RNA and RNA modification in the pathogenesis, diagnosis, and treatment of cancers volume II” focuses on the areas of RNA in cancer development, prognosis, and molecular signaling pathway. We selected seven manuscripts consisting of two review papers and five research papers aimed at introducing the latest findings in both basic research and clinical application in these research areas.This Research Topic “Liu et al. indicated the recent studies on m6A regulated by the m6A “writers” (m6A methyltransferases), m6A “erasers” (m6A demethylases), and m6A “readers” (m6A-binding proteins). m6A modification could affect the stability of RNA base pairing and control RNA splicing, exportation, and translation. “Writers” are composed of methyltransferase complexes, such as methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and METTL16, a newly discovered m6A writer and reader. As “erasers,” FTO, ALKBH5, and ALKBH3 can selectively remove methylation of m6A in RNA, while YTH, HNRNPs, eIF3, and IGF2BPs can specifically decode m6A-modified targeted RNA for the m6A group to trigger biological functions as “readers.” Since m6A has a key role in chromatin remodeling involved in almost all RNA metabolism, the alterations in “writers, erasers, and readers” eventually participate in different cellular biological processes including signaling pathway transduction, cell malignancy biology, hematopoietic function, and immunity, thus the dysregulation of m6A associated with cancer occurrence, metastasis, tumor self-renewal capacity, cancer metabolic reprogramming, drug resistance generation, and tumor immune microenvironment.N6-methyladenosine (m6A) modification has been found in almost all cellular transcripts as the most abundant internal modification of RNA . The revMETTL3 is an important m6A methylation modification gene facilitating tumor progression which promotes tumor proliferation, migration, angiogenesis, and other pathways. Peng et al. revealed the role of METTL3 in GC cells by employing m6A microarray and quantitative proteomics to explore its potential effect and mechanism. This study showed that METTL3 induced significant alterations in the protein and m6A modification profile in gastric cancer (GC) cells, and downregulated proteins, which were enriched in intracellular mitochondrial oxidative phosphorylation (OXPHOS), were significantly associated with oxidative phosphorylation in METTL3 overexpressing GC cells. Highly expressed and highly methylated molecules regulated by METTL3 exhibited a worse prognosis in GC patients with three  genes.Gastric cancer is one of the most common malignant tumors with a high mortality worldwide . As showJing et al.), the authors focus on the role of RNA modification in the tumor microenvironment and its mechanism of how different RNA modifications directly affect the tumor microenvironment. The study reveals RNA-modified signatures that may have a potential role in the TME and in predicting clinicopathological features by identifying three distinct RNA modification clusters and their role in different biological pathways and starkly correlate with the clinicopathological characteristics, immune cell infiltration, and prognosis of GC patients. An RM score system used to quantify and predict the prognostic value of RNA modification in GC was developed by conducting the principal component analysis, and it shows that patients with a high RM_Score were characterized by a higher tumor mutational burden, mutation frequency, and microsatellite instability which were more susceptible to immunotherapy and had a favorable prognosis.In another research on RNA modification in gastric cancer , including microRNA (miRNA), long non-coding RNA (lncRNA), circular RNA (circRNA), and ribosomal RNA (rRNA), which can regulate gene transcription and translation and their dysregulation, has been associated with cancer development and progression . RhabdomYe et al. investigated the relationship between lncRNA and gefitinib metabolism in non-small cell lung cancer (NSCLC). Metabolized in the liver by cytochrome P450, gefitinib is effective in the treatment of locally advanced or metastatic EGFR-mutated NSCLC patients  infection remains the leading cause of hepatocellular carcinoma (HCC), which is another common malignancy with poor outcomes worldwide. RNA-binding proteins (RBPs) are evolutionarily conserved proteins that can bind their RNA targets through their functional RNA-binding domains which regulate mRNA metabolic processes including pre-mRNA splicing, capping, polyadenylation, RNA modification, transportation, localization, translation, and degradation as well as transcription control by binding to chromatin . Xu et aZhang et al. found that a high expression of NAT10 was associated with poor prognosis in MM patients. BCL-XL (BCL2L1) was screened out as a significant downstream target of NAT10, and the increase of NAT can improve the stability of BCL-XL mRNA and promote protein translation to suppress cell apoptosis via activating the PI3K–AKT pathway.The maintenance of efficient translation and stability of mRNA is very important in the development of solid tumors, and it is also of great significance in hematological tumors. N-acetyltransferase 10 (NAT10) is an important regulator of mRNA acetylation involved in the regulation of telomerase activity, DNA damage repair, apoptosis resistance, and cell cycle regulation . Zhang eRNA and RNA modification in the pathogenesis, diagnosis, and treatment of cancers volume II” highlights multiple studies for developing RNA and novel therapeutics for cancer diagnosis and treatment.In conclusion, this Research Topic “QL wrote the first draft. LW, JZ, and D-HY revised the manuscript. All authors contributed to the article and approved the submitted version.


E. coli strains illustrates that RsuA renders a survival advantage to bacteria under streptomycin stress. The RsuA-dependent growth advantage for bacteria was found to be dependent on its pseudouridylation activity. In addition, the role of RsuA as a trans-acting factor during ribosome biogenesis may also play a role in bacterial growth under streptomycin stress. Furthermore, circular dichroism spectroscopy measurements and RNase footprinting studies have demonstrated that pseudouridine at position 516 influences helix 18 structure, folding, and streptomycin binding. This study exemplifies the importance of bacterial rRNA modification enzymes during environmental stress.Bacterial ribosome small subunit rRNA (16S rRNA) contains 11 nucleotide modifications scattered throughout all its domains. The 16S rRNA pseudouridylation enzyme, RsuA, which modifies U516, is a survival protein essential for bacterial survival under stress conditions. A comparison of the growth curves of wildtype and RsuA knock-out  Post-transcriptional ribonucleotide modifications are scattered in various biologically relevant RNAs in all three kingdoms of life. Currently, more than 170 post-transcriptional ribonucleotide modifications have been observed in nature. Although RNA nucleotide modifications are implicated in influencing local RNA structure and stability, RNA–RNA and RNA–protein interactions, and RNA folding and dynamics, the exact biological role of many RNA nucleotide modifications is unknown . HoweverE. coli numbering) of 16S helix 18  ha ha2+ contion 516 a. The usE. coli 16S helix 18 residues 511 through 540 with a pseudouridine at position 516, whereas the unmodified counterpart, h18-U model RNA, has the pseudouridine at position 516 replaced by uridine. CD spectra were obtained for both h18-Ψ and h18-U constructs at varying magnesium concentrations to track structural changes during helix 18 folding. Both of the helix 18 RNAs give rise to CD spectra typical for RNAs. In the absence of Mg2+ ions, both helix 18 model RNAs give rise to a dichroic maximum at approximately 266 nm in the CD spectra. However, the molar ellipticity value at the spectral peak at 266 nm for the modified helix 18 RNA is significantly higher than that for the unmodified helix 18 model RNA  of h18 RNA decreased as the Mg2+ was added up to 1 mM. However, when the Mg2+ concentration was increased beyond 1 mM, the shoulder started reappearing  for streptomycin binding with h18-Ψ and h18-U constructs were found to be 23 ± 3 µM and 63 ± 1 µM, respectively  in the presence of pseudouridine modification at position 516 suggests that pseudouridine preferably stabilizes a structure of helix 18 that favors streptomycin binding. On the other hand, streptomycin may bind to two different regions of the helix depending on the presence of pseudouridine.According to the X-ray crystal structure of the 30S ribosomal subunit bound to streptomycin, the antibiotic molecule interacts with C526 and G527 in the h18 upper hairpin . RNase TIn bacteria, the nucleotide modification process requires a set of unique modification enzymes . The rolE. coli cells lacking RsuA under streptomycin stress. Similar to previous bacterial growth studies for RsuA deletion strains, no significant growth defects were observed in the absence of streptomycin [This research clearly illustrates a growth disadvantage for ptomycin . The incptomycin . Howeverptomycin . It is aptomycin . Interesptomycin . The inaE. coli strain supplemented with the RsuA functional mutant compared with wildtype E. coli strain also supports the idea that the