KIT proto‐oncogene ligand (KITLG) is a pleiotropic factor which is found in diverse cancers and is involved in cell proliferation, differentiation, and survival. However, the value of KITLG in thymoma remains unclear.A total of 121 thymoma samples from The Cancer Genome Atlas Thymoma (TCGA‐THYM) dataset were used to analyze KITLG related genome‐wide expression profiles, and microRNA profiles and methylation alterations and a GEO dataset‐GSE29695, including 37 samples was used as verification. For cell‐based studies, specific small interfering RNA targeting KITLG or a KITLG overexpression vector were used to clarify the changes of the MAPK pathway in an AB thymoma cell line Thy0517.Both datasets showed that high expression of KITLG was significantly associated with type A and AB thymoma. Through multiomic analysis of the TCGA‐THYM, it was found that with the high expression of KITLG, there were 220 upregulated and 72 downregulated genes at the mRNA level, 79 positive and 78 negative miRNAs, 28 hypermethylation and 163 hypomethylation regions. In the thymoma cell line Thy0517, it was found that the expression of GRB2 and the phosphorylation levels of BRAF, MEK1/2, and ERK1/2 in the MAPK pathway were positively correlated with the change in KITLG.High expression of KITLG is a new hallmark of WHO type A and AB thymomas in which it might play a critical role through the activation of the MAPK signaling pathway. Additionally, it is hoped that KITLG will become a potential target for the diagnosis of type A and AB thymoma through further research in the future.KIT proto‐oncogene ligand (KITLG) is a new hallmark of type A and AB thymomas which induce a series of aberrant alteration of mRNA, miRNA and DNA methylation. The expression of KITLG is significantly higher in type A and AB than other subtypes of thymoma.KITLG activated the MAPK signaling pathway to promote type A and AB thymoma which might be a potential diagnostic biomarker or target. Thymoma is the most common anterior mediastinal tumor with a complex histological classification.KIT proto‐oncogene ligand (KITLG), also known as stem cell factor (SCF), is a ligand of the c‐KIT tyrosine kinase receptor with multiple biological functions.In this study, we used bioinformatics methods to search KITLG‐related genomics and methylation data for thymoma in TCGA and GEO databases. In addition, we used the AB type thymoma cell line Thy0517http://xena.ucsc.edu/public). We performed the analysis of RNA‐seq data of 121 thymoma samples. In addition, another independent cohort (GEO accession number: GSE29695) including 37 patients with thymic neoplasms from Gene Expression Omnibus (GEO) database was used as verification.We obtained RNA‐seq gene expression version 2 (RNA Seq V2) level 3 data  from post‐surgery thymoma tissue samples from thymoma patients of each WHO type from the UCSC XENA website . Upregulated genes were identified separately by applying a Benjamini‐Hochberg (BH) false‐discovery rate (FDR) of P < 0.01 with a log‐fold change (logFC) >1.5. Finally, we obtained the overlapping upregulated genes from both datasets using “Venny 2.1” (https://bioinfogp.cnb.csic.es/tools/venny/index.html) for further analysis. KITLG was selected from 58 overlapping upregulated genes after analyzing the significance of differential genes and the relationship with tumor growth. Additionally, the median value of KITLG expression was used to divide 121 patients into high expression of KITLG (KITLGhigh) and low expression of KITLG (KITLGlow) groups. We first sorted out the expression values of KITLG of 121 thymoma samples in the TCGA‐THYM data set, and then found the median of all these values. There were 61 samples in which expression of KITLG were greater than or equal to the median, classified as the group KITLGhigh, while another 60 samples with KITLG expression less than the median were classified as the group KITLGlow. KITLG‐related genomic, epigenetic and KEGG analyses were based on these two groups from TCGA‐THYM data. All experimental design, quality control, and data normalization were in line with the standard protocols. Finally, we conducted basic investigations into KITLG using the type AB thymoma cell line Thy0517 , as well as 70 B1, B2, B3, and C subtypes (B1 + B2 + B3 + C). The samples from GEO‐GSE29695 were also divided into two groups: 11 A + AB subtypes, as well as 26 B1 + B2 + B3 + C subtypes. First, we carried out an analysis of differentially expressed genes in these two groups of TCGA and GEO using R/Bioconductor package and GEO2R  was established from thymoma type AB patients by tissue explant and cultured by the Department of Cardiothoracic Surgery, Tianjin Medical University General Hospital. We own the patent of this cell line. Cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (Clark Bioscience), 100 U/mL penicillin, 100 μg/mL streptomycin (Sigma‐Aldrich), and 2 μg/mL amphotericin (Sigma‐Aldrich) at 37°C in the presence of 5% COKITLG‐siRNA were purchased from GenePharma . The oligonucleotide sequences were as follows: 5′‐GCGAGAUGGUAGUACA AUUTT‐3′ for the sense strand and 5′‐AAUUGUACUACCAUCUCGCTT‐3′ for the antisense strand. A negative control siRNA was used in parallel. Thy0517 cells were transfected with 25 μM siRNA against KITLG using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. The cells were cultured in Opti‐MEM (Gibco) at 37°C in the presence of 5% CO2 for 6 hours. Total RNA and total protein were prepared 48 hours post‐transfection and used for real‐time PCR and western blot analysis, respectively.pcDNA3.1(+)‐KITLG was obtained from GeneChem . Thy0517 was transfected with pcDNA3.1(+)‐KITLG to induce overexpression of KITLG and with the empty pcDNA3.1(+) vector as a control. Transfections were performed using Lipofectamine 3000 according to the manufacturer's instructions.Total RNA was extracted using TRIzol (TaKaRa) according to the manufacturer's instructions, and reverse‐transcribed using a reverse transcriptases M‐MLV kit (TaKaRa). qPCR was performed in a real‐time PCR system (Applied Biosystems) using TB Green Premix Ex Taq II (TaKaRa). Cycling conditions were 95°C for 2 minutes, followed by 40 cycles at 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 30 seconds. The primer sequences are shown in Table ‐△△Ct method.The reactions were conducted three times, and threshold cycle values were normalized to the expression of GAPDH mRNA. The specificity of the products was determined by melting curve analysis. Relative mRNA expression of the target genes was obtained by normalization to the control group and to GAPDH levels with the 2The harvested cells were suspended in cell lysis buffer (Beyotime). After 12% SDS‐PAGE electrophoresis, the proteins were transferred onto nitrocellulose membranes (Amersham), and blocked with 5% skim milk prior to primary antibody incubation overnight at 4°C. After washing, the membranes were incubated with HRP‐conjugated secondary antibodies  for 1 hour. The proteins were detected using ECL reagent  and images were captured by a chemiluminescent imaging system (Tanon). The list of antibodies are shown in Table t‐test and multiple hypothesis correction were used to identify genome‐wide differences in gene, miRNA, and methylation profiles between the KITLGhigh and KITLGlow groups. All analyses were performed using the R limma software package. qPCR and protein data were depicted as the mean ± SEM. Comparisons between groups were evaluated by one‐way ANOVA test using GraphPad Prism 7.0 software. Statistical significance was considered when P < 0.05.Student's P < 0.001; Fig P < 0.001; Fig Two public microarray datasets were used to compare KITLG expression between A + AB with B1 + B2 + B3 + C thymoma patients. KITLG was highly expressed in