Generally, RNA-seq protocols employ poly(A) selection for mRNA enrichment. However, poly(A) based enrichment is subject to potential bias depending on the poly(A) status of various mRNAs, which could be particularly undesirable in the context of studying post-transcriptional gene regulatory mechanisms, such as miRNA repression . Therefore, ribosomal RNA (rRNA) depletion is a desirable alternative strategy to enrich for mRNA sequences in RNA-seq sample preparation . However, currently available rRNA depletion toolkits were designed for either mammals or bacteria, and hence do not offer an efficient option for rRNA depletion of RNA samples from certain experimental organisms, such as C. elegans.RNA-seq is widely used for the quantitative analysis of transcriptomes in the context of studies of gene expression and regulation . We suggest that this rRNA depletion protocol is applicable to RNA-seq applications where the yield of mRNA enrichment should be independent of poly(A) status, or any application which benefits from the removal of rRNA sequences, such ribosome profiling, or sequencing of non-coding RNAs other than rRNA.Here we describe a rRNA depletion protocol based on RNase H digestion using antisense oligonucleotides (ASOs) specifically designed for C. elegans genotypes) prepared with our rRNA depletion protocol, the cytoplasmic rRNA  reads only comprised < 0.2 % of the total reads , indicating that our protocol is effective for removing rRNA. Highlighting the specificity of the depletion, we observed reads from rRNA precursor transcripts  and mitochondrial rRNA  due to the absence of ASOs against those sequences. As expected, the rRNA-depleted datasets contained a large representation of other non-coding RNA sequences, further supporting that our protocol enriches for RNAs lacking poly(A) . The performance of our depletion protocol appears to be similar to that of two independently developed similar protocols for C. elegans-specific rRNA depletion . Interestingly, we found that a large proportion of the non-coding RNA reads after RNA depletion correspond to the signal recognition particle RNA (srpR) . A similar  enrichment of srpR was also observed in the libraries of Barucci et al., 2020 and Arribere et al., 2016. The srpR reads were manually removed in our analysis ; however we suggest that later iterations of our protocol could include ASOs corresponding to srpR, as well as mitochondrial rRNA and cellular primary rRNA transcripts.We designed 120mer DNA ASOs complementary to 26S, 18S and 5.8S rRNA sequences and 60mer ASOs complementary to 5S rRNA . We used thermostable RNase H and optimized protocols at high temperature for the ASO-rRNA duplex annealing and digestion to reduce the formation of secondary structure of rRNA and off-target annealing of the ASOs. We found that among the 12 RNA-seq libraries . Interestingly, these two sites do seem to locate in regions of their respective transcriptional units where reads were relatively depleted locally, suggesting that the ASOs may have triggered off-target depletion at these sites . However, the apparent sequence depletion in these two instances was only restricted to regions around the ASO off-target match sites, and the alignment distribution for each gene in sequences other than the off-target sites seems to be relatively unaffected . From this analysis, we suggest that the ASO rRNA depletion method results in essentially negligible off-target mRNA depletion.Although we did not directly compare the genome-wide distribution of mRNA reads from this ASO protocol to that of mRNA reads from other approaches for mRNA enrichment, we did conduct a computational assessment of the potential for off-target effects of our ASO rRNA depletion. We adopted a conservative assumption that, for an ASO to trigger RNase H digestion of an mRNA sequence, it should match the mRNA with a melting temperature of no less than 20rRNA depletion and RNA-seqet al., 2012). The RNA-seq libraries were constructed by NEBNext Ultra II RNA Library Prep kit  and sequenced by Illumina NextSeq 500 system.Harvested worms were washed with M9 medium and flash-frozen in liquid nitrogen. The worm pellets were lysed by QIAzol  as previously described . ASOs and total RNA were mixed with approximate 2:1 molar ratio . Higher molar ratios of up to 6.5:1 were tested but did not result in observable improvement in depletion efficacy. Accordingly, a mixture containing final concentrations of 0.1 mM of each ASO, 50 ng/µl total RNA, 10 mM Tris-HCl (pH = 8.0), 100 mM NaCl, 1 mM EDTA and 0.8 U/µl RNase Inhibitor  was incubated at 95 °C for 2 min for denaturation and then 65 °C for 30 min for annealing. We have also tested annealing conditions with gradual decrease in temperature (0.5-2 °C per minute) but no apparent improvement in depletion efficacy was observed. To digest the rRNA, thermostable RNase H and the buffer  were preheated to 65 °C and added to the reaction (while maintaining the reaction at 65 °C) to obtain a final concentration of 0.2 U/µl RNase H and the reaction was further incubated at 65 °C for 40 min, and then chilled on ice. Other digestion temperatures from 30 to 85 °C were also tested, and 65 °C was determined to be optimal for reducing rRNA secondary structure without compromising overall RNA integrity of the samples. To digest the ASOs, Turbo DNase  and the buffer were added to a final concentration of 0.1 U/µl and the reaction was incubated at 37 °C for 25 min. mRNA was then purified by RNA Clean & Concentrator-5 Kit  as described in . For the analysis shown in Fig. 1F,the srpR reads were removed by initially mapping with Bowtie2/2.3.4.3 , and either total filtered reads  or the remaining reads after srpR removal  were mapped to C. elegans genome (WBcel235) by Star/2.5.3 with default parameters . Mapping data was sorted and processed by Samtools/1.9 . Gene counting was performed using featureCounts .Adaptor sequences were trimmed and reads shorter than 15 nt were filtered out from analysis by The ASO sequences in this research are available upon request.


Cells release nanometer-scale, lipid bilayer-enclosed biomolecular packages  into their surrounding environment. EVs are hypothesized to be intercellular communication agents that regulate physiological states by transporting biomolecules between near and distant cells. The research community has consistently advocated for the importance of RNA contents in EVs by demonstrating that: (1) EV-related RNA contents can be detected in a liquid biopsy, (2) disease states significantly alter EV-related RNA contents, and (3) sensitive and specific liquid biopsies can be implemented in precision medicine settings by measuring EV-derived RNA contents. Furthermore, EVs have medical potential beyond diagnostics. Both natural and engineered EVs are being investigated for therapeutic applications such as regenerative medicine and as drug delivery agents. This review focuses specifically on EV characterization, analysis of their RNA content, and their functional implications. The NIH extracellular RNA communication (ERC) program has catapulted human EV research from an RNA profiling standpoint by standardizing the pipeline for working with EV transcriptomics data, and creating a centralized database for the scientific community. There are currently thousands of RNA-sequencing profiles hosted on the Extracellular RNA Atlas alone (Murillo et al.,  Extracellular vesicles (EVs) are secreted, nanometer-scale genetic information carriers found in human biofluids. Aside from EVs, there are a number of other non-vesicular nanoparticles in circulation such as lipoproteins, RNA-binding proteins, and exomeres  vs. a eukaryotic cell of 4 × 10−15m3 (sphere of radius 10 μm), the volumes are different by a factor of 106 and only certain cellular sub-regions are necessarily sampled by the nature of EV biogenesis requiring a membrane. To this end, microscale sampling of membrane and cytosolic components along with stochastic variance in molecule distribution within the cell, and other biophysical and biochemical factors imply that differential composition of EVs relative to their parents is insufficient to claim organization. Active loading of EV-associated proteins is implied in limited instances, and we suspect they are those identified as biogenesis-related EV biomarkers such as CD63, CD9, CD81, Annexin A1, and TSG101  EVs  reporter suggests that a single cell can have between 1 and 15 multivesicular endosome-plasma membrane fusion events  per minute  considering variance within and between cell lines among the three human cell lines tested  embryo model system enabling in vivo live-tracking of single endogenous EVs  to address the research needs; these methods generally fall into four categories: centrifugation, chemical precipitation, microfluidics, and biochemical capture. MISEV2018 guidelines emphasize the adoption of the terms “separation” and “concentration” when discussing EV experimental methods. A separation technique selectively removes EVs from other fluid components, or one EV subtype from another . A concentration technique increases the EV concentration in the fluid and may not necessarily remove EVs from other fluid components . The MISEV2018 authors established a heuristic to conceptualize the efficacy of different methods by classifying extracellular particle recovery  and specificity  as either low, intermediate, or high efficacy. Each method has some component of separation and concentration regardless of whether the properties are explicitly stated. High-recovery and high-specificity together in a single method is unlikely to be achievable. EV size, density, surface markers, biofluid type, sample volume, product purity, and cost are all to be considered since every method has its own advantages and disadvantages  and product yield .Generally characterize that the sample contains vesicles and describe the purity of the preparation. Vesicle existence can be claimed by demonstrating that the sample contains intact lipid bilayers, that are unlikely the products of cell fragmentation. For example, the identification of at least one membrane-associated protein  and a transmembrane  or lipid-anchored  protein would be sufficient to meet this criterion. Investigate the presence and extent of non-EV contaminants e.g., lipoproteins (targets depend on EV source and preparation method).Employ a combination of single EV characterization techniques. Ideally use an optical  and a biophysical/biochemical  modality tailored to the EV product and considering the limitations and complementarities of each technique.When considering EV-associated biological functions, it is necessary to operate with the utmost detail and rigorous experimental design. Functional studies should be performed with strict biofluid fractions; select the appropriate method(s) to separate EVs and EV subtypes from non-EV contaminants. Test the purported activity with condition-matched controls  of the biofluid itself, the EV-depleted biofluid, crude EVs alone, and EV subpopulations if relevant. Evaluate the relevant molecular topologies  for membrane-bound molecules.A strategic approach to EV characterization is essential to advancing the field  is a mainstay EV separation technique. The operating principle of DUC is to apply step-wise increases in centrifugal force to a solution. The product of centrifugal force and time during each step then concentrates particles of a specific size and buoyancy. Ideally, using this principle, one can separate particles within a solution into discrete fractions. For example, a cell fraction, a large vesicle fraction, and a small vesicle fraction. Since DUC is a relatively simple and pure technique, it is the established EV isolation standard. The primary benefit of DUC is the ability to concentrate and separate EVs by simply subjecting the biofluid to high rotational speeds. Furthermore, DUC parameters such as rotor type, rotational force, solution density, and spin time can be finely adjusted to meet the needs of a specific application, for example a specific biofluid type and EV fraction. Most DUC protocols include some variation of a three-step method including a low-speed ~1,000 RCF spin to collect cells and other large particles, an intermediate-speed ~20,000 RCF spin to collect large EVs, and a high-speed ~100,000 RCF spin to collect small EVs. The most notable innovation/adoption among centrifugation-based protocols is iodixanol-based density gradient