Drosophila, Prmt5 acts as a type II arginine methyltransferase that catalyzes the symmetrical dimethylation of arginine residues in the RNA helicase Vasa. Prmt5 is critical for the regulation of spermatogenesis, but Vasa is not. To date, functional genetic studies of spermatogenesis in the lepidopteran model Bombyx mori has been limited. In this study, we engineered mutations in BmPrmt5 and BmVasa through CRISPR/Cas9-based gene editing. Both BmPrmt5 and BmVasa loss-of-function mutants had similar male and female sterility phenotypes. Through immunofluorescence staining analysis, we found that the morphs of sperm from both BmPrmt5 and BmVasa mutants have severe defects, indicating essential roles for both BmPrmt5 and BmVasa in the regulation of spermatogenesis. Mass spectrometry results identified that R35, R54, and R56 of BmVasa were dimethylated in WT while unmethylated in BmPrmt5 mutants. RNA-seq analyses indicate that the defects in spermatogenesis in mutants resulted from reduced expression of the spermatogenesis-related genes, including BmSxl, implying that BmSxl acts downstream of BmPrmt5 and BmVasa to regulate apyrene sperm development. These findings indicate that BmPrmt5 and BmVasa constitute an integral regulatory module essential for spermatogenesis in B. mori.In lepidopteran insects, dichotomous spermatogenesis produces eupyrene spermatozoa, which are nucleated, and apyrene spermatozoa, which are anucleated. Both sperm morphs are essential for fertilization, as eupyrene sperm fertilize the egg, and apyrene sperm is necessary for the migration of eupyrene sperm. In  Bombyx mori. Our genetic analyses revealed that both BmPrmt5 and BmVasa are essential for spermatogenesis. Combined with RNA-seq, genetic, immunofluorescence staining, and mass spectrometry analyses suggest that the BmPrmt5–BmVasa module regulates spermatogenesis in B. mori. This study expands our knowledge of reproduction biology in insects and identifies potential gene targets for pest control.In lepidopteran insects, males produce both nucleated and anucleated sperm. Both sperm morphs are essential for fertilization: The eupyrene sperm fertilizes the egg, and the apyrene sperm assist in transport of the eupyrene sperm to the female sperm-storage organs. In this study, we defined the function of the arginine methyltransferase Prmt5 and its substrate Vasa in the lepidopteran insect  Spermatogenesis is a vital process for sexually reproducing animals . During Bombyx mori (B. mori) displays dichotomous spermatogenesis. To date, the studies on dichotomous spermatogenesis in B. mori and other lepidopteran species have been limited mostly to aspects related to cytology and developmental timing carried out by microscopic observations. Only few genes such as poly(A)-specific ribonuclease-like domain-containing 1 (BmPnldc1), Hua enhancer 1 (BmHen1), Sex-lethal (BmSxl), Maelstrom (BmMael), and Polyamine modulated factor 1 binding protein (BmPMFBP1) have been experimentally linked to this process in B. mori [In pursuing novel insights into the diversity of spermatogenesis, Lepidoptera (moths and butterflies) is an emerging and notable taxon. Moreover, understanding reproductive processes in Lepidoptera is valuable because it includes both pest species and species of economic importance ,8. Lepid B. mori ,11,14–20Drosophila melanogaster, mutation of Dart5, a Prmt5 homolog, results in grandchild-less females and male spermatogenesis defects, as well as disruption of the circadian rhythms in locomotor activity [Prmt5 causes a reduction in germ cell number, leading to the failure of gonads to differentiate into normal testis or ovaries and eventual sterility [B. mori or other insects other than Drosophila. Given the precedent that Prmt5 plays a key role in development and gametogenesis in such diverse species, we hypothesize a similar role in B. mori, and in particular seek to assess if it regulates aspects of dichotomous spermatogenesis.Post-translational modifications play critical roles in diverse cellular events including the DNA damage response, chromosome condensation, and cytoskeletal organization during germ cell differentiation ,22. The activity –36. In zterility . The funDrosophila, Xenopus, and mouse [Drosophila and is required for the assembly and function of the pole plasm during oogenesis [Xenopus Vasa homolog is expressed in oocytes and embryos and is required for the formation of germ cells [Drosophila is unknown, and it is of particular interest to assess its role in the dichotomous spermatogenesis of Lepidoptera.A number of Prmt5 substrates have been identified, through biochemical and genetic studies, to function in regulating germ cell specification . Notablend mouse ,38. Of pnd mouse ; it is sogenesis –43. The rm cells –46. The rm cells . Like PrB. mori. We first generated loss-of-function mutants of BmPrmt5 and BmVasa through CRISPR/Cas9-based gene editing. The male BmPrmt5 mutants have severe defects in spermatogenesis, and both males and females are sterile. We showed by immunofluorescence staining that BmVasa is expressed in sperm cysts throughout spermatogenesis. The BmVasa mutants also have severe defects in spermatogenesis, and both sexes are sterile. To gain insights into the changes in global gene expression associated with spermatogenesis defects in BmPrmt5 and BmVasa mutants, we performed RNA-seq analysis and found that levels of mRNAs encoded by numerous cell-differentiation-related genes are significantly decreased. The combined results of our genetic, immunofluorescence staining, and RNA-seq analyses of BmPrmt5 and BmVasa mutants demonstrate that the observed defects in eupyrene and apyrene sperm result from disorganized cellular structural proteins and cell polarity disorders. Thus, in B. mori and likely other lepidopterans, BmPrmt5–BmVasa regulatory module is essential for spermatogenesis.In this study, we characterized the functions of BmPrmt5 and BmVasa in the regulation of spermatogenesis in Drosophila [BmPrmt5 has an organ-specific expression pattern in B. mori, we performed qRT-PCR analysis of samples prepared from different organs. BmPrmt5 was predominantly expressed in the gonads at all developmental stages analyzed  targeting exons 1 and 2 of BmPrmt5 were designed and synthesized, and BmPrmt5 mutants were generated through genetic crossing between the U6-sgRNA lines and the nos-Cas9 lines  females  according to the manufacturer’s instructions. An aliquot of 1 μg of the total RNA was used to synthesize cDNA using PrimeScript RT reagent Kit with gDNA eraser . qRT-PCR analysis was performed on a StepOnePlus Real-Time PCR system  with an SYBR green Real-Time PCR master mix . BmPrmt5 and BmVasa mutants. The nos-Cas9 transgenic silkworm lines (nos-Cas9/IE1-EGFP) express the Cas9 nuclease under the control of the B. mori nanos promoter [U6 promoter were constructed to generate BmPrmt5 and BmVasa mutants. Primers for plasmid construction and sgRNA targeting sequences are listed in A binary transgenic CRISPR/Cas9 system was used to construct promoter . The plaFor silkworm germline transformation, preblastodermal embryos were prepared and microinjected with transgenic plasmids (400 ng/μl) together with helper plasmids (200 ng/μl) and incubated in a humidified chamber at 25°C for 10–12 days until hatching ,63. G0 mBmPrmt5 and BmVasa mutants selected based on EGFP and DsRed fluorescence markers. Genomic DNA of the mutated animals was extracted by standard SDS lysis-phenol treatment, incubated with proteinase K, and purified for mutagenesis analysis via PCR amplification with specific primers , 1:1000; HRP Goat Anti-Rabbit IgG (H+L) , 1:1000; and HRP Goat Anti-Mouse IgG (H+L) , 1:1000. The Immobilon Western Chemiluminescent HRP substrate Kit  was used to detect the protein signal.BmPrmt5 mutants at the fourth day of the fifth larval stage . Ten paired testis of WT and BmPrmt5 mutants were combined as a sample, respectively. We then separated the immunoprecipitates by using 10% SDS-PAGE. After Coomassie Brilliant Blue staining , we excised the band corresponding to the BmVasa protein. Purified BmVasa protein was processed by 10 mM TCEP, 50 mM CAA, and then digested by Trypsin or Asp-N at 37°C for 4 h. The concentrated peptides were desalinated and analyzed using Q Exactive HF-X mass spectrometer . Raw LC-MS/MS data were analyzed using Proteome Discoverer 2.5  against BmVasa protein sequences. Two biological replicates of each sample were performed.To determine the arginine dimethylation sites of BmVasa, we first conducted co-immunoprecipitation to enrich BmVasa proteins from the testis of WT and BmPrmt5 mutant, and BmVasa mutant animals. The sperm bundles were released by tearing testis in PBS buffer. RNA was collected from six individual animals from each genotype, and samples from each genotype were pooled. The mRNA was enriched, and then fragmented and used for cDNA synthesis and library construction. The library was sequenced using Illumina Hiseq Sequencing 2000 System. The raw data were qualified, filtered, mapped, and quantified  to the reference silkworm genome database (http://silkbase.ab.a.u-tokyo.ac.jp/cgi-bin/index.cgi) [2(Y/X) > 1. Enrichment analyses of DEGs were conducted using the gene ontology (GO) analysis [Total RNA was extracted from the testes and sperm bundles from day four in the fifth larval stage WT, dex.cgi) –71, and analysis . The visAll data are expressed as the means ± standard error (SEM). Differences between groups were examined using either a two-tailed Student’s t-test or a two-way analysis of variance. All statistical calculations and graphs were made with GraphPad Prism version 9.S1 Table(XLSX)Click here for additional data file.S1 FigsDMA motifs in aligned sequences are denoted by red arrows.(TIF)Click here for additional data file.S2 FigΔBmPrmt5 represents BmPrmt5 mutants.The red arrow represents dimethylated sites of BmVasa peptide. (TIF)Click here for additional data file.S3 FigBmPrmt5 mRNA and B) BmVasa in testis from day one of the fifth instar to the third day of the pupal stage.(A and B) qRT-PCR analyses of levels of A) (TIF)Click here for additional data file.S4 FigGreen, BmVasa; Blue, Hoechst. Scale bar, 50 μm.(TIF)Click here for additional data file.S5 FigΔBmVasa represents BmVasa mutants.TE and OV denote testis and ovary, respectively. (TIF)Click here for additional data file.S6 FigBmVasa transcript in BmPrmt5 mutant. A) testis and B) ovary (C) Western blot analysis of BmVasa protein levels in testis and ovary of BmPrmt5 mutants. ΔBmPrmt5 represents BmPrmt5 mutants.(A and B) qRT-PCR analysis of (TIF)Click here for additional data file.S7 FigBmPrmt5 and BmVasa mutant testis and sperm samples. Red and blue represent up- and down-regulated DEGs, respectively, with a fold change > 1 and false discovery rate (FDR) < 0.05. Right: Venn diagram of the number of genes commonly and specifically differentially regulated in testis (green) and sperm (blue) samples of BmPrmt5 mutants and BmVasa mutants relative to WT. The “sperm-specific” DEGs were used for further analysis. ΔBmPrmt5 and ΔBmVasa represent BmPrmt5 and BmVasa mutants, respectively.Left: Volcano plots of DEGs in (TIF)Click here for additional data file.