pseudouridylation function of RsuA is also critical for the streptomycin resistance in bacteria. Interestingly, in M. tuberculosis, A514C and C517U mutations located in 16S helix 18 show resistance towards streptomycin, whereas, in T. thermophilus, C507A and G524U mutants are found to be streptomycin-dependent mutants [Streptomycin interacts with the 530 loop of 16S helix 18 . Interes mutants ,43,44. A mutants ,45 and cE. coli lacking RsuA were absent when catalytically inactive RsuA is overexpressed, indicating that the RsuA enzyme has a unique role in streptomycin resistance in addition to Ψ516. It is likely that the lack of nucleotide modification and their respective modification enzymes produce unique sub-optimally active ribosomes that are not sturdy enough to survive under various cellular stress conditions. Different subpopulations of ribosomes can exist in different stages of growth as well as various environmental stress conditions [E. coli that is triggered under stress conditions. MazEF-based cell death is considered a population phenomenon under stress conditions where it still results in the survival of a small subpopulation of cells. MazF leads to the selective synthesis of about 10% of total cellular proteins. The majority of the population has the expression of “death proteins”, while a small subpopulation will have “survival proteins” expressed [Some growth defects observed in nditions ,47,48,49xpressed . Interesxpressed ,47. An ixpressed . The absE. coli 16S rRNA at position 516 is likely to be critical for the structural integrity of helix 18 and its dynamics during mRNA decoding and may significantly influence bacterial growth in the presence of antibiotics that bind to its vicinity. In addition, it is also likely that the presence of RsuA, regardless of its activity, can influence the tolerance against streptomycin.In summary, pseudouridylation of E. coli numbering) model RNAs. The h18-Ψ model RNA contained a pseudouridine residue at position 516, whereas the h18-U model RNA contained uridine at position 516. These two model RNA oligonucleotides were purchased from Horizon Discovery in the 2′-ACE-protected form. RNAs were deprotected by incubating them for 30 min at 60 °C in a 2′-deprotection buffer  provided by Horizon Discovery. Deprotected model RNAs were then dried in vacufuge and stored at −20 °C until further use. RNA stocks for CD experiments were made by dissolving the deprotected aliquots in TE (1 M Tris–HCl pH 7.5 and 0.5 M EDTA pH 8.0) buffer. The concentrations of RNA stock solutions were calculated using A260 absorbance and appropriate extinction coefficients provided by the manufacturer. RNA samples used for RNase T1 footprinting experiments were radiolabeled at the 5′-end with 32P isotope using standard T4-polynucleotide kinase (PNK)-based end-labeling protocols. The radiolabeled RNAs were then purified using 16% denaturing polyacrylamide gel. RNA samples were electrophoresed for 1 h at 15W per gel. An autoradiograph was developed to identify the band representing the full-length RNA. The identified full-length RNA band was exercised, and the radiolabeled RNA was extracted onto 1 mL of TEN buffer  using the freeze–thaw method. Extracted RNAs were precipitated and dried before use.This study used two chemically synthesized 30 nt long 16S helix 18  or streptomycin concentration (0–0.5 mM). The averaged CD spectra were then smoothed using the Savitsky–Golay algorithm (n = 15). All CD experiments were performed in triplicate to ensure reproducibility. The fraction of model RNAs bound to streptomycin was calculated using CD at 265 nm and plotted against streptomycin concentrations. Equilibrium dissociation constants (Kds) for streptomycin–RNA complexes were obtained by least-square fitting of the fraction-bound versus streptomycin concentration curves of the binding isotherms, using the Origin program. CD spectra (200–320 nm) for model RNAs were obtained using a JASCO J-810 circular dichroism spectropolarimeter equipped with a water bath to control the temperature. Model RNA samples (5 μM) were resuspended in 500 µL of CD buffer . Three scans performed at 30 °C were averaged for each Mg2+ concentrations (0 mM–25 mM) or streptomycin concentrations (0 mM–0.5 mM). Each RNase T1/A digestion reaction was carried out by incubating 15 pmols of h18-Ψ/U RNAs and 1 μg of E. coli tRNA with 0.02 U of enzyme for 5 min at room temperature. Magnesium or streptomycin concentrations were adjusted with MgCl2 stock solutions (0–50 mM) or streptomycin sulfate stock solutions (0–2.5 mM), respectively. Digestion reactions were stopped by adding 1 mM aurin tricarboxylic acid and an equal volume of buffered formamide. Digestion products were run on a 16% polyacrylamide sequencing gel at 55W for 45 min. An autoradiograph was developed, and the intensities of RNase T1 cuts were quantified using the ImageJ software. The intensity of each band was normalized for the total intensity of the lane. The relative streptomycin cleavage versus streptomycin concentration curves was fitted to the binding isotherm as described in the previous section to obtain Kd for individual interaction.RNase T1 or RNase A footprints for the h18-Ψ and h18-U RNAs were obtained at various MgE. coli (E. coli K-12 BW25113: rrnB3 ΔlacZ4787 hsdR514 Δ(araBAD)567 Δ(rhaBAD)568 rph-1) were treated with varying concentrations of streptomycin ranging from 0–200 µg/mL in LB medium. They were grown at 37 °C for 18 h, and absorbance (at 600 nm) was measured with a SpectraMax4 spectrometer. The data were obtained in independent biological triplicates, and the average of the absorbance values  was plotted against (streptomycin) (µg/mL) and fitted with Origin to Equation 1 to determine the IC50 value.The RsuA knock-out (ΔRsuA) strain and the respective wildtype strain of E. coli DH5α competent cells. Plasmid DNA was extracted using QIAprep Spin Miniprep Kit  following the manufacturer-suggested protocol. The mutation was confirmed by DNA sequencing . The recombinant pCA24N vector containing the coding sequence for wildtype RsuA was purchased from the ASKA collection . The RsuE. coli strain ΔRsuA were prepared and transformed with recombinant pCA24N vector containing the coding sequence for wildtype RsuA (wtRsuA) and catalytically inactive RsuA (mutRsuA) to generate the ΔRsuA + wtRsuA, and ΔRsuA + mutRsuA E. coli strains, respectively. Transformed cells were then plated on LB agar plates containing 50 µg/mL chloramphenicol and incubated overnight at 37 °C. A single colony from each plate was used to prepare a glycerol stock of ΔRsuA + wtRsuA and ΔRsuA + mutRsuA E. coli. The expression of wtRsuA and mutRsuA (28 kDa) in E. coli (ΔRsuA) was confirmed by overexpression in the presence of 0–1 mM IPTG for 8 h followed by SDS-PAGE. Protein bands in the SDS-PAGE gels were visualized using coomassie staining. The overexpressed RsuA band intensity increased with the IPTG concentration. These gels were used to determine the IPTG concentration that gives the expected minimum level of RsuA overexpression. The selected IPTG concentration was used to obtain the growth curves for ΔRsuA + wtRsuA, and ΔRsuA + mutRsuA E. coli strains at varying streptomycin concentrations. Competent cells from the E. coli strains were obtained at 4 selected streptomycin concentrations from 0, 6, 11, and 13.5 µg/mL. Bacterial cultures from respective E. coli strains were started as a 100X dilution from an overnight culture in fresh LB media and were grown at 37 °C. The absorbance was recorded at different time intervals up to 24 h with a SpectraMax4 spectrometer. All experiments were undertaken in triplicate, and the average absorbance values for each streptomycin concentration were plotted against time (minutes). IPTG was added to the growth medium to a final concentration of 50 µM to obtain growth curves of the ΔRsuA + wtRsuA and ΔRsuA + mutRsuA E. coli strains.The growth curves for wildtype (Wt), ΔRsuA, ΔRsuA + wtRsuA, and ΔRsuA + mutRsuA 32P-labelled DNA primer (1.5 pmol) was mixed with total RNA (2 µg) in HK buffer , and the primer was annealed to rRNA by heating at 50 °C for 1 h and slow-cooling to room temperature. The samples were mixed with the non-denaturing loading buffer and run in an 8% non-denaturing PAGE gel. The intensities of the gel bands after phosphorimaging were measured to obtain the relative amounts of total rRNA (16S + 17S rRNA), 17S rRNA, and 16S rRNA . The total RNA, 17S rRNA, and 16S rRNA were plotted to investigate the influence of RsuA deletion for rRNA processing and, thereby, ribosome assembly.Bacterial pellets were collected from wildtype (Wt) and ΔRsuA strains of bacteria in the presence and absence of streptomycin (13.5 µg/mL) at 4, 8, 15, and 24 h of growth. Total RNA was extracted from each bacterial pellet using TRIzol Max Bacterial RNA Isolation Kit . Two DNA primers  that bind to mature 16S rRNA sequence and 17S leader sequence, respectively, were designed and purchased from Integrated DNA Technologies Inc. The Pseudouridylation enzyme RsuA and its activity can influence the growth of bacteria under streptomycin stress. The ability of Ψ516 to influence 16S helix 18 folding and streptomycin binding is partly the cause of streptomycin tolerance in bacteria. In addition, RsuA may function as a ribosome assembly factor that plays a role during streptomycin stress that is yet to be discovered.


