The chemical synthesis of modified oligoribonucleotides represents a powerful approach to study the structure, stability, and biological activity of RNAs. Selected RNA modifications have been proven to enhance the drug-like properties of RNA oligomers providing the oligonucleotide-based therapeutic agents in the antisense and siRNA technologies. The important sites of RNA modification/functionalization are the nucleobase residues. Standard phosphoramidite RNA chemistry allows the site-specific incorporation of a large number of functional groups to the nucleobase structure if the building blocks are synthetically obtainable and stable under the conditions of oligonucleotide chemistry and work-up. Otherwise, the chemically modified RNAs are produced by post-synthetic oligoribonucleotide functionalization. This review highlights the post-synthetic RNA modification approach as a convenient and valuable method to introduce a wide variety of nucleobase modifications, including recently discovered native hypermodified functional groups, fluorescent dyes, photoreactive groups, disulfide crosslinks, and nitroxide spin labels. N6-threonylcarbamoyladenosines  ,46O-silyO-phosphoramidites  and a 3′-O-DMTr-2′-O-TBDMS blockage of sugar residue. Activated 5′-O-phosphoramidite is coupled with a nucleoside bearing a 5′ succinate of ribonucleoside attached to a solid support leading (after oxidation) to the dimer formation with phophodiester linkage and a 3′-O-DMTr-protecting group. The removal of DMTr opens the next synthetic cycle. Oligonucleotide deprotection is performed using the same standard TBDMS chemistry currently utilized in the conventional 3′→5′ synthesis. Although the reverse RNA synthesis is used to a lesser extent than the conventional strategy, it offers a facile route to the assembly of 3′-conjugated RNA constructs of long and medium lengths. Importantly, it was demonstrated that the use of 3′-O-DMTr-5′-phosphoramidites enhances the coupling efficiency, which surpassed 99% per step leading to high-purity RNA products . Th. Th10 anO-DMTr-2′-O-TBDMS Verdine’s phosphoramidites were applied for the incorporation of photolabile 2-(2-nitrophenyl)propyl (NPP) and 2-(2-nitrophenyl)ethyl (NPE) “caging groups”  in methanol  or NPE-NH2 (26b) in acetonitrile . The use of sterically hindered amine 26b required an extra cleavage step with methylamine or aq. NH3 for complete base deprotection. In the case of inosine-containing oligomer 11 the conversion with NPE–NH2 turned out not to work. Finally, oligomers containing both NPP and NPE groups were desilylated using TEA⋅3HF/TEA/NMP mixture. An attempt to prepare 4-O-caged uridine- and 6-O-caged adenosine-containing oligomers by incubation of 10, 11 with NPP–OH nucleophilic reagent  in MeCN for 7 days at 42 °C  proved to be ineffective. The presented methodology enabled simplifying the synthesis of caged RNAs (compared to the standard monomer approach), since the commercially available Verdine’s phosphoramidite was used. The expected results of duplex destabilization caused by attached caging groups were achieved.Commercially available 5′- groups”  71]. So. SoO-DMT7 , respectively . Before conversion, the 6-methylthio group (6-SCH3) of support-linked 6-methylthiopurine-modified oligoribonucleotide 30 (31 and 32 decorated with 6-methylsulfoxide (6-S(O)CH3) and 6-methylsulfonyl (6-S(O)2CH3) groups, respectively. The oxidized oligomers 31, 32 (still attached to the support) were reacted with various primary alkylamines. To get oligomers modified with N6-methyladenosine or N6-isopentenyladenosine, 2 M MeNH2 in THF  and isopentenylamine hydrochloride with TEA in pyridine  were applied, respectively. Other amines, such as isoamylamine, neopentylamine, 1-methylpropylamine, 1-methylbutylamine, and propargylamine were used as a solution in acetonitrile (1:9 v/v) and left for 12 h at rt. After conversion, the samples were evaporated, and the aq. NH3/EtOH  was added for the complete removal of the base-labile protecting groups. After desilylation, the fully deprotected oligomers were purified on silica gel plates. The overall yields of N6-alkyladenosine-containing RNAs 33a–h were in the range of 40–77% (13–34% for 17-mers). It was indicated that the synthesis yields were not dependent on the position of convertible nucleoside in the RNA sequence but on the steric hindrance and the nucleophilic character of the amino group in alkylamine conversion reagents. In 2003, Kierzek’s group employed 6-methylthiopurine riboside 7 see  and 2-meectively  60]. Am. Am7  on the 1 µmol scale of the oligonucleotide synthesis. The trityl-off support-linked precursor 34  or isopentenylamine hydrochloride/TEA in pyridine . For complete removal of base-labile protecting groups, the samples were additionally treated with aq. NH3/EtOH . Displacement of the 6-chloro group with –NH2 involved the aq. ammonia and prolonged reaction time . After desilylation, the fully deprotected oligomers were purified on silica gel plates. The overall yields of 2-methylthio-N6-alkyladenosine-modified oligoribonucleotides 35a–c range from 20% to 65% (16–24% for 17-mers).To prepare the 2-methylthio-As 35a–c , severalde 8 see  exhibiteursor 34  was treaN6-alkylated adenosines and 2-methylthioadenosines elaborated by Kierzek’s group seems to be the method of choice for obtaining several homologous RNA oligomers, especially that the precursor synthons were stable during the synthesis of 3′-O-phosphoramidite/oligonucleotide and easily transformed to target N6-substituted adenosine-RNAs.The post-synthetic introduction of NAr reaction has shown to be attractive for the incorporation of 4-thiouridine (s4U) into RNA oligomers. The preparation of s4U-RNAs by the classical approach requires a special protection for 4-thiocarbonyl function in s4U-phosphoramidite to avoid the desulfuration by-product produced during the oxidation step . T. T4U-derfor 16 h B. Strong4U-RNAs by the post-synthetic conversion of oligomers containing 4-triazolyl-pyrimidine riboside 40 and 4-O-(2-nitrophenyl)uridine 41 with sodium hydrogen sulfide NaSH  to achieve complete displacement of the triazolyl group. Then, concentrated ammonia was added  to release the oligomers from the support and remove the base-labile protecting groups. The obtained trityl-on 2′-protected-RNAs were purified by RP HPLC and then treated with 10 mM aq. HCl , yielding ca. 10 OD of the final s4U-oligomer 42. It was found that the triazolyl derivatives (phosphoramidite building block and precursor oligomer 40) have a limited shelf life (<3 months), while the 4-O-(2-nitrophenyl)uridine derivatives appeared significantly more stable (shelf life > 3 years). In 2004, Avino and co-workers prepared side NaSH A 76]. T. T4U-RNA5, which was initially introduced by Shah and co-workers .,84.53a–bO-ACE , 2′-O-TBDMS , and 2′-O-TC (56) on 0.2 or 1 µmol scale. Iodo-oligomers were coupled with several tributylstannanes  such as vinyl-, furyl-, thienyl-, and benzothienyl(tributyl)-stannane. In contrast to Sonogashira post-synthetic cross-coupling, the Stille reaction was performed on column after the completion of automated RNA synthesis. The synthesizer column with fully assembled RNA was connected with the column-syringe system that loaded the solution of tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3, 15 equiv) and tri-2-furylphosphine (P(furyl)3, 30 equiv) in dry DMF and then a solution of alkylstannane (60 equiv) in DMF. The column with reaction solution was placed in the oven at 60 °C for 2–18 h. Then, the support-linked modified RNA (still closed in the synthesizer column) was washed, dried, and subjected to deprotection. The ACE-protected, polystyrene-bound oligomers were deprotected using the standard protocol. The TBDMS-protected, CPG-linked oligomers were treated with conc NH3/EtOH  to cleave the base-labile protecting groups and resin, and then, they were desilylated. The TC chemistry was tested exclusively for 5-iodouridine-RNA. The CPG-linked/TC-protected oligomer enclosed in the column was treated with 20% Et2NH/MeCN  to deprotect the phosphate groups. The residual blockage and CPG resin were cleaved with 50% solution of ethylenediamine in dry toluene , and finally, fully deprotected RNA oligomer was eluted from the column with 0.1 M TEAA buffer. All obtained oligomers 59a–d and 60a–d were purified by IE HPLC. In a few cases, the desired products of Stille cross-coupling reactions were accompanied by reduced deiodinated RNAs or methylated side products.The on-column concept was applied by the group of Engels to the post-synthetic C–C bond-forming reaction via palladium-mediated Stille cross-couplings  85]. Th. Th85]. Two types of post-synthetic cycloadditions were employed in the synthesis of nucleobase-modified oligoribonucleotides, Cu(I)-catalyzed cycloaddition of azides and terminal alkynes (CuAAC strategy) and inverse electron demand Diels-Alder cycloaddition (iEDDA).The CuAAC reaction , N-azidoacetyl-D-mannosamine (62b), 3′-azidothymidine , or azide of N-ethylpiperidine (62d). Click products 63a–d were generated by incubating an aqueous solution of RNA oligomer 61 with the water-soluble tris-triazol-4-yl)methyl]amine Cu(I)-stabilizing ligand (THPTA), CuSO4, and sodium ascorbate  species), and azide 62a–d (5 equiv) for 4 h at ambient temperature. Complete conversions were observed for primary azides 62b,62d; however, the use of secondary azides 62a,62c reduced conversion yields by 50%. Click products 63a–d were purified by denaturing PAGE and confirmed by ESI-MS. Azide 62b was also effectively employed for the post-synthetic preparation of the di-click 21-nt RNA oligomer. Thermal denaturation studies indicated that oligomers 63b–d showed similar base pairing stability and specificity to adenosine-containing counterparts. .,91.N2-mo64 was synthesized using standard phosphoramidite chemistry  with the coupling time prolonged to 25 min for a 7-ethynyl-modified monomeric unit . After deprotection, precursor oligomer 64 was incubated with aqueous solution of THPTA ligand, CuSO4, sodium ascorbate, and azide 65a–f for 6.5 h at rt or 35 °C. Azides were dissolved in aq. Tris-HCl buffer with pH 8.0 (65a), water (65b–d), or DMSO . Click products 66a–f were purified by denaturating PAGE and identified by MALDI-ToF MS. Study of the modification impact on RNA properties revealed a substantial destabilization of RNA duplexes modified with sterically demanding triazoles 66e and 66f. Oligomers with less hindrance (66a–d) indicated only a slight decrease of duplex stability and a little effect of modification on the base-pairing specificity.Further studies of the Beal group focused on the isertion of several 7-substituted 8-aza-7-deazaadenosines into 12-nt siRNA constructs by azide-alkyne cycloaddition  89]. 