While host miRNA usually plays an antiviral role, the relentless tides of viral evolution have carved out a mechanism to recruit host miRNA as a viral protector. By complementing miR-122 at the 5′ end of the genome, the hepatitis C virus (HCV) gene can form a complex with Argonaute 2 (Ago2) protein to protect the 5′ end of HCV RNA from exonucleolytic attacks. Experiments showed that the disruption of the stem-loop 1(SL1) structure and the 9th nucleotide (T9) of HCV site 1 RNA could enhance the affinity of the Ago2 protein to the HCV site 1 RNA (target RNA). However, the underlying mechanism of how the conformation and dynamics of the Ago2: miRNA: target RNA complex is affected by the SL1 and T9 remains unclear. To address this, we performed large-scale molecular dynamics simulations on the AGO2-miRNA complex binding with the WT target, T9-abasic target and SL1-disruption target, respectively. The results revealed that the T9 and SL1 structures could induce the departing motion of the PAZ, PIWI and N domains, propping up the mouth of the central groove which accommodates the target RNA, causing the instability of the target RNA and disrupting the Ago2 binding. The coordinated motion among the PAZ, PIWI and N domains were also weakened by the T9 and SL1 structures. Moreover, we proposed a new model wherein the Ago2 protein could adopt a more constraint conformation with the proximity and more correlated motions of the PAZ, N and PIWI domains to protect the target RNA from dissociation. These findings reveal the mechanism of the Ago2-miRNA complex’s protective effect on the HCV genome at the atomic level, which will offer guidance for the design of drugs to confront the protection effect and engineering of Ago2 as a gene-regulation tool. MicroRNAs (miRNA), which are short non-coding RNAs of ~22 nucleotides, could guide Argonaute (Ago) proteins to interact with target complementary RNAs, yielding the RNA-induced silencing complex (RISC) ,2. The fAmong them, Hepatitis C Virus (HCV) uniquely utilizes the liver-specific miRNA-122 for replication. MiRNA-122 could interact with the 5′ noncoding region of the HCV genome, which was the first direct viral RNA–host miRNA interaction to be discovered . An in vTwo complementary sites (site 1 and site 2) of miRNA-122 at the very end of the HCV genome have been reported . BesidesAlthough research has shown that this might be related to the unique mechanism of the exonuclease involved in the target RNA degradation , the undHere, we performed an extensive large-scale MD simulation to investigate how the T9 and SL1 structures of the HCV site 1 RNA affect the target RNA binding affinities. The results showed that the T9 and SL1 structures could induce the departing and less coordinated motion of the PAZ, PIWI and N domains, propping up the mouth of the central groove, which in turn causes the target RNA to be liable to dissociate. Moreover, combining with previous studies, we proposed a new model that the Ago2 protein could adopt a more constraint conformation with the proximity to and the more correlated motion of the PAZ, N and PIWI domains to protect the target RNA from dissociation. This study aims to shed light on the mechanistic understanding of the role of the T9 and SL1 structures, inspiring further research on developing anti-HCV strategies and engineering Ago proteins as gene regulation tools.Three systems were performed with the same Ago2: miRNA-122 complex and different target RNAs, including the wild-type target RNA (denoted as “WT”), the T9 base pair-depleted target RNA (denoted as “T9-abasic”) and the SL1-disrupted target RNA (denoted as “ΔSL1”). The SL1 disruption was achieved by the mutation of all of the paired nucleotides in the stem-loop to adenine. The RNA sequences in the three systems are shown in The systems were prepared with the LEaP program using ff14SB and “Rochester” torsions force field to describe the RNA–protein complexes ,36,37,38The principal component analysis (PCA), which is widely used in describing the kinetic processed that occur during simulations, can transform a series of potentially coordinated observations into orthogonal vectors. Among the vectors, the first two principal component (PC1 and PC2) provide the dominant motions during the simulation . In thisijGC) analysis was applied to calculate the correlated motion. Compared to the traditional Pearson correlation analysis, the ijGC analysis is independent of the mutual orientations of the atomic fluctuations and can distinguish between the linear and nonlinear contributions [MI) was introduced to reflect how much information of one atom’s position was provided by another:Generalized correlation  is a mathematical framework that is used to describe the dynamics of time-series data ,48,49,50τ). The ITS as a function of τ can be calculated by:iλ means the eigenvalue which was obtained from the MSM transition matrix of the ith process. The implied timescale plot can be drawn in which each curve represents the average transition time of one process. When the curve became approximately constant, the corresponding lag time was chosen for the following analysis and the system had a Markovian property. In this study, the lag time was set as 1 ns.An implied timescale (ITS) test was performed to check the Markovian property and select the proper lag time  with the frames in the same cluster was chosen as the representative structure.A total of three systems with different target RNAs were performed, one with the wild-type HCV site 1 RNA (WT), one with the T9 base pair depletion target RNA (T9-abasic), and the other with the SL1-disruption target RNA (ΔSL1). The RNA sequences of the three systems are shown in To reflect the local dynamics in the Ago2–RNA complex, the root-mean-square fluctuation (RMSF) of each residue (represented by the Cα atoms for Ago2 and P atoms for RNA) was calculated . To bettTo characterize the global conformational transition in the three systems, a principal component analysis (PCA) was performed . We projected the snapshots every 10 frames throughout the simulation of each system on the two-dimensional plot according to the first two principal components (PC1 and PC2) A, and thThe T9-abasic system contains one cluster of conformations with a PC1 value that is less than −10, which was not observed in the WT and ΔSL1 system, indicating a wider conformational space that was induced by the T9 base pair depletion. Along the PC1, the PAZ and N domains underwent a departing motion away from the MID and PIWI domains, widening the central groove transversely. The motion suggested that the Ago2 protein adopted a more constrained conformation in the T9-abasic system. Compared with the WT system, the PC2 value decreased in the T9-abasic system and the ΔSL1 system. Along the PC2, the PAZ and PIWI domains moved away from each other and drove the MID and PIWI domains closer together, yielding the longitudinal opening of the central groove which accommodates the target RNA. This indicates that the disruption of the T9 and SL1 structure could fasten the mouth of the central groove, thus, protecting the target RNA from dissociation. Taking the information provided by PC1 and PC2, we can conclude that the WT T9 and SL1 structures could induce the Ago2 protein in a more extended conformation, leading to a more opening mouth of the central groove which accommodates the target RNA. The opening of the mouth of the central groove could make the target RNA liable to dissociate, which is in line with the experiment results that the T9 or SL1 could decrease the Ago2 affinity to the target RNA.E333–S610 represents the distance between the PAZ and PIWI domains, while dE333–P63 represents the distance between the PAZ and N domains  was used to differentiate the characterized clusters in the free-energy landscape . To testify the Markovian property, the implied timescale test and the Chapman Kolmogorov test were applied  were observed with the dominant conformation of C5. In the ΔSL1 system, with the increase in the dE333–S610 parameter, the dominant C8 state and the minor C9 state were observed. Compared with the WT systems, the states in the T9-abasic system had relatively lower dE333–S610 and dE333–P63 parameters, indicating that there was a shorter distance between the PAZ, N and PIWI domains. The dE333–P63 parameter was reduced in the ΔSL1 system when it was compared with the WT system, reflecting a closer relationship between the PAZ and N domains, which was in accordance with the motion along PC2. In general, the landscape proved the conformational change that was revealed in the PCA analysis and suggested a more proximal relationship between the PAZ, N and PIWI domains with the disruption of the T9 and SL1 structure.Four states were observed in the WT system with the decrease in the dTo further illustrate the conformational transition, we superimposed the representative structures of the dominant state in the T9-abasic system (C5) and the dominant state in the ΔSL1 system (C8) with the dominant state in the WT system (C2), respectively D,E. ThouUsing a generalized correlation analysis, an overview of the inter-residue and inter-domain-correlated motions was provided . The results showed that the inter-residue correlation of regions A and B were significantly strengthened with the disruption of the T9 and SL1 A–C. The A community network analysis was performed to further uncover the network organization of the Ago2–RNA complex. Each community is shown as circles whose area is proportional to the number of residues that it contains. The strength of the inter-community correlation is represented by the width of the sticks connecting the communities. The community composition and connections are shown in In conclusion, the disruption of the T9 and SL1 structure induced a more correlated motion between the residues, nucleotides and domains  around the mouth of the central groove, which may be the cause of the shrinkage of the target RNA-binding pocket which was induced by the disruption of the T9 and SL1 structure.The Ago2 protein, as the core of RNA-induced silencing complex, plays a critical role in regulating gene expression and defending against exogenous pathogens. However, HCV has evolved a special mechanism which utilizes the complementary sites at the 5′ end of the genome to recruit the Ago2–miRNA complex to defend against the enzyme attacks. Here, using large scale MD simulations, we investigated how the special structures, T9 and SL1, in the site 1 HCV RNA affect the target RNA recognition at the atomic level, thereby providing guidance for the development of anti-HCV strategies and the engineering of Ago2 as a gene regulation tool.The T9 and SL1 could promote the Ago2 protein to adopt a more extended conformation. The PCA analysis and MSM analysis showed that the T9 and SL1 induce the departing motion of the PAZ, PIWI and N domains, propping up the mouth of the central groove which accommodates the target RNA, which may in turn make the target RNA liable to dissociate. Interestingly, we previously found a similar conformational change in another Ago2–RNA complex. To explore the underlying mechanism of how the miRNA extensions increased the target RNA affinity , we perfThe T9 and SL1 could affect the dynamics and correlated motion of the Ago2–RNA complexes. The RMSF analysis revealed that the SL1 region, PAZ and PIWI domains displayed a higher fluctuation in the WT system when they were compared with those of the T9-abasic and ΔSL1 systems, implying that the instability was induced by the T9 and SL1 structures. According to the GCCM and network analysis, the T9 and SL1 structures also weakened the correlated motion between the PAZ, PIWI and N domains which encompassed the mouth of the central groove. This may be related to the extended conformation which was induced by the SL1 and T9-abasic system.The modification of the Ago proteins into gene regulation and even editing tools has been a research hotspot ,54,55. HHCV infection is liable to become chronic due to it having multiple mechanisms of resistance to clearance , one of In summary, the collective sampling of the 15 μs MD simulations showed that the T9 and SL1 structure of the HCV site 1 RNA could induce the departing motion and the weakened correlation between the PAZ, PIWI and N domains, propping up the mouth of the central groove, which in turn causes the instability of the target RNA. This was in line with the experimental results that the T9 and SL1 structures were detrimental to Ago2 binding.Moreover, we proposed a new model that the Ago2 protein could adopt a more constrained conformation with the increased proximity to and more correlated motion of the PAZ, N and PIWI domains to protect the target RNA from dissociation. Thus, the modification of the residues to intensify the interaction between the PAZ, PIWI and N domains may be a feasible strategy to increase Ago’s binding affinity. These results shed light on the structural details of the assembly of the Ago–RNA complexes, which will help to offer guidance for the development of anti-HCV strategies and the engineering of Ago2 as a gene regulation tool.


