Caenorhabditis elegans is a round worm that has been successfully used to study synaptic transmission, the process by which neurons communicate with targets cells in the nervous system through the release of chemical neurotransmitters. Many synaptic proteins involved in this release process are highly conserved with some species-specific variations in individual components. Here, we examined how the C. elegans protein, clarinet (CLA-1), an elusive member of the Piccolo, Fife and Rab3-interactingmolecule (Rim) protein family, fits into the organization and function of synaptic release sites in the worm. Using CRISPR/Cas-9 to endogenously tag key synaptic players, we determined the functional hierarchy of these C. elegans synaptic proteins and provide insights into conserved design principles that govern nervous system function. Caenorhabditis elegans protein named Clarinet (CLA-1) based on homology to the AZ proteins Piccolo, Rab3-interactingmolecule (RIM)/UNC-10 and Fife. At the neuromuscular junction (NMJ), cla-1 null mutants exhibit release defects that are greatly exacerbated in cla-1;unc-10 double mutants. To gain insights into the coordinated roles of CLA-1 and UNC-10, we examined the relative contributions of each to the function and organization of the AZ. Using a combination of electrophysiology, electron microscopy, and quantitative fluorescence imaging we explored the functional relationship of CLA-1 to other key AZ proteins including: RIM1, Cav2.1 channels, RIM1-binding protein, and Munc13 . Our analyses show that CLA-1 acts in concert with UNC-10 to regulate UNC-2 calcium channel levels at the synapse via recruitment of RIMB-1. In addition, CLA-1 exerts a RIMB-1-independent role in the localization of the priming factor UNC-13. Thus C. elegans CLA-1/UNC-10 exhibit combinatorial effects that have overlapping design principles with other model organisms: RIM/RBP and RIM/ELKS in mouse and Fife/RIM and BRP/RBP in Drosophila. These data support a semiconserved arrangement of AZ scaffolding proteins that are necessary for the localization and activation of the fusion machinery within nanodomains for precise coupling to Ca2+ channels.Synaptic transmission requires the coordinated activity of multiple synaptic proteins that are localized at the active zone (AZ). We previously identified a  Although synapses vary in size and morphology, all AZs have electron-dense projections (DPs) packed with specialized proteins that recruit and prime SVs in close proximity to calcium channels. The probability of release has been shown to scale with the size of the AZ underscoring the importance of this proteinaceous scaffold for synaptic function /Liprin-α, uncoordinated-10 (UNC-10)/Rab3-interacting molecule (RIM), RIMB-1/RIM-binding protein (RIM-BP), ELKS-1/ELKS , UNC-13/Munc13, and UNC-2/CaV2 channels  that has homology to Piccolo, RIM and Fife (cla-1 gene was predicted to encode three isoforms based on size: long(L), medium(M), and short(S). Since the number and distribution of RNAseq splice junctions on Wormbase suggest there are only L- and S-isoforms, the M-isoform was not further explored in this study. CLA-1 L- and S- isoforms share and identical C-terminal PDZ and C2 containing domain, in common with Piccolo, Fife and RIM, which tethers CLA-1 at the AZ  (termed cla-1ΔS/M/L). For the current study we generated a CLA-1S mutant, cla-1(kur5), using Clustered Regularly Interspaced Palindromic Repeats/CRISPR-associated endonuclease (CRISPR/Cas9) (termed cla-1ΔS) and used a previously isolated mutant cla-1(ok560), which deletes CLA-1 long (CLA-1L) (termed cla-1ΔL) (cla-1ΔS deletion was verified (S1), we undertook a characterization of the cla-1(ΔL) and cla-1(ΔS) mutants and compared their phenotypes to the cla-1 null (cla-1ΔS/M/L).To better understand CLA-1 function, we compared the phenotypes of isoform-specific cla-1ΔL) A. After cla-1 mutants, which only reached significance in the cla-1(ΔS/M/L) and cla-1(ΔS) (cla-1(ΔS/M/L) exacerbates the synaptic defect of unc-10 mutants, we next performed thrashing assays on each of the cla-1 mutants in combination with the unc-10 null ;unc-10 and cla-1(ΔL);unc-10 double mutants exhibited more severe thrashing defects than unc-10 alone, whereas cla-1(ΔS);unc-10 mutants did not  B. Given -10 null . As expeild type B. In the did not B.cla-1 mutants. As we previously reported, the evoked amplitude in cla-1(ΔS/M/L) was not different from the wild type (cla-1(ΔL) nor cla-1(ΔS) exhibited reductions in evoked amplitude (cla-1(ΔS/M/L) and to a lesser extent the cla-1(ΔL) but not the cla-1(ΔS) mutants , though the long and short mutants trended lower (cla-1 mutants in the unc-10 background we saw that both cla-1(ΔS/M/L) and cla-1(ΔL) mutants significantly exacerbated the evoked and endogenous synaptic defects, whereas release in cla-1(ΔS);unc-10 double mutants was similar to unc-10  mutant, we can conclude that the short isoform (and possibly medium) also contributes to CLA-1 function at the NMJ.To determine whether the behavioral phenotypes were recapitulated at the level of the NMJ, we recorded evoked and endogenous release in dissected ild type C and D (ild type . Similarmplitude C and D.  mutants C and F. ed lower C and G. o unc-10 C–G. Minio unc-10 C and H. cla-1 null mutants have reduced DP size and fewer SVs (cla-1(ΔS/M/L);unc-10 double mutants could be due to additive effects of loss of CLA-1 and UNC-10 on synapse morphology. To test this, we compared the NMJ ultrastructure of the cla-1 mutants with and without UNC-10. We focused on the ventral NMJs from which the synaptic recordings were obtained. Worms were prepared using high-pressure freeze (HPF) fixation, a method that preserves synapse morphology in its near native state (cla-1(ΔS/M/L) mutants was significantly smaller than wild type, possibly indicative of reduced AZ protein content (cla-1(ΔL) nor cla-1(ΔS) mutants affected DP area, indicating that this phenotype requires loss of multiple isoforms ;unc-10 double mutants was no more reduced than cla-1(ΔS/M/L) alone, and neither cla-1(ΔS) nor cla-1(ΔL) mutants impacted the DP area in the unc-10 mutant background (cla-1(ΔS/M/L);unc-10 double mutants cannot be attributed to additive effects on DP morphology. Moreover, this result indicates that the major role CLA-1 plays in determining DP content, requires both long and short isoforms, suggesting that the common CLA-1 C-terminal localized at the AZ is responsible for this function.Given our previously published electron microscopy (EM) data showing that ewer SVs , we hypove state , 9. Consve state , the areve state A in cla- content B. Howeveisoforms B. Consisisoforms , the DP  altered B. Importckground B. Therefcla-1(ΔS/M/L) mutants have fewer total SVs at NMJs (cla-1(ΔS/M/L) mutants exhibited a reduction in SVs ;unc-10 double mutants, SV numbers were reduced compared to unc-10 and similar to cla-1(ΔS/M/L). The data suggest that CLA-1 isoforms impact SV density through a mechanism independent of UNC-10  mutants on the other hand have fewer total docked SVs, but the proximally docked SV pool is abundant (cla-1(ΔL) nor (ΔS) mutants exhibited a change in total number or distribution of docked SVs as compared to the wild type . We next asked whether the loss of CLA-1 isoforms and UNC-10 together further impacts docking. We found that the docked SV pool deficits of the cla-1 and unc-10 mutants were not exacerbated in the double mutants. In fact, in all three double mutants, SV docking trended higher than unc-10 alone (cla-1(ΔS/M/L);unc-10 double mutants incorporate changes seen in each single mutant but show no additivity, suggesting that CLA-1 and UNC-10 differentially regulate these aspects of the synaptic ultrastructure. Importantly, the dramatic release defects in the cla-1(ΔL) and cla-1(ΔS/M/L) double mutants are not attributable to further loss of docked SVs in the unc-10 mutant background.In previous studies, we have shown that o the DP . cla-1;unc-10 and cla-1(ΔL);unc-10 double mutants at the NMJ. We found that both double mutants exhibited robust hyperosmotic responses, indicating that the morphologically docked SVs observed are primed and fusion-competent and therefore, cannot account for the total loss of evoked release in the double mutants (S3).Docked SVs at  priming . Given tcla-1 mutants do not exacerbate the unc-10 SV docking phenotype, we hypothesized that the loss of release could be due to defects in calcium-secretion coupling in these mutants. In C. elegans, Ca2+ influx at the presynaptic terminal is largely mediated by the UNC-2 channel, the sole CaV2 channel a1 homolog in C. elegans , which exhibited a small reduction in UNC-2 peak number  suggesting both CLA-1 isoforms contribute to localization of UNC-2 (cla-1(ΔS/M/L) result was due to a background mutation in the cla-1(wy1048) allele, we examined a second cla-1(ΔS/M/L) mutant, (cla-1(ok2285), and demonstrated a similar reduction in UNC-2 levels (S4). In the cla-1(ΔS/M/L);unc-10 double mutants we saw a greater reduction in UNC-2 peak intensity compared to unc-10 single mutants, but no change in either cla-1(ΔL);unc-10 or cla-1(ΔS);unc-10 double mutants compared to unc-10 (As  elegans –16. Immu elegans . We rece by ~30% A–C 17)..cla-1 muof UNC-2 A–C. To ao unc-10 D–F. Thesrimb-1 null mutants also exacerbate the unc-10 mutant deficit in UNC-2 levels suggesting these two AZ proteins act redundantly to regulate UNC-2 ;unc-10 double mutants. While peak number was more severely reduced in rimb-1;unc-10 mutants, we found that UNC-2 peak intensity in rimb-1;unc-10 mutants had a similar reduction to cla-1(ΔS/M/L);unc-10 mutants (cla-1 mutants using endogenously GFP-tagged RIMB-1. In cla-1(ΔS) and cla-1(ΔL) RIMB-1 peak intensity and number were comparable to the wild type. However, we found a severe reduction in both peak number and average intensity in the cla-1(ΔS/M/L) mutants (cla-1(ok2285), as well as a second RIMB-1 fluorescent probe in cla-1(wy1048), both of which yielded similar results (S5). These data indicate that CLA-1 plays a major role in the localization of RIMB-1 at the synapse.How might CLA-1 impact UNC-2 in the absence of UNC-10? We previously demonstrated that te UNC-2 . Similarte UNC-2 . Therefo mutants D–F. Base mutants A–C. Herecla-1(S/M/L) mutants resembles that observed in the absence of the synaptic scaffolding protein SYD-2, suggesting that loss of SYD-2 may contribute to the RIMB-1 phenotype of cla-1(S/M/L) mutants (cla-1(S/M/L) mutants have a minor reduction (~20%) in synaptic levels of SYD-2  and cla-1(ΔS/M/L) mutant animals when compared to wild-type animals (S6). Together, these data demonstrate that CLA-1 is required for RIMB-1 localization at the AZs without dramatically impacting synaptic density.The reduction of RIMB-1 levels in  mutants . In factof SYD-2 . This raunc-10 mutants (unc-10 mutation did not cause any appreciable changes in RIMB-1 peak intensities and numbers in either cla-1(ΔL) or cla-1(ΔS) mutant animals ;unc-10 double mutants showed a further reduction in RIMB-1 puncta intensity with no change in number (cla-1(ΔS/M/L);unc-10 double mutants is less reduced than cla-1(ΔS/M/L) alone, implying that in the absence of CLA-1 and UNC-10, RIMB-1 is localized to the AZ by an alternate interaction. We have shown that both SYD-2 and ELKS-1 are involved in the localization of RIMB-1 (cla-1(ΔS/M/L);unc-10 double mutants either of these or other AZ proteins could partially compensate.Next, we examined whether UNC-10 and CLA-1 