The nucleophilic reactivity of RNA 2′-OH groupsin waterhas proven broadly useful in probing, labeling, and conjugating RNA.To date, reactions selective to ribose 2′-OH have been limitedto bond formation with short-lived carbonyl electrophiles. Here wereport that many activated small-molecule sulfonyl species can exhibitextended lifetimes in water and retain 2′-OH reactivity. Thedata establish favorable aqueous solubility for selected reagentsand successful RNA-selective reactions at stoichiometric and superstoichiometricyields, particularly for aryl sulfonyltriazole species. We reportthat the latter are considerably more stable than most prior carbonelectrophiles in aqueous environments and tolerate silica chromatography.Furthermore, an azide-substituted sulfonyltriazole reagent is developedto introduce labels into RNA via click chemistry. In addition to high-yieldreactions, we find that RNA sulfonylation can also be performed underconditions that give trace yields necessary for structure mapping.Like acylation, the reaction occurs with selectivity for unpairednucleotides over those in the duplex structure, and a sulfonate adductcauses reverse transcriptase stops, suggesting potential use in RNAstructure analysis. Probing of rRNA is demonstrated in human cells,indicating possible cell permeability. The sulfonyl reagent classenables a new level of control, selectivity, versatility, and easeof preparation for RNA applications. We introducesulfonyl reagents as a new class of RNA 2′-OHreactive small molecules. These molecules are stable, easy to synthesizeand purify, and useful for structure mapping and covalent labeling. Limitations of known RNA-reactivecarbonyl species include modest water solubility, rapid hydrolysislimiting yields and requiring elevated concentrations, and restrictedstrategies for preparation due to a lack of stability to column chromatography.20 Encouragingly, a recent studyhas also reported that RNA-binding proteins that are genetically encodedto possess fluorosulfate-containing modified tyrosine residues candirect the formation of sulfate ester linkages at RNA 2′-OH.21 Following these precedents, we undertook a studyof the potential for small-molecule sulfonylation in RNA. Our chiefmotivations were (1) finding reactive motifs beyond modified sulfurgroups on proteins, which may have different reactivities than smallalkyl/aryl sulfonyl compounds due to the macromolecular context; (2)discovering strategies to tune RNA reactivity of small-molecule sulfonylatingreagents and establishing the scope of effective structures; and (3)investigating the potential for further application in RNA chemicalbiology research . Importantly, to establish a general approach with wideapplicability, we tested small-molecule sulfonyl reagents whose reactivitywould be independent of any RNA-macromolecular interactions, to reducethe possibility of reactions occurring by proximity effects and toremove the requirement for genetic encoding.21 Given the literature examples and our motivation, the oxophilicnature of sulfur and the hydrophilicity of sulfonyl groups, we wonderedwhether there might exist a small-molecule sulfonylating reagent structurethat might enable aqueous reactivity with 2′-OH groups in RNA.In particular, activated sulfonyl groups have proven highly usefulrecently in protein profiling, showing reactivity at tyrosine andother side chains in water.invivo application, ease of synthesis and purification, versatilityof structural modification, and tunability of electrophilicity. Consideringthe versatility of known reactive sulfonyl reagents, it seemed possiblethat many of these features might be found in carefully designed electrophilicsulfur species.Desirable features for RNA 2′-OH-reactive reagents includesufficient reactivity to functionalize this relatively bulky secondaryhydroxyl in high yields, selectivity for 2′-OH groups in unpairedstructure over paired contexts, adequate lifetime and solubility inwater to remain available for RNA, cell permeability for With these issues in mind, we have undertakena study of a seriesof sulfur-based electrophilic reagents with the potential for usein functionalizing or mapping this nucleic acid. Here we report thatsulfonyl species with carefully tuned electrophilicity and leavinggroups can react successfully in high yields with the 2′-OHgroups of RNA. Notably, they can be prepared readily from sulfonylchlorides and purified easily via silica column chromatography. Weexplore the scope of varied reagent structure that can be incorporatedinto RNA via conjugation reactions. We further document a postsulfonylationconjugation reaction made possible by an azide-functionalized sulfonatereagent. Finally, we show that sulfonylation reactions applied intrace yields can potentially be applied to RNA structure mapping aswell, demonstrating selectivity for unpaired nucleotides over thosein a duplex structure. The results establish a new and versatile reagentclass for RNA research.23 A recent study of tyrosine sidechain phenolic modification in aqueous environments with sulfonyltriazoles confirmed good yields,18 andgiven the relative acidity of 2′-OH in RNA (pKa ≈ 12.5),24 a similarreaction in this distinct biopolymer seemed plausible. To begin ourinvestigations, we prepared the pyridyl imidazole compounds 1 and 2, inspired by the structure of the earliernicotinyl acylimidazole compound NAI7 ). We found that understandard RNA reaction conditions  with 20% DMSO, 24 h, 37 °C) both of these compounds demonstratedsignificant RNA 2′-OH reactions detectable by MALDI-TOF massspectroscopy with a short 16 nt single-stranded RNA . With 1, a modest6.4% of the test RNA strand underwent modification with a pyridine-3-sulfonylgroup, presumably generating an O-sulfonate ester (see below for confirmation).Encouragingly, with the stronger 2-chloroimidazole leaving group (2) we observed 54% conversion of the test RNA strand . Similar levels of modification werealso detected upon reaction of these molecules with other short RNAsequences and at varying DMSO composition percentages over 10–50%. Importantly, MALDI-TOF analysis of parallel reactionsof 1 and 2 with a DNA strand having thesame sequence as the test RNA gave no reaction, confirming that theRNA adducts occurred at 2′-OH groups rather than exocyclicamines . Based on these initial findings, we adopted ageneral scaffold for testing RNA 2′-OH sulfonylating agents,with a heteroaryl group linked to a leaving group through the sulfonylmoiety , under 10–50% DMSO conditions and the “TestDNA”control. Sulfonylation conversions were assessed by MALDI-TOF MS analysisafter ethanol precipitation of the RNA. To measure aqueous stability,we also followed hydrolysis of several of the species via 1H NMR measurements in deuterated buffer .The significant differenceobserved in sulfonylation yields between imidazole vs the less basic2-chloroimidazole leaving group led us to hypothesize that the yieldsmight be tuned by simply changing the leaving groups, with the expectationthat greater electron-withdrawing character might promote higher RNA2′-OH sulfonylation reactivity. However, countering this isthe likelihood that increasing the reactivity could also promote morerapid hydrolysis by water prior to reaction with RNA, leading to diminishedsulfonylation yield. To investigate this, we prepared a set of 3-pyridylsulfonylspecies having a range of leaving groups 1. The co25 For example, 1 and 2 displayed half-lives of nearly 7 daysand 8 h respectively in the aqueous buffer, as compared with a 34min t1/2 of acylimidazole reagent NAIand 14 s t1/2 of isatoic anhydride 1M7 resulted in shorter half-livesof ca. 4 h and 4 min respectively, consistent with expectation. 5 and 7  were found to be too reactive and were unstablein DMSO stock solution, and hence not used for further experiments.Our experiments revealed thatseveral of the compounds were notably more stable than prior RNA 2′-OHmodifying reagents in aqueous environments.ride 1M71B. More 26 and a recent report of engineered protein-directedRNA modification,21 we tested the simplepyridine-3-sulfonyl fluoride 9. In addition, we testedan NHS sulfonyl ester (10). Both 9 and 10 were found to be relatively stable in aqueous environments,with half-lives of approximately 6 h each, allowing sufficient availabilityin solution for RNA 2′-OH sulfonylation to occur, and bothdisplayed significant levels of RNA reaction (25% and 12% respectively),but were unable to surpass the 54% sulfonylation yield of 2. Considerably less reactive than these were imidazolium, pyridinium,and azide leaving groups (11–13),which gave no detectable yields with RNA  and 15  products; andreaction with 1,2,4-triazole yielded only 16 (N1-linkage).Encouragingly, 15 was found to achieve sulfonylationconversion with RNA of 70%; however, it was found to be particularlyunstable in aqueous solution, and its half-life could not be measured.Compounds 14 and 16 were both found to improveon that yield, affording 84% and 81% conversion, respectively. Importantly,both of these molecules also have aqueous half-lives of ca. 90 min,nearly three times that of the structurally similar acylimidazoleNAI.6 Again, control reactions with DNAwith these three sulfonyltriazoles revealed little or no reaction,confirming the reaction site of 2′-OH in RNA . Analyzing this information, moving forward weemployed 1,2,4-triazole as an optimal leaving group to maximize sulfonylationyield, avoiding the isomeric mixtures that result with 1,2,3-triazole.We followed up on this finding by testing a range of aryl and alkyl1,2,4-triazole-activated sulfonyl species , but the solubility and electron deficiencyafforded by the pyridine scaffold proved optimal, and subsequent experimentsand conjugations were carried out with this scaffold as a basis.Numerous reports in the literaturehave utilized sulfonyl triazoles as electrophilic covalent modificationagents for proteins (via sulfur(VI) triazole exchange, (SuTEx)),29 is also not amenable to chromatographicpurification techniques, and must rely on extraction. In general,improvements in stability of reagents (while maintaining tunable RNAreactivity) and amenability to purification are desirable. Our preparationstudies with test compound 16 (henceforth referred toas P3S) showed that it is readily accessible in near-quantitativeyield in one simple synthetic step from commercially available pyridine-3-sulfonylchloride and 1,2,4-triazole (Supporting Information). Importantly, we were able to perform silica chromatography withthe reaction mixture, enabling the isolation of