7-. 7-89]. 67 (prepared as synthetic RNA oligomers or yeast tRNAPhe in vitro transcript) were incubated with bifunctional azido-bromo-coumarin 68 in denaturating conditions  to provide products 69 with chemoselectively N3-alkylated uridine. The azidocoumarin-modified RNA 69 was subjected to click conjugation with fluorescent alkyne-containing AlexaFluor 647 (70a) or fluorescein (70b) in buffer of pH = 8, in the dark, using Cu+ (generated in situ from sodium ascorbate and Cu2+) with a THPTA ligand . Click products 71a–b were analyzed by PAGE electrophoresis.Kellner and co-workers employed click chemistry to label unmodified RNAs with fluorescent dyes  92]. Fo. Fo92]. O-DMTr-5-ethynyl-2′-deoxyuridine phosphoramidite  was effectively introduced to an RNA oligomer by conventional solid-phase synthesis. Using the “in-solution” approach, the fully deprotected oligomer 72 was conjugated with azide-functionalized nitroxide 73 with CuI instead of a Cu2+-ascorbate system  with the excess of THPTA in DMSO-H2O solution for 30 min at 37 °C. The spin-labeled oligomer was purified by HPLC to afford the product 74 in total 72% yield. The same oligomer spin labeling was demonstrated by Kerzhner and co-workers in solid phase amine (TBTA) for 1 h at rt. The RP HPLC purified product 77 was identified by MALDI-ToF MS.Recently, Nakamoto and co-workers have demonstrated the use of CuAAC reaction for the incorporation of biotin to oligoribonucleotide modified with a novel bifunctional nucleoside analog containing aryl trifluoromethyl diazirine and ethynyl moieties  93]. Pr. Pr93]. 78 with disubstituted 1,2,4,5-tetrazine-fluorophore derivatives 79a–d . Cycloaddition products were identified by PAGE and MS analysis. It was indicated that the overall yields of cycloaddition reactions were comparable regardless of norbornene position (the single- or double-stranded regions of RNAs). However, they were dependent on the steric effects of the disubstituted tetrazine derivatives with the RNA oligomer . A bioorthogonal reaction of iEDDA between tetrazine-fluorophore 79a and 21-nt norbornene-modified RNA 78 (prepared as a siRNA duplex) was effectively performed in mammalian cells. In contrast to already known techniques, the fluorophore can be attached to RNA after the target binding, which minimizes the distortion of RNA transport and the functions in the cells.Beyond the CuAAC cycloaddition, the inverse electron demand Diels-Alder reaction (iEDDA) was employed for the post-synthetic labeling of norbornene-containing RNA oligomer es 79a–d  94]. Th. Th78 wiN-hydroxysuccinimide (NHS), tetrafluorophenol (TFP), or N-hydroxybenzotriazole (HOBt). The active esters react selectively with an aliphatic amino function in solution, after the deprotection of RNA oligomers, without any risk of side reactions with 2′–OH and aromatic amino groups of nucleobases. Based on this strategy, efficient methods for the incorporation of fluorescent dyes, flavin mononucleotide derivative, as well as nitroxide and diazirine labels into RNA oligomers have been developed for their application in the structural and functional analysis of RNA molecules . Pr. Pr81a–cde (FAM) a–c. 81a–c and 82a,c the standard phosphoramidite chemistry was used with 5′-O-DMTr-2′-O-TBDMS-protected building blocks and CPG support . Oligomers were effectively deprotected  and purified by PAGE electrophoresis. To attach the fluorescent dyes, precursor oligomers 81a–c and 82a,c were dissolved in borax buffer at an approximate pH 9; then, freshly prepared solution of active TFP ester 83 (for Alexa488) or NHS ester 84 (for Cy5 and ATTO647N) in DMF was added. The labeling reactions were carried out at rt overnight in the dark. To conjugate the flavinmononucleotide derivative, the 5-aminoallyluridine-RNA 81a was treated with R4-NHS ester 84 generated in situ using TSTU condensing reagent and DIPEA in DMF. Oligomers containing Alexa488, Cy5, and flavin mononucleotide derivative were purified using denaturing PAGE (yields 15–54%), while ATTO647N-labeled oligomers were isolated by RP HPLC (yields 40–50%). The structures of target oligomers were confirmed by MALDI-ToF mass spectrometry.To prepare the amino-modified oligomers 87a , then Na–HEPES (pH 7.5) and a solution of diazirine NHS ester (>60 equiv) in DMSO were added. The reaction was vortexed and incubated at ambient temperature for 2–4 h in the dark. The mixture was purified by RP HPLC and analyzed by mass spectrometry. The developed transformation worked also for 3′-biotynylated 6-methyladenosine-modified amino RNA.Recently, post-synthetic transformations of amino-modified oligoribonucleotides to photo-crosslinkable diazirine-derivatives have been employed to investigate the regulation of protein-RNA interactions by 6-methyladenosine . For pre87a , (E)-5-(89) or C5 position of U (90). The precursor oligomers were prepared by the complementary addressed modification method (oligomer 89) . Am. Am69]. omer 89)  or by th6N-threonylcarbamoyladenosines containing a hydantoin ring  have been recently discovered at position 37 of anticodon loops in several prokaryotic and eukaryotic tRNA sequences .,64.N6-th5U) convertible nucleoside has been designed for preparing the 5-aminomethyluridine (R5U)-modified RNA oligomers 97a–e  and poor accessibility to oligomers containing these modifications . Th. Th5U) cications ,101. 5U to 5-aminomethyluridines . ThG-linked A or releG-linked B. The sy” method A, detritivatives . After d2→ges2 transformation was performed in solution  → P(V) oxidation step. s4U residue, the iodo-containing proxyl spin label was employed (4U-RNAs 104 (30- and 31-nt) were dissolved in 100 mM phosphate buffer (pH 8.0), treated with solution of 3-(2-iodoacetamido)proxyl 105 in H2O/EtOH (7:3 v/v), and incubated overnight at rt in the dark. S-Alkylated oligomers 106 were isolated (the NAP-10 column and ethanol precipitation) in 80% yields on average. Similar procedures of s4U-labeling were employed by others [In the studies of post-synthetic appending of nitroxide labels to the thiocarbonyl group of semployed A 112]. . 4U resiy others ,97,114 t4-thiocarbonyl function of s4U-RNA oligomers was achieved by applying the methanethiosulfonate spin label reagents 107a–c (4U-precursor RNAs 104 (23- and 57-nt) were incubated with dithiothreitol (DTT) reducing agent in 100 mM phosphate buffer (pH 8.0) for 30 min-2 h at rt. After the removal of DTT excess, the oligomers were treated with appropriate 1-oxyl-2,2,5,5-tetramethylpyrroline- methanethiosulfonates 107a–c in phosphate buffer overnight at rt to afford products 108a–c. Reagents 107a–c were found to be effective for the thioalkyl transfer to the sulfur of 4-thiouridine with formation of the disulfide tethered group. However, the labile nature of the disulfide bond requires the storage of labeled RNAs at −20 °C to minimize the nitroxide radical detachment. The fast attachment of nitroxide radical to the ss 107a–c B , wh, whS-prohemistry ,119. The acetate ,119 and 115 using Verdine convertible inosine nucleoside 6 (see 115 oligomer into full-length pre-mRNA molecules via the ligation procedure, the disulfide was reduced to thiol-RNA 117 using DTT. The RNA construct 117 was subjected to derivatization with highly effective thiol-specific benzophenone maleimide photoreagent in 50% aq. DMF for 1 h at rt.The thiol-adenine-containing oligomer homologous with the branched region of pre-mRNA was post-synthetically reacted with benzophenone reagent to indicate photo-crosslinks with proteins under splicing conditions . A two-sde 6 see , which pde 6 see . After t118 was incubated in HeLa nuclear extract under standard splicing conditions and crosslinked with proteins by irradiation with a 302 nm lamp.The obtained benzophenone-pre-mRNA Every year, new strategies are discovered to synthesize modified oligonucleotides. Nevertheless, there is still a growing demand for the development of new approaches, particularly toward RNA oligomers, the preparation of which is more challenging than their DNA counterparts. Unlike enzymatic procedures, the chemical synthesis of RNA oligomers offers the possibility of site-specific incorporation of modified nucleosides, making RNA synthesis an important tool for many interdisciplinary studies. The hereby presented concept of post-synthetic oligonucleotide modification represents an important alternative for standard phosphoramidite chemistry, particularly when target modifying groups/labels are unstable or reactive under conditions of solid-phase synthesis. The great advantage of post-synthetic reactions is that one building block with a reactive group can react with a wide variety of reagents, providing several variously modified oligomers of homologous sequences. In addition, reported precursor oligomers can be easily adapted to incorporate new modifications using already well-optimized post-synthetic reactions. Some of the post-synthetic transformations are bioorthogonal and have been successfully employed to study and monitor RNA nucleic acids in their native environment ,122,123.


Novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (coronavirus disease 2019 [COVID-19] pandemic) attacks the host cells modulating the local renin-angiotensin (RAS) system. Pending a definitive antivirus therapy, Fedson and colleagues propose in their recent article . All the1–1–There are, however, some intriguing characteristics blemishing the role of ACE2 and NEP. ACE2 is an ACE homologue, unaffected by ACEis. Unexpectedly, in the case of the pandemic, ACEis become the main gate of coronavirus entry into the host cells. ACEis, ARBs, and statins all overexpress ACE2 on the surfaces of infected cells, increasing the virulence . Althoug1–NEP has an equally complex role in the setting of HF patients infected by COVID-19. NEP catalyzes angiotensins to angiotensin 17 to a fa1–1–1–


Caenorhabditis elegans, the avoidance response to gentle body touch is mediated by six touch receptor neurons (TRNs), and is dependent on MEC-4, a DEG/ENaC channel. We show that hemichannels containing the innexin protein UNC-7 are also essential for gentle touch in the TRNs, as well as harsh touch in both the TRNs and the PVD nociceptors. UNC-7 and MEC-4 do not colocalize, suggesting that their roles in mechanosensory transduction are independent. Heterologous expression of unc-7 in touch-insensitive chemosensory neurons confers ectopic touch sensitivity, indicating a specific role for UNC-7 hemichannels in mechanosensation. The unc-7 touch defect can be rescued by the homologous mouse gene Panx1 gene, thus, innexin/pannexin proteins may play broadly conserved roles in neuronal mechanotransduction.Mechanosensation is central to a wide range of functions, including tactile and pain perception, hearing, proprioception, and control of blood pressure, but identifying the molecules underlying mechanotransduction has proved challenging. In  Like in vertebrates, where gap junctions are formed from unrelated proteins called connexins, each invertebrate gap junction consists of two innexin hemichannels, each of which is a hexamer of constituent subunits  in the relationships between partner cells  movement of ions and small signaling molecules between the cytoplasm of the connected cells, resulting in electrical coupling and the propagation of signals such as Casubunits . The inner cells .2+ concentration, or mechanical stimulation  or contralateral (i.e. the right side for ALML) stimuli . Howeverunc-7 and unc-9 in this electrical coupling, we re-examined the effects of innexin RNAi, distinguishing between ALM responses to ipsilateral and contralateral stimuli , while less than half of contralateral stimuli generated responses , suggestior TRNs . Thus, uunc-7 could rescue the unc-7 mechanosensory defect in touch neurons. As unc-7(e5) animals, as we observed previously for unc-7 RNAi. Expression of a wild type unc-7 cDNA  gentle touch response defect, indicating that the TRN defect is related to hemichannel rather than gap junction activity. Expression of mouse Panx1 (encoding Pannexin 1), but not Panx2, in the TRNs also successfully rescued the unc-7 mechanosensory defect  and unc-7(e5) (1.82 ± 0.32). Together, these results indicate that loss of UNC-7 does not affect touch neuron excitability, and its role is likely to be specific to mechanosensation. unc-7 is expressed in other sensory neurons, including the polymodal nociceptor PVD. PVD neurons respond to several aversive stimuli, including harsh touch and cold temperature  animals were severely defective in the Ca2+ response of the PVD neurons to harsh touch. In contrast (unc-7(e5) animals showed no significant difference compared to wild-type in the PVD response to cold (temperature shift from 22° to 15°). Thus, unc-7 is required for mechanosensory responses, but dispensable for thermosensory responses, in PVD, suggesting a specific role for UNC-7 in mechanotransduction.In principle, UNC-7 could affect mechanosensory responses by affecting the excitability of the touch neurons; alternatively, UNC-7 could play a direct role in mechanosensation. To address these possibilities, we examined the effect of mulation . To assemulation  suggestiperature . To examobserved  that unccontrast , unc-7 slower and longer-lasting than the responses observed for gentle touch stimuli  mutations eliminated virtually all TRN responses to harsh touch. Expression of cysless unc-7 in the TRNs significantly rescued the harsh touch response defect in unc-7 mutant animals, indicating that the loss of both transient and prolonged responses in the mutant was at least partially due to the cell-autonomous mechanosensory activity of UNC-7 hemichannels. unc-7 knockdown in combination with mec-4 null abolished both types of response, consistent with the hypothesis that UNC-7 and MEC-4 act at least partially independently in the touch neurons, with UNC-7 but not MEC-4 particularly important for prolonged responses to harsh touch.The TRNs also exhibit responses to fast, high-displacement stimuli (‘harsh touch’) that are distinct from those seen in response to low-displacement press or buzz stimuli (‘gentle touch’). Harsh touch responses are  stimuli . Indeed, stimuli . As obse stimuli  mec-4 muild-type , althougesponses , while umec-4(u253) animals (mec-4(u253) animals expressing unc-7 dsRNA in the TRNs. As expected, we observed  and unc-7 RNAi did not eliminate this response; indeed, the phenotype resembled mec-4(u253) alone. Likewise, while disruption of mec-4 or unc-7 alone results in a very substantial decrease in the magnitude of the gentle touch response  defect in the behavioural response to gentle touch. This suppression was independent of mec-10, the other DEG/ENaC known to function in the TRNs. Interestingly, unc-7 overexpression only restored touch responses to stimuli applied near the head . This suggests that UNC-7 overexpression only partially compensates for loss of MEC-4, leading only to local depolarization of the ALM axon. Such activation might be insufficient to generate calcium transients in the ALM cell body, and only sufficient to activate downstream neurons and thus induce a behavioural response when triggered in presynaptic regions near the nerve ring. Conversely, we also tested whether overexpression of MEC-4 could compensate for the absence of UNC-7. We observed  in response to either anterior or midbody touch stimuli. Thus, although both mec-4 and unc-7 are essential for the response to gentle touch in the TRNs, both can, at least to some extent, substitute for the other when overexpressed. This suggests that MEC-4 and UNC-7 indeed act independently as mechanotransducers in the TRNs.Like response . We reasthe TRNs  we obserhead a3, , and we observed  that wheunc-7 in the ASKs or the ASJs, two pairs of ciliated amphid neurons that do not respond to mechanical stimulation  animals, indicating that UNC-7 is not an essential component of this particular current . It is unclear how the pneumatic pressure stimulus used in these studies relates to externally-applied gentle touch, and one possibility is that different mechanically sensitive innexins function over different sensitivity ranges.Although these results are the first to implicate UNC-7 as a mechanosensory molecule, other results are consistent with a role for innexins in recently  that theunc-7 and mec-4 single mutants both are insensitive to gentle touch, overexpression of mec-4 can suppress the unc-7 defect, while unc-7 overexpression can partially suppress the gentle touch defect of mec-4. Third, mec-4 and unc-7 mutations have distinct phenotypes with respect to harsh touch responses in the TRNs; unc-7 affects and is required for large, long-lasting mec-4 independent responses, whereas mec-4 is required for small, transient responses that resemble responses to gentle touch. Thus, although the activities of both UNC-7 and MEC-4 appear to be essential for sensitivity to weaker stimuli, they function at least somewhat redundantly in the response to stronger stimuli such as harsh touch.Unexpectedly, we have found that two ion channels, UNC-7 and MEC-4, are both required for normal touch responses in the TRNs; loss-of-function of either UNC-7 or MEC-4 alone leads to significant touch insensitivity. Nonetheless, several lines of evidence suggest that UNC-7 and MEC-4 function independently in the touch neurons, rather than functioning together in a common mechanotansduction complex. First, although both UNC-7 and MEC-4 proteins are distributed in a punctate pattern along the TRN dendrite, UNC-7- and MEC-4-containing puncta do not colocalize, and therefore appear to represent physically-distinct complexes. Second, although What does this imply about the function of UNC-7 in mechanosensation? One possibility is that UNC-7 and MEC-4 are each mechanically-sensitive channels that respond to qualitatively distinct types of mechanical stimulation. MEC-4, for example, is believed to be tethered to both the extracellular matrix and the cytoskeleton , whereasmec-4 gentle touch phenotype by sensitizing or enhancing the mec-4-independent harsh touch mechanotransducer in the TRNs. Interestingly, UNC-7 appears to be necessary for the slow and prolonged time course typically observed in harsh touch responses; thus, perhaps this property results from prolonged opening of UNC-7 channels following the stimulus.Alternatively, UNC-7 might not itself be a mechanosensitive channel, but rather might modulate touch neuron responses by amplifying mechanoreceptor potentials or locally enhancing excitability. This accessory function might be essential to enhance MEC-4-dependent gentle touch responses, but partially dispensable, at least in the presence of MEC-4, for responses to stronger harsh touch stimuli. According to this model, UNC-7 overexpression might suppress the unc-7 mechanosensory phenotypes likewise imply expression in both ALM and AVM. Thus, for the ALM-AVM gap junctions, it seems likely that UNC-7 and UNC-9 contribute in both partner cells. Unlike UNC-7, the role of UNC-9 appears to be confined to gap junctions, since unc-9 mutations (like AVM ablations) only affect responses to contralateral stimuli. It is interesting that an analogous cooperative organisation (i.e. connection of PLML and PLMR via PVM) does not exist for the posterior TRNs, and our hypothesis that this results in a functionally distinct network is corroborated by our demonstration that PLM responses are significantly less robust when the neurons are far from the stimulation site. The difference in organisation presumably reflects the dominance of forward locomotion, and thus the greater selective pressure on the avoidance of anterior aversive stimuli.In addition to its role in mechanosensation, UNC-7, along with UNC-9, also contributes to gap junctions that functionally link the anterior TRNs. In the locomotion circuit, for example in the electrical synapses between the AVB premotor interneurons