independently regulate presynaptic RIMB-1 localization. As previously reported, RIMB-1 intensity, but not peak number, was considerably reduced in  mutants . Introdu animals D–F. But n number D–F. Intef RIMB-1 , therefocla-1(S/M/L) mutants, this cannot explain the severity of the evoked release defects observed in the cla-1;unc-10 double, as we have previously shown that evoked release in syd-2;unc-10 double mutants is no more severe than unc-10 mutants alone (cla-1(S/M/L);syd-2 double mutant exhibited evoked amplitudes similar to those of syd-2, the mini frequency of these double mutants was reduced beyond that of syd-2 suggesting additional roles for CLA-1 in synaptic release (S7).Though SYD-2 is reduced in ts alone . Furtherrimb-1;unc-10 double mutants to phenocopy cla-1(ΔS/M/L);unc-10 double mutants. To address this possibility, we compared the phenotypes of the two double mutants. We first performed a behavioral assay looking at thrashing rates. Although cla-1(ΔS/M/L) and rimb-1(ce828) single mutants exhibited mild but significant reductions in thrashing rates compared to the wild type, they were not different from each other and were less severe than the unc-10 mutants (cla-1(ΔS/M/L);unc-10 and rimb-1;unc-10 double mutants were more impaired than unc-10, the cla-1(ΔS/M/L);unc-10 thrashing defect was significantly worse than rimb-1;unc-10 ;unc-10 double mutants (3% of WT and 5% of unc-10 evoked amplitude) and mini frequency (9% of WT and 24% of unc-10). Consistent with these clear functional differences, when we compared the ultrastructure of cla-1;unc-10 and rimb-1;unc-10 double mutants, we also saw significantly fewer SVs and docked SVs in cla-1;unc-10 when compared to rimb-1;unc-10, though the distribution of these docked SVs is similar in the double mutants and much like that observed in rimb-1 and unc-10 alone . Given that these ultrastructural and physiological results mirror the differential severity of the behavior in the intact worms, these data support the interpretation that CLA-1 has roles in neurotransmission beyond the observed regulation of RIMB-1 and UNC-2 levels.To determine whether the above behavioral differences were recapitulated at the level of the NMJ, we recorded evoked and endogenous release in these mutants. Whereas the evoked amplitude in te refer C and G . cla-1(ΔL) and cla-1(ΔS/M/L) mutants showed a similar reduction in UNC-13 puncta number without changing puncta intensity ;unc-10, UNC-13 puncta were barely discernible (cla-1(ΔL), cla-1(ΔS/M/L) and unc-10 single and double mutants. Consistent with the endogenous data, both cla-1 and unc-10 single and double mutants reduced UNC-13L levels (cla-1(ΔL) and cla-1(ΔS/M/L) single mutants but cla-1;unc-10 double mutants had wild-type levels  mutants, in which only UNC-13S remains ..C. elegaL levels A–C. UNC-e levels D–F. The rimb-1 mutants. In rimb-1 single mutants the puncta intensity was not significantly affected, whereas the puncta number showed a ~30% reduction, possibly reflecting effects of RIMB-1 on a subset of synapses  double mutants we did not observe appreciable changes in UNC-13 levels. Thus, the additive effect on UNC-13 levels in cla-1(ΔS/M/L);unc-10 double mutants supports a role for CLA-1 in the regulation of UNC-13 that is independent of both RIMB-1 and UNC-10. Surprisingly, when we examined cla-1(ΔL);unc-10 double mutants, we found that the endogenous UNC-13 levels were not further reduced relative to unc-10, suggesting that in cla-1(ΔL);unc-10 mutants, RIMB-1 and the remaining CLA-1 isoforms are able to support UNC-13 synaptic expression in the absence of UNC-10. Together these data suggest that UNC-10, RIMB-1 and CLA-1 exhibit partial redundancy in regulating UNC-13 localization.Given that CLA-1 and, to a lesser extent UNC-10 regulate RIMB-1, we next examined endogenous UNC-13 labelling in synapses A–C. In ro unc-10 D–F. In rC. elegans synapses  mutant animals exhibit a negligible effect on UNC-2 localization relative to unc-10 mutants, the cla-1(ΔS/M/L);unc-10 double mutants exacerbate the UNC-2. The additivity of the UNC-2 localization defect in the cla-1(ΔS/M/L);unc-10 doubles likely results from the loss of RIMB-1, which we have shown is heavily dependent on CLA-1. Mammalian Bassoon interacts with RIM-BPs via a proline-rich (PxxP) SH3-binding motif  mutants (cla-1(ΔS/M/L) mutants would contribute to the reduction in DP size. Here, we found that cla-1(ΔS/M/L) mutants also exhibit a dramatic reduction in RIMB-1 levels. Using endogenously tagged proteins, we know from a previous study that RIMB-1 is more abundant than other key AZ components examined   . Thus, wand area . Based oC. elegans NMJs the morphologically docked SV pool is partially dependent on interactions between SV-associated RAB-3 and UNC-10/RIM , whereas cla-1(ΔS/M/L) mutants retain this proximal pool but show reductions in overall docking. We show here that, like unc-10 mutants, rimb-1 mutants exhibit a reduction in proximally docked SVs, suggesting that RIMB-1 also has a role in positional docking in C. elegans. Furthermore, since the rimb-1;unc-10 double mutants do not show additivity in proximal docking defects, their roles appear to be redundant. This would explain why in a cla-1(ΔS/M/L) mutant in which RIMB-1 levels are greatly reduced, proximal docking can still occur due to the continued presence of UNC-10. This interpretation is supported by the observation that in cla-1;unc-10 double mutants, the proximal SV pool is reduced to the same extent as rimb-1 and unc-10 single mutants as well as the rimb-1;unc-10 double mutants.At C-10/RIM . In unc-cla-1;unc-10 and rimb-1;unc-10 double mutants may contribute to the release defects observed in these strains. Indeed, redundant roles for RIMB-1 and RIM in synaptic SV docking appear to be well-conserved as knockouts of RIMs and RIM-BPs in mice exhibit severe reductions in SV docking and priming associated with a dramatic reduction of neurotransmitter release  mutants have reduced RIMB-1 levels, this presents a potential mechanism by which CLA-1 could affect UNC-13 localization. Indeed, we observe a reduction in endogenous UNC-13 levels in rimb-1 mutants consistent with this model. However, the observed reduction in RIMB-1 cannot fully account for the extent of UNC-13 loss in cla-1 mutants. Two lines of evidence support this conclusion. First, cla-1(ΔL) single mutants have wild-type RIMB-1 levels but exhibit reduced UNC-13 levels. Second, cla-1(ΔS/M/L);unc-10 exacerbates loss of UNC-13 beyond that of unc-10 alone, whereas rimb-1;unc-10 does not. Though these findings support RIMB-1-dependent and independent roles for CLA-1 in regulating the synaptic localization of UNC-13, the mechanism of the latter remains to be elucidated.How might CLA-1 regulate UNC-13? At hippocampal mossy fiber synapses, Munc13-1 is recruited to the AZ by RIM-BP2  and in D3 domain . Since wcla-1(ΔL), cla-1(ΔS/M/L) and unc-10 single and double mutants dramatically reduced UNC-13L fluorescence intensity, whereas UNC-13S was only reduced in cla-1(ΔL) and cla-1(ΔS/M/L) mutants. How might this contribute to the severity of the neurotransmission defect in the single and double cla-1 and unc-10 mutants? While UNC-13L and UNC-13S share a common C-terminal MUN domain required for priming, the N-terminal unique to UNC-13L is responsible for its enrichment at the AZ through interactions with UNC-10 , indicating these differential release kinetics are highly conserved ;unc-10 and cla-1(ΔS/M/L);unc-10, the persistence of docked and primed SVs in these double mutants is attributable to UNC-13S, as the unc-13 null mutants lack primed SVs  and cla-1(ΔS/M/L) mutants have relatively mild synaptic defects compared to unc-10 mutants despite a significant reduction in UNC-13 puncta number. Our data suggest that this release in cla-1 mutants is supported by the continued presence of UNC-10. The decrease in charge integral observed in cla-1(ΔL) and cla-1(ΔS/M/L) mutants is most likely attributable to reduced UNC-13S levels.Since UNC-13L is dramatically reduced in both  mutants . Likewis3B alone , or expr3B alone . Both clC. elegans synaptic components UNC-2, RIMB-1 and UNC-13. While CLA-1 plays a minor supporting role in UNC-2 localization in conjunction with UNC-10, its impact on UNC-13L appears to be the predominant role of CLA-1 in synaptic function. This has overlapping design principles with other model organisms (RIM/RBP and RIM/ELKS in mouse and Fife/RIM and BRP/RBP in Drosophila)  , 49 and channels , 50.cla-1(kur5) short isoform deletion allele, we used two guide RNAs to remove the intronic sequence between exons 32 and 33 of the cla-1a/long isoform, which serves as a promoter for the short isoform of cla-1. The resulting fusion can only express the M/L isoforms of cla-1. Guide RNAs were synthesized as ALT-R CRISPR (cr)RNAs and injected at 1.5 μM in a mix with 1.5 μM ALT-R Cas9 protein, 1.5 μM ALT-R tracRNA, 6 μM Ultramer DNA oligo repair template (197 bp), and cocrispr reagents including 1.5 μM dpy-10 crRNA and 6 μM dpy-10(cn64) repair template , a tracrRNA, a homologous repair construct, and Cas9 into the gonads of wild-type N2 animals. The repair construct was built by subcloning the 1.5 kb upstream and 700 bp downstream sequences from the unc-13 3′ end into the 5′ and 3′ ends of GFP in frame, respectively. Co-CRISPR (dpy-10) reagents were also included in the injection mixture. Progeny of F1 Roller animals were screened by PCR for GFP insertion.To generate endogenously tagged cla-1(S) deletion mutant were assessed through RT-PCR of complementary DNA (cDNA) from these mutants and compared with wild-type N2 levels .mRNA expression of the long and medium isoforms of cla-1 in the SI Appendix, Methods).Thrashes from a single worm after 1-min equilibration in M9 solution were scored as number of completed thrashing waves/min measured at the head . Prism (GraphPad) was used to graph data and for statistical analysis. .Electrophysiological methods were as previously described . Data weSI Appendix, Methods).Strains were prepared using HPF fixation and freeze substitution (FS) as previously described , 9. .Fluorescent microscopy was performed as described previously . Images SI Appendix, Methods).Confocal imaging of fluorescently-tagged RIMB-1::Skylan-S were collected from L4 animals anesthetized with 10 mM levamisole in M9 buffer. using a Zeiss Axio Observer Z1 microscope stage outfitted with a Plan-Apochromat 63× 1.4 NA objective and a Yokogawa CSU-W1 spinning disk confocal scanner attached to a Prime 95B camera. Maximum intensity projections of z-stack images were cropped and straightened around the dorsal nerve cord (DNC) using ImageJ/Fiji, and were analyzed using a Matlab script designed to identify puncta in individual TIFF files based on local means thresholding coupled with watershed segmentation. .Young adult worms (~10) were imaged on an Olympus Fluoview FV10i inverted laser scanning confocal microscope with the 60× (NA 1.35) oil immersion lens and optical zooming to a total magnification of 120×. Fluorescent analysis was conducted using NIH FIJI/ImageJ software in which max projections were created from obtained z-stacks; DNCs were straightened. Fluorescent levels and peaks from a 40-µm line along the nerve cord were identified from the Plot Profile data using peak finder in Matlab. Statistical analysis was conducted in Prism (GraphPad) using one-way ANOVA with Dunnett’s multiple comparison test. Click here for additional data file.