Dimensional models of personality disorders postulate interpersonal dysfunction as the core feature of personality pathology, and describe maladaptive personality traits that characterize the specific pattern of dysfunction that is experienced. Herein, we examined whether maladaptive traits predict prosocial and trusting behavior, both of which are highly relevant behaviors for interpersonal functioning. Specifically, we examined antagonism as a predictor of prosocial behavior in a dictator game, and suspiciousness as a predictor of trust in the faith game.https://osf.io/er43j. Data and code are available at https://osf.io/2rvbg/. Participants (N = 445) completed the German version of the Personality Inventory for DSM-5 to measure antagonism and suspiciousness. Additionally, they played the dictator game  and the faith game (choosing the sure choice instead of the faith choice indicates less trust). We conducted a linear regression model to test whether antagonism is associated with prosocial behavior in the dictator game and a logistic regression model to test whether suspiciousness predicts selection of the sure choice in the faith game.The study was preregistered and conducted online. The preregistration protocol is available at As hypothesized, higher levels of antagonism were associated with less prosocial behavior in the dictator game. The remaining hypotheses were not supported, as suspiciousness was not significantly associated with the likelihood of choosing the sure choice in the faith game. Exploratory analyses on participants’ estimates of the sure choice amount suggest successful experimental manipulation in the faith game.The results on antagonism and prosocial behavior are consistent with those of previous studies that used categorial classification systems of personality disorders or examined non-pathological personality traits. Potential explanations for the non-significant effects of suspiciousness are discussed, including the small size and range of the sure choice payoff and that the anonymity of the game may have precluded suspicious traits from expressing. Future research with higher stakes and known interaction partners is needed to further probe the effects of suspiciousness.The online version contains supplementary material available at 10.1186/s40479-022-00201-0. ICD-11 allows for the optional specification of five maladaptive trait qualifiers, whereas the AMPD requires that individuals must meet for one or more maladaptive traits. Both systems describe largely overlapping traits of negative affectivity (the tendency to experience a broad range of negative emotions), detachment , antagonism (DSM-5)/ dissociality (ICD-11) (the tendency to disregard the rights and feelings of others), and disinhibition . DSM-5 further specifies a psychoticism trait , whereas ICD-11 includes anankastia as a fifth trait .With the inclusion of the alternative model for personality disorders (AMPD) in the fifth edition of the diagnostic and statistical manual of mental disorders  and theMultiple personality theories postulate that personality traits express themselves more or less strongly depending on the situational affordance that is given, and thus conceptualize behavior as the outcome of a person×situation interaction  in the presence of specific situational affordances. In their n et al.  detail tAntagonism encompasses callous antipathy toward others and unawareness of others’ needs and feelings, as well as a willingness to use others in the service of self-enhancement [dictator game [Based on the trait and facet-level content of the maladaptive personality traits postulated for DSM-5 AMPD, we identified the antagonism trait  as a likely predictor for low prosocial behavior when given the possibility for exploitation, and the detachment facet suspiciousness for low trusting behavior in situations with dependence under uncertainty . This seancement , showed tor game , 9. The tor game , 11 is ator game  agreeabltor game , 14, as tor game  honesty-tor game , 17. TakSuspiciousness is a facet of the maladaptive trait detachment and encompasses mistrust and doubt with respect to the loyalty and fidelity of others [faith game [f others . Individf others , 19, shof others , 21, andf others . To testith game . In thisBased on the definition of the suspiciousness facet and previous findings on borderline personality disorder, we hypothesized that the level of suspiciousness is positively associated with the likelihood of selecting the sure choice over the faith choice in the faith game (Hypothesis 2). Additionally, we expected that the amount offered as the sure choice is positively associated with the likelihood of selecting the sure choice over the faith choice (Hypothesis 3). Lastly, we hypothesized that the association between the amount of money offered as the sure choice and the likelihood of selecting the sure choice is moderated by the level of suspiciousness. More specifically, we expected individuals high in suspiciousness to have a strong overall preference for the sure choice, thus their decision should be influenced less strongly by the exact amount offered as the sure choice. They should be more likely to select the sure choice even when the sure choice amount is small. In other words, individuals high in suspiciousness would rather accept a small amount of money that they will definitely receive (sure choice) than rely on someone else to share with them (faith choice). Accordingly, we hypothesized that the level of suspiciousness weakens the association between the amount of money offered as the sure choice and the likelihood of selecting the sure choice over the faith choice in the faith game (Hypothesis 4).www.soscisurvey.de; [https://osf.io/er43j. Study procedures were approved by the ethics committee of the medical faculty Mannheim at Heidelberg University (protocol no. 2021–524). All participants provided informed consent prior to their participation.We conducted the study in German language and online, hosted on the platform SoScisurvey  from FeN = 390 . Since we pre-registered a number of data exclusion criteria  to ensure data quality, we planned to oversample by approximately 15%. Eventually, a total of 460 individuals participated in the online study. Based on pre-registered exclusion criteria, we excluded nine participants (1.96% of the sample) because they spent less than 2 seconds on answering each item of the PID-5 questionnaire, four participants (0.87%) because they indicated that we should not use their data due to bad quality, and two participants (0.43%) because of repeated participation.We advertised the study as investigating the association between personality traits and decision-making. As we wanted to assess a wide range of maladaptive trait levels, including participants with high maladaptive trait levels, we recruited through Facebook groups, Instagram pages, and online forums dedicated to personality disorders and mental health topics in general. Additionally, we sent invitation emails to individuals that participated in previous studies on borderline personality disorder and childhood maltreatment within our clinic. Additionally, we recruited participants via student mailing lists at the University of Mannheim. Based on a-priori power-analyses, we pre-registered a requited sample size of M = 33.79, SD = 13.17). The diversity of the sample was limited in terms of gender, nationality, and migration background, as most participants were women , had a German nationality , and had no migration background . Furthermore, most participants had a university entrance qualification or university degree  and were currently employed  or studying . Almost all participants were native German speakers . Participants indicated an average monthly net income of 1509.00€ (SD = 1344.94), ranging from 0€ to 9500€. A total of 54 participants (12.13%) identified as belonging to a minority group  due to belonging to the queer community, 9 participants (2.02%) due to mental disorders, 5 participants (1.12%) due to religion and 28 participants (6.29%) due to other reasons). Detailed demographic data are shown in Table After applying the exclusion criteria, the final sample comprised 445 participants. Of these, 253 participants (56.85%) were recruited via advertisements in Facebook groups, online forums, Instagram channels, and websites dedicated to mental health topics. In addition, 81 participants (18.20%) who had participated in previous studies of borderline personality disorder and childhood maltreatment in our clinic were recruited via mailing lists. The remaining 111 participants (24.94%) were recruited via advertising in non-clinical settings . The age of participants in the final sample ranged from 18 to 73 years  and personality disorders . Details of these self-reported clinical characteristics are shown in Table Regarding clinical characteristics, 202 participants (45.39%) indicated that they had been diagnosed with at least one mental disorder in the past. The most frequently self-reported diagnoses were mood disorders  until after they had completed the dictator game, to avoid spillover effects.After providing demographic information, participants were asked to complete the German version of the PID-5  and the Both games were partially incentivized. This means that, for some participants, we actually paid out the money they earned in the dictator game and the faith game at the end of the study via an amazon voucher. The voucher was automatically displayed on the last study page, so complete anonymity was guaranteed to participants . Participants were informed that “every 10th player” would win their actual outcome. Randomly, 47 participants (10.22%) were selected to receive their actual payoff. This payoff could vary between 2€  and 18€ .We assessed maladaptive personality traits using the German version of the PID-5 . In detaIn the dictator game , particiIn the faith game , particihttps://osf.io/2rvbg/. Data were analyzed using R [https://osf.io/er43j). To test our hypotheses, we conducted a linear multiple regression and a logistic regression model. In all models, we adjusted for the pre-registered covariate gender , because meta-analytic results suggest cooperative behavior may vary as a function of gender [n = 428 participants, because the remaining 17 participants did not indicate their gender identity or reported a gender identity other than cis/trans male/female.1All data and analysis code is available at  using R  and all f gender . All anaTo test H1, we entered the PID-5-SF dimension antagonism (mean score) and the covariate gender into a linear regression model to predict prosocial behavior in the dictator game . To test H2, H3, and H4, we conducted a logistic regression model with the likelihood of choosing the sure choice (versus the faith choice) as outcome. Choice was coded as 1 for the sure choice and 0 for the faith choice. As predictors, the model included the PID-5 facet suspiciousness (centered on the grand mean) and the amount of money offered as the sure choice , as well as their interaction, and the covariate gender. The main effect of suspiciousness constitutes the test of H2. The main effect of the amount of money offered as the sure choice constitutes the test of H3. The interaction term constitutes the test of H4.M = 0.61, SD = 0.40), with 0 indicating a low level and 3 indicating a high level of antagonism. Compared with the normal distribution, the distribution of antagonism mean scores was highly right-skewed (skewness = 1.19) and leptokurtic (kurtosis = 2.09). Figure M = 1.08, SD = 0.70), with 0 indicating a low level and 3 indicating a high level of suspiciousness. Compared with the normal distribution, the distribution of suspiciousness mean scores was right-skewed (skewness = 0.43) and platykurtic (kurtosis = − 0.72). Figure The mean scores of the PID-5 dimension antagonism ranged from 0 to 2.54 . Compared to the normal distribution, the distribution of money taken away in the dictator game was right-skewed (skewness = 0.67) and leptokurtic (kurtosis = 0.20). A total of 184 participants (41.35%) divided the amount of money equally between themselves and player B (5€ each). In contrast, 92 participants (20.67%) took nothing from player B, whereas 43 participants (9.66%) took everything from player B. Figure The amount of money that participants took from player B in the dictator game ranged between 0€ an 8€ . Cohen’s f2 for the main effect of antagonism was 0.15, indicating a medium effect size [b = 0.37, β = 0.07, p = .138. A model plot for the multiple regression analysis is shown in Fig. We conducted all confirmatory analyses as pre-registered. Supporting H1, the main effect of antagonism