ultracentrifugation (DGUC)  which haCentrifugation-based protocols face a number of limitations that have driven innovation to create new methods:The amount of variance and lack of standardized protocols within the field presents a challenge for validating findings made between groups and a barrier to entry for new groups. Commercially available isolation kits  are one solution aimed at the reproducibility problem.DUC protocols for EV isolation are lengthy, taking at least several hours.Only a small fraction of EVs are captured by DUC, which can make investigations of rare samples difficult  which many people suggest may irreversibly damage EVs or change EV properties studied thereafter  centrifugation and take about 1 h to complete EV isolation from blood plasma with newer kits. PEG-based precipitation methods have been shown to yield hundreds of times more EVs than centrifugation and therefore can be used effectively in low sample input applications EV precipitation strategies are advantageous over ultracentrifugation because they often recover a larger fraction of EVs, and they can function with standard benchtop centrifuges. Reports describing chemical precipitation of virions and other nanoparticles by volume-excluding polymers e.g., polyethylene glycol (PEG) date as early as the 1970s. Alice Adams reported that the Epstein-Barr virus could be recovered with high yield and retained viral activity when separated directly from cell culture medium using PEG-based precipitation, while sucrose density gradient centrifugation had low recovery and substantially attenuated viral activity Adams, . The opeBiochemical capture techniques are most appropriate for investigators who seek to subset EV populations by specific membrane components and ascribe functions to those EV sub-populations . Biochemical capturing of EVs in most embodiments depends on immunoaffinity capture (IAC), where antibody-binding interactions dictate which particles become concentrated. The IAC approach uses antibodies conjugated either with the surface that the fluid sample passes over  technologies use physical properties to manipulate solutions on a small (μL-pL) scaleThe most simple manifestation of MF technology applied to EV separation/concentration is filtration. In this method, a solution is forced through a nanoporous filter material where particles larger than the filter's effective pore size are concentrated on the filter, while smaller particles pass through and remain in the filtrate solution. Particles of a specific size range can be separated from particles outside of that range by implementing a filter series where each filter has different effective pore sizes. Many groups use filtration alone or in combination with other techniques such as centrifugation or precipitation for greater power to select specific EV subsets. A device with low protein binding, track-etched polycarbonate membrane Apel,  filters,Chromatographic techniques, which can be further divided into size-exclusion and affinity modes, are widely employed for EV separation protocols and have a marked advantage over other methods we review here because they can yield very clean and highly-reproducible products. In traditional size-exclusion chromatography (SEC), a solution (the mobile phase) travels through a column packed with porous resin beads (the stationary phase). Small particles can enter the resin pores, and thus have a longer path to elution, while larger particles travel a more direct path outside of the beads and elute more quickly . For a pIn affinity chromatography, the mobile phase travels through a stationary matrix exhibiting an affinity for certain particle types; the interaction can be general  or specific . High affinity particles are retained non-covalently in the stationary phase upon sample application. Then, graduated elution of a solution with comparable affinity displaces adsorbed particles once the bonding interaction is overcome. Zeta-potential measurements of EVs by both resistive pulse sensing and electrophoretic mobility assays have indicated that EVs carry a negative surface charge  studies. The principle is to construct an obstacle network which selectively traps EVs while allowing undesired fractions to pass through. Then, the network integrity is dissolved or perturbed to release the trapped EVs. An early embodiment used ciliated silicon micropillars in a regularly-interspaced (0.9 μm) array with a tunable capturing range of 30–200 nm. The nanowire forest captured EV-like liposomes, while other cellular and protein debris flowed through. Then, the liposomes were released by incubating the MF channel with phosphate-buffered saline, thereby dissolving the silicon nanowires. This architecture was additionally flexible in that antibodies could be loaded on the nanowires; however, it was not demonstrated  is a promising technology which has already been used to concentrate EVs within human biofluids for RNA-seq analysis  has great potential for separating EVs from other nanoparticles though its implementation in EV research has thus far been limited. Field flow fractionation first was described in 1966  from large EVs (1–2 μm milk fat globules)  is now a mainstay for EV counting and sizing that is based on visible light microscopy. NTA devices shine a laser through the sample and record a video. Brownian motion of individual EVs, which can be inferred by the way light scatters through the sample over time, directly relates to their particle size . Therefo2 and 5 × 105 per μL based on the change in fluorescence polarization  functions by capturing EVs on an antibody microarray chip. Light is shone onto the chip and then the reflectance is measured. Interference between the reference light-field, produced by reflectance off of the silicon chip alone, and the scattered light field produced by reflectance off of the EV-bound microarray, gives a signal which corresponds to the EV size at each location on the chip  is another commonly implemented technique for EV counting, sizing, and surface charge characterization. RPS principally operates by electrophoretic translocation of particles through a conical pore; when within the pore, the particle increases electrical resistance across the pore and a current drop is detectable. The magnitude and frequency of current drops are proportional to particle volume and concentration, respectively , nucleic acid sequencing (NA-Seq), and antibody affinity-labeling techniques are all currently being implemented for high-throughput, multiplexed profiling of EV biochemistry. The advantages of EV-focused MS analysis have been made clear through a number of studies including the identification of GPC1 as a marker of pancreatic cancer-derived EVs, genetic renal disease markers in urinary EVs, and novel myokines secreted during exercise in mammals  conjugated with 1,2,4,5-tetrazine were added, the antibody-bead linkage was achieved by spontaneous cycloaddition, and the EV-antibody-MNP product was formed. The sample was finally washed and analyzed for protein content by nuclear magnetic resonance using a microcoil on the device. The NMR output corresponded to EV protein concentration and type. Proteins were detected with orders of magnitude higher sensitivity than traditional approaches, including flow cytometry, ELISA, and nanoparticle tracking analysis. Additionally, the NMR signature of four EV-associated proteins sensitively and specifically identified glioblastoma vs. healthy patients . For representative examples, see Ratajczak et al.  and ValaExtracellular RNAs in human serum are present in ng/mL concentrations and predominantly in the 20–200 nt size range  include riboswitch systems, ribozyme activities, cis-regulatory mRNA sequence presentation or concealment, and trans-regulatory behaviors, which are all driven by mRNA folding and/or processing  are the most heavily studied and strongly characterized extracellular RNA biotype contained within EVs. Hundreds of miRNA species can be detected in a given biofluid and there is longitudinal evidence that each fluid type has a distinct miRNA composition which can be changed by health status including pregnancy, urothelial cancers, neurological disorders, diet, and exercise  are a key component of EV-derived RNAs, taking large fractions of the RNA in most biofluids tested  are one of the most abundant RNA components of EVs, and specifically can dominate within blood  and circular RNA (circRNA) species are generally present to lesser extents in circulating biofluids . An RNA-seq study with 22 subjects identified 21 micro-RNA candidates that showed significant differential expression between those with beneficial vs. adverse remodeling outcomes. Top pathways targeted by these micro-RNAs include immune cell signaling, fibrosis, and apoptosis, indicating that specific inflammatory states lead to positive or negative resolutions of myocardial infarction (Danielson et al., in vitro by way of EVs (Balaj et al., The physiological role of exogenous EVs is being newly investigated. EVs are secreted by most organisms and accordingly, given the extensive population of humans by microbial species and viruses, we can predict a substantial number of exogenous EVs to exist in human biofluids. A large proportion of circulating RNAs, including miRNAs, are contributed by the microbiome. In some EV preparations, 75% of the total RNA reads originate exogenously (Wang et al., in vivo, and prodrug administration led to tumor death when at least 1% of the tumor cells in a given tumor received the minicircle DNA. Dysregulated EV signaling has also been implicated in tumor immunology (Ricklefs et al., EVs are particularly interesting for novel therapeutic strategies. Because EVs have a suspected mechanism for targeting and delivering their cargoes to certain cell populations (Hyenne et al., There are a number of clinical scenarios when it may be desirable to attain the genetic information of a developing fetus, such as when the pregnancy is at-risk for yielding a trisomy or other chromosomal abnormalitiesDeep and high-coverage sequencing studies of the fetal exome and genome have demonstrated the potential of extracellular RNA to make clinical assessments of the fetal genome at various time points (Fan et al., There are a number of online public databases which host EV-related gene expression data and serve as a reference for the research community . VesicleTo address the ERC program goal of generating an exRNA reference catalog, 2,270 extracellular small RNA-seq and 3,039 qPCR profiles across 19 studies and 23 health states were compiled in the ERC Consortium data repository, the exRNA Atlas (Murillo et al., In this review we have provided historical context for EVs and their rise to prominence in biomedical research, reviewed EV biogenesis and fates with perspective on functional outcomes, and identified some of the latest experimental approaches for preparing and analyzing EVs with a focus on clinical applications. We also discussed the physiology and biochemical content of EVs with a focus on RNA biotypes, and provided an overview of online databases hosting EV-related experimental findings. The research community has consistently advocated for the importance of EVs especially with regard to their RNA contents. EVs are hypothesized to act as intercellular communication agents that functionally transfer biological information between cells and tissues. EVs protect RNAs from degradation in the extracellular environment. Furthermore, RNAs contained within EVs encode biological state-specific information, and RNA-containing EVs can be easily and frequently collected in a clinical setting and analyzed. In the future, it is possible to envision noninvasive EV-based diagnostics enabling precision health applications–where sampling an individual over time is beneficial to gain insight into the evolution of a dynamic health or disease state (Chen et al., As we indicated throughout this review, there is a concerted effort toward single-EV characterization and a number of projects under the ERC commonfund have recently shifted to new stagesEV wrote and reviewed the manuscript and generated the figures. GM conceived of and supervised the project, reviewed, and edited the manuscript. All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.EV declares equity ownership in Gilead Sciences, Inc., an American biotechnology company. The remaining 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.


