The recognition properties towards three xanthine derivatives were then explored through thermophoresis. Modifying the aptamer at position G11 led to binding inhibition. Modification at G25, however, changed the selectivity towards theobromine (Kd≈160 μm), with a poor affinity for theophylline (Kd>1.5 mm) being observed. Overall, 8‐oxoG can have an impact on the structures of aptamers in a position‐dependent manner, leading to altered target selectivity.Aptamers are attractive constructs due to their high affinity/selectivity towards a target. Here 7,8‐dihydro‐8‐oxoguanosine (8‐oxoG) has been used, due in part to its unique H‐bonding capabilities (Watson–Crick or Hoogsteen), to expand the “RNA alphabet”. Its impact on the theophylline RNA aptamer was explored by modifying its binding pocket at positions G11, G25, or G26. Structural probing, with RNases A and T Oxidation of RNA in aptamers—8‐oxoG: Incorporation of the 8‐oxoG unit within the aptamer for theophylline altered its selectivity and affinity. Modification at position G25 changed the selectivity in favor of theobromine, whereas the G11‐modified aptamer experienced drastic structural changes that resulted in inhibited recognition of three xanthine derivatives. We are interested in probing the H‐bonding capabilities of modified nucleobases to control the function of aptamers and to diversify the “toolkit” to generate constructs with distinct selectivity and, ideally, increased affinity.An aptamer is the smallest fragment of a biopolymer that is able to recognize a particular target with high affinity and specificity, often with a sub‐micromolar/sub‐nanomolar dissociation constant.anti→syn conformational change around the glycosidic bond, and 2) the distinct H‐bonding patterns arising from each of these isomers  or pH (5–8) and is due to steric hindrance between the C5′‐position and the atom at the C8‐position.We opted to explore a modification that has been widely studied in other contexts: 7,8‐dihydro‐8‐oxoguanosine (8‐oxoG). Of the many oxidative lesions that have been characterized in DNA or RNA, purine‐derived modifications are expected to be the most abundant [redox potential trend=G<A<C<U].To probe for the impact that 8‐oxoG has on the structure and function of RNA, the aptamer for theophylline was used as model. This construct was originally selected with the development of SELEX over two decades ago.The construct is an RNA strand, 33 nt in length, with high selectivity for theophyllineIn brief, three levels of scaffolding provide support for the aptamer's binding pocket Scheme  B, which1), 33 nucleotides in length, was modified with 8‐oxoG at positions G25, G26, and G11, chosen on the basis of their roles in target recognition within the binding pocket, to yield RNA strands 2, 3, and 4 respectively , thus suggesting that these positions are in environments available for ribonuclease access and not restricted to H‐bonding interactions with other nucleobases. Cleavage at position C9 was unexpected; however, this marks the beginning of the internal loop (4×1), thus presumably making it more accessible. Interestingly, incorporation of 8‐oxoG at positions G25 or G26 gave rise to similar cleavage patterns, thus suggesting that modification at these sites does not affect the overall structure of the aptamer, or that these positions are cleaved because they are also exposed in another structural arrangement. The only observed difference was that cleavage around the C20–C22 region was increased slightly in the case of RNA 3 (G26‐modified), thus indicating that interactions amongst nucleobases in this section of the construct might be altered. The most striking difference was observed in the case of strand 4, modified at position G11, which displayed prominent cleavage at additional positions (C12 and C13), consistent with disruption of the stem in the upper hairpin and exposure of these nucleobases to the ribonuclease. Assignment of the bands was carried out by comparing the results against a hydrolysis ladder . The ladder works through cleavage of every nucleobase from the 3′‐end, thus allowing a band to appear at every position of the aptamer. A pattern in which doublet bands are observed in cases of shorter fragments is consistent with the formation of the 3′‐phosphate, along with the corresponding cyclic phosphate derivative that has been characterized/observed previously.To understand the impact of the modification on the structure, all strands were radiolabeled and treated with RNase A, which cleaves single‐stranded RNAs at both pyrimidine and 8‐oxoG sites into fragments containing 5′‐OH and 3′‐phosphate ends.4, which displayed cleavage at positions G14, G18, and G19. Consistent with the results obtained in the case of the RNase A cleavage, this observation points to disruption of the upper hairpin within the construct. Although the intensity of the cleavage bands was weak, the pattern was reproducible. Furthermore, in agreement with alteration at the binding site, cleavage at position G29 took place in the case of RNA strand 2, whereas position G25 (G26 was ruled out because of the reported inability of RNase T1 to cleave at 8‐oxoG sites) was accessible for ribonuclease activity in the case of modified aptamer 3.To complement these observations, experiments were also carried out with RNase T1, a ribonuclease that specifically cleaves at all G‐sites in a single‐stranded context. Interestingly, the only aptamer that showed some hypersensitivity to this ribonuclease was aptamer 1 matched relatively well with the known structure, with the only exception being the prediction of an A11:U24 base pair . Because 8‐oxoG is not available in this hybridization package, we reasoned that substituting the modification with uridine could provide a valid model, on the basis of the H‐bonding similarities between syn‐8‐oxoG and U. However, this approach predicted structural changes in the case of RNA strands 2/3 (modified at G25 and G26) that do not explain the observed RNase A cleavage patterns . The same analysis was carried out by substituting G11 with U, which resulted in disruption of the hairpin stem in this region but did not fully explain the RNase A cleavage pattern at positions C12 and C13. Overall, substitution of U in place of 8‐oxoG did not provide a good model in this context.In an attempt to interpret these changes and to explain the reactivity patterns, the UNAFold server was used to explore possible changes in structure .5 and 6, containing G or 8‐oxoG  and 8 (lacking a nucleotide) were also prepared, and displayed larger thermal destabilization in both cases. This result indicates that 8‐oxoG does interact with C at this position, albeit more weakly than in the case of the corresponding WC pair. The observed trends were in agreement with those predicted by use of UNAFOLD, albeit with values that were consistently lower than those observed experimentally  suggests that positioning 8‐oxoG in the aptamer at this position  between an aptamer and its cognate ligand in free solution and with minimal sample consumption.λabs=646 nm, λem=662 nm) at the 3′‐end were prepared via solid‐phase synthesis to yield RNA strands 9–12  to establish the selectivity and affinity of each construct towards the three xanthine derivatives. This technique was chosen because it allows for the determination of the dissociation constant  of (29±35) μm, a value consistent with the literature.1 to theophylline, to obtain a Kd value of 1.35 μm . Interestingly, modified aptamers 10 and 11 also displayed binding affinity towards theophylline, albeit with affinities approximately two orders of magnitude weaker  displayed a higher binding affinity towards theobromine, with Kd>160 μm , and with no binding of caffeine. This result highlights the ability of 8‐oxoG to have an impact on structure as well as on small‐molecule affinity and selectivity. Furthermore, consistent with large structural changes, aptamer 12 (modified at G11) did not display any binding affinity towards the three xanthine derivatives tested in this work. To provide better understanding of the impact of 8‐oxoG as a function of position, the thermal denaturation transitions were obtained  to show values for aptamers 9 and 10 that are equivalent, whereas aptamers 11 and 12 have increased thermal stability. As established previously, stabilization of structure can, in some cases, be directly related to decreased affinity in target binding.11 might be due to increased interactions between 8‐oxoG and other nucleobases, arising from extended H‐bonding networks formed from both Watson–Crick and Hoogsteen faces.Binding checks were performed in the presence of each target to validate interactions between the small molecule and the aptamer, or to rule out aptamers that do not display specific binding; these experiments were carried out by measuring the ON mobility either in the absence or in the presence of high concentrations of the small molecules. As depicted in Figure (Kd) of ±35 μm, a(Kd) of 9±35 μm, (Kd) of 9±35 μm, 4/12 (modified at position G11), the CD spectra showed that the extent of the duplex region is comparable to those of the other modified RNA strands, as well as that of the canonical aptamer .Attempts to record changes in structure by CD in the absence and in the presence of the xanthine derivatives did not show any appreciable differences . All aptamers displayed dichroic bands consistent with folding into A‐form duplexes. Although the structural probing experiments indicate that a different structure might be formed in the cases of RNAs 8 (8‐oxoG at position 25), which displayed Kd values in the micromolar range for theobromine and in the millimolar range for theophylline.The impact of 7,8‐dihydro‐8‐oxoguanosine (8‐oxoG) on RNA structure and function was explored by using the aptamer for theophylline as a model. The 8‐oxoG motif was independently incorporated onto ONs of RNA and its structural impact was assessed by electrophoretic analyses and circular dichroism, whereas its function was established by assessing the ability of the modified aptamers to bind three different xanthine derivatives: theophylline, theobromine, and caffeine. Thermophoretic analyses showed that the selectivity was altered in the case of modified