10 min. The eluted complex was analyzed with western blot.For DRB treatment, 12 million K562 cells were treated with 100 µM DRB for 24 h before subjected to nuclei extraction. For RNase A treatment, 10 million K562 cells were first harvested and washed with 1X PBS, and then treated with 1 mg/mL RNase A in 0.05% Tween-20 for 10 min at RT before subjected to nuclei extraction. The DRB/RNase A treated cells were lysed on ice for 5 min with cell lysis buffer  SUPERase-In was used in substitution of ribonucleoside Vanadyl complex from the cell lysis step. (B) Fixed K562 cells were lysed with Dounce homogenizer by 10 strokes using Pestle A and 60 strokes using Pestle B. (C)The nuclei were sonicated with Bioruptor (Diagenode) for 8 cycles . (D) Nuclear lysates were pre-cleared with Dynabeads protein G twice at 4 °C. (E) 5 µg of EZH2 or IgG isotype control antibodies were used in each RIP experiment. RIP experiments were performed in three biological replicates, and two of these replicates were independently sequenced. For sequencing, the immunoprecipitated RNA was quantified with the Qubit RNA HS assay kit (Thermo Scientific) before rRNA Removal with Ribo-Zero Gold rRNA Removal Kit (Illumina). 15 ng of the rRNA-depleted RNA was used for sequencing library construction. The stranded RIP-seq libraries were constructed with a Script-seq V2 RNA-seq library preparation kit (Illumina). The libraries were subjected to size selection (250–500 bp) on a 4–20% TBE PAGE gel (Thermo Scientific). The recovered libraries were sequenced on the Illumina MiniSeq platform using the MiniSeq High Output kit with 2 × 150 paired-end.RIP was performed as described previously36 with minimal modifications. Briefly, K562 chromatin was sonicated with Bioruptor (Diagenode) for 15–25 cycles , and immunoprecipitation was performed with Dynabeads protein G. For ChIP-sequencing, the libraries were constructed using a Thru-PLEX DNA-seq 12S kit (TakaraBio). The libraries were subjected to size selection (250–500 bp) on a 4–20% TBE PAGE gel (Thermo Scientific). The recovered libraries were sequenced on the Illumina Nextseq platform using Nextseq 500 high-output kit with 2 × 76 paired-end. For H3K27me3, H3K27me2, or H3K27Ac ChIP-seq, one replicate was sequenced, and the results were validated by ChIP-qPCR with independent biological replicates.ChIP was performed as described previouslyFractionated nuclear RNA was used to construct a cDNA library using a 1D sequencing kit (SQK-LSK108) according to the manufacturer’s instructions. In brief, first-strand cDNA was generated from 20 ng of rRNA-depleted nuclear RNA using SuperScript IV reverse transcriptase with 0.35 pmol/µl random hexamer at 52 °C for 60 min. Second strand cDNA was synthesized with the NEBNext second strand synthesis kit for 1 h at 16 °C. cDNA was purified with AMPure XP bead. Library was constructed with LongAmp Taq PCR for 18 cycles. The PCR product was purified with phenol:chloroform:isoamyl alcohol (25:24:1) and ethanol precipitated in the presence of glycogen and was finally dissolved in 10 mM Tris buffer (pH 8). The cDNA library was sequenced on a Nanopore flow cell (FLO-MIN106). Enriched polyA+ RNA was used to construct the library for direct RNA sequencing (SQK-RNA001) according to the manufacturer’s instructions. In brief, 400 ng of total polyA+ RNA was used for reverse transcription adapter (RTA) ligation before subjected to reverse transcription. RNA/DNA hybrids were purified with RNAClean XP beads and ligated with RNA adapters as direct RNA sequencing library. The constructed library was sequenced on the Nanopore flow cell (FLO-MIN106).37 with modifications. K562 cells were fixed as ChIP protocol except that the cells were fixed in 2% formaldehyde at the concentration of 1 million cells per mL for 10 min. Cells were lysed in complete lysis buffer ). The nuclei were pelleted, washed once with complete lysis buffer, and then lysed with 100 µl 0.5% SDS solution at 62 °C with 400RPM agitation for <10 min. 299 µl nuclease-free water and 20 µl of 20% Triton X-100 were added before 15 min incubation at 37 °C to quench SDS. 1X DpnII buffer (NEB), 0.8 U/µl DpnII restriction enzyme (NEB), and 0.08 mg/µl BSA were supplemented to the chromatin solution for DpnII digestion at 37 °C for 4 h with 1000 RPM agitation. Additional 400U of DpnII was added to the sample for overnight digestion. DpnII was heat-inactivated at 62 °C for <20 min. An aliquot of the digested chromatin was sampled for DNA purification followed by DNA quantification, agarose gel electrophoresis, and PCR to monitor digestion efficiency. Digested chromatin equivalent to a total 10 µg of DNA was ligated with 660U HC DNA ligase  in 1X ligation buffer  for 8–10 h at 16 °C without shaking. The reaction was incubated at room temperature for 30 min. The chromatin was reverse-crosslinked with 0.5% SDS and 50 µg/mL of Proteinase K (Invitrogen) at 64 °C overnight. RNA in the chromatin was digested with 2 mg/mL RNase A at 37 °C for 1 h. The ligated DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) followed by chloroform and then precipitated with ethanol (68% to avoid SDS