Retinoic acid inducible gene (RIG)-I-like receptors (RLRs), including RIG-I, melanoma differentiation associated-5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), play pivotal roles in viral RNA sensing to initiate antiviral interferon (IFN) responses.We previously reported that an RNA-silencing regulator, transactivation response RNA-binding protein (TRBP), up-regulates MDA5/LGP2-mediated IFN responses through interaction with LGP2. Here, we aimed to investigate the mechanism underlying the TRBP-mediated up-regulation of IFN response.Cardiovirus-triggered IFN responses. These results suggest that encephalomyocarditis virus (EMCV) attenuates the TRBP-mediated IFN response via TRBP phosphorylation, since EMCV infection activates the kinase responsible for TRBP phosphorylation for virus replication.Data indicated that phosphomimetic TRBP showed a modest effect, whereas the nonphosphorylated form exhibited hyperactivity in enhancing Furthermore, we found that TRBP-mediated up-regulation of IFN response required the ATP hydrolysis and RNA binding of LGP2. TRBP enhanced RNA-dependent ATP hydrolysis by LGP2 but not that by RIG-I or MDA5. Nonphosphorylated TRBP exhibited higher levels of activity than phosphomimetic TRBP did, suggesting its possible involvement in the mechanism underlying the up-regulation of IFN response. TRBP activated the ATP hydrolysis of LGP2 and RIG-I, but not that of MDA5, in the absence of RNA. Collectively, we showed that TRBP differentially regulated RLR-mediated ATP hydrolysis. Further elucidation of the mechanism underlying the regulation of ATP hydrolysis leading to IFN response and self- and non-self-RNA discrimination could advance the development of effective therapeutic agents against autoimmune diseases. Innate immune recognition of invading pathogens requires strict regulation for the discrimination of self- and non-self-antigens by pathogen recognition receptors (PRRs), such as retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs). A family of RLR proteins, consisting of RIG-I, melanoma differentiation associated-5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), recognizes double-stranded RNAs (dsRNAs) derived from replicating RNA viruses in the cytoplasm.RIG-I preferentially selects dsRNA with 5′-triphosphate (5′ppp) at the end of the viral RNA (5′ppp dsRNA), and MDA5 selects a long dsRNA region at the viral RNA stem . RIG-I aAlthough RLR proteins exhibit similar structural architectures in their RNA-binding domains, such as the helicase and C-terminal domains, the structure of the third RLR protein, LGP2, is unique. LGP2 lacks the caspase activation and recruitment domain (CARD) required for signal transduction to MAVS and, therefore, cannot activate antiviral gene induction alone . AnotherMechanistic analyses suggest that LGP2 stabilizes MDA5 RNA binding and the co-operative formation of ribonucleoprotein filaments for signal transduction and positive regulation . In addiWhile restricting viral replication is essential for immunity, accelerated or sustained IFN-stimulated antiviral gene expression is detrimental and contributes to autoimmune disorders , includiThe intrinsic ATP hydrolysis activity of RLRs is fundamental to discriminating between self- and non-self-RNA. As part of a proof-reading function, ATP hydrolysis of RIG-I dissociates self-RNA faster than it does 5′ppp dsRNAs and converts RIG-I to a signaling-active oligomeric state . The ATPIn addition to being involved in self- and non-self-RNA discrimination, RIG-I- and MDA5-induced ATP hydrolysis also contributes to displacing viral proteins prebound to dsRNA to assist antiviral proteins, including RNA-dependent protein kinase (PKR) and RIG-I for their RNA recognition . CompareCardiovirus-triggered immune responses through an association with LGP2, suggesting that TRBP is a cofactor for MDA5/LGP2-mediated immune response  = 1) in DMEM, supplemented with 2% serum, for 2 h; then, complete DMEM was added, followed by incubation for 14–17 h. We used a dual-luciferase reporter assay system (Promega) for luciferase assays and Renilla luciferase as the internal control to normalize luciferase activity.The 293FT cells were seeded onto a 48-well plate  at 30°C for 30 min. The dsRNA-TRBP complex was separated on a 5% acrylamide gel and stained with SYBR Gold (Thermo Scientific). Relative RNA binding was quantified as the ratio of the shifted and original RNA band intensities using the Quantity One software (Bio-Rad Laboratories).A sense strand 45-mer RNA (5′-UUACUAUUGACACUAUGGCUUGCAAACAUG GAUACCCAGAUGUGU-3′) and an antisense strand 45-mer RNA (5′-ACACAUCUGG GUAUCCAUGUUUGCAAGCCAUAGUGUCAAUAGUAA-3′) from the Theiler’s murine encephalomyelitis virus genomic sequence were synthesized by Japan Bio Services  and annealed for dsRNA preparation. The dsRNA (60 nM) was incubated with recombinant TRBP wild-type (WT) or TRBP SD protein  in buffer A  knockdown and TRBP-overexpression experiments [We previously reported that the RNA-silencing regulatory protein TRBP positively regulates eriments .Cardiovirus-triggered IFNβ promoter activity in 293FT cells. MDA5 alone enhanced both basal and EMCV-induced IFN response, and addition of LGP2 (MDA5/LGP2) further enhanced this activity compared with that in the control  also abolished TRBP-induced IFN responses in mock or EMCV-infected cells  and a cysteine residue in the C-terminal domain crucial for binding to the dsRNA-binding protein PACT and Zned cells C. Mutatied cells C.All these results suggest that the ATP hydrolysis and RNA binding of LGP2 are required for TRBP to up-regulate MDA5-mediated type I IFN induction.TRBP has four known phosphorylation sites at serine residues; the phosphorylation of TRBP enhances its activity for microRNA (miRNA) production and miRNA-targeted silencing . In addiCardiovirus-triggered IFN induction. Expression plasmids for phosphomimetic and nonphosphorylatable TRBP mutants with all four serine residues replaced with aspartic acid (SD) and alanine (SA), respectively, were generated  in a dose-dependent manner  with botBecause the reporter gene assay  showed tWe also tested the effect of TRBP on RLR-mediated ATP hydrolysis in the absence of RNA A. SurpriIn the present study, we investigated the mechanism underlying the effect of the RNA-silencing regulatory protein TRBP on MDA5/LGP2-induced IFN signaling. Our results demonstrated that TRBP requires LGP2-mediated ATP hydrolysis and RNA-binding for up-regulating the MDA5/LGP2-mediated IFNβ promoter activity.Since the phosphorylation status of TRBP impacts miRNA regulation  and EMCVTRBP is a dsRNA-binding protein that interacts with Dicer, a multidomain enzyme that consists of several RNA-binding domains, to enhance the processing of pre-miRNA into miRNA duplexes. TRBP is suggested to enhance the processing of a subset of miRNAs that bind to it through a miRNA-binding preference . In addiThe major antiviral system of mammalian innate immunity is the protein-based IFN system. In contrast, the primary antiviral defense strategy of plants and invertebrates is RNA-based antiviral immunity, wherein virus-derived siRNAs are loaded onto the RNA-induced silencing complex (RISC) and target complementary viral RNA to block virus replication. However, recent studies have suggested that small RNA-mediated antiviral systems are also active in mammals, depending on the cell type , althougLGP2-induced ATP hydrolysis is a requirement for the picornavirus-mediated IFN response ,25,26 anWe evaluated whether the RNA binding of TRBP is required for the up-regulation of ATP hydrolysis activity using mutants with possible defects in RNA binding  , since RUsing our real-time ATP hydrolysis assay, we demonstrated that TRBP positively enhanced the RNA-dependent ATP hydrolysis activity of LGP2 and, in parallel to IFN enhancement, phosphomimetic TRBP showed a lower effect than non-phosphorylated TRBP did. In contrast with its effect on LGP2, TRBP partially and strongly inhibited the RIG-I- and MDA5-induced RNA-dependent ATP hydrolysis, respectively, indicating that TRBP differentially regulates the activity of RLRs. Because TRBP is a negative regulator of RIG-I signaling , down-reIt is intriguing that TRBP differentially regulates RLRs depending on the presence or absence of RNA. TRBP up-regulated the activity of RIG-I and LGP2 in the absence of RNA, while it partially inhibited RIG-I and activated LGP2 in the presence of RNA. Drawing on this observation, we have substantiated the interaction between RLRs and TRBP through the utilization of purified proteins B. The enTRBP may contribute to the structural transformation of RIG-I and LGP2 to their active forms for ATP hydrolysis independent of RNA presence. The up-regulation of ATP hydrolysis activity in RIG-I, which occurs independently of RNA, has the potential to result in the constitutive activation of IFN by TRBP under certain conditions.Another dsRNA-binding protein, protein activator of interferon-induced protein kinase PACT (EIF2AK2 and PRKRA), which is structurally similar to TRBP, has also been reported to activate RIG-I-induced ATP hydrolysis in the absence of RNA, thereby enhancing the RIG-I-dependent antiviral response .It is also interesting that the effect of phosphomimetic TRBP on the ATP hydrolysis activity of LGP2 varied with the absence or presence of RNA. Our RNA-binding experiments demonstrated that the TRBP SD mutant had a greater RNA-binding activity than the nonphosphorylated TRBP did D. TherefThe strong inhibition of MDA5 and weak inhibition of RIG-I during RNA-dependent ATP hydrolysis activity might be due to the sequestration of RNA by TRBP according to the order of RNA-binding activity (LGP2 > RIG-I > MDA5) .The biological role of ATP hydrolysis modulation by dsRNA-binding proteins, such as TRBP and PACT, remains unclear. More specifically, we could not reveal the biological context of LGP2-mediated ATP hydrolysis and how the modulation of ATP hydrolysis contributes to the IFN response. However, we showed that TRBP up-regulated the ATP hydrolysis activity of LGP2, which is required to regulate the IFN response, and, in the phosphorylated state, differentially regulated RLRs depending on the presence of RNAs . FurtherClick here for additional data file.