aptamer 2/10 has features that suggest a structure similar to that of the canonical analogue 1/9, based on enzymatic degradation and Tm analyses, a change in the H‐bonding pattern of 8‐oxoG25 might induce a change in the binding pocket. It is plausible that the anti‐to‐syn flip for 8‐oxoG25 allows it to hydrogen bond with A10, in turn allowing the formation of WC base pair G26:C9, breaking interactions with C8, which ultimately disrupts the ceiling for the theophylline binding pocket . As mentioned above, A7 needs the ability to move out of the A‐form helix to create space for the small molecule, and this is unachievable if it is H‐bonding with 8‐oxoG26. This distinct H‐bond might also aid in the formation of a new ceiling for the small molecule, because G25 can now H‐bond with C8. The H‐bond between the 8‐oxoG and A7 might also disrupt the π‐stacking interactions of C21, A7, C22, and U6 by abolishing its ability to intercalate between C21 and C22; this could decrease the interaction between the upper and lower loop residues. Both RNAs 2/10 and 3/11 retain the ability to bind to theophylline, although weakly, because U24 is still conserved in the core and is potentially not affected by adverse H‐bonding or π‐stacking interactions, while still having an impact on the binding pocket.The results obtained with RNAs 4/12) would not greatly impact small‐molecule recognition. However, this RNA did not bind to any of the xanthine derivatives. This can be explained in terms of the large structural changes observed in the upper loop, evident from the distinct RNase A/T1 cleavage pattern and suggesting that 8‐oxoG11 disrupts the upper scaffolding of the aptamer overall. It is possible that 8‐oxoG11 could be involved in H‐bond interactions with A16 or A17, which would explain why both G18 and G19 are exposed to degradation. In addition, this could result in C21 and C22 being prevented from intercalating with A7 and U6, in turn affecting the role of U24 within the binding core and intercalation between G26 and A28, which would ultimately destabilize the S‐turn that is needed to maintain the integrity of the binding pocket. Attempts to explain the overall structure with the assistance of the UNAFold engine were carried out by substituting 8‐oxoG with U; however, the predicted structures, as well as other possibilities  the development of sequencing technologies that would enable a selection process that includes 8‐oxoG in RNA ,General: 8‐OxoG phosphoramidite was synthesized out according to a previous report.RNA synthesis: ONs were synthesized with a 394 ABI DNA/RNA synthesizer and use of CPG supports and 2′‐O‐TBDMS phosphoramidites (purchased from Glen Research). 5‐Ethylsulfanyl‐1H‐tetrazole (0.25 m) in acetonitrile was used as the coupling reagent, dichloroacetic acid in dichloromethane (3 %) was used for deblocking, a 2,6‐dimethylpyridine/acetic anhydride solution was used for capping, and an iodine/THF/pyridine solution was used in the oxidation step. Coupling times of 10 min were used. ONs were deacetylated/debenzoylated/deformylated and cleaved from the CPG support in the presence of 1:1 aq. methylamine (40 %) and aq. ammonia (40 %) with heating . A mixture of 1‐methylpyrrolidin‐2‐one/triethylamine/HF (3:2:1) was used for removal of the TBDMS groups , followed by purification by electrophoresis (20 % denaturing PAGE). C18‐Sep‐Pak cartridges were obtained from Waters and used to desalt the purified oligomers with NH4OAc (5 mm) as the elution buffer. ONs were dissolved in H2O and used as obtained for subsequent experiments. Unmodified ONs were purchased from IDT‐DNA or ChemGenes and, after quantification by UV/Vis, used without further purification. ONs containing a fluorescent Cy‐5 probe at the 3′‐end were synthesized on resin (purchased from Glen Research) containing this moiety and purified as described above.RNA characterization: MS (MALDI‐TOF) was used in the characterization of all modified ONs, with use of C18 Zip Tip pipette tips to desalt and spot each ON as follows: 1) wash tip with acetonitrile , 2) equilibrate tip with trifluoroacetic acid , 3) load tip with sample , 4) wash tip with TFA , 5) wash tip with water (10 μL×2), 6) elute sample into matrix , and 7) spot directly onto MALDI plate. All analyses were carried out with an ABI 4800 Plus MALDI‐TOF/TOF mass spectrometer in positive mode (see the Acknowledgements).UV/Vis spectroscopy: Concentrations of all ONs were determined by UV/Vis with a PerkinElmer λ‐650 UV/Vis spectrometer and quartz cuvettes (1 cm pathlength). General UV/Vis spectra were also taken with a 1 mm pathlength and 1 μL volumes (Thermo Scientific Nano Drop Nd‐1000 UV/Vis spectrometer). Origin 9.1 was used to plot and normalize spectra of monomers and ONs for comparison.TCircular dichroism (CD) spectroscopy and thermal denaturation transitions (m): CD spectra were recorded at various temperatures (PTC‐348W1 Peltier thermostat) with use of quartz cuvettes with a 1 cm pathlength. Spectra were averaged over three scans  and background‐corrected with the appropriate buffer or solvent. Solutions containing the RNA strands had the following compositions: RNA (1.5 μm), MgCl2 (5 mm), NaCl (10 mm), sodium phosphate . All solutions that had been prepared in order to record thermal denaturation transitions (Tm) were hybridized prior to the recording of spectra by heating to 90 °C followed by slow cooling to room temperature. Tm values were recorded at 270 nm with a ramp of 1 °C min−1 and step size of 0.2 with temperature ranges from 4 to 95 °C. A thin layer of mineral oil was added on top of each solution to keep concentrations constant at higher temperatures. Origin 9.1 was used to determine all Tm values and to plot CD spectra of RNAs with/without small molecules.ON labeling: T4 polynucleotide kinase (PNK) and γ‐32P‐ATP‐5′‐triphosphate were obtained from PerkinElmer. ONs were labeled by mixing PNK, PNK buffer, ATP, DNA, and water  according to the manufacturer's procedure followed by incubation at 37 °C for 45 min. Radiolabeled materials were passed through a G‐25 Sephadex column followed by purification by electrophoresis (20 % denaturing PAGE). The bands of interest (slowest) were extruded and eluted over a saline buffer solution (0.1 m NaCl) for 36 h at 37 °C. The remaining solution was filtered and concentrated to dryness under reduced pressure followed by precipitation over NaOAc and ethanol. Supernatant was removed and the remaining ON was concentrated under reduced pressure and dissolved in water. Activity was assessed with a Beckmann LS 6500 scintillation counter.Microscale thermophoresis (MST): This technique was used to establish binding affinities and potential for binding with the three xanthine derivatives. RNA strands modified with the Cy‐5 fluorophore at the 3′‐end were used.MST—small‐molecule binding studies: The small‐molecule binding studies were performed with 16 twofold dilutions of each small molecule as can be seen in Table Kd). The Monolith NT.115 MST system was used for these studies with concentrations of small molecules as shown in Table m range, so experiments were set such that the concentration of theobromine was 1 mm maximum.Kd determination. The binding checks were carried out to validate binding of the small molecule and aptamer. Eight samples were prepared—four that contained the RNA aptamer and four that contained the RNA and the small molecule. Both the RNA and the small molecule were kept constant at 10 nm and at the highest small‐molecule concentration . The prepared mixtures were incubated at room temperature for ≈10 min, captured in a capillary tube (as described below), and placed on the plate, with the RNA sample in rows 1–4 and the RNA/small molecule complex in rows 5–8.Binding checks were accomplished for all RNAs with each small molecule prior to ⋅HCl (50 mm), NaCl (150 mm)], whereas for theobromine the buffer was 1× TBS with DMSO (10 %). This was carried out in such a manner that sequential twofold dilution, with respect to the small molecule, was present in each tube. This was followed by addition of RNA  to each tube to accomplish 10 nm [RNA] (while keeping the content of DMSO at 5 % DMSO max.). The mixtures were then incubated in ice for 20 min in the cases of the mixtures containing theophylline or caffeine and at room temperature in those of the mixtures containing theobromine. Next, ≈10 μL of the mixture was captured in a capillary tube, placed on the plate, and covered with the magnetic strip to prevent movement. Each mixture was withdrawn and handled one at a time, with the solutions containing the highest concentration of small molecule placed at position “1”. The microtube and the capillary were held horizontally to prevent bubble formation, and the capillary was handled by the end of the tube. Each programmed experiment ran for approximately 20 min.Dissociation constants were obtained as follows. First, 16 microtubes (0.6 mL) were filled with 10 μL of the buffer containing the appropriate small molecule. For theophylline and caffeine, the buffer was 1× TBS  was added, and the mixture of interest (9–10 μL) was added to a denaturing PAGE. For shorter oligomers (<20 nt) a short gel was used. For longer oligomers a long gel was used. The gels were run until the methylene blue dye ran halfway to three quarters of the way down the gel.1RNase T: A cocktail solution of RNA (3000–5000 counts) in phosphate buffer  was made. A mixture of the RNA and the enzyme (1:1) was prepared and incubated at 50 °C for 45 min. After incubation, LB (7 μL) was added, and the mixture of interest (9–10 μL) was loaded onto a denaturing PAGE. For shorter oligomers (<20) a short gel was used. For longer oligomers (>20) a long gel was used. The gels were run until the methylene blue dye ran halfway to three quarters of the way down the gel.m NaHCO3, pH 9.1), which was used for band assignment. The ladder works through hydrolysis of every nucleobase from the 3′‐end and was produced by incubating the RNA of interest and the hydrolysis buffer at 90 °C for 12 min  followed by addition of loading buffer  prior to loading onto gel.The degradation patterns for all aptamers were compared against a hydrolysis ladder  should be addressed to the authors.SupplementaryClick here for additional data file.