precipitation) in the presence of glycogen. The ligated DNA product was analyzed by agarose gel electrophoresis, and the DNA concentration was determined by Qubit DNA HS assay kit.4C-seq was performed as described previouslyThree micrograms of the DNA product was further digested with 3U of specific second cutter . Restriction enzyme was heat-inactivated by incubating the chromatin at 65 °C for 20 min (MluCI was heat-inactivated in the presence of 1.17% SDS). The double-digested DNA was analyzed by agarose gel electrophoresis prior to phenol:chloroform:isoamyl alcohol (25:24:1) extraction and ethanol precipitation in the presence of glycogen. One microgram of purified DNA was ligated with HC DNA ligase  at 20 U/µg DNA in 1X ligation buffer (Invitrogen). For NlaIII or MulCI digested samples, the ligation was performed at 16 °C overnight. For MseI digested samples, the ligation was performed at 4 °C for 24 h. The ligated DNA was recovered by phenol:chloroform:isoamyl alcohol (25:24:1) extraction and ethanol precipitation. 800 ng DNA from each of the biological triplicates were pooled to prepare 4C library . The library for each viewpoint was constructed by PCR with 0.05 µM 4C adapter primers (see primers section) and 2 ng/µl DNA templates using KAPA HiFi HotStart ReadyMix (KK2602). The libraries were subjected to size selection (250–500 bp) on a 4–20% TBE PAGE gel (Thermo Scientific). The recovered libraries were sequenced on the Illumina Nextseq platform using Nextseq 500 mid-output kit with 2 × 150 paired-end.38 and were mapped by bowtie239 against human reference genome GRCh38. The PCR duplicates were removed by SAMtools rmdup40. The mapping results were converted into signals by BEDTools41 in bedGraph format. Then they were converted to bigWig format by bedGraphToBigWig43 for visualization on UCSC genome browser44. Peak calling was performed by MACS245.For ChIP-seq analysis, reads were trimmed of adapter sequences by TrimGalore46 against human reference genome GRCh38 with GENCODE transcriptome annotation (v26)47. RNA-seq reads were trimmed of adapter sequences by TrimGalore38 and then mapped by STAR to the human reference genome GRCh38 with reference gene annotation GENCODE 2647. PCR duplicates were removed in the paired-end alignments by SAMtools rmdup. Alignments with mapping quality <20 were removed. Gene expression levels in FPKM were determined by cuffdiff48. RNA-seq read counts are generated by featureCounts49.RNA-seq reads were mapped by STAR38 and were mapped respectively by STAR46 against the human reference genome GRCh38 with GENCODE47 transcriptome annotation (v26). The resulting alignments were separated into two parts. (1) The exonic part consisted of alignments belonging to GENCODE annotated transcripts. (2) The non-exonic part consisted of the other alignments. Based on the number of reads that can be mapped into the transcriptome, the larger data set was shrunk down to fit the size of the smaller one. Then the normalized read count of each genomic position was generated for the EZH2 RIP data set and IgG control data set, respectively. The read coverage of each position was defined as the average of normalized read counts within ±150 bp region. Based on the comparison of the read coverage between the EZH2 RIP and the IgG control, sites with ≥2-fold enrichment and Poisson distribution p-value ≤10−5 were defined as peaks. Each peak was extended to surrounding areas until the fold enrichment dropped below 2. Peaks that can be found in both replicates within 1000 bp range were merged and considered reproducible peaks. Sequences covered by reproducible peaks were extracted for further motif discovery analysis. The upstream and downstream sequences of reproducible peaks were also extracted and served as the background sequences in the motif finding analysis. Motif discovery is performed with the MEME suite using differential enrichment mode50.For RIP-seq, reads from the RIP data set and its control data set were trimmed of adapter sequences by TrimGalore51 with -ax splice -uf -k14. The bam files were converted to bed12 by bamToBed -bed 12 and were converted to bigBed file by bedToBigBed -type = bed12. Only uniquely mapped reads were retained. For cDNA sequencing, the reads were mapped to hg38 by LAST with lastal -d90 -m50 -D10 and last-split -d253. The maf files were converted to psl file with maf-convert -j1e6 psl. The psl files were subsequently converted to bam file with psl2sam.pl and cigar_tweaker scripts within the SAMtools package40. For visualization, the read alignments were converted into bedGraph by BEDTools42, and finally, into bigWig format by bedGraphToBigWig43.For Nanopore sequencing, raw read collected from MinKNOW were converted to FASTQ file by Albacore 2.3.3 with the following arguments: -k SQK-RNA001 -f FLO -MIN106 -o fastq for direct RNA sequencing; or the following arguments: Albacore 2.3.1 with - -flowcell FLO-MIN106 - -kit SQK-PCS108 - -output_format fastq for cDNA sequencing. For direct RNA sequencing, the reads were mapped to hg38 by Minimap254. Briefly, for each replicate, the raw reads were aligned to the masked