and the B-class motorneurons, UNC-7 and UNC-9-containing gap junctions appear to be asymmetric, with UNC-7 expressed in AVB and UNC-9 expressed in B motor neurons . LikewisAn important feature of any sensorimotor circuit must be to ensure that a given stimulus elicits an appropriate behavioural response. Encountering an innocuous obstacle requires a distinct response (brushing against it or a steering change) compared to a noxious stimulus (a rapid avoidance movement). We have seen previously in the nose touch circuit that electrical connections between sensory neurons can play an important role; when OLQ and CEP are activated by gentle nose touch, they facilitate gentle touch responses in FLP, which otherwise only responds to harsh touch, whereas if OLQ and CEP are inactive, they inhibit the activity of FLP through shunting . Since tunc-7(e5), the shorter SR isoform fails to rescue. Conversely, others  remain controversial . Our obsStrains used in this study are described in Plasmid constructs. Pmec-7::dsRNA plasmids for unc-7, unc-9, inx-7 were constructed by ligating a cDNA fragment of approximately 600 bp between the second and third multiple cloning sites of pPD117.01 (A gift from Andrew Fire). The complementary sequences used in the primers were as follows: unc-7: gttgctacgtcactatgctc and agtctatcgtcccttgaccg; unc-9: atgctattgtattatttcgcg and agtcgttgagaacttgcagtc; inx-7: tcgtgtcttaaacactgttcc and agaatcttgtgtggaactatc. An E. coli Cat1 (chloramphenicol acetyl transferase gene) dsRNA plasmid was constructed in a similar fashion. For each target, two plasmids, with sense and antisense orientations of the insert, were co-injected at 50 ng/µl each. Pnmr-1::dsRNA plasmids were constructed by replacing Pmec-7 in these plasmids with Pnmr-1 (1.7 kb). unc-7 rescue plasmids were made using the Multisite Gateway 3-Fragment Vector Construction Kit (Invitrogen). A 1078 bp mec-4 promoter fragment  cDNA were amplified from RB1 cDNA library (a gift from Robert Barstead) and cloned into pDONR 221. These were combined with pDONR P2R-P3/SL2::mcherry (a gift from Mario de Bono) in a derivative of pDEST R4-R3 into which unc-54 3’UTR had been inserted downstream of the recombination sites. Cysteine to arginine substitutions were made in the relevant pDONR 221 plasmid, using codon-optimised mutagenic primers, designed using C. elegans Codon Adapter . Thermal stimulation and nose touch stimulation were performed in CTX . Where appropriate, neurons were categorised into ipsilateral or contralateral, depending on the position of their cell body with respect to the site of stimulation and a hypothetical midline.Caeviously , using aeviously . Images eviously . Illumintrans retinal in ethanol and incubating overnight at 22°C), and grown at 22°C, then assayed as day one adults. Assay plates were prepared by seeding 30 mm NGM plates with 40 µl of a 1000:1 mixture of OP50 culture and 100 mM all-trans retinal in ethanol and incubating for 30 min at 22°C. Control plates contained ethanol without all-trans retinal. Animals were picked individually to assay plates using an eyelash hair, and acclimatised for 15 min. To stimulate the TRNs, lite-1(ce319) worms expressing Pmec-4::ChR2  for ImageJ. Object-based colocalisation of puncta was analysed using the JACoP plugin  for Imag In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:C. elegans dedicates a remarkable proportion of its nerve cells (at least ~10%) to perceiving such mechanical stimuli. Worms exhibit conserved molecular machineries for mechanosensation, like channel complexes of the DEG/ENaC and TRP families. Vertebrates also employ hemichannels of the pannexin family for mechanosensation; their invertebrate relatives, the innexins, however, were thought to act primarily as gap junctions. Here, Walker and Schafer show that the major innexins in C. elegans, termed UNC-7 and UNC-9, might serve indeed such dual roles. They report evidence suggesting that UNC-9 is involved in electrical coupling in a network of a few touch receptor neurons (TRNs), while UNC-7 also exhibits the role as a mechanosensitive hemichannel in TRNs. Strikingly, the unc-7 gene can be functionally substituted with the vertebrate pannexin gene Panx1, suggesting a common mechanism for mechanosensation shared between innexins and pannexins.Mechanosensation mediating touch- pain- or proprioceptive sense is essential for animals. The nematode Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Ronald Calabrese as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Mei Zhen (Reviewer #2).Thank you for submitting your article "Distinct roles for innexin gap junctions and hemichannels in mechanosensation" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:unc-7 gene in C. elegans mechanosensation. Based on their results they propose that unc-7 might serve as a hemichannel directly involved in primary sensory transduction, either alone or in complex with other yet un-identified components. In their model, innexin genes serve two roles in anterior touch receptive neurons (TRNs). (1) integration of bilaterally received stimuli via a gap junction network of ALMR-AVM-ALML, and (2) direct mechanosensation via unc-7 hemichannels. They collect support for this model via tissue specific dsRNAi and transgenic rescue experiments. Their model finds the strongest support in their finding that a mutant unc-7 variant, previously shown incapable of electrical coupling ("cysless unc-7"), can rescue some of the mechanosensory defects of unc-7 null mutants. Moreover, unc-7 is not required for TRN excitability via ChR2 activation suggesting specificity. Ectopic expression of unc-7 renders ASK neurons mechanosensitive, suggesting sufficiency in the ASK context. Interestingly, unc-7 function can be rescued with a mammalian pannexin1 gene (there is some evidence in the literature that pannexin1 might function as a stretch sensitive hemichannel). Direct electrophysiological and biophysical evidence that UNC-7 is indeed a hemichannel or mechanosensitive is missing in this study, therefore in the evidence is indirect. However, in total many lines of indirect come together to support the major claims. The results are surprising, considering that unc-7 expression encompasses a large portion of the worm's nervous system and that it was typically seen as a major gap junction gene. The work has impact on the interpretation of previous studies, assuming a sole role for unc-7 in electrical coupling. Moreover, it paves way for interesting future work studying how unc-7 exactly confers mechanosensitivity.In this manuscript Walker and Shafer study a potential role of the innexin Essential revisions:unc-7 seems not to be expressed in AVM and ALM in adult animals. This is confusing, and the authors ignore this recent study, not even citing it. Since the authors' conclusions strongly depend on endogenous unc-7 expression in anterior TRNs and since this seems controversial now it is necessary to validate expression of unc-7 in AVM and ALM in adult animals using proper reporter constructs, showing that they overlap with a well characterized TRN marker.1) Reviewer 1. The authors rely on previous gene expression studies , however, these data have been outdated by a recent state of the art expression study using fosmid reporters . Here, unc-7 and unc-9 are expressed in many neurons of the worm and ALM and AVM form gap junctions with other neurons, therefore non-cell autonomous effects of their gene knock-downs need to be controlled for. The authors apply a cell specific dsRNAi technique. dsRNAi, however, potentially could spread from cell-to-cell . Since the author's interpretations strongly depend on the assumption of TRN-specific knockdown, additional controls are needed. The most straight forward control would be to rescue the RNAi phenotype with a codon altered RNAi insensitive transgene in TRNs.2) Reviewer 1. unc-7 or unc-9 mutants.2b) Reviewer 2. The key evidence here was that RNAi against UNC-9 in TRN showed modest or no change in the calcium response of ALM and PLM, in contrast to the drastic effect of RNAi against UNC-7. My concern is the lack of information or control experiments that demonstrate the specificity of UNC-7 RNAi, and the robustness of UNC-9 RNAi. Both were driven by the same promoter. There is sequence similarity between the two cDNAs. It was unclear which 600bp sequences were selected for each innexin to address their specificity and efficiency. One useful control would be to compare the effect of both RNAi driven pan-neuronally, on how effective they are to mimic the phenotypes of unc-9 and unc-7 mutants. They argue that unc-9 mutation has a specific effect on contralateral responses while unc-7 affects both, ipsilateral as well as contralateral responses. They conclude right away that unc-9 is mainly involved in gap junction coupling and unc-7 additionally in primary mechano-transduction. Looking at the traces in 2A and scatter graphs in 2B, these statements are somewhat on weak grounds. All manipulations in 2B seem to follow the same trend, especially when examining the scatter graphs. The authors rely solely in their p-values obtained, but to be more convincing simply more repetitions are needed. In any case, it is possible that unc-7 and unc-9 function in a very similar manner, simply dsRNAi could be differentially effective. Without controlling that both are complete knockdowns, the authors should be more careful with concluding differential roles for unc-7 vs. unc-9.3) Reviewer 1. I find the AVM requirement for contralateral ALM responses quite interesting; this important result goes a bit under in the Discussion. However, in the remaining experiments shown in Figure 2, the authors claim a differential effect in unc-7 mutants, all touch response was abolished, including the response to harsh touch by all TRNs and PVD. MEC-4 specifically affects gentle touch and is the proposed gentle touch sensor. It is difficult to envision why overexpression of MEC-4 could robustly bypass the requirement of UNC-7, especially in the harsh touch response (and I am not sure if authors addressed this directly). A simpler interpretation of these results is that the expression of cation leak channels (at ER or plasma membranes) changes a neuron's intrinsic property. If authors have attempted to address if MEC-4 can specifically rescue the harsh touch-mediated response in PVD and ALM in the unc-7 mutants. MEC-4 is not responsible for harsh-touch mediated . If MEC-4 overexpression still rescues, then it is more likely that the cation leak non-specifically changed PVD's sensitivity to mechanical stimulation.4) Reviewer 2. I found some results here hard to interpret. In 5) Reviewer 2. Authors should address the impact of a recent paper , which suggest that both UNC-7 and UNC-9 are endogenously expressed in ASK, on the interpretation of this experiment. Calcium-imaging