Virus-derived siRNAs (vsiRNAs) generated by the host RNA silencing mechanism are effectors of plant’s defense response and act by targeting the viral RNA and DNA in post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) pathways, respectively. Contrarily, viral suppressors of RNA silencing (VSRs) compromise the host RNA silencing pathways and also cause disease-associated symptoms. In this backdrop, reports describing the modulation of plant gene(s) expression by vsiRNAs via sequence complementarity between viral small RNAs (sRNAs) and host mRNAs have emerged. In some cases, silencing of host mRNAs by vsiRNAs has been implicated to cause characteristic symptoms of the viral diseases. Similarly, viroid infection results in generation of sRNAs, originating from viroid genomic RNAs, that potentially target host mRNAs causing typical disease-associated symptoms. Pathogen-derived sRNAs have been demonstrated to have the propensity to target wide range of genes including host defense-related genes, genes involved in flowering and reproductive pathways. Recent evidence indicates that vsiRNAs inhibit host RNA silencing to promote viral infection by acting as decoy sRNAs. Nevertheless, it remains unclear if the silencing of host transcripts by viral genome-derived sRNAs are inadvertent effects due to fortuitous pairing between vsiRNA and host mRNA or the result of genuine counter-defense strategy employed by viruses to enhance its survival inside the plant cell. In this review, we analyze the instances of such cross reaction between pathogen-derived vsiRNAs and host mRNAs and discuss the molecular insights regarding the process of pathogenesis. RNA-triggered gene silencing is a highly conserved mechanism in eukaryotes. The key feature of RNA silencing is the production of 21–25 nucleotides (nt) long small RNAs (sRNAs)  downregutrans-acting siRNAs (ta-siRNAs), involved in endogenous gene regulation. Resistance against DNA viruses and regulation of transposons/or repetitive elements are conferred by 24-nt siRNAs produced by DCL3, whereas DCL2 produces 22-nt siRNAs and coordinates with DCL4 in conferring antiviral response. DCL1 is involved in the production of endogenous sRNAs, called miRNA duplex, in the nucleus . AGO 2/3/7 interact with vsiRNAs, miRNAs, and most importantly tasiRNAs and are involved in plant defense and morphogenesis. AGO 4/6/8/9 proteins bind to 24-nt siRNAs and other endogenous siRNAs and recruit DNA methyltransferase DRM2 to effect RNA-dependent DNA methylation (RdDM) (The trigger for RNA silencing is either double stranded RNA (dsRNA) or hairpin RNA (hpRNA) or fold-back structures formed by ssRNAs derived from the pathogens. These RNA structures are recognized by the plant host-derived ribonucleases, Dicer-like (DCL) enzymes, which cleave the dsRNA into siRNA duplexes . The Ara nucleus . The resn (RdDM) . The RISn (RdDM)  and in tRDR1 and RDR6 contribute to the accumulation of vsiRNAs in the infected cells , jasmonic acid (JA), ethylene, and nitric oxide, suggestive of its role in systemic acquired resistance (RDR1 expression in tobacco resulted in systemic spread of potato virus X (PVX) , whereas vsiRNAs associated with RDR6 were mapped to the 3′-ends in rdr1 mutant plants and appears to be RDR6-dependent  also play major roles by producing secondary siRNAs . The RDRed cells ; RDR2 antructure ; RDR6 prate RNAs . RDR1 hasistance . Downreg X (PVX) . RDR1 faependent . The rolependent . Thus, p∗) via precursor miRNA (pre-miRNA) intermediate. The mature miRNA duplex is exported from nucleus to cytoplasm via HASTY-1 (HST-1)  by the activity of plant RNA polymerase II. The coordinated activity of plant-derived enzymes such as DCL1, DAWDLE, HYPONASTIC LEAVES-1 (HYL1), SERRATE (SE) processes pri-miRNA into mature miRNA duplex (miRNA:miRNA (HST-1) . Either equences . Thus, mIt is increasingly evident that sRNAs, comprising mainly of siRNAs and miRNAs, at the virus–plant interface are the major determinants of an outcome of disease reaction  Figure . AdditioArabidopsis thaliana infected with tobacco mosaic virus–Cg (TMV-Cg) divulged multiple host genes as potential targets for the vsiRNAs. However, TMV-Cg-derived siRNA-mediated cleavage sites were detected only for two target genes, namely, cleavage and polyadenylation specificity factor (CPSF30), and a protein similar to translocon-associated protein alpha  using RLM-RACE  involved in chlorophyll biosynthesis resulting in yellow symptoms. Corroboratively, expression of RNA silencing constructs targeting Y sat RNA also caused yellowing symptoms. Moreover, incorporation of RNA silencing gene constructs with the chll sequence of Arabidopsis and tomato manifested typical yellow mosaic symptoms in an otherwise non-responsive host. Thus, this study demonstrated the role of sub-viral agent and associated vsiRNAs in inducing characteristic symptoms in the natural host  model. It was known that yellow mosaic symptom appears when CMV-Y sat infects ral host . Furthercumented . CMV-2b-rocesses .N. benthamiana model. Among the differentially expressed genes, 11 (nine of them were chloroplastic genes) were found to be involved in inducing chlorosis. Also, the eukaryotic translation initiation factor 4A (NbeIf4A) was downregulated resulting in leaf twisting and stunting symptoms associated in RSV infection. Interestingly, vsiRNA-4A derived from RSV showed sequence complementarity with NbeIf4A mRNA, and artificial miRNA expression of these vsiRNAs demonstrated silencing of NbeIf4A gene  in susceptible tomato cultivars. However, this sequence complementarity is lacking in the resistant plants due to the 14 nt deletion in the host genome. The viral siRNAs derived from the IR have the ability to silence the SILNR1 in susceptible cultivars actuating the stunting and curling leaf traits. Further, SILNR1 overexpressing lines showed reduced accumulation of TYLCV suggestive of the negative crosstalk between the vsiRNAs and the lncRNAs , causing characteristic symptoms and suppressing the host defense network  infecting N. benthamiana neither elicit the host DCL induction nor the RDR6 activity to mount antiviral defense; however, PLPV vsiRNAs, representing the entire genome of the virus, are abundant in the non-symptomatic plants. Despite the abundance of vsiRNAs, accumulation of the host mRNAs is not affected. Infection caused alterations in host miRNA concentrations; nonetheless, it is not correlated with any phenotypic abnormalities. Hence, it is pertinent to investigate other host factors that remain uncharacterized yet but could be associated with viral pathogenesis and symptom appearance  to nucleotide binding site–leucine-rich repeats (NBS-LRR) involved in host defense pathways  has the ability to overcome the vsiRNA-mediated targeting of NBS-LRR class R-genes. The wide range of host mRNA targets indicate that vsiRNAs are not mere pathogenicity factors but also are the determinants of the outcome of host–virus interaction (Laodelphax striatellus) were investigated to gain insights about the regulatory roles of vsiRNAs in different hosts. Among the downregulated genes of RSV-infected rice, 70% of the genes had potential vsiRNA target sites, and these genes were found to be enriched in carbohydrate binding and kinase activity. Similarly, 47.5% of downregulated genes in RSV-infected plant hoppers were potential targets for vsiRNAs and were involved in activities such as cuticle formation, peptidase, and oxido-reductase. vsiRNA profiles of the same virus instigate different molecular responses depending on the host system reflecting diverse biological requirements -derived siRNAs from infected tomato identified that 2,450 unique vsiRNAs have potential to interact with tomato transcriptome. It is noteworthy that tomato cultivars exhibited differential molecular response to the TSWV-derived vsiRNAs that potentially targeted the host mRNAs. Tomato cultivar (Red Defender) containing the virus resistance-conferring loci  involved in callose deposition in pollen mother cell walls and during pathogen infection  from the virulence modulating genomic region of PSTVd specifically targets multiple callose synthase genes of the host  involved in host defense process were identified. Transient expression of all the three vd-siRNAs using a VIGS model based on osa-MIR528 backbone mimicked viroid infection phenotypes (Understanding the interaction between enotypes .MYB TF caused enhanced (>threefold) CLCuD viral DNA accumulation (vsiRNAs from the antisense strand of sugarcane mosaic virus (SCMV) exhibited multiple target sites in the maize transcriptome compared to the siRNAs from the sense strand denoting the strand-specific role of vsiRNAs in host gene regulatory pathways. Furthermore, a broad range of host transcriptome targets involving various pathways such as gene regulation, energy metabolism, cellular defense, and plant signaling underlines the predominance of vsiRNAs in enhancing the virulence of pathogen . Cotton mulation . Though mulation . Thus, tN. benthamiana plants co-infected with a polerovirus, brassica yellows virus (BrYV), and an umbravirus, pea enation mosaic virus 2 (PEMV 2), showed differences in their target pathways enhancing the synergistic infection and virulence  and SCMV in maize revealed possible roles of vsiRNAs in synergistic infection . In SCMVirulence . Similarirulence . Chineseirulence . It estairulence .HDA6) led to the postulate that favorable transcriptional changes are induced in the viroid infected cells so as to recognize the pathogenic RNA as an RNA pol II template so that viroid replication is enhanced  (Viroid genomic RNAs are lncRNAs  that subenhanced . Infectienhanced . Additiopulus L. . Computapulus L. . Five ges (PARE) . These gFRL3 mRNA by PARE sequencing and RLM-RACE techniques. Thus, it provided the direct evidence for the role of a vd-siRNA in modulating the expression of host-mRNA wherein, vd-siRNA not only downregulated FRL3 mRNA but also induced early flowering in tomato  was shown to enter plant nuclei and viral genome-derived miRNA (hcrsv-miR-H1-5p) was identified to be involved in the regulation of viral replication  play main role in growth and development of organisms. Animal-infecting DNA viruses encode miRNAs that modulate the expression of host genes and to regulate self gene expression . miRNAs V genome . TuMV-desic acid . Similarlication . In anotredicted .Computational analysis of SCSMV also revealed the hairpin-RNA structured miRNA precursor in its genome. The predicted miRNA precursor, pre-miR-16, was present in the 3′UTR region of the viral genome and its expression was demonstrated. Furthermore, the reduced chlorophyll and carotenoids content in SCSMV-infected leaves was co-related with the virus-derived miRNA-mediated silencing of plastidial isoprenoid biosynthesis pathway . RecentlThese studies thus unravel hitherto an unknown mode of counter-defense strategy employed by the plant-infecting viruses. Plant virus-derived miRNAs is a rarity when compared to miRNAs of animal viruses . VirusesGeminiviridae) infecting Cassava and Jatropha revealed nine pre-miRNAs from the genomes of African cassava mosaic virus (ACMV) and five pre-miRNAs from East African cassava mosaic virus- Uganda (EACMV-UG). In addition, DNA A-derived ORFs AC1, AC2, AC4, and IR regions of the begomoviruses exhibited miRNA-coding potential. Viral -derived miRNAs were also validated through stem-loop RT-PCR  are considered to be of recent origin and exhibit wide