type A and AB thymoma  were identified as being significantly associated with KITLG expression . Pathways involved growth, proliferation, and differentiation of thymoma were upregulated, including pathways in cancer and pathway of hematopoietic cell lineage. Other molecule‐related pathways, such as axon guidance and regulation of actin cytoskeleton were also expressed at higher levels .vels Fig c.P < 0.05, Pearson R > 0.5 or < −0.5, Fig miRNA data was excavated from TCGA‐THYM to further explore the correlation with KITLG expression. A total of 79 positive and 78 negative miRNAs were identified . The level of DNMT1, DNMT3A, and DNMT3L appeared lower in the KITLGhigh group (P < 0.001), which was different from other tumors  along with a corresponding decrease in GRB2, which is also a critical upstream protein in the MAPK pathway (P < 0.001), whereas the mRNA levels of BRAF, MEK1/2, and ERK1/2 remained almost unchanged (P > 0.05)  , an NC‐siRNA group, and a KITLG‐siRNA group. Thymoma cells were transfected with NC‐siRNA or KITLG‐siRNA.Then, 48 hours post‐transfection, the mRNA and protein levels of KITLG as well as key factors of the MAPK pathway were analyzed. The results showed that both the mRNA and protein levels of KITLG was significantly decreased compared with the control groups (.05) Fig . However001) Fig b.P < 0.001). As expected, the mRNA and protein levels of GRB2 showed the same trends as KITLG (P < 0.001)    group, and a pcDNA(+)‐KITLG group. Thy0517 cells transfected with empty pcDNA(+) plasmid or pcDNA(+)‐KITLG were cultured for 48 hours following transfection. The levels of KITLG mRNA and protein were remarkably upregulated in the pcDNA3.1(+)‐KITLG group compared with the negative control groups (high group contained significantly more patients with the A and AB subtypes, suggesting KITLG might be another independent biomarker of type A and AB thymoma.Herein, we demonstrate that an important gene, KITLG, is a genomic hallmark of type A and AB thymomas. KITLG interacts with its receptor c‐KIT to perform important roles which has been found in various tumors.high could contribute to the pathogenesis of type A and AB thymoma. DNMT3B, as a DNA methyltransferase, was expressed at notably higher levels in the KITLGhigh group. We also found hypomethylated DMRs were more apparent in the KITLGhigh group compared with hypermethylated DMRs, which might result from the decline of tumor suppressors among the KITLGhigh expressers. This abnormal methylation may provide additional information regarding type A and AB thymoma.In addition, mRNA expression, as well as post‐transcriptional regulation by miRNA and DNA methylation, also have critical roles in thymoma genesis. Thus, we analyzed the effects of KITLG overexpression in thymoma from these three aspects. First, we investigated KITLG‐associated gene expression profiles and related cell signaling pathways. Second, we constructed an interaction network of differentially expressed miRNAs along with high expression of KITLG to reveal the complex associations between them. The ceRNA mechanisms could be predicted based on these miRNAs. Finally, we realized that differentially methylated genes related to KITLGAs previous studies reported, KITLG can result in the activation of several downstream signaling pathways, such as STAT and PI3K/AKT. In detail, overexpressed KITLG is released by keratinocyte‐activated melanocyte via the MAPK pathway.Thymoma has strong association with autoimmune diseases, especially myasthenia gravis. A previous study reported that KITLG as a critical mast cell growth factor, secreted by Th17 cell‐mediated keratinocytes, could stimulate the accumulation and proliferation of mast cells, and mast cells could act as antigen‐presenting cells or effector cells secreting various immune responses to promote immune disorders.With the development of precision medicine, accurate histological diagnosis of thymoma WHO subtypes will depend increasingly on molecular biology. The genomic data of different thymoma subtypes are also essential for drug development for this disease. However, characteristic biomarkers for thymoma are insufficient compared with other tumors because thymoma is a rare group of tumors driven by a limited number of genomic events. Fortunately, several molecules have been found to be closely related to thymoma, and PI3K pathway inhibitors as a novel therapy for relapsed or refractory thymoma have been in phase II clinical trials.The authors declare no potential conflicts of interest.


E.coli by expression vectors, which is a method to obtain genetically engineered RNAs. But the prepared RNAs by this method contain endotoxin, which limits their application in vivo and in cell experments. Here we improved SDS-NaCl filtration method based on SDS-NaCl centrifugation method. Endotoxin removal efficiency of SDS-NaCl filtration was nearly 4.2 times more than did SDS-NaCl centrifugation. Triton X-114 phase separation was used to reduce futher the endotoxin content of SDS-NaCI filtration-extracted RNA (from 11.25 EU/µg RNA/ml to 0.08 EU/µg RNA/ml). RNA prepared using the methods established in this paper meets the requirements for in vivo and cell culture experiments. Here we describe the process of preparing endotoxin-free B1as RNA from pET-B1as-DE3 E. coli (DE3 transformed by pET-B1as expression vector which containing a tandem SINE B1 elements) using SDS-NaCl filtration incorporating Triton X-114 phase separation.•The endotoxin removal efficiency of SDS-NaCl filtration is higher than that of SDS-NaCl centrifugation.•in vivo experiments on animals.RNA prepared by SDS-NaCl filtration incorporating Triton X-114 meets the requirements for RNAs have been elucidated to play the critical role in regulating gene expression and to be expected as effective drugs in the treatment of cancer and age-related diseases. RNAs are extracted by SDS-NaCl centrifugation after transformation of   Specification tableIt is necessary to obtain genetically-engineered RNA without endotoxin contamination for animal experiments. Here, we demonstrate that the SDS-NaCl filtration method incorporating Triton X-114 can be used to obtain endotoxin-free genetically engineered RNA.E. coli. RNAs are extracted by SDS-NaCl centrifugation after transformation of E.coli by expression vectors, which is a method to obtain genetically engineered RNAs. However, RNAs prepared from E. coli using the above method are plagued by endotoxin contamination that, even in small quantities, can result in fever, inflammation, sepsis, tissue damage and even death in vivo, which limits their use in vivoRNAs  have been elucidated to play the critical role in regulating gene expression The endotoxin removal efficiency of SDS-NaCl filtration was nearly 4.2 times more than did SDS-NaCl centrifugation. The supernatant could not be completely clarified after centrifugation when using SDS-NaCl centrifugation method . This SDS-NaCl solution has a high salt concentration (2 mol/L NaCl) and a high specific gravity, so it cannot be completely clarified during centrifugation, especially when it contains lipids in vivo experiments on animals.It has been proved that Triton X-114 phase separation is the effective way of removing endotoxin from recombinant proteins and plasmids Here we describe the process of preparing endotoxin-free B1as RNA from pET-B1as-DE3 bacteria (DE3 transformed by pET-B1as expression vector which contains a tandem SINE B1 elements) by SDS-NaCl filtration incorporating Triton X-114.Material•LB medium (containg 30 µg/ml kanamycin)•Competent DE3 cells•pET-B1 × 16 antisense expression vector •Kanamycin•LB agar plates containing 30 µg/ml kanamycin (the concentration of agar is 1.3%)•d-1-thiogalactopyranoside (IPTG) stock