Organellar gene expression (OGE) in chloroplasts and mitochondria is primarily modulated at post-transcriptional levels, including RNA processing, intron splicing, RNA stability, editing, and translational control. Nucleus-encoded Chloroplast or Mitochondrial RNA-Binding Proteins (nCMRBPs) are key regulatory factors that are crucial for the fine-tuned regulation of post-transcriptional RNA metabolism in organelles. Although the functional roles of nCMRBPs have been studied in plants, their cellular and physiological functions remain largely unknown. Nevertheless, existing studies that have characterized the functions of nCMRBP families, such as chloroplast ribosome maturation and splicing domain (CRM) proteins, pentatricopeptide repeat (PPR) proteins, DEAD-Box RNA helicase (DBRH) proteins, and S1-domain containing proteins (SDPs), have begun to shed light on the role of nCMRBPs in plant growth, development, and stress responses. Here, we review the latest research developments regarding the functional roles of organellar RBPs in RNA metabolism during growth, development, and abiotic stress responses in plants. Plant chloroplasts and mitochondria are thought to be derived from free-living cyanobacteria and α-proteobacteria, respectively ,2. DurinOGE is commonly regulated at the post-transcriptional level, including RNA processing, editing, stabilization, turnover, intron splicing, and translational control, all of which are crucial for a number of organellar processes ,11,12,13E. coli YhbY associated with pre-50S ribosomal subunits [Arabidopsis and rice [Single chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins were first studied in archaea and bacteria ,29, in wsubunits   was first observed in the ribosomal proteins S1 (RPS1) of E. coli. . E. colino acids . The strno acids . S1 domano acids  and havens EMB-5 , which pns EMB-5 ,49,50,51ns EMB-5 ,53, bactns EMB-5 , and thens EMB-5 . Furtherns EMB-5 . These fArabidopsis and rice genomes, respectively [Arabidopsis AtCRS1 [Arabidopsis AtCFM4 [16S and 23S rRNA processing, and that rice OsCAF1 [Arabidopsis mCSF1 [The latest studies have indicated the importance of nCMRBPs, including CRM, PPR, DBRH, and SDP, for organellar RNA metabolism during plant growth and development . Fourteeectively ,20. Prevs AtCRS1 , AtCAF1 s AtCRS1 , AtCAF2 s AtCRS1 , AtCFM2 s AtCRS1 , and rics AtCRS1 , and duas AtCRS1  in both s AtCFM4  is invole OsCAF1  and OsCFe OsCAF1  is imporis mCSF1  and CFM9is mCSF1  are invoArabidopsis OTP51 [Arabidopsis showed that these proteins play a role in the splicing of chloroplast introns through their role in the recognition of the specific RNA sequences. Arabidopsis HCF152 [Arabidopsis are involved in chloroplast rRNA processing. In addition, mitochondria-localized Arabidopsis OTP43 [Arabidopsis MISF 26, 68, 74 [Arabidopsis MTSF1 [As the structural characteristics of PPR motifs were first determined in plants, the functions of a large number of plant PPR proteins have been reported over the last 20 years ,26. As iis OTP51  and OTP7is OTP51 , are essis OTP51 , EMB2654is OTP51 , PBF2 [6is OTP51 , and SOTis OTP51  in Arabis HCF152 , MRL1 [7s HCF152 , PGR3 [7s HCF152 , BFA2 [7s HCF152 , and mais HCF152  and PPR1s HCF152  were shos HCF152 , SOT1 [7s HCF152 , and PPRs HCF152  in Arabiis OTP43 , BIR6 [7is OTP43 , TANG2/Ois OTP43 , and SLOis OTP43  are know, 68, 74 , and EMB, 68, 74  have alsis MTSF1  and PPR1is MTSF1  were fouArabidopsis and rice genome, respectively [Arabidopsis, chloroplast-localized RH3 [Arabidopsis RLSB [Nicotiana STF [Arabidopsis SDP is crucial for chloroplast rRNA processing during plant growth and development. Approximately 58 and 50 DBRH were annotated from the ectively ,87. Althectively ,89,90,91ized RH3  and ISE2ized RH3  and mitoized RH3  are invoized RH3 , RH39 [9ized RH3 , and RH5sis RLSB  and Nicoiana STF , play roImportantly, the aforementioned nCMRBPs are transported into chloroplasts and/or mitochondria, and mutations in these genes result in various phenotypes, including embryo lethality, albino, pale green, dwarfism, delayed growth, as well as impaired photosynthesis and mitochondrial respiration . This inAs sessile organisms, plants often face adverse environmental conditions, including extremes of temperature, high salinity, drought, and UV stresses, all of which can severely damage crop productivity and yield ,99. To sArabidopsis CRM-containing CFM4 (16S and 23S rRNA processing) has been determined as a positive effector in seed germination and seedling growth under low temperature and salt stress conditions [Arabidopsis CFM9, which is involved in the splicing of multiple mitochondrial introns, has demonstrated its positive role in seed germination and seedling growth in the presence of the abscisic acid (ABA) and under high salinity or dehydration stress [cfm4 and the cfm9 mutants grew slowly under normal conditions, the mutant characteristics of growth retardation and delayed germination were much more severe under abiotic stress conditions compared to those of the wild type. This indicates that organelle-targeted CFM4 and CFM9 also play a crucial role in plant responses to abiotic stresses. Chloroplast-localized nditions . Recent n stress . AlthougArabidopsis GUN1 was found to be hypersensitive to sucrose and ABA [rpl2 introns, enhanced seed germination and seedling growth in response to multiple environmental factors, such as glucose, ABA, and salinity, owing to its reduced translation efficiency [OsV4, OsRpoTp, V1, V2, RNRL, RNRS, 16S rRNA, rpl21, and OsDG2, and WSL5 are involved in the editing of rpl2 and atpA, as well as the splicing of rpl2 and rps12 intron2, are crucial for chloroplast biogenesis, the mutants of which lead to albino or pale yellowish phenotypes during cold stress [Arabidopsis PPR40 has been shown to promote seed germination in the presence of salt or ABA and improve seedling growth under conditions of high salinity by reducing reactive oxygen species (ROS) damage in the mitochondria [Arabidopsis PGN, which is involved in the expression of mitochondrial NAD1, RPL2, NAD9, and MATR genes, plays a role in both biotic and abiotic stress tolerance, and its loss-of-function mutants are susceptible to ABA, salt, and glucose, as well as necrotrophic fungal pathogens [Arabidopsis ABO5 and ABO8, which are involved in the splicing of mitochondrial nad2 intron3 and nad4 intron3, respectively, have been shown to have enhanced sensitivity to ABA during post-germination and root growth phase due to the accumulation of ROS in the mitochondria [Arabidopsis PPR96, which is thought to be involved in mitochondrial RNA editing, has a negative impact on seed germination and seedling growth [The diverse roles of organelle-localized PPR proteins have been demonstrated in the responses of plants to abiotic stresses. The loss-of-function mutant of chloroplast-localized  and ABA . Chloropficiency . Chloropd stress ,106,107.chondria ,109. In athogens . Arabidochondria ,112. Intg growth .Arabidopsis RH3, which is involved in the splicing of ndhA and ndhB introns, displays hypersensitivity to salt and cold stress, and to ABA [POR, RBCL, CLPB3, PSBA, and PETA transcripts, and cabbage RH22, which affects the translation of chloroplast RBCL, PSBA, and YCF3 genes, contributed to an enhanced tolerance to salt and drought stress in Arabidopsis by increasing the translational efficiency of chloroplast mRNAs [The organelle-localized DBRHs are essential for the responses of plants to environmental stresses. The loss-of-function mutant of chloroplast-localized d to ABA . Recentld to ABA . Moreovest mRNAs ,114.trnL intron and 5S rRNA processing in the presence of ABA [16S rRNA processing, enhanced seedling growth in response to cold stress [Chloroplast-localized SRRP1 harboring two S1 domains was shown to decrease sensitivity to ABA by impairing the splicing of the chloroplast e of ABA . Additiod stress . A recend stress . Beyond d stress ,120,121.d stress ,124,125.The mechanistic role of nCMRBPs in plant growth, development, and abiotic stress responses remains largely unknown. However, recent studies have revealed that nCMRBPs act as RNA chaperones in plant growth and development, as well as in stress adaptation processes.RNA molecules must adopt correct structures in order to maintain functional RNAs. However, RNA molecules are often misfolded into non-functional secondary or tertiary structures in cells, due to intrinsic thermodynamic and kinetic folding problems ,127. As E. coli Hfq and ProQ are important for stress responses because of their roles as RNA chaperones [Arabidopsis and rice, is involved in the splicing of U12-type introns as an RNA chaperone, and it is essential for the correct folding of introns during normal growth and development [Arabidopsis, rice, cabbage (Brasscia rapa), and wheat (Triticum aestivum) under various environmental conditions [Arabidopsis CFM4, RH3, SDP, and SRRP1 have RNA chaperone properties that are crucial for maintaining the structures of the precursor-RNA molecules suitable for splicing or rRNA processing [Arabidopsis and rice PPR4, containing both RRM and PPR motifs, possess RNA chaperone activity through its RRM motif, and thereby affect the trans-splicing of rps12 intron1 [Arabidopsis CFM9 was shown to affect the splicing of multiple mitochondrial introns, and the cfm9 mutant was found to be sensitive to abiotic stresses [Research conducted over a number of decades has demonstrated that RNA chaperones are crucial for diverse cellular processes in prokaryotic and eukaryotic organisms . It has aperones ,132. Stuaperones ,134,135.elopment ,137. Stunditions ,146,147.ocessing ,115,147. intron1 . Moreove intron1 ,114. Mitstresses . As such6A methylation patterns in the chloroplast and mitochondria RNAs have been reported [Although the functional roles of nucleus-encoded organellar RBPs are still not fully understood, the latest studies of the cellular and physiological functions of nCMRBPs has shed some light on the significance of nCMRBPs for organellar RNA metabolism during plant growth, development, and environmental stress responses. It has been demonstrated that chloroplasts- or mitochondria-localized CRM, PPR, DBRH, and SDP proteins play pivotal roles in organellar post-transcriptional RNA metabolism, including intron splicing, rRNA processing, and translational control under normal and stressful conditions . Since dreported . It woul