on dictator game contributions, adjusted for gender, was significant and positive, ect size . The covThe results for the test of H2, H3 and H4 are shown in Table b = − 0.27, β = −.10, p = .042), indicating that higher levels of suspiciousness were associated with lower estimates of the faith choice amount, though this effect was small f2 = 0.02.As described above, the pre-registered analyses for the faith game revealed that suspiciousness did not show a significant association with selecting the sure choice in the faith game. To further probe possible effects of suspiciousness, we conducted an exploratory analysis with participants’ estimates of the amount of money the other player left for them as the faith choice. In detail, we used a linear regression model with suspiciousness as a predictor of participants’ estimates of the faith choice amount, adjusted for gender. Results showed a significant and negative main effect of suspiciousness (p < .001). That is, the higher the payoff participants expected as the faith choice, the more likely they were to select the faith choice. In addition, the interaction between the faith choice estimate and the sure choice amount was significant , indicating that participants were particularly prone to select the sure choice when the sure choice amount was high and their expectation of the faith choice amount was low. A regression plot illustrating the interaction term of the logistic regression analysis is shown in the online supplement, Fig. SAdditionally, we conducted an exploratory analysis to further probe whether the faith game manipulation had the desired effect. For this purpose, we conducted a logistic regression model with the likelihood of choosing the sure choice, predicted by the faith choice estimate, the sure choice amount, and their interaction (adjusted for the covariate gender). Results showed a significant and negative main effect of the faith choice estimate . Yet, as shown in Table The present study assessed whether the maladaptive personality trait antagonism and the maladaptive trait facet suspiciousness, as conceptualized in the AMPD  and measSupporting hypothesis 1, the level of antagonism was positively associated with the amount of money participants extracted in the dictator game , 11. Thuwill show this behavior) and address them once they occur. Specifically, therapists could aim to help patients understand how others react to them when they behave in an exploiting way, in order to decrease interpersonal problems.In sum, the present findings provide reason to assume that individuals with high levels of antagonism may be prone to take advantage of situations in which they can exploit others, which may also occur in the therapeutic context. For instance, in group settings or inpatient care, where patients regularly have to take on responsibilities for and cooperate with the group, those high in antagonism may exploit others to evade unwanted activities or duties. This can have substantial negative effects for the group dynamic, the course of the patient’s own treatment, and that of others. Patients high in antagonism are likely to show this exploiting behavior with little insight into the potential consequences of it and may repeatedly experience rejection by others who feel exploited by them. This could lead to them being isolated from the rest of the group and disrupt the therapeutic progress. Naturally, the same behavior can also occur in interaction with the therapist and likely occurs regularly in their daily lives. As such, it may be a central factor underlying the disruption or rupture of interpersonal relationships. Clinicians should thus be mindful of the increased likelihood of individuals high on antagonism showing exploiting tendencies . We observed that participants were more likely to select the faith choice, the higher the amount they expected to receive. This underlines the validity of the faith game. Additionally, we observed a significant interaction between the amount of money offered as the sure choice and participants’ faith choice estimates. The interaction indicated that a higher sure choice amount decreased the strength of the association between participants’ guesses about the faith choice amount and their likelihood of choosing it. This suggests that, when the amount of the sure choice is small, the faith choice is more attractive and the guess about the amount of the faith choice becomes more relevant. However, when the safe choice envelope contains larger amounts, the expected amount of the faith choice becomes less important.them. They assume that there is some quality within themselves that invokes this negative behavior from others, rather than a general assumption that others necessarily treat everyone negatively. However, in our version of the faith game, Player A does not know Player B so they cannot specifically decide to exploit them because of any specific qualities of Player B. It is possible that, in order to use suspiciousness to predict the behavior of individuals in the faith game, the game would have to involve real interactions . Future studies could consider implementing such a version of the faith game.In addition to the possibility that the monetary stakes were too small and restricted in range, it is possible that restricted variance in suspiciousness scores contributed to the absence of the expected effects. However, the distribution of suspiciousness was only slightly right-skewed, and several participants showed the highest possible mean score on this facet, thus we do not deem this explanation very likely to hold. Instead, the anonymity afforded in the faith game may have rendered the situational context less relevant for the expression of suspiciousness. As described above, individuals high in suspiciousness assume that others want to specifically harm or exploit Additionally, future studies with more diverse samples are needed. We acknowledge that our sample diversity was restricted with regard to nationality, migration background, education level, and gender, which limits generalizability of the results largely to women without a migration background and with a higher education. As personality disorders affect individuals of all genders in the population , future In this pre-registered online study, we assessed associations between the maladaptive personality trait antagonism and prosocial behavior and between the maladaptive trait facet suspiciousness and trusting behavior in two partially incentivized economic games. Supporting our first hypothesis, higher levels of antagonism were associated with lower levels of prosocial behavior in the dictator game, which affords the situational possibility for exploitation. In exploratory analyses, this effect replicated when testing antagonism against the other four maladaptive traits, underlining the specificity of this effect to antagonism. Contrary to hypotheses, we did not observe a significant association between suspiciousness and the likelihood of selecting the sure choice in the faith game, a game that models the situational affordance of dependence under uncertainty. We discussed that this may have resulted from a low range and low absolute levels of the sure choice amounts that we implemented in the faith game and from the anonymity afforded by the game, which may have precluded the expression of suspiciousness. The results underline that maladaptive personality traits correlate with actual behavior in situations that afford their expression. Future studies are needed to further investigate the specific associations between a wider range of maladaptive traits and behavior relevant to interpersonal functioning.Additional file 1: Fig. S1. Regression Plot of the Interaction Effect of the Sure Choice Amount and the Guess About the Faith Choice in the Faith Game on the Likelihood of Choosing the Sure Choice in the Faith Game, Controlling for Gender. Table S1. Multiple Linear Regression Analysis for H1: Effects of the Mean Score of the PID-5-SF Dimension Antagonism on Prosocial Behavior in the Dictator Game (Amount of Money Taken Away), Controlling for Gender. Table S2. Logistic Regression Analysis for H2-H4: Effects of the Mean Score of the PID-5 Facet Suspiciousness and the Sure Choice Amount in the Faith Game on the Likelihood of Choosing the Sure Choice, Controlling for Gender. Table S3. Multiple Linear Regression Analysis: Effects of the Mean Score of the PID-5 Facet Suspiciousness on the Guess About the Faith Choice in the Faith Game, Controlling for Gender. Table S4. Logistic Regression Analysis: Effects of the Guess About the Faith Choice and the Sure Choice Amount in the Faith Game on the Likelihood of Choosing the Sure Choice in the Faith Game, Controlling for Gender.


Whole-cell RNA lysates were collected for sequencing of poly(A)-selected RNA 24, 48, and 72 h after infection. Three primary species of viral RNA were produced: genomic, subgenomic, and defective viral genomes (DVGs) encoding the RNA capping protein nsP1. Antibody treatment resulted in overall lower production of SINV RNA, decreased synthesis of subgenomic RNA relative to genomic RNA, and suppressed production of the nsP1 DVG. The nsP1 DVG was packaged into virus particles and could be translated. Because antibody-treated cells released a higher proportion of virions with noncapped genomes and transient transfection to express the nsP1 DVG improved viral RNA capping in antibody-treated cells, we postulate that one mechanism by which antibody inhibits SINV replication in neurons is to suppress DVG synthesis and thus decrease production of infectious virions containing capped genomes.Alphaviruses are positive-sense RNA viruses that are important causes of viral encephalomyelitis. Sindbis virus (SINV), the prototype alphavirus, preferentially infects neurons in mice and is a model system for studying mechanisms of viral clearance from the nervous system. Antibody specific to the SINV E2 glycoprotein plays an important role in SINV clearance, and this effect is reproduced in cultures of infected mature neurons. To determine how anti-E2 antibody affects SINV RNA synthesis, Oxford Nanopore Technologies direct long-read RNA sequencing was used to sequence viral RNAs following antibody treatment of infected neurons. Differentiated AP-7 rat olfactory neuronal cells, an  Togaviridae) are important causes of viral encephalomyelitis worldwide and a significant public health concern due to their high mortality rate, potential for permanent neurologic damage, and limited availability of vaccines and therapeutics  the full-length positive-sense genomic RNA (gRNA) that is eventually incorporated into released infectious virions, (ii) the negative-sense genomic RNA that serves as a template for viral RNA replication, and (iii) the shorter positive-sense subgenomic RNA (sgRNA), which includes the latter third of the genome encoding the structural proteins . The relUpon infection, the positive-sense capped SINV RNA genome from the infecting virion is directly translated by host machinery to produce the nonstructural polyprotein P123 or P1234, depending on read-through of an opal stop codon between the nsP3 and nsP4 genes (10% occurrence) . nsP1 wiIn humans, SINV infection typically results in rash, fever, arthritis, and myalgia with persistent musculoskeletal pain . In rodeFollowing intranasal or intracranial infection with TE, a strain of SINV that does not cause fatal encephalomyelitis, weanling mice develop transient kyphoscoliosis, hind-limb paralysis, and muscular atrophy but recover in 2 to 3 weeks without lasting clinical or histological evidence of neuronal damage –8. Peak –In vitro studies with primary rat dorsal root ganglion (DRG) neurons  mice that lack functional adaptive immune responses are unable to clear SINV from the CNS and continue to produce infectious virus . Adoptiv neurons –13 and ineurons –, 15 furtsurvival , 14–16.In differentiated rat AP-7 olfactory neuronal (dAP-7) cells , 18, antUsing high-throughput direct Oxford Nanopore MinION viral RNA sequencing, we evaluated the effect of anti-E2 antibody on RNA in SINV-infected dAP-7 olfactory sensory neuronal cells and found that antibody treatment induces changes in RNA abundance and type. Anti-E2 treatment improved production of rat RNAs and decreased production of SINV RNAs relative to untreated virus-infected cells. Antibody further affected the types of SINV RNAs produced, specifically decreasing sgRNA synthesis relative to gRNA synthesis and suppressing production of two previously unrecognized SINV RNAs containing the 5′ end, the nsP1 gene, and the 3′ untranscribed region (UTR) characteristic of deletion-type defective viral genomes (DVGs) . AntibodTo