It has been found that m6A modification occurs in microRNA, circRNA, rRNA, and tRNA, etc. The m6A modification plays an important role in regulating gene expression, and the abnormality of its regulatory mechanism refers to many human diseases, including cancers. Pitifully, as it stands there is a serious lack of knowledge of the extent to which the expression and function of m6A RNA methylation can influence prostate cancer (PC). Herein, we systematically analyzed the expression levels of 35 m6A RNA methylation regulators mentioned in literatures among prostate adenocarcinoma patients in the Cancer Genome Atlas (TCGA), finding that most of them expressed differently between cancer tissues and normal tissues with the significance of p < 0.05. Utilizing consensus clustering, we divided PC patients into two subgroups based on the differentially expressed m6A RNA methylation regulators with significantly different clinical outcomes. To appraise the discrepancy in total transcriptome between subgroups, the functional enrichment analysis was conducted for differential signaling pathways and cellular processes. Next, we selected five critical genes by the criteria that the regulators had a significant impact on prognosis of PC patients from TCGA through the last absolute shrinkage and selection operator (LASSO) Cox regression and obtained a risk score by weighted summation for prognosis prediction. The survival analysis curve and receiver operating characteristic (ROC) curve showed that this signature could excellently predict the prognosis of PC patients. The univariate and multivariate Cox regression analyses proved the independent prognostic value of the signature. In summary, our effort revealed the significance of m6A RNA methylation regulators in prostate cancer and determined a m6A gene expression classifier that well predicted the prognosis of prostate cancer.RNA methylation accounts for over 60% of all RNA modifications, and N Prostate cancer (PC), namely prostate adenocarcinoma (PRAD), is a great threat to the male reproductive system . Althoug6A mapping has been disclosed to provide the landscape of m6A RNA methylation for its crucial function in cellular differentiation, cancer progression and other processes.RNA modification participates in many biological processes in vital movement, counting regulation of RNA post-transcriptional stability , localiz6A modification mainly occurs on adenine in RRACH  sequence, the functions of which are regulated by an expanding list of writers, readers and erasers  and nuclear heterogeneous proteins HNRNP family (HNRNPA2B1 and HNRNPC) (M erasers . The m6A HNRNPC) .6A is thought to be closely linked to various cancer types, including gastric cancer  database was explored. Afterward, consensus clustering was conducted according to the gene expression levels of m6A RNA methylation regulators with principal components analysis (PCA), survival analysis, Gene Ontology (GO), and Kyoto Encyclopedia Genes and Genomes (KEGG) for evaluation. Finally, we formed an efficient prognosis indicator comprising five pivotal genes related to m6A RNA methylation by the last absolute shrinkage and selection operator (LASSO) Cox regression.Herein, we comprehensively investigated the role of RNA m1, cBioPortal for Cancer Genomics2, and UCSC Xena browser3, including gene expression datasets (RNA-seq) on PRAD patients, as well as corresponding demographic (age and gender), clinicopathological  and survival [overall survival (OS) and disease-free survival (DFS)] information. Anatomic stage or prognostic group was determined by American Joint Committee on Cancer (AJCC) Cancer Staging Manual 8th edition (2017) (The data were gathered from TCGA projectn (2017) . Patient6A RNA methylation regulators were selected from articles and reviews manually  curve. Univariate and multivariate Cox regression analyses were performed to validate the independent role of the risk signature. The analyses above were performed by R 4.0.2. Finally, the relevance of risk score and clinicopathological features was evaluated by Prism 7.04 .Univariate Cox regression was performed to evaluate the correlation between OS or DFS and the transcriptome levels of differentially expressed genes (DEGs) above. The inclusion genes with a cut-off criterion of analysis . Five m6® reagent . The concentration and purity of the RNA solution were detected using a NanoDrop 2000 spectrophotometer . Extracted RNA was then reverse transcribed into cDNA using PrimeScriptTM RT Master Mix  according to the manufacturer’s protocols. The reaction conditions were as follows: 37°C for 15 min; 85°C for 5 s. Subsequently, the cDNA was subjected to qPCR using AceQ® qPCR SYBR Green Master Mix  on CFX Connect Real-Time PCR Detection System  according to the manufacturer’s protocols. The qPCR conditions were as follows: pre-denaturation at 95°C for 5 min; 40 cycles of denaturation at 95°C for 10 s; annealing and extension at 60°C for 30 s. The housekeeping gene, GAPDH, was used to normalize the relative expression of HNRNPA2B1, NXF1, RBMX, YTHDF1, and TRMT112 as an endogenous control by the comparative Ct (threshold cycle) method (2Ct–ΔΔ). All qRT-PCR reactions were performed in duplicate. The primers used to amplify target genes and GAPDH were chemically synthesized by TSINGKE, China. The primer sequences were listed in Total RNA was isolated from tissues using TRIzol6A RNA methylation regulators in patient with PC, we first considered the genome information. The profiles of m6A RNA methylation related genetic alteration, including mutation and putative copy-number alteration (CAN), were accessed. Of 499 PRAD patients, 368 (73.7%) harbored at least one type of genetic alterations, including inframe mutation, missense mutation, truncating mutation, amplification, and deep deletion, as well as transcriptomic changes  and para-cancer tissue (n = 52). The information of m6A RNA methylation regulators was extracted and analyzed for DEGs. As shown in 6A regulators expressed differently with the significance of p < 0.05, including 14 up-regulated genes  and 10 down-regulated genes , indicating the obvious variation of m6A modification in tumorigenesis.Then transcriptome profile of m6A, the interaction among all m6A regulators was explored. As shown in 6A RNA methylation regulators correlated with others. Thereinto, RBMX, HNRNPA2B1, RBM15B, PRRC2A, YTHDF1, YTHDF2, RBM15, KIAA1429, YTHDF3, YTHDC2, YTHDC1, CPSF6, SRSF10, METTL14, NUDT21, ZC3H13, FTO, ZCCHC4, FMR1, XRN1, and SETD2 positively correlated with other regulators while TRMT112 and ALKBH3 negatively correlated. However, some regulators such as WTAP, IGF2BP1, IGF2BP2, IGF2BP3, ALKBH5, and PCIF1 showed poor correlation with other regulators, which may result from their versatility in multiple biological processes. 6A methylation regulators, suggesting a close connection between them. A novel algorithm of Maximal Clique Centrality (MCC) of CytoHubba mode in Cytoscape was performed to filter the hub genes of the network based on known or predicted PPIs  of the consensus cluster for k = 2 to 9 and increment in the AUC were shown in 6A RNA methylation regulators that is high within groups and low between groups, k = 2 was determined, namely the number of clusters  in comparison with the low-risk group  caused by long-term use of drug is calling for new therapeutic targets.6A RNA methylation, the most frequent modification in RNA, closely relates to many types of cancers  infection, non-alcoholic fatty liver disease (NAFLD); down-regulated DEGs not only affected some classic cancer-related biological processes, such as PI3K-Akt pathway and focal adhesion, but may also participate in the onset of cardiomyopathy. According to the literatures, AD and PC shared genetic etiology via gene expression regulators PA2 and HNR(N)PB1, is an RNA binding protein and complex with heterogeneous nuclear RNA (hnRNA). HNRNPA2B1 mostly binds a set of mRNAs in nuclear and elicits alternative splicing effects but it also works in part on primary microRNA . HNRNPA26A readers, YTHDC1 and SRSF3, for mRNA export promotion , cBioPortal , and TCGA project .Publicly available datasets were analyzed in this study. This data can be found at Xena browser (JX and YL designed the study. JX, JL, TX, GC, YS, JT, and LL carried out data acquisition and analysis. JX, YL, XZ, and KC wrote the manuscript. JX, WX, ZX, CY, and ZC collected the clinical samples and managed the clinical data. HY and HL contributed to bioinformatics analysis. XZ and KC were involved in project management and contributed to preparing and making figures and tables. KC and XZ supervised the study. All authors read and approved the final 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.