purified P3S as a white powder without detected impurities. Conveniently, gram-scalesynthesis of P3S is also feasible, with 89% preparativeyield (Supporting Information).Previously reportedand commonly used RNA 2′-OH modifying acylimidazole reagentsrequire either cold storage in frozen solution in their activatedforms, or fresh synthesis of each batch of active molecule from inactiveprecursor. The standard preparation of the acylimidazoles directlyin dry DMSO stock solution also generates one equivalent of imidazole,and although the imidazole is not known to interfere with applications,it can affect buffering power of solutions in which it is applied.Another commonly used class of RNA 2′-OH modifying agents,isatoic anhydrides,P3S and supported by the extended hydrolysis half-life,wefurther probed its stability under laboratory storage conditions.We found that when it is stored under Ar at −20 °C, itis completely stable for at least 8 months with no hydrolysis (tothe sulfonic acid) detected by 1H NMR . Finally, we also found that P3S adducts on Test RNA are stable for at least 8 days inwater at 37 °C , whereassome acyl adducts of RNA are hydrolyzed after a few hours on RNA,thus requiring rapid analysis before the adduct is gone.28 Adduct stability is also important in covalentlabeling, where extended stability of the conjugate can render itconsiderably more useful without risk of diminishment of the labeledsignal due to hydrolysis of the linkage.Encouraged by such ease of synthesis and purificationfor y 1H NMR 2B and su31 or with biotinfor pull-down and separation protocols.32 However, mostRNA covalent labeling methods involve enzymatic nucleotide incorporationprotocols or engineered RNA sequences,37 and only a few reagents based on short-lived activatedcarbonyl species have been tested for high-yield labeling.11 Given the stability and ease of synthesis of sulfonyltriazoles,we asked whether such species could be used to introduce a reactivehandle in RNA. For our first test, we designed a sulfonyl triazole-containingreagent functionalized with an azide functional group that could potentiallyreact with RNA 2′-OH groups, potentially allowing for furtherRNA conjugation. We synthesized derivative 26 .Remarkably, the sulfonyltriazole moiety remained stable to Pd-mediatedSonogashira chemistry and to silica chromatography. Encouragingly,we found that AzP3S sulfonylates an 18-nucleotide tRF3RNA with 87% conversion at 50 mM concentration over 24 h at 37 °C. Thisis similar conversion as has been achieved with carbon-based electrophiles,30 albeit requiring longer time due to the lowerreactivity of the sulfonyl species. To introduce a fluorescent label,we precipitated the RNA to remove residual small-molecule speciesand then redissolved and performed a strain-promoted cycloadditionreaction, employing commercial TAMRA-DBCO and Cy5-DBCO for 2 h inPBS buffer . Fluorescencemeasurements confirmed the presence of the dye on the RNA and therequirement for sulfonylation by AzP3S for covalent labeling;and TAMRA-labeled RNA was detectable by eye over a transilluminator.Chemicalagents that can covalently modify RNA offer the potential for conjugationwith labels. For example, RNA can be covalently tagged with fluorophoresfor detection, imaging, and FRET analysis,19 . Encouraged by reports of SNAr strategies applied toward covalent tagging of nucleic acids usingincorporated halopurine nucleotides, and cysteine modification inproteins using 2-sulfopyridine electrophiles,39 we hypothesized that C-2 of our pyridyl reagent might be subjectto nucleophilic substitution via a SNAr reaction with anappropriate nucleophile . Afterscreening a small panel of simple thiols, we found that a short 18-nucleotide“tRF” hairpin RNA sulfonylated with CP5S could be further functionalized with thiophenol or a PEG thiol over24 h at 25 °C in the presence of Et3N base, with >95%yield for these thiols . Although further work is required to establish the scope and reactivitycharacteristics of this class of novel sulfonyl electrophile on RNA,we note that such SNAr conjugation reactions were not previouslypossible without the use of solid phase synthesis to incorporate theelectrophilic center into nucleic acids.Prompted by the versatility and ease of synthesisof the abovesulfonyltriazole reagents, we investigated the possibility of a secondcovalent conjugation strategy that could further exploit the particularchemical characteristics of an electron-deficient pyridinesulfonylgroup. Compound 45 One of the most widely used empirical techniques is SHAPE, whichyields structural information at single nucleotide resolution.6 SHAPE exploits the preference of RNA 2′-OH acylating agentsto preferentially covalently modify RNA 2′-OH at unpaired locationson folded RNA molecules over paired residues, allowing RNA secondarystructure readout after analysis of subsequent reverse transcriptase(RT) stops, which are commonly read by gel electrophoresis.7 In contrast to high-yield conjugation reactionson RNA, SHAPE probing employs only trace levels of reaction.Since most RNA functions are dependent on secondary/tertiary structureand on dynamic structural changes, methods of assessing folded structureare important tools for research.P3S isstructurally similar to commonly used pyridine-based acyl SHAPE reagentssuch as NAI, NAI-N3, and 2A3,47P3S might also demonstrate structure-sensitive2′-OH modification on folded RNAs. As an initial test of thispossibility in vitro, we chose the well-studied 157nt E. coli FMN riboswitch RNA,49 which possesses a variety of secondary structural elements  afforded RT-stop bands that suggeststructure-sensitive RNA modification. Experiments also revealed thatthe level of RNA modification increased over time from 5 to 40 min,while maintaining structure sensitivity . Specifically, we found that the established unpairedregions of the FMN riboswitch including hairpin loops , multibranched loops  and internal loops  underwent higher levels of modificationcompared to other regions of the RNA  as well as lower reactivityof the sulfonyl molecule could be responsible for the differencesin patterns. Overall, the data reveal clear selectivity for sulfonylationof unpaired regions over helices, and also confirm that the sulfonyladduct, like acyl, causes reverse transcriptase stops, both of whichenable structural mapping.We hypothesized that since the sulfonyl species elements 5A.50 Encsitivity S24. Specsitivity S24. SpecP3S to probe a 121nt segment of human 5S rRNA in HeLa cells, as its structure has beenwidely studied in the literature and its interactions with proteins in vivo well-documented (54 Using a range of P3S concentrations (50–100 mM) and limiting DMSO to10% of total volume of the cell incubation mixture, we found that1 h incubation at 37 °C with HeLa cells afforded sufficient rRNAmodification for structure mapping  while retainingthe ability to react with RNA 2′-OH. We find that they arestable in storage for extended periods even at room temperature. Importantly,they are readily synthesized, and can be easily purified by silicagel chromatography. Such facile purification and storage are significantlylimited with most current RNA acylating agents; for example, mostacylimidazoles used previously are best stored at low temperaturesto prevent hydrolysis, and are commonly applied as a stoichiometricmixture in DMSO solution with imidazole, due in part to their lackof stability to column chromatography.AzP3S-sulfonylated RNA. AzP3S is synthesized in only twosteps, and multiple strained-alkyne reagents are commercially availableas potential reactive partners for AzP3S-modified RNA,likely allowing labeling future application with dyes of varied emissionproperties as well as affinity labels. In addition, we show that a2-chloropyridine sulfonate adduct on RNA can be conjugated in highyields with simple alkyl and aryl thiols via a SNAr reaction.The sulfonyl reagentsoffer a practical alternative for covalentlabeling of RNA. We successfully utilized the copper-free strain-promotedclick strategy to attach TAMRA and Cy5 dyes onto P3S is able to performRNA 2′-OH sulfonylation in vitro with a reactivitypreference for unpaired RNA regions that is similar to that for carbon-electrophileRNA SHAPE reagents. While development of RNA 2′-OH sulfonylationis still at an early stage when compared to other well-establishedRNA reactive molecules, this suggests the potential for the use ofthese easy-to-handle sulfonylating agents for future RNA structuralmapping studies. Efforts are currently underway to determine the reactivitycharacteristics of these sulfonylating agents toward a wider varietyof RNA secondary structures. Encouragingly, we also observed structure-sensitive2′-OH reactivity of P3S with a rRNA in HeLa cells.The lower reactivity of sulfonylating molecules compared to cell-permeableacylimidazoles  necessitates the use of a longer incubationtime (1 h) in cellular experiments for a sufficient signal in gelanalysis, which may interfere with cell biology during the time ofthe experiment. However, the results do suggest useful levels of cellpermeability of the reagent tested here, and further studies withmore reactive sulfonyl species are merited in the future for in vivo application in RNA research.The current experiments also suggest the potential for sulfonyltriazolecompounds as alternatives for RNA structure mapping. Like common isatoicanhydride and acylimidazole structure-mapping reagents, we show thatthe sulfonyl reagents react preferentially at 2′-OH groups.Here we demonstrated with a folded riboswitch RNA that the simplepyridine sulfonyl triazole molecule 56 raising this future possibility for RNA.The sulfonylreagent class offers new chemistry for RNA functionalization.To date, all RNA 2′-OH reactive small molecules reported inthe literature have possessed just one class of functional group—activatedcarbonyl—as an electrophile to allow for bond formation withthe nucleophilic ribose 2′-hydroxyl. The use of carbonyl electrophileshas restricted the adducts to ester and carbonate linkages with RNA2′-OH. This is in sharp contrast to the protein literature,which documents a wide variety of electrophilic modifications at aminoacid side chains. Our new findings diversify the type of selectivesmall-molecule covalent linkages possible at RNA 2′-OH, generatingO-sulfonate ester linkages at RNA ribose 2′-carbon using avariety of different sulfonyl species. The considerably differentchemical properties of the tetrahedral sulfonate ester group (comparedto the planar ester/carbonate groups) could also be potentially exploitedin future studies to develop new chemistries and applications forRNA. For example, sulfonate esters are well-known as intermediatesfor substitution and elimination reactions,