nucleotide sequence diversity across the virus species. VSRs interfere in all the crucial steps of RNA silencing right from the recognition of viral RNA , dsRNA d genomes . Thus, Vin vivo conditions is the prevalence of VSRs. Many VSRs possess sRNA sequestration capability so that it bind siRNA duplexes or miRNA:miRNA∗ thereby limiting the active pool of siRNAs in effecting trans-silencing of host transcripts  efficiently affects host miRNA and siRNA pathways. Correspondingly, sub-cellular localization of a 126-kDa replicase protein of severe and mild isolates of pepper mild mottle virus (PMMoV) has been attributed with differential symptoms expression in N. benthamiana. Although, the authors reasoned that interaction of 126 kDa protein with unknown host factors for differences in the intensity of symptoms, differential impact of the proteins on the PMMoV-derived siRNAs cannot be ruled out. Moreover, suppressors enhance the virulence of viral pathogens by aiding in the accumulation of viral nucleic acids. Nonetheless, as already stated, viral genomic RNA titers do not always correlate with the symptom severity even in the presence of a strong or a weak suppressor protein  in the susceptible cultivar whereas manifestation of resistance trait of a tomato cultivar is due to the deletion of vsiRNA target site in SILNR1  thereby affecting the callose deposition in pollen mother cells disrupting not only the reproductive pathways of hosts but also augmenting the entry point of secondary pathogens -derived sRNAs selectively target immunity-conferring genes of hosts, Arabidopsis and tomato  technique revealed that long dsRNAs are precursors of vsiRNAs . MoreoveFeatures like 5′terminus nucleotide of siRNA also play important role in the efficacy of RNA silencing activity of vsiRNA. AGO 1 preferentially incorporates siRNAs with nucleotide “U” at 5′ terminus. Furthermore, recruitment of vsiRNAs onto AGO and RISC lacking proper ‘slicer’ activity also diminishes the cleavage potential of the sRNAs. Above all, the degree of vsiRNA activity against the host transcripts is also influenced by its relative abundance . DiffereEnormous quantum of data is being generated concerning the nature of vsiRNAs and vd-siRNAs encompassing wide variety of plant and pathogen species. It is also possible for vsiRNAs to act on host chromosome in TGS manner involving RNA directed DNA methylation. However, little research evidences are available to prove this probability, even though sequence-specific epigenetic modification of viral genomic regions mediated by siRNAs is common . DevelopFunctional miRNAs derived from viral genomes are generally identified in DNA viruses  not in RSVR, SY, PG, SC, SW, and HRP performed the data mining and drafted the various sections of the manuscript. All the authors have read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Non-coding small RNAs play important roles in virus–host interactions. For hosts, small RNAs can serve as sensors in antiviral pathways including RNAi and CRISPR; for viruses, small RNAs can be involved in viral transcription and replication. This paper covers several recent discoveries on small RNA mediated virus–host interactions, and focuses on influenza virus cap-snatching and a few important virus sensors including PIR-1, RIG-I like protein DRH-1 and piRNAs. The paper also discusses recent advances in mammalian antiviral RNAi. The study of host–virus interaction is critical for developing effective antivirus strategies and cures. Its importance is fully exemplified in the current emergency of Coronavirus disease 2019 (COVID-19). Viruses interact with hosts using diverse mechanisms across infection stages. A typical mechanism involves protein–protein or protein–lipid interaction when viruses search target cells, such as in the cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus infections ,4. Once Host cells have developed multiple layers of antiviral mechanisms in the endless battles against viruses throughout evolution. In mammals, cellular immunity and humoral immunity based on T, B and other immune cells play critical roles. Host immunity can also be classified as innate and adaptive immunity. Innate immunity constitutes the first layer of immune responses and utilizes physical, chemical, cellular and molecular mechanisms to clear invading viruses while adaptive immunity involves the development of antibody and long term memory primarily involving T and B immune cells. In innate immunity, the interferon (IFN) I, II and III signaling pathways play critical roles in defending host cells against viruses. In these pathways, short double-stranded RNAs (dsRNA) are recognized by the Retinoic acid-inducible gene I (RIG-I) family proteins and stimulate the expression of interferons and downstream factors to clear viruses ,23.The recent discoveries in RNA interference (RNAi) and CRISPR demonstrate a novel but long awaited mechanism to clear viruses, i.e., using small RNA sensors to recognize viral nucleic acids and using enzymes to destroy them ,24. In RHere, we discuss recent advances in host–virus interactions via small RNAs, primarily focusing on IAV cap-snatching, virus sensors, and mammallian antiviral small RNAs.The IAV genome is composed of eight negative-sense viral RNAs (vRNA) for generating mRNAs encoding polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix protein (M including M1 and M2), and nonstructural protein (NS including NS1 and NS2). PA, PB1 and PB2 constitute the IAV RNA-dependent RNA polymerase (RdRP) complex responsible for RNA transcription and replication; glycoprotein HA and NA on the IAV virion surface are responsible for target cell interaction ; M plays roles in virion assembly and budding; NP binds/protects vRNAs in the virion; NS1 inhibits host RNAi and interferon-mediated innate immunity, and NS2, also called nuclear export protein (NEP), is involved in IAV virion export from host cell nuclei ,33,34. ITo make viral proteins, most viruses hijack host translation systems, which usually utilize capped RNAs, i.e., mRNAs, as substrates. To make capped viral mRNAs, viruses can utilize host capping enzymes, which reside in nuclei. However, many RNA viruses have to encode their own RNA capping enzymes since they perform transcription only in cytoplasm, which lacks host RNA capping enzymes . Influen7G) cap instead of a 2,2,7 trimethyl Guanosine  cap (mature snRNAs), are the authentic donors. Their speculation was based on two previous observations: (1) the m2,2,7G cap on mRNA has a lower affinity to translation factor eIF4E than m7G cap; and (2) the ratio of U1 snRNA cap to U2 cap on IAV mRNAs corresponds well with the transcription rate (representing pre-RNA levels) of U1 and U2 snRNAs rather than the steady-state levels (primarily representing mature RNA levels) [2,2,7G caps may help virus-specific translation such as in human immunodeficiency virus (HIV) translation [2,2,7G immunoprecipitation, can address whether cap-snatching utilizes mature U1 and U2 snRNAs or only pre-U1 and U2.Host non-coding (nc) small RNAs likely serve as the major cap donors in IAV cap-snatching. For decades, host ncRNAs have not been within the radar range of cap-snatching studies. Based on limited sequencing data and annotations, host mRNAs were initially identified as the major cap source ,37,38. H levels) ,45,46. T levels) ; (2) m2,nslation ; and (3)nslation . This diCap-snatching may prefer host capped ncRNAs as substrates. Unlike the other two groups, which only sequenced viral capped RNAs, Gu et al. simultaneously obtained host and viral capped RNAs, allowing them to obtain unique matches to host capped RNAs (substrate) for the capped small RNA parts on IAV mRNAs (product) in the same samples and to obtain cap-snatching rates (product/(product + substrate)). U1/U2 snRNAs combined provided ~7% caps on IAV mRNAs; all known ncRNAs including U1/U2, other snRNAs and snoRNAs provided at least 55% caps; pre-mRNAs provided less than 45% including ~7% snatched from sense promoter-associated small RNAs (PASR), a class of small ncRNAs associated with Pol II transcription initiation  or NCRs: one attached to in-framed IAV-encoded proteins and the other attached to out-of-frame IAV-coded “novel proteins” F. AlthouLi and Hui et al. recently reported that IAV utilizes non-canonical cap-snatching to diversify its mRNAs and ncRNAs . CanonicPattern recognition receptors (PRR) play important roles in host immunity against microbial pathogens and viruses. PRRs specifically recognize/bind shared pathogen structures and molecules, collectively called pathogen-associated molecular patterns (PAMP) . In otheRIG-I, another type of PRRs, serves as a virus RNA sensor that recognizes several RNA viruses in mammalian cells and stimulates IFN pathways to clear viruses ,62. RIG-C. elegans Dicer related helicase 1 (DRH-1) is homologous to mammalian RIG-I. However, DRH-1 only contains an RNA helicase domain while lacking a triphosphate binding domain [C. elegans virus, the Orsay virus [C. elegans, since DRH-1 is only required for antiviral RNAi.g domain . Althougg domain ,65. Howeay virus ,67. DRH-ay virus . Howeveray virus . WhetherUnlike its paralogs, including the triphosphatase domains of RNA capping enzymes, PIR-1 as an RNA polyphosphatase removes both the β and γ phosphates rather than just the γ phosphate of triphosphorylated RNAs (ppp-RNA) A 69,70,,73,74,75C. elegans PIR-1 interacts with Dicer [th Dicer  and is rth Dicer , which rth Dicer ,80,81,82th Dicer . These oth Dicer ,77. Inteth Dicer ,83. Techth Dicer .C. elegans RNAi pathways to silence Orsay virus, a naturally occurring RNA virus originally discovered in a wild isolate of C. elegans and thereafter used as a virus model [C. elegans, RNA interference and other small RNA pathways play critical roles in silencing viruses [C. elegans serves as a perfect model system to study antiviral small RNAs since it lacks other antiviral pathways including cellular and humoral immunity, interferon pathways, etc. Both viral transgenes and natural viruses have been used to investigate the antiviral RNAi mechanisms [Our unpublished data suggests that PIR-1 and Dicer function together in us model . In C. e viruses ,66,68,84chanisms ,66,68,84chanisms ,86. Whenchanisms . Intereschanisms ,88. The In conclusion, PIR-1 may serve as a sensor of ppp-dsRNAs including viral transcription/replication intermediates and dsRNA intermediates in the 26G-RNA pathway. Interestingly, both types of intermediates are likely short dsRNAs instead of long dsRNAs synthesized using full-sized template RNAs. In antiviral pathways, these dsRNAs may be “abnormal” RdRP products including abortive transcription and erroneous initiation. However, in the 26G-RNA pathway, it is apparent that the mechanism is designed on purpose by the cells.C. elegans do not target transposons. Instead, at least some of them target endogenous RNAs via basepairing with mismatches (C. elegans germline cells are protected via a CSR-1 (Argonaute)/22G-RNA mediated mechanism [C. elegans germline cells (Orsay virus only infects intestine cells), the model has not been tested with any live viruses. It is also interesting to see whether the piRNAs of low abundance can protect hosts from viruses since there is no evidence suggesting that the expression of those piRNAs can be induced in response to foreign RNA invasion.In animal germlines, piRNA, a class of 20 to 30 nts long ncRNAs play critical roles in silencing