solution (store at −20 °C)20 mg/ml isopropyl β- •500 ml Erlenmeyer flasks•Shaker•High speed centrifuge•50 ml plastic centrifugal tubes with pointed bottom•SpectrophotometerNote: All operations need to be sterile to prevent contamination of miscellaneous bacteria1.Competent DE3 cells (0.1–0.2 ml) are transformed with 1 µl pET-B1as expression vector (0.1 µg/µl), and selected on LB agar plates containing 30 µg/ml kanamycin and cultured overnight. The bacteria transformed with pET-B1as expression vector are named pET-B1as-DE3 bacteria.2.E.coli bacterial colonies. Wash these colonies down with LB medium (containing 30 µg/ml kanamycin).Choose 10–20 correct single 3.Place 200 ml LB medium (containg 30 µg/ml kanamycin) into one 500 ml Erlenmeyer flask and total need 6 Erlenmeyer flasks.4.Divide the washed bacterial suspension evenly among 6 Erlenmeyer flasks.5.600 of 1.0.Shake these flasks at 120 rpm at 37 °C about 3 h until the pET-B1as-DE3 bacteria are cultured to an OD 6.Add 2 ml IPTG stock solution into each Erlenmeyer flask and shake the flasks for another 2 h to produce B1as RNA.7.Centrifuge the cultured bacterial suspensions at 10,000 rpm for 5 min at 4 °C in 50 ml plastic centrifugal tubes with pointed bottom.8.After centrifugation, discard the supernatant and add the remaining cultured bacterial suspensions again until all the cultured bacterial suspensions are centrifuged. This approach typically yielded approximately 0.5 g of wet bacteria per 200 ml of cultured bacterial suspensions.Material•E. coli DE3 transformed by pET-B1as expression vector)pET-B1as-DE3 bacteria •Diethy pyrocarbonate (DEPC)•Absolute ethyl alcohol and 75% ethanol•RNase inhibitor (40 U/µl)•RNase-free DNase I kit )•Slow filter paper (10–15 µm)•Double-distilled H2O (DDW)•50 mg/ml lysozyme (store at −20 °C)•Normal saline (0.9% NaCl)•4 mol/L NaCl•High speed centrifuge•50 ml plastic centrifugal tubes with pointed bottom•SpectrophotometerProcedure1.After the bacterial suspensions are centrifuged, the pellets are made into 50% bacterial suspension (in volume) using normal saline. The specific method is as follow: the pellets in the six 50 ml plastic centrifugal tubes are suspended with 2 ml normal saline, determine the volume, and then add the appropriate amount of normal saline to make the final concentration of wet bacteria is 50%.2.Divide 1 ml 50% bacterial suspension into one 50 ml plastic centrifugal tube, add 20 µl 50 mg/ml lysozyme to one tube and place the tubes on ice for 5 min to get the lysed bacteria.3.Add 9 ml 1.5% SDS solution  to the lysed bacteria.4.Add 6 µl DEPC to prevent RNA degradation at room temperature.5.Add 10 ml 4 mol/L NaCl to each tube and mix it.6.Use slow filter paper to filter the bacterial lysates at 4 °C.7.Use three volumes of absolute ethyl alcohol to precipitate the filtered solution for 30 min at room temperature and then centrifuge for 10 min at 10,000 rpm.Note: Precipitating the filtered solution process should not be carried out at 4 °C, otherwise a large amount of salt precipitation will occur, interfering with the further purification process.8.Use 75% ethanol to wash the precipitate twice.9.Use hot air from a hair dryer to dry the RNA.Note: The drying process cannot be performed via natural evaporation because of each tube having a large amount of precipitation and the long time being required in natural evaporation drying. After the ethanol volatilization during the natural drying process, the water-soluble RNA state will be formed, leading to RNA degradation.10.Prepare 2 ml 0.5 × DNase I digestive solution: DDW 1.775 ml, 0.1 ml 10 × DNase I buffer, 0.1 ml RNase-free DNase I , 25 µl RNase inhibitor 11.Add the above 2 ml 0.5 × DNase I digestive solution into the dried RNA, mix, keep for 30 min at 37 °C, collect the supernatant after centrifugation .12.In addition to RNA, the supernatant (DNase I digested supernatant) contains degraded DNA and residual proteins. All DNase I digested supernatant obtained is treated with SDS hot-phenol method Note: If further Triton X114 removal of endotoxin is required, the following steps are performed: take out 0.1 ml of the supernatant, use SDS hot-phenol method Material•Triton X-114•3 mol/L sodium acetate (pH 6.0)•Absolute ethyl alcohol and 75% ethanol•10% SDS•Water-saturated phenol•Chloroform•Thermostatic metal bath•Shaker•High speed freezing centrifuge1.Add one-tenth volume of 3 mol/L sodium acetate (pH 6.0) into the above 1 mg/ml RNA solution.2.Add one-thirtieth volume of Triton X-114 and mix.3.Incubate and shake the mixture for 20 min at room temperature.4.Move to a metal bath at 50 °C for 10 min.5.Keep at room temperature for 5 min.6.Centrifuge the mixture at 10,000 rpm 10 min at 23 °C using high speed freezing centrifuge.7.Collect the supernatant, remove the residual proteins using SDS hot-phenol methods 8.Collect the supernatant, precipitate the supernatant with three volumes of absolute ethyl alcohol for 30 min at 4 °C9.Centrifuge for 10 min at 10,000 rpm and wash the precipitate twice using 75% ethanol.10.Dry the precipitate using hot air from a hair dryer to get endotoxin-free RNA.E.coil transformed by expression vectors and effectively remove endotoxin at the same time. SDS-NaCl filtration was performed to extract RNA from pET-B1as-DE3 bacteria and endotoxin content of RNA was 11.25 EU/1 µg RNA/ml. The OD260 / OD280 ratio of the RNAs extracted by SDS-NaCl filtration was approximately 2.0, which suggests that the method is capable of producing RNA with sufficient purity and implies that contamination by DNA and protein can be ignored.The novel combination of SDS-NaCl filtration and Triton X-114 phase separation can extract engineered RNA from SDS-NaCl filtration is the effective way of removing endotoxin. However, RNA extracted by SDS-NaCl filtration still contains some endotoxin (11.25 EU/ 1 µg RNA/ml). We next sought to test whether Triton X-114 could be incorporated into the SDS-NaCl filtration protocol to remove residual endotoxin and found that Triton X-114 dramatically reduced the endotoxin content of RNA samples (from 11.25 EU/1 µg RNA/ ml to 0.08 EU/1 µg RNA/ml) extracted by SDS-NaCl filtration.in vivo experiments.RNA extracted by SDS-NaCl filtration with Triton X-114 phase separation was injected intravenously into three New Zealand rabbits (50 µg RNA/kg). The total increased body temperature of three rabbits was 0.09 °C. Based on the injected dose of RNA, this increase suggests that the endotoxin content of the RNA was lower than 1 EU/50 µg RNA. BALB/c mice injected intravenously with RNA extracted by SDS-NaCI filtration with Triton X-114 had no any adverse effect. Therefore, this new protocol-extraction of genetically engineered RNA by SDS-NaCl filtration combined with Triton X-114-meets the requirements for The effects of Triton X-114 treatment on RNA integrity and B1as RNA yield were examined using northern blotting. Total RNA was treated without or with Triton X-114, electrophoresed and transferred into nylon membrane. The ratio of 23S to 16S without A, lane 1The authors declare that they have no conflicts of interest related to this work.


Since the end of 2019, a novel coronavirus disease 2019 (COVID-19) caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been developing in Wuhan, Hubei Province, China , and health emergencies. School closures were among the first measures which had been adopted. According to UNESCO monitoring, over 160 countries have implemented nationwide closures, impacting over 87% of world's student population. Together these hard interventions seem to give encouraging results : reducinSP drafted and wrote the paper. GS and FL contributed to the discussion. GP reviewed and approved the paper.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.