The rapid identification of pathogen clones is pivotal for effective epidemiological control strategies in hospital settings. High Resolution Melting (HRM) is a molecular biology technique suitable for fast and inexpensive pathogen typing protocols. Unfortunately, the mathematical/informatics skills required to analyse HRM data for pathogen typing likely limit the application of this promising technique in hospital settings.https://skynet.unimi.it/index.php/tools/meltingplot/.MeltingPlot is the first tool specifically designed for epidemiological investigations using HRM data, easing the application of HRM typing to large real-time surveillance and rapid outbreak reconstructions. MeltingPlot implements a graph-based algorithm designed to discriminate pathogen clones on the basis of HRM data, producing portable typing results. The tool also merges typing information with isolates and patients metadata to create graphical and tabular outputs useful in epidemiological investigations and it runs in a few seconds even with hundreds of isolates. Availability: The analysis and result interpretation of HRM typing protocols can be not trivial and this likely limited its application in hospital settings. MeltingPlot is a web tool designed to help the user to reconstruct epidemiological events by combining HRM-based clustering methods and the isolate/patient metadata. The tool can be used for the implementation of HRM based real time large scale surveillance programs in hospital settings. As stated above, The melting temperatures of each interrogated genomic region depend on its nucleotide composition. Consequently, melting temperatures can be used to cluster the isolates in a n-dimensional space. Previous works suggested that HRM data can be used for fast pathogen typing [Klebsiella pneumoniae [K. pneumoniae typing protocol with Multi Locus Sequence Typing (MLST) and Whole Genome Sequencing (WGS) approaches: HRM typing protocol showed a discrimination power comparable to MLST on clinical K. pneumoniae isolates [The rapid typing of pathogens is pivotal to perform fast epidemiological investigations and to detect and block outbreaks. High-Resolution Melting (HRM) analysis is a single-step molecular biology technique able to determine the melting temperature of a PCR amplicon. The main output of an HRM assay is the melting curve, which indicates the denaturation level of the DNA amplicon in relation to temperature. The melting temperature is defined as the temperature in which half of the DNA duplex is dissociated. Considering that the melting temperature depends on the nucleotide composition of the amplicon, melting temperature can be used to discriminate sequence alleles. For each isolate, HRM analysis interrogates n typing –4. Receneumoniae . In the isolates .Here we present MeltingPlot, a tool for rapid epidemiological investigation using HRM data. The tool implements an evolution of the clustering algorithm we already published . Moreove: HRM-based clustering/typing of isolates, prevalence analysis and transmission analysis. HRM-based clustering/typing is computed only on the basis of the High Resolution Melting (HRM) temperatures of the isolates amplicons, that are the only inputs needed for this step. Actually, the tool does not use any other information that can be derived from the melting curves, like the shape or the height of the curve. Indeed, in our experience, these features are more subjected to experimental noise than melting temperature so we are not using them to type the isolates. After the computation of the average HRM temperature of the technical replicates, the isolates are organized in a graph where the vertices represent the isolates and two vertices are connected if the difference of their average HRM temperatures is less or equal to 0.5 °C for each PCR primer set used in the HRM typing method. The graph is then decomposed into separate components (groups of connected vertices) and each one is then divided in clusters using the Edge Betweenness Clustering algorithm [The flow of MeltingPlot can be divided in three main stepslgorithm  implemenlgorithm . BrieflyFurthermore, the betweenness centrality of a vertex was computed as the number of graph short paths passing that vertex. Hence, vertices with higher betweenness centrality values are those that connect two or more clusters. We used this parameter to identify vertices that were not strongly associated with a single cluster. Thus, vertices with normalized betweenness centrality values above a threshold were not assigned to any cluster and they were classified as “undetermined” by the tool .Unfortunately, HRM-based clustering results obtained from different datasets are not directly comparable. To obtain comparable HRM typing results, the user can include in the analysis the HRM temperatures of a collection of reference strains: isolates previously analysed by the same HRM protocol and for which typing annotation is known (e.g. Sequence Type). When a reference collection is provided, MeltingPlot labels each cluster with the annotation of the reference isolates contained in it. For details see the Additional file Prevalence analysis and transmission analysis steps can be performed only when patients/isolates metadata is provided. In these steps the tool joins the HRM clustering results with the isolates’ metadata to create various outputs that depict the spreading of pathogen clones among wards and patients over time. For more details see the output files section below or the Additional file HRM_temperatures, Isolates_metadata, Reference_isolates and an HELP_notes sheet:HRM_temperatures: in this sheet the user has to report the high resolution melting (HRM) temperatures of the study isolates. This is the only mandatory data and it is used to perform the HRM-based clustering/typing analysis. If HRM experiments were performed using technical replicates, the users have to report all the replicate temperatures;Isolates_metadata: in this sheet the users can provide patients/isolates metadata, e.g. isolation date, isolation location  and an ID for the patients . This information is not mandatory for HRM isolates typing but it is required to perform the complete epidemiological investigation ;Reference_isolates: this sheet contains the HRM temperatures of the reference isolates and their annotation (e.g. Sequence Type). The reference isolates annotation will be used to label the clusters, making the obtained HRM typing results portable when the same reference collection is used.HELP_notes: this sheet contains important information about the rules for each column of the spreadsheet.Users are required to download and fill an xls template spreadsheet that contains four sheets: All the templates  are available on the MeltingPlot webpage.MeltingPlot creates three groups of plot files (in PDF and PNG format), one for each step of the analysis: HRM-based clustering/typing, prevalence analysis and transmission analysis. The HRM-based clustering/typing plot group includes the isolates graph (where each isolate is colored on the basis of its cluster) and a heatmap showing the HRM temperatures and the isolates clusters. In the isolates graph each vertex is an isolate and two isolates are connected as described above. The last two groups are created when metadata of the isolates is provided. The prevalence analysis plot group includes bar plots showing the distribution of the clusters over time in the different locations. The transmission analysis plot group contains a patient timeline and a patient-to-patient graph. In the latter, two patients are connected when two isolates belonging to the same HRM cluster were collected from both patients. The edge is thicker when the isolates were collected in the same location (e.g. ward) within a number of days set by the user (7 by default). Thus, thicker edges highlight most probable transmission events. MeltingPlot also produces xls spreadsheets containing the isolates HRM clusters and metadata. See Additional file Klebsiella pneumoniae nosocomial outbreak (100 outbreak isolates) sustained by multiple clones spread in different wards, a situation comparable to real large nosocomial outbreaks [K. pneumoniae isolates previously analyzed in our laboratory. We simulated an outbreak scenario sustained by five different clones: three major clones that caused the outbreak and two sporadic clones. Thus we included in the dataset the temperature of previously HRM typed isolates belonging to five highly epidemiologically relevant clusters: wzi173_(ST307), wzi154_(ST512/ST258), wzi89_(ST15), wzi137_(ST101)/wzi24_(ST11) and wzi56_(ST147)/wzi95_(ST10); the first three causing the outbreaks and the last two being sporadic. We also simulated the metadata of the isolates to obtain a complex outbreak with three wards showing different epidemiological scenarios  (in red), wzi154_(ST512/ST258) (in green) and wzi89_(ST15) (in violet) using the annotation given by the user for the reference isolates. Most of the infections are caused by two pathogen clusters (green and red), each one associated with a single ward: the green one with Ward A and the red one with Ward B. A smaller cluster (in violet) caused an outbreak in Ward C at the beginning of the investigated period. The patient’s timeline and the patient-to-patient graph clearly show that two patients (Pz15 and Pz17) were infected by isolates of both the red and green clusters and they also crossed the wards A and B: this highlights two possible pathogen transmission routes among the wards. A complete description of each output file is available in the Additional file https://skynet.unimi.it/index.php/tools/meltingplot/. The source code for the stand alone version is available at https://github.com/MatteoPS/MeltingPlot.To show the capability of MeltingPlot, we simulated a complex large utbreaks . The isoHigh Resolution Melting (HRM) is a fast and inexpensive molecular biology technique  applicabThe tool implements an algorithm for the HRM-based clustering that groups isolates on the basis of their melting temperatures. Unfortunately, the HRM-based clusters obtained from different collections of isolates are not directly comparable. To overcome this limitation we made MeltingPlot able to include in the clustering analysis the melting temperatures of a collection of reference isolates. MeltingPlot uses the reference isolates as a guide to label the obtained isolates clusters. On this way, MeltingPlot results obtained from different isolates collections become comparable.Furthermore, MeltingPlot performs complete epidemiological investigations merging HRM clustering results with isolates/patients metadata. It produces easy-to-read graphical representations and tabular files (see Results section) useful to reconstruct epidemiological scenarios and to identify pathogen transmission routes.The HRM typing is cost saving and it can be carried out using instruments usually present in hospital microbiology laboratories and by not highly specialized personnel. MeltingPlot also eases the HRM data analysis for epidemiological investigation. In our opinion, the implementation of HRM typing can improve nosocomial surveillance programs with a limited impact on the hospital policies in terms of costs, workload and personnel involved.MeltingPlot is available in stand-alone and web versions. The web interface makes the tool user-friendly and the user has only to upload the data into an xls template spreadsheet. MeltingPlot analyses hundreds of isolates in a few seconds.HRM technique allows pathogen typing in a few hours and ~ 5 euros per sample. Despite this, the mathematical/informatics skills required for the analysis and interpretation of HRM results limit the application of HRM typing protocols in hospital real time surveillance. MeltingPlot is a user-friendly tool that facilitates the application of HRM to real time large scale surveillance programs in hospital settings.Project name: MeltingPlot.Project home page: https://skynet.unimi.it/index.php/tools/meltingplot/Operating system(s): Platform independent.Programming language: R, PHP.Other requirements: Any web browser.License: GPL.Any restrictions to use by non-academics: none.Additional file 1: This file contains a detailed description of the clustering algorithm, of the input file and of each output file created by the tool.