version of the reference human genome  downloaded from the R Bioconductor repository (BSgenome.Hsapiens.UCSC.hg19.masked). The chromosome 11 was selected as the viewpoints, and DpnII was used as the restriction enzyme to digest the genome. A non-overlapping fragment was selected to identify the interaction regions. BAM files were converted to read coverage by bedtools genomecov. The read coverage was normalized according to the sequencing depth. The read coverage difference of the treatment and the control was calculated based on the normalized read coverage. The resulting bedGraph files were converted to bigWig format by bedGraphToBigWig. The genome version of bigWig files was then converted to hg38 by CrossMap55. The profile of read coverage difference on genomic intervals was plotted by deepTools56.For 4C-seq analysis, the long-range genomic interaction regions generated by the 4C-Seq experiment were analyzed by the R package r3CSeq v1.30.0R was used for statistical analysis. Details about statistical methods and sample numbers used in each experiment were elaborated accordingly within each figure legends.Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting Summary


Here, we present the synthesis of this nucleosideand its phosphoramidite, allowing first time access to c1G-modified RNA. Thermodynamic analyses revealed the base pairingparameters for c1G-modified RNA. Furthermore, by NMR spectroscopy,a c1G-triggered switch of Watson-Crick into Hoogsteen pairingin HIV-2 TAR RNA was identified. Additionally, using X-ray structureanalysis, a guanine–phosphate backbone interaction affectingRNA fold stability was characterized, and finally, the critical impactof an active-site guanine in twister ribozyme on the phosphodiestercleavage was revealed. Taken together, our study lays the syntheticbasis for c1G-modified RNA and demonstrates the power ofthe completed deazanucleoside toolbox for RNA atomic mutagenesis neededto achieve in-depth understanding of RNA recognition and catalysis.Atomic mutagenesisis the key to advance our understanding of RNArecognition and RNA catalysis. To this end, deazanucleosides are utilizedto evaluate the participation of specific atoms in these processes.One of the remaining challenges is access to RNA-containing 1-deazaguanosine(c Furthermore, we describe the impactof c1G on the thermodynamic stability of RNA double helices.Moreover, we found evidence for Hoogsteen base pair formation of c1G with protonated cytosine in HIV-2 TAR RNA by nuclear magneticresonance (NMR) spectroscopy. The study is complemented by the crystalstructure of a c1G-containing RNA hairpin to shed lighton a specific guanine N1–phosphate backbone interaction observedin the wild-type RNA, and finally, we evaluate the crucial role ofthe guanosine N1 atom in catalysis of phosphodiester cleavage by thetwister ribozyme.Thus far, diverse deazanucleosides have beenutilized for RNA atomicmutagenesis experiments; these are 3-deazacytidine (c27 DNA containing 1-deaza-2′-deoxyguanosine(c1dG) is unstable toward acids, and this feature has beenutilized to generate abasic sites.28 Accessto the naked ribonucleoside 1-deazaguanosine was first reported inthe nineteen eighties,30 employing rather harsh nucleosidationreactions involving mercury cyanide and based on O6-benzylated 1-deazaguanine, which itself requires laboriousmultistep synthesis.31 Later, access to1-deazaguanosine was demonstrated via 5-amino-1-β-d-ribofuranosylimidazole-4-carboxamide (AICA-riboside) as the keyintermediate.32 We, however, decided toput efforts into a direct, more efficient, and unprecedented routefrom readily available starting materials.To date,synthetic routes to 1-deazaguanine nucleoside buildingblocks for oligonucleotide synthesis have been described for DNA only.1G nucleoside 6 and the phosphoramidite precursor 5 acetamide (BSA) and trimethylsilyltrifluoromethanesulfonate (TMSOTf) provided nucleoside 2 in good yields. The exchange of acetyl to tert-butyldimethylsilyl(TBS) protection of the ribose hydroxyl groups (compound 3) was required to enable efficient copper-catalyzed coupling of thearyl iodide with benzyl alcohol to furnish nucleoside 4, inspired by the work of the Buchwald laboratory.33 Then, site-specific nitration of the O6-benzyloxy-1-deazapurine moiety was accomplished usingtetrabutylammonium nitrate (TBAN) and trifluoroacetic anhydride (TFAA)to obtain nucleoside 5, in analogy to work by Koomen.36 Finally, selective reduction of the nitro group was conducted byHSiCl3 referring to Benaglia and coworkers,37 followed by the cleavage of the benzyl groupby hydrogenation, and cleavage of the silyl ethers to give 1-deazaguanosine 6 in 22% overall yield in six steps with five chromatographicpurifications; in total, 0.4 g of 6 was obtained in thecourse of this study.For ccursor 5 1, we sta1G building block 12 from precursor 5 with the reduction of the nitro groupusing HSiCl3, followed by the cleavage of the benzyl groupvia hydrogenation providing nucleoside 7 ethyl(NPE) moiety applying Mitsunobu reaction conditions, followed by protectionof the exocyclic NH2 group using