Summarized, all these criteria were met, and thus, our analytical platform is considered fit-for-purpose for microRNA biomarker discovery from biofluids in the setting of any diagnostic, prognostic or patient stratification need. The established miND assay was tested on serum, cerebrospinal fluid (CSF), synovial fluid (SF) and extracellular vesicles (EV) extracted from cell culture medium of primary cells and proved its potential to be used across different sample types.The plasma levels of tissue-specific microRNAs can be used as diagnostic, disease severity and prognostic biomarkers for chronic and acute diseases and drug-induced injury. Thereby, the combination of diverse microRNAs into biomarker signatures using multivariate statistics seems especially powerful from the perspective of tissue and condition specific microRNA shedding into the plasma. Although next-generation sequencing (NGS) technology enables one to analyse circulating microRNAs on a genome-scale level, it suffers from potential biases  and lacks absolute transcript quantitation as well as tailor-made quality controls. In order to develop a robust NGS discovery assay for genome-scale quantitation of circulating microRNAs, we first evaluated the sensitivity, repeatability and ligation bias of four commercially available small RNA library preparation protocols. The protocol from RealSeq Biosciences was selected based on its performance and usability and coupled with a novel panel of exogenous small RNA spike-in controls to enable quality control and absolute quantitation, thus ensuring comparability of data across independent NGS experiments. The established  MicroRNAs (miRNAs) are small endogenous non-coding RNAs of 17–25 nucleotides in length that regulate gene expression in mammalian cells. MicroRNAs are produced by virtually all cell types, and their expression can be changed in response to physiological stimuli and pathological processes. MicroRNAs can be measured in both tissues as well as liquid biopsies, including serum and plasma . FurtherThe minimally invasive analysis of microRNAs in liquid biopsies allows for rapid, economical, and repeated sampling. This provides an opportunity for the development of screening programs and close monitoring of treatment response and disease progression . The levThe Translational Safety Biomarker Pipeline (TransBioLine) Project of the IMI2 consortium aims to discover and qualify novel microRNA safety biomarkers for five organ systems  by 2024. Furthermore, TransBioLine aims to characterize inter- and intra-individual variability of circulating microRNAs through investigation of healthy volunteer populations. In order to achieve this goal, a robust assay for identification and quantitation of microRNAs shall be established.Small RNA sequencing is a commonly used next-generation sequencing (NGS) technology for various types of short non-coding RNAs, including microRNAs in tissues and biofluids ,16. SeveThe detection of microRNA by NGS can be affected depending on the technical methods used for library preparation, which will bias the relative abundance of the selected microRNAs across different samples ,21,22. TMicroRNA NGS Data Analysis (miND) spike-in) for microRNA biomarker identification and absolute quantitation in plasma. We then implemented the conversion of read counts to the absolute amounts (amol or molecules/µL) in our analysis and selected the NGS protocol that fulfilled defined criteria including sensitivity, consistency, and ac-curacy of microRNA quantitation. The selected protocol was further optimized and subsequently validated for relative accuracy, analytical measurement range, precision and ligation bias. Finally, the established miND assay was tested in serum, cerebrospinal fluid (CSF), synovial fluid (SF) and extracellular vesicles (EV) extracted from cell culture media of primary cells.In order to reach these aims, we comparatively evaluated four commercial small RNA library preparation protocols in combination with a novel exogenous spike-in control , RealSeq-Biofluids Plasma/Serum miRNA library kit , NEXTFLEX small RNA-seq kit v3  and CleanTag small RNA library prep kit  . These kThree of the selected kits, CleanTag, NEXTFLEX and QIAseq, relied on sequential 5′ and 3′ adapter ligation to microRNAs followed by reverse transcription and PCR amplification. Two of these kits, NEXTFLEX and QIAseq, used random nucleotide sequences that serve as unique molecular indices (UMIs). UMIs enable the removal of duplicated reads introduced during amplification and increase the precision especially for low RNA input samples that require extensive library amplification . The QIATwo types of reference samples were selected for the kit evaluation: RNA isolated from a pool of platelet-poor plasma (PPP) samples obtained from normal healthy volunteers (NHVs) and miRXplore Universal Reference , a synthetic miRNA reference pool comprising 1006 synthetic RNA oligonucleotides in equimolar concentrations matching mature microRNAs annotated in miRBase 9.2 database. PPP samples were used since they represent high quality biological samples  that areOn the basis of these data, we aimed to select the commercial protocol with the best performance in order to establish absolute quantitation of microRNAs using a set of exogenous spike-in RNAs B.In order to evaluate the sequencing bias of the selected protocols, the synthetic equimolar pool of 1006 microRNAs, miRXplore Universal Reference, and the pool of plasma samples were used. First, we compared the sets of microRNAs that were identified by each protocol and assessed their relative values (RPMs). The results of unsupervised clustering analysis demonstrated that the samples were grouped by sample type as well as library preparation protocols A.Although equimolar pooling was performed, the number of absolute reads varied between