transposons, maintaining germline genome integrity ,28,29,31smatches  [18,19,8smatches  [13,18,8echanism . This piechanism . HoweverIFN (interferon) signaling has been established as a textbook model for clearing viruses in mammalian cells and has withstood scrutiny for several decades. In these pathways, RIG-I related proteins and other virus sensors detect invading viruses and then induce the expression of IFN. IFN engages IFN receptors to activate signal transducer and transcription activator 1 and 2 (STAT1/STAT2), which promote the expression of IFN-stimulated genes (ISG) ,94. AmonThe discovery of virus-derived siRNAs (vsiRNA) in mammalian cells by Mallard et al. and Li et al. not only further expands the functions of RNAi but also challenges the canonical view on mammalian antivirus ,99, i.e.RNA viruses often encode proteins antagonizing host immunity. Many viruses including Nodamura virus (NoV), influenza virus, and Dengue virus-2 utilizes viral suppressors of RNAi (VSR), protein B2, NS1 and 2A respectively, to inhibit Dicer activity  98,99,1,103. In While there are multiple independent experiments confirming the existence of antiviral vsiRNAs, others failed to detect vsiRNAs . Many faOne of the core questions that remains unanswered in antiviral RNAi is whether vsiRNAs are functionally relevant in clearing viruses in differentiated cells in which IFN signaling is fully functional, playing predominant roles. A recent study by Han et al. found that vsiRNAs generated in differentiated mouse cells (mouse embryonic fibroblast cells or MEF) infected by NoV are indeed loaded to AGO2, forming functional RNA-induced silencing complexes (RISC), as demonstrated using in vitro slicer activity assays . Surpris-/- mice  exhibited similar levels of vsiRNAs as Rag1-/- mice . However, a previous study using artificial dsRNAs suggested that siRNA biogenesis was inhibited by IFN signaling [In addition, Han et al. presented three other discoveries , which wignaling ,99. In aThis finding that mammalian cells are able to use RNAi to fully clear RNA viruses is exciting. Targeting VSRs to inhibit virus infection in mammalian cells may prove feasible. Even if this strategy may not eradicate viruses completely, it could slow down virus infection and alleviate clinical symptoms. The discoveries presented by Han et al.  will cerSmall RNAs play important antiviral roles, as exemplified by the CRISPR Nobel Prize this year and RNAi Nobel prize in 2006. Ideally, the small RNA based sensors are more specific and easy to design than conventional chemical and antibody drugs. However, viral RNAs are usually protected by proteins and may not be accessible. It is still challenging to design more target-specific and efficient strategies for delivering small RNA based drugs. Since many RNA viruses encode RNAi inhibitors, small molecule drugs targeting these inhibitors have potential in inhibiting virus infection. IAV strictly utilizes a WG (W represents A or U) motif to initiate mRNA transcription . This mo


Zoonotic coronavirus (CoV) infections, such as those responsible for the current severe acute respiratory syndrome-CoV 2 (SARS-CoV-2) pandemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). Although double-membrane vesicles (DMVs) appear to be a pan-CoV RO element, studies to date describe an assortment of additional CoV-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labeling of newly synthesized viral RNA followed by quantitative electron microscopy (EM) autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs Middle East respiratory syndrome-CoV (MERS-CoV) and SARS-CoV and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in CoV infection. Coronaviruses drastically remodel the cellular endomembrane landscape, generating particularly complex replication organelles. This study shows that previous disparate observations converge on a unified model for the coronavirus replication organelle and establish which elements serve as central hubs for viral RNA synthesis. The RNA synthesis of all positive-stranded RNA (+RNA) viruses of eukaryotes occurs in the cytoplasm of the host cell, in conjunction with modified endomembranes that are often referred to as viral replication organelles (ROs) –3. ROs aTwo main RO prototypes have been discriminated: small spherular invaginations and large(r) vesiculotubular clusters consisting of single- and/or double-membrane structures, to which viral replicative proteins and specific host factors can be recruited. The formation of invaginations can occur at the membrane of various organelles, including endoplasmic reticulum (ER), endolysosomes, and mitochondria –9. The lBetacoronavirus), raised a variety of functional considerations. Intriguingly, while double-stranded (ds) RNA, a presumed intermediate and marker for vRNA synthesis uridine) and applied the classic and highly sensitive technique of EM autoradiography uridine) for subsequent detection by EM autoradiography [3H]uridine label when compensating for its size and somewhat higher relative uracil content.To investigate the subcellular localization of vRNA synthesis in cells infected with different CoVs, we metabolically labeled newly synthesized vRNA by arresting cellular transcription with actinomycin D and using a radiolabeled nucleoside precursor  but sho3H-UTP in the cell, and its incorporation into vRNA. In order to explore the practical limits of the approach, we tested different labeling pulses in Vero E6 cells infected with SARS-CoV, measuring the amount of radioactive label incorporated into RNA  is that detection of the label does not rely on immunolabeling. This makes autoradiography compatible with high-contrast EM sample preparation protocols that provide excellent morphology at the price of epitope integrity. Moreover, as the signal derives from radioactive disintegrations, EM autoradiography is a very sensitive technique, which, in principle, should allow for short labeling pulses, essential to minimize the chance of migration of labeled vRNA products from their site of synthesis . Neverthinto RNA . AlthougAbundant autoradiography signal was detected by EM in SARS-CoV-infected cells pulse-labeled for 20 minutes . The sigImportantly, establishing the association of autoradiography signal with specific subcellular structures requires a detailed quantitative analysis, as the autoradiography signal can spread up to a few hundred nanometers from the original radioactive source see . This tyn = 36)  are directly involved in vRNA synthesis. A first answer to this question became readily apparent as regions containing DMVs, but not either of the other RO structural elements, were densely labeled in cells infected with SARS-CoV  or MERS-n = 36) , aligninNext, we specifically investigated the possible involvement in vRNA synthesis of CM and/or DMSs , which wTo explore whether these observations could be extended to distantly related CoVs, we expanded this type of analysis to the gamma-CoV IBV. IBV-induced DMSs are particularly abundant, and a large proportion of them have an open configuration, which contributed to the hypothesis that these open DMSs could be engaged in vRNA synthesis . HoweverIf CM and DMSs are not involved in vRNA synthesis, what is the role (if any) of these RO structural elements in CoV replication? To investigate this, we analyzed the subcellular location of different viral markers in MERS-CoV-infected cells by immunoelectron microscopy (IEM) .CoV-induced DMSs, with their electron-dense content and their remarkably regular size, are particularly intriguing structures. Revealing DMSs in IEM samples, however, was challenging and, in our hands, required a modified protocol for the preparation of thawed cryo-sections  that, unPreviously, the CM induced by SARS-CoV and MHV were shown by IEM to accumulate viral nsps, whereas dsRNA signal was primarily found inside the DMVs ,20. SimiIn summary, for the antibodies tested , the labeling pattern in MERS-CoV-induced DMSs closely resembled that of the CM, from which they seem to derive. The absence of labeling for key proteins in virus assembly, like the M and S proteins, strongly suggest that DMSs do not represent (spurious) virus assembly events.The comprehensive analysis presented here demonstrates that viruses across different CoV genera induce essentially the same type of membrane structures. After somewhat disparate observations ,26–27,47Our results clearly confirm and extend DMVs as the primary—if not only—site of vRNA synthesis within the coronaviral RO. This point has been subject to quite some speculation, due in part to the limited experimental data directly addressing this question. An early study using Br-UTP and IEM to detect newly synthesized vRNA mapped signal in DMV regions of MHV-infected cells , but theWhile the finding of open DMSs, similar to the invaginations that many +RNA viruses use as replication sites, made them attractive candidate sites for vRNA synthesis ,57, we cGiven the prevalence of CM and DMSs across different CoV genera, it is tempting to speculate that these structures must play a role in virus replication. In particular, for the highly regular DMSs, it is hard to imagine that they would lack a specific function; however, their role remains elusive. A suggestive possibility was that DMSs represent nonproductive virus assembly events, but the lack of DMS labeling for key structural proteins seems to rule out this option. In fact, for the viral markers tested, no differences were apparent in the labeling patterns of DMSs and the CM from which they seem to derive. Although their distinct morphology implies that DMSs likely contain specific host or viral proteins, these factors—which may give important clues about DMS function—remain to be identified. Another possibility is that CM and DMSs represent DMV precursors. In this scenario, ER membrane pairing would first give rise to CM, which would then produce DMSs that could eventually expand into DMVs. Pairing of ER membranes, which appears to be driven by 2 key transmembrane viral proteins (nsp3 and nsp4), indeed, is the likely first step in CoV DMV biogenesis, as suggested by studies in cells ectopically expressing these proteins –23,27,58Nidovirales. In contrast with the DMVs induced by other +RNA viruses ), briefly washed with 0.1 M PHEM buffer, and stored at 4°C in 3% (wt/vol) PFA in 0.1 M PHEM buffer until transfer from the biosafety level 3 facility for further processing. To prepare EM samples, the cells were first pelleted and embedded in 12% (wt/vol) gelatin. Cubes of approximately 1 mm3 in size were cut from these pellets, infiltrated with 2.3 M sucrose for cryo-protection, snap-frozen in liquid nitrogen, and sectioned by cryo-ultramicrotomy. Thawed cryo-sections (70-nm thick) deposited on EM grids were incubated first with the corresponding primary antibody and then with protein A coupled to colloidal 10-nm gold particles. Virus-induced membrane modifications, however, were not discernible in these samples, and clear signs of membrane extraction were present. To tackle this issue, we used a previously described modification of the protocol that includes sequential poststaining steps with 1% (wt/vol) OsO4, 2% (wt/vol) uranyl acetate, and Reynold’s lead citrate after immunogold labeling and prior to the final embedding in a thin layer of 1.8% methyl cellulose ) or a OneView 4k high-frame rate CMOS camera (Gatan), respectively. Mosaic EM images of large grid areas were generated for the quantitative