trifluoroacetic anhydride(TFAA), resulting in derivative 8. By the cleavage ofthe silyl ethers with tetrabutylammonium fluoride (TBAF), triol 9 was quantitatively obtained. Next, the 5′ and 3′OH groups were simultaneously protected using di-tert-butylsilyl bis(trifluoromethanesulfonate) (tBu2Si(OTf)2),39 followed by silylationof the 2′-OH group with tert-butyldimethylsilylchloride (TBS-Cl) and subsequent removal of the 5′-O and 3′-O protection clamp witha solution of HF in pyridine to give compound 10. Thefunctionalization of the 5′-OH group with 4,4′-dimethoxytritylchloride was conducted under standard conditions and yielded compound 11. Finally, the phosphoramidite 12 was generatedby treatment with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite(CEP(NiPr2)2) in the presenceof 5-benzylthio-1H-tetrazol (BTT). Starting fromprecursor 5, the target compound 12 wassynthesized in six steps, with six chromatographic purifications andan overall yield of 33%; in total, 1.1 g of 12 was obtainedin the course of this study.We startedthe synthesis toward ceoside 7 2. Then, 1G modifications wasperformed using the new building block 12 together with2′-O-TBS protected A, C, G U phosphoramidites,or alternatively, with 2′-O-[(triisopropylsilyl)oxy]methylprotected (TOM) amidites.41 The novel buildingblocks were coupled with yields higher than 98% according to the tritylassay. The cleavage of the oligonucleotides from the solid supportand deprotection were conducted using methylamine/ammonia in water(AMA), followed by treatment with tetra-n-butylammoniumfluoride (TBAF) in tetrahydrofuran. Salts were removed by size-exclusionchromatography, and RNAs were purified by anion-exchange chromatographyunder denaturating conditions . The molecular weights of the purifiedRNAs were confirmed by liquid chromatography (LC) electrospray-ionization(ESI) mass spectrometry (MS). The sequences of c1G containingRNAs synthesized in the course of this study are listed in Supporting Table S1. Notably, HPLC analysis of the crudedeprotected c1G containing RNAs displayed a second productthat was migrating slower, in particular, when TOM amidites were used.Isolation of this product and mass spectrometric analysis using ahigh-resolution Fourier-transform ion cyclotron resonance (FT ICR)spectrometer suggested RNA dimers that were cross-linked between twoc1G nucleosides by a CH2 bridge, most likelybetween their exocyclic amino groups . Such a linkage most likely formsduring deprotection of the TOM group where formaldehyde emerges asa byproduct.The solid-phasesynthesis of RNA with site-specific c1G, tautomeric formsand distinct rotamers have to be considered. An earlier study reportedthe energy differences of 9-methyl-1-deazaguanine tautomers and rotamersestimated by ab initio calculations.27 Itwas found that the c1G tautomer/syn-rotamerthat we show in anti-rotamerwith 6-OH providing H-donor properties at the Watson Crick face, being4.7 kcal/mol less stable. Importantly, the N3–H pyridone tautomeris 20.4 kcal/mol less stable.27Inprinciple, for the nucleobase of c1G is expected toimpair Watson–Crick pairingbecause the central N1–H of G is replaced by C–H, therebydepriving the capability for the formation of strong hydrogen bonds(1G modification in the center. The second motif (Type II) isa hairpin with a GCAA loop (extrastable GNRA) and c1G residingin the center of its short stem. The third RNA motif (Type III andIII′) consists of palindromic RNA of eight base pairs and equivalentpurine–pyrimidine stacking patterns with the c1Gseither directly stacked toward each other, or separated by two standardbase pairs. The type III/III′ design is very sensitive forthe impact arising from a modification on base pairing. With onlytwo or three regular Watson–Crick base pairs next to the modification,the nucleation of such duplexes can become significantly hindered.43 Thus, these RNA palindromes are anticipated to significantly respondto the c1G modification reflected in changes of the thermodynamicpairing parameters .cen bonds2A. The dFigures S2 to S11).45 The native type I RNA I melts at 66.7 °C (Ia) destabilizes the duplex by 15.8 °C. Destabilization is evenmore pronounced if U, G, and A are the mismatch partner . Forthe hairpin RNA (Type II), c1G opposite of C (IIa) also decreases the melting temperature compared to the native hairpin II (by −27.9 °C)  2D. We nond 18 °C2C. This 1G on base pairing,we investigated the short palindromic RNAs that are particularly sensitiveto double helix nucleation as mentioned above.43 Indeed, for c1G, the destabilization in both palindromicRNAs was large, reflected in −35.9/–36.5 °C reduced Tm values (IIIa and III′a), accounting for −17.9/–18.3 °C destabilizationper single modification which is higher compared to the destabilizationthat we observed for a single c1G–C base pair inthe bimolecular 9 bp duplex Ia with four regular –and hence nucleation-supportive – Watson–Crick basepairs at both 5′ and 3′ directions to the modificationsite.To furtherelucidate the impact of csynG•A Hoogsteen base pair of a GNRA loop in hairpin IIb was tolerated with significantly less decrease in