So far, numerous studies have reported on how coronaviruses affect the human nervous system. However, these studies mainly focused on the impact of a single coronavirus on the nervous system, and failed to fully report the invasion mechanisms and the rules of symptoms of the seven human coronaviruses. This research can assist medical professionals in identifying the regularity of coronavirus invasion into the nervous system by examining the impacts of human coronaviruses on the nervous system. Meanwhile, the discovery also helps humans to prevent the damage to the human nervous system caused by the more novel coronavirus in advance, thus reducing the rate of disease transmission and fatality caused by such viruses. In addition to describing the structures, routes of infection, and symptomatic manifestations of human coronaviruses, this review also finds that the structures of human coronaviruses correlate with virulence, pathways of infection, and blocking mechanisms of drugs. This review can provide a theoretical basis for the research and development of related drugs, promote the prevention and treatment of coronavirus infectious diseases, and contribute to global epidemic prevention. Human coronaviruses refer to single-stranded RNA viruses with a protein envelope, including Severe Acute Respiratory syndrome virus (SARS-CoV), Middle East Respiratory Syndrome virus (MERS-CoV), HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1. Many researchers regard these coronaviruses as potent respiratory viruses, ignoring their ability to invade the nervous system (NS). The emergence of a new coronavirus, SARS-CoV-2, which also belongs to the human coronavirus, is brought about by the novel coronavirus disease (COVID-19). Numerous studies have elaborated on the mechanisms, pathways, and scopes of SARS-CoV-2 infestation in humans, and found that it can cause not only intestinal and respiratory diseases in humans , but alsThe seven human coronaviruses are classified as beta coronaviruses or alpha coronaviruses. Among them, SARS-CoV-2, MERS-CoV, HCoV-HKU1, HCoV-OC43, and SARS-CoV belong to the branch of beta coronaviruses. Genome analysis showed that SARS-CoV-2 and SARS-CoV had a highly homologic sequence . FurtherTherefore, this paper will describe the influence of the above seven coronaviruses on the NS, summarize and analyze the mechanisms of action and symptoms of these coronaviruses attacking the NS, as well as the connection between these coronaviruses and disease prevention and treatment. By exploring the influence of different coronaviruses on the human nervous system, the regularity of coronaviruses’ invasion into the nervous system of the body can be found. This discovery can provide a reference for the prevention and control of the more novel coronaviruses that may appear in the future. Meanwhile, it also can help humans to prevent the damage to the human nervous system caused by the more novel coronavirus in advance, thus reducing the rate of transmission and fatality of disease caused by such viruses, and contributing to global epidemic prevention and control.Coronaviruses are named because of their coronal structure under electron microscopy . The posThe S protein is a critical mediator of coronavirus infection of the host cell, like SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoV-NL63 require binding to the receptor with the help of the RBD on the S protein. Additionally, the combining capacity of RBD is used to judge the virulence of coronavirus, and the stronger the combining capacity, the greater the virulence. For example, the binding of the S protein to ACE2 relies on the cleavage by the host cell proteases at the S1/S2 cleavage sites, and the process requires the assistance of transmembrane serine protease (TMPRSS2) . The S1 When coronaviruses infect cells, there are two ways to enter the cell with the help of the S protein: Firstly, after the S1 protein binds to the receptor, the coronavirus uses TMPRSS2 on the host cell to cleave the S protein, and its outer membrane fuses with the host cell membrane, thereby infecting the host cell. Secondly, the coronavirus enters the host cell directly with the receptor through endocytosis. The above processes are inseparable from the S protein. The ability of the coronavirus to combine with host cells will be severely compromised if the S protein is inactivated, which lowers the infectiousness and virulence of the coronavirus. The S protein hence can be regarded as a crucial indicator of the virulence of coronavirus. Therapy of the coronavirus can be achieved by disrupting the structure and activity of such proteins. However, it has been found that TMPRSS2 can be inhibited by protease inhibitors, thus reducing the infectivity of coronaviruses. Camostat is the first serine protease inhibitor proven to inhibit TMPRSS2 . Soon afThe HE protein can bind to sialic acid on the surface glycoproteins of coronavirus particles  , and it The E protein has ion channel activity and is a “target” for calcium ions. Under the action of calcium ions, the E protein stimulates the inflammasome, prompts the inflammasome to secrete inflammatory factors, enhances the inflammatory response, and finally destroys the NS . MoreoveAfter learning the destruction mechanism of the above coronavirus structural proteins in the human body, people can reduce the transmission rate of coronaviruses by disrupting or binding the chemical structure of these structural proteins with drugs. Therefore, before the coronavirus invades the body, spraying 75% of the volume fraction of alcohol can destroy the structure and properties of the coronavirus protein, and achieve the effect of eliminating the coronavirus.The NS includes the CNS and the PNS. The CNS controls brain movement and the body’s homeostasis, while the PNS is closely related to the sensations and movements of the body and internal organs. When the coronavirus enters the body and attacks the NS, it is more susceptible because the peripheral nerves (PNs) are directly connected to the local tissues. Further, the coronavirus can cross the blood-brain barrier  to reach and destroy the CNS. The primary function of BBB is to maintain a relatively constant brain environment and protect the brain tissue and the CSF. However, many studies have found coronavirus particles in human cerebrospinal fluid, suggesting that coronaviruses can destroy and cross the BBB. The following section is a comprehensive description of the main pathways and mechanisms of the CNS and PNS damage caused by coronaviruses.The CNS consists of the brain and spinal cord, the central part of the NS. The brain is composed of the brain stem, cerebellum, etc., and there are glossopharyngeal nerve and vagus nerve on the side of the medulla oblongata in the brain stem. The cerebellum receives information from the brain stem, spinal cord, and cerebral cortex, and communicates with all three through the feedback loops. This process can not only coordinate the voluntary movement initiated by the cerebral cortex, but also regulate the activities of motor neurons in the brain stem and the anterior horn of the spinal cord, thus coordinating the contraction activity and muscle tension of all muscle groups in the body. Correlatively, the spinal cord includes gray matter and white matter. The gray matter is the site of an extensive collection of the nerve cell body. It innervates skeletal muscle, smooth muscle, cardiac muscle, and glands. The white matter consists of nerve fibers that can carry afferent impulses from the spinal cord to the brain, or efferent impulses from the brain to the spinal cord to form various conduction tracts, such as the corticospinal tract. Because the operation of the aforementioned human physiological structure can affect human behavior and state, clinical patients will exhibit related movement changes and mental states due to the NS damage. Therefore, a preliminary diagnosis of the link between the coronavirus and the CNS can be made based on patient symptoms. The cellular condition of damaged tissue needs to be detected by the CSF or other brain tissue material, or by electron microscopy, which can further confirm the mode of the coronavirus invasion of the CNS. Recently, the symptoms  and testing (presence of COVID-19 RNA in the CSF) of the COVID-19 patient also support this assertion. Numerous investigations have also demonstrated that diverse coronavirus types penetrate the central nervous system (CNS) via multiple paths, triggering various neurological disorders. The characteristics of coronavirus invasion into the CNS can be summarized as four pathways: olfactory system pathway, receptor binding infection of cerebral vascular cells, blood circulation infection of mononuclear/macrophages (Trojan horse mechanism), and neurons retrograde across synapses.The olfactory system pathway mainly involves the transmission and destruction of the CNS by a coronavirus from olfactory epithelial cells through the olfactory nerve and the olfactory bulb. Studies have shown that after the administration of drugs in the nasopharynx of mice, the fluorescent-labeled drugs formed a transmission route. This pathway travelled from the nasopharynx to the olfactory bulb, its leading edge, the interior of the olfactory bulb and tissues, and eventually the central nervous system . TherefoThe receptor binding pathway is the approach through which virus particles contact and invade host cells by combining surface proteins with host cell receptors, thus infecting the human body. These seven human coronaviruses can affect the CNS by receptor binding pathway, but their receptors are not identical. Coronavirus receptors primarily include protein receptors and non-protein receptors. The aminopeptidase N, ACE2, and dipeptidyl peptidase 4  are the most common protein receptors. Also known as APN, the aminopeptidase N is a metal transmembrane glycoprotein of zinc ion-dependent membrane-bound type II and adhesive the receptor for all coronaviruses . SARS-CoThe virus infects mononuclear/macrophages through blood circulation, and the immune defense cells can naturally cross the BBB and move uncontrollably toward the brain tissue in a process called Trojan horse mechanism. Immune cells are constantly multiplied by the stimulation of the coronavirus, exerting an immune effect and destroying the structure and function of the NS. Also, the activation of coronavirus particles in the circulatory system enhances the release of inflammatory cytokines. This reaction can disrupt the integrity of the blood-brain barrier and affect its permeability, prompting T-cell invasion into the CNS, which can trigger inflammation of the brain tissue and further develop encephalitis . Xu et aAfter infection of peripheral neurons, coronaviruses can infect synaptic neurons connected to central neurons or other peripheral neuron cell bodies by retrograde transsynaptic neuron propagation (reverse movement from postsynaptic neuron to presynaptic neuron) . HCoV-OCThe above content describes the main ways and mechanisms of the human CNS damaged by coronaviruses, which helps people to understand the invasions of coronaviruses more accurately. The types, names, and routes of entry into the CNS of various coronaviruses are summarized in The PNS is composed of cranial and spinal nerves. In terms of function, the PNS can be divided into sensory nerves and motor nerves, which have different functions due to the different distribution locations of neurons. The peripheral protrusions of sensory neurons are distributed throughout the body along with the spinal nerves, and their endings form various receptors that sense various stimuli. Meantime, motor neurons can be found in skeletal muscle, heart muscle, smooth muscle, and glands. These neurons primarily control the human body’s movement and the voluntary movement of internal organs, such as the movement of the skeletal muscles and the peristalsis of the intestinal tract. According to the state of coronavirus invasion of the PNS, the ways of invading peripheral neurons can be classified into two pathways: direct destruction and indirect destruction.Neuromuscular symptoms were reported in patients with SARS-CoV from Taiwan, China . This syThis view provides a new idea for the treatment and research of peripheral nerve diseases caused by coronavirus infections in the future, and can be more accurately understood through the clinical manifestations of patients.The ability of coronaviruses to infect NS is related to structural proteins on the envelope, such as S proteins and HE proteins, which can bind to receptors on the surface of host cells. And the virulence of coronaviruses carrying these proteins changes as their activity varies. In short, the stronger