Background: Emerged in December 2019, coronavirus disease 2019 (COVID-19) is one of the largest pandemics ever. During the early phase, little was known about public knowledge, attitudes, and practices (KAP) relating to coronavirus disease. This study was designed to determine KAP of Iranians toward COVID-19.Methods: A cross-sectional online survey was carried out in Iran from February 25 to April 25 using a self-administered questionnaire on 1,480 people. COVID-19-related KAP questions were adapted from other internationally validated questionnaires specific for infectious diseases.Results: All participants were aware of COVID-19. When asked unprompted, 80% of respondents could correctly cite fever, difficulty in breathing, and cough as signs/symptoms of COVID-19. Most of our sample population knew that staying at home and isolated (95.3%) as well as constant handwashing and using disinfectants (92.5%) could prevent COVID-19. However, there were also widespread misconceptions such as the belief that COVID-19 can be transmitted by wild animals (58%) and by air (48.3%). Unprompted, self-reported actions taken to avoid COVID-19 infection included handwashing with soap and water (95.4%), avoiding crowded places (93%), cleansing hands with other disinfectants (80.), and covering mouth and nose when coughing or sneezing (76.1%). The Internet and social media (94.5%) were the main coronavirus information sources. However, the most trusted information sources on coronavirus were health and medical professionals (79.3%). The majority of participants (77.0%) wanted more information about coronavirus to be available.Conclusion: Our findings suggest that people's knowledge and attitude toward COVID-19 at the time of its outbreak was at a high level. Having emerged in the last months of 2019, coronavirus disease 2019 (COVID-19) spread beyond an unimaginable rate, imposing a heavy burden on worldwide healthcare systems forcing almost all countries to apply quarantine rules which were unprecedented over the last century  of people so that the authorities know which areas of KAP need enhancing . TherefoThis cross-sectional survey was conducted from February 25 to April 25, to assess the public knowledge, attitudes, and practices regarding the coronavirus outbreak in Iran.d (margin of error) = 0.02. The calculated sample size of this study was 1,233 participants, by design effect = 1.2 reaching a sample size of nearly 1,480 participants.Convenient sampling was employed in this study. The estimation of the sample size was done by assuming a minimum disease prevalence of 15%, confidence level = 95%, and The survey questionnaire included both prompt direct questions as well as unprompted open-ended questions that allowed for multiple responses. Coronavirus KAP domains in the questionnaire included sources of information, knowledge about coronavirus, behavioral intentions, prevention practices, and attitudes toward survivors. The indicators used to assess coronavirus KAP were informed by lessons learned from similar KAP studies on other communicable diseases, especially MERS, SARS, HIV/AIDS, and Ebola –13. Befohttps://survey.porsline.ir/#/). The survey started with a sentence that “completing the questionnaire by the participants is considered voluntary participation.” After confirmation that participants understood this, people were guided to complete the self-report questionnaire. On average, questionnaires took ~10–15 min to complete. The ethics committee of Birjand University of Medical Sciences (BUMS) approved this study before starting the formal survey .Due to maintaining social distance, the survey was done online in all provinces of the country. The questionnaire was designed on Porsline (similar to SurveyMonkey) shared on social media such as Telegram, WhatsApp, LinkedIn, and Facebook. We also tried to distribute this questionnaire on social media channels related to all provinces and cities to increase the response rate. People who were of Iranian nationality, aged 10 years or more, and agreed to participate in the survey were directed to complete the questionnaire via clicking the link . Descriptive statistics were then generated for national-level estimates (proportions) and their 95% CIs.A total of 1,480 people responded to the questionnaire, yielding a response rate of 70.2%. This response rate is based on the number of views by participants and the number that fulfilled the survey. The mean age of participants was 31.29 years. Among the respondents, 797 (57.2%) were female. Moreover, 782 (53.1%) were married. Of all respondents, 41.9% were between the ages of 20–31 years. Also, above 80% of respondents hold degrees of higher education .The majority of respondents (84.5%) had heard of coronavirus disease 2019 (COVID-19) before the interview. Overall, 84.5% of respondents were aware that it is possible to survive and recover from COVID-19. Approximately 60% of respondents (59.6%) perceived themselves to be at some risk of contracting COVID-19.In an open-ended question, the most common perceived cause/origin of coronavirus was “virus” (94.4%); only 31.7% linked coronavirus to a “bats, monkeys, and wild animals.” Very few respondents mentioned that coronavirus is caused by “Bacteria” (6.7%), “Evildoing/Sin” (4.1%), or “Parasites” (3.7%).In response to an unprompted open-ended question, the most frequently cited modes of transmission were shaking hands with an infected person (91.9%), kissing and hugging (90.1%), and being in contact with the saliva of an infected person (87.2%) .Overall, 80% of respondents could state, without prompting, three key signs/symptoms of Coronavirus: “difficulty breathing” (97.7%), “fever” (97.6), and “cough” (94.3%) .Knowledge of coronavirus prevention and treatment was high among respondents in that nearly everyone knew that coronavirus can be prevented by staying at home and reducing contact with people (95.3%) as well as constant hand washing and using disinfectants (92.5%).Furthermore, 75.1% of respondents expressed that early treatment of coronavirus could increase survival and reduce the chance of transmission within the household .There were widespread misconceptions about coronavirus transmission, prevention, and treatment. Nearly half (47%) of respondents said they could protect themselves from coronavirus by washing hands with salt and hot water solution and nearly half again (48.3%) said coronavirus is transmitted by air while 58% of the respondents expressed that coronavirus is transmitted by wild animals. Moreover, 15.1% of respondents perceived that traditional healers could successfully treat coronavirus .Most of the respondents (75.2%) reported that they would go to a health facility if experiencing a high fever. Nearly all respondents (97%) reported taking some preventive action since learning about coronavirus. Handwashing with soap and water was the most prevalent behavior reported in unprompted response (95.4%), followed by avoiding crowded places (93%), cleaning my hands with other disinfectants (80.9 %), and covering mouth and nose when coughing and sneezing (76.1%).Only 10.6% reported that they would not welcome back a coronavirus survivor into the community through 49.5% would refuse to buy fresh vegetables from a shopkeeper who survived coronavirus.Of respondents, 73.2% reported they would accept an approved coronavirus vaccine if it were to become available, while, only 26.1% said they would accept experimental treatments that have not been trialed .The Internet and social media (94.5%) is the primary coronavirus information channel mentioned by respondents in an open-ended question, followed by television (70.7%), relatives and friends (40.7%), and house visits by health workers (35.3%) .The most trusted information sources on coronavirus were health and medical professionals (79.3%), the Government/Ministry of Health (55.7%), and the Internet and social media (44.6%) .The majority of participants  wanted more information about coronavirus and specifically mentioned wanting to know more about providing medical care and treatment for infected people  as well as how to prevent the disease , while about half of the respondents wanted more information on coronavirus' signs/symptoms and its cause/origin .This study aimed to investigate the level of KAPs of the general population around COVID-19 in Iran. In this regard, the KAP findings suggest that while during the outbreak, awareness and preventive behaviors related to COVID-19 were promoted well in Iran, misconceptions and discriminatory attitudes to those who survived coronavirus were common.The study also highlighted the gap between the perceived susceptibility of catching the virus and reality. For example, while almost all adults were at risk of catching coronavirus, only 60% of people considered themselves at risk . TherefoKnowledge about the signs and symptoms of coronavirus disease was at a high level among people with over 80% providing correct answers. This compares with studies in various countries, including China , USA 1717, and IMoreover, according to the results, 95% of people have stated that staying at home and obeying health protocols will prevent them from being infected. In this regard, studies have also illustrated that staying at home and maintaining social distance has a positive effect on reducing disease transmission . CountriIn line with this, some misconceptions about the disease persisted. For instance, 47% of people believe the virus is killed by the saline solution, and 58% believe that the virus is transmitted through wild animals . Any misHowever, positive key findings show that 75% of people stated they would go to a health facility if they had a fever, and 90% of people say they have taken preventive measures since the onset of coronavirus such as washing their hands with soap and water and avoiding crowded areas.One area of concern revealed by the study is the stigma of coronavirus survivors. Some 10% of people said they would not welcome a survivor of coronavirus into the community while 49.5% stated they would refuse to buy fresh vegetables from shopkeepers who had survived coronavirus. This may raise main concerns about the stigma attached to patients who have the disease among their community. Therefore, measures should be taken to increase awareness at the community level to reduce stigma , 21.From the publicly available information sources, the most important source for acquiring knowledge for most people is social media. Therefore, it is necessary to provide accurate, precise, and timely information to the public , 23. Of Finally, more than 50% of people stated a need for more information and knowledge about the treatment and diagnosis of the virus. Since this is a new epidemic, it is important to have reliable and up-to-date information on all aspects of the disease; from prevention to diagnosis and treatment to acquire coherence and cohesion in people's behavior using scientific protocols, not gossip or fake news in social media and on the Internet.This study is the first to be conducted nationally in Iran about the KAPs of the general population regarding coronavirus and has a high sample size. Since the study was conducted online only and people who have access to social media networks have been included in the study, it would not be prudent to generalize the results to the whole community (people without smartphones and less literate).In conclusion, people's knowledge, attitudes, and performances about the disease are at a high level, but there are misconceptions about the disease. Also, the fear of dealing with recovered patients and the lack of communication and knowledge around recovered patients are at a high level. However, the disease is unlikely to be transmitted to a person after recovery, so training and information via the media can help reduce misconceptions and the stigma around recovered patients in the community.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Birjand University of Medical Sciences, Ethical Committee . Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.MA-Z and EK: idea, design, and editing. MA-Z, EK, DG-N, KM, and ZC: preparing the questionnaire. KM, ZC, HA, SH, and HS: data collection. SH, EK, and HS: analyses. All authors: writing draft and approval of final draft.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.