The chemical modification of RNA bases represents a ubiquitous activity that spans all domains of life. Pseudouridylation is the most common RNA modification and is observed within tRNA, rRNA, ncRNA and mRNAs. Pseudouridine synthase or ‘PUS’ enzymes include those that rely on guide RNA molecules and others that function as ‘stand-alone’ enzymes. Among the latter, several have been shown to modify mRNA transcripts. Although recent studies have defined the structural requirements for RNA to act as a PUS target, the mechanisms by which PUS1 recognizes these target sequences in mRNA are not well understood. Here we describe the crystal structure of yeast PUS1 bound to an RNA target that we identified as being a hot spot for PUS1-interaction within a model mRNA at 2.4 Å resolution. The enzyme recognizes and binds both strands in a helical RNA duplex, and thus guides the RNA containing the target uridine to the active site for subsequent modification of the transcript. The study also allows us to show the divergence of related PUS1 enzymes and their corresponding RNA target specificities, and to speculate on the basis by which PUS1 binds and modifies mRNA or tRNA substrates. TruA, was identified, and found to modify tRNA in bacteria [6-methyladenosine (m6A) modifications [The cellular transcriptome throughout all domains of life displays a highly complex regulatory network of more than 150 known post-transcriptional RNA modifications that modulate RNA biogenesis, function, specificity, and stability . Pseudoubacteria , 8. Moreications –14. DespTwo unique classes of pseudouridine synthase enzymes, that are jointly responsible for pseudouridylation of RNA, differ in the way that they target their RNA substrates. Guide RNA-dependent PUS enzymes in eukaryotes and archaea (such as Cbf5 in yeast and dyskerin in humans) are part of a box H/ACA ribonucleoprotein complex  that utilize small nucleolar RNAs (snoRNAs) as guides that recognize and base-pair with its substrates, which are mostly non-coding RNAs (ncRNA) . AlternaEukaryotes generally contain multiple unique stand-alone PUS enzymes , which differ in their substrate preference and localization in the cell. Each of them modifies specific sites in tRNAs, snRNAs, and rRNAs by targeting a sequence and/or structural element in their respective substrates , 20. TheIt remains somewhat unclear which PUS enzymes are responsible for the pseudouridylation of messenger RNA (mRNA). Various studies have indicated that more than half of the Ψ modifications in mRNA are catalyzed by the tRNA-specific PUS4 (a member of the divergent TruB family of PUS enzymes) and PUS7 (TruD family) through recognition of tRNA-like structures  and TruB, each bound to tRNA [E. coli tRNA-TruA complex [Only a few structures of eukaryotic stand-alone PUS enzymes have been solved: the catalytic domains of human PUS1 and PUS10 –43, as w to tRNA , 46. Des complex .S. cerevisiae PUS1 and a catalytically inactive PUS1 mutant in complex with short mRNA fragments. The structures, from two unrelated crystal forms, represent the first structures of a eukaryotic stand-alone PUS enzyme bound to a target RNA. The structures illustrate how PUS1 recognizes and binds a helical RNA duplex and enable the identification of RNA-contacting amino acid residues that make extensive contacts with each RNA strand to orient and guide the RNA into the active site of the enzyme. Additional examination and comparison with previously described structural and biochemical studies indicate that a) while PUS1 and TruA use the same protein surface to interact with their respective target-RNAs, the position and identity of the corresponding contact residues and the position of the bound RNAs differ significantly from one another, and b) PUS1 likely binds and acts on at least one tRNA target in a manner that is closely related to how it engages with its docking site in mRNA.Because visualization of how PUS1 binds to mRNA might provide new insight into the basis of its action on such substrates, we generated a pair of crystal structures of wildtype S. cerevisae PUS1  was used for most biochemical assays. A catalytically inactive PUS1 was generated by single point mutation of active site residue aspartate 134 into an alanine (PUS1D134A). For biochemical characterization, PUS1 expression plasmids (E. coli (NEB # C3010) and grown in Luria Broth media at 37°C. Expression of the PUS1 variants was induced by the addition of 0.4 mM IPTG at OD600 0.6. Cells were harvested after incubation for two hours at 37°C, washed with cold 1x PBS, resuspended in HisTrap binding buffer  + 1x protease inhibitors  and stored at -20°C. Cells were thawed, sonicated, and after centrifugation, the supernatant was saved and applied to a 5 ml HisTrap HP column (Cytiva) calibrated with HTBB. PUS1D134A was eluted with HisTrap elution buffer . Fractions containing PUS1 were pooled and dialyzed into HiTrap Heparin binding buffer .N-terminally HIS-tagged plasmids  were tra2HPO4, 2 M NaCl, 20% glycerol, 1 mM DTT). The fractions containing PUS1 were pooled and dialyzed into HiTrap SP binding buffer . The dialyzed PUS1 was applied to a 5 ml HiTrap SP HP cation exchange column and eluted with HiTrap SP elution buffer . Finally, fractions containing PUS1 were pooled and dialyzed into storage buffer  and stored at—20°C.The dialyzed protein was applied to a 5 ml HiTrap Heparin column (Cytiva) and eluted with HiTrap Heparin elution buffer  using a Gibson Assembly cloning kit and protocol (NEB # E5510S) and sequence verified. A clone encoding tagless wild type PUS1 was then generated from PUS1D134A by converting alanine 134 back to wild type aspartate 134 using a QuickChange II XL kit and protocol (Agilent).To generate an untagged version of the PUS1 enzyme for crystallography trials, PUS1Sequence-verified constructs were transformed into BL21-CodonPlus(DE3)-RIL competent cells (Agilent) and expressed using a previously described autoinduction protocol . Bacteri5’ - gcgaaattaatacgactcactataggg – 3’). 1 μg of the duplex was in vitro transcribed using the HiScribe™ T7 Quick High Yield RNA Synthesis Kit (NEB # E2050S) following the standard IVT protocol and purified using Monarch® RNA Cleanup columns (NEB # T2030).A DNA duplex containing the T7 promoter followed by firefly luciferase (Fluc) mRNA  coveringin vitro transcript (IVT) in 1x NEB buffer 1.1 for 90 min at 30°C in a 100 μL reaction. Reactions with full length Fluc mRNA contained 2.1 μM PUS1 and 8.3 nM Fluc mRNA). Reactions were stopped by adding 0.8 units of Proteinase K (NEB # P8107S) followed by incubation for 10 min at 37°C. The modified RNA was subsequently column-purified (NEB # T2030), 2 pmol were digested to single nucleosides using the Nucleoside Digestion Mix (NEB # M0649), and the ratio of Ψs versus uridines was determined via tandem quadrupole mass spectrometry  PUS1 was incubated with 84 pmol  synthetic RNA oligos . Drops that generated visible crystals were then used to set up subsequent screening and expansion trays by hand, with 2 microliter drops equilibrating against 1000 microliter reservoir volumes.D134A) at 11.6 mg/mL and a slight molar excess of RNA  crystallized at 4° C after equilibration against a reservoir containing 0.05 M ammonium sulfate, 0.05 M Bis-Tris pH 6.0, and 26% pentaerythritol ethoxylate. In contrast, a mixture of wildtype PUS1 (PUS1wt) at 11 mg/mL in the presence of a similar RNA construct harboring a 5’-fluorouracil moiety at a position corresponding to potential enzymatic modification  crystallized at 25°C after equilibration against a reservoir containing 0.2 M potassium sodium tartrate and 18% PEG 3350 at 25°C.A mixture of catalytically inactive PUS1   as a seain vitro transcribed Fluc mRNA  that was unable to pseudouridylate RNA  that is strictly required for activity , 55. Sublate RNA .wt or PUS1D134A either. We then focused on RNA oligo R263  . Unfortunately, neither of these oligos formed crystals with PUS1in vitro , it was in vitro . When tes (EMSA) . We therD134A inactive enzyme, were successfully grown and found to belong to space group C2  of 0.222 and 0.265 respectively, with tight protein geometry . The average B-factor values for the protein and the bound RNA were comparable . The electron density maps from molecular replacement and subsequent rounds of refinement displayed well-ordered density for most of the protein chain  and the first 12 bases of the RNA substrate .PUS1-RNA co-crystals that diffracted to 2.4 Å resolution, containing the R263 RNA construct and the PUS1The contents of the crystallographic asymmetric unit correspond to a single protein subunit and a single strand of the bound RNA ligand . That coIn contrast to the extensive base-paired contacts between the two crystallographically related RNA strands, the corresponding pair of bound protein molecules do not display significant contacts with one another; the few contacts between the two bound protein subunits are limited to two surface-exposed loops (residues 86 to 89 and 95 to 98) in the N-terminal region of the enzyme. Therefore, we believe that the two copies of PUS1 that are associated with opposite sides and ends of the symmetric RNA duplex are bound as individual monomers, independently of one another. That conclusion agrees with the solution behavior of the purified enzyme, which eluted from a size exclusion column as a monomeric species at high micromolar concentrations of protein .Within the complex between a single bound protein subunit and the RNA duplex, at least thirteen amino acid side chains contact numerous atoms on each of the two RNA strands . The resH98A and PUS1K363A still modified their substrate  or cluster B  that are in close proximity to the RNA strands. While replacing histidine 89 with alanine only had a weak effect on PUS1 activity and/or specificity when tested with the R169 RNA oligo (~ 29% reduction of activity), mutating arginine 132 to alanine dramatically reduced pseudouridylation of the R169 RNA oligo by approximately 87%, suggesting an important functional role of this amino acid side chain in coordinating the RNA in the active site of the enzyme . Despiteubstrate .To test whether mutations that significantly reduced PUS1 activity affected substrate binding, we performed EMSAs of wildtype and mutant PUS1 enzymes with the RNA substrate used to generate the crystal structures (R263) as described in Materials & Methods. Neither of the catalytically impaired cluster A and B mutants showed a reduction of substrate binding . While tD134A bound to the R263 duplex, the unpaired 5’ base (A1) is positioned within 4.5 angstroms of residue 134  and appears to be within potential distance to rotate into the enzyme active site  belonged to a different space group (P6122) and diffracted to 2.9 Å resolution (PDB 7R9F). Although the space group and corresponding lattice packing arrangement of the protein-RNA enzyme complex was completely unrelated to the packing of the C2 space group of the PUS1D134A:RNA complex, the same RNA duplex, and the same position of two independently bound protein subunits was observed  by the 5’ 5FU nucleobase of the RNA . HoweverE. coli TruA and their respective RNA substrates revealed that the orientation of the TruA-bound leucyl tRNA results in a clash of the tRNA with a yeast specific large insertion, spanning residues S206 to approximately L279  and the structure of bacterial tRNA pseudouridine synthase TruA (PDB 2NR0), with a rmsd across all comparable alpha carbons of approximately 2 Å in both pairwise superpositions . PUS1 an279 Figs  and 6.Previous biochemical studies have provided solid evidence indicating how PUS enzymes might target and interact with their ncRNA substrates , 14, 40.While bacterial TruA has been reported to crystalize as a homodimer , other PUS enzymes–including human PUS1 , 42–funcA question that immediately arises from our analyses is if the base-paired RNA duplex observed in the crystal structures represents a physiological and/or mechanistical state that influences PUS1 specificity and activity on mRNA. The Schwartz laboratory recently reported the importance of double-stranded stem RNA-structures for mRNA pseudouridylation by TruB1 and PUS7 through high-throughput Ψ mapping . Their din vitro. Assuming that pseudouridylation occurred at specific sites rather than randomly throughout the oligo, this suggests that either one (R167/R168) or two specific uridines (R169) were modified by PUS1. Accordingly, PUS1 substrate selectivity–at least in vitro–may be less restricted than recently reported.In contrast, PUS1 and its bacterial counterpart TruA are promiscuous in their target selection, modifying multiple RNAs with divergent sequences in the region of modification . IlluminThe crystal structure presented here provides mechanical insight into why PUS1 requires a double stranded RNA stem for target recognition, binding, and activity. Our data show that each individual PUS1 subunit makes extensive contacts to both strands in the RNA duplex. Those RNA-contacting residues, which are well-conserved across PUS1 homologues, appear to enable binding of an RNA double helix and position the 5’ flanking region of the bound RNA strand close to the active site in PUS1. We find that alanine mutations of PUS1 residues arginine 132 and arginine 362 significantly reduce PUS1 activity. Interestingly, these two residues contact opposite strands in the RNA double helix, further supporting the hypothesis that an RNA double helix structure is important for binding of the RNA substrate and/or the positioning of the pseudouridylation site in the active site of the enzyme.One explanation for yeast PUS1’s less restricted mode of structure recognition could be the location of its thumb loop (Tyr 83 –Thr 99). In the PUS1:mRNA structure, the loop is ordered and pointing away from the active site. This orientation results in a significantly wider opening of the active site cleft compared to TruA or human PUS1 , which mIn agreement with our finding that PUS1 does not significantly modify the R263 substrate, none of the uridines within the crystallization oligonucleotide are located near the enzyme’s active site in the crystal structures but are instead involved in duplex formation via base pairing interactions between RNA strands. The structure suggests that an RNA-duplex with a minimum length of 10 to 12 nt is required for all RNA:PUS1 interactions to stably form and to position the RNA correctly in the active site. This agrees with a 11 bp median length of double stranded stem structures found in PUS1 mRNA targets . Our PUSH89GMQYNPPN97 in PUS1; Y24GWQRQNEV32in TruA) that contact unique nucleotide identities and conformations in their corresponding RNA targets. In addition, the orientation of the TruA-bound leucyl tRNA causes a significant clash with two helices in a yeast specific insert (S206 –L279), providing further evidence for divergent evolution of these enzymes.Comparison of the structures solved in this study with that of bacterial tRNA pseudouridine synthase TruA  (a repreIle from Staphylococcus aureus (PDB ID: 1QU3), which in eukaryotes is known to be modified within its anticodon loop by PUS1 [Ile substrate, via its anticodon stem loop, onto the RNA duplex from the crystal structure positions the tRNA anticodon loop and the uridines at positions 34 and 36 adjacent to the enzyme’s active site  and catalytically inactive PUS1  run as monomers on a HiLoad 16/60 Superdex 200 column. Standards (Bio-Rad Gel Filtration Standards # 1511901) are overlaid in dotted gray and appropriate peaks labeled. (inset) SDS-PAGE of the final purification product of PUS1wt without (-) or with (+) dithiothreitol.Wildtype PUS1 (PUS1(PDF)Click here for additional data file.S2 Fig(a) Schematic view of the RNA substrates generated and tested to determine the optimal RNA substrate for crystallography. The black bar represents the full-length Firefly luciferase (Fluc) mRNA sequence. The six bars above Fluc represent ~ 300-mers of the Fluc sequence. The respective start- and end-positions are shown above the segments, the number of total uridines per segment (x U) and the percent of uridine-to-Ψ conversion in a PUS1 reaction after two hours (data from two independent replicas) are shown inside the bars. The bars below Fluc represent ~ 60-mer substrates between Fluc positions 691 and 993. (c) Table showing the sequence of the RNA oligos tested (uridines are highlighted in red), the average percent of uridine-to-Ψ conversion from at least two independent experimental replicates, the number of uridines per RNA oligo, and its respective size in nucleotides. (d) RNA secondary structure predictions of the RNA oligo substrates by MXfold2 . Uridine(PDF)Click here for additional data file.S3 FigPercent of uridine-to-Ψ conversion as determined by LC-MS/MS of increasing concentrations of PUS1 wildtype (left) and D134A mutant (right) with the RNA oligo substrate R168 after two hours. Data of two replicas are shown.(PDF)Click here for additional data file.S4 FigD134A (right) as shown above the gels with 100 nM of RNA oligo R263. Free R263 RNA oligo and the PUS1:RNA complex are indicated.Electrophoretic mobility shift assays of increasing concentrations of wild type PUS1 (left) and PUS1(PDF)Click here for additional data file.S5 FigAmino acid residues that interact with the substrate RNA are highlighted by red boxes.(PDF)Click here for additional data file.S6 Figwt reaction. (b) Protein gel comparing the input amount and purity of the PUS1 variants in the in vitro reactions. (c) Electrophoretic mobility shift assays of the mutant PUS1 enzymes .(a) Table showing the average percent of uridine-to-Ψ conversion of the reactions shown in (PDF)Click here for additional data file.S7 FigD134A / RNA complex (colored protein backbones) solved in crystallographic space group C2 and wild type PUS1 / RNA complex (grey protein backbones) solved in an unrelated, P6122 space group. In both structures, two monomers of the enzyme are independently bound in a symmetric arrangement to an RNA duplex that is generated via crystallographic 2-fold symmetry. (b) Model and electron density for wild type PUS1 and the region of the RNA containing the 5’ 5-fluorouracil base. A 2Fo-Fc map (gray) is contoured at 1s. In the corresponding refinement, the base is estimated to display greater than 90% occupancy of the unflipped conformation, as modeled and shown.(a) Superposition and side-by-side comparison of PUS1(PDF)Click here for additional data file.S8 FigThe conserved catalytic aspartate at position 134 in PUS1 and position 60 in TruA is indicated by a yellow box. RNA-interacting residues are highlighted in green (PUS1) and magenta (TruA) (top panel). Distribution of RNA-contacting residues in the protein-subunit interface for PUS1 (left) and TruA .(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Access to comprehensive sexual and reproductive health (SRH) services remains a challenge worldwide. Describing community pharmacists’ SRH services in countries with different scopes of practice will aid in understanding how pharmacists view their roles and how to support them in providing needed services. A cross-sectional web-based survey was administered to pharmacists working in community pharmacies in Japan, Thailand, and Canada. The survey covered 7 SRH categories: pregnancy tests, ovulation tests, contraception, emergency contraception, sexually transmitted and blood-borne infections, maternal and perinatal health, and general sexual health. Descriptive statistics were used to analyze the data. A total of 922 eligible responses were included in the analysis . Most Thai and Canadian participants reported dispensing hormonal contraceptives  and emergency contraceptive pills . Most Japanese participants provided patient education on barrier contraceptives for men (56%) and information on the safety of medications in pregnancy (74%) and breastfeeding (76%). The majority of participants expressed interest in additional training and expanding their roles in SRH. Sharing international experiences can guide challenges faced by the evolution of pharmacists’ practice in SRH. Providing pharmacists support could help their readiness for this role. Access to comprehensive sexual and reproductive health (SRH) services remains a challenge worldwide. According to the World Health Organization (WHO), 4.3 billion people of reproductive age worldwide will not have access to at least one SRH intervention in their lifetime . More reThe evolution of pharmacy practice is an international phenomenon. Development, adoption, and integration of new roles and responsibilities by pharmacists have been the trend over the past few decades ,12,13. BSeveral factors prevent the translation of contemporary patient-oriented pharmacy practice models globally ,18. One Given the prominent role in communities as part of primary health care, describing community pharmacists’ SRH services in countries with a different scope of practice will aid in understanding how pharmacists view their roles, see themselves as SRH providers, and what support they need to provide SRH services. To our knowledge, there is limited information regarding pharmacists’ experiences as SRH providers in Canada ,22,23,24The general characteristics and differences among educational programs, pharmacy regulations, and scope of practice between Japan, Canada, and Thailand are highlighted in The number of years required to obtain a pharmacy degree is similar in these countries. In Japan, completing the extended Bachelor of Pharmacy (6 years) is mandatory to qualify for the national examination for entry to practice. Influenced by the US-Thai consortium, Thailand developed their first Doctor of Pharmacy (PharmD) program in 1999. Since 2014, a 6-year PharmD program has been compulsory for national licensure . PharmacThe scope of practice differs in each country. In Canada, Alberta is the province with the broadest scope of practice and was the first to implement prescribing authority for pharmacists in 2007 . LegislaPharmacy practice in Japan is principally focused on dispensing medications and providing patient counselling . HoweverIn the last decade, Thailand has expanded community pharmacists’ roles to meet the needs of the