the protein activity, the more virulent the coronavirus. Because the affinity between SARS-CoV-2 S protein and ACE2 is higher than that of SARS-CoV, the former has a higher ability to infect host cells than the latter. Especially in the “receptor binding pathway” in which coronavirus invades the CNS, as the “right-hand man” of coronavirus, the S protein can constantly bind new receptors and destroy host cells with the spread of coronavirus. Additionally, it is crucial to recognize the role that the HE protein plays. It can be used as both a mediator and a cofactor to bind receptors, and can also promote the binding of S protein to receptors, speeding up the rate of virus invasion. Furthermore, the E protein induces an immune response that “accidentally wounds” the normal cells, causing them to be damaged or die. The N protein can participate in and promote viral replication, and accelerate the adaptability of the virus to the host. As a result, several drug research and development teams are attempting to stop the proliferation and pathogenicity of coronaviruses by destroying the glycoproteins from their surface. At the same time, these proteins have become the markers for identifying coronaviruses. It can be considered whether the specific recognition of immune cells promotes the occurrence of human-specific immunity, and finally eliminates the coronaviruses. It is also a crucial idea for vaccine research.After entering the body, coronaviruses can rely on unique structural proteins to invade host cells  and affect the NS function. The mechanisms of coronavirus invasion into the CNS and the PNS are comprehensively described in this article. The patient’s performance varies as a result of the coronavirus infecting the two nervous systems. Thereby, the infection routes and clinical manifestations of the CNS and the PNS caused by coronaviruses are analyzed and compared, as shown in As shown in For the combination of coronaviruses (such as SARS-CoV-2) and the olfactory epithelial cells in the nasopharynx, humans can prevent the invasion of coronaviruses on the epithelial mucosa and olfactory epithelial cells by covering the nose and mouth. With this measure, humans can effectively prevent the coronavirus from entering the blood circulation and prevent a range of respiratory and nervous system diseases. If a person is already infected with such a virus, he or she will need to take the medication as prescribed by his or her doctor. The action mechanisms of drugs usually prevent coronaviruses from binding to ACE2 receptors on host cells, by destroying the structure of viral proteins or binding to viral proteins. The result is to reduce the infectivity of coronaviruses to host cells and achieve the therapeutic effect. Currently, it is known that the antibiotic ceftazidime could bind to the RBD domain of S protein and inhibit the binding of S-RBD to ACE2, thus blocking the invasion of coronaviruses . AnotherAccording to the inhibitory effects of drugs on coronaviruses, the inhibitory pathways of drugs can be plotted, as shown in The infectivity of a virus is determined by both the route of transmission and the intensity of infection. Among the seven human coronaviruses, SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoV-OC43 have the most transmission paths, so they are more obvious in patients with more diverse symptoms. Nonetheless, unlike the high fatality rates of viruses such as SARS-CoV and MERS-CoV, the mortality rates of patients infected with SARS-COV-2 and HCoV-OC43 are not high. This means that the transmission path is not directly related to the fatality rate, which is more due to the infectivity of the coronavirus and the patient’s pre-existing illness. SARS-CoV-2, for example, worsens or kills patients mainly by inducing or aggravating their existing diseases, so the fatality rate among middle-aged and elderly patients is much higher than among teenagers. In contrast, the remaining common coronaviruses  are less infectious because of their limited transmission routes and infectivity, and the diseases they cause are mostly mild and usually present with symptoms associated with respiratory tract infections.This article reviews the mechanisms of coronavirus infection in the NS, and thoroughly explains the impacts of the viruses on the PNS and the CNS, which can effectively help people to prevent the infection of the coronavirus, reduce the damage of the coronavirus to the human body, and contribute to the prevention of the infection of the coronavirus. Further, according to the NS invasion mechanisms of different coronaviruses, the consequences and symptoms of individuals infected with the coronavirus may be assessed, and all diseases brought on by coronaviruses can be purposefully treated. Therefore, the exploration of the mechanisms of coronavirus invasion into the NS can provide a theoretical basis for the research and development of related drugs, further promote the prevention and treatment of coronavirus infectious diseases, and contribute to the global epidemic prevention cause.AS conceived and planned the overall structure of the review, wrote the manuscript, and approved the final manuscript. HC collected the references and wrote the manuscript. AS and HC collaboratively finished the revision of the manuscript. Both authors contributed to the article and approved the submitted version.


Carcharhinus melanopterus) to simulate potential predatory threats to mesopredatory reef fishes . The responses of these reef fishes to the models and the snorkelers were compared to those generated by three non-threatening controls . A Remote Underwater Stereo-Video System (Stereo-RUV) recorded the approach of the different treatments and controls and allowed accurate measurement of Flight Initiation Distance (FID) and categorization of the type of flight response by fishes. We found that mesopredatory reef fishes had greater FIDs in response to the approach of threatening models  compared to the controls (706 ± 151–896 ± 8963 mm). There was no significant difference in FID of mesopredatory fishes between the shark model and the snorkeler, suggesting that these treatments provoked similar levels of predator avoidance behaviour. This has implications for researchers monitoring behaviour in situ or using underwater census as a technique to estimate the abundance of reef fishes. Our study suggests that, irrespective of the degree to which sharks actually consume these mesopredatory reef fishes, they still elicit a predictable and consistent antipredator response that has the potential to create risk effects.Both sharks and humans present a potentially lethal threat to mesopredatory fishes in coral reef systems, with implications for both population dynamics and the role of mesopredatory fishes in reef ecosystems. This study quantifies the antipredator behaviours mesopredatory fishes exhibit towards the presence of large coral reef carnivores and compares these behavioural responses to those elicited by the presence of snorkelers. Here, we used snorkelers and animated life-size models of the blacktip reef shark ( These risk-induced trait responses include modifications in prey behaviour5 (referred to hereafter as antipredator behaviours). Predation avoidance behaviours can result in reduced access to food and mates and can ultimately influence the demography, fitness, growth and morphology of prey8. Such non-consumptive effects of predators are known as predation-risk effects9.Predators are a key determinant of the structure of ecosystems, impacting energy flows and nutrient cycling both through the consumption of prey and by inducing trait responses in prey species that mitigate predation risk10. As humans are free from many of the energetic and physical constraints facing other nonhuman predators, they can exploit large-bodied mesopredator and carnivore guilds at very high rates, which has resulted in them being termed “super-predators”12. In terrestrial systems, where humans and large carnivores compete for the same prey, the presence of humans or human cues can have pervasive effects on these ecological communities. For example, the sound of human vocalisations can trigger avoidance responses in large carnivores and reduce foraging in medium-sized carnivores, which in turn benefits small mammals that increase foraging and habitat use13. In marine systems, where large predators are highly mobile14 and have been severely depleted15, the extent to which humans and large carnivores contribute to ecosystem function and ultimately the expression of life history traits in prey species is largely unknown.In both terrestrial and marine ecosystems, the guild of predators at or near the top of the food chain includes humans17. Furthermore, the presence of an observer may also be perceived by prey fishes as a threat, confounding responses to predators and reducing the likelihood of predatory interactions18. To circumvent these issues, researchers have used life-sized models of predators to evoke antipredator responses in reef fishes. This is typically combined with the use of remote cameras to avoid disturbance from an on-site observer. To date, these studies have used only static models20, which realistically mimic ambush predators such as coral trout and groupers (Serranidae20), but do not imitate the behaviour of many of the large predators in the reef systems, such as sharks (but see19). These large animals are highly mobile in both horizontal and vertical planes22 and move over extensive home ranges23. For this reason, a static model may not offer a realistic simulation of predatory threat because, unlike a live shark, the model is continually present within the experimental arena and thus might offer a greater degree of threat to prey species. Additionally, because the model is inactive, prey may habituate to its presence24. One effective solution to this issue is the use of animated models that can provide a predator threat in a more realistic manner to prey species. Such an approach has been used in the study of bird ecology, where researchers have animated models of hawks and other apex predators to examine the anti-predatory responses of prey25.In most coral reef ecosystems, quantifying the relative impacts of different fish predators on their prey has been problematic. Large mesopredatory and apex species such as sharks that prey on other fishes are highly mobile and interactions with their prey occur unpredictably and often in a transitory manner29. For example, flight initiation distance  is much greater in areas where fish are frequently targeted by spearfishers than in places where spearfishing does not occur31.The use of animated models of fish predators offers the opportunity to compare impacts of fish and human predators in a single experiment. Here, we examine the behavioural responses of mesopredatory reef fishes to the presence of shark models and human snorkelers in a coral reef ecosystem. Spearfishing by snorkelers is an important means of harvesting species for recreational, artisanal and small-scale fisheries in these environments. Unlike many other types of fishing, this activity is known to have predictable impacts on the behaviour of reef fishes that increase their responses to reduce capture risk32. We used an experimental approach to compare the threat response of these reef fishes to animated models of sharks and to the presence of snorkelers. We used standardized metrics (including FID) to quantify the response of reef fishes to these threat treatments compared to animated models that did not pose a predation threat (sea turtles and two object controls). As prey individuals of large body sizes tend to display the greatest responses to predatory threat34, we predicted that FID would be greater for larger than smaller mesopredators in the presence of both animated models of sharks and snorkelers. Additionally, we used the difference of swimming speed prior to treatment approach and during flight response to calculate speed of flight as a measure of threat response to the different treatments. We predicted a higher difference in swimming speed during the approach of threatening treatments compared to the approach of the non-threatening animated models and controls and expected a greater difference in swimming speed for larger mesopredatory fishes during the two threatening treatments.Mesopredators, including lethrinids, haemulids, lutjanids and serranids , are relatively large reef fishes that are an important target for spearfishing in coral reef systems and at the same time show threat responses to sharks as a potential predator and/or a lethal competitorThe study was conducted in November and December of 2017 on the fringing reefs surrounding Lizard Island  a no-take