analysis of autoradiography samples, using overlapping automatically collected images  that were subsequently combined in a composite image as described in .Prior to the poststaining step, semithin sections (150-nm thick) of CoV-infected cells were incubated with protein A coupled to 10-nm colloidal gold particles that served later as fiducial markers for alignment. Dual-axis tilt series were collected in a Tecnai12 BioTwin microscope using Xplore 3D acquisition software (Thermo Fisher Scientific), covering each 120°–130° around the specimen with an angular sampling of 1° and a pixel size of 1.2 nm. The alignment of the tilt series and tomogram reconstruction by weighted back-projection was carried out in IMOD . The dia2 were randomly selected from the mosaic EM maps, and the autoradiography grains present in those areas were manually assigned to the underlying cellular structures. The abundance of the different types of subcellular structures was estimated through virtual points in a 5×5 lattice superimposed to each selected area, which were also assigned to the different subcellular classes. Regularly along the process, the annotated data per condition were split into 2 random groups, and the Kendall and Spearman coefficients, which measure the concordance between 2 data sets [Large mosaic EM maps containing dozens of cell profiles were used for the quantitative analysis of the newly synthesized RNA autoradiography signal see . For eacata sets , were caata sets .d1) and the distance to the opposite CM edge (d2) were measured. The relative distance to the CM edge was then calculated as d1/(d1+d2) and expressed in percentages. All the measurements in different DMVs, DMSs, and CM were made using Aperio Imagescope software (Leica) and pooled together into 3 single data sets.For the analysis of the association of vRNA synthesis with each of the different ROs motifs, the specific DMVs, DMSs, and CM included in the analysis were carefully selected. Only individual DMVs that were at least 1 μm away from any other virus-induced membrane modification were selected. For every grain present in an area of 750-nm radius around each DMV, the distance to the DMV center was measured. In the case of DMSs, which were always part of clusters of virus-induced membrane structures, only DMSs in the periphery of these clusters were selected. The quantified signal was limited to subareas devoid of other RO motifs, which were defined by circular arcs  opposite to the RO clusters. CM are irregular structures that appear partially or totally surrounded by DMVs. Only large CM (>0.6 μm across) were selected in order to make more apparent (if present) any decay of the autoradiography signal as the distance to the surrounding DMVs increased. For each autoradiography grain, both the distance to the closest CM boundary (S1 VideoAnimation illustrating the tomography reconstruction and model presented in (MP4)Click here for additional data file.S1 FigAnalysis of previously described samples of CoV-infected cells, prepared for EM either by HPF (A) or cryo-plunging (B). A targeted search revealed the presence of DMSs (white arrowheads) in close association with CM. In comparison with the chemically fixed samples used in this study, the superior ultrastructural preservation of cryo-fixation results in less distorted membranes, but also in a denser cytoplasm and darker CM that makes DMS less apparent. (A) Example from a MERS-CoV-infected Vero cell (16 hpi) in a sample used in . (B) Reg(TIF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S1 DataDMS, double-membrane spherule; DMV, double-membrane vesicle.(XLSX)Click here for additional data file.S2 DataSARS-CoV, severe acute respiratory syndrome-coronavirus.(XLSX)Click here for additional data file.S3 DataMERS-CoV, Middle East respiratory syndrome-coronavirus; SARS-CoV, severe acute respiratory syndrome-coronavirus.(XLSX)Click here for additional data file.S4 DataDMV, double-membrane vesicle; MERS-CoV, Middle East respiratory syndrome-coronavirus.(XLSX)Click here for additional data file.S5 DataCM, convoluted membranes; DMS, double-membrane spherule; MERS-CoV, Middle East respiratory syndrome-coronavirus.(XLSX)Click here for additional data file.S6 DataDMS, double-membrane spherule; DMV, double-membrane vesicle; IBV, infectious bronchitis virus.(XLSX)Click here for additional data file. 10 Mar 2020Dear Dr Bárcena, Thank you for submitting your manuscript entitled "A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis" for consideration as a Research Article by PLOS Biology.Your manuscript has now been evaluated by the PLOS Biology editorial staff, as well as by an academic editor with relevant expertise, and I'm writing to let you know that we would like to send your submission out for external peer review. I should warn you that we're not wholly persuaded of the strength of advance, so we'll be looking for some enthusiasm from the reviewers.However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.Please re-submit your manuscript within two working days, i.e. by Mar 12 2020 11:59PM.https://www.editorialmanager.com/pbiologyLogin to Editorial Manager here: http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF when you re-submit. During resubmission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. Once your manuscript has passed all checks it will be sent out for review. plosbiology@plos.org if you have any queries relating to your submission.Feel free to email us at Kind regards,Roli RobertsRoland G Roberts, PhD,Senior EditorPLOS Biology 9 Apr 2020Dear Dr Bárcena,Thank you very much for submitting your manuscript "A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis" for consideration as a Research Article by PLOS Biology. As with all papers reviewed by the journal, yours was evaluated by the PLOS Biology editors as well as by an Academic Editor with relevant expertise and in this case by three independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we will probably accept this manuscript for publication, assuming that you will modify the manuscript to address the remaining points raised by the reviewers. Please also make sure to address the Data Policy and other policy-related requests noted at the end of this email.We expect to receive your revised manuscript within two weeks. Your revisions should address the specific points made by each reviewer. In addition to the remaining revisions and before we will be able to formally accept your manuscript and consider it "in press", we also need to ensure that your article conforms to our guidelines. A member of our team will be in touch shortly with a set of requests. As we can't proceed until these requirements are met, your swift response will help prevent delays to publication.*Copyediting*Upon acceptance of your article, your final files will be copyedited and typeset into the final PDF. While you will have an opportunity to review these files as proofs, PLOS will only permit corrections to spelling or significant scientific errors. Therefore, please take this final revision time to assess and make any remaining major changes to your manuscript.NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:https://journals.plos.org/plosbiology/s/supporting-information*Published Peer Review History*Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/*Early Version*Please note that an uncorrected proof of your manuscript will be published online ahead of the final version, unless you opted out when submitting your manuscript. If, for any reason, you do not want an earlier version of your manuscript published online, uncheck the box. Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.*Protocols deposition*https://journals.plos.org/plosbiology/s/submission-guidelines#loc-materials-and-methodsTo enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: *Submitting Your Revision*https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include a cover letter, a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable), and a track-changes file indicating any changes that you have made to the manuscript. To submit your revision, please go to Please do not hesitate to contact me should you have any questions.Sincerely,Roli RobertsRoland G Roberts, PhD, Senior EditorPLOS Biology------------------------------------------------------------------------DATA POLICY:http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:1) Supplementary files . Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to  using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).2) Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication. Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are needed for readers to assess your analysis and to reproduce it: Figs 1D, 3ACD, 4CD, 5BDE, and S3CDE. NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived .Please also ensure that figure legends in your manuscript include information on where the underlying data can be found, and ensure your supplemental data file/s has a legend.Please ensure that your Data Statement in the submission system accurately describes where your data can be found. ------------------------------------------------------------------------BLOT AND GEL REPORTING REQUIREMENTS:https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirementsFor manuscripts submitted on or after 1st July 2019, we require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare and upload them now. Please carefully read our guidelines for how to prepare and upload this data: ------------------------------------------------------------------------REVIEWERS' COMMENTS:Reviewer #1:This manuscript by Snijder and co-workers entitled "A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis" describes elegant studies aimed at discerning if double membrane vesicles or other ER sites modified by viral proteins are the site of coronavirus RNA synthesis during viral replication. The authors describe that an early study using immuno-EM revealed that virus-induced double membrane vesicles (DMVs) were the sites of viral RNA synthesis . With the advancements in EM techniques and particularly EM tomography, researchers appreciated that coronavirus replication induced reorganization of host membranes, generating both convoluted membranes (CM) and DMVs . In addition, the term double membrane spherules (DMS) was introduced to describe membrane rearrangements detected during the replication of infectious bronchitis virus , but it was not clear if these structures were unique to avian coronaviruses, or if they played a role in RNA synthesis. In this study, the authors used radiolabeling of viral RNA and detected the labeled RNA in association with the DMVs. Importantly, they also determined that CMs, DMSs, and DMVs can be visualized for all the coronavirus specimens they studied, including MERS-CoV, suggesting that these structures are generated during the assembly of the viral replication complex and the DMVs necessary for viral RNA synthesis. The technical work here is tremendous and the images of the viral protein-modified membranes are stunning. However, the manuscript seems have an inordinate focus on the DMSs, potentially implicating these as unique  entities. Isn't is possible that the DMS detected at the moment of fixation is in fact on its way to becoming a DMV? Alternatively, some of these DMSs could be unable to progress to DMV status, for reasons that are not yet clear. In the absence of live-cell EM, it is not possible to