stability was obtained, attributed to theprotonation of N7 . The second value of 9.10 ± 0.10 (pKa 2) was attributed to the deprotonation of the 6-OH group.The pKa values are thus comparable tothe ones of guanine, which range from 9.2 to 9.6  and 3.2 to 3.3 , respectively.46To understandbase-pairing and catalytic properties of nucleobases in functionalRNA, knowledge about their acid–base properties is crucial.1Gin RNA, we aimed at a high-resolution X-ray crystallographic analysis.We utilized the 27 nt fragment of the E. coli 23S rRNA sarcin/ricin loop (SRL), which is a frequently appliedcrystallization scaffold  (Supplementary Table S2). X-ray structure determination demonstratedthat the c1G nucleobase is well defined in the electrondensity maps for the c1G-modified RNA (1G-modifiedRNA structure superimposed with the unmodified RNA displayed a root-mean-squaredeviation (rmsd) of 0.09 Å (within the errors on coordinatesof 0.09 Å). Direct comparison of the base triples U2656-A2665-G2655.A comparison of the melting profiles of wild-type and c1G does notsignificantly affect an RNA fold as long as itis not replacing G in a Watson–Crick base pair. The weakening(or loss) of an H-bond to the phosphate backbone seems better toleratedand allowed crystallization and structure solution.Taken together,our crystallization experiments imply that c1G26), the formationof a HG base pair with C39 occurs  is sufficientto switch the pairing mode -C39H+ forms in acomparable manner. Characteristically, we observed a downfield shiftedimino proton at ∼15 ppm  and downfield shifted amino protons  base pairs are energetically disfavored relativeto Watson–Crick (WC) pairs. With 1-deazaguanosine in our hands,however, we were wondering if stable HG pairing might become favorable.We thereby focused on the human immunodeficiency virus type 2 (HIV-2)transactivation response element (TAR) RNA, where a G26-C39 WC bpis adjacent to a dinucleotide bulge 5A. It wa9 occurs 5B, C.49 ing mode 5B, C. In ∼15 ppm S14 and d ∼15 ppm S14 and d50 In particular, atomicmutagenesis experiments led to an in-depth understanding of generalacid–base catalysis of small nucleolytic ribozymes that cleavetheir phosphodiester backbone, revealing the functionally crucialimino groups of purines and pyrimidines in the active site. For instance,this concerns the twister ribozyme51 whereproton transfer from the (protonated) N3 of a conserved adenine (A6)at the cleavage site to the 5′-O leaving groupsignificantly contributes to reaction catalysis; the replacement ofthis adenine by c3A or c1c3A renderedthe twister ribozyme inactive.52 Another example isa phosphodiester cleavage by the pistol ribozyme.53 Replacements of active site purines by c3A,c1A, and c7G revealed the key residue –a highly conserved guanine (G33) – that serves as inner spherecoordination site for a hydrated Mg2+ ion, thereby likelyproviding a 1st shell water molecule as general acid for protonationof the 5′-O leaving group in the course ofthe reaction.17Deazanucleobase-modified RNAs are frequently applied in atomicmutagenesis studies of ribozymes to shed light on the mechanism ofthe chemical reactions they catalyze.1G-modified RNA now allows evaluation of active site guanines thatare suspected to be involved in reaction catalysis via their Watson–Crickface. To exemplify this, we picked the three-way junctional twisterribozyme,53 for which several structuresof precatalytic states were solved by X-ray crystallography58 and structural dynamics elucidated by smFRET imaging.59 Bases U5 and A6 at the cleavage site are splayed apart, with a guanine(G48 for PDB ID 4RGE54 and 5DUN15) in a hydrogen bond distance (2.6 Å) betweenthe N1 atom and the nonbridging pro-R oxygen of thescissile phosphate .With c to refs , 62). In1G48-modifiedtwister ribozyme. We further note that the 6-OH group of c1G (possessing a comparable pKa to N1–Hof G) is dislocated in comparison with N1 (in G) and therefore isnot likely to be able to efficiently take over its role. However,the 2-NH2 group of guanine is present also in c1G, and therefore, this NH2 group can contribute to stabilizationof the phosphorane transition state (together with a remaining weakstabilization originating from a C1–H interaction with phosphorane).The remaining cleavage activity indicatesthat the other catalyticdeterminants 62)) are 1G, the correspondingphosphoramidite, and c1G modified RNA. The synthetic foundationenabled comprehensive analysis of the biophysical properties of suchmodified RNA, and furthermore, enabled c1G atomic mutagenesisin functional RNA assays. This led to evidence for c1Gswitching the mode of base pairing from Watson–Crick to Hoogsteen.Moreover, the approach allows direct evaluation of ribozyme mechanisticproposals that claim a catalytic role for guanosines via their N1position, the central H-donor of their Watson–Crick face. Beyondtwister, such guanosines are found in many ribozymes including twistersister, pistol, hatchet, and the most recently discovered RNA methyltransferaseribozymes.63 Functional atomic mutagenesis relying on c1G RNA modificationswill contribute to achieve an in-depth understanding of RNA catalysisof ribozymes that exhibit a much broader reactivity scope than previouslyanticipated.Our study introduces robust syntheses of c