results from this experiment appeared highly variable . An overexpression of UNC-7 may alter ASK's property, and its network property . The authors may want to select another neuron that does not have UNC-7 expression to test their hypothesis again.unc-7 acts independently of mec-4, why isn't there any evidence of mechanoreceptor current in the absence of mec-4 in electrophysciological recordings . Also, no calcium transients were observed in mec-4 null mutants in other works , even with Ca indicators superior to the one used in this present study. A potential explanation would be that unc-7 is only able to generate local transients and thus remain undetectable for stimuli far away from the cell body, however, in previous studies neither were current or transients recorded after stimuli close to the cell body.6) Reviewer 3. Please discuss a potential for controversy with previously published data: one of the major questions that remained unanswered, and to my surprise aren't even discussed, is the following: If mec-7p is not specific to touch receptor neuron but also expressed in head neurons and probably PVD. Thus, the statement of cell-specificity needs to be toned down and concerns arise that the observed effects are due to defect in inx of other neurons. The authors addressed this in parts by using a mec-4p construct already.7) Reviewer 3. mec-4 under the presented experimental conditions for gentle touch? How do the authors distinguish between these two modalities – any quantitation would help to reconcile their conclusion.8) Reviewer 3. How often has the same animal been tested? Are there still calcium signals in animals lacking mec-4 still causes a response to gentle touch  but seems to habituate after the first or second touch. This is puzzling as no mechanoreceptor currents could be measured using electrophysiology or visualized in dendrites with calcium reporter. The authors also report behavioral response in the mec-4 mutation, consistent with a function of another channel in this response – did they try the mec-4/unc-7 double mutation to see if the animals did not respond at all?9) Reviewer 3. In previously published data, deletion of  Essential revisions:1) Reviewer 1. The authors rely on previous gene expression studies , however, these data have been outdated by a recent state of the art expression study using fosmid reporters . Here, unc-7 seems not to be expressed in AVM and ALM in adult animals. This is confusing, and the authors ignore this recent study, not even citing it. Since the authors' conclusions strongly depend on endogenous unc-7 expression in anterior TRNs and since this seems controversial now it is necessary to validate expression of unc-7 in AVM and ALM in adult animals using proper reporter constructs, showing that they overlap with a well characterized TRN marker.unc-9 was shown to be expressed in the anterior touch neurons in the Altun et al., 2009 paper (which used short promoter fusions). The expression of unc-7 in anterior touch neurons  was reported in Starich et al., which used a cosmid-based reporter which  contained even more upstream sequence than the fosmid-based construct used by Bhattacharya. The expression pattern was specifically shown in that paper to mirror the staining pattern of an anti-unc-7 antibody. Bhattacharya also did not report expression in some other neurons that expressed long form-specific reporters in the Starich manuscript. Thus, we suspect that the Bhattacharya construct lacks some upstream elements necessary for expression of the long form of unc-7. Single cell transcriptional profiling  also provides evidence that unc-7 is expressed in the TRNs. We note that the fact that touch neuron-specific RNAi experiments (but not off-target controls) phenocopy the unc-7 loss-of-function mutant, which also implies that unc-7 is endogenously expressed in touch neurons. It is interesting that according to the Starich paper PVD also expresses only the long-form of unc-7, which we show to be the only isoform of unc-7 rescues mechanosensory phenotypes.We apologize for the confusion here; in fact only We have fixed the citations in the Introduction (fourth paragraph) and added a discussion of the expression patterns of various isoforms and reporters (including the results of Bhattacharya) and their relation to mechanosensory activity .2) Reviewer 1. unc-7 and unc-9 are expressed in many neurons of the worm and ALM and AVM form gap junctions with other neurons, therefore non-cell autonomous effects of their gene knock-downs need to be controlled for. The authors apply a cell specific dsRNAi technique. dsRNAi, however, potentially could spread from cell-to-cell . Since the author's interpretations strongly depend on the assumption of TRN-specific knockdown, additional controls are needed. The most straight forward control would be to rescue the RNAi phenotype with a codon altered RNAi insensitive transgene in TRNs.unc-7 RNA in other neuron types (premotor neurons) does not lead to touch phenotypes. This is consistent with our expectations since the SID complex required for intracellular uptake of small RNAs  is not expressed in neurons , and other investigators have found RNAi to act cell-specifically within the nervous system .We have added off-target RNAi controls  showing that expression of double-stranded unc-7(e5) loss-of-function mutant. We have expanded the text  to make it clearer that we have used Pmec-7 for RNAi and the more specific Pmec-4 for the rescue experiments. We have also edited the description of the initial RNAi results to avoid giving the impression that we are claiming this is evidence of a cell-autonomous role in the TRNs (subsection “The mechanosensory function of UNC-7 is gap junction-independent”).We note also that the cell-specificity of the mechanosensory phenotypes in ALM  and PVD  were also shown by cell-specific rescue of the 2b) Reviewer 2. The key evidence here was that RNAi against UNC-9 in TRN showed modest or no change in the calcium response of ALM and PLM, in contrast to the drastic effect of RNAi against UNC-7. My concern is the lack of information or control experiments that demonstrate the specificity of UNC-7 RNAi, and the robustness of UNC-9 RNAi. Both were driven by the same promoter. There is sequence similarity between the two cDNAs. It was unclear which 600bp sequences were selected for each innexin to address their specificity and efficiency. One useful control would be to compare the effect of both RNAi driven pan-neuronally, on how effective they are to mimic the phenotypes of unc-7 or unc-9 mutants.unc-9 loss-of-function mutant; as with the unc-9 RNAi line, these animals showed no abnormality in touch response per se, but a defect coupling between the ALM lateral pair . We have included the primer sequences in the Materials and methods (subsection “Plasmid constructs”). Contiguous sequence conservation between unc-7 and unc-9 is not substantial; for the unc-7 construct, the longest contiguous stretch of conserved bases is 9, for unc-9 it is 17.To address this question, we have included imaging experiments on the 3) Reviewer 1. I find the AVM requirement for contralateral ALM responses quite interesting; this important result goes a bit under in the Discussion. However, in the remaining experiments shown in Figure 2, the authors claim a differential effect in unc-9 and unc-7 mutants. They argue that unc-9 mutation has a specific effect on contralateral responses while unc-7 affects both, ipsilateral as well as contralateral responses. They conclude right away that unc-9 is mainly involved in gap junction coupling and unc-7 additionally in primary mechano-transduction. Looking at the traces in 2A and scatter graphs in 2B, these statements are somewhat on weak grounds. All manipulations in 2B seem to follow the same trend, especially when examining the scatter graphs. The authors rely solely in their p-values obtained, but to be more convincing simply more repetitions are needed. In any case, it is possible that unc-7 and unc-9 function in a very similar manner, simply dsRNAi could be differentially effective. Without controlling that both are complete knockdowns, the authors should be more careful with concluding differential roles for unc-7 vs. unc-9.unc-7 and unc-9 null mutants. As outlined in the response to reviewer 2, comment 2b above, the unc-9(e101) mutant  shows the same phenotype as unc-9 RNAi. We confined our statistical comparisons to the proportion responding because we were concerned that disruption of gap junctions could have confounding effects (i.e. a lack of shunting through gap junctions might result in an increase in amplitude) which could mask the low proportion of responders.The possibility that differential RNAi efficacy could explain the differences between ipsilateral and contralateral responses has been addressed by comparing the phenotypes of the unc-9 in touch neuron gap junction connectivity has indeed been somewhat ignored in the Discussion! We have inserted an additional section (subsection “Coordination of the anterior TRNs via gap junctions”) and reorganised the section headings to address this.The role of 4) Reviewer 2. I found some results here hard to interpret. In unc-7 mutants, all touch response was abolished, including the response to harsh touch by all TRNs and PVD. MEC-4 specifically affects gentle touch and is the proposed gentle touch sensor. It is difficult to envision why overexpression of MEC-4 could robustly bypass the requirement of UNC-7, especially in the harsh touch response (and I am not sure if authors addressed this directly). A simpler interpretation of these results is that the expression of cation leak channels (at ER or plasma membranes) changes a neuron's intrinsic property. If authors have attempted to address if MEC-4 can specifically rescue the harsh touch-mediated response in PVD and ALM in the unc-7 mutants. MEC-4 is not responsible for harsh-touch mediated . If MEC-4 overexpression still rescues, then it is more likely that the cation leak non-specifically changed PVD's sensitivity to mechanical stimulation.mec-4 overexpression does not rescue the harsh touch defect of the unc-7 mutant. We have not included these data in the figure since the reciprocal experiment (overexpression of UNC-7 in a mec-4 mutant) would not be meaningful given the robust harsh touch response of mec-4 single mutants, but in total only 14.3% responded, all with a low amplitude “transient” response, N=14. We have not tried this experiment in PVD because neither MEC-4 nor many of its required cofactors (e.g. MEC-9) are natively expressed there and it would therefore be difficult to interpret the results.This is an interesting point. We have tried the experiment suggested by the reviewer in the TRNs and found that mec-4 overexpression results do not argue conclusively that UNC-7 itself