However, as DRS is designed to profile intact RNA, expression quantification may be more heavily dependent upon RNA integrity than alternative RNA sequencing methodologies. It is currently unclear how RNA degradation impacts DRS or whether it can be corrected for. To assess the impact of RNA integrity on DRS, we performed a degradation time series using SH-SY5Y neuroblastoma cells. Our results demonstrate that degradation is a significant and pervasive factor that can bias DRS measurements, including a reduction in library complexity resulting in an overrepresentation of short genes and isoforms. Degradation also biases differential expression analyses; however, we find that explicit correction can almost fully recover meaningful biological signal. In addition, DRS provided less biased profiling of partially degraded samples than Nanopore PCR-cDNA sequencing. Overall, we find that samples with RNA integrity number (RIN) > 9.5 can be treated as undegraded and samples with RIN > 7 can be utilized for DRS with appropriate correction. These results establish the suitability of DRS for a wide range of samples, including partially degraded  Cellular fate and function are tightly regulated by networks of expressed genes. Therefore, accurate quantification of the transcriptome is crucial in understanding how cells behave in physiological and disease contexts. RNA sequencing (RNA-seq) has become the prevailing method for quantifying the abundance of RNAs and elucidating the complex and dynamic nature of the transcriptome .Numerous factors affect RNA abundance, including the rates of transcript production and decay. RNA degradation has a substantive impact on the transcriptome and can occur both through regulated processes within living cells to remove unwanted RNAs and due to cellular damage and death . Regulatin vivo levels. Profiling samples with low or variable RNA quality requires accounting for the confounding effects of RNA quality on gene and isoform quantification . RIN is a global measure of RNA quality  defined by the ratio of 28S to 18S ribosomal RNA . More reRecently, third-generation long-read sequencing technologies such as Oxford Nanopore Technologies (ONT) and PacBio have grown in popularity for expression profiling. Long-read methods can sequence entire mRNAs in a single read, enabling improved identification and quantification of genes and isoforms . Howeverin vitro polyadenylation or sequence-specific adaptors are utilized  almost completely removed the impact of degradation on differential expression (DE) analyses. Comparison to ONT PCR-cDNA sequencing demonstrated that DRS is not overtly more sensitive to RNA degradation, while DRS of human post-mortem brain confirmed that useable results can be obtained from partially degraded 2, 37°C) with DMEM + GlutaMAX , supplemented with 10% foetal bovine serum  and 1% penicillin–streptomycin (10 000 U/ml) .Human SH-SY5Y cells were cultured in growth media under standard conditions  and combined into one 50 ml falcon tube, spun gently at 800 rpm to form a cell pellet and supernatant was removed. Two additional wash steps in DPBS  solution removed traces of culture media, TrypLE and cell-free RNA without disturbing the cell pellet. The clean cell pellet was then resuspended by gentle pipetting in 250 μl of DPBS. Two 40 μl T0 control/non-degraded sample aliquots were then collected and placed into 1.5 ml Eppendorf tubes, 1 ml of QIAzol (QIAGEN) was added and pipette mixed to disrupt the cells, and samples were incubated for 5 min and placed on dry ice.Next, we implemented our degradation method, which we optimized to degrade RNA to a RIN range between 7 and 10. This range retains enough long RNAs to make DRS worthwhile, while at the same time provides a broad enough range to measure the effects of degradation and provides a framework to compare ideal samples (RINs between 9.5 and 10) with high-quality samples that have experienced some RNA degradation (RINs of 7–9).To degrade RNA in the remaining cells, cells were freeze–thawed by placing the 50 ml falcon tube on dry ice for 10 min before returning it to room temperature. Duplicate 40 μl samples were collected at the following time intervals: 0.5, 1, 2, 3, 4, 6 and 8 h. The cell suspension was gently agitated before sample collection to ensure that sampling was from a homogeneous cell solution. Immediately after collection, cells were disrupted using 1 ml of QIAzol and incubated for 5 min before being placed on dry ice. RNA extractions were performed using RNeasy Lipid Tissue Kit  according to the manufacturer’s instructions. A maximum of six samples were extracted at any one time and samples were randomly assigned to extractions to reduce extraction times and minimize batch effects. RNA quality (RIN) was assessed on a TapeStation 4200 (Agilent) and RNA purity on a NanoDrop (Thermo Fisher), while RNA concentration was assessed using a Qubit fluorometer (Thermo Fisher). QC data are available in To prepare RNA for sequencing, a minimum of 15 μg of total RNA from each sample was polyA+ purified using 50 ml of NEXTflex polyA+ beads (Bioo Scientific). Samples were randomized, purified and sequenced in groups of 3–5 to reduce batch effects. The concentration of purified RNA was assessed using a Qubit fluorometer. Sequencing libraries were prepared immediately thereafter using the SQK-RNA002 kit (ONT) using a minimum input of 120 ng of polyA+ RNA . SynthetRNA from 250 mg of post-mortem cerebellum tissue (obtained from the Victorian Brain Bank) was extracted and polyA+ purified according to the methods stated above. SQK-RNA002 library preparation was performed with 500 ng of polyA RNA as input and sequenced on a PromethION flow cell (FLO-PRO002). FAST5 files were basecalled with Guppy (v3.5.2) (ONT). Nanopore PCR-cDNA sequencing was also performed on four samples, two control (T0) and two 6 h post-degradation treatment, using the SQK-PCS110 kit (ONT) as per the manufacturer’s instructions. Sequin controls  were incQ) score ≥7] unless otherwise stated.Initial data analysis was performed using pycoQC  to gathe-ax splice -uf as recommended. The transcriptome alignments were performed using the long-read mode for ONT data -ax map-ont -k14 to retain multiple secondary alignments.FASTQ files containing ONT pass reads were aligned to the human (GRCh38)  and synthttps://github.com/josiegleeson/BamSlam)  . Read co   against all other time points (T1–T5). Next, we fit a linear regression and calculated Pearson’s and Spearman’s correlation coefficients. Quantification was performed with the same input bam files for both NanoCount and Salmon.Quantification at the gene level was performed using featureCounts (v1.6.5) . Isoformv1.32.0)  utilizinTo identify genes and isoforms that could no longer be detected at 8 h post-freeze–thaw treatment, we used normalized gene and isoform counts obtained from the DESeq2 pipeline. Undetectable genes and isoforms were defined as per the following criteria:Five or more normalized counts in at least four of the first five samples (T0 and T1).Zero normalized counts in both samples at 8 h.r) ≥ 0.5.We fit an exponential decay curve to each gene or isoform and selected those with a Pearson’s correlation coefficient  apparent upregulation (upregulation and increased relative abundance of highly stable RNAs); (ii) stable (i.e. that maintain or slightly increase in apparent abundance); (iii) slow degradation; and (iv) fast degradation. Log fold change values were normalized using the centroid values from cluster 2. This normalization accounts for relative changes in abundance that may make stable genes appear upregulated as genes or isoforms degrade throughout the time series. To compare gene/isoform architecture between the degradation clusters, we extracted gene and isoform feature information  from the comprehensive GENCODE v31 annotation file. We extracted %GC content from the BioMart database v2.0  terms among each cluster was performed with Goseq (v1.44.0)  in R implementing a ase v2.0 . The fulGoseq v1..0 (44) ain vivo samples, including loss of external nutrients, cellular stress and loss of membrane integrity. Three replicates of the degradation time series were performed. We selected 15 samples for DRS from six time points [0 (control), 0.5, 1, 3 or 4, 6 and 8 h), with at least 2 sample replicates included at each time point.As RNA from post-mortem, clinical or field samples is often partially degraded prior to sequencing, we set out to examine the effects of this degradation on long-read DRS. We implemented a degradation time series on SH-SY5Y neuroblastoma samples Figure  , r = 0.83 and P < 0.001; percentage of full-length transcripts, r = 0.68 and P < 0.001; and median coverage fraction at both the read, r = 0.76 and P < 0.001, and transcript isoform levels, r = 0.84 and P < 0.001  and has the largest impact on isoforms between 750 and 2000 bp long  alignments. Supporting this, we observed a correlation between the proportion of reads with five or more secondary alignments and decreased RIN  at T0, demonstrating that genes and isoforms that are no longer detectable at 8 h are likely to be those that are lowly expressed to begin with and therefore fall below the detection threshold for DRS during the time series. Almost all genes and isoforms can still be detected at a RIN of ∼7.We wanted to determine whether there were any genes and isoforms we could no longer detect at 8 h post-degradation treatment. Nearly all genes and isoforms with detectable expression at T0 and/or 0.5 h were detected throughout the entire time series, with only 2 genes  and 34 iK-means clustering, we grouped log fold change data (relative to 0 h) into four groups (each group outlining how gene/isoform abundances change over time): (i) Genes or isoforms that have apparent upregulation. This may seem counterintuitive given that most changes in abundance are likely the result of RNA degradation; nevertheless, as our experimental design treats cells and does not inhibit transcription, it is possible for some cells to be transcriptionally active throughout the time series. (ii) Genes or isoforms that are stable (i.e. that maintain or slightly increase in apparent abundance). (iii) Genes or isoforms that degrade slowly. (iv) Genes or isoforms that degrade rapidly. Of the 7864 genes clustered, we identified 4.9% (383) that show apparent upregulation, 