Our findings highlighted the essential role of ac4C in EV71 infection and provided insights into potential antiviral treatments.Chemical modifications are important for RNA function and metabolism. N4-acetylcytidine (ac4C) is critical for the translation and stability of mRNA. Although ac4C is found in RNA viruses, the detailed mechanisms through which ac4C affects viral replication are unclear. Here, we reported that the 5′ untranslated region of the enterovirus 71 (EV71) genome was ac4C modified by the host acetyltransferase NAT10. Inhibition of NAT10 and mutation of the ac4C sites within the internal ribosomal entry site (IRES) suppressed EV71 replication. ac4C enhanced viral RNA translation via selective recruitment of PCBP2 to the IRES and boosted RNA stability. Additionally, ac4C increased the binding of RNA-dependent RNA polymerase (3D) to viral RNA. Notably, ac4C-deficient mutant EV71 showed reduced pathogenicity  Chemical modifications are critical for RNA metabolism and function, including RNA splicing , localizDiscovered in the 1960s , ac4C isUsing mass spectrometry or RIP-seq with anti-ac4C antibodies, the ac4C modification has been detected in several RNA viruses, including Zika virus, Dengue virus, hepatitis C virus, poliovirus, human immunodeficiency virus type 1 (HIV-1) , and infEnterovirus genus within the Picornaviridae family  at the necessary concentration was initiated 24 h prior to EV71 infection. Control cells were treated with the same volume of DMSO.Vero , RD , and HEK293T  cells were maintained in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (Gibco). All cells were cultured with 5% CO50) assays with the Reed–Muench formula (EV71 (strain XF) was obtained from the Microorganisms & Viruses Culture Collection Center, Wuhan Institute of Virology (WIV), Chinese Academy of Sciences (CAS) and amplified in Vero cells. EV71 wild-type (WT) and ac4C mutants were rescued from infectious DNA constructs in our laboratory. All viruses were titrated using 50% tissue culture infectious dose  was constructed by inserting the coding sequence (CDS) of NAT10 from ac4C mutation EV71 infectious DNA constructs were generated by mutating nt 331 (pMut-331), 350 (pMut-350), both (pMut-331–350) or 383 from C to T, with the base pairing restored by G-A mutation at position 356, 343 or 390 Figure  in the iEV71 5′ UTR-enhanced green fluorescent protein (eGFP) reporter plasmids were generated by inserting the EV71 WT or ac4C mutant 5′ UTR between the cytomegalovirus (CMV) promoter and eGFP in pEGFP-N1  on EV71 WT  based on the AB Sciex QTRAP 6500 LC–MS/MS platform.UPLC-MS/MS was performed by Wuhan Metware Biotechnology Co., Ltd. Briefly, EV71 supernatants were concentrated by ultracentrifugation in a SW28Ti rotor (Beckman Coulter) at 26 000 rpm for 2 h at 4°C. EV71 RNA was extracted using an RNeasy mini kit  and digested into nucleosides using S1 nuclease , alkaline phosphatase (Takara), and phosphodiesterase I . The nucleosides were extracted with chloroform and analyzed using a UPLC-ESI-MS/MS system . RNA modifications were detected using MetWare (in vitro transcribed from a HindIII (ThermoFisher)-linearized infectious DNA construct  using standard protocols. Signals were detected using a ChemiDoc MP imaging system .Anti-ac4C dot blot assays were performed as previously described . Briefly−△△Cq method with Bio-Rad CFX Manager 3.0. The primers used in qRT-PCR were as follows: EV71 VP1 , EV71 5′ UTR , NAT10 , glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for humans and monkeys , and GAPDH for mice . The EV71 genome was detected using the EV71 VP1 primer, and the WT- or Mut-VP4/2-Flag RNA in the in vitro translation system was detected using the EV71 5′ UTR primer.RNA was extracted from cells using TRIzol reagent (Invitrogen), and qRT-PCR was performed using a HiScript II One Step qRT-PCR SYBR Green Kit (Vazyme) and Hard-Shell PCR Plates  on a CFX Connect Real-Time system (Bio-Rad Laboratories). The thermocycling parameters were as follows: 55°C for 5 min, 95°C for 30 s, and 40 cycles of 95°C for 10 s and 60°C for 45 s. For detection of EV71 positive- or negative-strand RNA, reverse transcription was first performed using HiScript 1st Strand cDNA Synthesis Kit (Vazyme) with the EV71 VP1 forward qPCR primer (for negative-strand RNA detection) or reverse primer (for positive-strand RNA detection) and qRT-PCR was then carried out using Hieff qPCR SYBR Green Master Mix (Yeasen). The thermocycling parameters were as followed: 95°C for 5 min, followed by 40 cycles of 95°C for 10 s, 55°C for 20 s, and 72°C for 20 s. Gene expression was quantified using the 2in vitro-transcribed EV71 RNA was incubated with anti-ac4C antibodies (Abcam) or IgG antibodies  in 500 μl IP buffer  for 4 h at 4°C. The mixtures were incubated with 30 μl anti-rabbit antibodies conjugated with magnetic beads  for 2 h at 4°C and washed six times with 1 ml IP buffer. RNA was extracted using TRIzol reagent and quantified