distinguish these possibilities, but certainly the possibility the DMS to DMV transition could be considered as the authors develop their "unifying structural and functional model" of coronavirus RNA synthesis. Currently, there is a significant emphasis on the DMS in the manuscript, although this structure is not the take home message, and there is no functional role given to these structures, even as potential intermediates. If there is evidence that a DMS can not continue to grow into a DMV, then this should be made clear in the manuscript. Overall, the authors provide important results using state-of-the-art techniques that confirms and extends the early studies that that DMVs are the site of coronavirus RNA synthesis. Comments for the authors' consideration:1. Lines 375-378: Consider changing the order of this description to 1) CM and zippered membranes; ii) DMS and iii) DMVs. This would allow you to speculate in a unifying model that coronaviruses modify membranes to promote "zippering", resulting in the formation of spherules that may mature into double membranes vesicles, the site of viral RNA synthesis. This "progression" is still hypothetical because it has not be visualized in real time, but certainly all the data presented here are at least consistent with this unifying model. One of the challenges is that researchers tend to see all of these structures simultaneously, and not a neat progression from one form to another. The discussion lines 410-413 seems to discount a progression model because of a lack of intermediate, but why couldn't a small double membrane spherule progress into a DMV? One could even speculate that the open DMS allows for capturing of a RNA that "seeds" the DMV to become a site of RNA synthesis. It is reasonable to speculate about potential models in the discussion section, and to be open to multiple interpretations of the current findings.2. Line 381: change "establish" to confirms and extends. This manuscript works very hard at presenting uncertainty or controversy about coronavirus replication sites. However, the questions that arose about the sites of RNA synthesis came about as the techniques to visualize the modified membranes were improved over time, suggesting the potential for more than the originally documented DMVs were involved RNA synthesis. Perhaps this is a stylistic point, but pointing out that the results here confirm and extend earlier studies is important and accurate. 3. The text could be much more concise by focusing on the idea that this work is evaluating all structures in multiple coronaviruses, which has not been done before in one manuscript, and seeking to determine if one or multiple sites are used for viral RNA synthesis. The "controversy" seems vastly over emphasized here. 4. The images in the supplementary section are striking also. If PLoS Biology allows it, try to include all figures in the body of the paper and have only the movie in the supplement.Reviewer #2: Coronaviruses have emerged as important zoonotic viruses with major threat to human health. In this paper, the authors characterized the ultra structures of human cells infected by different coronaviruses including MERS, SARS-CoV1, MHV, HCoV-229E and IBV. They have identified three different membranous structures, DMVs, CM and DMS, which have all been observed before in various coronavirus infections. The detection of DMS is new for MERS infection. Importantly, they were able now to provide a more universal picture that all these viruses induce the formation of all these RO structures. By using metabolic labeling of viral RNAs, after shutting down cellular transcription, they were able to demonstrate that DMVs are the likely places for coronavirus RNA synthesis and replication. Somewhat surprisingly, they find no significant roles for CM and DMS in coronavirus RNA synthesis and replication. This conclusion was further supported by detection of viral dsRNAs within DMVs. The findings are potentially important, especially the metabolic labeling data are quite exciting. I found the manuscript is fun to read with beautiful images. The manuscript requires the following changes. Fig. 1 shows images after 12 h incubation with many DMSs. What time point are these DMSs first appear?Fig. 1 and Fig. 6. Please replace the arrowheads with different colors to aid the readers. Gray and black arrowheads could be quite similar on different computer screens.Fig. 1F: please define what are arrows pointing at? Fig. 1F: Are the outer DMVs and the ER membranes are continuous membranes or are there gaps between them. It seems that they are continuous based on the presented images here.Fig. 4B: it would be useful to pinpoint DMVs, since it is very crowded RO is shown here.Fig. 5. It would be useful to know what % of label was inside versus outside of DMVs? It looks more label is on the outside of DMVs . What is the possible explanation? Lane 155: based on the findings in this work, I doubt it is correct to call zippered ER and CM as ROs, since no viral RNAs were detected in those structures.L407: CM and DMS biogenesis does look very abundant at 12 time point in Fig. 1A-B, contradicting that these structures form late during infection. What is the first time point when CM and DMS structures are detected? The time of the vents for the biogenesis of various RO structures is confusing in this work.Please discuss the origin of DMVs, since the ER origin of CM and DMS is discussed nicely in the discussion part. I note that DMSs are possibly too small to harbor the large coronavirus RNAs, possibly dsRNA (if that is the template for RNA synthesis), sgRNAs and the replicase complex, while copying the RNA templates. Even the 6x times smaller tombusviruses and nodaviruses form almost that size spherules to support replication as the observed size of DMS in this work.Is it possible that DMSs are high-density membrane-protein  storage granules to reduce ER stress in the cells?Do the authors have data on how much percentage of the label is incorporated into genomic versus subgenomic RNA species during metabolic labeling? That would be useful information.Reviewer #3:In this manuscript, the authors performed a detailed structural and functional characterization of the replication organelles (ROs) of coronaviruses. Coronavirus ROs are composed of predominantly double membrane vesicles (DMVs). Through comparative analysis, the authors demonstrate that convoluted membranes, zippered ER and double-membrane spherules are conserved across alpha-, beta- and gamma- CoVs and altogether form the viral ROs. Additionally, the authors employed a classical, yet elegant approach that combines ultrastructural analysis with RNA labeling using radiolabeled nucleosides to demonstrate that viral RNA synthesis is associated with double membrane vesicles. This finding provides best available evidence so far that DMVs are the site of viral RNA replication. This is a timely study that sheds light on the role of the different membranous structures observed during CoV replication, although these structures have been described in several earlier reports. The main point of the present study is the clarification of a long-standing debate by showing that DMVs most likely are the site of viral RNA replication. Major comment:The results of this study are clear and the author´s claims are supported by their data. One of the aims was to clarify the apparent differences reported in the ultrastructural architecture of the coronaviral ROs. The authors achieved their goal by showing that the ROs of the different CoVs are essentially the same. One of the limitations is that the authors provide a single snapshot looking at a single time point for the different viruses, often in different cells and using different MOIs. Moreover, the authors do not provide a growth curve of the viruses in the different cell lines. This information would be useful to allow for a direct comparison between the different conditions. The authors could at least comment if the selected time points reflect the initial phase of the viral infection or a later phase.Additionally, what is the order of appearance of the different structures forming the CoV ROs and is this order conserved among the different CoVs? This could support the idea of a general mechanism used by all CoVs and the regulation of the biogenesis of their ROs. If such information is already present in the literature the author should comment on this point in the discussion. Minor points:For the autoradiography experiments time points, MOIs and cell types differ between MERS-CoV and SARS-CoV infections. Are the time points selected by the authors comparable in terms of progression of viral infection of the two viruses in the different experimental conditions? In other words, are the selected time points within the exponential phase of viral replication or at the plateau phase of viral replication? The authors should comment on this.Line 413 - there is no reference to backup this claim. Since this study does not analyze how the RO architecture changes during infection, the claim that DMV formation precedes the appearance of CMs should come from a previous study. The authors should add the reference of the paper to which they are referring.FigS2- the figure legend states that Huh7 cells were infected with MERS-CoV while the figure annotation says Vero cells. The authors should clarify.Line 177 - truncated sentence 7 May 2020AttachmentResponseToReviewers.docxSubmitted filename: Click here for additional data file. 14 May 2020Dear Dr Bárcena,On behalf of my colleagues and the Academic Editor, Andrea Cimarelli, I am pleased to inform you that we will be delighted to publish your Research Article in PLOS Biology. The files will now enter our production system. You will receive a copyedited version of the manuscript, along with your figures for a final review. You will be given two business days to review and approve the copyedit. Then, within a week, you will receive a PDF proof of your typeset article. You will have two days to review the PDF and make any final corrections. If there is a chance that you'll be unavailable during the copy editing/proof review period, please provide us with contact details of one of the other authors whom you nominate to handle these stages on your behalf. This will ensure that any requested corrections reach the production department in time for publication.Early VersionThe version of your manuscript submitted at the copyedit stage will be posted online ahead of the final proof version, unless you have already opted out of the process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.PRESS biologypress@plos.org. If you have not yet opted out of the early version process, we ask that you notify us immediately of any press plans so that we may do so on your behalf.We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with http://www.plos.org/about/media-inquiries/embargo-policy/.We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit Thank you again for submitting your manuscript to PLOS Biology and for your support of Open Access publishing. Please do not hesitate to contact me if I can provide any assistance during the production process.Kind regards,Vita Usova,Publishing Editor PLOS Biologyon behalf ofRoland Roberts,Senior EditorPLOS Biology