Since the infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in China during December 2019, the coronavirus disease 2019 (COVID-19) has spread on a global scale, causing the World Health Organization (WHO) to issue a warning. While novel vaccines and drugs that target SARS-CoV-2 are under development, this review provides information on therapeutics which are under clinical trials or are proposed to antagonize SARS-CoV-2. Based on the information gained from the responses to other RNA coronaviruses, including the strains that cause severe acute respiratory syndrome (SARS)-coronaviruses and Middle East respiratory syndrome (MERS), drug repurposing might be a viable strategy. Since several antiviral therapies can inhibit viral replication cycles or relieve symptoms, mechanisms unique to RNA viruses will be important for the clinical development of antivirals against SARS-CoV-2. Given that several currently marketed drugs may be efficient therapeutic agents for severe COVID-19 cases, they may be beneficial for future viral pandemics and other infections caused by RNA viruses when standard treatments are unavailable. On February 11, 2020, the WHO officially renamed this infectious disease coronavirus disease (COVID-19). The coronavirus study group within the International Committee on Taxonomy of Viruses also renamed 2019-nCoV, as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, the COVID-19 pandemic is spreading all over the world, with cases reported in China -5-hydroxy-1-methyl-2-[(phenylthio)methyl]-indole-3-carboxylate hydrochloride monohydrate; trade name Arbidol) is a potent Russian-made broad-spectrum antiviral agent, that is used to treat influenza A and B viruses and hepatitis C virus (HCV) . AlthougRecently, a trial involving the use of lopinavir/ritonavir (LPV/r), which are protease inhibitors used to treat HIV, in adults hospitalized with severe COVID-19, showed no observable benefit of LPV/r treatment beyond the standard of care .Another retrospective cohort study tested umifenovir combined with LPV/r, versus LPV/r alone, against COVID-19 . The resIt was reported that chloroquine phosphate, a well-established drug used to treat malaria, was shown to have apparent efficacy, and was acceptably safe, when used against COVID-19 in multicenter clinical trials conducted in China . In ChinChloroquine, which has been used since 1934, has several anti-inflammatory and antiviral effects that have been reported by previous studies . For insDuring a viral infection, the immune response is activated and the production and release of pro-inflammatory cytokines, TNFα, IL-1, IL-6 and interferon-gamma (IFNγ) is increased. Chloroquine, however, blocks these events . AccordiPrevious studies showed that the inhibitory effects involve the inhibition of autophagy . AutophaA recent study using cancer stem cells demonstrated that mefloquine hydrochloride, an antimalarial drug used to treat patients with resistance against chloroquine, efficiently eliminated colorectal cancer stem cells by disrupting endolysosomal proteins RAB5/7 . Given tPrevious studies indicated that the angiotensin-converting enzyme 2 (ACE2) is the functional receptor for SARS-CoV , determiThe latter study demonstrated that the receptor-binding domain (RBD) of the viral spike (S)-protein in SARS-CoV-2 shows a strong interaction with human ACE2 molecules, despite its sequence diversity . The autIn many human and rodent studies, ACE2 expression is induced by treatment with ACE inhibitors (ACEIs) or angiotensin II receptor blockers (ARBs), which are commonly used as antihypertensive drugs . The expA recent retrospective study indicated that COVID-19 patients with cardiovascular disease (CVD) have a higher risk of mortality . Lower lIn addition, it has been reported that the small intestine is the organ expressing ACE2 most highly . Given t0AT1, also known as SLC6A19, and that their bindings affect the binding between ACE2 and S-protein [The crystal structures of S-protein binding to ACE2 has been revealed as an important interaction between host and SARS-CoV-2 ,29. In a-protein ,31. More-protein . Among t-protein . Many thIt was reported that dipeptidyl peptidase 4 (DPP4) is a functional receptor for the emerging human coronavirus via S-protein, as well as ACE2 . The intIt was previously reported that aminopeptidase N (APN) is involved in broad receptor engagement, which promotes the cross-species transmission of COVID-19 .Interestingly, previous studies identified APN as a surface marker for cancer stem cells in the human liver . RepurpoAlthough the modification of RNA by methylation is critical in biology, methylation is also important for the process of RNA capping in coronaviruses .Like the coronaviruses that cause SARS and MERS, the mechanism of RNA capping may also be a draggable target in SARS-CoV-2. RNA capping plays a role in the transcription of viral RNA, as well as stability, replication, and evasion from the host’s immune response.O-position of the first nucleotide. This mechanism plays a critical role in pre-mRNA splicing, mRNA export [Many RNA viruses, including the coronaviruses, have evolved mechanisms for generating their cap structures with methylation at the N7 position of the capped guanine, and the ribose 2’-A export , RNA staA export , translaA export , and escA export .7GpppN) [O-MTase, to form the cap-1 (m7GpppNm) and cap-2 (m7GpppNmpNm) structures [O-MTase are catalyzed via the transfer of a methyl group from S-adenosyl-methionine (SAM) to the RNA substrate through the DxGxPxG/A SAM-binding motif. During the methylation process, S-adenosyl-homocysteine (SAH) is generated as a byproduct.In general, 5’-end-capped mRNAs are produced through several steps . Althoug7GpppN)  in the pructures . Both N7Given the proposed function of the viral RNA cap structure in preventing 5’-triphosphate terminal (5’-pppN) from activating the host innate immune response (cap-0 and cap-1) ,46,47,48Although specific treatments, including vaccines, have not yet been developed for COVID-19, effective prevention methods are now recommended on a global scale. Accordingly, to overcome this pandemic, developing specific inhibitors for viral entry and replication, as well as drug repositioning, will be necessary. As above, several clinical trials and drug repositioning studies are currently ongoing. Eventually, new studies will allow us to better control this pandemic and identify new treatments. Computational calculation and artificial intelligence would help the rapid development of a therapeutic method. On the other hand, accurate crystal structure determination and much drug-response data are necessary for its success. The efficient sharing of information will be important for overcoming this pandemic in the era of globalization.


Perionyx excavatus, has the most powerful regeneration capacity, which can completely and regenerate an amputated head and tail. Earthworm regeneration is a form of epimorphosis, a simple mode of development in adults that occurs around the sites of damage rather than throughout the body. In order to achieve this process, the earthworm must have molecular tools via which a variety of cell and tissue types can be precisely recovered from the pluripotent (or possibly totipotent) blastemal cells. Adenosine to inosine (A-to-I) RNA editing catalyzed by adenosine deaminases acting on RNA (ADAR) can generate substantial transcriptome and proteome variability and provide an ideal tool for cell and tissue re-specification. To understand the role of ADAR during earthworm regeneration, the molecular characteristics of an ADAR gene identified from P. excavatus (Pex-ADAR) were analyzed, and its spatial and temporal expression patterns were observed during regeneration. Domain analysis showed that Pex-ADAR is a member of the ADAR1 class. Its expression level primarily increases when and where muscle redifferentiation is actively taking place, suggesting that the RNA-editing enzyme Pex-ADAR is involved in muscle redifferentiation.Among the animal species capable of regenerating missing body parts, a species of earthworm, Perionyx excavatus is an earthworm with potent regenerative potential; it can regenerate the head and tail and is an advantageous model system to investigate the molecular mechanisms of regeneration. During RNA sequencing analysis of P. excavatus regenerates, we identified an ADAR homolog (Pex-ADAR), which led us to examine its spatial and temporal expression to comprehend how Pex-ADAR is linked to regeneration. At first, in domain analysis, we discovered that Pex-ADAR only has one double-stranded RNA-binding domain (dsRBD) and a deaminase domain without a Z-DNA-binding domain (ZBD). In addition, a comparison of the core deaminase domains of Pex-ADAR with those of other ADAR family members indicated that Pex-ADAR comprises the conserved three active-site motifs and a glutamate residue for catalytic activity. Pex-ADAR also shares 11 conserved residues, a characteristic of ADAR1, supporting that Pex-ADAR is a member of ADAR1 class. Its temporal expression was remarkably low in the early stages of regeneration before suddenly increasing at 10 days post amputation (dpa) when diverse cell types and tissues were being regenerated. In situ hybridization of Pex-ADAR messenger RNA (mRNA) indicated that the main expression was observed in regenerating muscle layers and related connective tissues. Taken together, the present results demonstrate that an RNA-editing enzyme, Pex-ADAR, is implicated in muscle redifferentiation during earthworm regeneration.Adenosine deaminases acting on RNA (ADAR) catalyze the hydrolytic deamination of adenosine (A) to produce inosine (I) in double-stranded RNA substrates. A-to-I RNA editing has increasingly broad physiological significance in development, carcinogenesis, and environmental adaptation.  