evaluate changes in SINV RNA at the nucleotide level following anti-E2 antibody treatment of infected dAP-7 cells, nanopore sequencing was employed. Nanopore sequencing directly sequences RNA to avoid potential biases from either PCR or reverse transcription and provides long reads for read-through of full-length viral genomic RNA (11.7 kb).AP-7 cells were differentiated for 7 days into their nondividing form that models mature neurons (dAP-7 cells) , 20, 21 Sequencing reads were aligned to the rat and SINV genomes to assess host and viral gene expression. By 24 h after infection, 76% of the total RNA reads were aligned with the SINV genome in untreated virus-infected cells , reflectTo evaluate changes in relative production of the different SINV RNA species with antibody treatment, we calculated the sequencing depth coverage across the SINV genome reference normalized to the total number of SINV reads in each run for each time point . We obseTo determine how the SINV RNAs were individually affected by antibody treatment, a viral RNA abundance ratio was calculated by taking the coverage differences at each junction site and dividing by the total of these differences . In geneP < 0.0001; 48 h, P < 0.001; 72 h, P < 0.0001) and lower subDVG levels at 24 h (P < 0.01) than the untreated cells. These data suggest that in addition to antiviral antibody affecting the relative proportions of different SINV RNAs present, production of the nsP1-containing RNAs was especially sensitive to the effects of antibody.Antibody treatment also had a pronounced effect upon levels of the nsP1-containing DVGs. The antibody-treated cells had significantly lower DVG levels at all time points assessed  and treated with medium (untreated) or 5 μg/mL anti-E2 antibody 4 h after infection . At 0, 1P = 0.25) (P = 0.11). However, by 48 h, gRNA production declined in both the antibody-treated and untreated cells and the levels were roughly equivalent. sgRNA production over the 2-h pulse period was 2-fold lower at 12 h after infection in the antibody-treated versus untreated cells (P = 0.18), and at 24 h, production was 1.2-fold lower. By 36 and 48 h, levels of synthesis of sgRNA were comparable. To assess the relative proportions of viral RNAs being synthesized, the ratios of sgRNA to gRNA band density at each time point were compared between SINV-infected, untreated, and antibody-treated cells (P < 0.0001).Two distinct bands corresponding to the SINV gRNA and sgRNAs were detected  and quan = 0.25) . By 24 hed cells . While tTo better quantify changes in RNA synthesis induced by antibody treatment, cells were prelabeled with 5-ethynyl uridine for 15 h. RNA was extracted and nascent RNA isolated at the times indicated by reaction with biotin azide and streptavidin bead capture. Bead-captured viral RNAs were quantified by quantitative reverse transcription-PCR (qRT-PCR) using nsP2 primers (gRNA) and E2 primers (gRNA plus sgRNA), and ratios of nsP2 and E2 RNA expression were compared for treated and untreated infected cells . At earlThe nanopore sequencing data identified DVG-like SINV RNAs containing the nsP1 gene that were 1 to 2 kb in length and increased in abundance over time . To furtTo confirm the identity of the PCR bands, 4 bands  from the untreated and antibody-treated 6- and 12-h lanes were excised and submitted for Sanger sequencing . Three oTo quantify levels of DVG and full-length SINV genomic RNA during infection, we designed a BRYT Green-based qPCR assay using primer sets that either span the deleted portion of the SINV genome for DVG amplification or are within the deleted portion (nsP3 gene) for SINV genomic RNA detection . Using t−ΔΔCT) method  or AT3-bcl-2 adenocarcinoma cells, it is not protective in cell types such as BHK-21, which are interferon-deficient, highly susceptible to SINV infection, and undergo rapid cell death following infection , 15, 19.) method .Infections of all three cell types generated detectable levels of nsP1 DVGs that correlated with the viral life cycle kinetics and levels of virus production. BHK-21 cells generated the highest relative amount of nsP1 DVG followed by cAP-7 and finally dAP-7 cells . AntibodBecause nsPs are translated from gRNA as a polyprotein, they are present in equal amounts after processing. To determine whether the ratio of nsP1 to the other nsPs is affected by translation of the nsP1 DVG, lysates of infected dAP-7 cells with and without antibody treatment were probed for expression of nsP1, nsP2, and nsP3 . The proDVGs are incomplete forms of the viral genome produced during infection due to errors in the viral genome replication process . The mosThe SINV packaging signal required for viral RNA interaction with the capsid protein for assembly into viral particles includes nucleotides 945 and 1076 of the nsP1 gene , which aTo further confirm whether the nsP1 viral RNAs are encapsidated into viral particles, we treated culture supernatant fluids from SINV-infected and antibody-treated cells with RNase A to degrade unprotected single-stranded RNA leaving only encapsidated RNA. Following RNase treatment, RNA was extracted with TRIzol reagent that inhibits RNase activity for analysis. As a positive control for RNase activity, the TRIzol-extracted RNA was also directly treated with RNase A under the same conditions and then reextracted with TRIzol. The final isolated RNA was then assessed for nsP1 DVG using the RT-PCR approach from before.For both the untreated and antibody-treated supernatant fluid samples, RNase A treatment did not completely remove all detectable nsP1 RNA, indicating that some of the nsP1 viral RNA was packaged . The posin vitro transcription and protein expression. Only the shorter of the two nsP1 DVGs (subDVG) without a poly(A) tail was successfully cloned due to Escherichiacoli toxicity. This DNA sequence was in vitro transcribed to RNA, and a poly(A) tail was subsequently added using poly(A) polymerase. The resulting RNA was transfected into uninfected undifferentiated cAP-7 cells that are more efficiently transfected than dAP-7 cells. RNA transcribed from the pTE plasmid that contains the complete cDNA sequence of SINV TE was transfected in parallel as a positive control, and the nsP1 RNA sequence without a poly(A) tail was included as a negative control. Twenty-four hours after transfection, protein lysates were collected and evaluated by immunoblotting using polyclonal antibody against SINV nsP1.Because the nsP1 DVG sequence can include the SINV 5′ terminus, 3′ UTR, and a poly(A) tail, components necessary for recognition by the host translation machinery, we next asked whether the DVGs could be translated into a viral protein. To answer this question, nsP1 DVG cDNA sequences were cloned into the pcDNA3.1/V5-His-TOPO expression vector with an upstream SP6 promoter for in vitro-transcribed RNA induced robust expression of SINV nsP1 protein at the expected molecular weight of ~60 kDa (−) did not induce protein expression and was identical to the mock-transfected cells. The polyadenylated nsP1 RNA-transfected cells uniquely produced a protein band detectable at a significantly lower molecular weight (15 kDa) than expected (60 kDa), suggesting that the shorter nsP1 DVG results in production of a truncated form of nsP1. Why the protein differs so much in size from the full-length nsP1 protein despite the DVG containing most of the nsP1 sequence is unclear and may be due to the absence of other components from the SINV genome.pTE  ~60 kDa . The ligWe next explored what biological function the nsP1 DVG protein product might have in SINV infection. Due to the correlation between decreased viral replication and decreased nsP1 DVG production, we hypothesized that the nsP1 DVG may be playing a proviral role that is inhibited by anti-E2 antibody.7meGpppA cap required for translation , suggesting that approximately 50% of the packaged SINV RNA was not capped.To assess whether antibody treatment affects the proportion of viral particles that have capped genomes, we performed an exoribonuclease degradation assay utilizing the enzyme XRN-1, which selectively degrades noncapped RNAs. Virus particles from the supernatant fluids of SINV-infected, untreated, and antibody-treated dAP-7 cells 24 h after infection were purified by ultracentrifugation. The RNA was isolated from the virus particles using TRIzol reagent and treated with XRN-1 for 1 h at 37°C to degrade noncapped RNAs. The remaining XRN-resistant RNAs were extracted using phenol-chloroform and reverse transcribed to cDNA for SINV RNA qRT-PCR analysis. The ratio of SINV nsP2 and E2 RNA with or without XRN-1 treatment was compared. Levels of nsP2 and E2 RNA from the SINV-infected, untreated cells were not affected by XRN-1 treatment (ratio of ~1) , suggestTo confirm the effects of supplemental nsP1 on genome capping and assess the effects of additional nsP1 expression in antibody-treated cells, dAP-7 cells were transiently transfected with lentiviral vector plasmids expressing nsP1 or the nsP1 DVG , infecteIn vitro treatment of SINV-infected neuronal cells with bivalent antibody to the E2 glycoprotein inhibits virus replication, restores cellular functions, blocks virus release, and improves neuron viability  tail. Similar deletion-type DVGs have been reported for many families of RNA viruses, including alphaviruses , 40, 41.nsP1 has an amphipathic peptide segment and is palmitoylated for binding to the plasma membrane where it induces formation of spherules that organize replication complexes for viral RNA synthesis. nsP1 oligomerizes to form a dodecameric ring at the neck of the spherules to cap RNAs as they are exported –46. Geno–In vivo, antibody clears infectious virus from SINV-infected brain and spinal cord neurons of mice within 8 days after infection, but viral RNA is eliminated much more slowly and continues to be detectable for months  (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) , 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM glutamine (Gibco). At 25% confluence, cells were differentiated for 7 days by shifting the cultures to 39°C and 5% CO2 and supplementing the medium with 1 μg/mL insulin, 20 μM dopamine, and 100 μM ascorbic acid (Sigma). Cells were routinely tested for mycoplasma using the MycoAlert PLUS mycoplasma detection kit (Lonza).AP-7 rat olfactory sensory neuronal cells immortalized with a temperature-sensitive simian virus 40 (SV40) T antigen   were groStocks of the TE strain of SINV  were genGACCATGCTAATGCCAGAGC-3′) and 11655R (5′-GTTATGCAGACGCTGCGTGG-3′). For sequencing, the band of interest was extracted using the QIAquick gel extraction kit (Qiagen) and submitted to the Johns Hopkins Synthesis and Sequencing Facility. To quantify levels of SINV gRNA versus nsP1 DVG, qRT-PCR was performed on the cDNA samples using the GoTaq master mix (Promega) and the following primers on a 7500 Fast real-time PCR system: for detection of the nsP1 defective genome, 1672F (5′-TCGGAGCAGCATTAGTTGAA-3′) and 11615R (5′-ATTATGCACCACGCTTCCTC-3′); for detection of the SINV genomic RNA, 4449F (5′-GGAAAAGACCGCCTTGAAGT-3′) and 4585R (5′-ACAGACTCCTTAAGTTGGAGTGC-3′). To quantify levels of the SINV nsP2 and E2 genes, qRT-PCR was performed using EagleTaq universal master mix (Roche) and the following sequences: for SINV nsP2, nsP2 3373F, 5′-CCG CAA GTA TGG GTA CGA TCA-3′, and nsP2 3454R, 5′-GTG CCC TTC CCA GCT AGC T-3′; for TaqMan probe nsP2 3317, 5′-FAM (6-carboxyfluorescein)-CCA TTG CCG CCG AAC TCT CCC-TAMRA (6-carboxytetramethylrhodamine)-3′; for SINV E2, E2 8732F, 5′-TGG GAC GAA GCG GAC GAT AA-3′, and E2-8805R, 5′-CTG CTC CGC TTT GGT CGT AT-3′; and for TaqMan probe E2 8760, 5′-FAM-CGC ATA CAG ACT TCC GCC CAG T-TAMRA-3′.Cellular RNA samples were collected by direct lysis in TRIzol reagent (Thermo Fisher Scientific), and supernatant fluid RNA samples were collected by adding 1 mL TRIzol to 100 μL culture fluid. RNA was purified according to manufacturer’s instructions and reverse transcribed into cDNA using (i) the Maxima H minus first strand cDNA synthesis kit (Thermo Fisher Scientific) with oligo(dT) primers for RT-PCR or (ii) a high-capacity cDNA reverse transcription kit (Applied Biosystems) with random primers for qRT-PCR. For PCR amplification of SINV RNA for gel electrophoresis, Phusion polymerase (Thermo Fisher Scientific) and the following primers were used for PCR: 171F (5′-NC_001547.1). The junction sites of subgenomic RNA and the DVGs were determined to be positions 7609 and 1751, respectively, and the abundance of each species was defined as the coverage difference at these junctions. The abundance of genomic RNAs for each sample was defined as the coverage at position 1752. Because the subDVG does not have a sharp 3′ junction, the