zone or IUCN II Marine Park, in the northern Great Barrier Reef (GBR), 30 km from the mainland  , which is a locally abundant predator on the Great Barrier Reef35. A snorkeler was used as the second threatening treatment . Finally, a piece of transparent Perspex  during each trial. These treatments consisted of a life-sized  fiberglass taxidermic casting of a blacktip reef shark , which consisted of two cameras (GoPro HERO3 Silver) mounted 0.7 m apart on a steel frame, facing 8° inwards to achieve the optimal field of view and calibrated before and after measurements, as perDeployments of treatments were randomised with at least 20 min between deployments. Three replicate trials were conducted per treatment at each of the five pulley systems , resulting in a total of 75 videos. A minimum wait of 24 h separated any treatment replicates within a pulley system. One replicate trial was discarded due to the presence of a live blacktip reef shark appearing in the video.37) of the RUVS to ensure accuracy of measurements of lengths or distances38. For each video the reactions of individual fishes to the approach of the treatments were categorized into four responses: C-turn, flight, hide, and no response, with the latter indicating that the fish did not move or change its position at any point during approach of the object on the pulley system or the snorkeler . Mesopredators on coral heads were mostly representatives of the reef fish families Haemulidae, Serranidae and Lutjanidae (see Table 39 on the eye40 or tail of the fish (perspective dependent) at three, six and nine seconds prior to flight. A small piece of Blu Tack was placed on the screen to mark the position coordinate and the distance was measured between two consecutive position coordinates40. These distance measurements were converted to speed using the Eq. , it was excluded from the analysis as distance swum could not be calculated accuratelyLutjanus, Diagramma, Plectorhinchus and Plectropomus), body length (continuous), and number of fish  were included as fixed factors. Body length was log10 transformed for all analyses to ensure that the data were normally distributed  with a full-subsets information theoretic approach was used to examine the influence of treatment, treatment size, fish body length, fish genus and number of fish on FID and difference in swimming speed of fish before and after flight was initiated. The effect of VHB was only examined on FID, as measurement of VHB after flight had begun was logistically challenging to standardize. The five treatments , genus of mesopredator  and AICc weights (wAIC43) were used for model selection. Models containing variables with correlations > 0.28 were excluded from the analysis to eliminate strong collinearity (as per the recommendations of Graham)44. Models with AICc values that differ by less than two units show weak evidence for favouring one over the other43. For this reason, the most parsimonious model was considered the one with the fewest variables and within two AICc units of the lowest AICc value45. The wAICc were used to assist in the interpretation of the best models. The summed wAICc across all subsets of models was used to obtain the relative importance of each variable45. Models were fitted to a Gaussian distribution and identity-link function, as all response data were approximately normally distributed. All analyses and graphical representations were conducted using R46 with the packages dplyr47, ggplot248 and Patchwork49.The package 50) for the most parsimonious models for both FID and speed of flight. The PERMANOVA was analysed using PRIMER v6 and the add-on package PERMANOVA+; with 9999 permutations51. For this analysis four fixed factors were included: treatment , genus of mesopredator , body length (continuous) and number of fish  and one random factor: pulley system (five levels), which was nested within site.In addition to the information-theoretic approach, a univariate permutational analysis of variance (PERMANOVA) was used to independently test for significance (95% confidence) and appropriate pair-wise comparison  and were conducted within University guidelines. Informed consent was obtained from all human subjects.Overall, 90% of mesopredatory fishes displayed an escape response  when approached by the shark model, and 96% displayed an escape response to a snorkeler. In contrast, the turtle, pipe and Perspex treatments had very high proportions of fish displaying no response  included treatment and body length, which collectively explained 21% of the variation Table . Model pgth Fig. b.Table 2This result was corroborated by a PERMANOVA, which found significant effects of treatment  and body length on FID . Pairwise comparisons indicated a significant effect of body size on FID for the smallest size class, with a larger FID in mesopredatory fishes > 200 mm  than individuals ≤ 200 mm in length . Pairwise comparisons found no difference in the speed of flight of mesopredators in shark and snorkeler treatments . There was, however, a significant increase in the speed of flight in the shark compared to the pipe treatment , but no difference between shark and turtle  or shark and Perspex  treatments. The speed of flight differed significantly between the snorkeler and turtle , snorkeler and pipe  and snorkeler and Perspex  treatments.Our study found that large mesopredatory reef fishes responded to the approach of non-human and human threats (animated shark model and snorkeler) by displaying greater flight initiation distances (FIDs) and faster speeds of flight compared to responses to non-threatening models (turtle) or object controls. The approach of the model shark and snorkeler elicited similar FID and speed of flight reactions, although it was not clear whether mesopredatory fishes might regard these threats as potential predators or lethal competitors.53, can occur across the guild of large predatory reef fishes. Reef sharks, such as the blacktip, are known to feed on large predatory teleosts54, but are also known to compete with these fishes for smaller prey56; thus the escape response triggered by the shark model could be interpreted as an anti-predatory behaviour and/or a response to a potentially lethal competitor. Regardless of the motivation for the escape response, our key finding is that the approach of a threat elicited a flight response in mesopredatory fishes and one that may plausibly result in an individual forgoing the opportunity to forage, interact or compete with other individuals of the same or other species in the vicinity. This adds to the evidence that the presence of larger predators such as reef sharks and humans may ultimately create risk effects within reef fish populations.Lethal competition or intraguild predation, defined as the killing and consumption of a potential competitor58. Earlier studies that focused on spearfishers as a threat have also found a similar correlation with size in many29, but not all coral reef fishes31. Gotanda et al.33 attributed this positive relationship to the lower risk-taking associated with the higher reproductive value of larger fish, consistent with predictions of the asset-protection principle, which states that as reproductive value increases, risk-taking should decrease59. Another (non-exclusive) possibility is that larger fish are older and have more experience with potential predators, thus are more wary. There are many field studies that show that prey can learn from earlier attempts at predation directed towards them and their conspecifics63. Additionally, larger mesopredatory fishes are more likely to be competitors with reef sharks, so may be under greater threat as they increase in size.Body length was positively correlated with FID in mesopredatory fishes, as has also been seen in birds64. However, our experiment took place in a no-take zone, where there is theoretically zero fishing pressure, and all fish are protected from spearfishing regardless of their size. This implies that other factors might explain the greater FIDs seen in larger fish. Interestingly, a recent meta-analysis conducted on FID studies failed to detect any effect of habitat protection status as an explanation of the body length-FID relationship29. Although our experiment was conducted immediately adjacent to coral heads, the proximity of shelter can also influence FID65. For example, Lester et al.17 found that mesopredatory fishes took longer to feed in the presence of a shark model at greater distances from the shelter of a patch reef. Larger fish may require relatively large crevices and holes in which to shelter from predators, which may force them to move earlier in response to a threat than smaller species if these refuges are rare on the reef. It is notable that the FIDs we recorded in this study were only a third of the distances reported for large mesopredators on the reef slopes of coral reefs with only moderate complexity26. This implies that reef slopes may offer less shelter than the coral heads in the lagoon and/or that there may be higher predation pressure on large mesopredators on the reef slope. Certainly, relative densities of sharks as measured by BRUVS and underwater visual surveys are higher in the reef slope habitat than the shallow lagoon or backreef66. Rigorous comparisons of FIDs across habitats will require both empirical measures of habitat complexity and of the abundance and distribution of shark populations.Other studies have concluded that the increased FIDs with size could be attributed to higher spearfishing pressure on larger fishes68. Visual acuity is expected to increase in larger fish69, allowing them to detect the approaching threat before smaller fishes. In addition, larger fishes may have better sensory systems to detect cues that prompt anti-predatory behaviour70, such as smell71, electrosensory and pressure fields72, alarm calls from other conspecifics and predator calls73. Future experiments that involve other sensory systems could provide a more comprehensive approach to threat detection and the associated anti-threat behaviours of mesopredatory fishes.The ability of prey to detect an approaching threat can influence the level of wariness and willingness to take risks34, who found that smaller fish had faster escape speeds, as is also the case with size in lizards76. These earlier studies suggested that their results were due to trait compensation, whereby individuals with a trait that made them more vulnerable to predation had stronger antipredator behaviours than those that lacked this trait. In our study, the positive relationship between size and escape speeds may be an example of co-specialisation of anti-predator behaviours77, similar to other fishes such as sturgeon (Acipenser fulvescens) where larger body size is also positively correlated with stronger and more sustained escape responses78.We found that speed of flight increased with body size of mesopredators. These results contrast to those of Miller et al.18 found that reef fishes showed the same response to the threat of a model of a large coral trout that was 78 cm in length and a model of a black tip reef shark of 170 cm in length, despite the over two-fold differences in relative size of these predators. In this case, the control treatment (a pipe) was the same size as the large coral trout. Such differences in relative sizes of predators have also been found to have little effect on anti-predator behaviour of prey of fishes in other experimental systems79, suggesting the effect of predator size in evoking antipredator behaviours may not be as significant as predator identity and other visual cues61. Fish are well known to recognize features, such as body shape, presence and position of eyes and shape of mouth to identify potential predators80, although disentangling the effect of size and visual cues in prey recognition has proven challenging to date.It is important to note that our object controls were smaller than either the model reef shark or the snorkeler. Although it is possible that the differences in reaction between our controls and threatening treatments were a response only to relative differences in sizes of the objects approaching the reef, size was not included within the most parsimonious model in our analysis. Similarly, an earlier study81.Finally, our results have implications for researchers seeking to survey reef communities or monitor behaviour, since they show that the presence of an observer could induce behaviours in reef fishes that seek to reduce potential predatory threat. This might provide an explanation as to why techniques such as underwater visual census often underestimate densities of the larger, mobile reef fishes within coral reef ecosystemsSupplementary Information.