public. After more than 10 years since transitioning to a 6-year PharmD program , communiThis was a descriptive, cross-sectional international study using survey methods. Results were analyzed and reported following the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for cross-sectional studies .An anonymous, web-based survey was distributed electronically in Japan, Thailand, and Canada (Alberta) using REDCap. REDCap is an electronic data capture tool hosted by the Women & Children’s Health Research Institute at the University of Alberta ,51. The The survey was conducted from June 2020 to June 2021, with each country distributing the survey at different times. The period to collect responses was 1 to 6 months; in Thailand and Japan, the survey was open for 6 months due to slower response. Details about the strategies used are provided in The development and content of the survey have been described previously ,53. The The survey covered 7 common SRH categories: pregnancy tests, ovulation tests, contraception , emergency contraception, sexually and blood-borne transmitted infections, maternal and perinatal health, and general sexual health, and included 6 sections: demographics, provision of SRH services, attitudes towards the provision of SRH services, confidence when educating patients on SRH topics, factors influencing the provision of SRH services, and additional training preferences . The surA 5-point Likert-type scale was used to assess attitudes towards  and factors influencing the provision of SRH services . The primary outcomes were the proportion of participants providing SRH services, the proportion of participants agreeing (or disagreeing) with a series of statements regarding pharmacists as SRH providers and the influence of factors in the provision of SRH services. Secondary outcomes were the proportion of participants interested in expanding their roles in SRH and additional training preferences, including SRH knowledge and competencies.Survey responses were anonymous. A neutral language was used when designing the survey questions to minimize response bias. Increasing the number of days the survey was open and sending invitation and reminder emails were strategies used to reduce non-response bias and improve response rates ,55,56.There was no sample size set a priori.For analysis, eligibility was defined as submitted survey responses. Partial and unsubmitted responses were not included. Descriptive statistics were used to analyze the variables. Analysis was performed using Microsoft® Excel v.16.70 (Microsoft Corporation 2018).This study received approval from the Josai International University Research Ethics Review Committee (10M200001), the Chulalongkorn University Research Ethics Review Committee (114.1/63), and the University of Alberta Health Research Ethics Board (Pro00095881).A total of 1,265 pharmacists attempted the survey , and 922 eligible responses from community pharmacists were included in the analysis . Participants were primarily female and between 20 and 40 years of age . RegardiAll categories of SRH services were, to some extent, provided in the three countries . HoweverOverall, participants’ attitudes in all three countries were positive toward pharmacists’ roles in SRH . Most paIn general, most participants from the three countries responded that all factors included in the survey influenced the provision of SRH services to some extent . In JapaMost respondents expressed interest in additional training in SRH services . More than half of the participants in Japan (54%) and about three-quarters of the participants in Thailand and Canada  were interested in expanding their roles in SRH.Participants’ preferences varied between countries. For Japanese participants interested in additional training, the top preferences were emergency contraception (77%) and hormonal contraceptives (63%), followed by pregnancy/postpartum/breastfeeding (59%). Additionally, the preferred areas for Thai participants were sexual dysfunction (68%), pregnancy/postpartum/breastfeeding (59%), and sexual health concerns of LGBTQ+ patients (55%). In Canada, approximately half of the participants or more preferred more training in the areas of STBBI treatment (64%), STBBI prevention (55%), and sexual health concerns of LGBTQ+ patients (50%) .In terms of SRH competencies, more than 60% reported interest in additional training related to the use of appropriate and straightforward language when counselling patients . This was the preferred choice for participants in Japan and Thailand, while for Canadian participants, their top selection was additional training on providing referrals (75%). Less than half of the participants reported interest in additional training related to confidentiality and privacy . A higher proportion of Japanese participants considered approaching individuals nonjudgmentally a relevant topic for training (55%) .This multi-country study examined SRH services provided by pharmacists from regions with different regulations around pharmacy practice and perspectives regarding SRH. Results showed that pharmacists were involved in providing SRH services and had a positive attitude towards pharmacists’ roles in SRH. Participants’ perceptions of factors influencing the provision of SRH were similar in the three countries, as well as the willingness to expand roles and receive additional SRH training. The findings from this study can be used to inform education and training needs as well as potential barriers to the adoption of SRH services offered by community pharmacists.Overall, a high percentage of Alberta pharmacists reported providing patient education across several SRH areas in addition to the provision of emergency contraception. This may be a reflection of changes in the scope of practice  and healWe found that community pharmacists from countries with different scopes of practice see themselves as SRH providers and consider offering advice on SRH topics an essential part of their roles. Still, they face similar challenges when providing SRH services. Regardless of differences in educational, economic, and healthcare system contexts, a set of competencies essential for patient care practice are similar in all regions ,61. ProvOne of the common findings was the willingness to receive more SRH training. However, the top preferences differed between countries, which can be influenced by the role participants associate with being an SRH provider. The focus of Japanese participants was emergency contraception, one of the main SRH discussion topics on the public agenda in Japan . It is nTo position pharmacists as SRH providers and increase access to SRH services, it is relevant that they see themselves as SRH providers. Recognizing that pharmacists’ process to identify as SRH providers is dynamic, complex, and influenced by many factors will help to find the best tools and strategies to support their identity formation. Sharing international experiences on the influence of the changes in the profession can help to face challenges brought by this process. Exploring what different countries do to support their pharmacy workforce to prepare them to be SRH providers can inform the development of training programs and implementation strategies customized to the practice environment. Expanding the offer of continuing education programs and creating pharmacist-specific guidelines and practice tools based on the context of each jurisdiction are some of the recommendations of this work.The strengths of this study include the description of community pharmacists’ perspectives regarding the provision of SRH services in Canada, Japan, and Thailand. This study is the first to characterize perceptions of SRH practice in several topics from pharmacists in countries with different scopes of practice. Despite these strengths, several limitations need to be considered. This study may not be generalized to other regions in the selected countries and nations. Due to our study design, the results are limited due to volunteer, acquiescence, social desirability, and non-response bias. The sample size was not calculated a priori, and the number of participants varied between countries. No current detailed pharmacy workforce statistics are available to consider the representativeness of the population we aimed to survey in Japan and Thailand. However, the representativeness of this sample of the Canadian (Alberta) pharmacists’ population has been described before . The surCommunity pharmacists in countries with different scopes of practice and pharmacy regulations reported providing services in several SRH areas. While the extent to which pharmacists self-report the provision of SRH services varied in each country, pharmacists were interested in expanding SRH services and additional SRH training. Addressing factors that influence the provision of SRH services and supporting pharmacists is essential for expanding access. Sharing international experiences can guide challenges faced with the evolution of practice in SRH. Introducing support strategies based on local needs and international experiences, such as creating continuing education opportunities and practice tools, could help support pharmacists in providing care for individuals seeking SRH services.


Published predictions of 5-methylcytosine at GCU sites inferred from direct RNA sequencing and the Tombo Alternative Model should be reconsidered unless further validated with orthogonal methods. 5C) modifications is Tombo, which uses an “Alternative Model” to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including viruses, bacteria, fungi, and animals. The algorithm consistently identified a m5C at the central position of a GCU motif. However, it also identified a m5C in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this is a frequent false prediction. In the absence of further validation, several published predictions of m5C in a GCU context should be reconsidered, including those from human coronavirus and human cerebral organoid samples.RNA modifications, such as methylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m Oxford Nanopore Technologies (ONT) direct RNA sequencing  enables 5C) modifications, including two for detecting m5C in a single sample: “Alternative Model” and “de novo Model” (Preprint). The Alternative Model detects signals that fall within the expected range for a m5C modification. In contrast, the de novo detection method identifies differences from a canonical base model and is unable to predict the type of putative modification. Given that these methods can be run on a single sample, they have been used to predict modifications in many biological samples including Arabidopsis thaliana . Sample Compare can detect differing levels of modification between two biological samples, or it can detect putative modifications in a single sample through comparisons with unmodified RNA generated by in vitro transcription (IVT) or knockout/knockdowns of modifying enzymes. However, this is limited by the ability to generate IVT RNAs or knockout/knockdowns. Sample Compare has been used to detect native SARS-CoV-2 modifications relative to an IVT control, but the Sample Compare predictions differed from those generated with the Alternative Model , Drosophila ananassae Hawaii , Candida albicans SC5314 , and Escherichia coli K-12 MG1655   tail for annealing, E. coli total RNA was first polyadenylated using E. coli poly(A) polymerase before library preparation. This resulted in a larger proportion of ribosomal RNA present in the E. coli data (Table S1). Sequencing resulted in 822 Mbp B. malayi, 809 Mbp D. ananassae, 2.4 Gbp C. albicans, and 216 Kbp E. coli mapped reads (Table S1).To broadly assess RNA modification patterns, RNA was isolated or existing data were used from diverse organisms including 3 genes)  .The Tombo Alternative Model detection method  was usedStatistics for the 10-nt sequences surrounding the top 1,000 5-methylcytosine fractions for each sample.Table S2. 