the NGS protocols. Absolute read counts for the miRXplore samples varied from 10 million to 60 million reads (avg = 22.2 million reads) between the samples and NGS protocols , but theNone of the tested protocols was able to detect all microRNAs that were present in the miRXplore Universal Reference samples. The average number of the detected unique microRNAs varied between the kits, ranging from 501 for CleanTag to 616 for NEXTFLEX (RPM > 0) . The varn = 6) detected for each microRNA in the miRXplore Universal Reference samples and expressed it as the relative fold change in comparison to the median RPM for each protocol  allowing for one mismatch. We selected seven core sequences that demonstrated the lowest overlap with the selected genomes. Each core sequence was flanked by four randomized nucleotides on the 5′ and 3′ ends , each being a 21 nucleotide (nt) long RNA, based on the methodology that was published by Lutzmayer et al., 2017 [ 3′ ends , resultiSpike-ins were then mixed in specific molar ratios to cover the dynamic range of endogenous microRNA concentrations in human plasma samples and diluted into working stocks, which were added to the RNA samples before library preparation and processed with the RealSeq protocol , and miND spike-in counts (RC) and absolute concentrations (molecules/μL) demonstrated high correlation   .The miND spike-ins showed compatibility with both RNA extraction protocols, as the measurement range covered a dynamic range of 5 log10 s for observed RPM values and showed good overlap with read counts from endogenous microRNAs B. The hit-test, p-value < 0.0001). This difference in microRNA abundance was not detectable using relative normalisation (RPM data), emphasizing the advantages of absolute normalisation. The lower RNA extraction efficiency was confirmed by RT-qPCR analyses of 13 low to high abundant microRNAs, which showed that Cq-values from Promega-extracted samples were on average 1.61 Cq-units higher compared to the values from Qiagen-extracted samples  and patients with acetaminophen-induced liver injury (APAP). The APAP samples served as an example of patient samples with perturbed microRNA expression profiles .The plasma pools of NHV and APAP samples were either directly analysed, or APAP plasma was spiked into the NHV samples in the following proportions: 1:1, 1:5 and 1:10 in triplicates. This approach allowed us to simultaneously determine the titration response of multiple microRNAs, thus providing an opportunity to draw a reliable conclusion about the relative accuracy of the assay for different microRNAs.The differential gene expression analyses identified 266 differentially regulated microRNAs (FDR < 0.05) between NHV and APAP samples . Thirty-Next, reproducibility and repeatability of the established NGS protocol were evaluated. We used a pool of RNA isolated from NHV plasma samples that was processed by three operators on three independent days. Thus, a total of nine libraries were generated to assess intra-operator repeatability and inter-operator reproducibility. We found that the coefficient of variation (CV%) across all nine replicates was associated with the mean concentration C. RepeatThe analytical measurement range of the NGS protocol was defined by concentrations of the highest  and the lowest  miND spike-ins. The microRNA profile data, which were generated to evaluate reproducibility and repeatability of the NGS protocol, were used to investigate the average portion of endogenous microRNAs within the analytical measurement range. The miND spike-in E with the lowest concentration was detected in eight out of nine technical replicates. On average, 69.56% of the detectable microRNAs (RPM > 0) were within the analytical measurement range as defined by the miND spike-in D.Next, the sequencing bias of the established NGS protocol was investigated, since the analysis of miRXplore Universal Reference indicated that the RealSeq as well as other tested protocols for small RNA sequencing exhibited sequencing bias B. This bThe calculated increase in concentration that was detected in the spiked-in samples demonstrated variations between the selected microRNAs E. The miThe obtained results allowed us to validate the relative accuracy, precision, and analytical measurement range. Furthermore, it enabled us to characterize the sequencing bias of the established NGS protocol. A better understanding of these parameters of the miND assay ensures the correct interpretation of the obtained results.In order to evaluate the performance of the miND spike-in in different types of biological samples, we analysed plasma, serum, cerebrospinal fluid (CSF), synovial fluid (SF) and extracellular vesicles (EV), which were isolated from cell culture medium of primary human cells in triplicates.All miND spike-in sequences were detected in each of the analysed samples. Furthermore, the selected range of the miND spike-ins covered the range of endogenous microRNAs A. Next, Finally, the established miND spike-in assay also allowed us to estimate the mean number of microRNA molecules detected in 1 µL of biofluid by adjusting to the biofluid input volumes .This experiment demonstrated the broad application spectrum of miND spike-ins for absolute quantitation of small RNAs in various sample types, specifically samples with low RNA input, such as biofluids and EVs. Importantly, the miND spike-ins enabled us to directly compare the absolute abundances of microRNAs extracted from different sample types and improve our understanding of microRNA compositions in different sample types.Here, we reported on the development and characterization of a microRNA Next-Generation-Sequencing Discovery Assay (miND) for the identification of circulating microRNA biomarkers in liquid biopsies using absolute quantitation. Based on the methodology that was developed by