Using putative m6A/m6Am sites called from 8 profiling approaches, we illustrate that iMVP enables comprehensive comparison of different approaches and advances our understanding of the difference and pattern of true positives and artifacts in these methods. Finally, we demonstrate the ability of iMVP to analyze an extremely large human A-to-I editing dataset that was previously unmanageable. Our work provides a general framework for the visualization and interpretation of epitranscriptomic data.Advances in sequencing technologies have empowered epitranscriptomic profiling at the single-base resolution. Putative RNA modification sites identified from a single high-throughput experiment may contain one type of modification deposited by different writers or different types of modifications, along with false positive results because of the challenge of distinguishing signals from noise. However, current tools are insufficient for subtyping, visualization, and denoising these signals. Here, we present iMVP, which is an interactive framework for epitranscriptomic analysis with a nonlinear dimension reduction technique and density-based partition. As exemplified by the analysis of mRNA m The current available tools lack the ability to accurately classify and visually represent epitranscriptomic profiling data. Here, the authors provide a framework that offers a general solution for the visualization and interpretation of such data. Accordingly, RNA modification writers are spatiotemporally controlled, and their misregulation leads to a number of pathologies. For example, the writers of m6A and A-to-I editing are misregulated in multiple types of cancers and have been identified as promising therapeutic targets of cancers5, with related cancer treatments in pre-clinic or pre-investigational new drug (IND) stages. Catalytic inhibitor of METTL3 STM2457 leads to impaired engraftment and prolonged survival in various mouse models of AML5. ADAR1p150 inhibitor Rebecsinib prevents malignant A-to-I editing-mediated leukemia stem cell self-renewal in completed pre-IND studies6.The recent discovery of various RNA modifications in the transcriptome has given rise to the fast-growing field of epitranscriptomics. Individual RNA modification sites have been found to regulate nearly all aspects of RNA metabolism and are involved in a wide range of biological processes11). Putative RNA modification sites called by these strategies from a single high-throughput experiment contain one type of modification deposited by different writers  or different types of modifications . Meanwhile, various levels of noise are present in such putative RNA modification sites because most methods have limited accuracy and specificity17. Despite the importance of the precise classification of modification types and their corresponding writers, the currently developed tools are insufficient to address this issue.Methods for transcriptome-wide RNA modification detection at the single-base resolution are undergoing rapid development. The common strategies are to convert RNA modification signals into next-generation sequencing-detectable signals, such as mutation and truncation  for epitranscriptomic subtyping, visualization, and denoising, with demonstrations of its utility on various kinds of high-throughput experiments.Here we developed a framework termed 27 or discovering motifs by canonical methods (e.g. MEME28), were performed.Our strategy is based on two principles. (1) Most RNA modifications are deposited within a specific sequence context, which is determined by the biophysical interactions between writers and their substrates. This is presumably also true for most artifacts introduced by the enzymic and chemical processes in modification detection. (2) The input sequences only account for a tiny subset of the k-mer space. Accordingly, our iMVP framework consists of five steps for the analysis and visualization of the topological distribution of RNA modifications, as follows Fig. : (1) the30, especially t-distributed Stochastic Neighborhood Embedding (t-SNE)31 and Uniform Manifold Approximation and Projection (UMAP)30. To benchmark the candidate algorithms, we selected m5C profiling data in fly embryos, which consisted of experimentally validated NSUN2-dependent  sites and NSUN6-dependent  sites with clear motif preferences  than PCA, Isomap34, and DensMAP35, generating projections much closer to their corresponding centroids. Compared with t-SNE and Open t-SNE, UMAP had a better discernibility , making the boundaries of Type I and Type II sites much clearer  treated HeLa cells, in which at least 4 motifs, including two minor motifs, are present  and numeric labeling. No other methods with better performance than that of the one-hot encoding method were found  and graph-based algorithms  were able to recognize distinct clusters from UMAP projections .Since the majority of reported RNA modification motifs are within a range of 15 nucleotides (nt)To compare the performance of iMVP with other traditional tools in motif search, we analyzed two simulation datasets that comprised 100,000 and 200 sequences with 12 and 5 heterogeneous motifs, respectively , we extensively optimized the parameters of the traditional tools for the larger dataset, resulting in the generation of 200 parameter sets (see ”Methods” section). We evaluated the performance of different methods by analyzing their computational time and accuracy in motif searching. Overall, our findings revealed that iMVP surpassed existing tools in terms of both computational time and precision . With these methods, modification caused errors and disturbances have been found to occur from the −3 to +3 positions relative to the modification sites46. Because of the position-aware nature of iMVP, we proposed a phase-matching strategy to solve such a “phase mismatching” issue, thus broadening the applications of iMVP.In addition to the scenario in which positions with modification-induced signals matched the coordinates of RNA modification sites themselves, signals generated due to the modified bases can be shifted to adjacent positions. For example, such an issue is common in signals from Nanopore direct RNA-seq, which shows promise for discriminating and identifying different RNA modifications in native RNA. Several methods based on base-calling errors, altered current intensities, or trace profiles of Nanopore direct RNA-seq data have been developed to identify signatures that represent RNA modifications  of sites as authentic m6A sites within RRACH motifs 48 or BBCABW49, as METTL3 is exclusively associated with m6A modification, these sites are likely to be false positives. Consistently, when examining the locations of CAR sites relative to transcription start site (TSS), no enrichment around TSS was observed . A winscore-based method50 was used to quantify the m6A/m6Am peaks in our m6A-seq dataset. Winscore quantifies the enrichment of modified reads within a 50-nt window relative to gene expression and modified reads across the entire gene. As a positive control, we used m6A/m6Am sites identified by m6ACE-seq  generating sliding windows on input sequences; (2) performing iMVP on all the windows to extract clusters; and (3) conducting motif alignment and comparison on the extracted clusters to identify the patterns. To test this strategy, we utilized a simulation dataset comprising 500 PDX1 motifs randomly distributed in a 50-bp sequence, along with 500 random noise sequences. We first generated 20-bp windows with a 1-bp step, resulting in a total of 30,000 windows. Then we applied iMVP to these 30,000 windows  was found in both early developmental stages in vertebrates , was also found  from six species to generate a combined iMVP map. Next, we projected the sites of individual species onto the combined map for comparison. Interestingly, despite all three types of sites being highly consistent among all six species  and 2 (#4 and #5) minor clusters that were likely false positive, respectively  were spiked into the putative RNA modification-induced variants to label the motif preferences of known RNA modifications   Fig. . In the #2) Fig. . Of the ure Fig. . These hves Fig. : some we6A/m6Am at single-base resolution58. A systematical evaluation and comparison of sites called from different methods may shed light on our understanding of the comprehensive methylomes and the pattern of true positives and artifacts in these methods. However, such a comparison is missing due to the lack of tools. To bridge this knowledge gap, we collected m6A/m6Am sites from 7 studies with 8 different m6A/m6Am profiling methods . m6A-label-seq, which is based on metabolic incorporation, had a unique enrichment in cluster #6. Although both MAZTER-seq and m6A-REF-seq utilized the MazF endoribonuclease strategy, sites identified by MAZTER-seq were more enriched in cluster #1. DART-seq, which is based on the hitchhiking of APOBEC C-to-U editing, had a strong bias in cluster #4 (NNACU).We next performed iMVP on the aggregated 74,179 mity Fig. . Cluster6A positions, antibody-independent approaches have become the recent focus of method development. Because the sensitivities of different methods vary and are dependent on the local sequence features of the modified bases, we evaluated the reliability of m6A-antibody-independent approaches by comparing the winscores of individual clusters with the background winscores using m6A-seq peak data (see ”Methods” section). As a positive control, we used clusters identified by the m6A-antibody-dependent m6ACE-seq. DART-seq had the lowest m6A-peak enrichment among all methods   Fig. . After drs) Fig. . Repetit59. We found that both sets of sites had decreased editing levels . Third, in the clustering step, the cluster output may vary with different parameters, especially when extracting clusters from the condensed density tree. Although this issue may be partially solved by introducing a hyperparameter optimization strategy with DBCV scores61, prior knowledge is still required for the selection of the proper parameters.It should be noted that iMVP has some limitations. First, iMVP is not suitable for application to datasets containing too few sites or sites without sequence preferences. Second, iMVP is a position-sensitive strategy, and even with a phase-matching strategy, the current version may have poor performance when dealing with datasets with high numbers of phases. Future improvements, such as the use of parametric UMAPGiven the recent rapid progress in the use of Nanopore direct RNA-seq to discover RNA modifications, it is expected that more tools will be developed to compare native and unmodified RNAs and identify differential sites, i.e., putative modification sites deposited by various writers. Hundreds of thousands to millions of sites may be found in a single Nanopore run. As we have shown that iMVP can handle the extremely large number of A-to-G variant sites, we expect iMVP to greatly aid in the visualization and interpretation of the Nanopore direct RNA-seq data in future studies.Taken together, we anticipate that iMVP will be a valuable tool that can be rapidly adopted by the epitranscriptomics community and facilitate epitranscriptomic research in the future.HeLa cells were purchased from Cell Bank, Type Culture Collection, Chinese Academy of Sciences (CBTCCCAS). HeLa cells have been identity verified using short tandem repeat (STR) analysis by CBTCCCAS. Cells were maintained in DMEM  supplemented with 10% FBS . Cells have been checked for mycoplasma contamination by CBTCCCAS and are routinely tested for mycoplasma by PCR detection of conditioned medium.For Nocodazole treatment, wild type or knockout HeLa cells were first seeded and grown to 50% confluency. For Nop2 knockdown cells, cells were treated with siRNA for 48 hours before nocodazole treatment. Otherwise, cells were treated with 0.1 μg/ml nocodazole  for 48 hours and then collected for further experiments.19. In brief, a gRNA sequence was designed using CRISPR-ERA (http://CRISPR-ERA.stanford.edu). The sgRNA template oligonucleotide was synthesized and cloned into lentiCRISPR v.2 plasmid (Addgene no. 52961). The plasmid was transfected into the cells using Lipofectamine 3000  following the manufacturer’s instructions. Transfected cells were selected using puromycin. Western blot with NSUN5 antibody  was used to verify the loss of NSUN5 protein. NSUN2 and NSUN6 knockout cells generated previously were used in this study21.NSUN5  knockout cells were generated via CRISPR/Cas9-induced mutagenesisFor NOP2 knockdown, NOP2 siRNAs  were transfected using Lipofectamine RNAiMAX  following the manufacturer’s instruction. 