19.1% (1500) that are stable, 48.6% (3820) with slow degradation and 27.5% (2161) with rapid degradation , UTR lengths (5′ and 3′) (P < 0.001), CDS lengths (P < 0.001) and total gene length (5′ UTR + CDS + 3′ UTR) (P < 0.001)  Figure  and long) Figure  and D, C) Figure  when com) Figure  and in s) Figure , though ) Figure  suggeste) Figure . Since DHIF1A, a gene known to regulate cellular responses to hypoxia  confirmed that RNA integrity was the major contributor to sample variance at both the gene and transcript levels  Figure . PC2 sepGiven the significant effects of RNA degradation on the transcriptome, we investigated what impact this had on differential gene and isoform expression analyses. Genes and isoforms identified as DE should largely be a result of degradation treatment rather than true biological signal. We assessed gene and isoform DE with DESeq2  accountiWe did not detect any DE genes between the 0 h control and 0.5 h post-treatment, further demonstrating that the effects of degradation at this early time point are minimal. At 1 h post-treatment, we detected 36 DE genes. Both the number of DE genes and the magnitude of expression changes increased as RNA degrades reaching 381 DE genes by 8 h, with a similar trend evident at the isoform level Table . IsoformWe investigated the impact degradation had on NanoCount and Salmon quantification by comparing isoform counts at 0 h to all other time points . ResultsOur GLM framework shows clear evidence of a degradation bias as detection of DE genes and isoforms is largely a result of degradation and not a real biological effect. We therefore tried to account for these degradation biases by adding sample RIN as a covariate to our model (∼RIN + replicate effects + time). We also compared our GLM to the DegNorm pipeline  for genet = 0) samples received Mix A, while all other samples received Mix B. Mix A and Mix B contain the same synthetic genes and isoforms in different concentrations to simulate changes in expression. Visualizing the sequin data using a PCA demonstrated separation of Mix A and Mix B samples  to assess the impact of RIN on the GLM. We added synthetic mixes into our samples prior to library preparation after the degradation time series; therefore, sequins should not show degradation over time. Control . We were interested in comparing these two sequencing approaches to assess which would be most suitable for profiling partially degraded RNA. The PCR-cDNA protocol should enrich for full-length transcripts, and we hypothesized that this would make it less susceptible to RNA degradation. Our results show that PCR-cDNA produced a greater percentage of full-length transcripts as expected, but that read lengths were shorter for both intact and partially degraded RNA Table . StratifDRS offers the unique ability to measure RNA as it exists in the cell, including sequencing complete RNA molecules and accurate quantification of gene and isoform expression levels . As applOur time series of RNA degradation focused on samples with a RIN between 7 and 10. We chose this range because clinical and/or post-mortem samples commonly fall within this range and we hypothesized that long-read sequencing of native RNA would be highly sensitive to degradation. As reasonably intact RNA is required to enable many of the advantages of long-read RNA-seq methods, the RIN range tested in this study is smaller than previously examined for short-read RNA-seq where highly degraded RNA (RIN < 4) was also utilized ,31,46, wDE analyses are central to studies investigating the transcriptome but can be limited by confounding factors. Degradation or sample quality is a factor that can falsely implicate genes and isoforms as DE. Previous studies using short-read RNA-seq have demonstrated the need to correct for sample quality, employing a wide array of correction strategies to minimize these impacts . We findin vitro polyadenylation on samples with lower RIN scores. We speculate this would increase 5′ coverage by making mRNAs without intact 3′ ends sequenceable, but would also result in more short reads that may be difficult to assign to isoforms. A combination of polyadenylation and 5′ capture (Previous studies have shown that RIN alone is not sufficient to explain the non-random nature of decaying genes and isoforms ,9. Altho capture  could alIt is likely that RNA decay differs under different degradation conditions. Degradation of transcripts due to ischaemic cell death likely differs from that induced by freeze–thaw. The former is more closely associated with biologically mediated processes , while tWe compared the impact of RNA degradation on both long-read Nanopore DRS and PCR-cDNA as this has not been previously established. We found that PCR introduces amplification biases and that DRS provides a more accurate representation of the native molecules present within samples. DRS was more suitable for the identification and quantification of longer molecules even in the context of partially degraded samples. It is important to note that PCR-cDNA sequencing allows sample multiplexing, lower inputs and also produces ∼10× more reads. This results in increased detection of genes and isoforms, making PCR-cDNA more suited to experiments requiring higher throughput. Furthermore, the number of cDNA datasets we generated was limited, and further research is necessary to fully understand the impact of RNA degradation on this sequencing method.Previous studies assessing the impacts of RNA degradation on short-read sequencing have recommended a range of RIN values that can be used for RNA-seq ,16,50. DObtaining such high-quality samples is not always feasible; however, our results suggest that useful results can still be obtained from DRS in samples with a RIN > 7 if appropriate correction steps are implemented. We highly recommend NanoCount filtering in conjunction with explicit RIN correction to mitigate confounders due to degradation. Although our study did not test DRS on samples below a RIN of 7, the increasingly significant and pervasive changes that degradation introduces means we would recommend approaching data derived from such samples with caution, though useable data are likely obtainable below this range.In summary, we show that degradation induces widespread changes to the transcriptome and can have negative effects on DRS results. Nevertheless, explicitly accounting for sample quality is an effective approach for reducing the confounding effects of degradation and restoring meaningful biological signal. We conclude that DRS can be applied to samples with a range of RIN values from 7 to 10 and provide a framework to correct for the impact of degradation on DRS results.https://github.com/Sefi196/RNA_Deg. BamSlam is available at https://github.com/josiegleeson/BamSlam. Permanent DOI: 10.5281/zenodo.7970821.FAST5 and FASTQ files are publicly available to download from ENA: PRJEB53210. FASTQ files from the post-mortem brain sample are available at EGA: EGAS00001006542. All code used to perform the analysis is available on GitHub: lqad060_Supplemental_FilesClick here for additional data file.


Mycobacterium tuberculosis has been a historically challenging task for researchers due to the thick lipids associated with the cell wall of this “notorious” pathogen that is responsible for Tuberculosis (TB) outbreaks. Several studies have successfully extracted RNA from M. tuberculosis using a Trizol reagent combined with organic solvents. Recently, our laboratory has successfully extracted high quality total RNA using a commercial kit from clinical strains belonging to F15/LAM4/KZN, Beijing and F11 strain families and H37Rv laboratory strain by exploiting high speed homogenizer for cell lysis and spin columns for RNA purification. The quality and integrity of the extracted RNA was analyzed and confirmed through the Nanodrop, Bioanalyzer and RNA 3-(N-morpholino) propanesulfonic acid (MOPS) gel electrophoresis. Furthermore, to confirm the integrity of small RNA (sRNA) molecules due to their vulnerability to degradation, the RNA samples were converted to cDNA and sRNAs were amplified and confirmed through PCR. This detailed RNA extraction protocol proposes to carve a new path into TB transcriptome research without the use of organic solvent for downstream purification steps while yielding high quality RNA that can be used to understand M. tuberculosis transcriptome regulation.RNA extraction from  Mycobacterium tuberculosis has been history challenging due to the thick lipids associated with the cell wall of this etiological agent of TB disease. Due to high demand of DNA in TB molecular diagnosis assays, its extraction has been optimized since the early 1900s [M. tuberculosis has been evolving with new optimized protocols for high quality DNA that can be used in sequencing studies [M. tuberculosis samples without the use of organic solvents [Nucleic acid extraction from ly 1900s . However studies –8. RecenM. tuberculosis [M. tuberculosis transcriptome studies [M. tuberculosis, however, the stability of non-coding RNAs were not evaluated in these methods. The current protocol provides a detailed extraction method of high-quality total RNA from laboratory and clinical strains M. tuberculosis using a commercial spin-column kit. Furthermore, the extracted total RNA using this protocol can be used to study regulatory non-coding RNAs such as small RNAs (sRNAs) that can provide new insight into regulatory mechanisms of M. tuberculosis transcriptome.RNA extraction using a Trizol method has been optimized for variety of samples , 11 inclrculosis , 13. Lys studies –16 have  studies , 18 haveM. tuberculosis Beijing, F11 and F15/LAM4/KZN (KZN) strains were isolated from sputum specimens of patients in the province of KwaZulu-Natal, South Africa [M. tuberculosis H37Rv  used as a virulent control was obtained from the culture collection in Department of Medical Microbiology and Infection control, University of KwaZulu-Natal (UKZN) South Africa. Strain identity was confirmed by means of IS6110-restriction fragment length polymorphism analysis (RFLP). Drug-resistance profiling confirmed that F15/LAM4/KZN and F11 were a representative of XDR-TB, while the Beijing and H37Rv represented MDR-TB and drug-sensitive TB, respectively. All M. tuberculosis strains were resuscitated from storage at -80 °C, thawed at room temperature, and cultured in Middlebrook 7H9 broth supplemented with 0.5% glycerol and 0.05% Tween-80 and 10% Oleic acid Albumin Dextrose Catalase (OADC) at 37 °C to an OD600nm of 0.8-1 (1 × 108) colony