RNA editing, a post-transcriptional modification of RNA molecules, is a widespread phenomenon in all metazoans. The most common RNA editing event is the chemical conversion of adenosine to inosine (A-to-I) catalyzed by adenosine deaminases acting on RNA (ADAR) enzymes, which mainly act on double-stranded RNA (dsRNA) substrates. This base modification in RNA molecules confers substantial transcriptome and proteome variability and impacts protein function, microRNA (miRNA) biogenesis or target spectrum, and RNA fate , and it is thought to be associated with mammalian brain and neural crest cell development ,2, carciADAR gene to be identified was ADAR1 from oocytes of vertebrate Xenopus laevis [ADAR2 was described, which is critically required for GluR-B Q/R editing, and several other editing events that largely affect protein recoding [Capitella teleta (Polychaeta) and Helobdella robusta (Hirudinea) are thought to possess ADAR-like genes, consisting of one ADAD, one ADAR, and two ADAR2 in C. teleta and only one ADAR2 in H. robusta [The first s laevis . Subsequrecoding ,11. Thro robusta . HoweverPerionyx excavatus has the most powerful regenerative capacity and can completely regenerate an amputated head and tail within approximately 3 weeks post amputation. In particular, during complete anterior regeneration, P. excavatus can reconstruct the essential body parts, including the brain, heart, and reproductive organs  [ADAR1 and ADAR2 messenger RNA (mRNA) are differentially expressed during different phases of regeneration in multiple tissues [Earthworms show a wide spectrum of regenerative potential capable of reconstructing body parts lost due to injury. Among the earthworm species, itellum) , and theitellum) . Therefo tissues .P. excavatus, we identified a full-length complementary DNA (cDNA) sequence showing significant homology to mammalian ADAR1. To our knowledge, this is the first report on the molecular characterization of an earthworm (Oligochaete) ADAR and its possible implication in muscle redifferentiation during regeneration.Through RNA sequencing analysis of the regenerates of P. excavatus obtained from a commercial source  were reared using a method previously described [Pex-ADAR was predicted by PSORT II [Sexually mature escribed . Before PSORT II .http://www.uniprot.org/). Amino-acid sequence alignment was carried out using ClustalX software . Phylogenetic analysis was performed via the neighbor joining (NJ) method, using the NJplot. (http://doua.prabi.fr/software/njplot). Bootstrap analysis was performed with 1000 replications. The phylogenetic tree was built with ADAR1 orthologs of metazoan animal models (Lophotrochozoa: Perionyx excavatus (MT905407), Pecten maximus (XP_033751634), Mizuhopecten yessoensis (OWF54145), Haliotis diversicolor (A0A5B7L1W7), Octopus vulgaris (XP_029636176), and Lingula anatina (XP_013411771); Deuterostomia: Homo sapiens (P55265), Rattus norvegicus (P55266), Mus musculus (Q99MU3), Gallus gallus (A0A1D6Y7R2), Xenopus laevis (O12982), Danio rerio (E9QIW2), and Takifugu rubripes (H2TCA4)), ADAR2 orthologs (Homo sapiens (P78563), Rattus norvegicus (P51400), Mus musculus (Q91ZS8), and Danio rerio (Q90VY9)), and ADAR3 orthologs (Homo sapiens (Q9NS39), Rattus norvegicus (P97616), Mus musculus (Q9JI20), and Danio rerio (X2BQ53)).Amino-acid sequences of ADAR1 were retrieved from the GenPept Database via protein Basic Local Alignment Search Tool (BLASTP) and UniProt  at the times indicated. We selected mRNA from total RNA using oligo (dT) primers  and then reverse-transcribed the mRNA into cDNA . Quantitative PCR (qPCR) was performed using WizPure™ qPCR Master (SYBR)  with specific primer pairs on an Applied Biosystems Stepone plus real-time PCR System. The sequences of primer pairs were as follows: Pex-ADAR (forward) 5′–TCCGTGTGGAGATGGTTCAC–3′ and (reverse) 5′–CGCCACGAAGAATTCCATCC–3′; Pex-GAPDH (forward) 5′–TCGGTCGTTTGGTGATGAGA–3′ and (reverse) 5′–TTCCATCGTGGTGGACTTCA–3′. Relative quantification of mRNA was conducted using the comparative 2−ΔΔCt method with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference gene. All data are expressed as the mean ± the standard error of the mean (SEM) and were analyzed using GraphPad Prism 6.01 . Differences between groups were tested by one-way ANOVA.Total RNA was isolated from both the head and the tail regenerates of Fluorescent in situ hybridization (FISH) on cryosections was performed as previously described  using a ADAR found in the earthworm P. excavatus (Pex-ADAR) are shown in The nucleotide and predicted amino-acid sequences of Pex-ADAR consists of 2571 bp corresponding to a polypeptide of 857 amino-acid residues. Pex-ADAR has only one dsRBD and a deaminase domain without a Z-DNA-binding domain (ZBD), and its subcellular localization was predicted to be nuclear. Because Pex-ADAR has a single dsRBD, we compared the sequence of the domain with those of dsRBDs at the equivalent position of diverse ADARs. All ADAR1 dsRBD sequences analyzed are highly conserved in size (65 amino acids) and residues, encompassing the KKXXK(R) motif (X is any amino acid and R indicates the conservative replacement of K), which is important for dsRNA binding in this enzyme  and a glutamate (E) residue in the first active-site motif for catalytic activity. It is also noted that Pex-ADAR shares 11 conserved residues, a characteristic of ADAR1, distinct from those of ADAR2 and 3, and the core deaminase domain of Pex-ADAR exhibits 61.7% sequence identity with hADAR1, while less than 50% for hADAR2 and hADAR3. Furthermore, phylogenetic analysis revealed that Pex-ADAR can be clustered with ADAR1s from other lophotrochozoans, including mollusks, which were grouped separately from those of deuterostomes , especially in the regenerating tissue of the head  . During Z-DNA with high affinity and probably functions to facilitate the binding of ADAR1 to transcriptionally active sites by localizing this enzyme to sites of active transcription [Caenorhabditis elegans with a single dsRBD displays RNA editing activity and has a distinct role from ADR-1, which has two dsRBDs [All metazoan ADAR proteins share a common domain architecture consisting of two or three N-terminal dsRBDs and a C-terminal catalytic deaminase domain that often has N-terminal ZBDs . Howevercription ,23. Howecription . In manycription . The numcription . As an eo dsRBDs .Pex-ADAR belongs to a class of ADADs comprising a single dsRBD and a deaminase domain. However, the sequence difference in the deaminase core region between ADARs, particularly close to the catalytic site, enables us to assign newly sequenced ADARs to corresponding orthologs [Pex-ADAR with that of other ADAR1 proteins shows the conservation of three active-site motifs and a glutamate (E) residue for catalytic activity. Additionally, in phylogenetic analysis, Pex-ADAR was grouped together with another metazoan ADAR1. These findings strongly support that Pex-ADAR is a member of the ADAR1 family.ADAR family proteins are classified into ADAD, ADAR1, and ADAR2 on the basis of the number of DNA- and RNA-binding domains: ADAD has one RNA-binding domain, ADAR2 has two RNA-binding domains, and ADAR1 has at least one DNA- and one RNA-binding domain ,28. In lrthologs . A compaP. excavatus head regeneration were reported, mainly focusing on nerve regeneration. Its head regeneration primarily accompanies wound healing and blastemal cell proliferation at 1–3 dpa, followed by ventral nerve cord elongation, brain reformation, and segmental ganglion appearance by 10 dpa [Eisenia andrei, blastema forms beneath the wounded dermis at 1–3 dpa, and segmentation occurs within 7 dpa, when redifferentiation is not yet dynamic [Pex-ADAR mRNA indicated that its transcription was inactivated in the early stages (1–7 dpa) when blastemal cells proliferate and the central nerve cord is reconstructed. Expression then suddenly rebounded at subsequent stages (10 dpa) when diverse cell types or tissues were regenerated in each segment. This suggests that ADAR activity is unrelated to the molecular mechanisms of dedifferentiation and is probably implicated in the generation of molecular diversity required for the re-specification of various cell types during redifferentiation [Very recently, the morphological processes involved in y 10 dpa . Earthwo dynamic ,31. The ntiation .Pex-ADAR mRNA expression was mainly observed in gut epithelia, the ventral nerve cord, and the dorsal vessel. Similarly, the ventral nerve cord of C. elegans showed intensive expression of adr-1, whose function is pertinent to normal chemotaxis behavior [Pex-ADAR mRNA expression was mainly observed in the regenerating muscle layers and related connective tissues, suggesting the possibility that the RNA-editing enzyme Pex-ADAR is involved in muscle redifferentiation during earthworm regeneration. During mouse development, ADAR1 shows tissue-specific roles in skeletal myogenesis, and ectopic expression of ADAR1 retards the myotube fusion of myoblasts and muscle development. It is thought that ADAR1 expression displays programmed alteration that is coordinated with differentiation cues [In intact earthworms, behavior . In addibehavior . During ion cues .Pex-ADAR, belonging to the ADAR1 family, is probably linked to the re-specification of muscle cells and tissues. The RNA editing activity of the enzyme may contribute to the generation of molecular diversity, which is required for muscle redifferentiation. Our subsequent research will focus on uncovering the molecular mechanisms behind how Pex-ADAR activity contributes to muscle cell re-specification from blastemal cells.Taken together, earthworm


DNA encodes RNA and is responsible for protein production in cells. RNA editing is the process by which genetic information is altered in the RNA molecule. RNA editing in cancer initiation, progression and development has been well documented and play an important role in tumorigenesis. Studying RNA editing and its application to change genetic information after transcription, RNA-editing technology could be an important innovation in cancer and has the potential for more effective precision treatment. Bioengineering integration approach and artificial intelligence could revolutionize the entire field of RNA editing for early detection of cancer. RNA, a short-intermediate of DNA, is responsible for protein production in cells ,2. RNA eRNA editing involves the insertion, deletion or substitution of nucleotides within parent RNA, and alters RNA sequence without altering the sequence of genomic DNA ,5. UnlikMultiple studies have recently linked RNA editing to cancer development and in metastasis of breast and many other types of cancer. Cancer is the leading cause of deaths worldwide ,7. One sPublicly available, DARNED and RADAR are the two main databases of RNA editing ,9,10. ThRNA editing could be used to unravel dormant cancer stem cells that often escape chemotherapies ,3,5. In We propose that engineered-guided-RNAs that bind ADAR and direct it to fix RNA mutations would bring new opportunities to identify cancer biomarkers at early stage ,5,6. Usi