48 hours after the transfection, the knockdown efficiency was examined by RT-qPCR with NOP2 primers  and GAPDH (as control) primers .19. In brief, total RNA was isolated with TRIzol reagent and Direct-zol RNA MiniPrep kit. poly(A) + RNA was separated from total RNA using Oligo dT Magnetic Beads . 100 ng–1 μg of poly(A) + RNA was converted using the EZ RNA methylation kit  with a modified high-stringency conversion condition  70 °C, 10 min; (2) 64 °C, 45 min; Desulfonation: 25 °C for 30 min). The converted RNA was fragmented into 150 − 200 nt fragments in fragmentation buffer  at 94 °C for 8 min. The fragmented RNA was then used for library construction using NEBNext Ultra II Directional RNA Library Prep Kit. Libraries were sequenced on Hiseq X10 (Illumina) to produce paired-end 150 bp reads  in IPP buffer  overnight at 4 °C. The m6A-Ab mixture was then immunoprecipitated by incubation with protein-G magnetic beads at 4 °C for another 2 hours. RNA was then eluted from the beads with IPP buffer containing 0.5 mg/ml N6-methyladenosine . VAHTS stranded mRNA-seq library prep kit  was used for library construction. Libraries were sequenced on HiSeq X (Illumina) to produce paired-end 150 bp reads , was fragmented in 1X NEB Next Magnesium RNA Fragmentation Buffer at 94 °C for 5 min. 10 ng of fragmented RNA was saved as input. 10 μg fragmented RNA was further incubated with 5 μg rabbit anti-mA summary of the source of data is in Supplementary Data 5C sites analyzed were obtained with standard BS-seq library preparation and analysis workflow32. These m5C sites were in GRCh37 or GRCm38 coordinates.All m26. We retrieved the strand information of the sites based on the provided gene ID.The processed data of HEK293T (in GRCh38 coordinates) were obtained from the supplementary table of xPore51.We downloaded the site list from the supplementary table of Koh et alThe site lists of ModTect in hg19 (GRCh37) coordinates were obtained from the supplementary table. Only sites with no ambiguous strand direction were used in our analysis. We spiked 14 known modified sequences , Shu et al55. (m6A-label-seq), Pandey et al56. (MAZTER-seq), Zhang et al57. (m6A-REF-seq), Meyer58 (DART-seq), and Pratanwanich et al26. (xPore). For m6A-label-seq data, we retrieved the genomic coordinates based on the RefSeq id and sequences. For DART-seq, we assigned the m6A sites adjacent to the C-to-U signals. For sites in hg19 format, we used UCSC liftOver tool to convert them to Ensembl GRCh38 format. The summarized site list is in Supplementary Data We performed the m25. We grouped the sites into Alu, repetitive non-Alu, and nonrepetitive sites based on the annotations in the original table.The site list (hg38) was downloaded from REDIportalhttps://github.com/sysu-zhanglab/iMVP). Our package (including helper functions and the interactive interface) is available on Python Package Index (Pypi): iMVP-utils. Documents of our analysis and the package can be found on https://imvp.readthedocs.io/. In brief, we encoded the 10 nt flanking sequences of the sites into one-hot encoded format, and then projected the encoded sequences onto a 2-D plane with UMAP. We used HDBSCAN to split the projections based on the density contours. If stubborn clusters  encountered, we further split those clusters with HDBSCAN and merged the small clusters manually based on the density contours. Density Based Cluster Validity (DBCV), which is implemented in the HDBSCAN API, may be used to search for suitable combination of the parameters. For Leiden, the Significance of Hierarchical Clustering (SHC) may be used to calculate the quality of clustering62.For each analysis, we provided the codes, example inputs, and example outputs in detail in Juypter-Notebook .We set up our analysis (CPU-based) with Python 3.7, and the following core packages were used: Pandas (v1.3.4), Numpy (v1.20.0), Scipy (v1.5.1), Scikit-learn (v0.23.1), biopython (v1.77), hdbscan (v0.8.27), umap-learn (v0.5.2), openTSEN (v0.6.1), louvain (v0.7.1), leidenalg (v0.8.8), dash (v2.2.0), dahs-bio (v0.9.0), imageio (v2.13.5), weblogo (v3.7.0), and opencv-python (v4.5.5). For GPU-based analysis, we used the Docker container provided by NVIDIA: RAPIDS release 22.02 in Ubuntu 18.04, Python 3.8, and CUDA 11.5. The workflow was run on a RTX2080Ti with 11 Gb memory. The parameters and operations of each analysis were summarized in Supplementary Data x and y are the X-Y coordinate of a data point, and Outgroup-ingroup score was defined as the coefficient of variation (CV) ratio between outgroup distances and ingroup distances. Ingroup and outgroup distances were defined as the Euclidean distances between a data point to the centroid of a group: P) of the neighbors that were not in the same group of the data point. Last, all proportions were aggregated to draw a cumulative distribution  to 6 and the number of motifs (nmotifs) to 10. Additionally, we explored various values for the maximum motif width (maxw), including 8, 12, and 15. We also varied the Markov model orders (markov_order) from 0 to 3. The mod parameter encompassed zoops, anr, and oops schemas, while the objfuns parameter encompassed the classic, de, se, cd, nc, and ce algorithms. This exploration resulted in the generation of 192 parameter sets for comprehensive testing.For HOMER, to address the constraints of HOMER, which is limited to searching for motifs of a single specific length, we performed an analysis using a set of seven length parameters, spanning from 6 to 12. We also set the number of motifs to 10. The mean values were used to represent the performance of HOMER.For STREME, we adjusted the two parameters: maxw and objfun. The maxw parameter was varied across three values: 8, 12, and 15. while the objfun parameter included two options: cd or ce. This led to 6 parameter sets being tested.E-values, p-values, and q-values of the motifs presented in the study were shown in Supplementary Data E-values of the motifs were calculated by Simple Enrichment Analysis (SEA) of MEME (v5.0.0). All 63. A customized script based on Biopython.motifs API was used to generate the PWM format input for MotifSuite.KL divergences were calculated by MotifSuiteWe used Weblogo (v3.7.0) to plot the motifs of selected sequences. MEME/ STREME (v5.3.3) and HOMER (v4.11) were used in motif discovery.5C sites were called as we previously described19. We first trimmed adapters, the first 10 bp of the reads, the last 6 bp of the reads, and the low-quality bases using Cutadapt (-e 0.25 -q 25 -trim-n)64 and Trimmomatic65. Then clean reads were mapped to the in silico converted genome by HISAT2 66 to obtain unique alignments. The remaining unmapped and multiple mapped reads were further mapped to the in silico converted transcriptome by Bowtie2 67. Alignment results were merged, and only bases with high quality (Q ≥ 30) were used for the variant calling. Last, the sites were called using a series of filters as previously described19. The methylation level (mismatch frequency) is defined as the number of reads with C divided by the number of reads with C or T.mRNA m68 with default settings. The m5C sites and 50 nt flanking sequences were extracted from the genome. The folding frequencies of each base of the sites with their 25 nt flanking sequences were shown.RNA secondary structure was predicted using RNAfold (2.4.12)64 (-e 0.1 -q 20 -m 20 --trim-n). Cleaned reads were then mapped to GRCh38 (Ensembl release 104) genome and transcriptome by HISAT2 with default settings in strand-specific mode66. Only unique alignments were transformed into bedGraph for further analysis. m6A peak analysis was performed as previously described with some modification50. In brief, each gene was split into 50-nucleotide sliding windows and an enrichment fold (winscore) was calculated for each window:We first trimmed adapters using CutadaptFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Reporting SummarySource Data


Response evaluation of carbon-ion radiotherapy poses a diagnostic challenge. Due to its functional aspects, fluorodeoxyglucose positron emission tomography (FDG/PET) has a role in the diagnosis of photon radiation therapy. In addition, several studies suggested that FDG/PET may be useful to select the optimal site for performing a diagnostic biopsy. Here, we report a 73-year-old female in which FDG/PET was effective in determining the recurrence of liposarcoma and the therapeutic effect. Based on the results of FDG/PET, we could make a pathologic definitive diagnosis and selectively performing carbon-ion radiotherapy for active tumors. Liposarcoma is one of the most common malignant soft tissue tumors. Liposarcoma tends to recur in various areas, and it is important how to determine recurrence and therapeutic effect in order to perform carbon-ion radiotherapy (CIRT) with an appropriate irradiation range.Historically, surgery is one of the popular treatments for liposarcoma, but the number of cases treated by CIRT for recurrence of liposarcoma has also increased The patient was a 73-year-old female who underwent wide excision for pleomorphic liposarcoma of her left humerus. About a year and a half after surgery, metastasis to her proximal eighth rib was found. CIRT (70.4 GyE/16FR) was performed for this metastasis. Subsequent follow-up FDG/PET showed no significant uptake in the post CIRT region, but significant uptake was observed in the eighth vertebra approximately 1 year after CIRT. The maximum standardized uptake value (SUVmax) of the eighth vertebra was 10.94 . On the Response evaluation of CIRT poses a diagnostic challenge. CIRT may allow dose escalation to tumors compared to photon radiation therapy while reducing radiation dose to adjacent normal tissues for its favorable dose distribution As Nomia et al. CIRT is frequently considered for re-irradiation therapy due to their more favorable radiation dosimetry, which can often improve normal tissue sparing of organs at risk from additional radiation FDG-PET/CT was effective in determining the recurrence of liposarcoma and the therapeutic effect. FDG/PET was useful for making pathologic definitive diagnosis and performing CIRT for active tumors selectively.Written informed consent was obtained from the patient for their anonymized information to be published in this article.