Although the current study is just the tip of an iceberg, these new evidences will provide new insights for possible GBM treatments. In this review, we summarize the recent studies about RNA modifications, such as N6-methyladenosine (m6A), N6,2′O-dimethyladenosine (m6Am), 5-methylcytosine (m5C), N1-methyladenosine (m1A), inosine (I) and pseudouridine (ψ) as well as the corresponding RMPs including the writers, erasers and readers that participate in the tumorigenesis and development of GBM, so as to provide some clues for GBM treatment.Glioblastoma (GBM) is a grade IV glioma that is the most malignant brain tumor type. Currently, there are no effective and sufficient therapeutic strategies for its treatment because its pathological mechanism is not fully characterized. With the fast development of the Next Generation Sequencing (NGS) technology, more than 170 kinds of covalent ribonucleic acid (RNA) modifications are found to be extensively present in almost all living organisms and all kinds of RNAs, including ribosomal RNAs (rRNAs), transfer RNAs (tRNAs) and messenger RNAs (mRNAs). RNA modifications are also emerging as important modulators in the regulation of biological processes and pathological progression, and study of the epi-transcriptome has been a new area for researchers to explore their connections with the initiation and progression of cancers. Recently, RNA modifications, especially m Glioblastoma (GBM), a World Health Organization (WHO) grade IV glioma, is the most prevalent, malignant and lethal intrinsic tumor in the central nervous system . It accoTo know how GBM initiates and develops, the molecular characteristics of GBM should be accurately determined. Actually, many genetic, epigenetic, metabolic and immunologic profiles in GBM have been described in recent years ,6,7,8,9.6-methyladenosine (m6A) modification, are shown to be essential for tumor development [Recently, RNA modifications, as known as RNA epigenetics, were found to be present in almost all the cellular RNAs in archaeobacteria, bacteria, prototists, plants, fungi, animals and viruses. Even in mitochondria, there are also some chemical modifications in mitochondrial DNA-encoded RNAs . More thelopment ,23,24,256A writers, erasers and readers, and their molecular mechanisms of action in GBM, so as to provide some clues for the development of new strategies for GBM treatment.In this review, we summarize the most recent advances in RNA modifications, especially the functions of m7G  and 3′ poly(A) tail are well-known modifications in mRNA [RNA modifications refer to dynamic, reversible covalent alterations present in the oligonucleotides of RNAs, including mRNAs, tRNAs, rRNAs and spliceosomal RNAs (sRNAs). These changes are commonly present in the transcriptome. Since 1957, when pseudouridine  was discovered by Davis and Allen , at leas in mRNA . In 2012 in mRNA .6-methyladenosine (m6A), N6, 2′O-dimethyladenosine (m6Am), 5-methylcytosine (m5C), 5-hydroxymethylcytosine (hm5C), N1-methyladenosine (m1A), inosine (I), N4-acetylcytidine (ac4C), ψ, uridylation (U-tail), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2i6A), 3-methylcytosine (m3C), m7G, 7-methylguanosine cap (m7Gpp(pN)), queuosine (Q), wybutosine and derivatives (yW), 5-methyluridine (m5U), 5-carbamoylmethyluridine (ncm5U), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxy-carbonylmethyl-2-thiouridine (mcm5s2U), dihydrouridine (D), 2′-O-methylnucleotide (Nm), N6-isopentenyladenosine (i6A), and 5′-phosphate monomethylation (m(pN)) [In eukaryotic RNA, the most commonly found modifications are N (m(pN)) ,29,30. M (m(pN)) . rRNAs a (m(pN)) , thereby (m(pN)) .Each modification has its writers, the enzymes that deposit RNA chemical marks; erasers, the enzymes that remove these marks; and readers, the proteins that selectively recognize and bind to these marks . Collect6A is the best-known RNA mark, it is highly connected with GBM progression and aggressiveness and some regulators may be potential drug targets. Recently, other kinds of RNA marks, including m6Am, m1A, m5C, hm5C, I and ψ, as well as their modulators are also emerging to be correlated with GBM progression  [6A modification of mRNA usually occurs in nuclear speckles where the methyltransferases and demethylases are concentrated [6A-METTL associated complex (MACOM), that can mediates the m6A methylation. Meanwhile, two demethylases, Fat mass and obesity-associated protein (FTO) and ALKBH5, catalyze the m6A demethylation [RNA m(snRNAs) . m6A modentrated  and are entrated . Generalhylation ,40,41,426A-binding proteins such as YTH N6-methyladenosine RNA binding protein 1/2/3 (YTHDF1/2/3) and YTH domain-containing 1/2/3 (YTHDC1/2/3), which have the YTH domain, are readers that mediate the degradation of m6A-labelled RNAs [6A marks, too [6A reader proteins can recognize these marks and also can play key roles in the RNA stabilization of the writers [6A marks recognized by different readers with different sub-locations may exert different functions: the readers in the nucleus, such as hnRNPC, hnRNPA2B1 and YTHDC1, are responsible for RNA structure switching, alternative splicing, microRNA maturation, RNA stability, RNA export and X chromosome inactivation; while readers in the cytoplasm, such as YTHDF1/2/3 and YTHDC2, are mainly responsible for mRNA translation and decay [Several mled RNAs ,44. Otherks, too . These m writers . m6A marnd decay .6A affects RNA stability, processing, splicing, translation, and the epigenetic function of some non-coding RNAs (ncRNAs) [6A also emerges as an important modulator. For instance, METTL3 drives tumorigenicity and metastasis through suppressing suppressor of cytokine signaling 2 (SOCS2) expression in hepatocellular carcinoma [As RNA m(ncRNAs) ,48, it p(ncRNAs) ,52,53,54arcinoma .6A methylomes of brain tissues are highly specific [6A may be different in GBM. A bioinformatic analysis in The Chinese Glioma Genome Atlas (CGGA) microarray and RNA sequencing databases [6A writers, including METTL3, METTL14, WTAP, RBM15 and RBM15B, as well as m6A readers including YTHDC2, YTHDF1, YTHDF2, YTHDF3, hnRNPA2B1 and hnRNPC are elevated, while m6A writer ZC3H13 and m6A eraser FTO are decreased in gliomas. In GBMs, m6A writers WTAP and RBM15, m6A eraser ALKBH5, and m6A reader YTHDF2 are significantly elevated, while m6A writers METTL3, VIRMA and ZC3H13, m6A eraser FTO, m6A readers YTHDC2 and hnRNPC are significantly decreased, compared with low-grade gliomas (LGGs). In addition, four lncRNAs including MIR9-3HG, LINC00900, MIR155HG, and LINC00515 seem to be related with m6A. Another bioinformatic analysis [6A RNA methylation regulators are tightly correlated with pro-oncogenic genes in gliomas. These evidences imply that m6A modifications are tightly correlated with GBM progression. According to present studies in GBM, m6A modification seems to play pivotal roles since both m6A writers and erasers contribute to the tumorigenesis of GBM  and total RNA-Sequencing (RNA-seq) of METTL3-silenced GSCs revealed that METTL3 is the most important writer responsible for m6A modification in GSCs [6A modification in some GSCs-specifically-expressed genes [6A modification of mRNA of SRY-box transcription factor 2 (SOX2), a pluripotent gene, at three METTL3/m6A sites present in its 3′ untranslated region (UTR), leading to the recruitment of human antigen R (HuR) to SOX2 mRNA to enhance its stability, thereby resulting in GSC maintenance and dedifferentiation [The expression of METTL3, the most important mntiation . Elevatentiation . An inte in GSCs . Moreove in GSCs ,57. Furt in GSCs . Mechanied genes . Particuntiation .6A-specific methylated RNA immunoprecipitation with NGS (MeRIP-Seq or m6A-Seq) analysis also shows that m6A modification peaks seem to be enriched at transcripts responsible for metabolic pathways in GSCs, compared with neural progenitor cells [6A modification also affects the levels of serine- and arginine-rich splicing factors (SRSFs) through upregulating BCL-X  or nuclear receptor corepressor 2 (NCOR2) isoforms, which are important to prevent YTHDC1-dependent nonsense-mediated mRNA decay (NMD) [6A marks is also highly expressed in GSCs, compared with that of protein-coding genes [6A marks within 3′UTRs seem to hinder the miRNA binding in GSCs [In addition to directly affecting the pluripotent genes, an mor cells . METTL3 or cells . Furtheror cells . Additioay (NMD) . lncRNAsng genes . m6A mar in GSCs .6A sequencing and molecular experiments implies that METTL3 or METTL14 knockdown induced alterations in mRNA m6A enrichment and the mRNA expression of critical factors, such as a disintegrin and metallopeptidase domain 19 (ADAM19), that participate in modulation of GBM stem cells. In contrast, overexpression of METTL3 renders GBM stem cells growth and self-renewal. Another study in established GBM cell line U251 cells also shows the similar results: METTL3 overexpression reduces migration and proliferation ability and induces cellular apoptosis possible by downregulating heat shock protein 70 (HSP70) expression [6A