forming units (CFU/mL) [The current study was approved by the Biomedical Research Ethics Committee (BREC) at the University of KwaZulu-Natal (reference no. BREC/00002909/2021). All methods were performed in accordance with the relevant guidelines and regulations from the University of KwaZulu-Natal BREC. h Africa  and the M. tuberculosis cells were initially grown in standard Middlebrook 7H9 media and pelleted when the log phase OD (0.8-1 600 nm) was achieved. The cells were thereafter washed twice and resuspended with minimal media that was deficient in supplemented cholesterol. This resuspension was used as the inoculum for the cholesterol-rich media, to a growing final volume of 100 mL. This was followed by total RNA extraction as described in detail:To encourage the expression of sRNAs implicated to growth and cholesterol metabolism, the laboratory H37Rv strain was cultured in an in vitro cholesterol model using the protocol described by Pandey and Sassetti  and ChanSpin 100 mL of culture (OD ~ 0.8-1) into two 50 mL tubes for 15 min at 1481 Relative Centrifugal Force (RCF).Discard supernatant.Resuspend pellet in 1 mL TriZol reagent.Transfer 1 mL of sample into an O-ring tube.Add ~ 300 µL of zirconia beads to the O-ring tube.Balance bead beater, press protocol 1 (3366 RCF for 30 s X3) and accept .Keep on ice for 2 min.Repeat step 6 and 7 two times (only for Precellys 24).Centrifuge samples for 1 min at 17,640 RCF.Transfer ~ 800 uL of supernatant to a 2 mL Eppendorf.11.Add 1 mL of molecular grade ethanol (95–100%) and mix thoroughly.12.Zymo-spin IIICG Column in a Collection Tube and centrifuge at 17,640 RCF for 30 Sec. Transfer 1 mL of mixture to a 13.Discard flow-through, add the rest of the mixture (~ 800 ul) and centrifuge at 17,640 RCF for 30 Sec. 14.new collection tube and discard flow-through.Transfer column into a NB: DNase I Treatment (Keep DNase on ice and store at -20°C)15.RNA Wash Buffer to the column and centrifuge at 17,640 RCF for 30 Sec. Add 400µL 16.DNase I (5 U/µl) and 75 µl DNA Digestion buffer per reaction and mix.In an RNase-free tube: add 35 µl 17.Add 80 µl of DNase I - DNA Digestion buffer mixture directly to the column.18.Incubate at room temperature for 15 min.19.Direct-Zol RNA PreWash to the column and centrifuge at 17,640 RCF for 30 Sec. Add 400 µL 20.Discard flow-through and repeat the wash step (400 μL Direct-Zol RNA PreWash and centrifuge).21.RNA wash buffer to the column and centrifuge at 17,640 RCF for 2 min.Add 700 µL 22.Transfer column to a 1.5 mL eppendorf.23.DNase/ RNase-free water to column matrix and centrifuge at 17,640 RCF for 30 Sec to elute.Add 35 µl of 24.Optional: Repeat step 23 with the eluted 35 µL (do not add more DNase/RNase water because it will dilute the RNA concentration).25.Make 10 μL aliquots into RNase free Eppendorfs to be used for MOPS gel and nanodrop analysis and keep the rest in -80°C to avoid freezing and thawing the RNA.RNA quality was evaluated through 3-(N-morpholino) propanesulfonic acid (MOPS) gel electrophoresis following Good, Winget  recommenTotal RNA extracted from 7H9 broth and cholesterol rich minimal media were used for cDNA synthesis following iScript cDNA synthesis protocol. Briefly, RNA was initially heated at 70 °C for 10 min followed by immediate cooling on ice. Thereafter, RNA was reverse transcribed using the iScript cDNA Synthesis Kit containing 5x iScript Reaction Mix, iScript Reverse Transcriptase and nuclease-free water. The reaction was carried out in a thermal cycler (Bio-Rad) as follows: priming for 5 min at 25 °C, reverse transcription for 20 min at 46 °C and reverse transcriptase inactivation for 1 min at 95 °C. The cDNA samples were then stored at -20 °C and used for polymerase chain reaction (PCR).www.snapgene.com) (v6.1.1) for sRNAs linked to cholesterol metabolism in M. tuberculosis to evaluate their integrity using the modified Zymogen spin column extraction method. The Thermo Scientific DreamTaq DNA Polymerase kit (#EP0703) was used for amplification of sRNAs in the C1000 Touch™ Thermal Cycler (#) following initial denaturation of 95 °C for 3 min, denaturation at 95 °C for 30 s, primer specific annealing temperature  Table , were de.com) v6..1 for sRM. tuberculosis. RNA was successfully extracted from clinical strains of M. tuberculosis and a laboratory strain using a modified Zymogen spin-column kit. Shown in Fig. ASdes (Lane 1), ASpks (Lane 2), MTS2823 (Lane 3) and Mcr11 (Lane 4) at 195, 80, 300 and 100 bp, respectively. The Nanodrop 260/280 and 260/230 ratios revealed no contamination of the extracted RNA with ratios between 1,88 − 2,17; and the Bioanalyzer RNA integrity number (RIN) was between 7,1 and 8.6, which can be used for further downstream application, including next generation sequencing (Table The current protocol exploits the use of high-speed homogenizers and spin-column kit to extract good quality RNA from pks Lane , MTS2823M. tuberculosis has been successfully performed using a traditional Trizol-organic solvent extraction protocol over the years [M. tuberculosis transcriptome research.RNA isolation in he years –16, 24. he years , 25–27 hhe years . The curM. tuberculosis transcriptome studies. This protocol reduced the use of toxic organic solvents since it only requires Trizol and provides good yields and pure RNA samples. Furthermore, non-coding small RNAs extracted using this protocol can be used in sequencing studies to unravel complex mechanisms in gene regulation of this infectious pathogen; as well as transcriptome research as recently published by our team [M. tuberculosis followed by RNA purification and other commercial kits can be used, provided they are able to stabilize non-coding RNA.This protocol exploit high speed homogenizer with microbeads and the commercially available spin-column kit to extract quality RNA samples that can be used in our team . It shouMycobacterium tuberculosis.The limitation in the current study is the need for the homogenizer, which can be a high-speed disruptor such as Precellys or low speed one such as the Disruptor Genie for the lysis of a thick cell wall of


Immune checkpoint inhibitors (ICIs) are increasingly being used in the treatment of advanced human malignancies. ICIs-related adverse events, including pancreatitis and diabetes, have been individually characterized in the literature. The co-occurrence of ICIs-related pancreatitis with diabetes is rare and easily overlooked, but it is often severe or fatal. We present a patient with renal tumor resection who was treated with injection of the PD-L1 inhibitor toripalimab and eventually developed acute pancreatitis and fulminant type 1 diabetes mellitus. In addition, we conducted a literature review of ICIs-related pancreatitis with diabetes. The case in our report presented with paroxysmal abdominal pain and loss of appetite. Intravenous fluids and insulin infusion improved the patient’s pancreatitis and explosive hyperglycemia. This article suggests that ICIs can affect endocrine and exocrine functions of the pancreas, while providing information and new perspectives for the diagnosis and treatment of this challenging rare disease, helping inspire clinicians for the early identification and effective management of similar cases. TherefoHowever, the widespread use of ICIs has been followed by a series of immune-related adverse events (irAEs) associated with their mechanism of action. These irAEs can influence almost all organ systems, from minor self-limiting symptoms to severe life-threatening incidents, including the dermatologic, gastrointestinal, hepatic, pulmonary, and endocrine systems . ICIs-as22) diagnosed with clear cell renal cell carcinoma (WHO/ISUP nuclear grade was 4) underwent left nephrectomy in August 2021. In December 2021, the patient began immunotherapy with toripalimab (240 mg/dose), a humanized antiprogrammed cell death 1 receptor. Eleven weeks after initiating toripalimab treatment, he visited the hospital with a seven-day history of paroxysmal abdominal pain and the resulting loss of appetite. He had a 30-year history of smoking but had quit smoking for a year and had no history of alcohol consumption. Furthermore, he had no personal or family history of endocrine or autoimmune diseases.A 58-year-old Chinese man  , 9. Afte2.3According to the Common Terminology Criteria for Adverse Events (CTCAE 5.0) and the National Comprehensive Cancer Network (NCCN) guidelines , the pat3The incidence of ICIs-related pancreatic irAEs  is relatively low, with an incidence of 0.5%-4%, but can lead to poor quality of life and even be life-threatening . ICIs-P Of the three cases collected, one patient had pancreatitis and diabetes mellitus simultaneously, similar to our case , a case The mechanism by which ICIs are associated with pancreatic injury remains unclear, although several factors may be involved. However, excessive activation of T-lymphocytes, leading to the destruction of pancreatic cells, may be an important part of the pathogenesis .PD-1 negNCCN guidelines for the treatment of ICIs-P state that for grade 2 pancreatitis (moderate), ICI treatment should be discontinued and 0.5-1 mg/kg/day prednisone/methylprednisolone should be administered until symptoms improve to grade ≤ 1 . For pan4Although the simultaneous occurrence of ICI-induced endocrine and exocrine pancreatic injuries is rare, it still requires more attention due to its life-threatening nature. Any new symptoms of pancreatic injury should be considered clinically significant for immunotherapy patients. Therefore, careful recognition and accurate diagnosis are extremely important. Despite the effectiveness of fluid therapy and insulin supplementation, the underlying mechanisms of immune pancreatic injury remain unclear and require further investigation.The original contributions presented in the study are included in the article/The studies involving humans were approved by the Medical Ethics Committee of Chengdu Shuangliu Hospital of Traditional Chinese Medicine. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. Written informed consent was obtained from the participant/patient(s) for the publication of this case report.WF, YG, and XS contributed equally to this work and share first authorship. YG and HW conceived and designed the study. WF were involved in clinical management. XZ, SZ, HZ, WY, and HW contributed to the literature search, data acquisition, and data analysis for the review. YG, WF, and XS drafted and revised the paper. YG contributed to all the figures. All authors contributed to the article and approved the submitted version.