Escherichia coli, endoribonuclease RNase E initiates degradation of many RNAs and represents a hub for post-transcriptional regulation. The tetrameric adaptor protein RapZ targets the small regulatory RNA GlmZ to degradation by RNase E. RapZ binds GlmZ through a domain located at the carboxyl terminus and interacts with RNase E, promoting GlmZ cleavage in the base-pairing region. When necessary, cleavage of GlmZ is counteracted by the homologous small RNA GlmY, which sequesters RapZ through molecular mimicry. In the current study, we addressed the molecular mechanism employed by RapZ. We show that RapZ mutants impaired in RNA-binding but proficient in binding RNase E are able to stimulate GlmZ cleavage in vivo and in vitro when provided at increased concentrations. In contrast, a truncated RapZ variant retaining RNA-binding activity but incapable of contacting RNase E lacks this activity. In agreement, we find that tetrameric RapZ binds the likewise tetrameric RNase E through direct interaction with its large globular domain within the catalytic amino terminus, independent of RNA. Although RapZ stimulates cleavage of at least one non-cognate RNA by RNase E in vitro, its activity is restricted to GlmZ in vivo as revealed by RNA sequencing, suggesting that certain features within the RNA substrate are also required for cleavage. In conclusion, RapZ boosts RNase E activity through interaction with its catalytic domain, which represents a novel mechanism of RNase E activation. In contrast, RNA-binding has a recruiting role, increasing the likelihood that productive RapZ/GlmZ/RNase E complexes form.In  In diverse bacteria, endoribonuclease RNase E usually initiates decay of bulk RNA , particiNTD; aa 1–529)  is intrinsically disordered and contains binding sites for RNA and proteins as well as a membrane attachment site  , which ca 1–529) . The lara 1–529) . The carent site . RNA helradosome . These aNTD. This allosteric interaction converts RNase E into a closed conformation enabling appropriate orientation of the substrate and boosting enzyme activity  connected by a linker. The tetramer is maintained through homotypic  as well as heterotypic (NTD/CTD) self-interactions. Their disruption abolishes glmS regulation suggesting that tetrameric RapZ and RNase E form an encounter complex to achieve cleavage of the presumably sandwiched sRNA GlmZ. The RNA-binding domain is apparently formed by the 19 carboxy-terminal residues, which are surface exposed and enriched in positively charged amino acids  with RNase E as observed previously . These observations suggest that virtually the complete RapZ sequence is required for interaction and that removal of few residues from either terminus abolishes binding to RNase E.In order to pinpoint a region in RapZ that binds RNase E, we first assessed the interaction properties of the separated globular domains of RapZ using the bacterial adenylate cyclase two-hybrid system (BACTH). BACTH relies on reconstitution of cAMP synthesis through interaction of candidate proteins fused to the separately encoded T25- and T18-fragments of a split adenylate cyclase . β-Galaceviously , whereasl plates B. Analyses (rapZ B. In con1–279) retained interaction with RNase E . We previously showed that RapZ requires oligomerization for activity, that is, substitutions of residues involved in inter-domain interactions concomitantly abrogate regulation of glmS . To dissect the roles of residues 279 and 278 further, each of them was substituted with a glycine in the context of the RapZ1–279 variant. As expected, these substitutions did not affect self-oligomerization confirming that both residues are dispensable for RapZ multimerization . Reporter gene assays addressing the ability to complement an endogenous rapZ deletion showed that the RapZ mutants are largely inactive , which was confirmed by analyzing GlmS protein levels in total protein extracts . These results corroborate that undisturbed interaction of RapZ with RNase E is essential for regulation of GlmS synthesis. However, the identified substitutions scatter across the RapZ sequence and fail to form a defined surface . Interestingly, the Val29 and Leu36 residues hit in the screen were previously shown to contribute to the RapZ NTD/NTD self-interaction , collectively corroborating the idea that loss of interaction with RNase E might be an indirect consequence of a disturbed self-assembly of the various RapZ mutants. We conclude that interaction with RNase E requires undisturbed multimerization of RapZ and that residues from its NTD as well as CTD including Leu279 contribute to RNase E binding.In an attempt to identify additional residues involved in binding RNase E, we randomly mutagenized eraction . Moreove1–597 and RNase E1–762) still comprising the entire catalytic domain . Removal of subdomains from the large domain abolished interaction (We previously demonstrated interaction of RapZ with an RNase E variant (aa 1–597) comprising the amino-terminal catalytic domain . To map c domain E, as experaction E. Thus, NTD were mutagenized. Altogether, 10 RneNTD mutants carrying different single amino acid substitutions and exhibiting diminished interaction with RapZ were isolated . Five of these mutants carried proline substitutions suggesting that structural distortion abrogated interaction. This may also hold true for the G66S and L68F substitutions, which were previously isolated in an independent study and implicated in destabilization of the S1 subdomain, conferring loss of RNase E functionality at high temperature . These residues are part of the 5′-monophosphate sensing pocket and Arg169 directly engages in binding this moiety , leaving open whether RapZ/RNase E interaction is direct or mediated through bound RNA. For clarification, we tested the ability of selected RapZ variants to bind RNase E and sRNA GlmZ by pull-down experiments using a strain lacking endogenous rapZ and carrying a chromosomally encoded RNase E (aa 1–598)-FLAG (Rne1–598) variant. A control experiment shows that Rne1–598 cleaves GlmZ in a RapZ dependent manner in vivo with virtually no difference to full-length RNase E . Accordingly, the scaffolding domain of RNase E and degradosome formation do not play a role in the GlmY/GlmZ/glmS circuit. Among the various truncations, only RapZ1–279 preserved interaction with RNase E , which carries a quadruple substitution in the RNA-binding domain  abolishing RNA-binding activity as demonstrated previously . Nevertheless, this residual RNA-binding activity is not sufficient to bind GlmZ in vivo as indicated by the copurification experiment  only occurs in the presence of both RapZ and RneNTD , and in the case of the uspF mRNA it even appeared to act inhibitory on degradation , 29 genes were identified whose expression was altered in the ΔrapZ mutant . The rapZ transcript was strongly down-regulated confirming its absence in this strain. Accordingly, GlmZ was ninefold up-regulated reflecting block of its degradation and concomitantly glmS was strongly up-regulated as expected. Northern blot analysis confirmed selective up-regulation of glmS in the ΔrapZ mutant . Across strains, 15 additional genes were exclusively affected in the ΔrapZ mutant suggesting that they are regulated by RapZ through the sRNA(s) similar to glmS. In addition to ybhH, opgB, trxB, hcaB, and lysU, this group included several transcripts related to iron uptake . However, entC and cirA mRNAs, which were selected for validation by northern blotting, were only detectable under iron starvation conditions and unaffected by RapZ . Hence, only basal expression levels of iron acquisition genes might be affected, perhaps through pleiotropic effects generated by chronic overproduction of GlmS in the ΔrapZ mutant. Additionally, transcripts for type 1 fimbriae (pili) synthesis (fimAICDFG) were up-regulated in the ΔrapZ mutant and surprisingly even stronger in the ΔglmY ΔglmZ double mutant . However, the fimA promoter is known to undergo spontaneous site-specific inversion (fimA promoter from the “OFF” state as observed in the wild type ancestor strain into the “ON” configuration. This switch occurred either in a minority (ΔrapZ strain) or majority (ΔglmY ΔglmZ strain) of cells, correlating with the mean read numbers obtained by RNA-seq . Consequently, up-regulation of the fimAICDFG operon cannot be attributed to RapZ or the sRNAs. Finally, yeeR and flu were the only transcripts, which changed in the ΔrapZ and the ΔrapZ ΔglmY ΔglmZ mutants, but remained unaffected in the ΔglmY ΔglmZ strain, as expected for transcripts directly regulated by RapZ not involving the sRNAs . This result shows that RapZ does not have a prominent role in turnover of transcripts that are degraded by RNase E beyond GlmZ, at least when produced at physiological levels.Our results have shown that RNA-binding activity is not strictly required for RapZ to stimulate cleavage of GlmZ and the non-cognate in vitro A,B, 6A. nversion . Indeed,leuE and maltose  utilization genes , but northern blot analysis of malQ could not confirm such a change . Moreover, a couple of cold-shock-related transcripts  were up-regulated in the ΔglmY ΔglmZ strain and flagellar-related transcripts  were down-regulated in both the double and triple mutant strain . Northern blot analysis could not detect the fliA transcript (data not shown), but confirmed accumulation of cspB and cspH mRNAs in the ΔglmY ΔglmZ mutant . Thus, it is possible that GlmY/GlmZ play a role in motility as previously suggested  in the two mutants, respectively, exhibiting an overlap of 1021 genes . A severe impact brought about by plasmid constructs, regardless of their genetic content, on host cell