CRISPR-based RNA-United Interacting System (CRUIS), which captures RNA–protein interactions in living cells by combining the power of CRISPR and PUP-IT, a novel proximity targeting system. In CRUIS, dCas13a is used as a tracker to target specific RNAs, while proximity enzyme PafA is fused to dCas13a to label the surrounding RNA-binding proteins, which are then identified by mass spectrometry. To identify the efficiency of CRUIS, we employed NORAD (Noncoding RNA activated by DNA damage) as a target, and the results show that a similar interactome profile of NORAD can be obtained as by using CLIP (crosslinking and immunoprecipitation)-based methods. Importantly, several novel NORAD RNA-binding proteins were also identified by CRUIS. The use of CRUIS facilitates the study of RNA–protein interactions in their natural environment, and provides new insights into RNA biology.No RNA is completely naked from birth to death. RNAs function with and are regulated by a range of proteins that bind to them. Therefore, the development of innovative methods for studying RNA–protein interactions is very important. Here, we developed a new tool, the  RNA-binding proteins (RBPs) play important roles in various biological processes such as regulation, splicing, modification, localization, translation and stabilization of RNAs. Many RBPs, including some proteins that lack the classical RNA-binding domains, have distinct spatial and temporal distributions in cells and tissues. The malfunction of RBPs is responsible for many human diseases . In ordeThe protein-centric class involves all immunoprecipitation-based methods, targeting to a protein of interest and analyzing the bound RNAs. Initially, the RNA immunoprecipitation (RIP) assay, which was adapted from the chromatin immunoprecipitation assay (ChIP) , was usein vivo labeling without in vitro manipulation remains unfulfilled.The RNA-centric class is oligo-capture based approach, which targets to a given RNA and captures its bounding proteins. The examples include RNA antisense purification-mass spectrometry (RAP-MS) and comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) ,8, both CRISPR-based RNA-United Interacting System (CRUIS), a new RNA-centric method for studying RNA-binding proteins on specific RNAs using engineered dCas13a and PUP-IT. Recently, a number of CRISPR-based RNA-targeting Cas nucleases have been reported. These nucleases specifically bind and cleave target RNAs in the presence of guide RNA. This feature, which involves the tracking of target RNAs and the editing of specific bases of RNAs, allows researchers to manipulate specific RNAs . The pro2. All constructs were prepared using E.Z.N.A.® Endo-free Plasmid DNA Mini Kit  and transfected with Lipofectamine 2000 . The sequence of CRUIS is available in the supplementary information. Stable cell lines were generated with the piggyBac transposon system, which is widely applicable to various cell lines including non-mammalian cell lines. GFP-positive cells were enriched by flow sorting after transfection. Single colonies were picked, expanded, and tested via PCR, western blot, and enzyme activity identification for PafA. The HEK293T cell line with the best inducibility (referred to as 293T-CRUIS) was expanded and used for all subsequent experiments.HEK293T cells were grown in DMEM (Hyclone) supplemented with 10% FBS  in a humidified incubator at 37°C with 5% COdLwaCas13a–PafA-P2A-EGFP) was generated by subcloning dLwaCas13a fused with PafA at the C-terminus and a self-cleaving P2A peptide-linked EGFP (enhanced green fluorescent protein) into a piggyBac transposon backbone. dLwaCas13a was obtained by introducing two point mutations (R474A and R1046A) in the LwaCas13a (Addgene plasmid #90097) HEPN domains. The PafA was obtained from pEF6a-CD28-PafA (Addgene plasmid #113400). ClonExpress MultiS One Step Cloning Kit  and Mut Express II Fast Mutagenesis Kit V2  were used for construct generation. The CRUIS plasmid has been deposited to the open-access platform Addgene.The CRUIS construct , and anti-myc primary antibody . Cells were then incubated for 2 h at room temperature with secondary antibody and mounted using the anti-fade mounting medium.293T-CRUIS cells were plated in 24-well tissue culture plates on poly-d-lysine coverslips and transfected with 500 ng 5 293T cells were extracted with Trizol  and RNA concentration were determined by NanoDrop 2000c (Thermo Fisher). cDNA was synthesized using 1 μg RNA by the reverse transcription kit PrimeScript™ II 1st Strand cDNA Synthesis Kit  according to the manufacturer's instructions. Each qRT-PCR reaction was performed with cDNA transcribed from 25 ng RNA in a final volume of 20 μl with ChamQ™ SYBR Color qPCR Master Mix (Vazyme Cat. # Q431-03), assayed by QuantStudio™ 7 Flex (Life Technologies). The qPCR data were normalized to GAPDH expressions by relative quantification (ΔΔCt) method. The primers used were: CXCR4 , p21 ; NORAD , β-actin . GAPDH .Total RNAs from 5 × 10293T-CRUIS cell lines transfected with or without pCMV-bio-pupE were analyzed by western blot. About 2 million cells were harvested and washed with cold PBS. Lysis buffer  with 100× protease inhibitor was added to the pellet. Cells were resuspended and incubated on ice for 1 h. Then the lysate was spun down and the supernatant collected with the addition of protein loading buffer. The samples were boiled at 100°C for 10 min and loaded on 4–20% SDS-PAGE gels, followed by immune-bolting with anti-myc antibody and streptavidin-HRP  to identify the expression of dCas13a-PafA fusion protein and the activity of PafA ligase.For the enrichment of Bio-PupE modified proteins by streptavidin magnetic beads. Thirty-six hours after transfection with sgRNA or non-target sgRNA into the 293T-CRUIS cell line, the treated cells were harvested and lysed using cell lysate buffer. 20 μl streptavidin magnetic beads used for capturing labeled proteins from cell lysate supernatant and washed three times by wash buffer . The obtained proteins were boiled at 100°C for 20 min and used for western blot to analyze whether HNRNPK was modified by Bio-PupE, HNRNPK was identified by specific antibody .About 30 million cells transfected with pCMV-bio-pupE and sgRNA were used for the mass spectrum. Cells were harvested and washed with cold PBS, then incubated with 2 ml lysis buffer at 4°C. After shaking for 1 h, the lysate was spun down at 4°C for 10 min. The supernatant was transferred into new tubes, with the addition of urea and DTT to a final concentration of 8 M and 10 mM. The lysate was incubated at 56°C for 1 h, then treated with 25 mM iodoacetamide in the dark for 45 min to aminocarbonyl modify the Cys site of proteins. 25 mM DTT was added to terminate the modification. Streptavidin–biotin magnetic beads were washed with 500 μl PBS three times and then resuspended in lysis buffer with an equal volume of beads. The lysate was then added 50 μl beads and it was incubated on a rotator at 4°C overnight. The beads were washed with the following buffers: twice with buffer 1 , once with buffer 2 , twice with buffer 3 , three times with buffer 4 (100 mM ammonium carboxylate), and finally the beads were resuspended in 100 μl buffer 4. Trypsin, 4 μg  was added to digest overnight at 37°C. The peptides were collected with ziptip by the addition of 1% formic acid, then washed with 0.1% TFA  and eluted in 50 μl of 70% ACN  –0.1% TFA. The peptides were analyzed on an Orbitrap Fusion.t-test  and fold change (fold change > 3). Previously reported RNA binding proteins were obtained from StarBase v2.0 (http://starbase.sysu.edu.cn/) . The subcellular localization of the identified RBPs was analyzed by an online gene annotation & analysis resource ‘Metascape’ (www.metascape.org). All data visualization was implemented in R using the ggplot2 package.For statistical analysis, the R package Limma  was appledu.cn/) . The R p® Ribonuclease Inhibitor (Promega cat. # N2515). Cells were allowed to lyse on a rotator for 20 min at 4°C and then sonicated for 2 min with a 30 s on/30 s off cycle at low intensity on a Bioruptor sonicator (Diagenode) at 4°C. Insoluble material was pelleted by centrifugation at 16 000g for 10 min at 4°C, and the supernatant containing the clarified lysate was split into two portions for pulling down with anti-HA magnetic beads (bimake cat. # B26202) or mouse IgG-conjugated magnetic beads overnight in a rotator at 4°C. After incubation with sample lysate, beads were pelleted, washed three times with RIPA buffer, and then washed with 1× DNase buffer (RNase-free). Beads were resuspended in 100μl DNase buffer (RNase-free). DNase I (RNase-free) was added, followed by incubation at 37°C for 30 min on a rotator. Proteins were then digested by the addition of Proteinase K (Takara cat. # 9034) for about 2 h at 37°C with rotation. After that, MicroElute RNA Clean Up Kit (Omega cat. # R6247-01) was used for RNA purification. Purified RNA was reverse transcribed to cDNA using PrimeScript™ II 1st Strand cDNA Synthesis Kit , and pulldown was quantified with qPCR using ChamQ™ SYBR Color qPCR Master Mix (Vazyme cat. # Q431-03) and the Life Technologies QuantStudio™ 7 Flex. Enrichment was quantified for samples compared with their matched IgG antibody controls. The primers used for RIP-qPCR were: forward primer, 5′-GACAGGCCGAGCCCTCTGC-3′; reverse primer, 5′-GGCTTCAAGGTCTGGGCACAGC-3′.For RNA immunoprecipitation experiments, HEK293T cells were plated in a 6-cm dish and transfected with target protein expression plasmid (labeled with HA-tag at the C-terminus). Thirty-six hours after transfection, proteins were crosslinked to RNA by adding formaldehyde drop-wise directly to the medium to a final concentration of 0.75% and rotating gently at room temperature for 15 min. After crosslinking, 125 mM glycine in PBS was used for quenching, and the cells were incubated for 10 min at room temperature. Cells were washed with ice-cold PBS, harvested by scraping, and the cell suspension was centrifuged at 800 g for 4 min to pellet the cells. Cells were lysed with RIPA buffer supplemented with Protease Inhibitor Cocktail, EDTA-free and Recombinant RNasinHuman cells encode a large number of RNAs, including many non-coding RNAs. These RNAs are expressed differentially in various cells and physiological conditions. However, the functions and regulatory mechanisms of the majority of these transcripts remain unknown. One potential key to understanding is the RNA-binding protein, which is a feature throughout the entire life cycle of RNA , indicating the importance of the study of detailed RNA–protein interactions.We used the CRISPR-based RNA-target Cas nuclease as an RNA tracker to bring the proximity-labeling system to the designated target RNA ,18. CRUIUsing this strategy, proteins that interact with specific RNAs can be labeled in living cells, which avoids the risk of RNA degradation introduced by processing RNA–protein complexes in vitro. In addition, this method avoids over-expressing the target RNA with the MS2-tag sequence in the cell, so the abundance of the target RNA in the cell is in a natural state and the acquired RNA represents the real situation. In short, we capture RNA–protein interactions of specific RNAs via Pup ligase PafA fused to the dLwaCas13a and targeting specific RNA loci with single guide RNAs (sgRNAs) Figure .in vitro and decreasing the impact of RNA degradation.In comparison to the existing methods, CRUIS shows quite a few advantages. First, it provides a simple and effective way to obtain potential RNA-binding proteins of target RNA. Second, CRUIS can identify RNA–protein interactions in a natural state. Finally, CRUIS can label potential RNA-binding proteins in living cells, thereby avoiding the manipulation of RNA dLwaCas13a and PafA, and then cloned the fused dLwaCas13a–PafA gene in-frame with the self-cleaving P2A peptide sequence and EGFP, and the fusion gene driven by a CAG promoter (dLwaCas13a-PafA (referred to as 293T-CRUIS) by the piggyBac transposon system. For 293T-CRUIS cells, it is only necessary to transfect an expression vector of sgRNA and PupE to achieve the labeling of the RNA-binding proteins of target RNAs  compared with the non-targeting sgRNA control group , mRNA processing (GO:0006397), and RNA splicing via transesterification reactions (GO:0000375). Furthermore, the subcellular localization analysis of the identified NORAD-binding proteins also shows a significant enrichment of nuclear proteins . Fu. FuNORADs Figure –C.p21 mRNA and applied CRUIS. Our data from mass spectrometry retrieved putative RBPs for p21 mRNA, some of them are known RBPs of p21 mRNA (marked in red)  . We veri in red) . The enrHere we have described a new effective tool for capturing RNA–protein interactions. The proximity targeting system PUP-IT is brought into contact with an RNA by dCas13a to label the surrounding proteins, providing a powerful tool for finding potential RNA-binding proteins of specific RNAs. This RNA-centric RBP-capturing strategy overcomes the limitations of the existing RNA-centric methods for studying RNA–protein interactions.in vitro, which maximally avoids the possible disruption of RBP complexes. And the low off-target characteristics of LwaCas13a provide a guarantee for CRUIS to accurately binding to the target RNA -tagged dCas9 protein and introduced BirA, which marks FB-tagged dCas9 by biotin for subsequent purification and sequencing . In addiImportantly, our results also show that CRUIS can be universally used for different types of RNA, including lncRNA and mRNA. This characteristic is dependent on dCas13a, which does not show selectivity for the type of RNA. This indicates that CRUIS has broad applicability potentials.Since the guide RNA activity remains difficult to predict and the secondary structure of RNAs remains incomplete, we recommend testing multiple guide RNAs to select potent guides for analysis. Given the differences in length of RNA, theoretically, one sgRNA does not represent the intact pattern of a whole RNA. Nevertheless, CRUIS provides a potential tool for studying RNA-binding proteins at specific positions on target RNAs. For the working window of CRUIS, without considering the secondary structure of RNA, the labeling radius is controlled by the linker length between dCas13a and PafA. In the case of CRUIS, we used a 19-aa linker linked to the C terminus of dCas13a, ∼7 nm. Considering the size of Cas13a, we estimate the labeling radius of dCas13a-PafA is 17 nm, covering about 50 bases if in a stretched conformation.CRUIS still faces some challenges as the spatial structural uncertainty of the target RNA and the efficiency of sgRNAs. Understanding the secondary structure of the target RNA is useful for the effective implementation of CRUIS. The efficiency of sgRNA is an important factor, therefore, efficient sgRNA must be selected before applying CRUIS. Because the CRISPR complex is large, CRISPR-based targeting might affect the structure of the targeted RNA, which in turn changes the patterns of interacting proteins.CRUIS is a hybrid of two molecular machineries, CRISPR-based RNA-targeting Cas nucleases (dCas13a) and a proximity-labeling system (PUP-IT). Our results demonstrate that CRUIS is feasible and specific for the identification of RBPs of both non-coding RNAs and mRNAs. The universality of dCas13a and PUP-IT in mammalian cells promises a broad application prospect. Individual sgRNAs can be used to guide CRUIS to label the RBPs at a certain position on a target RNA. Therefore, CRUIS can potentially ‘cruise’ along the target RNA molecule to generate the RNP atlas of an RNA.gkaa143_Supplemental_FileClick here for additional data file.