is a MEC-4-independent mechanotransduction channel. In the revision, we have modified the text in many places to clarify that UNC-7 hemichannels might play other roles in touch sensing, including as a channel that alters local excitability as suggested by the reviewer. We have particularly addressed this with regard to the overexpression experiments in the subsection “Coordination of the anterior TRNs via gap junctions”).We agree that the 5) Reviewer 2. Authors should address the impact of a recent paper , which suggest that both UNC-7 and UNC-9 are endogenously expressed in ASK, on the interpretation of this experiment. Calcium-imaging results from this experiment appeared highly variable . An overexpression of UNC-7 may alter ASK's property, and its network property . The authors may want to select another neuron that does not have UNC-7 expression to test their hypothesis again.unc-7 expression in the Bhattacharya paper. We did observe small but significant nose touch responses in the unc-7-expressing lines that were absent in control animals. These results have been added to revised Figure 7. Since these responses were small and therefore not inconsistent with other interpretations, we have added a caveat in the subsection “Heterologous expression of UNC-7 hemichannels in olfactory neurons confers touch sensitivity”. As we have shown that at least one of the shorter unc-7 isoforms does not confer mechanosensitivity, expression of such an isoform in ASK may explain the native touch-insensitivity of ASK neurons .To address this point, we expressed UNC-7 in the ASJ neurons, one of the few chemosensory neurons that show no evidence of 6) Reviewer 3. Please discuss a potential for controversy with previously published data: one of the major questions that remained unanswered, and to my surprise aren't even discussed, is the following: If unc-7 acts independently of mec-4, why isn't there any evidence of mechanoreceptor current in the absence of mec-4 in electrophysciological recordings . Also, no calcium transients were observed in mec-4 null mutants in other works , even with Ca indicators superior to the one used in this present study. A potential explanation would be that unc-7 is only able to generate local transients and thus remain undetectable for stimuli far away from the cell body, however, in previous studies neither were current or transients recorded after stimuli close to the cell body.unc-7 mutants, UNC-7 overexpression did not restore cell body calcium transients to mec-4 null mutants and only rescued the behavioural phenotype for stimuli applied near the animal’s neck. As suggested by the reviewer, a simple explanation of this is that UNC-7 overexpression only allows local depolarization of the ALM dendrite and that MEC-4 is necessary for cell-body responses to gentle touch. We have modified the text to make this point more explicitly (subsection “UNC-7 and MEC-4 act independently in touch neuron mechanosensation”f).This is a good point, and conforms quite closely to our interpretation of the results in Figure 6. Specifically, while MEC-4 overexpression was able to compensate for the cell body calcium imaging as well as the behavioural phenotype of mec-4 null mutants appear to lack mechanosensory responses to gentle touch in ALM, both our lab  and Hang Lu’s lab  have observed robust responses to stronger mechanical stimuli in mec-4 null animals .  So if unc-7 encodes either another mechanosensor or a specific amplifier of mechanotransduction currents, its overexpression could potentially enhance sensitivity of those harsh touch responses. We have added a comment to this effect to the text (subsection “UNC-7 and MEC-4 function independently in touch neurons”).We note that while 7) Reviewer 3. mec-7p is not specific to touch receptor neuron but also expressed in head neurons and probably PVD. Thus, the statement of cell-specificity needs to be toned down and concerns arise that the observed effects are due to defect in inx of other neurons. The authors addressed this in parts by using a mec-4p construct already.mec-7 promoter in initial RNAi experiments because it is a very strong promoter that expresses in touch neurons, though as the reviewer notes it is less selective than mec-4. We have clarified this in the text . With regard to the cell autonomy of the unc-7 mechanosensory phenotypes, as noted in the response to reviewer 1 comment 2 we have confirmed all the key experiments using an unc-7 loss-of-function mutant rescued using the mec-4 promoter.We used the 8) Reviewer 3. How often has the same animal been tested? Are there still calcium signals in animals lacking mec-4 under the presented experimental conditions for gentle touch? How do the authors distinguish between these two modalities – any quantitation would help to reconcile their conclusion.Care was taken that animals were not repeatedly stimulated; where they were stimulated more than once, a wait ensured that we avoided habituation effects. The protocols for gentle and harsh body touch are now explained in the Materials and methods section  (subsection “Plasmid constructs”).mec-4 null animals following gentle touch stimulation .We have included calcium imaging for gentle touch for strains used in Figure 5, in Figure 5—figure supplement 4. In agreement with previous reports, we observe only occasional calcium responses in 9) Reviewer 3. In previously published data, deletion of mec-4 still causes a response to gentle touch  but seems to habituate after the first or second touch. This is puzzling as no mechanoreceptor currents could be measured using electrophysiology or visualized in dendrites with calcium reporter. The authors also report behavioral response in the mec-4 mutation, consistent with a function of another channel in this response – did they try the mec-4/unc-7 double mutation to see if the animals did not respond at all?mec-4 mutant , We carried out the experiment suggested by the reviewer, assaying the gentle touch avoidance response in mec-4 null mutant animals in which unc-7 was knocked down by RNAi. We observed that these mec-4/unc-7 doubly-deficient animals still responded to gentle touch at frequencies similar to the mec-4-single mutant, indicating that the MEC-4-independent behavioural response is not mediated by UNC-7; this suggests a role for another neuron class in this behaviour. We have presented behavioural data in Figure 5—figure supplement 5 and discussed this in the Results section .This is a really interesting point. In common with these previous reports, we also see a residual behavioural response in the


FurNAD-RNA) as a new research tool for the identification and characterization of novel NAD-RNA decapping enzymes. These compounds are found to be suitable for various enzymatic reactions that result in the release of a fluorescence quencher, either nicotinamide (NAM) or nicotinamide mononucleotide (NMN), from the RNA which causes a fluorescence turn-on. FurNAD-RNAs allow for real-time quantification of decapping activity, parallelization, high-throughput screening and identification of novel decapping enzymes in vitro. Using FurNAD-RNAs, we discovered that the eukaryotic glycohydrolase CD38 processes NAD-capped RNA in vitro into ADP-ribose-modified-RNA and nicotinamide and therefore might act as a decapping enzyme in vivo. The existence of multiple pathways suggests that the decapping of NAD-RNA is an important and regulated process in eukaryotes.The complexity of the transcriptome is governed by the intricate interplay of transcription, RNA processing, translocation, and decay. In eukaryotes, the removal of the 5’-RNA cap is essential for the initiation of RNA degradation. In addition to the canonical 5’-N7-methyl guanosine cap in eukaryotes, the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD) was identified as a new 5’-RNA cap structure in prokaryotic and eukaryotic organisms. So far, two classes of NAD-RNA decapping enzymes have been identified, namely Nudix enzymes that liberate nicotinamide mononucleotide (NMN) and DXO-enzymes that remove the entire NAD cap. Herein, we introduce 8-(furan-2-yl)-substituted NAD-capped-RNA ( In eukaryotes, the removal of the 5’-cap is essential for 5’ to 3’ RNA decay. RNA capping is considered to be a hallmark of eukaryotic gene expression, in which a canonical 5’-N7-methyl guanosine cap . The discovery of NAD-capped RNA [E. coli provided an unexpected link between redox biology, metabolism, and RNA processing [In 2009, the ubiquitous redox coenzyme nicotinamide adenine dinucleotide (NAD) was discovered as a new RNA modification in bacteria [hia coli . NAD washia coli ,4. To idhia coli . Using tpped RNA  in the bocessing ,7. Saccharomyces cerevisiae, and in the model plant Arabidopsis thaliana [Initially assumed to be prokaryote-specific ,8,9, thithaliana ,11,12,13thaliana . While sthaliana , the quethaliana .7G cap and thereby trigger different RNA decay pathways [Canonical capping and decapping are highly regulated processes, until recently thought to be exclusive for eukaryotes. Multiple decapping enzymes, such as Dcp2  or DXO/Rpathways . E. coli, we identified the Nudix NADH-pyrophosphohydrolase NudC as the first bacterial decapping enzyme, which converts 5’-NAD-RNA into 5’-P-RNA, thereby triggering RNase E-mediated RNA decay [Bacillus subtilis, the Nudix hydrolase BsRppH was found to perform the same reaction [7G-cap—the NAD modification. In contrast to the Nudix hydrolases, the DXO enzymes cut off the complete NAD cap including the adenosine, generating a 5’-P-RNA shortened by one nucleotide [The discovery of NAD-capped RNAs in bacteria and eukaryotes laid the foundation for the discovery of decapping enzymes that are able to remove these caps. In NA decay . In Gramreaction . Both Nucleotide ,21. In consideration of the variety of mammalian proteins that show decapping activity on canonically capped RNAs , we assu32P-radioactively labelled capped RNA substrates [The discovery and characterization of decapping enzymes has so far been time-consuming and laborious. Most commonly, bstrates  are addebstrates ,24 or thbstrates ,25. Altebstrates ,27. All Thus, the field would benefit from a robust, fluorescent real-time assay that i) reports immediately the removal of the NAD-cap, ii) can be parallelized in a high-throughput manner using standard laboratory equipment, and iii) enables the discovery as well as characterization of NAD-RNA decapping enzymes.FurNAD-RNA). The design of the assay is shown in FurNAD-RNA, which is generated either by solid-phase oligonucleotide synthesis (SPOS) or in vitro transcription, is used as a substrate for putative decapping enzymes. In its intact form, the nicotinamide moiety efficiently quenches the fluorescence of the 8-(furan-2-yl)-adenosine. If an enzyme removes the quencher, a fluorescence increase can be detected in a standard