abundance of subDVG RNA is calculated as the difference between the coverage at position 1110 and the minimum coverage between 1111 and the DVG junction.For direct sequencing of the RNA, poly(A) RNAs were isolated from frozen cell pellets using TRI-Reagent (Invitrogen), following the manufacturer’s instructions, with 1-bromo-3-chloro-propane for phase separation. Five to 50 μg of total cellular RNA diluted in 50 μL of nuclease-free water was poly(A) enriched using the NEXTflex poly(A) beads (BIOO Scientific). The resulting poly(A) RNAs were eluted in nuclease-free water and quantified using the Qubit 4 fluorometer (Thermo Fisher Scientific). One hundred forty to 800 ng of RNA was prepared for nanopore direct RNA sequencing following the ONT SQK-RNA002 kit protocol, including the optional ONT recommended reverse transcription step using Superscript IV (ThermoFisher). RNA sequencing was performed on the Oxford MinION platform using ONT R9.4.1 flow cells. The ONT Guppy workflow (version 4.0.11) was used for RNA base calling. Strand reads that had an average sequence quality of 7 or higher were classified as passing quality reads. Minimap 2 version 2.17 was used to align the nanopore RNA reads to the rat genome Rnor_6.0 and SINV TE strain genome (3H]uridine (20 μCi/mL) (Perkin Elmer) and dactinomycin (1 μg/mL) (Sigma) at 39°C for 2 h. Cellular lysates were collected with TRIzol and RNA purified as described above. RNA concentrations were determined by NanoDrop spectrophotometer. Two-microgram RNA samples were prepared for electrophoresis with RNA gel loading dye (Thermo Fisher Scientific) and separated on a 1% formaldehyde-agarose gel. Fluorography was performed with 2.5% 2,5-diphenyloxazole as previously described, and the gels were dried and autoradiographed at −80°C  was used. In brief, dAP-7 cells were cultured with DMEM containing 5-ethynyl uridine (0.2 mM) for 15 h, and at indicated times after infection with or without antibody treatment, RNA was isolated and biotin azide was clicked onto nascent RNA for capture with streptavidin. One microgram of RNA was heated at 68 to 70°C for 5 min and incubated with Dynabeads MyOne streptavidin T1 for 30 min at room temperature with low vortexing. Beads were captured with a magnet and washed twice before cDNA synthesis with the SuperScript VILO kit. (Invitrogen) and quantitative PCR (qPCR) for gRNA (nsP2) and gRNA plus sgRNA (E2).g for 5 min to pellet cellular debris. Cell-free supernatant fluids were collected and treated with 10 μg/mL RNase A (Thermo Fisher Scientific) for 1 h at room temperature. Following treatment, RNA was isolated from the samples with TRIzol reagent and reverse transcribed to cDNA using Maxima H minus first strand cDNA synthesis kit with oligo(dT) primers and assessed by PCR as described above.Supernatant fluid samples from dAP-7 cells infected with SINV and treated or untreated with anti-E2 antibody were centrifuged at 1,200 × ATTGACGGCGTAGTACACACTA-3′) and oligo(dT)18. The PCR products were separated by gel electrophoresis, and the band of interest was excised and purified using the QIAquick gel extraction kit (Qiagen). Following sequencing confirmation, the defective genome sequence was cloned into the pcDNA3.1/V5-His-TOPO vector (Thermo Fisher Scientific) and the plasmid was amplified. For production of RNA for transfection, the plasmid was linearized using XhoI (New England Biolabs) and in vitro transcribed and capped using the mMessage mMachine SP6 transcription kit (Ambion). The transcribed RNA was precipitated with LiCl and polyadenylated using poly(A) polymerase (New England Biolabs). The RNA was repurified by phenol-chloroform extraction and ethanol precipitation prior to transfection.The nsP1 defective genome was PCR-amplified from the cDNA of SINV-infected dAP-7 cells 24 h after infection using Easy-A PCR cloning polymerase (Agilent Technologies) and the primers SINV 1F . dAP7 cells were established as above and incubated with lipofectamine and 1 μg of plasmid for 24 h before confirmation of transcription and translation by qPCR and immunoblotting. Transfected cells were then infected followed by antibody treatment or not as described. Cell lysates were collected in TRIzol for quantification of E2 and nsP2 RNA by qPCR and in radioimmunoprecipitation (RIPA) buffer for protein by immunoblotting.6 undifferentiated AP-7 cells were transfected with 2.5 μg RNA and 10 μL of Lipofectamine 2000 reagent (Thermo Fisher Scientific). Twenty-four hours after transfection, protein lysates were collected for immunoblot analysis with radioimmunoprecipitation (RIPA) buffer  containing protease and phosphatase inhibitor cocktails (Roche) as previously described  for 1 h at 37°C to degrade noncapped RNAs. The treated RNAs were repurified by TRIzol extraction and reverse transcribed to cDNA using an Applied Biosystems high-capacity cDNA reverse transcription kit with random primers. The cDNA was assessed for SINV nsP2 and E2 gene abundance by qRT-PCR as described above.Extracellular virus particles were isolated from cell culture supernatant fluids by one-step sucrose cushion ultracentrifugation, as previously described , or usinP value of <0.05 being considered significant. Differences between two groups were analyzed by unpaired, two-tailed Student's t test. Multiple comparisons between groups were made using two-way analysis of variance (ANOVA) with Sidak’s posttest.Data are expressed as means ± standard deviation (SD). Statistical analyses were performed using Prism Software v8.3 (GraphPad), with a PRJNA720710. All code can be found at https://github.com/mbartl13/mbartl13 (mbartl13/GriffinLab-Sindbis).Data related to this article were deposited in SRA under BioProject no.


Saccharomyces cerevisiae an IFIT1-based mRNA pull-down with yeast targeted rRNA depletion by the RiboMinus method. IFIT1-based RNA capture depletes rRNA more effectively, producing high quality RNA-seq data with an excellent coverage of the protein coding transcriptome, while depleting cap-less transcripts such as mitochondrial or some non-coding RNAs. We propose IFIT1 as a cost effective and versatile tool to prepare mRNA libraries for a variety of organisms with cap 0 mRNA ends, including diverse plants, fungi and eukaryotic microbes.Analysis of the protein coding transcriptome by the RNA sequencing requires either enrichment of the desired fraction of coding transcripts or depletion of the abundant non-coding fraction consisting mainly of rRNA. We propose an alternative mRNA enrichment strategy based on the RNA-binding properties of the human IFIT1, an antiviral protein recognizing cap 0 RNA. Here, we compare for   RNA sequencing (RNA-seq) is an approach to profile transcriptomes with deep sequencing technologies. RNA-seq allows detection and analysis of a variety of RNA species within a sample, including mRNA, long and small non-coding RNA, as well as pathogen RNA. Transcriptomic analysis gives insight into many cellular processes and provides information about gene expression level, gene fusions, alternative splice variants, mutations, transcript isoforms in terms of their 5′ and 3′ ends, and many other features. In order to perform RNA-seq-based analysis of the protein coding transcriptome, mRNAs need to be efficiently separated from other RNA species, especially from the highly abundant ribosomal RNA (rRNA) which account for the majority (as much as 80–90%) of total RNA in the cell . This is7G(5′)ppp(5′)N cap of eukaryotic mRNA   . For inss laevis . Convenis laevis  or TOP ms laevis . Recentls laevis . eIF4E i7G-capped RNA without further 2′O-methylation of the first and second nucleotide; Figure 7GpppNm-RNA) (7GpppRNA in a largely sequence-independent manner (in vivo (Characterized over a decade later than eIF4E(K119A) , InterfepNm-RNA) ,17,20,21pNm-RNA) , which h shows that IFIT1-based RNA purification effectively depletes rRNA and tRNA and produces high quality RNA-seq data. Moreover, we are reporting on artifacts generated by the use of RM showing depletion of some mRNAs, whereas such an artifact was not observed for the IFIT-based approach. Importantly, eIF4E(K119A) or yeast cap-binding proteins failed to enrich yeast mRNA, leaving IFIT1 as the only viable tool for 5′ end-dependent RNA capture in this species that can effectively replace eIF4E in such applications. Due to the high specificity of IFIT1 for cap 0, we believe that our method could be particularly useful for transcriptomic analysis in model and non-model lower eukaryotes or those relying on template-switching reverse transcription , Cbp80  and Cbp20  in PCR with primer pairs: CDC33_F1 and CDC33_R1, CBP80_F1 and CBP80_R1, CBP20_F2 and CBP20_R2, respectively  was transformed with SLIC products. Positive clones were selected in standard LB medium containing kanamycin (100 μg/ml) and recombinant plasmids isolated with the use of Plasmid Mini kit  were validated by digestion with restriction endonucleases and Sanger sequencing.cDNA obtained with SuperScriptO vector . EscheriE. coli BL21-CodonPlus(DE3)-RIL strain  was transformed with pET28M N-6xHis-SUMO vector derivatives carrying Ccd33 or Cbp80-Cbp20 inserts. Transformants were grown in a standard Luria-Broth (LB) medium supplemented with 50 μg/ml kanamycin and 34 μg/ml chloramphenicol overnight. Subsequently, 1 l of Auto Induction Medium (AIM) Super Broth Base including Trace elements  containing 2% glycerol and both antibiotics, was inoculated with 30 ml of the starter culture. Bacteria were grown for 72 h at 18°C with shaking (150 rpm) and eventually collected by centrifugation at 5000 rpm in a Sorvall H6000A/HBB6 swinging-bucket rotor for 15 min at 4°C.Bacterial pellet was resuspended in 70 ml of lysis buffer , 0.02 μM pepstatinA, 0.02 μg/ml chymostatin, 0.006 μM leupeptin, 20 μM benzamidine hydrochloride), incubated with lysozyme  for 30 min in a cold cabinet, and then broken in an EmulsiFlex-C3 High Pressure homogenizer at 1500 Bar. The homogenate was centrifuged in a Sorvall WX Ultra Series ultracentrifuge (F37L rotor) at 33 000 rpm for 45 min at 4°C.280 nm absorbance were collected after gel-filtration and pooled together for coupling with the appropriate resin.The supernatant was used for protein purification using the ÄKTA Xpress system , employing nickel affinity chromatography on an ÄKTA-compatible 5 ml column that was manually filled with Ni-NTA Superflow resin (Qiagen). The column was equilibrated with 25 ml of low-salt (LS) buffer  prior to extract loading. After protein binding, the resin was sequentially washed with 40 ml of LS buffer, 25 ml of high-salt (HS) buffer , and again with 20 ml of LS buffer. Bound proteins were recovered by elution with 30 ml of buffer E . Pooled eluate fractions (approximately 5 ml) were directly used for coupling with appropriate resins or optionally dialyzed overnight at 4°C against 2 l of LS buffer in the presence of 50 μg of home-made SUMO protease. In the latter case, the mixture was afterwards subjected to second round of purification on the nickel resin, performed using ÄKTA Purifier system  and employing LS buffer for collection of the flow-through, containing protein of interest devoid of the tag, and buffer E2  for elution of 6xHis-tagged SUMO or TEV protease and cleaved-off 6xHis-SUMOTag. Further purification of epitope-containing or cleaved-off target proteins from contaminating chaperones and degradation products was achieved by separation of the eluate from the first-round of affinity chromatography (no SUMO protease cleavage) or pooled flow-through obtained in the second-round of affinity chromatography (SUMO protease cleavage step included) on size exclusion Superdex 75 10/300 GL (Cdc33) or Superdex 200 10/300 GL (Cbp80-Cbp20) column  using 1.2 column volumes of gel-filtration (GF) buffer . Fractions corresponding to the maximum of ARecombinant Cdc33 and Cbp80-Cbp20 dimer lacking or containing N-6xHis-SUMO tag were coupled to CNBr-activated SepFast MAG resin , following the manufacturer's instructions. Coupling of Cdc33 and Cbp80-Cbp20 to Dynabeads™ His-Tag Isolation & Pulldown magnetic beads  was carried out according to the manufacturer's recommendations, except for the omission of the elution step.AK314588.1) was previously sub-cloned into the prokaryotic expression vector pETG10a  at OD600 of 0.6 and conducted overnight at 25ºC with shaking at 200 rpm. The cells were harvested by centrifugation at 4,000 × g, at 4°C for 20 min. The cell pellet was resuspended in buffer containing: 50 mM Tris pH 7.5, 0.5 M NaCl, 20 mM imidazole, 10% glycerol, 0.5 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), Complete protease inhibitor cocktail (Roche Life Science), DNase I and lysozyme. The suspension was lysed by sonication and the cell debris