expression has also been noted previously . Interestingly, glmS and GlmZ levels were down-regulated regardless whether RapZ or RapZquad was overproduced , confirming our observation that strong overproduction may compensate for loss of RNA-binding activity of RapZquad . To determine whether the 36 transcripts are enriched in RNase E substrates, we compared them with two data sets reporting RNase E target transcripts in E. coli-K12. The first data set comprises transcripts up-regulated in the absence of RNA pyrophosphohydrolase RppH activity, that is, RNAs likely cleaved by RNase E via the 5′-monophosphate dependent pathway . However, there was no prominent overlap between the two latter data sets and the 36 candidate transcripts regulated by RapZ . Taken together, RapZ is not able to stimulate RNase E activity globally through protein–protein interaction, even when overproduced. These results show that stimulation of RNase E activity by RapZ affects only GlmZ but virtually no other RNase E substrate in the cell.Altogether, we retrieved 939 transcripts that changed greater than or equal to twofold in at least one of the three overproducing strains and were not affected by the empty vector D. Among  pathway . The secIn this work, we reveal a novel mode for regulation of RNase E. We demonstrate that activity of the RNase E catalytic domain can be boosted by direct interaction with an accessory protein, RapZ, bypassing the need of an RNA 5′ monophosphate group for allosteric activation of the cleavage reaction. This conclusion is drawn from the characteristics of RapZ variants that are still able to stimulate RNase E activity, albeit their RNA-binding activity is impaired. Nonetheless, RapZ is highly specific for GlmZ in vivo, as revealed by RNA-seq, suggesting that transcripts must possess certain properties to be cleaved by the RapZ/RNase E complex.glmS  or identified residues  previously shown to contribute to RapZ inter-domain contacts , supports a direct interaction of RapZ with the latter domain. However, substitution of key residues Arg169 or Thr170 employed by RNase E for 5′ monophosphate sensing does not compromise RapZ-stimulated cleavage of GlmZ in vitro or interaction with RapZ as measured by BACTH . This observation rules out that RapZ acts by making direct contact to one of these residues. Alternatively, RapZ could stimulate RNase E activity by interaction with an autoinhibitory pocket that is formed by four acidic residues in the large domain , and the changes observed for the pili-related fimAICDFG transcript must be ascribed to spontaneous inversion of the fim promoter . Among the 36 transcripts collectively affected in the various strains overproducing RapZ, only seven were previously annotated as RNase E substrates and also down-regulated as expected for enhanced turnover . Thus, even though a tripartite encounter complex may form, certain features within the transcript are also required for cleavage. This is supported by previous findings that sRNA GlmY, albeit perfectly bound by RapZ, is not cleaved by RNase E  contains many functions related to the cell envelope, some of which are likely transcribed from promoters controlled by RpoE, the sigma factor governing the cell envelope stress response . How caoli K-12 . Hence, NTD, but whether CsrD also binds this domain is unknown. Interestingly, CsrD binds RNA only nonspecifically, emphasizing that specific RNA-binding activity is dispensable for this “adaptor” function as observed for RapZ in the current study. The phage T4 protein Srd accelerates host mRNA degradation by stimulating cleavage activity of RNase E  and expression of mal genes was induced by addition of 0.2% maltose. Established alleles tagged with a resistance marker were moved between strains by general transduction using phage T4GT7 , served as negative control. The positive control was provided by cells carrying plasmids pUT18C-zip and pKT25-zip, which encode the leucine zipper of yeast protein Gcn4 fused to T18 and T25, respectively.The bacterial two-hybrid (BACTH) system allows monitoring of protein–protein interactions in vivo . Candidaβ-Galactosidase activities were determined as described . Unless rapZ DNA fragment obtained by PCR using primers BG168/BG639, was treated with NEBNext dsDNA Fragmentase (NEB) for 10, 15, and 20 min at 37°C according to manufacturer's instructions and reactions were stopped by addition of EDTA to a final concentration of 0.1 mM. Obtained DNA fragments were blunted using the Fast DNA End Repair Kit (Thermo Fisher Scientific) and subsequently ligated with SmaI-digested plasmid pKT25, resulting in three libraries containing arbitrarily truncated rapZ variants fused to the T25 sequence. These libraries were subsequently used to transform strain BTH101 carrying plasmid pYG99 encoding T18-RneFL. Recombinants were screened on X-gal plates and the rapZ inserts of candidate colonies were PCR-amplified using primers BG646/BG647.An amount of 500 ng of a rapZ and rne were randomly mutagenized by error prone PCR (rapZ DNA fragments were inserted between the XbaI/KpnI sites of plasmid pKT25. The mutagenized rne fragments were digested with XbaI and AflII and used to replace the XbaI-AflII fragment comprising aa 1–497 of RneNTD in BACTH plasmid pYG101. The mutant plasmid libraries were used to transform strain BTH101 carrying plasmid pYG99 encoding T18-RneFL (for screening of rapZ mutants) or plasmid pBGG349 encoding T18-RapZ (for screening of RneNTD mutants), respectively. Plasmids were extracted from colonies exhibiting less intense blue coloration on X-Gal plates and used to transform BTH101 once again to ensure persistence and uniformity of the phenotype. Plasmids passing this test were sequenced and subjected to quantitative BACTH measurements.Genes rone PCR  using prNTD were performed as described previously . Eluates derived from StrepTactin affinity chromatography were analyzed by SDS-PAGE/Coomassie blue staining for visualization of the prey proteins and by western blotting for detection of copurifying RneNTD-FLAG. Half of the eluates were used for RNA extraction as described previously and samples of the isolated RNAs  were subjected to northern blotting for detection of GlmZ.Copurification experiments addressing interaction of Strep-tagged RapZ variants with GlmZ and FLAG-tagged Rneeviously . Briefly600 units of cells per sample were separated on 12.5% SDS-PAA gels. For detection of RneNTD-FLAG 8% SDS-PAA gels were used. Proteins were either visualized by Coomassie Brilliant Blue R-250 staining or transferred to a PVDF membrane (Amersham) via semi-dry blotting (Peqlab) for 90–120 min at 120 mA. The membranes were incubated overnight at 4°C with the desired primary antiserum . Signal detection was achieved by incubation with anti-rabbit IgG AP-conjugated secondary antibodies (Promega) diluted 1:100,000 and using CDP* (Roche Diagnostics) as substrate.Usually, total protein extracts corresponding to 0.0625 OD+, GE Healthcare) by electroblotting  using 0.5× TBE as transfer buffer and crosslinked by UV radiation. For detection of longer transcripts, 7.5 µg total RNA was separated by formaldehyde agarose gel electrophoresis  and transferred to Hybond+ nylon membrane via vacuum blotting  at 85 mbar for 4 h. To obtain loading controls, 23S and 16S rRNAs were visualized by ethidium bromide staining before transfer. Probes were synthesized and labeled with digoxigenin (DIG) by in vitro transcription using T7 RNA polymerase, the DIG RNA labeling mix (Roche Diagnostics) and PCR-generated DNA templates. Primers to generate the PCR templates were BG230/BG231 (glmZ), BG260/BG261 (glmY), BG149/BG150 (glmS), BG1915/BG1916 (entC), BG1911/BG1912 (cirA), BG1917/BG1918 , BG1942/BG1943 (cspB), BG1927/BG1928 (cspH), BG1919/BG1920 (fimA), and BG287/BG288 (rrfD). Detection of specific signals was achieved by incubation with anti-DIG AP-conjugated antibodies and CDP* as substrate (Roche Diagnostics).Total RNA was extracted using the ReliaPrep RNA Cell Miniprep System (Promega). Usually, 5 µg total RNA or designated volumes of the RNA solutions derived from ligand fishing experiments  were mixed with 2× RNA loading dye  and separated on denaturing gels  using 0.5× TBE as running buffer. RNA was transferred to a positively charged nylon membrane  was coupled to 12.5 µL magnetic Ni-NTA beads (Qiagen) by incubation in 250 µL interaction buffer  for 1 h at 4°C. Subsequently, the supernatant was removed and 3.5 µg of the candidate protein provided in 250 µL interaction buffer was added. Following an additional incubation at RT for 1.5 h, beads were 4× washed using 500 µL interaction buffer each. For analysis of the bound protein fraction, the beads were dissolved in 50 µL Laemmli buffer and 10 µL were separated by SDS-PAGE followed by Coomassie blue silver staining  in 1× reaction buffer for 1 h at 37°C and subsequently used for in vitro pull-down assay.Purified Hisstaining . For RNA2, and 1 mM DTT) in 10 µL volume, as described previously (32P-UTP labeled GlmZ was mixed with 1 µg yeast tRNA (Ambion), heat-denatured, chilled and incubated for 5 min at 30°C. Subsequently, required amounts of the respective RapZ variant were added and following an additional incubation for 10 min, the assay was started by adding RneNTD . In end point assays, reactions were stopped following the indicated incubation time  by addition of 0.2 U of proteinase K (NEB) and further incubation for 30 min at 50°C. In the case of time course analyses, assay volumes were scaled up accordingly and 10 µL aliquots were removed at indicated times to stop reactions by proteinase K treatment. Finally, samples were mixed with 2× RNA loading dye  and separated on denaturing polyacrylamide gels  using 1× TBE as running buffer. Gels were analyzed by phospho-imaging except for assays using cold GlmZ RNA, which were visualized by ethidium bromide (0.5 mg/mL) staining. Cleavage assays using glyX–glyY RNA were performed at 37°C.RNase E cleavage assays were carried out in 1× reaction buffer (25 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM MgCleviously . α-32P-USupplemental Material.The whole transcriptome analysis is described under Supplemental material is available for this article.