Increasing evidence reinforces the essential function of RNA modifications in development and diseases, especially in the nervous system. RNA modifications impact various processes in the brain, including neurodevelopment, neurogenesis, neuroplasticity, learning and memory, neural regeneration, neurodegeneration, and brain tumorigenesis, leading to the emergence of a new field termed neuroepitranscriptomics. Deficiency in machineries modulating RNA modifications has been implicated in a range of brain disorders from microcephaly, intellectual disability, seizures, and psychiatric disorders to brain cancers such as glioblastoma. The inaugural NSAS Challenge Workshop on Brain Epitranscriptomics hosted in Crans-Montana, Switzerland in 2023 assembled a group of experts from the field, to discuss the current state of the field and provide novel translational perspectives. A summary of the discussions at the workshop is presented here to simulate broader engagement from the general neuroscience field. Hundreds of RNA modifications, which occur in mRNA, tRNA, and rRNA, exert control over almost every facet of RNA metabolism, including splicing, export, stability, and translation . Recent 6N-methyladenosine (m6A), and its functions in the nervous system. He presented evidence demonstrating that depletion of m6A and its reader protein Ythdf in Drosophila led to axonal overgrowth at the neuromuscular junction and in mushroom bodies -derived forebrain organoids via cell cycle and mRNA decay regulation  first discussed the role of mgulation . She thed humans . Depleti6A modification has minimal effect on mRNA stability in differentiating postmitotic neurons. Instead, it was observed that m6A modification promotes the distal localization of mRNAs associated with cytoskeletal rearrangement and cell adhesion. As a result, depletion of Mettl14 or Ythdf2 led to impaired axonal projections and growth cone advancement in the developing mouse cortex. Additionally, Kate Meyer  presented data that showcased the consequences of genetic loss of m6A or reader proteins, such as YTHDF2 or YTHDF3, on the altered subcellular localization of mRNAs within the neurites of mouse hippocampal neurons  presented findings indicating that m neurons .Collectively, these findings underscore the diverse functions and mechanisms of epitranscriptomic regulation during the development of the nervous system, from flies to mammals.6A modification during mouse peripheral nervous system (PNS) nerve injury , Parkinson’s disease, and amyotrophic lateral sclerosis ALS; . It has e injury . In this6A hypomethylation in mouse brains of AD models led to a reduction in local translation within synapses, resulting in impaired neuronal plasticity  presented findings demonstrating that masticity . Althoug1A methylated by TRMT61A and demethylated by ALKBH3. The m1A modification in the CAG repeat RNA can bind to TDP-43 and result in its truncation, cytoplasmic redistribution, and aggregation, which recapitulated TDP-43 proteinopathy observed in neurodegenerative diseases, including ALS. Shuying Sun  highlighted the m6A dysregulation in C9ORF72 repeat expansion-linked ALS and frontotemporal dementia  discussed how adenosine in the CAG repeat RNA can be mtia FTD; . Both iPJunmin Peng  demonstrated the use of the latest TMT-LC/LC–MS/MS technology in analyzing brain proteomics samples from a mouse model of AD and hundreds of AD patients. This approach led to the discovery of a large set of novel protein components of amyloid plaque, including Aβ-correlated proteins and numerous RNA-binding proteins . His lab6A selective allyl chemical labeling  discussed the use of chemical-based RNA modification sequencing techniques, such as ming SAC; , eTAM-seing SAC; , and pseing SAC; . BID-seq6A modifications exhibit heterogeneity across cells, both in terms of the proportion of cells in which a particular RNA is methylated as well as the abundance of m6A at individual sites. In addition, mRNAs contain many more sites than previously thought; however, most of these sites occur relatively rarely within individual cells of a population. This argues for the importance of regions of methylation within RNAs as opposed to individual sites. She also introduced a new DART transgenic mouse line that expresses the APOBEC1-YTH protein, enabling in vivo single-cell mapping of m6A in the brain and other tissues of interest. In vivo single-cell DART-seq in the mouse cortex identified over 170,000 m6A sites across different cell types, and revealed that microglial cells have substantial hypomethylation of their mRNAs compared to other cell types in the cortex. Lastly, she presented a new technology which combines the APOBEC1-YTH system with a fluorescent reporter mRNA to create a genetically encoded m6A sensor capable of detecting m6A dynamics in live cells.Kate Meyer utilized deamination adjacent to RNA modification targets (DART) sequencing in neural cells. Unlike antibody-based approaches that require large amounts of RNA, DART-seq can be applied to small samples, even at the single-cell level. Interestingly, m5C and pseudouridine, which are installed by NSUN6 and PUS7, respectively, on intellectual disability and aggressive behavior, utilizing multiomics analysis. In particular, the lack of PUS7 in Drosophila led to aggressive behavior and metabolic changes, which were rescued by overexpressing tRNAAsp. It has been suggested that epitranscriptomic dysregulation of metabolism may contribute to mental disorders  explored the involvement of small noncoding RNAs, such as the Piwi-interacting RNAs (piRNAs), in adult neurogenesis . Depleti1G methyltransferase, and FTO, which influences the m6A methylation levels of a subset of messenger RNAs , which synergistically promotes translation efficiency.Kathy Fange Liu  proposed the concept of direct and indirect coordination of RNA modifications between mRNA and other RNAs. She highlighted the interaction between TRMT10A, the tRNA mger RNAs . Interes6A deficiency in human microglial cells. He highlighted that m6A deficiency leads to the repression of cell growth and the activation of a viral mimicry response through reprogramming of the RNA G-quadruplexes (rG4) landscape. The increased presence of rG4 structures promoted ZBP1 dimerization, which in turn resulted in cell death.Yi-Lan Weng  discussed the impact of mΔNLS mouse model, similar to the electrophysiological defects in myasthenic syndromes , where mutations in glycyl-and tyrosyl-tRNA synthetases (GlyRS and TyrRS) inhibit mRNA translation, leading to peripheral neuropathy phenotypes . This results in insufficient production and supply of glycyl-tRNAGly to the ribosome and stalling of the ribosome on glycine codons  discussed the molecular mechanisms underlying two neurodegenerative diseases and their clinical implications. Firstly, he described how ALS-associated mutations in the nuclear localization signal (NLS) of the FUS protein are intrinsically toxic to both motor neurons and skeletal muscles, leading to structural and functional neuromuscular junction defects and motor neuron degeneration , 2017. Ayndromes . This suenotypes . Mechanie codons . Ribosome codons . The traapproach .6A modification in GSC self-renewal and GBM progression. m6A methylation in GSCs is catalyzed by METTL3 and METTL14 proteins. Dysregulation of these proteins leads to hypomethylation of transcripts in GBM, promoting the growth and self-renewal of GSCs. Treatment with an FTO inhibitor was shown to increase m6A levels in GSCs that resulted in reduced GSC self-renewal and suppressed GBM progression, suggesting that targeting the m6A machinery can be a viable approach to treat GBM  presented two distinct molecular mechanisms involved in glioblastoma (GBM) progression, glioblastoma stem cell (GSC) self-renewal, and potential clinical approaches to target GBM using inhibitors of RNA modification machinery. She discussed the role of mreat GBM . She alsigenesis . These rigenesis .The workshop participants had an active discussion of the future translational potential of neuroepitranscriptomic research and highlighted a few areas .Epitranscriptomic dysregulation has been found in a number of brain disorders, raising the possibility of its use as a biomarker for diagnosis and treatment efficacy of these disorders. Currently, traditional methods of detecting RNA modifications have limitations in terms of resolution, quantitative analysis with stoichiometry, available material quantity, and large sample requirements. However, recent advances in the field have introduced novel methods that use fusion proteins and chemical-based sequencing, allowing for analysis at the single base pair and single-cell level . FurtherWhen dealing with the diagnosis, prognosis, and treatment of diseases related to RNA modifications, embracing a comprehensive approach that encompasses various mechanisms from different RNA modifications (on different RNA molecules) has potential for novel approaches to personalized medicine. By deciphering the regulation of gene expression through RNA modifications and identifying biomarkers that arise from perturbations of the epitranscriptome in pathological conditions , we can develop customized drugs tailored to individual patients and identify specific RNA biomarkers for diverse diseases.To unravel the intricate “epitranscriptome” as an additional layer of the biological system, a future large-scale human cohort study is indispensable for comprehensive exploration. Additionally, the use of disease models such human NPCs  and iPSC6A demethylase inhibitors for various diseases, including glioblastoma and neurodegenerative conditions  in mRNA vaccines enhances their stability and translation, reduces the natural immune response to uracil, and facilitates more efficient transportation and storage of the vaccines (Kobuvirus enhances mRNA stability by interacting with ZCCHC2 and TENT4 proteins to form a mixed tailing, which is elongated with mixed sequences, thereby delaying deadenylation (The utilization of mRNA vaccines and drugs offers a promising avenue to stimulate adaptive immunity for targeting infectious diseases . For insvaccines . Becausenylation . By straThis workshop explored innovative approaches in neuroepitranscriptomic research, highlighting the potential of RNA modifications as biomarkers, and in disease modeling and therapies. Recent advances in high-resolution RNA modification analysis, coupled with integration into other “omics” disciplines, illuminate prospects for personalized medicine. Therapeutically, attention is directed toward inhibiting downstream reader proteins of RNA modification for better specificity and employing combinatorial therapies to enhance treatment outcomes. With the ongoing progression of the field, adopting a multidisciplinary strategy becomes imperative to effectively leverage these breakthroughs. In addition, collaboration between research and clinical communities will play a pivotal role in translating these concepts into tangible benefits for patients.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.S-ML: Writing – original draft. BK: Writing – original draft. CC: Writing – review & editing. AF: Writing – review & editing. CH: Writing – review & editing. AK: Writing – review & editing. KL: Writing – review & editing. KM: Writing – review & editing. G-lM: Writing – review & editing. JP: Writing – review & editing. J-YR: Writing – review & editing. ES: Writing – review & editing. SS: Writing – review & editing. DD: Writing – review & editing. YW: Writing – review & editing. Y-LW: Writing – review & editing. LP: Writing – review & editing. YS: Writing – review & editing. K-JY: Writing – review & editing. HS: Writing – review & editing.