cuvette photometer or a fluorescence microplate reader in real-time. In addition to characterizing several known NAD-RNA decapping enzymes, we report here the discovery of a novel decapping enzyme and chemistry, namely the removal of nicotinamide (NAM) by the human glycohydrolase CD38. Here, we report the development of a highly sensitive, fluorescent system to identify novel NAD-RNA decapping enzymes based on a 8-(furan-2-yl)-NAD-RNA  and used without further purification. Synthetic RNAs were purified by reversed-phase HPLC and their identities confirmed by MALDI-MS (oligonucleotides) or ESI-MS (initiator dinucleotides). DNA oligonucleotides (IVT templates) were purchased from Integrated DNA Technologies .2a, 2b) was performed in dry DMF at room temperature in the presence of excess MgCl2. Experimental details are highlighted in the The 8-substituted adenosine monophosphates were synthesized based on published protocols ,29 with 9) is described in the Oligonucleotides were synthesized on an ExpediteTM 8909 automated synthesizer  using standard reagents from Merck  in a 1 µM scale. The synthesis of 8-(furan-2-yl)-adenosine-3’-phosphoramidite  was coupled as the last nucleobase to the still immobilized, fully protected RNA strand. In the final step of the synthesis, commercial bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite  was used to generate the required 5’-phosphate. Oligonucleotides were deprotected and cleaved from the solid support with concentrated ammonia  for 4 h at 40 °C, followed by three washes using 1 mL of water each time. The combined liquid phases were lyophilized. TBDMS-deprotection was carried out with a 600 µL mixture of 3HF*Et3N/DMF (1:1) and the oligonucleotides were precipitated with 1.2 mL isopropoxy-TMS and 5 mL Et2O and washed again with Et2O. Further reversed-phase HPLC purification resulted in the isolation of 5’-FurAMP-RNAs , using standard conditions for RNA synthesis. The synthesis was carried out with commercial 5’-DMT-2′-O-TBDMS-(tac)-3’-phosphoramidites of all four canonical RNA nucleobases. For sequence information, see RNA-1b - RNA-4b, see 2 (50–100 mM) at 50 °C [2 for 2 h at 50 °C. The concentration of Im-NMN should be kept constant, using 1 mg/50 µL reaction volume, as higher concentrations did not lead to a higher conversion. Pure 5’-FurNAD-RNAs were obtained by reversed-phase HPLC purification. In most cases, two purification runs were necessary to obtain the required purity. The oligonucleotides were identified via MALDI-MS or ESI-HRMS for 5’-NAD-dinucleotides. 5’-NAD-RNAs  equipped with a diode array detector using a Phenomenex Luna 5 μm C18(2) 100 A (250 × 15 mm)  for oligonucleotides obtained from solid-phase synthesis or a Phenomenex Luna 5 μm C18(2) 100 A (250 × 4.6 mm) column for oligonucleotides generated by in vitro transcription using initiator nucleotides. The applied buffer system was 100 mM triethylammonium acetate pH 7.0 (buffer A) and 100 mM triethylammonium acetate in 80% acetonitrile (buffer B).Small molecules were analysed with a Bruker microTOF-Q II ESI mass spectrometer. MS experiments for oligonucleotides were performed on a Bruker MicroFlex MALDI-TOF mass spectrometer  using 3-hydroxypicolinic acid (3-HPA) as matrix. For desalting oligonucleotides, ZipTip C18 pipette tips  were used. DN-1a–DN-3a) (the corresponding triphosphate was used in 1.5 mM concentration) 4 mM NTPs, 40 mM Tris-HCl pH 8.0, 1 mM spermidine, 22 mM MgCl2, 0.01% Triton X-100, 5% DMSO, 10 mM DTT, 0.25 µg/μL T7 RNAP and incubated for 4 h at 37 °C. RNA was purified by analytical reversed-phase HPLC.The sense and antisense ssDNA template strands . The DNA sequence encoding for the hNUDT5 gene was ordered from IDT and also amplified by PCR as well. pET28a-hDcp2 was ordered from Addgene (72214) . Restriction sites were introduced during amplification and resulting sequences are listed in E. coli One Shot BL21 (DE3) . The genes encoding for dB, nudC , nudD, nE. coli One Shot BL21 (DE3) containing the respective plasmid were induced at OD600 = 0.8 with 1 mM IPTG. Bacteria were harvested after 3 h at 37 °C by centrifugation and lysed by sonication  in HisTrap buffer A . For the preparation of NudC, 1 M urea was added to HisTrap buffer A and B to remove contaminating RNA [g, 30 min, 4 °C) and the supernatant was applied to a Ni-NTA HisTrap column . The protein was eluted with a gradient of HisTrap buffer B (HisTrap buffer A with 300 mM imidazole) and analysed by SDS–PAGE. Fractions of interest were concentrated in Amicon filters . The enzymes were additionally purified via size exclusion chromatography with a HiPrep 16/60 Sephacryl S-200 High Resolution column   and stored at −20 °C in 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 50% glycerol. All purified protein samples were 95% pure, according to SDS–PAGE.ting RNA . The lysFurNAD (4.5 µM) or FurNAD-RNA (4.5 µM), 25 mM Tris-HCl pH 7.5, 50 mM NaCl, 50 mM KCl, 10 mM MgCl2, 1 mM DTT.Fluorescence time course and fluorescence spectra were measured with a FP-6500 spectrofluorometer  at 37 °C. Reactions were performed in a 20 µL scale in the presence of exc/em: 304/380 nm; 5 nm excitation/emission bandwidth, response: 0.5 s; at 37 °C.The following settings were applied for fluorescence measurements: sensitivity mode: manual 350 V; λAll enzymes were applied in low µM concentrations (4 µM) except for CD38 (2.5 nM), which was highly active on modified NAD as well as NAD-RNAs.FurNAD (4.5 µM) or FurNAD-RNA (4.5 µM), 25 mM Tris-HCl pH 7.5, 50 mM NaCl, 50 mM KCl, 10 mM MgCl2, 1 mM DTT and an appropriate enzyme. Enzymes were either cloned, expressed and purified as described and their concentration determined using Nanodrop, or obtained from commercial sources , RppH , polyphosphatase , ADP ribosyl cyclase (ADPRC) ).Reactions were performed in a 20 µL scale using a 384 well-plate format  in the presence of exc/em: 304/380 nm; 20 nm excitation/emission bandwidth; measurement position: bottom of the well plate; gain 75; 40 µs integration time; measurement every 20 s; at 37 °C. Time course measurements of fluorescence intensity were conducted using a Tecan Sapphire 2 fluorescence microplate reader  in 1 x reaction buffer B and 250 μCi 32P-γ-ATP . The reaction was incubated at 37 °C for two hours and purified by phenol-chloroform/diethyl ether extraction and isopropanol precipitation. To convert the purified 5’-32P-RNAs into 5’-32P-NAD-capped RNAs, 5’-32P-RNAI was incubated with 1000-fold excess of Im-NMN in the presence of 50 mM MgCl2 and at 50 °C for 2 hours [32P-NAD-capped RNAs were purified from remaining 5’-32P-RNAs by acryloylaminophenyl boronic acid (APB) gel electrophoresis [5’-radioactively labelled NAD-RNAI was prepared by IVT as described in . Briefly 2 hours . 5’-32P-phoresis  and isop2, 1 mM DTT, 50 nM 5’-32P-NAD-RNAI and 3 nM CD38. Enzymatic reactions were stopped by heating the samples to 98 °C for 30 s and RNA digested to single nucleotides by Nuclease P1  treatment (0.1 U/µL). CD38-mediated conversion of 32P-labelled NAD-RNA was analysed by TLC using 4:6 1 M ammonium acetate (pH 5.5)/ethanol as mobile phase as previously described [Enzyme kinetics of CD38 were performed in the presence of 25 mM Tris-HCl pH 7.5, 50 mM NaCl, 50 mM KCl, 10 mM MgClescribed . 1 μL ofFor a fluorescent turn-on NAD-RNA decapping assay, we first had to develop fluorogenic NAD-RNA derivatives that are accepted as substrates by putative decapping enzymes. We were inspired by earlier work from the Wagner group who developed fluorogenic substrates for studying NAD hydrolysis and related reactions . These aBased on these findings, we considered it reasonable to assume that the installation of a similar fluorescent substitution at the 5’-end of NAD-RNA should also lead to turn-on probes that might report the action of decapping enzymes. In contrast to the previously mentioned design in which the fluorophore is inactivated by one quencher, we expected that the fluorophore will interact with two quenching moieties in NAD-RNA, namely the nicotinamide “upstream” and the following nucleobase “downstream” .FurAMP, 2a) 2-pyrrole , 2-thiophene , 2-, and 2-   and recoAMP, 2e) . Only coFurAMP , while the emission maxima varied between 374 and 405 nm. We therefore synthesized 8-(furan-2-yl)-NAD  and the previously described 8-(pyrrol-2-yl)-NAD  . AMP  had theNAD, 3b) . After tn maxima 0–308 nm,g effect . The locFurNAD and PyrNAD as promising candidates at the nucleotide level, we had to develop methods to synthesize these as 5’-cap structures of longer RNA strands. The direct incorporation of 3a by T7 RNA polymerase during in vitro transcription was considered [9) (FurAMP at the 5’-terminus. Four 5’-FurAMP-RNAs of different length and nucleotide sequence (RNA-1a–RNA-4a) were synthesized, deprotected, and purified by HPLC  . During  by HPLC . Finally by HPLC B. FurNAD by HPLC .RNA-1b–RNA-4b) bearing a 5’-FurNAD modification were obtained in moderate yields (30–60%) and their identities confirmed by MALDI-MS  was revisited. As neither FurNAD (see above) nor FurAMP were accepted as a substrates by T7 RNA polymerase (data not shown), we modified the approach using initiator dinucleotides, which we had previously employed to solve the problem of “unincorporatable” 5’-moieties [FurAA (DN-1a), FurAG (DN-2a) and FurAU (DN-3a), were synthesized by SPOS (FurAMP-RNAs (FurAA and FurAG were incorporated with efficiencies of 45% and 37% respectively (FurAMP-RNAs were purified by HPLC. MALDI-MS of full-length RNA transcripts and respective controls (5’-pppRNA) confirmed the synthesis of pure FurAMP-RNAs -adenine is quenched by one moiety (nicotinamide) from one side in the first case, and potentially by two (nicotinamide plus the neighbouring nucleobase) from both sides in the latter , fluorescence intensities were in the same range as determined for the non-modified FurAMP-RNAs or FurNAD-RNAs . The main difference between e latter . Thus, aFurAMP-dinucleotides FurAA (DN-1a), FurAG (DN-2a), FurAU (DN-3a), and FurAC (DN-4a) revealed the influence of the neighbouring base: Quenching was strongest for G , followed by C and U (33%), while A showed the weakest quenching (69%)  . Attachmleotides D,E. FurNAD-RNA appears to be suitable for turn-on decapping assays. To demonstrate this property, we analysed the well-described NAD-RNA decapping activity of the bacterial Nudix hydrolase NudC [FurNAD-RNA as a substrate. This enzyme hydrolyses the pyrophosphate bond within the NAD (or here FurNAD) moiety, thereby releasing the quencher NMN and FurAMP-RNA. Indeed, the fluorescence time traces (FurNAD-RNA (FurNAD (blue trace) is in agreement with our previous findings that NudC prefers NAD-RNA over NAD [Thus, ase NudC ,37 usinge traces  and  S10e traces B. Comparover NAD .FurNAD-RNA system with a known NAD-RNA decapping enzyme, we now investigated its capacity to discover novel decapping enzymes. To this