5C motif that spans multiple kingdoms of life. The latter would be similar to the DRACH motif observed for N6-methyladenosine (Preprint) and in vitro transcribed RNA from A549 cells (Preprint) also indicated methylation of the GCU motif in both samples  RNA genome was in vitro transcribed and compared with native viral RNA from infected JW18 insect cells, which contains modified bases , 2022. Aexamined  and S11.modified .Quantification results from LC-MS/MS analysis of Sindbis virus in vitro transcription RNA.Table S4. 5C-specific pretrained model has made the Tombo Alternative Model an attractive tool for epitranscriptomics analysis. Using this method, a GCU motif is consistently predicted to contain a central m5C in our data across diverse taxa and in data reported by others  Research Protocol 8.4 (www.filariasiscenter.org). The L3 larvae were injected into the peritoneal cavities of Mongolian gerbils , and female adult worms were harvested from the peritoneal cavities after euthanizing the gerbils. The worms were washed in warm RPMI media containing 0.4 U penicillin and 4 μg streptomycin per ml, flash-frozen, and stored at −80°C. RNA was isolated using TRIzol  and homogenized in a TissueLyser (QIAGEN), followed by a PureLink RNA Mini column (Ambion). After isolation, RNA was treated with the TURBO DNA-free kit  according to the manufacturer’s protocol.RNA was isolated from adult female  methods . BrieflyC. albicans strain SC5314 was grown overnight in YPD at 30°C at 225 rpm on an Excella E25R rotary shaker (New Brunswick Scientific). After RT centrifugation at 3,500 rpm using an SX4750 rotor in an Allegra X-14R centrifuge (Beckman Coulter) for 10 min, the cell pellet was resuspended in 1 ml TRIzol  reagent per 100-mg pellet. β-Mercaptoethanol was added at a ratio of 1:100, and the pellet was homogenized using a bead beater and TissueLyser (QIAGEN) at 30 s−1 for 8 min. After centrifugation at 12,000g for 5 min at 4°C, followed by incubation at RT for 5 min, 0.2 ml chloroform was added for every 1 ml TRIzol. The sample was shaken by hand for 15 s, incubated at RT for 3 min, loaded into a prespun Phase Lock Gel heavy tube (5Prime), and centrifuged at 12,000g for 5 min at 4°C. The upper phase, along with 1 vol of 100% ethanol, was loaded onto a PureLink RNA Mini column (Ambion). The remaining steps were performed according to the Pure Link RNA Mini Kit protocol, and the RNA was eluted in 30 μl RNase-free water and stored at −80°C. Before sequencing, poly(A) selection was performed according to the Dynabeads mRNA Purification Kit protocol (Thermo Fisher Scientific).A single colony of E. coli K12 MG1655 was grown overnight in LB broth at 37°C at 200 rpm on an Excella E25R rotary shaker (New Brunswick Scientific). The overnight culture was diluted 1:100 in fresh LB broth, incubated at 37°C with aeration at 200 rpm on an Excella E25R rotary shaker (New Brunswick Scientific), and harvested at OD600 0.50. RNA was isolated from pelleted cells using RNeasy Mini Kit (QIAGEN) with QIAGEN RNAprotect Bacteria Reagent and on-column DNase digestion following the manufacturer’s instructions.To create a starter culture, a single colony of Wolbachia (strain wMel) colonized, Drosophila melanogaster–derived JW18 cells grown in serum-free Shields and Sang media at MOI of 10. Infected JW18 cells were harvested 5 d post-infection, and total RNA was extracted using TRIzol reagent (Invitrogen), followed by RQ1 RNase-free DNase (NEB) treatment using the manufacturer’s protocol.SINV stocks were generated as previously described . Briefly4 acetate, and 2 vol ethanol, followed by chilling at −20°C for 15 min. The DNA was pelleted for 15 min at max speed, and the supernatant was removed, followed by resuspension in 2 μl water. SINV RNA was in vitro transcribed from the linearized DNA template according to the Invitrogen MEGAscript SP6 Kit (Thermo Fisher Scientific Inc.) protocol with an incubation time of 2.5 h and addition of 2 μl m7G RNA cap analog (NEB), followed by precipitation with the Lithium Chloride Precipitation Solution provided in MEGAscript SP6 Kit.IVT was carried out using the TE12 BC 4.10 plasmid received from the Hardy Lab at Indiana University Bloomington. The pGEM-based plasmid contains a previously described SINV TE12 clone  with theE. coli K12 RNA was performed using E. coli poly(A) polymerase (NEB). The reaction was incubated for 30 min at 37°C and stopped by the addition of EDTA to a 10 mM final concentration.Poly(A) tailing of isolated For all samples, RNA was quantified and assessed for quality on DeNovix DS-11 Spectrophotometer (DeNovix), a Qubit fluorometer using the Qubit RNA High Sensitivity kit (QIAGEN), and/or Agilent TapeStation (Agilent). SQK-RNA002 Direct RNA Sequencing Kit (ONT) was used for library preparation, and sequencing was performed using ONT MinION with an R9 flow cell. Reads were basecalled using Guppy v6.4.2 with the “rna_r9.4.1_70bps_hac.cfg” model and filtered using a minimum quality score cutoff of 7.D. ananassae , and curlcake synthetic sequences  using the SGNex_A549_directRNA_replicate1_run1 dataset (Preprint). SARS-CoV-2 native and IVT RNAs (https://osf.io/8f6n9/).Publicly available direct RNA sequencing data were downloaded from the NCBI Sequence Read Archive database for  dataset PreprintIVT RNAs  were dowB. malayi (GenBank: GCF_000002995.4), D. ananassae (GenBank: GCA_017639315.2), C. albicans (GenBank: GCA_000182965.3), SARS-CoV-2 (GenBank: NC_045512.2), and human (GenBank: GCF_000001405.40) reference transcriptomes. The published curlcake sequences (E. coli reference transcriptome was produced from the E. coli genomic reference (GenBank: GCF_000005845.2) using the BEDOPS suite v2.4.36 , and the BAM file was converted to FASTQ format with “samtools fastq.” To determine reads mapped to rRNA, GFF files for each organism were filtered to include only rRNA regions. The BEDOPS suite “gff2bed” tool was used to create a BED file from the filtered GFF file, and rRNA reads were pulled from the BAM file after mapping to the reference genome with SAMtools view, using the “-L” option and the BED file containing rRNA regions.The number of reads sequenced and mapped was determined for each sample using SAMtools v1.17  with the5C RNA modifications were detected using the Tombo Alternative Model. Multi-read fast5 files were converted to single-read fast5 files using ONT fast5 API software and reannotated with Tombo preprocess annotate_raw_with_fastqs. The raw signal was then assigned to each base using Tombo resquiggle with the --rna option, and modified bases were detected with Tombo detect_modifications alternative_model.mUsing Tombo, the 10-nt regions surrounding each of the top 1,000 modified cytosines were obtained with the “tombo text_output signif_sequence_context” tool with options “--num-regions 1,000 and --num-bases 10” specified. The output was a FASTA file of the 1,000 regions, and this was analyzed with MEME suite v5.5.1  using th5C [M+H]+ at m/z 258.1→126.1, and m6A [M+H]+ at m/z 282.1→150.1. External calibration curves with known amounts of the nucleosides were used to calculate their ratios within the sample.SINV IVT RNA (650 ng) was digested overnight to single nucleosides using Nucleoside Digestion Mix (NEB). LC-MS/MS analysis was performed by injecting digested RNA on Agilent 1290 Infinity II UHPLC equipped with a G7117A diode array detector and a 6495C triple quadrupole mass detector operating in the positive electrospray ionization mode (+ESI). UHPLC was carried out on a Waters XSelect HSS T3 XP column  with a gradient mobile phase consisting of methanol and 10 mM aqueous ammonium acetate (pH 4.5). MS data acquisition was performed in the dynamic multiple reaction monitoring mode. Each nucleoside was identified in the extracted chromatogram associated with its specific MS/MS transition: rC [M+H]+ at m/z 244.1→112.1, rU [M+H]+ at m/z 245.1→113, rG [M+H]+ at m/z 284.1→152.1, rA [M+H]+ at m/z 268.1→136.1, Ψ [M+H]+ at m/z 245.1→209, mhttps://www.ncbi.nlm.nih.gov/sra) and assigned the BioProject identifier PRJNA944578 (Table S1). All commands and scripts are available on GitHub: https://github.com/Dunning-Hotopp-Lab/Common-Analysis-of-Direct-RNA-Sequencing-Misidentification-of-5mC-Modifications-at-GCU-Motifs.All raw and processed sequencing data from this publication have been deposited in the NCBI Sequence Read Archive database (


Caenorhabditis elegans, to investigate the mechanisms that protect against dysregulated homeostasis. We report that the stomatin homologue UNC‐1 activates neurohormonal signalling from the sulfotransferase SSU‐1 in ASJ sensory/endocrine neurons. A putative hormone, produced in ASJ, targets the nuclear receptor NHR‐1, which acts cell autonomously in the muscles to modulate polyglutamine repeat (polyQ) aggregation. A second nuclear receptor, DAF‐12, functions oppositely to NHR‐1 to maintain protein homeostasis. Transcriptomics analyses of unc‐1 mutants revealed changes in the expression of genes involved in fat metabolism, suggesting that fat metabolism changes, controlled by neurohormonal signalling, contribute to protein homeostasis. Furthermore, the enzymes involved in the identified signalling pathway are potential targets for treating neurodegenerative diseases caused by disrupted protein homeostasis.Alzheimer's, Parkinson's and Huntington's diseases can be caused by mutations that enhance protein aggregation, but we still do not know enough about the molecular players of these pathways to develop treatments for these devastating diseases. Here, we screen for mutations that might enhance aggregation in  Caenorhabditis elegans ASJ neurons modulates protein homeostasis in the body wall muscles. The signal depends on the stomatin homologue UNC‐1 in neurons and the nuclear receptor NHR‐1 in muscles.A neurohormonal signal emanating from  In these animals, 40Q aggregates in an age‐dependent manner and forms inclusion bodies that can be observed and counted under a dissecting microscope. Though several mutants were identified from this screen (Appendix Table unc‐1 is responsible for the enhanced aggregation phenotype of unc‐1(vlt10) mutants, we examined the effect of other mutant alleles of unc‐1 (e719 and e1598) on animals expressing two polyQs with different