Affiliations 1 and 2. Instead of “Department of Pharmacy, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai, China,” affiliation 1 should be “Department of Pharmacy, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China.” Instead of “Department of Pharmacy, Shanghai East Hospital, Tongji University, Shanghai, China,” affiliation 2 should be “Department of Pharmacy, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, China.”In the published article, there was an error in The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Education evolves and progresses globally in a technology-driven world, highlighting the integration of VUCA elements of volatility, uncertainty, complexity, and ambiguity in lessons. Learners are molded to equip themselves with the world and real-life knowledge in their readiness to adapt to the VUCA world. Global education consists of preparing learners for the VUCA world. This study aimed to investigate the current English as a Second Language (ESL) teaching practices and the ESL teachers’ incorporation of VUCA elements in ESL lessons. This study was conducted quantitatively using a survey questionnaire. This study was administered to 30 ESL teachers from different secondary schools in a district in Malacca. The results from the questionnaire revealed that the ESL participants had positive perceptions toward the adaptation of VUCA in the current ESL lessons. Most of the ESL teachers agreed that they adapted VUCA elements into the activities during lessons, although some showed uncertainty about their knowledge and understanding of VUCA. From the high agreement levels in the findings, it can be concluded that the ESL teachers agreed that VUCA elements through problem-based, digital-based, collaborative, and challenging activities in English lessons are beneficial to assist students’ meaningful and autonomous learning. Based on these findings, implications were made for enhancing ESL teachers’ knowledge, understanding, and skills in adapting VUCA in lessons in response to global education demand. Globalization plays a dominant role in shifting ESL lessons from a traditional approach to a more modern setting . Orazbaythe preparation of individuals to live, work, and cooperate with others,the development of students’ global competence, andthe development of individuals with creative and reflective personalities.Generally, it affects the teaching and learning process in the new era, resulting in a beneficial situation for teachers and learners . SimultaIn today’s so-called global age, English represents a medium of interconnectivity. Proficiency in the language profoundly influences individuals and communities worldwide . EnglishAccording to This study focuses on the teaching and learning English as a second language (ESL) in Malaysia as there has been a lack of discussion on the integration of VUCA in ESL lessons in Malaysia. VUCA is the acronym for volatility, uncertainty, complexity, and ambiguity. According to Developing English proficiency is crucial, as it has become the language to gain excellent knowledge of the world . BesidesTo ensure VUCA’s smooth implementation, ESL teachers must be proactive  and implWhat are the current ESL teachers’ teaching practices in association with VUCA elements?What are the ESL teachers’ perceptions on integrating VUCA elements in ESL lessons?Today, VUCA elements are integrated into 21st-century teaching and learning for learners to work collaboratively, use technological tools, and develop their higher-order thinking skills (HOTS) in lessons . AccordiThe integration of VUCA elements should begin at the school level to ensure learners’ readiness to immerse themselves in the VUCA environment. This is concurrent with global education, which aims to prepare students to live, cooperate, and collaborate with people from diverse backgrounds of a larger community . AccordiOECD has envisioned for future generations to develop skills and attitudes that will assist them to succeed in a VUCA world . Hadar eMalaysian ESL teachers must be confident and well-prepared to design the new VUCA environment to meet the current educational trends. Besides, it reflects the country’s education and management . AdditioThe study was conducted using a quantitative approach, using a survey questionnaire to collect data in accordance with the purpose of the study. A purposive sampling approach was employed to get better insights on the topics from the ESL respondents, who were the practitioners. The study results were not meant to be generalized to a larger population. This is due to the small sample size . The parThe questionnaire was designed according to the study’s aims to investigate ESL teachers’ perceptions on their teaching practices in relation to VUCA adaptation and their perceptions on the importance of integrating VUCA elements in ESL lessons. The questionnaire was developed through an online platform; Google Form, since the MoE has provided a Google account for all Malaysian teachers. The instrument consisted of 36 items, with 30 items on a 5-point Likert scale agreement. The ESL teachers were required to identify their perceptions on the topic using the Likert scale, 1 for Strongly Disagree, 2 for Disagree, 3 for Uncertain, 4 for Agree, and 5 for Strongly Agree. These items were divided into three sections: demographic information, the perceptions on current ESL teaching and lessons, and the perceptions on the importance of integrating VUCA in ESL lessons.Prior to the distribution of the study, the questionnaire went through content and face validity checking by an expert in the area of study and the researcher’s supervisor. It was then pilot-tested on four teachers. The ESL teachers involved in the pilot study were not involved in the actual study. The purpose of the pilot test is to provide an initial evaluation of the items’ internal consistency, assess the duration of answering the questionnaire, and identify potential concerns . After ovia social networking sites from the district ESL teachers’ group on Telegram. An explanation of the survey study was provided together with a Google Form link to the questionnaire. It was vital to provide the respondents with sufficient information on the area of interest and on VUCA. Considering the ESL secondary school teachers’ heavy workload and other professional commitments, the respondents were given 2 weeks to complete the questionnaire. Their participation was not made compulsory as the study required willing respondents who would like to contribute to the findings by sharing their views quantitatively. The survey questionnaire using online Google Form was employed as it was easy for the ESL teachers to access.The actual participants were contacted The accumulated data were analyzed using Statistical Package for Social Sciences (SPSS 22.0) through descriptive analysis of mean score and standard deviation. The overall mean score was interpreted in four categories of the agreement level; low, medium-low, medium-high, and high , “authentic materials” (M = 4.13) and “project” (M = 4.03) in the English language tasks. The results show that to introduce VUCA elements in ESL lessons, most of the teachers in this study carried out classroom projects based on the real-life situations that required students to use authentic materials. Such projects give students exposure to challenging real-life situations and their ways to respond to them. The teachers also added the elements such as “meaningful learning” (M = 4.37), teacher’s comfortability in teaching and learning style (M = 4.13), student’s comfortability in teaching and learning style (M = 4.10), and “enjoyable lessons” (M = 4.03) in their teaching approaches. These are indirect evidences that the teachers attempt to vary their teaching approaches that train students to adapt to the VUCA environment.Based on the mean scores above, the teachers generally adopted VUCA elements in their teaching practices. First, the teachers included “real-life situation” (M = 4.27), “process of solving the problem” (M = 4.17), and “critical thinking skills” elements (M = 4.13) in students’ learning. These results confirm that the teachers are aware of the VUCA elements where they employed problem-based learning in their ESL lessons. They viewed that this integration could enhance students’ critical thinking and problem-solving skills. Apart from that, the teachers also promoted collaborative learning in ESL lessons (M = 4.23) and projects (M = 4.10). In the 21st-century teaching and learning, using technology in VUCA environment is necessary. In that regard, both teachers (M = 3.77) and students (M = 4.07) used “technology in lessons” (M = 4.07). The teachers perceived that their students showed “better understanding” when they used technology in the ESL lessons (M = 3.93).Students also find classes enjoyable and relevant as the teachers incorporated “problems” . This response explains the teachers’ willingness to explore VUCA and the information from the ministry would assist them in its implementation. The teachers also agreed that VUCA elements are to assist learners dealing with uncertain situations (M = 3.63) and a part of the 21st-century learning (M = 3.60). This agreement shows that the teachers want VUCA to be part of students’ learning due to its ability in preparing students for the unexpected challenges. The teachers also understood what VUCA stands for (M = 3.30) and this is an indication that the teachers are familiar with its concept.The results show that the teachers had positive perceptions of the knowledge, attitudes, and practices of VUCA elements. In terms of knowledge on VUCA, the teachers mostly agreed that the Ministry of Education Malaysia should provide sufficient information on the integration of VUCA in lessons (M = 3.83), positively viewed VUCA’s integration in ESL lessons (M = 3.80), and implemented VUCA to prepare students for the real-working and volatile world (M = 3.77). These results show that most of the teachers prepared students with challenging and practical activities in the ESL classrooms that aimed to strengthen their problem-solving skills. However, the teachers showed a moderately positive response to the negative statement, “I do not think the VUCA elements suit my lessons and activities” (M = 2.30). This result indicates that the teachers carefully selected the ESL activities to ensure they suit students’ learning needs.Furthermore, the teachers also have optimistic attitudes on the integration of VUCA elements in their teaching approaches. The teachers applied VUCA to enhance students’ problem-solving skills , National University of Malaysia/ Universiti Kebangsaan Malaysia with grant no. GG-2022-031.