Major depressive disorder (MDD) is a major health problem with significant limitations in functioning and well-being. The World Health Organization (WHO) evaluates MDD as one of the most disabling disorders in the world and with very high social cost. Great attention has been given to the study of the molecular mechanism underpinning MDD at the genetic, epigenetic and proteomic level. However, the importance of RNA modifications has attracted little attention until now in this field. RNA molecules are extensively and dynamically altered by a variety of mechanisms. Similar to “epigenomic” changes, which modify DNA structure or histones, RNA alterations are now termed “epitranscriptomic” changes and have been predicted to have profound consequences for gene expression and cellular functionality. Two of these modifications, adenosine to inosine (A-to-I) RNA editing and m6A methylations, have fascinated researchers over the last years, showing a new level of complexity in gene expression. In this review, we will summary the studies that focus on the role of RNA editing and m6A methylation in MDD, trying to underline their potential breakthroughs and pitfalls. Major depressive disorder (MDD) is characterized by symptoms such as profound sadness, a decrease in vitality, loss of interest in normal activities and negative and pessimistic thoughts. It is a very frequent and widespread disorder. The World Health Organization (WHO) evaluates MDD as one of the most disabling disorders in the world with a lifetime prevalence ranging from 12% to more than 20% and with a very high social cost .Beside genetic variations, other alterations that might underlie the pathological behaviors observed in depressed individuals are stress-induced epigenetic modifications . Indeed,Recently, “omics” approaches have revealed that RNA molecules are broadly and dynamically altered by many different mechanisms. More than 100 RNA modifications have been identified . SimilarOne of the most studied processes occurring at the RNA level is the co-transcriptional event of “RNA editing” . The firIn addition to RNA editing, RNA molecules are altered by a variety of reversible chemical modifications . Among tIn this revision, we will try to summarize the state of art of the studies that focus on the role of RNA modifications in MDD trying to underline their potential breakthroughs and pitfalls.Adenosine to inosine (A-to-I) editing is the most frequent form of RNA editing, widespread in the majority of human genes. The conversion of adenosine to inosine is mediated by enzymes named Adenosine Deaminases Acting on RNA (ADAR); ADAR enzymes are able to bind to double stranded RNA (dsRNA) and modify A nucleotide into I by deamination. The mammalian genome encodes for three members of the ADAR family: ADAR1 (encoded by the gene ADAR), ADAR2 (encoded by ADARB1) and ADAR3 (encoded by ADARB2). ADAR1 and -2 are active enzymes, whereas ADAR3 lacks the enzymatic activity and is mainly expressed at low-level in brain regions ,16. ADARADAR enzymes edit millions of sites in the mammalian transcriptome. The majority of these sites is located in non-coding sequences: 5′UTRs, 3′UTRs and in intronic retrotransposons, such as ALU-inverted repeats . In prinMany re-coding sites are phylogenetically conserved and affect transcripts important for neuronal functions . For exaOver the decades, RNA editing studies in MDD have focused mainly on the analysis of the serotonin 2C receptor (5-HT2c-R) and glutamate receptor transcripts, in line with the serotonergic and glutamatergic hypothesis of mood disorders ,35.The 5-HT2c-R is a G-protein coupled receptor inducing the activation of phospholipase C and the generation of inositol phosphates and diacylglycerol ,37. ThisThe main studies tackling the association between MDDs and RNA editing in the serotonin receptor 2C are summarized in As already noted , Table 1Finally, the link between serotonin transmission, 5-HT2c-R editing and the risk of suicide is further strengthen by the observation that polymorphisms located in the ADARB1 (coding for ADAR2) and HTR2C genes where associated with suicidal attempts  and ADARRNA editing modifies three alfa-Amino-(3-Idrossi-5-Metil-4-isoxazole) propionate (AMPA)  and two kainate (GluK1 and GluK2) glutamate receptor subunits in different positions . In partResearch focusing on glutamate receptor RNA editing dysregulation in human post-mortem brains of MDD patients is lacking, or positive results are lacking. At present, only one study reported a modest decreased of R/G editing levels of AMPA receptors in patients with mood disorders, correlated with the downregulation of ADAR2 expression .Physical and psychological distress, if repeated, strongly affects genetically predisposed subjects and might be a main risk factors for neuropsychiatric disorders such as MDD . ProgresAs for human studies, the first reports correlating modifications in the editing reaction with stress events focused on the analysis of specific and well-known re-coding sites, such as those modulating serotoninergic  and glutMany studies reported that common stress paradigms ,57,58,59The first report linking glutamate receptor editing modifications and stress took advantage of fear conditioning, a behavioral test in which animals are able to foresee aversive stimuli. This stress paradigm induced a decrease in the RNA editing level of kainate GluK1 Q/R site in the amygdala . FurtherAs previously mentioned, for a long time the analysis of changes in the RNA editome remained restricted to re-coding sites. Recently, thanks to Next Generation Sequence (NGS) technology, the analysis of RNA editing of a large number of sites located throughout the transcriptome has become feasible and allowed having a more detailed picture of the involvement of RNA editing in stress and stress-related disorders. A recent work analyzing chronic social defeat stress (CSDS) quantified RNA editing at hundreds of sites, using a microfluidics-based multiplex polymerase chain reaction and a deep sequencing (mmPCR-seq) analysis revealed that CSDS induced moderate changes in RNA editing in a set of mRNAs within the Basal lateral amygdala (BLA) and medial PFC, including modest alterations of the 5-HT2c-R transcript . The autFinally, it has been reported that if adolescent female rats are exposed to chronic unpredictable stress ahead of reproduction , RNA editing is affected in the PFC and amygdala of the offspring for two generations. The editing level of the five 5-HT2c-R sites resulted to be affected by stress and led to the expression of different 5-HT2c-R isoforms in stress-exposed offspring versus naïve females . In partDue to the central role of RNA editing in the proper function of the brain and its involvement in neuropsychiatric disorders ,52, largAs for MDD and stress paradigms studies, the majority of the analyses correlating RNA editing and antidepressants were focused on the 5-HT2c-R . ChronicIntriguingly, in rats, maternal treatment with fluoxetine reduced 5-HT2c-R editing in the amygdala of the offspring at birth , upregulated the expression of 5-HT2c-R mRNA and ADAR enzymes in PFC and rescued pre-reproductive stress modifications on 5-HT2c-R editing in the same region. Furthermore, maternal treatment with fluoxetine increased alterations of glutamate receptors editing levels between offspring of stress-exposed rats and their controls, leading to enhanced social preference in adult offspring .Therefore, it is difficult to have a clear view of antidepressant modifications on 5-HT2c-R editing levels as results largely vary among antidepressant treatments, brain area and animal strains analyzed.2+ influx and kinase phosphorylation [It has been shown that chronic treatments in rats with fluoxetine and desipramine, a tricyclic antidepressant (TCA), both normally used among the first medications for MDD, induced specific but modest effects on the expression level and RNA editing level at R/G site of GluA2 and at Q/R site for GluK2 . Afterwarylation ,80, indirylation  and naïvNew indications suggest that several chemical modifications on mRNA, now called epitranscriptomic modifications, can affect every aspect of RNA biology, including stability, transport, splicing, and translation ,8,9. AmoIn addition to association studies on humans, other studies on animal models tested the effect of m6A in the brain under different conditions. Engel et al.  investigFinally, a very recent paper revealed that circSTAG1 is able to weaken depressive-like behaviors through the modulation of m6A/m of fatty acid amide hydrolase (FAAH). CircSTAG1 is a circular-RNA that was found downregulated in chronic unpredictable stress (CUS)-treated mice and in the blood of MDD patients. Huang et al. showed that circSTAG1 overexpression inhibited the ALKBH5 translocation into the nucleus, leading to an increased m6A methylation of FAAH mRNA and its degradation in astrocytes. This in turn determined an improvement of astrocyte functionality and a reduction of depressive-like behaviors induced by CUS .This very recent data suggest that the m6A modification dynamically responds to environmental stimuli in a brain-area and gene-specific manner and that its dysregulation may contribute to stress-related disorders .Up to date, the vast majority of RNA editing studies in mood disorders have focused mainly on few re-coding sites in serotonin receptor 2C and glutamate receptors mRNAs. The comparison of the reported results, however, is extremely difficult due to the large heterogeneity in experimental designs . In addition, some studies suffer from low statistical power  that impact on the reproducibility of the results.Considering the 5-HT-2c receptor RNA editing, converging analyses reported a trend of increased editing mainly in the PFC of depressive patients that committed suicide. We might speculate that lower receptor activity, induced by higher editing in specific brain areas, could be a risk factor for suicide in MDD patients. Nevertheless, a clear and final link between 5-HT-2cR RNA editing dysregulation and MDD is still missing. It is of note that 5-HT2c-R RNA editing has been studied mainly in HI and PFC, two of the main areas involved in depressive disorders; no study at present has investigated the RNA editing level of this receptor in the nucleus accumbens, the brain area where it is expressed at the highest levels . Future As concern glutamate receptors, RNA editing has been extensively studied in many animal models of stress paradigm and after antidepressant treatments with preliminary results that, although not always concordant, seem to suggest an involvement of this mechanism in depression. Unfortunately, these data are not reflected in humans, where the absence of studies suggests a lack of positive results.Therefore, the role of RNA editing remains extremely unexplored in the contest of MDD and a more comprehensive picture is needed to shed light on the complex pattern of modifications that underly the onset and the progression of this multifaceted disorder. Some attempts in this sense have already been reported for other neurological and psychiatric disorders with interesting results. Indeed, global analysis of RNA editing using NGS technology have shown a general dysregulation of RNA editing and ADAR activity in multifactorial diseases such as schizophrenia , autism,Finally, the emerging field of m6A modification open new and unanswered questions about its role in MDD. Here, we are still limited by available technologies. Future studies are needed to shed light on the epitranscriptome that dynamically regulate gene expression in our brains.