modifications regulated by METTL3 in GBM should indeed be further confirmed.Nevertheless, an opposite study shows that METTL3 and METTL14 may be a tumor suppressor in GBM . Their rpression . The con6A methyltransferase complex. It is also highly expressed in glioma, closely correlated with pathological grade and negatively correlated with postoperative survival in glioma patients [WTAP is also an important part of the mpatients ,62. WTAPpatients . In GSCspatients .ZC3H13, as a zinc-finger protein, is also an essential regulator in the MACOM complex, which can anchor WTAP, Virilizer, and Hakai in the nucleus . A recen6A writers are important for GBM progression. Most of them are upregulated in GBM, and show oncogenic roles by regulating specific signaling pathways, especially factors that contribute to stemness maintenance. However, some opposite results show that the expression alterations of some writers in GBM are downregulated and they may pose anti-cancer properties, indicating that much more work should be done to confirm their expressions and functions in GBM.Collectively, m6A erasers also play essential roles in GBM. Inhibition of FTO, an m6A eraser, also impedes the self-renewal ability and tumorigenecity of GBM stem cells both in vitro and in mice models [6A modification in cells is important and FTO may be a promising drug target of GBM.Like the writers, me models . Other se models . Besidese models . These e6A demethylase, ALKBH5, is also highly expressed in patient-derived GSCs. Silencing ALKBH5 inhibits cell proliferation of GSCs. An integrated transcriptome and m6A-seq analysis identified several ALKBH5 target genes, including forkhead box M1 (FOXM1), a transcription factor that is critical for GSCs. ALKBH5 demethylates FOXM1 nascent transcripts, thereby enhancing its expression. A lncRNA forkhead box M1-antisense RNA (FOXM1-AS), also enhances the interaction of ALKBH5 with FOXM1 nascent transcripts [6A erasers may also play pro-oncogenic roles in GBM and may be potential drug targets for GBM treatment. If both m6A writers and erasers are confirmed to be upregulated in GBM, it is probable that the modulatory capacity of m6A in GBM is enhanced, indicating that GBM may be more flexible to response to complex extracellular or intracellular stresses.Another mnscripts . These r6A-modified RNA is determined by m6A readers, so they may be more important factors for cancer treatment, because when functions of m6A writers and erasers are settled, the functions of readers depend on what kinds of readers to carry out the effects of m6A marks. Importantly, the aberrant expression of m6A readers may contribute to GBM tumorigenesis independently of m6A alterations to some extent. This means that even in the condition that m6A is not changed, the effect of m6A may be altered by the abnormal expression of m6A readers. Based on current study, several families that exert as m6A readers are tightly related to the tumorigenesis of GBM.The fate of m6A readers that contain an YTH domain that can bind to RNAs. They exhibit different functions: YTHDC1 mediates mRNA splicing; YTHDF2, YTHDF3, and YTHDC2 mediate mRNA decay; YTHDF1, YTHDF3, and YTHDC2 mediate translation; and YTHDC2 mediates RNA structures [6A marks dependent on its tryptophan 377 (W377) or W428 sites [6A binding activity [YTHDF and YTHDC families are the most important mructures . YTHDC1 28 sites . Recentlactivity .6A reader protein family, the IGF2BP family, including IGF2BP1/2/3, inhibits the degradation of m6A-modified transcripts and facilitates their translation [6A readers and oncogenic roles in GBM. For example, IGF2BP1 promotes cell proliferation and invasion in GBM cells through stabilizing the mRNA transcripts of its target genes, including c-Myc, marker of proliferation Ki-67 (MKI67), phosphatase and tensin homolog (PTEN) and CD44 [6A reading remains unknown.Another mnslation . For a land CD44 ,67. HoweIGF2BP2 is also upregulated in GBM tissues. It supports GBM cell proliferation, migration, invasion and epithelial-to-mesenchymal transition (EMT) via regulating the activity of insulin-like growth factor 2 (IGF2), which further activates phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Additionally, inhibition of IGF2BP2 sensitizes GBM to TMZ treatment . AnotherIGF2BP3 mRNA and protein levels are also upregulated in GBMs but not in lower grade astrocytomas . Particu6A readers, and can directly, physically and functionally interact with METTL3, thereby enhancing translation, and the formation of densely packed polyribosomes through recognizing m6A modification at the 5′ UTR of mRNA [eIF3a/b/h, also function as m of mRNA ,103,104. of mRNA . eIF3b s of mRNA . These e6A readers that can bind to unfolded RNA through an m6A structural switch mechanism, since the m6A marked RNA cannot efficiently form secondary structures because the base pairing of m6A-U is weaker of than that of A-U [During nuclear RNA processing period, hnRNPC and hnRNPA2B1 are other types of mt of A-U ,106. A st of A-U , suggestActually, hnRNPC is one of the important physiological and cancer-related regulators (including GBM) of 3′ UTR processing and miRNA maturation . hnRNPC 6A readers that contribute to GBM pathogenesis.hnRNPA2B1 is also an important modulator of pre-mRNA processing, mRNA metabolism and transportation in cells. hnRNPA2B1 protein is highly expressed in glioma tissues, correlated with glioma grades and related to poor prognosis . Mechani6A RNA modification. Whether other readers’ functions are m6A-dependent remains unknown. However, most of their functions are dependent on their RNA binding capacity, implying that m6A in RNAs may be involved in their binding and their functions in GBM. This hypothesis needs to be further confirmed. As an important mark, m6A may have more readers than we can imagine. These readers are like messengers of m6A marks but beyond that, because they not only deliver message of m6A modified mRNA, but also determine their fate.Collectively, from the current study, YTHDC1 is the only confirmed m6A reader that exerts its function in GBM dependently on its biological function in m6,2′-O-dimethyladenosine (m6Am) is a terminal modification at mRNA cap  cap) in higher eukaryotic mRNAs and ncRNAs, expecially snRNAs, as well as viral RNAs [6Am plays essential roles in RNA splicing [6Am writers, the m6A eraser FTO also functions as an m6Am eraser, and mRNA-decamping enzyme 2 (DCP2) may be one of the m6Am readers [Nral RNAs . m6Am plsplicing , snRNA bsplicing , mRNA stsplicing  and cap-splicing . In huma readers ,114,115.6Am demethylation is an important regulatory mechanism in adjusting the stem-like properties of colorectal cancer cells [6Am also emerges as an essential regulator in GBM (6Am part). For instance, an integrated proteogenomic analysis in 17 GBM patient samples reveals that METTL4 is one of the top-ranked missense mutation genes [6Am probably participates in the development of GBM.Recent evidence shows that FTO-mediated mer cells , and manon genes . DCP2 ison genes . DCP2 ison genes . These r5C) in DNA has been well described, however, it is not well understood in RNAs. Actually, m5C is not only confined within tRNAs and rRNAs, but also present in mRNA and ncRNAs [5C usually responsible for stabilization of tRNA molecules via affecting Mg2+ binding, regulating mRNA translation, preventing recognition of endogenous RNAs by the innate immune system, and modulating RNA-dependent inheritance of certain phenotypes [5C writers, while ALY/REF export factor (ALYREF) is an m5C reader [5-Methylcytosine H quinone dehydrogenase 1 (NQO1) [5C reader, ALYREF, may be an overexpressed antigen in GBM, according to a study with a nanobody-based anti-proteome approach [5C and its RMPs may also play important roles during the tumorigenesis of GBM (5C part), indicating they are promising pharmaceutical targets for GBM treatment.As a target of proto-oncogene c-Myc , NSUN2 h1 (NQO1) ,82. The approach  and is uapproach , implyin5C are subject to oxidative processing by the ten-eleven translocation (TET) family (TET1/2/3), forming 5-hydroxymethylcytidine (hm5C) and 5-formyl-cytidine (f5C), which are considered as mediating substances during the demethylation pathway [5C oxidation is a conserved process that could have critical regulatory functions inside cells [5C is distributed at lncRNA loci, involved in long-range chromatin interactions and positively correlated with lncRNA transcription [5C modification in RNA has never been explored in GBM (5C part).In mammals, both DNA and RNA m pathway . Howeverde cells . For exacription . TET1 iscription , while Tcription , and TETcription . 