by qRT-PCR.RNA was extracted from EV71-infected Vero cells and concentrated viral supernatants using TRIzol reagent (Invitrogen) or transcribed by T7 polymerase. For acRIP, 300 μg total RNA, 10 μg supernatant RNA, or 10 μg acRIP-Seq was performed by Cloudseq Biotech Inc.  according to a previously published protocol . BrieflyCells infected with EV71 or transfected with plasmids were harvested and lysed at the indicated times. The cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Protein detection was performed with anti-NAT10 antibodies , anti-GAPDH antibodies , anti-VP1 antibodies , anti-GFNAT10 , green monkey NAT10 , and green monkey monkey poly(C) binding protein 2 . The shRNAs were cloned into pLKO.1-TRC (Addgene) using EcoRI (ThermoFisher) and AgeI (ThermoFisher) and packaged into lentiviruses using psPAX2 and pMD2.G. Stable knockdown cell lines were screened by puromycin after lentiviral infection .The shRNAs specific to each gene were as follows: human RIP-qRT-PCR was performed as described previously . BrieflyImmunofluorescence assays were performed as described previously . Briefly2, 1% Triton X-100, 2 mM DTT, 100 μg/ml cycloheximide, 100 U/ml RNase inhibitor, and 1 μg/ml proteinase inhibitor). The lysates were sheared using a 26-gauge needle and clarified by centrifugation at 2000 × g for 10 min at 4°C. One-tenth of the supernatant was used as the input sample. The remaining supernatant was loaded onto a 10–45% sucrose gradient and centrifuged at 30 000 × g for 3 h at 4°C. RNA was extracted from the input or ribosomal pellet using TRIzol and quantified by qRT-PCR.Ribosome-loaded EV71 RNA was quantified as previously described . Briefly2), followed by three washes with binding buffer. Subsequently, GST-PCBP2-beads and 0.05 nmol EV71 genome were mixed in binding buffer and incubated at 37°C for 5 min, followed by six washes with binding buffer. The RNAs were then extracted with TRIzol reagent and quantified using qRT-PCR.The EV71 genome was transcribed using a T7 Kit (Ambion) with CTP or ac4CTP added or extracted from the supernatant of EV71 WT- or Mut-331–350-infected Vero cells. EV71 RNA levels were quantified using qRT-PCR, and 5 μg GST-PCBP2 (Proteintech) was mixed with Glutathione High Capacity Magnetic Agarose Beads (Millipore) for 1 h at 4°C in binding buffer -linearized EV71 cDNA plasmids or from gel-purified PCR product of full-length GAPDH mRNA sequence with CTP (ThermoFisher) or ac4CTP (MedChemExpress) as substrates using a MEGAscript T7 Kit (Ambion). The in vitro translation assay was performed using a Retic Lysate IVT Kit (ThermoFisher) according to the manufacturer's instructions. The translation reaction was carried out for 1.5 h, and western blot was performed.The RNA templates were 2) in a total volume of 100 μl, denatured at 75°C for 10 min, slowly annealed, and then cooled to room temperature. The annealed solution and His-3D  and heated at 37°C for 1.5 h. The mixture was then incubated with anti-His tag antibodies (Proteintech) overnight at 4°C, then incubated with protein-G agarose beads for 1 h and washed six times with binding buffer. RNA was extracted with TRIzol and quantified using qRT-PCR.The EV71 genome was transcribed using a T7 Kit (Ambion) with CTP or ac4CTP added or extracted from the supernatant of EV71 WT-infected Vero cells. EV71 RNA levels were quantified using qRT-PCR. RNA primers (0.5 nmol) and the EV71 genome (0.05 nmol) were mixed in RNA annealing buffer . Mouse morbidity was monitored by daily weighing. For histology, immunohistochemistry (IHC), and viral RNA quantification, mice were sacrificed at 2 or 3 days postinfection. All tested organs, including the heart, liver, spleen, lungs, kidneys, brain, intestines and thigh muscles, were harvested.AG6 mice were inoculated via the intraperitoneal (i.p.) route  overnight at 4°C. After three washes with PBS, the sections were incubated with secondary antibodies (labeled with horseradish peroxidase) at room temperature for 50 min, and diaminobenzidine chromogenic agent was used for IHC according to standard protocols. Samples were counterstained with hematoxylin stain solution for approximately 3 min. The images were collected using Pannoramic DESK, P-MIDI and P250 (3D HISTECH).P values of 0.05 or less were considered significant.All data were analyzed using GraphPad Prism software (version 8). The details of the statistical tests used in this study are specified within the corresponding figure legends. Data are presented as the means ± standard error of the means (SEMs) or means ± standard deviations (SDs). Results with in vitro-transcribed EV71 genomic RNAs, as evidenced by dot blot with an anti-ac4C antibody  within the IRES, which is crucial for viral protein synthesis ; therefoA Figure , which lA Figure , was notA Figure . These rin vitro transcription with CTP or ac4CTP  protein, but is able to initiate on RNA primer-template pairs using purified 3D in vitro . To expls Figure . 