was removed by centrifugation at 48 880 × g for 30 min. The supernatant was applied on a HisTrap HP column  equilibrated in the same buffer. IFIT1 protein was eluted from the HisTrap HP column with a gradient of 20 mM to 500 mM imidazole in the buffer. The fractions containing purified protein were combined and diluted 5× with the buffer containing: 50 mM Tris pH 7.5, 10% glycerol, 0.5 mM TCEP. IFIT1 was next purified by heparin affinity using a HiTrap Heparin HP column , pre-equilibrated in buffer containing: 50 mM Tris pH 7.5, 100 mM NaCl, 10% glycerol, 0.5 mM TCEP. The protein was eluted with a gradient of 100 mM to 1 M NaCl in the buffer. Finally, IFIT1 was purified by size exclusion chromatography on a Superdex 200 Increase column  pre-equilibrated in 50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 0.5 mM TCEP buffer. The eluted protein samples were aliquoted and flash frozen in liquid nitrogen, and kept at –80ºC until use.The human IFIT1 gene -RIL cells as described above with some modifications: after purification on a HiTrap Heparin HP column, His-SUMO tag was removed by incubation with SUMO protease with dialysis into 50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1 mM DTT, 10% glycerol buffer and size exclusion chromatography was performed in PBS, 5% glycerol, 0.5 mM TCEP buffer.E. coli BL21-CodonPlus(DE3)-RIL strain by affinity chromatography on Glutathione Sepharose 4B resin and ion-exchange on ResourceS column, as described previously (GST-eIF4E(K119A) was purified following expression autoinduction in eviously .The 4 different RNA molecules  were 5′-terminal fragments of a sequence antisense to 7SK (7SK-as) of 80 nt, 100 nt, 135 nt and 160 nt respectively . The secin vitro from PCR-amplified templates using the HiScribe T7 High Yield RNA Synthesis Kit (New England BioLabs), according to the manufacturer's protocol. The RNA intended for microscale thermophoresis (Cap 0–100-mer) was body-labeled with Cy5 by the addition of 0.75 mM Cy5-UTP (Cytiva) to the transcription reaction. Obtained RNAs were loaded on a 5% (or 10% for Cy5-labeled RNA) polyacrylamide (PAA) gel containing 8 M urea and resolved in 0.5x Tris-borate-EDTA (TBE) buffer at 450 V for 4.5 h. The separated RNA samples were visualized using a UV imager. RNA-containing bands were excised from the gel and the Elutrap Electroelution system (Whatman) was used to purify RNA in 1× TBE at 100 V at 4ºC overnight. Cy5-labeled RNA was purified with Zymoclean Gel RNA Recovery Kit (Zymo Research) after excision from the gel.The RNAs were transcribed The purified 5′ triphosphate RNA 135-mer was left unmodified . Other purified RNAs were further modified enzymatically on the 5′ ends according to the enzyme manufacturer's protocols. The 80-mer was dephosphorylated with alkaline phosphatase  in order to obtain 5′OH-RNA. The 100-mer was treated with RNA 5′ pyrophosphatase  to obtain 5′p-RNA of 100 nt. The Vaccinia Capping System (New England BioLabs) was used to generate the 5′cap 0-RNA of 160 nt (160mer) or 100 nt . The modified RNA was purified with Rneasy Mini Kit (Qiagen) or in the case of Cap 0–100-mer with Monarch® RNA Cleanup Kit (New England BioLabs). The 4 RNA mixture for a pull-down reaction was prepared by combining 70 picomoles of each RNA .S. cerevisiae strain BY4741, using a published protocol (RNA was extracted from the protocol  with modFor pull-downs of RNAs using eIF4E(K119A), Cbp20/80 or Cdc33, yeast cell pellets were resuspended in 400 μl of TES buffer , supplemented with 400 μl of phenol solution saturated with 0,1 M citrate at pH 4.3 (Sigma-Aldrich P4682) and vortexed for 40 and then 20 minutes at 65°C. Between incubations samples were centrifuged at 14 krpm for 10 min at 4°C. The aqueous phase was washed with 400 μl of chloroform, centrifuged as previously and precipitated with 45 μl of 2 M LiCl in 1 ml of 96% ethanol at -80°C for at least 30 min. RNAs were pelleted, washed for 15 min with 80% ethanol, dried and resuspended in RNase free water. Human RNAs were extracted by resuspending cell pellets in 1 ml of Tri Reagent (Sigma-Aldrich T9424) and incubating for 5 min at room temperature. Subsequently 0.2 ml of chloroform was added and the samples were vortexed for 3 minutes and incubated for 10 min at room temperature. The samples were centrifuged for 15 min at 14 krpm at 4°C and the aqueous phase was precipitated with 0.5 ml of isopropanol. The pellets were washed with 75% ethanol, dried and resuspended in water for further use.2+-Sepharose 6 Fast Flow  slurry beads was washed with RNase-free water and equilibrated in a chilled Binding Buffer . Beads were sedimented by centrifugation at 500 × g for 1 min at 4°C and the supernatant was discarded. A portion of 2–3 μg (35–55 pmol) of 6xHis-IFIT1 was immobilized on the beads under rolling mixing for 0.5–1 h at 4°C. The beads were sedimented as before and washed with 1 ml of the Binding Buffer. The binding of IFIT1 with RNA (a mixture of 4 of the in vitro transcribed RNAs (2). Selected RNA was released by proteolysis with Proteinase K (Invitrogen) for 1 h at 37°C. The eluted RNA was precipitated with ethanol, re-suspended in RNase-free water and used for further reactions. RNeasy Mini Kit (Qiagen) was used for RNA clean-up prior to RNA-seq.Two alternative pull-down protocols were used, ‘Complex on Beads’ Con-B  and ‘Combed RNAs  or 10 μgYeast RNA samples  were analyzed by high-resolution automated electrophoresis with Agilent Bioanalyzer 2100 and Agilent RNA 6000 Pico Kit (according to the manufacturer's protocol).in vitro transcribed mix of the four RNAs were separated in a 12% PAA gel containing 8 M urea, stained with SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific) and visualized on a ChemiDoc MP Imaging System (Bio-Rad).RNA oligonucleotides recovered after the pull-down (Con-B protocol) of the 50 μl of MagneGST magnetic beads (Promega V8600) per 1 sample were washed 3 times with 1 ml of PBS. The GST binding capacity declared by the manufacturer at 1 mg and is far thus below the amounts of GST-eIF4E(K119A) that were subsequently applied to the resin  and bound for 1 h in PBS at 4°C on a rotating wheel. 200 μl of CNBr-activated SepFast MAG resin with coupled Cdc33 or Cbp80-20 dimer (encompassing or lacking N-6xHis-SUMO tag) or 40 μl of Dynabeads™ His-Tag Isolation & Pulldown magnetic beads with bound N-6xHis-SUMO tagged Cdc33 or Cbp80/20 were used in parallel, at approximately 0.1 mg protein/ml. The resins were washed 3 times in 1 ml PBS and then 3 times in 1 ml IP buffer . 120 μg of total RNA in 50–90 μl water were denatured at 70°C for 10 min and cooled for 2 min on ice, The RNAs were supplemented to 500 μl with IP buffer and incubated with the MagneGST-eIF4E(K119A) resin for 90 minutes at 4°C on a rotating wheel. The flow-through was collected for control analyses. The resin was washed 3 times with 1 ml of IP buffer, 2 times with IP buffer containing 0.5 mM GDP, which should reduce background levels, and again 2 times with 1 ml of IP buffer. The resin was resuspended in 500 μl of IP buffer for elution. Elution was performed with the protocol for RNA extraction using 400 μl of phenol solution saturated with 0.1 M citrate at pH 4.3 and following all the steps described above.Depletion of rRNA with a commercial RiboMinus™ Transcriptome Isolation Kit, yeast (Invitrogen) was performed according to the manufacturer's protocol. Samples for small-scale trial sequencing were sent to Eurofins Genomics, where polyA-based enrichment was performed according to the Standard eukaryotic library service and compared with our Con-B and RiboMinus ribodepleted samples. mRNA was fragmented and cDNA synthesis was performed using random hexamer priming, and analyzed by Illumina NovaSeq 6000 sequencing using 150 bp paired-end reads.Saccharomyces cerevisiae rRNA with bowtie2 v2.4.4, then resulting sequences were counted and removed from further analysis. All remaining reads were aligned to the genome of S. cerevisiae (NCBI: GCF_000146045.2_R64) using STAR v2.7.9a, allowing up to 4% of mismatches per read length  reaction using First Strand cDNA Synthesis Kit (Roche Life Science) and random hexamers, according to the manufacturer's protocol). The resulting cDNA was used for real-time quantitative PCR (qPCR). qPCR was performed in a final volume of 25 μl containing 1x HOT FIREPol EvaGreen qPCR Mix Plus (ROX) (Solis BioDyne), 0.8 μM forward primer and 0.8 μM reverse primer, according to the manufacturer's protocol. Three technical replicates were performed for each qPCR reaction. Primers used for the qPCR are listed in the Supplementary material . ThermocMST experiments were performed on a Monolith NT.115 system (Nanotemper Technologies) at 22°C with 60% IR-laser power and 60% LED in MST buffer  using premium capillaries. The concentration of Cy5-labeled cap 0–100-mer RNA was kept constant at 10 nM. Unlabeled protein was titrated in 1:1 dilutions with the highest concentration of 1 μM IFIT1 or 50 μM eIF4E(K119A). The recorded fluorescence was normalized to the fraction bound and analyzed using GraphPad Prism 9.3.0.Saccharomyces cerevisiae but is rare for human RNAs   .This high selectivity of IFIT1 towards RNAs modified with cap 0 allowed us to apply an IFIT1-based pull-down to much more complex samples to achieve mRNA enrichment prior to a transcriptomic analysis. To this end, we isolated total RNA from yeast and captured its cap 0 fraction in two alternative approaches . To optiIn order to identify the right benchmarking method, we started our NGS analyses with a small-scale trial experiment comparing two commercially available ribodepletion approaches, RiboMinus (RM) and polyA enrichment, with the Con-B approach applied to the total yeast RNA (raw data not included). The Principal Component Analysis (PCA) for RM, polyA and Con-B showed PC1 and PC2 contributions of 63% and 20.41%, respectively . The polWe compared the RNA samples recovered from the IFIT1 pull-down (Cin-S and Con-B) with those obtained using the commercially available RiboMinus (RM) standard ribodepletion procedure applied to total yeast RNA. Triplicates for each approach were prepared and sequenced using Illumina NovaSeq 6000 PE100. We received 406.7 million paired-end raw reads in total (ranging from 25.6M to 88.9M reads per sample) with a mean length of the read of 101 bp. In the first stage, data was trimmed and filtered to remove low-quality reads. On average, about 38% of sequences contained adapters. After adapters removal and verification of the data quality , 6.808 to 76.621 sequences per sample were deleted . In the 32,2,7GTP) post-transcriptionally  and non-coding transcripts  as compared to RM Figure . The CinWe next validated these differences for selected genes using RT-qPCR on the total RNA, Cin-S and RM samples Figure . We confxrn1- strain , which - strain  18), we, wexrn1-- strain . Notably- strain , but at - strain . At the - strain . We coul- strain . We concmRNA enrichment using the cap-binding eIF4E protein is commonly used for transcriptomic analyses, especially those focusing on 3′ ends . Often, Surprisingly, in our attempt with human or yeast eIF4E, its binding to yeast RNA did not perform as well as for another less known cap-binding protein, IFIT1. This might have resulted from unsuccessful purification or instability of eIF4E. However, since eIF4E(K119A), in our expert hands, was able to pull down human mRNA, we concluded that the quality of the protein sample was not the source of the poor binding to yeast mRNA. Nonetheless, problems with protein production or stability may also regard other protein affinity-based transcriptomic analyses, and it is worth noting that under the same experimental conditions, IFIT1 performed more robustly than eIF4E (nor could we find published studies of yeast mRNA capture by eIF4E). Another explanation is that poor capture efficiency by eIF4E may stem from the nature of 5′ ends of the analyzed RNA, for example due to obstructive or missing RNA modifications, such as defective N7-methylation of the cap. Unlike eIF4E, IFIT1 has no dependence on N7-methylation, which could facilitate mRNA capture in suboptimal or degraded samples (in which N7-methyl group may be lost or inefficiently introduced). This property of IFIT1 could also be advantageous in studies using synthetic RNA . This in32,2,7GpppRNA, or, more likely, detect the nascent cap 0 ends of snRNAs and snoRNAs prior to their post-transcriptional trimethylation in the cytosol . The seThe 5′-end capture, ribodepletion and polyA-dependent methods are known to produce slightly different results ,30,31 anRaw data is available under accession number ENA: PRJEB49214 and GEO: GSE210198.gkac903_Supplemental_FilesClick here for additional data file.