5-hydrocription ,132,133,1-methyladenosine (m1A) is a reversible modification in tRNA, mRNA and lncRNAs, even in mitochondrial transcripts, and is critical for tRNA stability, and translation of mitochondrial DNA-encoded proteins [1A modifications in pre-tRNA and pre-mRNA are catalyzed by tRNA methyltransferase 6/61A (TRMT6-TRMT61A) catalytic complex and are erased by ALKBH1 and ALKBH3, respectively [1A methylation in mt-tRNA and mt-mRNA are installed by tRNA methyltransferase 61 homolog B (TRMT61B) and tRNA methyltransferase 10C, mitochondrial RNase P subunit (TRMT10C) [1A, both writers and erasers, seem to involve in the progression of GBM (1A part). TRMT6 and TRMT61A expression is significantly upregulated in highly aggressive GBM compared with grade II/III gliomas [Nproteins . In nuclectively ,136. WheTRMT10C) . Recentl gliomas . TRMT61A gliomas . The era gliomas .A-to-I RNA editing is abundant in the human transcriptome and plays essential roles in RNA processing, such as post-transcriptionally altering codons, introducing or removing splice sites, and affecting the base pairing of the RNA molecule with itself or with other RNAs . All 3 ePseudouridine (ψ), the most abundant RNA modification, is occurs in almost all kinds of RNAs, including tRNA, mRNA, snRNA, nucleolar RNA (snoRNA), and rRNA ,142. Ψ iAlthough most of the other RNA modifications are not well described, there are also some studies regarding the effect of these RNA modifications on GBM progression. For instance, ADAR2 and ADAR3 binds to the pre-mRNA of glutamate receptor subunit B, glutamate receptor ionotropic AMPA (GRIA2), contributing to a RNA editing at the Q/R site by modifying a codon replacing the glutamine (Q) with arginine (R). ADAR2/3 deficiency in GBM leads to increased unedited GRIA2 subunits, thereby leads to a calcium-permeable glutamate receptor, which can promote cell migration and tumor invasion . CleavagThe descriptions above indicate that RNA modifications are important for tumorigenesis and aggressiveness in GBM. Chemical modifications in both DNA and proteins have been well studied in cancer biology, and many chemical modifications such as DNA methylation, histone modification and EGFR post-translational modifications, as well as some of their modification enzymes have been shown to have clinical significance in both the diagnosis and therapy ,154,155.6-methylguanine–DNA methyltransferase (MGMT) significantly affects the TMZ treatment of GBM [Epigenetic alterations are considered as promising markers for GBM diagnosis ,161,162,t of GBM . Therefot of GBM .6A in in peripheral blood RNA has shown to be a predictive biomarker for gastric cancer [6A erasers ALKBH5 and FTO are also shown to be prognostic biomarkers in patients with clear cell renal carcinoma [As a kind of chemical modifications in RNA, the alterations of their regulations may also predict the prognosis or be used for diagnosis of GBM. Actually, mc cancer . Methylac cancer . The m6Aarcinoma . From TaCovalent modifications in both DNA, histones and other proteins have shown promising probability for cancer treatment. Actually, some epigenetic drugs such as azacitidine, 5-aza-2′-deoxycytidine, suberoylanilide hydroxamic acid (SAHA), romidepsin, belinostat, panobinostat, has been approved by the United States Food and Drug Administration (FDA) and chidamide has been approved by China FDA to treat some T-cell lymphoma or multiple myeloma, and many other epigenetic drugs on cancers are ongoing clinical trials . BesidesAs kinds of covalent modifications in RNA, their potentials in cancers like malignant GBM are being explored. Since more than 170 kinds of modifications, it is a wider world for us to explore. Specific antagonist against writers, erasers and readers of RNA modifications needs to be screened, designed and identified, as well as their biological functions and clinical applications in cancer treatment also need to be extensively studied. In fact, several pharmaceutical companies are planning to develop small-molecule inhibitors of the METTL3–METTL14 complex and ADAR . RecentlFrom the collective evidence, we can conclude that RNA modifications play increasingly important roles in the tumorigenesis and development of GBM. Some of the writers, erasers and even readers of RNA modifications are potential biomarkers for the diagnosis and drug targets for treatment of GBM . DespiteNevertheless, most of these studies show that no direct connections of their pro-oncogenic or anti-oncogenic roles are dependent on their capacities of RNA modifications. That is to say that the functions of these modulators in GBM may be not dependent on its function in RNA modifications. Therefore, the RNA modifications and their detailed targets regulated by RMPs should be comprehensively explored in the future. To explore these, much fundamental work should be done before a better understanding of RNA modifications in cancers.6A can be detected by fast-developing antibody-based NGS through a mechanism of capturing and sequencing of immunoprecipitated RNA targets [1A to m6A [The first task is to identify the physiological and biological function of more than 170 kinds of RNA modifications and their RMPs. However, it is a very challenging and difficult work to decode the complex layer of the epitranscriptome. Although some modifications such as m targets ,174,175,A to m6A . TherefoAfter solving these problems, exploring the connections of RNA modifications with GBM pathogenesis, prognosis and diagnosis, digging the mechanisms underlying their functions, designing of specific small molecules target RNA modifications, and detecting these targeted drugs penetrating the blood-brain barrier for GBM treatment can be possible. The results of basic research about RNA modifications in GBM will also soon be tested in clinical trials and be applied in the clinic.


Mycobacterium smegmatis, designated MazEF-ms. We first enlisted a specialized RNA-seq method, 5’ RNA-seq, to identify the primary RNA target(s) of the MazF-ms toxin. Just two tRNA species, tRNALys-UUU and tRNALys-CUU, were targeted for cleavage by MazF-ms at a single site within their anticodon sequence (UU↓U and CU↓U) to render these tRNAs nonfunctional for protein synthesis. The 5’ RNA-seq dataset also revealed hallmarks of ribosome stalling predominantly at Lys AAA codons even though both Lys tRNAs were cleaved by MazF-ms. Stalled ribosomes were then cleaved on their 5’ side by one or more RNases, resulting in very selective degradation of only those mRNAs harboring ribosomes stalled at Lys codons. This highly surgical, codon-dependent degradation of mRNA transcripts was validated using quantitative mass spectrometry of proteins that were newly synthesized during MazF-ms expression. The M. smegmatis proteome was altered as predicted, Lys AAA codon-rich proteins was downregulated while Lys AAA codon deficient proteins were upregulated. Analysis of specific subsets of proteins that were upregulated or downregulated was consistent with the growth-arrested phenotype of MazF-ms expressing cells. Curiously, the tRNA target and mechanism of action of MazF-ms paralleled that of one atypical MazF toxin in M. tuberculosis, suggesting manipulation of the levels of lysine tRNAs as the preferred conduit for reprogramming the proteomes via ribosome stalling at rare AAA codons in these GC-rich mycobacteria.Survival of mycobacteria, both free-living and host-dependent pathogenic species, is dependent on their ability to evade being killed by the stresses they routinely encounter. Toxin-antitoxin (TA) systems are unique to bacteria and archaea and are thought to function as stress survival proteins. Here, we study the activity of the endoribonuclease toxin derived from the MazEF TA system in  Escherichia coli TA systems  systems are believed to act as one line of defense enlisted by free-living bacteria to survive the constant barrage of environmental assaults in their native habitats . We found that the primary RNA targeted for cleavage by this endoribonuclease toxin was tRNALysUUU, identical to one of the 11 MazF toxins in M. tuberculosis, MazF-mt9 (M. tuberculosis (megmatis  vs. ~90 rculosis ]. In thiMazF-mt9 . We thenrculosis . This stMycobacterium smegmatis strain mc2 155 (ATCC 700084). M. smegmatis cells were grown at 37°C in Difco Middlebrook 7H9 media (BD) supplemented with 5 g/L albumin, 2 g/L dextrose, 0.085 g/L NaCl, 0.05% Tween 80 and 25 µg/ml kanamycin (for plasmid selection), under constant shaking at 200 rpm.All experiments were performed using M. smegmatis genomic DNA using the oligos NWO2791 (5’-AGA TAC ATA TGC GGC GCG GCG ATA TCT ACA CCG CGG-3’) and NWO2792 (5’-AGA TAA AGC TTC ACC CGG CGA TTC CCA GAA AAA CC-3’). The amplified DNA was cloned into the anhydrotetracycline (ATC)-inducible plasmid pMC1s  and transferred to 2 ml lysing kit tubes (Bertin Corp.) containing 0.1 mm glass beads. Cells lysis was performed on a Precellys Evolution homogenizer (Bertin Corp.) by three consecutive 30-s pulses at 9,000 rpm, with 1 min cooling periods on ice in between each cycle. The samples were centrifuged for 5 min at 14,000 rpm at 4°C, and RNA was isolated from the supernatant using the Direct-zol RNA Miniprep Plus extraction kit (Zymo Research). After isolation, the samples were treated with 1 U of Turbo DNase as an extra genomic DNA removal step, purified using the RNA Clean and Concentrator kit (Zymo Research) and eluted in 40 μl of RNase-free water. RNA concentration was measured