3D can s Figure . Interess Figure  and H ans Figure  and I sh1 Figure , which w1 Figure . These rMethylation modification of IAV has been shown to be linked with disease . Therefoin vivo.Methylation modifications, such as m6A, m5C, and Nm, have been extensively studied in retroviruses, simian virus 40, Kaposi's sarcoma-associated herpesvirus, influenza virus, flavivirus, EV71, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,40,56–66The distribution of ac4C in EV71 was different from that in mRNA  or in HIEV71 acetylation was catalyzed by host NAT10, which in turn promoted viral infection. NAT10-mediated acetylation of tRNA requires the help of the adaptor protein THUMPD1, which harbors an RNA binding motif ,23. WhetKnockdown or inhibition of NAT10 and mutation of the ac4C site resulted in a moderate decrease in the titer of the progeny virus, suggesting that ac4C promoted virus replication. ac4C modification is one of the key factors to regulate virus replication. In the absence of ac4C, other compensatory pathways may also be activated to facilitate virus replication.ac4C in mRNA promotes translation and stability , whereasin vivo and in vitro. When ac4C was paired with the last base at the 3′ end of the 331-primer in vitro, the affinity of 3D to viral RNA was obviously enhanced. Notably, ac4C promoted viral translation when 3D was mutated by frameshift  used in this work has been deposited with National Center for Biotechnology Information under accession number JQ804832.gkac675_Supplemental_FileClick here for additional data file.


Ubiquitin-fold modifier 1 (Ufm1) is a ubiquitin-like protein that can also be conjugated to protein substrates and subsequently alter their fates. Both UFMylation and de-UFMylation are mediated by Ufm1-specific proteases (UFSPs). In humans, it is widely believed that UFSP2 is the only active Ufm1 protease involved in Ufm1 maturation and de-UFMylation, whereas UFSP1 is thought to be inactive. Here, Liang et al. provide strong evidence showing that human UFSP1 is also an active Ufm1 protease. These results solve an age-old mystery in the human Ufm1 conjugation system and could have a greater impact not only on Ufm1 biology but also on the translation of genes employing nontraditional start codons.The  Ubiquitin-fold modifier 1 (Ufm1) is a ubiquitin family protein that can be conjugated to its protein substrates in a process termed ‘UFMylation’ via a classic E1-E2-E3 enzymatic cascade ( cascade . Like ma cascade .Ufm1-specific proteases or UFSPs (Both Ufm1 maturation and de-UFMylation are mediated by or UFSPs . UFSPs aor UFSPs ; howeveret al. (A recent discovery by Liang et al.  showed smyc uses both near-cognate CUG and cognate AUG as translational initiation codons to synthesize c-myc1 and c-myc2 oncoproteins, respectively (et al. selected four candidate CUG codons based on predicted molecular weights of polypeptides and their abilities to synthesize active UFSP1 enzymes. Using serial mutagenesis-based approaches, the authors found that 217CUG is the only potential start codon. To provide even more evidence, they turned to mass spectrometry. The authors engineered a FLAG-tag at the carboxyl terminus of UFSP1 using a CRISPR/Cas9-based knock-in method and then purified the UFSP1-FLAG fusion proteins from engineered cells. Six peptides covering more than 90% of the predicted N-terminal peptide sequences from 217CUG to the canonical 445AUG codons were identified using this method. In addition, using in vitro assays with purified recombinant proteins, the authors further consolidated UFSP1 as a protease in the maturation of Ufm1 precursor and de-UFMylation. Even more surprisingly, they provided some evidence suggesting UFSP1 as an even stronger enzyme for both Ufm1 maturation and de-UFMylation of Ufm1-conjugated ASC1 compared to UFSP2.Besides the cognate AUG start codon, eukaryotic cells also employ other near-cognate start codons to initiate translation , 8. For ectively . In factCUG is the most common near-cognate start codon employed in eukaryotes, but the mechanism behind its use is still unclear. The authors of this study discovered that the 5′-UTR sequence is essential for UFSP1 expression in humans; in particular, they identified an E-box motif, CAGCTG, which plays an important role in human UFSP1 expression. Furthermore, they confirmed the critical role of the eukaryotic translation initiation factor eIF2A in translation of UFSP1 from the CUG codon using both chemical and RNAi approaches.et al. (in vitro assays, although it appears that UFSP2 is better at de-UFMylation in cells. The question is, does the recombinant UFSP2 protein lose some activity during the purification process? Would the HA-tag sequence, if fused at the carboxyl terminus of UFSP2, lead to stronger cellular activity when expressed in cells, since the localization of UFSP2 to the endoplasmic reticulum membrane is potentially critical for its maximal activity? Furthermore, the authors only used ASC1 as a substrate in their assay, but these two proteases might behave differently toward other Ufm1 substrates. It is worth mentioning that a similar study by Millrine et al. (Ultimately, Liang et al.  providede et al.  answeredThe authors declare that they have no conflicts of interest with the contents of this article.