Most patients with nonsmall cell lung cancer (NSCLC) were initially diagnosed with distant metastasis. At present, there is no study to clarify the correlation between the primary location of the tumor and the metastasis pattern in advanced NSCLC. So we conducted this study to explored the relationship between the tumor primary location and metastasis pattern in stage IV NSCLC. P values were two-sided, and it was considered statistically significant when P ≤ 0.05.  A total of 19,295 eligible patients were identified from 2010 to 2012 in the SEER database. The main endpoint of our study was overall survival (OS). The survival curves were created by using the Kaplan–Meier method and compared by the usage of the Log Rank test. The clinical variable characteristics were compared by the chi-square test, and multivariate logistic regression analyses were used to evaluate the risk factors on metastasis patterns. All statistical P < 0.001), upper lobe (P < 0.001), lower lobe (P < 0.001) , and middle lobe (P = 0.005). Besides, there was no significant OS difference for patients whose primary location was overlapping lesion (P = 0.226). The results also demonstrated that compared with patients with primary tumor located in the main bronchus, those in the upper lobe were more likely to have brain metastasis (P = 0.01) and lung metastasis (P = 0.024), those in the middle lobe were more prone to develop lung metastasis (P = 0.035) and those in the lower lobe were more apt to cause bone metastasis (P = 0.005) and lung metastasis (P = 0.001). In addition, there was no statistical difference in metastasis patterns among patients with overlapping lesions (P > 0.05).  We found that different proportions of metastatic sites could be found in different tumor primary locations. In addition, the prognosis of lung metastasis was relatively good in patients with tumor location in main bronchus ( Different primary tumor locations might affect the metastasis pattern in patients with stage IV NSCLC. Lung and bronchus cancer is one of the most common types of malignant neoplasms as well as the leading cause of cancer mortality . NonsmalMost patients with NSCLC were initially diagnosed with distant metastasis , 7. BoneIn esophageal cancer and colon cancer, studies , 22 had ∗Stat 8.3.5 software. The inclusion criteria for this study were as follows: only one primary tumor, definite primary location, confirmed bone, brain, liver, or lung metastasis, active follow-up, and complete clinical information, such as age, race, sex, stage, and survival time.The Surveillance Epidemiology and End Results (SEER) program, which is supported by the Surveillance Research Program in the National Cancer Institute's Division of Cancer Control and Population Sciences (DCCPS), provides information on cancer statistics in order to reduce the cancer burden among the US population . We collThis study was primarily based on the SEER database and did not require informed consent as personal identifying information was not included. Our study was approved by the ethics committee of the Shandong Cancer Hospital and was conducted in accordance with the Declaration of Helsinki. We accessed the data from the SEER database with reference number 12356-Nov2017.P values were two-sided and it was considered statistically significant when P ≤ 0.05. The Statistical Product and Service Solutions 22.0  software package was applied for all statistical analyses.The main endpoint of our study was overall survival (OS). The survival curves were created by using the Kaplan–Meier method and compared by the usage of the Log Rank test. We compared the clinical variable characteristics through a Chi-square test and multivariate logistic regression analyses were used to evaluate the relationship between different prognostic factors and metastasis patterns. All statistical T1, T2, T3, and T4 were 11.5%, 28.8%, 25.7%, and 34.0%, respectively. The highest proportion of N stage was N2, which accounted for 47.2%, while the lowest proportion was N1, which merely accounted for 8.2%. As for primary location, the main site was the upper lobe (59.1%), followed by the lower lobe (29.2%), and the rest were the main bronchus (5.5%), middle lobe (5.0%), and overlapping lesion (1.3%) successively. In addition, the number of patients with bone metastasis and lung metastasis was similar, and the least was liver metastasis. Detailed data are shown in In this study, there were 11,360 (58.9%) patients who were ≥65 years old, and nearly three-quarters of patients were a white race. More than half of all patients were male gender. Among these patients, 13,745 (71.2%) were diagnosed with adenocarcinoma, and 5,550 (28.8%) were diagnosed with squamous cell carcinoma. The proportions of the As can be easily seen from the histogram , if the P < 0.001). The curves of patients with brain, liver, or bone metastasis, whose primary location was the main bronchus, almost overlapped (P < 0.001) . The result of P=0.226).Survival curves were generated by the Kaplan–Meier analysis and compared by the usage of the Log Rank test. We found that patients with lung metastasis whose primary location was the main bronchus had a better OS  and lung metastasis  were more likely to occur when the primary site was in the upper lobe than in the main bronchus, but there was no statistical difference between bone metastasis (P = 0.057) and liver metastasis (P = 0.463). Patients with primary site in the middle lobe were more prone to develop lung metastasis  than those in the main bronchus, while there was no difference in bone metastasis (P = 0.139), brain metastasis (P = 0.062), and liver metastasis (P = 0.384). In addition, the results also indicated that bone metastasis  and lung metastasis  more tended to the occurrence of tumors with the primary site in the lower lobe than those located in the main bronchus. However, there was no such trend in brain metastasis (P = 0.149) and liver metastasis (P = 0.550). At the same time, we found that there was no difference in bone metastasis (P = 0.342), brain metastasis (P = 0.252), liver metastasis (P = 0.495), and lung metastasis (P = 0.402) in patients whose primary location was overlapping lesion.Multivariate logistic regression analysis was used to evaluate the relationship between tumor primary location and metastasis pattern. Detailed data are displayed in In this study, we explored the prognostic significance of primary tumor location and metastasis pattern in patients with stage IV NSCLC. Meanwhile, we elucidated the prognostic relevance between both. The results demonstrated that the prognosis of lung metastasis was relatively good in patients with tumor location in the main bronchus, upper lobe, middle lobe, and lower lobe, while the prognosis of liver metastasis was the worst. Compared with patients with primary tumor located in the main bronchus, those in the upper lobe were more likely to have brain and lung metastasis, those in the middle lobe were more prone to develop lung metastasis, and those in the lower lobe were more apt to develop bone and lung metastasis. In addition, there was no statistical difference in metastasis patterns among patients with overlapping lesions.It had been proved that the metastatic site of NSCLC was related to the primary site , 24. GrbA large cohort analysis indicated primary tumor location (main bronchus or nonmain bronchus) played an important role in predicting the metastasis site of lung adenocarcinoma (ADC), which showed that main bronchus location was a predictor of lung ADC metastasis and prognosis . ResearcP=0.033), while liver metastasis was more apt to occur in the lower segment than in the upper segment (P=0.014) [A study had constructed a nomogram that can predict the risk factors of liver and lung metastasis in patients with colon cancer, in which tumor location was an independent risk factor for metastasis . In esopP=0.014) . The ana+T cell infiltration, which might be a potential mechanism for reduced response to immune checkpoint inhibitors (ICIs) monotherapy. In contrast, a meta-analysis [Several studies reported liver metastasis was associated with impaired survival , 29, 30.analysis  showed tanalysis . This miThe advantage of our study is its large population size, which leads to enough power to detect small differences in the results. However, this study had certain limitations. Firstly, as retrospective research of metastatic NSCLC cases, it had its intrinsic shortcomings. Secondly, the information about the therapeutic regimen and progression of metastatic disease was absent. Thirdly, some other variables that might affect survival and prognosis could not be extracted from the SEER database, such as smoking history, performance status, tumor differentiation, and laboratory parameters. In addition, gene mutations were not covered in this database, which might be related to different metastasis patterns and prognosis. Furthermore, we only collected information about lung, liver, brain, and bone metastases but did not count other metastatic patterns, such as adrenal gland, which may lead to the underestimation of other metastatic patterns. Therefore, large-scale prospective studies are needed to further clarify these results.In summary, different primary tumor sites might affect the metastasis pattern of patients with stage IV NSCLC and the prognosis of lung metastasis was relatively good in patients with tumor location in the main bronchus, upper lobe, middle lobe, and lower lobe. The primary site of the tumor was used to predict the metastatic risk and prognosis of patients so as to avoid unnecessary delays in treatment and make full use of medical resources. In addition, prospective studies are needed to fully verify the role of primary tumor location in predicting metastatic patterns.