in a BioSpectrometer (Eppendorf) with a µCuvette.In order to extract total RNA, ~50 ml of 5’ OH libraries were constructed as previously described . BrieflyNote that our 5’ RNA-seq method specifically selects for RNA molecules with 5’ hydroxyl ends created upon cleavage by MazF-ms. Based on the detailed schematic of our method in M. smegmatis genome (NCBI accession: CP000480.1) using bowtie 1.2 applying the parameters –n 0–l20  and uninduced samples. In order to label newly synthesized proteins, the methionine analog azidohomoalanine  was added to the media at 50 µM after 4.5 h of MazF-ms induction. After 1.5 h of incubation with AHA, the cells were pelleted by centrifugation at 2000 g at 4°C for 10 min and washed with PBS. The cells were resuspended in a 2% CHAPS, 8M Urea buffer and lysed as described in the “RNA isolation” section, using Precellys Evolution homogenizer (Bertin Corp). The cell lysate was centrifuged and the AHA-containing proteins in the supernatant were captured using Click-iT™ Protein Enrichment Kit (ThermoFisher) followed by in-column trypsin digestion.To identify and quantify which proteins are translated after induction of MazF-ms (MSMEG_4448), three biological replicates were grown to an ODDigests were analyzed in two separate runs and combined. The data was analyzed as described previously , consideM. smegmatis reference genome harbors only one gene from the MazF toxin family, annotated as MSMEG_4448 (here referred to as MazF-ms). To establish whether or not MazF-ms is toxic  when expressed in M. smegmatis, we cloned the gene under the control of an ATC-inducible promoter in the pMC1s plasmid and transformed M. smegmatis mc2 155 cells. The expression level from this plasmid is modest, a 4.5-fold induction, based on measurement of mCherry fluorescence (According to the Toxin-Antitoxin Database (TADB 2.0) , the M. rescence Figure 1All members of the MazF family reported to date are single-strand, sequence-specific endoribonucleases. To help elucidate the molecular mechanism by which the ribonuclease activity of MazF-ms is able to regulate growth, we applied our specialized RNA seq method, 5’ RNA-seq, to find its RNA target(s) . This teLys isoacceptors  annotatLys identified here as MazF-ms targets only differ by one nucleotide in the ± 5 nt region surrounding the cleavage site  paused at the rarer Lys AAA codon rather than the more frequent Lys AAG , followed by recruitment of another RNase that cleaves 5’ of the stalled ribosome . AHA is incorporated into nascent peptides during translation, therefore only marking proteins that were synthesized after its addition to the media. Due to its azide moiety, proteins containing AHA residues can be captured using an alkyne-containing resin through a Cu(I)-catalyzed click reaction and then analyzed by quantitative mass spectrometry. We added AHA to the cultures after 4.5 h of MazF-ms induction, tagging only proteins that were newly translated.In contrast to a translation shut-off model proposed for other tRNA-cleaving toxins , global In TA systems, specific stresses trigger degradation of the cognate antitoxin by a protease, freeing toxin to act within the cell . TherefoAmong the 171 statistically significant downregulated proteins from Escherichia coli , an ATP-dependent RNA helicase DEAD/DEAH box family protein (MSMEG_5042), sigma factor SigH (MSMEG_1914) and alkylhydroperoxide reductase (MSMEG_4891). Lon is a stress-responsive protease. Since it is known to degrade all TA system antitoxins in hia coli , it may e stress  while UnConsistent with the sustained protein synthesis while MazF-ms was being expressed, we observed new synthesis of a subset of elongation factors and ribosomal proteins. Four elongation factors were upregulated: EF-Ts (MSMEG_2520), EF-Tu (MSMEG_1401), EF-P (MSMEG_3035) and BipA/TypA ribosome-binding GTPase (MSMEG_5132). Many of these elongation factors are associated with the bacterial stress response. Finally, 18 of the 51 ribosomal proteins were also upregulated. The other 33 ribosomal protein levels were stable.M. tuberculosis, the M. smegmatis genome harbors just three TA systems  as one facet of its stress survival armamentarium. A thorough understanding on how a TA system acts to protect its host from stress requires determination of the function of the toxin, and the function of the toxin is informed by determining its intracellular target. Since all MazF toxins are generally single-strand and sequence-specific endoribonucleases, in this work we identified the RNA target of the MazF-ms toxin using 5’ RNA-seq which revealed its detailed mechanism of action. To our surprise, MazF-ms did not behave like the vast majority of MazF toxins that appear to predominantly cleave mRNAs  are deficient in AAA Lys codons, allowing for their sustained translation while the cell can save energy by not synthesizing proteins that do not contribute to this endpoint. Indeed, as discussed above, many of the upregulated proteins annotated in the stress response category have direct roles in one or more stress responses: cold shock, heat shock, oxidative stress and nitric oxide stress. The stress-specific sigma factor SigH was also upregulated. Therefore, it is implicated as the primary RNA polymerase sigma factor enlisted for the sustained transcription of M. smegmatis genes whose proteins were upregulated after toxin expression. However, other upregulated proteins in our dataset that are not commonly associated with the stress response, i.e. at least some elongation factors, appear to have indirect roles  and SRA under the accession number PRJNA564437 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA564437), respectively.The proteome data and RNA-seq data have been deposited and made public in the Massive database under the accession number MSV000084300 (VB performed all experiments. Data analysis/interpretation was performed by VB. NW directed the project and acquired funding. NW and VB wrote the manuscript.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.


Colorectal cancer (CRC) is the most familiar malignancy worldwide. Hence, searching for novel therapeutic options is of highest priority. Therefore, it is of interest to design inhibitorsto the protein target importin-11, which transports β-catenin linked to colon cancer cells. However, the structure of importin-11 is not known. Hence, we use a homology model ofimportin-11 to dock potential interactions with five phyto compounds using molecular interaction features for further consideration. Compared to all cancer types, colorectal cancer is most frequent form of cancer in oncologic pathology . At presIn the current examination, the protein sequence of of Importin-11 was retrieved from Uni-protKB/Swissprot database ID Q9UI26)[UI26[9].TThe details accuiquired from sequence alignment was helped to build the 3D model of Importin-11 protein using educational edition of MODELLER .Five 3D With the intention to evaluate the consistency of the Importin-11 modelled structure, structural analysis and verification server (SAVES) (http://servicesn.mbi.ucla.edu/SAVES/) wasused. The phi (Φ) and psi (Ψ) torsion angles of protein was calculated using PROCHECK program, as firmed by Ramachandran plot statistics .The rooThe structures Thalicarpine (CID:21470) Tylocrebrine (CID: 246845) Emetine(CID: 10219) Kamebanin(CID: 12004580) Helenalin(CID: 23205) were downloaded from the PubChem database. Fordocking studies, these downloaded compounds structures were used. All the compounds were downloaded in SDF format and it was converted as PDB format using Online Smiles Translator.Before to docking analysis, important active site of modeled Importin-11 protein was identified using CASTp server  Key bindMolecular docking studies were carried out using Patch Dock server .Both proThe drug likeness calculation of Thalicarpine, Tylocrebrine, Emetine, Kamebanin and Helenalin was performed by Lipinski filter (http://www.scfbio-iitd.res.in/software/drugdesign/ lipinski.jsp),according to which an orally active drug should obey at least of four of the five laid down criterion for drug likeness namely: molecular mass, cLogP, hydrogen donor and acceptor and molarrefractive index .The crystallographic structure of Importin-11 was not found in the PDB, however the sequence similarity between Importin beta-like protein KAP120 [Saccharomyces cerevisiae S288C (PDBID: 6FVB) and Importin-11 (swissprot id: Q9UI26) was 27% which in turn has made it a reasonable template for modeling a three dimensional structure of Importin-11. showed tIn order to identify the binding conformation of selected five compounds with Importin-11 molecular docking studies was carried out. Docking of selected five compounds with the targetprotein of Importin-11 was carried out with respect to interacting amino acids, ligand and protein atoms participated in hydrogen bonding, and docking score with ACE area .AnalysiWe report the modeling docking analysis of importin-11 homology model with five phyto compounds in the context of colorectal cancer for further consideration.