The restoration of genetic code by editing mutated genes is a potential method for the treatment of genetic diseases/disorders. Genetic disorders are caused by the point mutations of thymine (T) to cytidine (C) or guanosine (G) to adenine (A), for which gene editing (editing of mutated genes) is a promising therapeutic technique. In C-to-Uridine (U) RNA editing, it converts the base C-to-U in RNA molecules and leads to nonsynonymous changes when occurring in coding regions; however, for G-to-A mutations, A-to-I editing occurs. Editing of C-to-U is not as physiologically common as that of A-to-I editing. Although hundreds to thousands of coding sites have been found to be C-to-U edited or editable in humans, the biological significance of this phenomenon remains elusive. In this review, we have tried to provide detailed information on physiological and artificial approaches for C-to-U RNA editing. E. coli [RNA editing is a biological process or tool for repairing or altering RNA in a mitochondrion-encoded mRNA of a kinetoplastid trypanosome. RNA editing was first introduced to describe a process that occurs in trypanosomes and involves the insertion and deletion of uridine monophosphate (UMP) inside nascent transcripts after transcription . Since tE. coli .RNA editing tools are basically categorized into two types depending on their response mechanisms. For example, insertion/deletion RNA editing, involves the insertion or deletion of targeted nucleotides with the aim of changing the codon sequence of the targeted mRNA . HoweverRNA editing as a therapeutic approach was first conceptualized and utilized as a therapy in 1995. The main purpose of this method is to restore RNA sequences in order to treat genetic diseases caused by point mutations. Advanced research has enhanced and established this technology, which is now known as artificial site-directed RNA editing for restoring RNA. This unique therapeutic approach has the potential to be utilized to cure diseases, such as numerous neurological maladies in humans, by restoring the mutated A or C in mRNA without changing or affecting the genome sequence of the mRNA target .RNA editing in C-to-U is a process or therapeutic approach that converts a single or multiple C-to-U nucleotides in transcript sequences. C-to-U RNA editing can generate start or stop codons that can change the encoded amino acids depending on preferences towards the splice site . The C-tBoth C-to-U and A-to-I conversions are included in enzymatic site-directed RNA editing. Recently, artificial site-directed RNA editing of A-to-I has been successfully carried out in vitro and in cells as well as in vivo ,10. HoweThe artificial or enzymatic approach of deamination from C-to-U is mainly dependent on the enzymes from the apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) family proteins. Eleven genes code for members of the APOBEC family that have been discovered to date . They all have a zinc-dependent deaminase domain (ZDD) ,12. AmonFor the enzymatic approach, the MS2 system along with the APOBEC family protein enzyme has been a very promising technique for the therapeutic RNA editing technique. C-to-U editing  using thOnly vertebrates have APOBECs, and this type of RNA editing is the second most common after ADAR (Adenosine Deaminase acting on RNA) editing. Unlike ADARs, the APOBEC family of proteins, including the Alu (Arthrobacter luteus) sequence, primarily affects non-coding and intronic sequences ,20. SurpThe APOBEC family of proteins plays a vital role in the introduction of mutations in cancerous tissues ,23,24,25ApoB-100, the full-length form of ApoB protein, is expressed in hepatic cells (hepatocytes) in the liver. APOBEC-1 RNA editing, on the other hand, causes an early stop codon in ApoB mRNA in the small intestine, resulting in the premature termination of translation. Consequently, another isoform of ApoB-48 was created. The full-length form (ApoB-100) carries cholesterol in the bloodstream, whereas the shortened form (ApoB-48) transports triglycerides .There are seven APOBEC-3 paralogs in the human genome . Although all of these paralogs bind to RNA , only thThe restoration of genetic code from A-to-I and C-to-U are the two most common RNA editing processes in mammalian cells ,34. DeamIn the C6802U editing site, the threonine codon (ACA) is changed to an isoleucine (AUA). Because the C6802U editing event occurs concurrently with the C6666U editing event, C6802U is not expressed in the truncated ApoB48 protein but in the mRNA . The RNAThe editosome of C-to-U editing consists of a minimum of three protein components: ApoB1 and two essential cofactors, ApoB1-complementary factor (ACF) and RNA binding motif 47 (RBM47) . The cytWhile ACF knockout animals may die during early pregnancy, ACF+/mutant mice show a higher editing efficiency, contradicting the idea that cofactors are essential for editosome editability in vitro. Despite the abundance of scientific evidence for cofactors and C-to-U APOBEC-derived deaminase editing in vitro, there is no strong proof that cofactors are essential for C to-U RNA editing in vivo. As a result, the function of cofactors (ACF) in vivo remains unclear . C-to-U RNA editing alters the predicted nucleotide sequence in RNA molecules, resulting in a divergence from the genomic sequence of mRNA information for a protein. RNA editing converts C-to-U in the chloroplasts and mitochondria of flowering plants, and converts U-to-C in ferns and mosses. Specific proteins address almost 500 sites of editing in the mitochondria and only 40 sites in plastids of plants with flowering ability, whose genes are increased in plant species with organellar RNA editing . RNA ediAlthough there are significant distinguishing characteristics between mammals and plants C-to-U RNA editing, it may be technically possible to replace the mammalian APOBEC1 enzyme in human therapy applications by combining elements of RNA editing apparatus of plants with guide RNA (a complementary sequence of mRNA that binds with the targeted sequence). However, since C-to-U editing occurs automatically (endogenous property) in plants, the enzymes responsible for the editing/editors can be replaced in plants . The devIn plants, C-to-U and U-to-C are the two commonly found types of RNA editing events. Among them, C-to-U RNA editing is more commonly and precisely found than U-to-C. The RNA editing events of C-to-U occur in both the plastids and mitochondria of plants ,60,61,62Higher plant plastids have circular genomes that are 120–130 kb in size, with an expectation of 20–30 cytidines to be converted or altered to uridines, implying a 0.02% editing frequency in the plastid genome . BecauseA. thaliana, there are approximately 450 members of the PPR family, with roughly 250P and 200PLS subfamily members, respectively [A. thaliana. The cytidine deaminase 1 (At-CDA-1) protein from A. thaliana has been discovered; however, it does not show any RNA editing capacity [A. thaliana seedlings identified specific U-to-C RNA editing events [A. thaliana.Numerous pentatrico peptide repeat (PPR) motif proteins have been discovered as a part of the trans-acting components required for RNA editing (C-to-U) in both chloroplasts and mitochondria. These PPR motif proteins are the factors responsible for recognizing the site-specificity of cytidines that directly bind to cis-components. PPR motif proteins degenerate 35 repeats of amino acids, numbered from two to thirty ,78,79. Bectively ,81  and celIt has been kept in mind that when considering the impact of RNA editing in certain illnesses, most of the deaminases may be used for both DNA and RNA editing. Although APOBEC1 is now thought to be expressed in the outer part of the human digestive system, surprisingly, all the genetic illnesses associated with it have recently been found in the human brain. GlyR modification is linked to the onset of temporal lobe epilepsy (TLE) . In the The terminals of presynaptic regions of hippocampal neurons contain RNA-edited GlyRs, and even slight modifications in editing can cause malfunction ,106. APORNA editing was more likely to be observed in stem-loop structures with target C included in the stem loop, wherein the levels of RNA editing are proportional to the stability of the stem loop structure . HoweverAccording to the findings presented in this review, RNA editing levels, as well as the expression of A and C deaminases and specifically altered genes (tumor suppressors and oncogenes), could be important predictors of cancer pathogenesis and progression ,122,123.Although initial evidence on RNA editing events in humans was first published in the late 1980s, in respect of hepatocytes of the liver (C-to-U editing in ApoB mRNA was published in 1987), this topic has only recently received further attention. The role of RNA editing in the initiation, progression, and metastasis of cancer has been extensively researched in a range of cancer types, with a growing body of evidence pointing in that direction. However, many concerns remain unanswered, and the importance of RNA editing processes in cancer and other human diseases remains unclear. Furthermore, substantial RNA editing in transcripts encoding cancer-related proteins increases the likelihood of neoantigen formation. More research into RNA editing could lead to advancements in cancer immunotherapy and/or targeted anti-cancer drugs, as lymphocytes can swiftly recognize such neoantigens.