Flavivirus genus and occur both in mature virions and within infected cells. We provide an overview of these sequence motifs and RNA structures involved in circularization, describe their linear and circularized structures, and discuss the proteins that interact with these circular structures and that promote and regulate their formation, aiming to clarify the key features of genome circularization and understand how these affect the flaviviruses life cycle. The circularization of viral genomes fulfills various functions, from evading host defense mechanisms to promoting specific replication and translation patterns supporting viral proliferation. Here, we describe the genomic structures and associated host factors important for flaviviruses genome circularization and summarize their functional roles. Flaviviruses are relatively small, single-stranded, positive-sense RNA viruses with genomes of approximately 11 kb in length. These genomes contain motifs at their 5′ and 3′ ends, as well as in other regions, that are involved in circularization. These motifs are highly conserved throughout the  Flavivirus genus include yellow fever (YFV), tick-borne encephalitis (TBEV), Japanese encephalitis (JEV), Zika (ZIKV), West Nile (WNV), and dengue (DENV) viruses [Aedes spp. female mosquitoes , is estimated to infect approximately 400 million people per year, with some cases progressing to hemorrhagic dengue fever, leading to over 20,000 deaths worldwide every year [Flaviviruses comprise a number of arthropod-borne infections, most of which are prevalent in tropical and subtropical regions around the globe ,2,3,4. P viruses ,6. DENV,ery year ,8. ZIKV,ery year . Both DEery year ,11,12. Tery year ,15,16,17Flaviviruses contain single-stranded (+)-sense RNA genomes approximately 11 kilobases (kb) in length, with a single open reading frame encoding a genomic polyprotein that is post-translationally processed into its constituent parts. These genomes encode three structural proteins at the 5′ end, namely the capsid (C), precursor membrane, and the envelope protein, plus seven nonstructural proteins (detailed ahead). All flavivirus genomes include a short 5′ untranslated region (UTR) of approximately 100 nucleotides (nt) and a variable-length 3′ UTR containing a number of crucial RNA motifs broadly conserved across the family. As shown in The 3′ UTR  is an apCyclization, defined as the process of circularization that occurs in the flavivirus genome, is discussed in the context of this review. Here, both terms (cyclization and circularization) are used interchangeably. The most conserved 3′ UTR structures and sequences reside in a core region where RNA motifs directly participate in flavivirus genome replication. A single copy of the 3′ conserved sequence (CS1 or 3′ CS) is located upstream of the 3′ stem loop (3′ SL) A. CS1′s Flavivirus, as there is high sHP sequence conservation throughout the genus as a whole. Mutational and knockout analysis determined the 3′ SL is a cis-acting regulatory element essential to virus replication and lethal to infectious flavivirus clones if deleted, but it does not influence translation [The 3′ terminal stem-loops (3′ SL) A, 1B wernslation ,26. Likenslation .Tertiary interactions between the 3′ SL and sHP have been proposed for WNV , but mutThe long-range 3′-5′ RNA–RNA base-pairing interaction of complementary cyclization motifs results in formation of the 5′-3′ panhandle structure, which is a signature of circular flavivirus genomes, and it is a required precursor for negative-strand synthesis. The circular structure initiates the replication of negative-sense genomic progeny RNAs via NS5 C-terminal binding at the last two nucleotides of the 3′ SL. The native sequences of these motifs (3′ CYC and 5′ CYC) are critical to flavivirus replication, as alternative base-pair substitutions were found to reduce virus replication efficiency . IntroduThe 5′ variable region (VR) of the flavivirus 3′ UTR contains two independently folded domains, each comprised of several direct sequence repeats. Studies focusing on duplicated RNA structures derived from these repeats support a model in which the presence of redundant elements allows the virus to accommodate mutations beneficial in one host, but deleterious in the other, thereby increasing the robustness of flavivirus genomes . The flaThe dumbbell region (DBR), first identified in the 3′ UTR of DENV genomes, is so named due to one or two tandemly repeated stem-loops with a characteristic dumbbell structure. Initial investigations, spurred by flaviviruses’ curious maintenance of duplicate RNA structures, attempted to explain the biological function of each element. Dumbbell 1 (DB1) and dumbbell 2 (DB2) were initially believed to act primarily as viral replication augmenters . HoweverDespite this information, explicit DBR functions may be challenging to elucidate, as the DB structures, pseudoknots, and RCS2/CS2 sequences all influence genome folding. As discussed elsewhere , more reFlavivirius genus with the ability to calibrate viral RNA accumulation in response to host cell type during infection. Based on local sequence conservation, the VR can be divided into 5′ hypervariable (HVR) and 3′ semi-variable (SVR) regions. In 2007, Tajima et al. reported that deletions occurring within either or both subregions reduced the growth of recombinant DENV-1 virions in mammalian cells, but reverse sequence alterations in the SVR alone were able to influence viral replication in host cells, but not in mosquito cells [Spanning approximately 84 nucleotides, the variable region (VR) is situated immediately 3′ of the stop codon and exhibits comparatively low sequence conservation within the 3′ UTR. VR size varies: similar to DBR, the VR may contain one to three conserved direct repeat sequences. In WNV, these are known as conserved and repeated conserved sequence 3 . Large sequence alignment analysis suggests that short direct VR repeats are remnants of a larger ancient long repeat sequence . VR lengto cells ,49. WherIn addition to genome replication, evidence from studies in mouse models suggests the VR is also a critical flavivirus virulence factor. Using chimeric TBEV constructs derived from strains of disparate pathogenicity, a highly pathogenic Sofjin-HO Far-Eastern subtype VR was transposed with the VR from a low pathogenic strain (Oshima 5–10) . Oshima-VR stem-loops vary considerably both in size and number between flavivirus species; the stem-loops of the WNV VR are the most well-characterized, comprising four stem-loops, designated SL-I through SL-IV. SL-II and SL-IV of WNV may provide resistance to host nuclease activity . SL-II, VR stem-loops formed from direct repeat sequences  garner mutations that can provide host-specific adaptations. One study revealed a hot spot for sequence variations in the 3′ UTR in DENV populations restricted to replicate in either mosquito or human cells . SubsequThe flavivirus genome begins with the approximately 100 nucleotide long, highly conserved, 5′ UTR . In relaThe roughly 70-nucleotide long SLA A, 1B is cis-acting element involved in circularization. The lower stem of SLB is a U-rich region known as the 5′-UAR-flanking stem (UFS). Here, base-pair identity acts to lock 5′UAR/SLB conformation, which is an essential recognition structure for NS5 recruitment and, also, a switching mechanism for viral RNA synthesis [A small stem-loop present in most flavivirus species, known as stem-loop B (SLB) A,B, resiynthesis . These cynthesis . Neutralynthesis ,68. FuncThe cHP is a stable and well-conserved hairpin that follows downstream of SLB and covers the first nucleotides of DENV and WNV capsid coding regions A. Along cis-replicating element in both WNV and DENV genomes, distinguishing it as an RNA domain with multifunctional influence over the flavivirus life cycle [The cHP also acts as fe cycle . Translafe cycle . Thus, nThe occurrence of 5′-3′ circularization through intramolecular duplexes formation is conserved across the flaviviruses family, and it is effected by three complementary regions: (i) the DAR, (ii) the UAR, and (iii) the circularization sequence (CS) ,73. The The UAR is located closer to the 5′ and 3′ ends than the CS. The UAR and DAR are mostly contained in structured elements in the linear form of the genomic RNA, whereas the CS is located in a single-stranded stretch between 3′ DB and sHP. Consequently, these structural elements undergo rearrangement upon circularization. Such a feature has been previously observed in different (+)-sense RNA viruses. Olsthoorn et al. described a conformational change in the 3′ region of plant viruses of the Alfamovirus and Ilarvirus families that is necessary to initiate viral replication . In partCircularization of the genome is essential for its replication ,77,78,84Lott and Doran  suggesteIn addition to 5′-3′ circularization, recent flaviviruses studies revealed internal long-range interactions of functional relevance. These motifs involve the flaviviral genome-coding region and form persistent long-range interactions. Some interactions are exclusive to the packaged state of the genome within virions, whereas others are found in the virion and within infected cells. Chemical cross-linking techniques used to reveal these interactions include COMRADES (cross-linking of matched RNAs and deep sequencing)  and SPLAThe functional role of these internal circularization motifs is particularly interesting as the associated sequence elements are subject to two distinct sources of evolutionary constraint: when sequences that include a coding region change their nucleotides and evolve, the respective translated protein regions must maintain protein function while simultaneously maintaining a functional level of the interactions that are promoted by the nucleotide sequence. In addition, a nucleotide-level constraint exists to maintain RNA complementarity. DENV was particularly suitable to study these interactions, as it has four distinct serotypes that have been surveilled throughout lengthy periods of time and wide geographical areas. This has enabled the gain of a relatively comprehensive collection of viral sequences that lend themselves to statistical analysis for covariation. Covariation in genome sequences allows us to identify evolutionary constrained base pairs in genome structures. The basic approach behind this analysis is to assess complementary mutations for variation at each position. If the maintenance of a base pair is functionally important, then mutation in a specific base will induce a compensatory mutation at the paired base with higher than expected likelihood, which can be statistically evaluated. This statistical evaluation of covariation is implemented in the R-scape tool . The keyPrevious studies of internal circularization motifs have revealed competing structures within the genome of flaviviruses . Only abThe functional relevance of these internal circularizing interactions has been established by mutational studies. These show severe attenuation of viral fitness even if preserving both the protein sequence and avoiding rare codons that might reduce translation efficiency. Moreover, compensatory mutations at partner sites were able to restore viral fitness, hence demonstrating convincingly that the structure at the RNA level is indeed functionally necessary. The precise function of these structural motifs in the coding sequence remains elusive. Several possible explanations for their biological importance exist. One hypothesis is that these structural motifs promote viral packaging into nascent virions by compacting the genome after replication. In a similar vein, these structural motifs, together with other shorter-range structures, may form anchor points for host or viral protein interactions involved in viral replication and/or packaging. Unfortunately, current experimental data are not able to answer whether these motifs are of a dynamic or static nature. Evidence of competing interactions suggest that alternate conformations are possible but, at present, it is unclear whether these interactions will: (i) be formed in a “thermodynamic funnel”, where the genome folds into a local minimum and remains in a specific conformation until disturbed ; or, (ii) whether these motifs are dynamic and can rearrange in response to environmental conditions, possibly performing a regulatory function.Selisko et al., [2+ ions [The importance of the circularization motif for self-primed genome replication has been demonstrated by You et al. ,91. They et al.,  who demo[2+ ions .The C protein plays a key role in the formation of the circular form of the RNA genome. Ivanyi-Nagy et al.  demonstrBidet et al.  providedFlavivirus, which include several human pathogens, is one of the best-described genera exhibiting genome circularization and long-range RNA–RNA interactions . Several sequences have been identified as essential for these processes. It was demonstrated that genome circularization is a regulatory mechanism essential for the synthesis of the negative strand during genome replication. Studies in which long-range RNA–RNA interactions were impaired have shown drastic decreases in viral fitness, suggesting their importance in the assembly and encapsidation (packaging) of the new virions. It is also proposed that these interactions generate secondary and tertiary structures that act as anchor points for viral and/or cell proteins that facilitate virion packaging. Both mechanisms provide ideal conditions for viral life cycle steps. Several host proteins have been identified as essential to the circularization process and necessary for stabilization of the circular form . Viral proteins also participate in circularization, such as the C protein (RNA chaperone), which increases the genome circularization rate.Viruses have evolved several mechanisms in order to increase their protection against host cellular surveillance and attack mechanisms, as well as to manipulate cellular pathways to their own benefit, subsequently increasing their survival and persistence in the host. Genome circularization is one such mechanism that has been reported in a broad range of viruses. The flavivirus capsid protein is proposed to bind the RNA genome and promote its packing within the tight space of the virion. This function would not be possible without a concomitant condensing of the genome via circularization and long-range interactions. Mechanistically, specific loops, hairpins, and tertiary structure elements may coordinate RNA binding to the protein in addition to specific sequence motifs. However, the absence of circularization and long-range interactions would make viral genome packing extremely difficult. This is supported by the observation that such interactions are seen in genomes of other viruses. For example, long-range RNA–RNA interactions have also been reported for coronaviruses, being involved in essential steps of the viral life cycle. It is possible that if flaviviruses evolved to infect a single host, circularization and evolution into a latent virus form might have occurred. However, since flaviviruses infect several hosts, and given that several regions involved in circularization are also involved in virulence and host adaptability, it follows that it is more difficult for a virus that switches between hosts to develop and evolve into a fully circular latent genome form. Guided by these limitations, flaviviruses take advantage of circularization and long-range RNA–RNA interactions. While interesting from an evolutionary perspective, this knowledge is also relevant in the medical context. This is clear evidence that circularization is extremely important to the flavivirus life cycle and is therefore an interesting target that should be thoroughly explored for therapies against flaviviruses.