repeat lengths, 35Q and 40Q . In both cases, analysis of the inclusion bodies produced by polyQ aggregation showed that mutations in unc‐1 (e719 and e1598) phenocopy the aggregation pattern of vlt10 worms  would modify the late aggregation phenotype of a strain expressing 35Q fused to YFP (35Q::YFP) in body wall muscles, which produces both a later and weaker phenotype than animals expressing 40Q and inclusion bodies at late adult stages  animals, the vlt10 allele enhanced and accelerated the phenotype of these animals to a similar extent as other unc‐1 alleles, e179 and e1598  mutation, and as expected, unc‐1(vlt10) increased neuronal aggregate formation in the ventral nerve cord of young adults, further demonstrating the requirement of unc‐1 in protein homeostasis  allele into transgenic worms expressing α‐synuclein::YFP (referred to as α‐syn) and worms expressing human β‐amyloid in body wall muscles. In contrast with worms expressing polyQs, which show aggregation early in the life cycle of worms, both α‐syn and β‐amyloid aggregates do not appear until later stages of development (2‐day‐old adults). Quantifying the aggregate numbers showed that the loss of unc‐1 function increased both α‐syn and β‐amyloid aggregation in day 2 adults compared with controls  animals could be rescued via the expression of unc‐1 cDNA in the muscles. Indeed, expressing unc‐1 under the control of the muscle‐specific myo‐3 promoter (myo‐3p) appeared to partially rescue polyQ aggregation, since we observed a decrease in YFP‐labelled aggregates  animals had substantially reduced polyQ aggregation in all stages  using transgenesis . This silencing of unc‐1 across the nervous system increased the number of inclusion bodies compared with 40Q and 40Q; AMPr(RNAi) animals  animals  by expressing the unc‐1 cDNA under the control of the trx‐1 promoter, which expresses only in ASJ neurons. However, we did not observe any change in 40Q aggregation (65.2 ± 9.1 (unc‐1 mutants) vs. 64.1 ± 7.5 (rescued unc‐1 mutants), Mean ± SD).Previous reports showed a genetic interaction between et al, unc‐1 may be required in neurons that connect with ASJ through gap junctions. As the electrical and chemical synaptic connections of the entire C. elegans nervous system have been precisely characterised  in ASJ, knockdown of unc‐1 in ASJ had no effect, supporting the idea that ASJ is not the site of function. In contrast, knockdown in the IL2 neurons significantly increased polyQ aggregation to the same level as the unc‐1 mutants  animals using two different IL2‐specific promoters, oig‐1p and osm‐3p. Though the expression patterns of these promoters cover several neurons, they overlap exclusively in IL2 neurons, meaning that the RNAi will occur only in IL2 neurons. These animals showed reduced polyQ aggregation in muscle cells and neurons  from IL2 neurons, which are electrically coupled to the ASJ neuron, modulates polyQ aggregation. UNC‐1 may also function by modulating gap junctions  and unc‐24 , showed that only unc‐24 mutants have a phenotype comparable to unc‐1 mutants, suggesting that not all neuronal stomatin‐like proteins are involved in the regulation of polyQ aggregation , and others that are expressed in a restricted set of neurons (inx‐2 and inx‐6), into a polyQ background  that did not modify the aggregation pattern ; unc‐7(e5) produced similar levels of aggregation to the unc‐1 worms ; inx‐2(vlt22) animals showed an additive effect, suggesting that unc‐1 and inx‐2 operate in parallel pathways to modulate protein homeostasis  animals by expressing the genetically encoded ratiometric calcium indicator Cameleon in ASJ. We did not see any difference in the fluorescence ratio, either at the starting temperature of 23°C or after shifting to 17°C  on signalling from ASJ does not stem from a general increase in ASJ excitability, so cannot simply be explained by a disruption in the gap junctions eliminating current shunting to IL2.To further investigate this, we used the fact that the ASJ neurons are sensitive to temperature, with previous studies showing that calcium concentrations in these neurons increased upon warming and decreased upon cooling  and unc‐1(e580). ssu‐1 single‐mutant animals did not exhibit a modified aggregation phenotype  mutants using the trx‐1 promoter. As expected, reducing the expression of ssu‐1 in ASJ partially rescued the aggregation phenotype of unc‐1(vlt10)  Fig . These dunc‐1 mutants and that this sulfonated molecule promotes the aggregation of polyQs in muscle cells. Sulfonated molecules are more water‐soluble, and thus may more easily diffuse into organs and tissues than non‐sulfonated signals. However, to bind nuclear receptors, the sulfonated molecules need to lose their sulfonate moiety. Hence, we hypothesised that removing sulfonate from the signalling molecule may alter the aggregation phenotype. To test this, we disrupted the sulfatase enzymatic activity of the only three genes encoding enzymes with predicted sulfatase activity in C. elegans: sul‐1, sul‐2 and sul‐3. sul‐1 encodes a predicted protein similar to mammalian 6‐O‐endosulfatases  animals did not have reduced inclusion bodies , and observed that ablation of sul‐2 partially reduces the aggregation phenotype of unc‐1 animals  worms as an extrachromosomal array. We then analysed the daf‐36 homozygous worms that lost the array, and these animals showed a substantially higher number of inclusion bodies than their siblings carrying the array  worms, we used CRISPR to generate the mutation vlt16, which emulates nhr‐1(n6242). Additionally, we isolated and characterised a new allele of nhr‐1, vlt15, which was produced by an abnormal homologous recombination and which is still likely a null allele because it causes a frameshift in the gene to produce a truncated protein  animals with 2 mM metformin, an anti‐diabetic drug that activates AMPK to reduce polyQ aggregation  animals, suggesting that the motility rescue is due to decreased polyQ aggregation  worms, and deactivate these pathways. Hence, we performed real‐time PCR for selected genes related to the UPR of the mitochondria (UPRmt), endoplasmic reticulum (UPRER) and cytosol (HSR)  animals with mild amounts of tunicamycin  and compared them with wild‐type worms. After treatment, unc‐1(vlt10) animals showed higher hsp‐4::GFP expression levels than wild type animals which further shows that vlt10 animals have ER‐stress . To do this, we used the expression data from our transcriptomic analysis of the 40Q, 40Q; unc‐1, 40Q; nhr‐1 and 40Q; unc‐1; nhr‐1 worms (see below). This analysis showed that daf‐36 is clearly downregulated in 40Q; unc‐1 worms compared to 40Q animals, while daf‐9 tended to also be downregulated but less clearly , 40Q; nhr‐1(vlt16) and 40Q; unc‐1(vlt10); nhr‐1(vlt16). Here, unc‐1(vlt10) mutants exhibited a distinct transcriptomic signature compared to 40Q, and introducing the nhr‐1 mutation in 40Q; unc‐1 mutants restored the transcriptomic signature to the 40Q background  mutants, we have identified 532 genes whose expression is rescued by the nhr‐1(vlt16) mutation  analysis , biosynthesis (KEGG:cel00061), degradation (KEGG:cel00071) and fat content increase (WBPhenotype:0001184), UPR response and immune response, among others.To elucidate the mechanism behind the effect of NHR‐1 function, we performed transcriptomic analysis on the following strains: 40Q, 40Q; pod‐2 and fatty acid synthase FAS encoded by fasn‐1 show lower expression levels in unc‐1 mutants compared with double mutants  also had altered expression in unc‐1 mutants compared to the 40Q worms , which are involved in removing hydrogen atoms from carbon to produce double bonds  animals  to provide a semi‐quantitative measure of total neutral lipids. We observed a significant enhanced staining in 40Q; unc‐1 mutants compared to 40Q worms  worms  enhanced polyQ aggregation; adding dafachronic acids produced a protective effect  V; unc‐1(e580) X was kindly provided by Phillip G. Morgan . AX5828: dbEx804 was kindly provided by Mario de Bono. Appendix Table All strains were maintained under standard conditions Brenner, . Some stC. elegans, we took advantage of Gateway® MultiSite Pro system . Primer sequences for cloning are listed at Appendix Table rab‐3 gene using specific primers wild type gDNA. We cloned the attB1‐B2‐flanked PCR product into pDONR™ 221‐P1P5r to generate pDONR 221‐P1P5r‐rab‐3p (pAPG1). We amplified the cDNA of unc‐1 to clone it into pDONR 221‐P5P2‐unc‐1 (pAPG2). We used pDONR 221‐P1P5r‐myo‐3p kindly provided by Robyn Branicky. Plasmids containing each promoter region, were recombined with the cDNA of unc‐1, within a destination vector that contains the transcription terminator from the gene unc‐54, pHP2  and pDEST HP2‐myo‐3p::unc‐1::unc‐54t (pAPG4). To induce nhr‐1 expression in muscle cells and neurons we used pVD105 and pVD106, respectively  (pAPG5). Next, we used pAPG1 and pAPG5 as donor vectors and pHP2 as destination vector, to generate pAPG6 (rab‐3p::unc‐1(n494)::unc‐54t). To rescue unc‐1 in IL2 neurons we obtained fusion PCR products using osm‐3p and oig‐1p ], a DNA marker  at 2.5–5 ng/μl, together with the empty yeast plasmid pYES  as carrier DNA, at a total final concentration of 120 ng/μl.To produce a dominant negative form of rab‐3, trx‐1, oig‐1, osm‐3, glr‐1, gpa‐9) to allow us to express dsRNA in a tissue‐specific manner  , adding a sequence complementary to the specific promoter that we wanted to fuse with it  as negative control in the tissue‐specific RNAi experiments. We injected 2.5 ng/μl of each construct (sense/antisense) together with pCFJ90 (encodes a pharynx‐expressed mCherry as a transgenic marker) as a reporter, and pYES (Thermofisher) as carrier DNA until 120 ng/μl.To induce tissue‐specific RNAi we developed PCR fusion products using a promoter region to sense and antisense fragments of the interest gene  and specific primers  were grown in liquid Luria‐Bertani medium containing 50 μg/ml carbenicillin over night at 37°C. Next, bacteria culture was induced by IPTG for 2 h at 37°C, before seeding RNAi plates. After drying plates, synchronised animals  were feed with HT115 bacteria containing empty vector or RNAi vectors until reaching L4 stage to be scored.To induce ubiquitous and constitutive RNAi, we feed the animals with modified HT115 nhr‐1 and deletions into inx‐2 and daf‐12 sequences. We based on our design and strategy from Julián Ceron Lab     for RNA extraction  using the M165FC dissecting microscope . To evaluate expression of genes in mutants, we collected a synchronised population of young adult worms in RNA lysis buffer. Extracts were frozen at −80°C before RNA extraction. RNA samples were treated by DNAase treatment . cDNA synthesis was done using 0.1–1 μg approximately of RNA following the manufacturer protocol . We used the 2xPrimeTime® Gene Expression Master Mix (Takara Bio), and the PCR program was as follows: 1 cycle of 10 min at 50°C; 1 cycle of denaturalization at 95°C 15 s; 40 cycles of polymerisation at 60°C 1 min. To normalise relative expression, we used the housekeeping control, pmp‐3 . To measure expression of interest genes or transgenes