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Identifying drug–target interactions (DTIs) plays a key role in drug development. Traditional wet experiments to identify DTIs are costly and time consuming. Effective computational methods to predict DTIs are useful to speed up the process of drug discovery. A variety of non-negativity matrix factorization based methods are proposed to predict DTIs, but most of them overlooked the sparsity of feature matrices and the convergence of adopted matrix factorization algorithms, therefore their performances can be further improved.In order to predict DTIs more accurately, we propose a novel method iPALM-DLMF. iPALM-DLMF models DTIs prediction as a problem of non-negative matrix factorization with graph dual regularization terms and The online version contains supplementary material available at 10.1186/s12859-023-05496-6. Determining the drug–target interactions (DTIs) is a key step in drug development process . HoweverLigand-based models assume that similar ligands would interact with similar proteins .The ligand based models require that a certain number of binding ligands of a given protein target should be known . DockingTo avoid above difficulties, chemogenomics based models use known target-drug interactions, chemical structures of drugs, genomic sequences of target proteins, and/or other related information of targets and drugs to predict potential target-drug interactions. The chemogenomics based models  usually In addition to chemical structures of drugs and genomic sequences of target proteins, some works have incorporated multiple types of information, such as side-effects , 18, proAs a kind of machine learning method, matrix factorization has also been used to predict DTIs and has achieved better performance than other machine learning methods . In DTIs2,1-GRMF  and SRCM2,1-GRMF . Recentl2,1-GRMF  proposedTo solve matrix factorization problems, the above methods used either the alternating least squares algorithm  or the mIn this paper, we propose a novel method iPALM-DLMF. iPALM-DLMF models DTIs prediction as a problem of non-negative matrix factorization with graph dual regularization terms and (NNDSVD)  is used Improving the non-negative matrix factorization model by adding graph dual regularization terms and The inertial proximal alternating linearized minimization algorithm with fast convergence is used to solve the matrix factorization.Extensive experimental results show that iPALM-DLMF has better performance than other state-of-the-art methods. In case studies involving the drug gabapentin and the target prostaglandin-endoperoxide synthase 2, 46 of the 50 highest-scoring highest-scoring targets predicted to interact with gabapentin and 47 of the 50 highest-scoring drugs predicted to interact with prostaglandin-endoperoxide synthase 2 have been validated by wet experiments. The case studies show that, for drugs that do not have any known target proteins and for proteins that are so far not approved as drug targets, iPALM-DLMF also has good prediction performance.The main contributions of iPALM-DLMF are as follows: http://web.kuicr.kyoto-u.ac.jp/supp/yoshi/drugtarget/. The known interactions between n drugs and m proteins are recorded by a Z. If the ith drug is approved to target the jth protein, In order to evaluate prediction performance of the proposed iPALM-DLMF, we used the same four benchmark datasets as used by most similar works. The information of the four datasets are shown in Table n drugs are stored in a The structural similarities between drugs are calculated using SIMCOMP  accordinSW be the original Smith-Waterman alignment score. The similarity information of m target proteins are denoted by a The normalized version of the Smith-Waterman score is used to calculate the sequence similarity of the target proteins . Let \doZ, drug similarity matrix Z with graph dual regularization terms and iPALM-DLMF models DTIs prediction problem as a non-negative factorization problem with graph dual regularization terms and mentclass2pt{minimX and Y represent the latent feature matrices of drugs and targets, respectively. k is the rank of X and Y, X and Y.In DTIs prediction, the non-negativity matrix factorization (NMF) of the DTI matrix is widely used to obtain low-dimensional feature representations of drugs and targets in the DTI space. The general form of the NMF is as follows:As a embedding model, the learning performance of NMF can be greatly improved if the geometrical information has been taken into account . Cai et K-nearest neighbor graphs In order to obtain geometric information of drugs and targets, two i and j in graph K most similar drugs of drugs For two drugs Similarly, the weight matrix Z is formulated as follows.The normalized graph Laplacian forms of X and Y into GDNMF optimization model, and the optimization model of GDNMF with X and Y, respectively, and In order to ensure sparsity of the matrices obtained by non-negative matrix factorization, we introduce the Z. Z equals to the sum of k leading singular factors, Z.To provide better and explainable initial component matrices for matrix factorization, non-negative double singular value decomposition (NNDSVD)  is adoptz, z, z, i-th column of X. i-th column of Y. Otherwise, For a vector or matrix mentclass2pt{minimBolte et al.  proposedG with respect to X.Model  can be tf: x is a fixed point, t is a constant, Define the proximal map of  formula  is as foLet formula  is transAlvarez and Attouch  first prG to denote the object function of the model  and area under the precision-recall curve (AUPR) to evaluate performance of methods.The cross-validation experiments are conducted under the following two scenarios . \documeiPALM-DLMF are compared with the following eight methods, namely BLM-NII , WKNKN , RLS-WNNk is 26 on NR. k is 49 on GPCR. rank k is selected from According to the original literature , 33, 34 K, we change the values of K and show the corresponding AUC and AUPR of iPALM-DLMF under the K, the performance of iPALM-DLMF can not maintain stability on different datasets. As shown in Fig. K. Therefore, based on [In order to explore the effect of performance of iPALM-DLMF with different values of entclass1pt{minimaentclass1pt{minimabased on , we set Under the entclass1pt{minimaUnder the In order to determine the effect of several techniques on performance in our proposed iPALM-DLMF, we separately assess the performance of iPALM-DLMF, iPALM-DLMF , iPALM-DLMF  is an antiepileptic drug, which is an amino acid. In the mechanism of action, gabapentin (GBP) is different from other anticonvulsant drugs which makes identifying interaction target for GBP more complicated . The knoIn the second case study, we predicted candidate drugs for the target prostaglandin-endoperoxide synthase 2 (PTGS2) on the E dataset and aimed to assess the ability of iPALM-DLMF to predict candidate drugs for targets with no known targeting drugs. PTGS2 expression has been validated to be associated with colorectal cancer. However, PTGS2 and prostaglandin-endoperoxide synthase 1 are confused in colorectal cancer pathology and therapy. The known interactions of PTGS2 with drugs is essential in clinic . The knoAccording , the whoIt is important to ensure sparseness of the matrices obtained by non-negative matrix factorization to find the novel usage of drugs in drug research. In this paper, we propose a matrix factorization based method, iPALM-DLMF, to predict interactions between drugs and targets. iPALM-DLMF uses graph dual regularization terms to capture structural information from the drug similarity matrix and the target similarity matrix. At the same time, As a kind of gradient descent methods, iPALM-DLMF can converge to KKT point. In the future, we are interested in using the idea of multi-objective particle swarm optimization  and fixeAdditional file 1. The supplementary material for iPALM-DLMF.Additional file 2. iPALM-DLMF + appendix.


Adenium obesum, that was sent for virus screening at Australian Government post-entry quarantine (PEQ) facilities after being imported into Australia from China. The AobVX genome is 6781 nucleotides in length excluding the poly(A) tail and is predicted to encode conserved potexvirus proteins and sequence motifs across five open reading frames. The RNA-dependent RNA polymerase of this virus shares the highest amino acid sequence similarity with that of nerine potexvirus 1 (58.7% identity) and nerine virus X (58.58% identity). This is the first report of a positive-sense single-stranded RNA virus in A. obesum related to members of the genus Potexvirus in the family Alphaflexiviridae.Here, we report the detection and complete genome sequence of a novel potexvirus, tentatively named “Adenium obesum virus X” (AobVX), isolated from The online version contains supplementary material available at 10.1007/s00705-023-05871-x. Adenium obesum (Forssk.) Roem. and Schult. (family Apocynaceae), commonly known as desert rose, is a perennial succulent shrub native to southeast Africa. It is a popular exotic ornamental that is gaining prominence due to its drought resistance, ease of maintenance, and the variety of colours and shapes of its abundant and long-lasting flowers [ flowers . It is a flowers  and toma flowers , but not flowers .A. obesum, which were growing in an approved arrangement at PEQ Nambour Queensland .Total RNA was extracted from a symptomatic plant, and a small RNA (sRNA)-Seq library was generated and sequenced as recommended by Lelwala et al. . De novoirReport  enabled irReport . The AobAdenium specimens tested by coupling rapid amplification of cDNA ends (RACE) [The complete sequence of the sRNA-based genome assembly, including the 5’ and 3’ untranslated regions (UTRs), was independently confirmed in one of the s (RACE)  and Oxfos (RACE) . The scas (RACE)  resultedThe genome sequence of AobVX is 6781 nt long and predicted to contain five ORFs, which are flanked by a 91-nt 5’ UTR and a 50-nt 3’ UTR  and a C-terminal RdRp domain. The region between the putative methyltransferase and the helicase domains shows the most variation both in terms of length and sequence similarity, as has been observed in other potexviruses [ORF1 (4833 nt) encodes an RdRp of 1610 aa that is predicted to contain the three conserved replicase domains of the “alphavirus-like” supergroup of RNA viruses: (a) an N-terminal putative methyltransferase domain, (b) a helicase domain beginning with the NTP-binding consensus motif “GXxviruses . ORF2  and nerine virus X (58.58%) (Supplementary Table s X Fig. . Based oBelow is the link to the electronic supplementary materialSupplementary Material 1