end, we applied a library of different Nudix hydrolases, deglycosylases, and other enzymes to FurNAD and FurNAD-RNA, and monitored the increase of the fluorescent signal in real time on a fluorescence microplate reader. Using FurNAD, the assay revealed a fluorescence increase for all enzymes known to hydrolyse NAD into AMP or ADP-ribose, namely the human cluster of differentiation 38 (CD38) glycohydrolase [E. coli NudE [Aplysia californica [Having established the feasibility of the oli NudE , and witifornica , and humifornica  . CD38 was originally described to modulate NAD concentration by hydrolysing NAD into ADPR or cADPR at very low µM concentrations, using a fluorescence-based enzymatic assay using 1,N6-ethenonicotinamide adenine dinucleotide (ε-NAD) and nicotinamide guanine dinucleotide (NGD) as substrates, respectively [To explore the sensitivity of our turn-on ectively ,48.FurNAD was added to CD38, and the initial conversion rates were determined at varying substrate concentrations, yielding a curve that showed Michaelis-Menten saturation. The decline of the initial rates at high substrate concentration was suggestive of substrate inhibition  or NAD (from unconverted substrate RNA). Separation by TLC and densitometric analysis by phosphorimaging allowed quantification  theyThe application of synthetic light-up NAD-RNA presented in this article will facilitate and promote the elucidation of the biological relevance of NAD capping in prokaryotes and eukaryotes, the identification of binding partners, players and mechanisms in decapping and RNA turnover.FurNAD-RNAs, we discovered that the eukaryotic glycohydrolase CD38 processes NAD-capped-RNA in vitro into ADP-ribose-modified-RNA and nicotinamide and therefore might act as a decapping enzyme in vivo. The existence of multiple decapping pathways suggests that the removal of the NAD-cap is an important and regulated process in eukaryotes.Using


Members of the APOBEC family of cytidine deaminases show antiviral activities in mammalian cells through lethal editing in the genomes of small DNA viruses, herpesviruses and retroviruses, and potentially those of RNA viruses such as coronaviruses. Consistent with the latter, APOBEC-like directional C→U transitions of genomic plus-strand RNA are greatly overrepresented in SARS-CoV-2 genome sequences of variants emerging during the COVID-19 pandemic. A C→U mutational process may leave evolutionary imprints on coronavirus genomes, including extensive homoplasy from editing and reversion at targeted sites and the occurrence of driven amino acid sequence changes in viral proteins. If sustained over longer periods, this process may account for the previously reported marked global depletion of C and excess of U bases in human seasonal coronavirus genomes. This review synthesizes the current knowledge on APOBEC evolution and function and the evidence of their role in APOBEC-mediated genome editing of SARS-CoV-2 and other coronaviruses. -SARS-CoV-2 sequence variants contain an overabundance of C- > U transitions-C- > U transitions are the hallmark of the activity of APOBEC cytosine deaminases-Further work is needed to determine APOBEC's role in coronavirus evolution This mutational pattern is similar to that induced by the cytidine deaminases of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of proteins. The ability of this enzyme family to modify retroviruses, retroelements, and some DNA viruses is well established, while their potential to modify RNA viruses is exploratory. This review will provide an overview of the current knowledge of APOBEC function and evolution, the evidence for this mutational phenomenon in coronaviruses, and its potential evolutionary consequences for RNA viruses.The sustained spread of SARS coronavirus 2 (SARS-CoV-2), coupled with readily deployable and accessible sequencing technologies, has given researchers an unprecedented ability to track virus evolution from spillover through early adaptation to human hosts. Mining this data, several authors have identified an overabundance of homoplastic cytidine to uridine transitions in the SARS-CoV-2 genome, particularly in the context of 5′-U2Apolipoprotein B mRNA Editing Catalytic Polypeptide 1 (APOBEC1)  . Since tAPOBEC1) . The proAPOBEC1) .323-28-Pro-Cys-X2-4-Cys that is present in the cytidine deaminase fold (CD) (Members of the APOBEC family of proteins are readily recognized through their possession of a highly conserved ZDD motif His-X-Glu-Xold (CD) . In thosold (CD) . A3B, A3old (CD) . In humaold (CD) .4C-3′ with the mooring sequence 4–6 nt downstream of the target and is primarily found in the cytoplasm but can traffic to the nucleus. However, A1 is the exception and not the rule to this family, as most APOBEC proteins are regarded for their ability to deaminate C to dU in ssDNA rather than RNA.A1 primarily functions to increase diversity in host-expressed RNAs in a tissue-specific manner . The RNAC-3′ context  and is basally present in the cytoplasm but traffics to the nucleus to perform immunoglobulin gene editing.AID and AID-like enzymes are an essential component of the adaptative immune system in all jawed vertebrate species . In humain vivo function or is an artifact of the overexpression model. A4 was discovered in humans through bioinformatic identification and contains orthologs in other mammals, chickens, and frogs . The inhibition of HIV-1 replication through the function of A3D, A3F, A3G, and A3H is the most extensively studied of the viral restriction phenotype of the APOBEC family and has been well reviewed elsewhere . The early 2000s contained a cascade of information regarding the interaction between Vif and A3G . Per theRetrotransposons and Mobile Genetic Elements. Transposable elements (TEs) are selfish genetic elements that are able to move within the genome via one of two methods  is significantly depleted, consistent with long-term adaptation to infection of A3 expressing tissues  in the DNA viruses parvoviruses, herpesviruses, and papillomaviruses, there was little evidence for this in RNA virus genomes with the exception of the seasonal coronaviruses HCoV-NL63, HCoV-OC43, HCoV-HKU1, and HCoV-229E   . Howeverufacture .Deaminase-Independent Restriction and Insensitivity. Deaminase-independent restriction has been demonstrated primarily using overexpression models of catalytically inactive A3 mutants. This phenomenon has been reported for many viruses, including HIV-1, measles, mumps, respiratory syncytial virus, HCoV-NL63, adeno-associated virus, enterovirus A71, and minute virus of mice Origin. Tive site . Anotherive site . The AIDive site . A1 and ive site . A1 has ive site . A3 emerive site .Fig. 1EmA3 paralog expansion. A3 homologs are not present in marsupials or monotreme genomes and appear to be exclusive to placental mammals  and which may be involved in host-pathogen interactions . Current evidence for the overall evolutionary trajectory is summarized in 4.3C→U Mutational Asymmetry in SARS-CoV-2.Coronaviridae are a family of single-stranded, positive-sense RNA viruses, of which there are seven known to infect and transmit in human populations   . Analysi (PBMCs) . Tissue  (PBMCs) . While tEvolutionary consequences of C→U mutational bias in coronaviruses. Irrespective of the underlying mutational mechanisms, we have reviewed the substantial evidence for a potent editing process that drives C→U mutations into the genomes of coronaviruses and potentially other RNA viruses in certain favored sequence and RNA structural contexts. In evolutionary terms, this driver of virus sequence change is quite distinct from the occurrence of random mutations through misincorporation errors during transcription that is assumed in standard evolutionary models. Apart from the restriction of mutations to C→U changes (or G→A if the negative-sense strand is edited), sequence changes are also primarily distributed in mutational hotspots that may lead to the appearance of convergent changes between otherwise genetically unlinked strains. For example, several C→U mutations occurred independently among variants of SARS-CoV-2 that were distributed into different parts of the tree  to synonymous substitutions (dS) occurring during virus evolution. Most viral sequence diversity is generated through fixation of neutral substitution that do not influence the fitness of the virus. These typically are synonymous, and comparative analyses of virus sequence datasets typically reveals low dN/dS ratios indicative of purifying selection, such as the 0.1 ratio found in a dataset of Ebolavirus genome sequences during its recent epidemic spread in Central and West Africa . ContrasFurthermore, a substantial proportion of amino acid changes that evolve within SARS-CoV-2 populations originate from non-synonymous changes induced by C→U transitions , rather The long-term effects of the (C→U)/(U→C) transition asymmetry on virus diversification are difficult to predict. However, the compositional abnormalities of other coronaviruses, particularly human seasonal coronaviruses, might plausibly be attributed to the long term outcome of prolonged C→U editing . For exaDownstream induction of inflammatory cytokine and adaptive immune system responses may contribute the exaggerated and damaging inflammatory pathology observed in COVID-19 cases. The pronounced activity of APOBEC in human cells may derive from an inability of a bat-adapted virus to counteract the human APOBEC editing pathway. The varying degrees of base asymmetry and G + C contents of human seasonal coronaviruses may then be indicative of the varying times they have circulated in humans following their acquisition from bats . SARS-Co5in vivo model for APOBEC function in whole host defense possesses only one A3 gene compared to seven or more in primates and bats.APOBECs have been shown to be important for host and retroviral function, and the possibility that they play a role in RNA viral evolution would substantially expand the scope of their antiviral activities. However, part of the difficulty in studying the process functionally is the plethora of different APOBECs in human with likely different virus, RNA, and DNA targeting and biological activities. Furthermore, the number and identities of APOBECs in high variable in mammals; for example, the mouse which might otherwise represent a suitable trans complementation of the editing phenotype by gene transfection may be difficult to achieve in vitro if there are functionally required interacting cofactors or activated intracellular states. Further research into the protein family is warranted .The work was supported by a Jeremy Ratcliff: Conceptualization, Writing - original draft, preparation, Writing - review & editingWriting – review & editing. Peter Simmonds: Writing - original draft, Writing - Draft extension, Writing - review & editing, Funding acquisition.