we designed customised probes from IDTDNA. We considered all isoforms for each gene in the customised design. We included at least three technical replicates for each measuring.The relative expression of genes was measured by RT–qPCR using the ViiA7 thermal cycler from Applied Biosystems  using Taqman™ probes. To verify the expression of genes included within extrachromosomal arrays, we selected by hand worms expressing reporter  and transferred to polyvinylidene di‐fluoride (PVDF) membranes  by semi‐dry blotting . Membrane blocking was done with 5% non‐fat milk, according to the specification of the following primary antibodies: mouse anti‐polyQ  and mouse anti‐actin  to normalise the results. PVDF membranes were stripped after detecting actin. Primary antibody incubation was done at 4°C and next we incubated the second antibody anti‐mouse conjugated to HRP at room temperature . PVDF membranes were stripped after detecting actin and next, polyQ was detected in the same membrane. Images were obtained using NZY Advanced ECL . Quantification was done using the ImageJ software.et al, E. coli at 0.5 Optic density. Dafadine A  was added at three dose  Animals were growth in liquid medium at 60°C and moderate shaking until young adults. We evaluated the effect of both compounds at least three times in independent experiments over polyQ aggregation pattern.Briefly, we grown synchronised L1 animals on Dafachronic acid 1 μM (DA dissolved in ethanol) (Martin X‐34 dye (Sigma‐Aldrich‐Merck) stains specifically amyloid deposits. One‐day synchronised adults were incubated in a drop containing 1 mM X‐34 and 10 mM TRIS pH 7.5 solution. Stained animals were washed with PBS‐Tween and transferred to fresh NGM plates. We used the Oil Red O (Sigma‐Aldrich‐Merck) dye to stain triglycerides and lipoproteins for semi‐quantitative purposes. Young adults were stained with diluted 3:2 Oil Red O stock solution for 2 h at room temperature in a rotator. After this, animals were washed two times with PBS‐Tween (0.1%) for 30 min. Stained animals were mounted in 2% agarose pads with sodium azide (0.05 M) to count amyloid deposits using a DM2500 fluorescent microscope (Leica). To quantify the stained area of worms, with the Oild Red O stain, we used a DMD108 microscope (Leica). We analysed the pictures obtained from these animals using the Image J software.We generated NGM plates containing 2 mM oleic acid, which was added to molten NGM medium. Plates were conserved in the dark and seeded with OP50 before use it. Synchronised L1 animals were grown until they reached the L4 stage to score polyQ inclusion bodies.40Q::YFP transgene produces body inclusion formation in muscle cells in an age‐dependent manner. The average number of inclusion bodies were obtained by counting total number of polyQ::YFP inclusion bodies in muscle cells per animal in vivo using a dissecting microscope equipped with fluorescence . PolyQ aggregation happens in an age‐dependent manner, so we analysed aggregation in specific developmental stages . Animals were analysed from a heterogeneous population and, after scoring, they were removed to avoid undesired duplicates. Scoring was from at least three independent experiments, in which we counted at least, 10 animals per genotype and stage. Thirty animals or more were analysed for each genotype and stage in total.The expression of the F25B3.3 gene. Neuronal aggregation is more complex to follow, in whole animals, so we selected the Ventral Nerve Cord only to count inclusion bodies. Sixty young adults were analysed for each genotype in total, from three independent experiments. The number of neuronal polyQ aggregates was scored using the DM2500 Leica vertical microscope, equipped with fluorescence.To score neuronal polyQ aggregates we used a model that expresses 40 glutamines under the control of the promoter of the unc‐54p::α‐synuclein::YFP transgene induces late aggregation, in contrast with polyQ aggregation. Moreover, unlike 40Q::YFP inclusion bodies, which are visible using a dissecting microscope, α‐synuclein::YFP aggregates are only detectable using upright microscopes . Since it is not feasible to perform a complete count of whole muscle tissue, we selected the area located between the positions of the two pharyngeal bulbs, to evaluate the aggregation pattern of α‐synuclein. Sixty 2‐day‐old adults were analysed for each genotype in total, from three independent experiments.The C. elegans model that expresses the human Aβ peptide constitutively in muscle cells. To detect β‐amyloid deposits, we stained 1‐day‐old adults with X‐34 dye (Sigma‐Aldrich‐Merk). Stained 2‐day‐old animals were analysed using an upright microscope equipped with fluorescence . We selected the area between the positions of the two pharyngeal bulbs to evaluate the number of β‐amyloid deposits. Sixty 2‐day‐old adult animals were analysed for each genotype in total, from three independent experiments.To evaluate β‐amyloid protein aggregation, we used a We evaluated motility capacity of young adults by thrashing. This assay consists of scoring the number of thrashes that show an animal when swimming in M9 buffer. We have considered a thrash when animal moves simultaneous head and tail. Animals were acclimated for 30 s before scoring number of thrashes for the following 30 s. Each animal was collected independently in a well. The average number of thrashes was extrapolated for 1 min to show the mean of the values. At least 15–20 animals were analysed for each genotype and transgenic line in three independent experiments at 20°C. Animals damaged during handling were discarded in this study.et al, trx‐1 promoter . Images were recorded at 5 Hz using a Dualview beam splitter  to simultaneously record two wavelengths and an iXon EM camera with IQ1.9 capture software (Andor Technology). Fluorescence was quantified and analysed in Matlab (Mathworks) using SpikeFinder and NeuronTracker, custom analysis scripts written by Rabinowitch et al , 40Q; nhr‐1(vlt16) and 40Q; unc‐1(vlt10); nhr‐1(vlt16)) were grown in NGM plates at 20°C until they reached young adult stage. They were subsequently collected in M9 buffer, frozen, thawed and mechanically lysed with 200 mg of glass beads (Sigma‐Aldrich‐Merck) for 30 s using a Fastprep shaker apparatus . RNA was extracted using NZY total RNA isolation kit, Nzytech. Supernatant was recovered from lysate samples by centrifuging extracts at maximum speed for 1 min. The purified RNA was treated with DNAse, quantified and sent for sequencing by Novogene . Six biological replicates, consisting of pooled bulk nematode RNA were sequenced for each genotype. Briefly, after quality control, mRNA was enriched using oligo(dT) beads and randomly fragmented. cDNA was synthesised using random hexamers and reverse transcriptase. After first‐strand synthesis, a custom second‐strand synthesis buffer (Illumina) was added with dNTPs, RNase H and E. coli polymerase I in order to generate the second strand by nick‐translation. To prepare the final cDNA library, a round of purification, terminal repair, A‐tailing, ligation of sequencing adapters, size selection and PCR enrichment were performed. Library concentration was first quantified using a Qubit 2.0 fluorometer (Life Technologies). Insert size was checked on an Agilent 2100 and quantified using quantitative PCR (Q‐PCR).Synchronised L1   animals were collected in RIPA 1× buffer (Sigma‐Aldrich‐Merck) to obtain protein extract. We collected six biological samples per genotype in each independent experiment (n = 3 experiments and n = 10 cultures of worms per genotype). Total protein extract was quantified using the commercial kit  to normalise the lipid abundance of the samples. Samples were analysed using a liquid chromatography equipment coupled to a high‐resolution mass spectrometer with an orbitrap detector (UPLC‐Q‐Exactive Plus) and an electrospray source (ESI) following the procedures optimised in the Analytical Unit . Samples and quality controls were randomly injected into the chromatographic system to avoid variability within the analytical sequence, as well as to improve the quality and reproducibility of the study. Data were acquired in in Full MS, DIA  and DDA  scan modes and processed using an in‐house script in the R software (v.3.6.1) with the XCMS and CAMERA packages for detection, filtering and alignment of spikes. The LipidMS library was used to identify candidate lipids . Oil Red O images to analyse stained area per animal were taken with a digital dissecting microscope DMD108 (Leica). In all cases, animals were mounted on agarose pads (2% on water) containing one drop of sodium acid (0.05 M) to anaesthetise them. Nematode selection and manipulation were performed using dissecting microscopes without or with fluorescence .t‐test to obtain the statistical significance of the data. In the graphs, we show the mean ± standard error (SEM). SEM is displayed as a bar, while asterisks show significant of the data  . The abbreviation “ns” means not significant.Statistical analysis was performed using an analysis of variance test (one‐way ANOVA) combined with a Tukey test to perform multiple comparisons of different strains and/or conditions. To perform comparisons between two conditions we used a Mann Whitney Rafael P Vázquez‐Manrique: Conceptualization; data curation; supervision; funding acquisition; investigation; visualization; methodology; writing – original draft; project administration; writing – review and editing. Ana P Gómez‐Escribano: Conceptualization; data curation; formal analysis; validation; investigation; visualization; methodology; writing – original draft; writing – review and editing. Carlos Mora‐Martínez: Conceptualization; resources; software; formal analysis; methodology; writing – review and editing. Marta Roca: Software; formal analysis; investigation; methodology; writing – review and editing. Denise S Walker: Formal analysis; investigation; methodology; writing – review and editing. Joaquín Panadero: Data curation; software; formal analysis; investigation. Maria D Sequedo: Investigation; methodology. Ratni Saini: Investigation. Hans‐Joachim Knölker: Investigation. Jose Blanca: Investigation. Juan Burguera: Conceptualization; investigation. Agustin Lahoz: Conceptualization; validation; investigation; writing – review and editing. Joaquin Cañizares: Investigation. José M Millán: Conceptualization; investigation; writing – review and editing. Nick O Burton: Conceptualization; investigation; writing – original draft; writing – review and editing. William R Schafer: Investigation; writing – review and editing.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Source Data for Expanded View and AppendixClick here for additional data file.PDF+Click here for additional data file.Source Data for Figure 1Click here for additional data file.Source Data for Figure 2Click here for additional data file.Source Data for Figure 3Click here for additional data file.Source Data for Figure 4Click here for additional data file.Source Data for Figure 5Click here for additional data file.Source Data for Figure 6Click here for additional data file.