It is a very old conjecture that emotion governs human choice. Early philosophers like Epicurus  and ArisBritish thinkers of the 18th and 19th century were among the most important exponents of psychological hedonism. In a well-known dictum, Bentham , p. 1 leFamously affirming that human rationality or reason could never on its own control behavior, Hume  was equaIn the second half of the 20th century, psychologists and neuroscientists tended  to stayDamasio  argued cThough demonstrating that choice can be controlled by emotions, Damasio  held thaFurthermore, says Damasio , p. 177:These aspects of Damasio's hypothesis are worth discussing, since the SMH, though controversial, has been so influential—and the man behind the hypothesis has not changed his mind. A recent paper (Verweij and Damasio, If we are to believe its originator, the SMH is unable to explain important aspects of the process behind human choice. What Damasio says about the SMH's limitations leaves open the possibility that another neurobiological mechanism will explain how reasoning determines choice. With regard to “sublime achievements,” this may not be the case, however. As we saw, some such deeds may result from the rejection of biological and cultural causation Damasio, , p. 177.Darwin's thinking, for example, has been called sublime (e.g., Vucinich, There is also something in the reasoning process that can make a choice impervious to the effect of emotions, if we are to believe Damasio . He doesDamasio  does claThe present author admires Damasio for his important work in many fields (e.g., Damasio and Maurer, Says Hume , p. 414,No author can answer every query a reader might have, and it is not incumbent upon Damasio to catalog all things that may affect a choice. Yet few questions in psychology have greater fundamental importance than those regarding how we choose. It may be reasonable, then, to ask why emotion can bias choice but not, in Damasio's view, determine it.some room for such freedom.” It may appear from these assertions that Damasio wants to avoid challenging the doctrine of free will. If that is the intention, Damasio should state clearly that in certain situations, a choice is exempt from causality—and ideally, he should explain when this is the case, and why.Furthermore, Verweij and Damasio , p. 3 apAlternatively, Damasio may believe that when, “on the basis of reasoning” (Verweij and Damasio, Damasio , and toA goal is a state we want to reach. Not surprisingly, all generally accepted definitions of “problem” (e.g., Mayer, a problem is an emotional state—it is a state of wanting. Reaching or not reaching a goal state are also typically described as events that elicit emotion [see Overskeid (Robinson et al.  use “wanverskeid ].Reasoning is a way of solving problems. But reason, says Hume , is the Damasio , on the Some people oversell their insights. But might this be a story of underselling? Can the SMH explain more than its originator will admit?GO confirmed being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RNA molecules, not included in the original ‘protein-only hypothesis’, are also being recognized as important factors contributing to the ‘prion behaviour’, that implies the transmissibility of an aberrant fold. In particular, an association has recently emerged between aggregation and the assembly of prion-like proteins in RNA-rich complexes, associated with both physiological and pathological events. Here, we discuss the historical rising of the concept of prion-like domains, their relation to RNA and their role in protein aggregation. As a paradigmatic example, we present the case study of TDP-43, an RNA-binding prion-like protein associated with amyotrophic lateral sclerosis. Through this example, we demonstrate how the current definition of prions has incorporated quite different concepts making the meaning of the term richer and more stimulating. An important message that emerges from our analysis is the dual role of RNA in protein aggregation, making RNA, that has been considered for many years a ‘silent presence’ or the ‘stone guest’ of protein aggregation, an important component of the process.The study of prions as infectious aggregates dates several decades. From its original formulation, the definition of a prion has progressively changed to the point that many aggregation-prone proteins are now considered  This review deals with the concept of prion-like proteins and/or domains. This concept dates the 90s when Wickner found proteins in yeast with a behaviour similar to that observed for vertebrate prions . The defIn our review, we retrace the concept of prion-like proteins to understand its meaning, applications and role in normal and pathologic functions. We explore the concept of prion-like proteins and their link to RNA-binding properties and liquid-liquid phase separation. The view that comes out from our analysis is the richness of meanings that the prion-like term has now incorporated with a direct link to essential metabolic pathways that, when altered, may directly lead to pathology. Using the paradigmatic example of the TDP-43 protein, we discuss the link between prion-like proteins and RNA, a component previously banned from the definition of prions. We could thus say that RNA has acted in prion and prion-like proteins as the ‘stone guest’ of the Mozart's opera Don Giovanni. This metaphorical expression indicates an impending presence (person or object) known by all but not explicitly acknowledged, that is invisible, silent and, consequently, rather disturbing and unpredictable. Accordingly, we discuss in this review how the role of RNA is not unique: we show how it can be both beneficial or detrimental for aggregation depending on the sequence and composition of RNA. Keeping this message in mind, we suggest that RNA could be used as a powerful way to interfere with protein self-assembly or aggregation if the synthax of protein–RNA interactions was fully decoded.C) , is a normal component of both infected and uninfected mouse and hamster brain tissues (C) that could misfold into a pathologic form (PrPSc), by a conformational change of α-helices into a β-rich structure prone to undergo a liquid-to-solid phase transition, which is often called in the field ‘protein aggregation’. These β-rich aggregates were supposed to have structural features similar to those of ‘amyloids’ found in proteins associated with numerous other protein misfolding disorders  with infectious properties that would exclusively consist of a single protein without the involvement of a nucleic acid genome . The conC) . In the  tissues ,11. In 1isorders . Amyloidle hosts . Thus, tle hosts .C into the β-rich (ca. 47% β-sheet content) PrPSc is considered the key event in the transmission of prion disease or, in other words, of prion infectivity  to a β-rich structure ,14, evenThe use of software able to search prion-like regions was particularly helpful in finding functional correlations amongst prion-like sequences. Gene ontology (GO) annotations indicated that ∼30% of human proteins with prion-like domains function in RNA binding  and ∼33%et al. (2018)   explicitRNA-binding prion-like proteins have more recently been linked to processes other than toxicity, such as the formation of SGs or other non-pathologic coacervates that are involved in the formation of membraneless organelles ,88. ThisS. cerevisiae  thus supporting a possible cooperation between these domains in the phase separation process. Finally, it was hypothesized that the self-interaction capacity of prion-like domains can lead to solid-like aggregation in given conditions. As a large poly-anionic molecule involved in the maintenance of fluidity, RNA could counterbalance the effect of prion-like domains, shifting the equilibrium towards a more dynamic organization . T. TcatGRAe Figure . The prie Figure  The impoWe recently proved that, contrary to what previously postulated, other regions of TDP-43 are prone to misfolding and promote the formation of aggregates also in the absence of the prion-like domain ,106: conin vitro aggregation of the TDP-43 construct 1–269 (comprising the N-terminus up to the end of RRM2) was abrogated already at equimolar protein:RNA ratio; instead, the presence of non-UG-rich RNA sequences could induce faster aggregation of the same protein fragment (Figure We reasoned that, according to its role as an RNA-binding protein, aggregation of TDP-43 could be modulated by RNA. This view was comforted by several considerations. We had previously shown that native interactions could be used as a powerful and specific means to inhibit protein aggregation . Other at Figure . This mein vitro, aggregates are readily generated with the mutated protein because RNA is unable to contrast protein aggregation.To support these results, an increasing number of TDP-43 mutations are being identified in regions other than the prion-like. Among these, the most interesting ones are Lys181Glu and Lys263Glu mutants that affect two residues directly implicated in RNA binding . These mTogether these considerations may help us to understand the mechanisms going on in the triggering of ALS and how any misregulation of RNA binding may affect TDP-43 aggregation Figure . They alet al. (In summary, we have given here a historical perspective of prion-like proteins Figure . We haveet al.  have hypet al. , would p