Gene fusions can generate immunogenic neoantigens that mediate anticancer immune responses. However, their computational prediction from RNA sequencing (RNA-seq) data requires deep bioinformatics expertise to assembly a computational workflow covering the prediction of: fusion transcripts, their translated proteins and peptides, Human Leukocyte Antigen (HLA) types, and peptide-HLA binding affinity. Here, we present NeoFuse, a computational pipeline for the prediction of fusion neoantigens from tumor RNA-seq data. NeoFuse can be applied to cancer patients’ RNA-seq data to identify fusion neoantigens that might expand the repertoire of suitable targets for immunotherapy.https://icbi.i-med.ac.at/NeoFuse/.NeoFuse source code and documentation are available under GPLv3 license at Bioinformatics online. Besides being major determinants of response to immune checkpoint blockers, neoantigens are at the basis of other cancer immunotherapies like personalized cancer vaccines and adoptive T cell therapy  data have been proposed recently (https://sylabs.io) and Docker (https://www.docker.com) images to simplify installation and analysis.Here, we present NeoFuse, a user-friendly pipeline for the prediction of fusion neoantigens from tumor RNA-seq data. NeoFuse is available as Singularity (https://github.com/suhrig/arriba), considering both fusion junctions and 3’ out-of-frame sequences. We chose Arriba because it outperformed competitor prediction methods in the DREAM Somatic Mutation Calling–RNA Challenge (https://www.synapse.org/SMC_RNA). Moreover, it computes a confidence score reflecting the likelihood that a fusion is caused by a tumor-specific genomic rearrangement and is not due to technical artifacts. The third module uses MHCflurry . Although not distributed as part of NeoFuse, netMHCpan  [project n. T 974-B30 to F.F.] and by the European Research Council [advanced grant agreement n. 786295 to Z.T.].Conflict of Interest: none declared. btz879_Supplementary_DataClick here for additional data file.


Penicillium digitatum is the most damaging pathogen provoking green mold in citrus fruit during storage, and there is an urgent need for novel antifungal agents with high efficiency. The aim of this study was to investigate the antifungal effects of peptide thanatin against P. digitatum and the molecular mechanisms. Results showed that peptide thanatin had a prominent inhibitory effect on P. digitatum by in vitro and in vivo test. A total of 938 genes, including 556 downregulated and 382 upregulated genes, were differentially expressed, as revealed by RNA-seq of whole P. digitatum genomes analysis with or without thanatin treatment. The downregulated genes mainly encoded RNA polymerase, ribosome biogenesis, amino acid metabolism, and major facilitator superfamily. The genes associated with heat shock proteins and antioxidative systems were widely expressed in thanatin-treated group. DNA, RNA, and the protein content of P. digitatum were significantly decreased after thanatin treatment. In conclusion, thanatin could inhibit the growth of P. digitatum, and the underlying mechanism might be the genetic information processing and stress response were affected. The research will provide more precise and directional clues to explore the inhibitory mechanism of thanatin on growth of P. digitatum. Penicillium species are particularly serious  and further cause cell agglutination . The duamembrane . HoweverP. digitatum using high-throughput RNA-seq technique.RNA-seq, with an advantage of high sequencing accuracy and depth, can analysis the whole genome scale of samples to reveal changes in cellular processes or metabolic pathways when exposed to variable environmental conditions, which has been successfully used in the gene transcriptional analysis of plant pathogens under fungicide stress . The aimP. digitatum were scraped from potato dextrose agar plates  after cultured 7 day at 25°C. The spore suspension was filtered through a gauze, washed with sterile water, and counted with a hemocytometer. The mycelia were obtained by adding 1 mL fungal spore suspension (1 × 105 CFU mL–1) to 100 mL potato dextrose broth , shaking it (160 rpm), and culturing it at 25°C for 48 h.The fresh spores of –1) was prepared in ultrapure water and adjusted to the suitable concentration before the test and stored at −20°C.Thanatin (GSKKPVPIIYCNRRTGKCQRM) with >95% purity was synthesized by GenScript Corporation  through the solid-phase method using N-(9-fluorenyl) methoxycarbonyl (Fmoc) chemistry. The peptide was provided as white lyophilized (freeze-dried) powder in multiple vials. The stock solution (1 m mol L4 CFU mL–1) were mixed with 20 μL thanatin solution at the final concentrations of 0, 0.5, 1, 2, 4, 8, 16, or 32 μmol L–1, respectively, and the mixture was incubated at 25°C for 48 h. Absorbance was measured at OD600 using the Multiskan Spectrum microplate spectrophotometer . The minimum inhibitory concentration (MIC) value was defined as the lowest concentration of peptide at which no visible growth of spore can be observed in well after 48 h.The germination inhibition effect of thanatin on spores was determined by a dose–response curve using a 96-well assay plate as previously described . In each3 CFU mL–1) were exposed to the peptide at final concentration of 0, 2, 4, 8, 32, 64, and 128 μmol L–1 for 16 h in a 2 mL sterile microcentrifuge tube. The treatment solution 50 μL was evenly spread on PDA solid medium. After incubation at 25°C for 36–48 h, the spore survival rate (%) was calculated by counting conidia of each plate using the following formula.The inhibition effect of conidia survival was conducted as previously described with a few modifications . The spo3 CFU mL–1) and thanatin of 2, 4, 8, 32, 64, and 128 μmol L–1 were incubated for 15, 30, 60, 120, 240, 360, and 540 min; the control group (shown as 0 min) was treated with sterile water and then spread on PDA plates and incubated at 25°C for 36–48 h to calculate the survival rate. All the determinations were performed in triplicate. The minimum fungicidal concentration (MFC) value was defined as the lowest concentration of peptide at which the spore survival rate <1%.The time-kill kinetic assays were carried out using the above-mentioned counting method. The tubes containing spore suspension (2 × 10Citrus sinensis (L.) Osbeck) as previously described with a few modifications (P. digitatum spore suspensions (5 × 104 CFU mL–1) were treated with thanatin at 0 (sterile water), 8 μmol L–1, and 128 μmol L–1 for 2 h at room temperature. Afterward, 10 μL mixture was inoculated into each fruit wound. When the mixture was absorbed, the fruit was individually packed with plastic bags and then stored at 25°C and at 90–95% relative humidity. The disease incidence and lesion diameter were recorded after 3–6 days of inoculation. There were 15 fruit in each treatment, and the experiment was conducted with three replicates.The fruit decay test was carried out on olinda valencia oranges (ications . Fruit w–1) in PBS for 2 h. Total RNA extraction and RNA-seq analysis were carried out by Beijing Genomics Institute (BGI) Co., Ltd. The total RNA was extracted by TRIzol regent  according to the manufacturer’s instruction. The purity was quantified by a NanoPhotometer spectrophotometer . The degradation was checked using 1% agarose gel electrophoresis. The RNA integrity number (RIN) and concentration were measured by Agilent 2100 Bioanalyzer . Three biological replicates for each group were performed for RNA extraction and RNA-seq analysis.The mycelia cultured in PDB at 25°C for 2 day were washed twice with phosphate buffer saline  on a sterile funnel. In order to prevent RNA degradation and to establish a cDNA library, the mycelia were rapidly frozen in liquid nitrogen immediately after the treatment with PBS (as control) or with thanatin .The mRNA was enriched from total RNA by using the oligo (dT) magnetic beads and being exposed to divalent cations and then randomly divided into small fractions. The fragmented mRNAs were collected and carried out following the manufacturer’s instructions of NEBNextP. digitatum Pd1 1. The expression levels were normalized as Fragments Per Kilobase of exon model per Million mapped reads (FPKM) by featureCounts software (version 1.5.0-p3). The correlation coefficient of all genes among the three biological replicates in each group was performed with Pearson method on FPKM value. For selection of differentially expressed genes (DEGs), a normalization step was firstly performed by the DESeq2 software (version 1.16.1) with the read count as the inputting data. The p-value was based on negative binomial distribution model, and the false discovery rate  was calculated by BH method, which was used to select the DEGs. Finally, the following criteria of DEGs were used: |log2(Fold Change)| > 0 and padj < 0.05. . The clusterProfiler software  was applied for the functional enrichment analysis of DEGs mapped into the database of GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes).Quality control for the raw sequencing data was checked by filtering low quality reads, checking for sequencing error, and censoring rate of GC content distribution. The clean reads were mapped to reference genomes using hisat2 software (version 2.0.5), and annotated with P. digitatum treated at 0 or 4 μmol L–1 were used for extracting total RNA for qRT-PCR analysis using the above-mentioned method, and qualified by Nanodrop 2000 Spectrophotometer . The cDNA was reverse transcribed from total RNA at the same insert amount (600 ng) using a kit of PrimeScriptTM RT reagent Kit . The reaction mixture was prepared in a 384-well plate containing 1 μL of cDNA template, 1 μL primer sets (10 μmol L–1), 10 μL of iTaqTM Universal SYBR® Green Supermix  and 7 μL double distilled water. The thermal cycling conditions were as follows: 95°C for 60 s, followed by 40 cycles of 95°C for 5 s and then 60°C for 30 s. The melt curve was performed in a 65–95°C range with increments of 0.5°C every 5 s. The relative expression level was validated on CFX Real-Time PCR Detection Systems , analyzed by the 2Ct–ΔΔ method. Briefly, the Δ Ct value of target gene was normalized by the Ct mean value of the endogenous control, the ΔΔ Ct was calculated, respectively, by the subtraction from the Δ Ct between the control and treated samples. The experiments were performed in triplicate, and results were represented as mean values of 2Ct–ΔΔ ± SD. The primer pair of actin gene was obtained  were incubated for 12 h at 25°C and then treated with thanatin  at 25°C for 2 h and 6 h, respectively. The 50 μL reaction mixture with an equal volume of DAPI  was added into a well of fluorescence plate and incubated for 10 min in dark. The fluorescence of binding of DAPI to DNA and RNA in cells was monitored using the Multiskan Spectrum microplate spectrophotometer  with the following program: excitation wavelength of 364 nm and 400 nm, respectively, and emission at 460 nm. The experiment was repeated three times.The DNA and RNA contents were quantified by 4′,6-diamidino-2-phenylindole (DAPI) binding method according to the methodology P. digitatum spore suspensions  were cultured for 12 h at 25°C, and then incubated with tahantin treatment  at 25°C for 2 h and 6 h, respectively. Cell lysis was performed by adding 0.4 g glass beads in each mixture (600 