EIF2S3B, which regulates initiation and maintenance of spermatogenesis in males, is responsible for C/T expression. Because both male and females have tumors that express C/T antigens activation of spermatogenesis genes in tumors must involve a non-sex specific pathway. This can be explained by the copy number of EIF2S3 genes uniquely present within the human genome.Cancer/Testis (C/T) antigens are a group of antigens, expressed in almost all types of cancers, which can elicit an immune response in patients whose cancers express these antigens. They are currently of great interest as targets for the development of cancer biomarkers and the creation of immunotherapies that directly target tumors in patients. Currently there are 280 C/T antigens and their variants listed on the C/T antigen data base. All known C/T antigens are encoded for by genes which are normally only expressed in the male testis; specifically during the process of spermatogenesis. They are therefore only expressed in germ cells that are in the process of differentiating into sperm. Expression of C/T antigens in tumors is thus a biological anomaly as, with the exception of germ cell tumors, cancers arise from somatic tissues which are not known to express any of the genes specifically involved in spermatogenesis. How and why C/T antigens are expressed in tumors remains an enigma. In this paper we present a hypothesis which proposes a mechanism for the activation of C/T antigen encoding genes in tumors. We propose that aberrant activation of the human autosomal retrogene,  There has been an explosion of interest in the use of these antigens as cancer biomarkers and therapeutic targets , increased protein synthesis, avoidance of the immune system, recruitment of blood vessels and an ability to spread beyond the site of origin. Of particular interest is the aberrant expression of genes that are normally associated with the process of spermatogenesis that typically only occurs in the male testis. The expression of spermatogenesis associated genes in a cancer was first noted in 1991 . Current targets –8. In spA direct link between gametogenesis and cancer was first proposed by Old in 2001 . Severalvia expression of spermatogenesis specific genes. This is particularly the case if these antigens are released into the blood stream by the tumor and give rise to the C/T antigens.Testis display a phenomenon known as “immune privilege” whereby spermatogenesis is protected from the immune system. This means that antigens produced by meiotic and haploid germ cells, that are present in the testis of males post puberty, are protected from autoimmune attack even though they are produced long after the formation of systemic self-tolerance. In contrast any spermatogenesis associated proteins that are produced by tumors are not protected from the immune system and, as such, are immunogenic and can be recognized as being outside of systemic self-tolerance which is established during fetal development. Thus cancerous cells, which are derived from somatic tissues, can illicit an immune response to spermatogenesis associated proteins which may be produced EIF2S3Y is primarily responsible for the initiation of spermatogenesis in the testis of juvenile male mammals and its subsequent maintenance in the adult. Most mammals carry two versions of EIF2S3 within their genomes, one on the X chromosome (EIF2S3X) and the other on the Y chromosome (EIF2S3Y). Mouse models have firmly established that the initiation of spermatogenesis in the juvenile testis, and its subsequent maintenance in the adult, is regulated by the Y chromosome linked gene EIF2S3Y (EIF2S3Y from the human Y chromosome (EIF2S3X that is located on human chromosome 12 and known as EIF2S3B (EIF2S3 gene (two on the X chromosome and two on chromosome 12) versus males that carry three copies (one on the X chromosome and two on chromosome 12). This contrasts with the ancestral situation in most mammals where both males and females carry two copies of the EIF2S3 gene, with females having two copies (one on each X chromosome) and males having two copies (one on the X chromosome and another on the Y chromosome). Thus, unlike most other mammals, initiation and maintenance of spermatogenesis in humans is regulated by the activation of an autosomal retrogene (EIF2S3B) that is shared by both males and females. It is this uniquely human situation that could lead to activation of EIF2S3B during oncogenesis and cancer development and provide an explanation for the expression of C/T antigens in the tumors of both human sexes.Recently it has been demonstrated that activation of the gene  EIF2S3Y –17. Thisromosome . Its fun EIF2S3B . This isEIF2S3B, in somatic cells, pre-cancerous cells, or tumors results in the expression of genes normally involved only in spermatogenesis. This in turn results in the production of the C/T antigens by human cancers.It is our hypothesis that activation of the autosomal retrogene, EIF2S3X is an essential housekeeping gene that is ubiquitously expressed in all male and female mammals. EIF2S3 is a subunit of the eukaryotic initiation factor 2 (eIF2) that is involved in the initiation of protein synthesis. When bound to GTP, eIF2 forms a tertiary complex with tRNA Met-tRNAi which subsequently binds to the 40S ribosomal complex. These interactions are rate limiting in eukaryotic protein synthesis. The 40S ribosomal complex binds to the start codon of the protein encoding mRNA to form the 43S pre-initiation complex. Cleavage of the GTP precedes junction with the 60S ribosomal subunit and formation of the 80S ribosome that translates the bound mRNA. Hydrolysis of the GTP results in the release of the eIF2-GDP binary complex, which is recycled. Expression of EIF2S3X in mammalian cells is therefore essential for protein synthesis. EIF2S3Y presumably has a similar function in spermatogonial stem cells. In this case it is responsible for the initiation and maintenance of mRNA translation that is associated with the expression of spermatogenesis-specific genes during male gametogenesis.EIF2S3Y in the initiation of spermatogenesis was first identified, and subsequently explored, in mouse models. In 2001 Mazeyrat et al.  results in the development of testis populated with spermatogonia. In mice carrying the X linked EIF2S3Y transgene (XEO mice), addition of the SRY transgene (XEOSry mice) results in testis development, initiation of spermatogenesis and development of sperm up until the round spermatid stage. In 2016 Yamauchi et al. (SOX9 transgene (XOSox9 mice) that develop testis. When these mice were made transgenic with overexpressing EIF2S3X transgenes  spermatogenesis preceded up to the round spermatid stage. This clearly demonstrated that regulated over expression of the X chromosome linked gene, EIF2S3X, can substitute of EIF2S3Y in the process of initiating spermatogenesis in testis. It also shows that EIF2S3X and EIF2S3Y share functional equivalence with regard to the initiation and regulation of protein synthesis. In addition it has now been definitively established that a high level of EIF2S3 expression is required for the initiation of spermatogenesis in the juvenile testis and its maintenance in the adult male. Depending on the developmental stage EIF2S3Y expression during spermatogenesis is 4–39 times higher than of EIF2S3X in the testis  which in the male is characterized by profound intellectual disability, epilepsy, hypogonadism, hypogenitalism, microcephaly, obesity and growth retardation that may be lethal. Female carriers are unaffected and several different family specific mutations in EIF2S3X have been linked to this disorder . However, activation of EIF2S3B appears to be sample specific so that this cannot be considered a universal characteristic of all tumors. This observation is consistent with the fact that C/T antigens have been documented in many different types of cancers, but not all cancer samples display C/T antigen production. To test our hypothesis there is a need to determine if there is any correlation between EIF2S3B activation and C/T antigen production in individual tumor samples. A systematic approach to this is needed rather that the present random sampling currently available in the literature. It is possible that even transient activation of EIF2S3B could result in the expression of C/T antigens. In addition transfection of somatic cells, such as human fibroblasts could provide insight into how aberrant expression of EIF2S3B in cells modifies their phenotype and directly demonstrate if spermatogenic pathways are activated. Similar experiments involving transfection of fibroblasts with the testis specific human PIWIL2 gene have previously demonstrated transformation into a cancer stem cell like phenotype (Activation of henotype .Identifying the pathways that regulate C/T antigen activation can lead to a better understanding of tumor initiation and development. It could also determine if C/T antigen activation, in the many different tumor types it has been documented in to date, is the result of a shared pathway. This may yield new insights into how subsets of human tumors which express C/T antigens arise and develop.https://asia.ensembl.org/Homo_sapiens/Info/Index.Publicly available datasets were analyzed in this study. This data can be found here: JR and CE contributed equally to the conception of the article. JR wrote and submitted the article. All authors contributed to the article and approved the submitted version.This work was funded by internal funds.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.