μL). The sample was then placed on ice and vortexted for 30 s periodically for 15 min, until the mixture was clear. After centrifugation at 10,000 × g for 20 min at 4°C, the 100 μL supernatant was stained with an equal volume of Coomassie Brilliant Blue  for 5 min. The mixture was measured at 595 nm using the Multiskan Spectrum microplate spectrophotometer . The soluble protein contents were determined via a Bradford assay using BSA as the standard. The experiment was repeated three times.The test was conducted with some modifications . The P. p < 0.05. The figures were produced using GraphPad Prism 8.0 .Apart from the experiments analyzed by the bioinformatics software, other data were expressed as mean ± SD by measuring three independent replicates using SPSS 20 software . The differences of the data were compared with the variance (ANOVA), using Duncan’s multiple range test at P. digitatum spores as affected by peptide thanatin were shown in –1 , and the total mapping ratio of each sample was about 94%. The uniquely and exon regions mapping portion of each sample were more than 93% and 75%, corresponding to the standard for transcriptional analysis.The RNA-seq primary quality results of control and treatment groups were summarized in R2) corresponding to each biological replicates of control were over 0.93 , cellular component (CC), and molecular function (MF). The top 10 GO terms in the three categories of downregulated DEGs  and upre3 , the tra3 . A totalKyoto Encyclopedia of Genes and Genomes pathway analysis was performed to uncover enriched biochemical pathways mapped by DEGs. The complete data of the top 20 pathways were shown in the The KEGG subcategories of genetic information processing were annotated to downregulated DEGs, including the following: folding, sorting and degradation , transcription , and translation (ribosome biogenesis in eukaryotes). For the metabolism, four subcategories were clustered by 15 down-pathways and grouped into amino acid metabolism . Additionally, the subcategories of carbohydrate metabolism were inhibited, including glyoxylate and dicarboxylate metabolism, propanoate metabolism, pentose and glucuronate interconversions, pentose phosphate pathway, fructose and mannose metabolism, and butanoate metabolism.The up-pathways of the overexpressed DEGs were mainly assigned to a wide variety of metabolic processes in the sub-classification of lipid metabolism . Some DEGs were upregulated in carbohydrate metabolism, such as amino sugar and nucleotide sugar metabolism, fructose and mannose metabolism, starch and sucrose metabolism, galactose metabolism, and glycolysis/gluconeogenesis.The scatter plots showed the enrichment degree of the top 20 down-  and up-pNine DEGs mapped to the primary pathways were analyzed by qRT-PCR to validate the expression changes of genes. The expression of genes involved in the transcription , translaP. digitatum, the DAPI dye was used to quantify DNA and RNA content  after the incubation of 2 h.To further investigate whether protein synthesis was impaired by the thanatin, we determined the soluble protein content of Penicillium digitatum is one of the most destructive pathogens in citrus fruit. Like other peptides, the insect peptide thanatin exerted prominent inhibition and lethal effect on P. digitatum spores in vitro test (–1) was effective to decrease the fruit decay caused by green mold , TFIIE2 (26232826), TFIIH1 (26232621), and TAF3 (26228750). Among them, TFIIE2 is the small beta submit of TFIIE binding to the core region of promoter upstream of the transcription initiation site, and it can be firmly combined with RNA polymerase II or other BTFs (TFIIE2 (26232826) was significantly suppressed by thanatin based on the qRT-PCR results  encoding some complex functions of RNA polymerase  were downregulated as well. Kti12 is a protein related to RNA polymerase II elongator complex, it is responsible for the transcription elongation and tRNA modification activity (Kti12 (26230750) was obviously downregulated, implying the disfunction of RNA polymerase II occurred by thanatin  related to the 90S pre-ribosome components were downregulated 1.87- to 5.30-fold in thanatin-treated P. digitatum. We analyzed these genes more deeply and found that all the genes encoded U3 small nucleolar RNA-associated proteins (U3 snoRNPs). U3 snoRNP is the core component of 90S particle and can induce the first step to stabilize the ribosome primary structure and then recruit proteins for the structure of 90S particle  involved in the ribosomal biogenesis are responsible for encoding pre-60S particles or pre-40S particles. Protein kinase RIO1 is essential for the final maturation step of ribosomal small subunit converting from the pre-rRNA into the mature 18S rRNA (RIO1 (26236159) was found to be 2.98-fold reduced by thanatin. Only three genes  encoding for the mature structure of ribosome large subunit were found to be affected by thanatin, implying that thanatin mainly targeted on the construction of primary structure of ribosome, rather than the assembly process of mature subunits. As the translation initiation is the rate-limiting factor of the entire translation process in eukaryotes (HexA (26233464), the DEGs related to translation initiation factor  and aminoacyl-tRNA biosynthesis (26230368 and 26232778) were downregulated. In addition, more than 20 downregulated genes participated in various pathways with amino acid biosynthesis and metabolism, such as valine, leucine, and isoleucine degradation; glycine, serine, and threonine metabolism; and alanine, aspartate, and glutamate metabolism. The results indicated the possible mechanisms of thanatin to disturb the normal translation procedure of P. digitatum.Additionally, the other downregulated genes , an ABC transporter ATP-binding protein that functions as an ATPase involving in the 40S and 60S ribosomal biogenesis  are important in stabilizing proteins, and they are recognized as molecular chaperones in response to stressful environmental conditions  in the cells may rapidly increase, and the excessively generated toxic oxygen radicals can react with the essential intracellular biomolecules such as nucleic acids, proteins, lipid causing oxidative injury, and further leading to cell damage and death. Moreover, ROS also act as a signaling molecule in cascades : Penicillium digitatum raw sequence reads, and the six SRA accession numbers were listed as follows: SRR12245251, SRR12245252, SRR12245250, SRR12245253, SRR12245249, and SRR12245254 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA646579/).All the raw data were deposited in NCBI’s Sequence Read Archive. The six sequences were grouped to a BioProject . GF and WW: writing (review and editing). XL: visualization. KZ: supervision. KZ and LD: project administration. KZ and LD: funding acquisition. All authors have read and approved this version of the article.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.


METTL16 also controls a significant portion of m6A epitranscriptome by regulating SAM homeostasis. Multiple molecular structures of the N-terminal methyltransferase domain of METTL16, including apo forms and complexes with S-adenosylhomocysteine (SAH) or RNA, provided the structural basis of METTL16 interaction with the coenzyme and substrates, as well as indicated autoinhibitory mechanism of the enzyme activity regulation. Very recent structural and functional studies of vertebrate-conserved regions (VCRs) indicated their crucial role in the interaction with U6 snRNA. METTL16 remains an object of intense studies, as it has been associated with numerous RNA classes, including mRNA, non-coding RNA, long non-coding RNA (lncRNA), and rRNA. Moreover, the interaction between METTL16 and oncogenic lncRNA MALAT1 indicates the existence of METTL16 features specifically recognizing RNA triple helices. Overall, the number of known human m6A methyltransferases has grown from one to five during the last five years. METTL16, CAPAM, and two rRNA methyltransferases, METTL5/TRMT112 and ZCCHC4, have joined the well-known METTL3/METTL14. This work summarizes current knowledge about METTL16 in the landscape of human m6A RNA methyltransferases.Methyltransferase-like protein 16 (METTL16) is a human RNA methyltransferase that installs m N6-methyladenosine (m6A), has been identified in all RNA classes. m6A affects multiple aspects of RNA processing, including biogenesis, stability regulation, half-life control, pre-mRNA splicing, export, and translation . . 10 (η) 6A methyltransferase activity, indicating their crucial role in methylation  of METTL16 alone shows methyltransferase activity towards the identified RNA substrates of METTL16, hairpins (hp1-hp6) of MAT2A mRNA and (less efficiently compared to full-length METTL16) U6 snRNA ,36. Even1 aa, MTDSuperposition of METTL16-MTD apo form (PDB ID: 6b91), METTL16-MTD/SAH complex (6gfn), and METTL16-MTD/RNA complex (6du4) reveals structural differences in the 163–167 region, the so-called K-loop between the β3 and β4 strands. The K-loop actually forms a short α helix (αC) in structures non-bound to RNA substrate , while in the METTL16-MTD/RNA complex (6du4), it functions as a loop A,B,E. Im6A methylation PP[YFW] located near the SAM binding pocket and the methylated adenosine [6A methylation, as METTL16 N184A, PP185/186AA, and F187G mutants were not able to methylate U6 and MAT2A RNA substrates in vitro  patients [METTL16, may lead to tumorigenesis via their inactivation [6A regulatory genes, including METTL16, influence overall survival rate of patients with soft-tissue sarcoma [METTL16 is considered a protective gene, suppressing the development of OC, HCC, and endocrine system tumors [METTL16 is an essential protein for mammals. Attempts of the ectively . Severalectively ,93,94. Tpatients . In CRC tivation . Copy nu sarcoma . Recentl sarcoma ,99. Decr sarcoma . Thus, Mm tumors ,99,100. m tumors . Finallym tumors .6A methyltransferase. It installs m6A marks on at least two substrates, U6 snRNA and 3′ end hairpins of MAT2A pre-mRNA. METTL16 manifests a rigorous sequential and structural basis of substrate recognition. New structural information regarding this enzyme has been recently revealed, for instance, discovering its autoregulatory function. Last year, we got our first insight into the structure of the METTL16 C-terminal domain. Despite all these fascinating discoveries, METTL16 still keeps secrets, and numerous questions about this protein need to be addressed. For instance, how to explain the broad METTL16 impact on m6A pattern of transcriptome, not entirely mediated by the SAM level regulation? Are there other RNA substrates of METTL16? May unidentified cellular factors expand substrate specificity of METTL16? Does METTL16 universally function as both modification writer and reader, e.g., in splicing regulation? Are there other, methylation-independent, biological functions involving METTL16/RNAs interactions? These and many other questions concerning METTL16 are awaiting answers, opening a broad field for further studies.Our knowledge about METTL16 has expanded significantly during the last five years. We have learned that this protein interacts with multiple RNAs: pre-mRNAs, ncRNAs, lncRNAs, rRNAs. METTL16, next to the four other enzymes: METTL3/METTL14, CAPAM, METTL5/TRMT12, and ZCCHC4, functions as a human SAM-dependent m