Lutzmayer et al. , we geneOur results suggest that the miND assay allows users to determine absolute concentrations of microRNAs in total RNA samples obtained from plasma as well as other liquid biopsies  and, therefore, enables the comparison of data within or across sample types irrespective of differences in RNA composition that would affect relative normalisation.To achieve this, we first performed a systematic comparison of four commercially available small RNA library preparation protocols in terms of sensitivity, consistency, and ligation bias . The kitThe results obtained during the development of this workflow clearly show the impact that individual sample preparation steps can have on the final NGS data. For instance, different library preparation protocols resulted in different sequencing biases B,C and dThe use of spike-in controls to monitor data quality is a common strategy for messenger RNA (mRNA) sequencing ,32 but hIn order to meet this need, we designed and developed the miND spike-ins and demonstrated their compatibility with a commercial small RNA sequencing protocol. The broad concentration range that covers a significant range of concentrations of the endogenous microRNAs enables the application of the miND spike-in with various sample types that exhibit different microRNA concentration ranges . By perfMost importantly, however, absolute quantitation is required to obtain a realistic view of the true amounts of microRNA present in a given biological sample. For example, it allowed us to confirm RT-qPCR results that suggested lower microRNA extraction efficiency for a bead-based protocol compared to precipitation- and column-based RNA purification also by NGS , which wTo verify the utility of this NGS workflow and miND spike-ins, their incorporation into the analysis of larger sample cohorts will be required, and benchmarking against relative normalisation strategies needs to be performed. Nevertheless, our results demonstrate the high potential of spike-ins for quality control of small RNA sequencing experiments and absolute quantitation of microRNAs across different sample types, biological conditions and datasets. Furthermore, the absolute quantitation of circulating microRNAs detected by the miND assay will be leveraged in the TransBioLine project in order to identify sensitive and specific microRNA biomarkers for individual disease states or drug-induced injuries.g for 10 min and 10,000× g for 10 min at 4 °C). The samples were stored at −80 °C.Platelet-poor plasma (PPP) samples from 16 healthy individuals were collected and pooled. The samples were obtained from the placebo period of a clinical study investigating the effects of desmopressin on endothelial activation . The indThe miRXplore Universal Reference 25 , the reference pool comprising more than 950 single-stranded synthetic RNA oligonucleotides in equimolar concentrations matching mature microRNAs annotated in the miRBase 9.2 sequence database, was used. The lyophilized oligonucleotides were dissolved in 28 μL sterile RNase-free water and stored at −80 °C.Usage of −80 °C stored serum and CSF samples for the purposes of this study was approved by the ethics committee of Charité—Universitätsmedizin Berlin (EA1/258/19). Written informed consent was obtained from all subjects.Synovial fluid samples were obtained in accordance with the ethical guidelines of the Declaration of Helsinki. This study was approved by the institutional ethics committee of the Vinzenz Group (registration number: EK10/2020). Informed consent was obtained from all subjects. The samples were stored at −80 °C.g and 2000× g, respectively. After the centrifugation, the medium was filtered through a 0.45 µM filter and concentrated to a volume of around 2.5 mL using Amicon Ultra-15 30 kDa filters . Two millilitres of the concentrated conditioned medium was applied to a qEV2 35 nm size-exclusion chromatography column . Fractions containing extracellular vesicles were collected according to the manufacturer’s recommendation, concentrated using Amicon Ultra-15 10 kDa filters and used for isolation of RNA. The samples were stored at −80 °C.Primary human cells were cultivated in DMEM  supplemented with 10% foetal bovine serum (FBS) and 4 mM L-Glutamine . For generating cell conditioned medium, cells were incubated serum-free for 24 h using DMEM supplemented with 4 mM L-Glutamine. Sixty-five millilitres of conditioned medium was harvested and sequentially centrifuged for 10 min each at 200× The miND spike-ins were designed following the protocols described by Lutzmayer et al. .The spike-ins were synthesized in collaboration with Integrated DNA Technologies (IDT). The lyophilized oligonucleotides were dissolved in sterile RNase-free water to 100 μM. Next, the oligonucleotides were diluted and mixed to obtain the miND spike-in solution containing corresponding attomolar concentrations for each spike-in Total RNA was extracted from plasma, serum, SF, and CSF samples using the miRNeasy Mini kit following the manufacturer’s protocol . Briefly, aliquots of 200 μL were lysed in QIAzol Lysis reagent, followed by incubation with chloroform. After the phase separation, the upper aqueous phase was mixed with glycogen  and subjected to automated RNA purification in a QIAcube. Total RNA was eluted in 30 μL of nuclease-free water and stored at −80 °C.g for 5 min to remove cellular debris. For each sample, 200 μL of plasma were consequently mixed with the following reagents: 200 μL of homogenization solution, 200 μL of lysis buffer and 15 μL of Proteinase K. Next, samples were incubated for either 15 min or 2 h on a heat block at 37 °C, 300 rpm. After the incubation, samples were transferred to RSC cartridges followed by automated RNA extraction with the Maxwell instrument. Finally, total RNA was eluted in 50 μL nuclease-free water and stored at −80 °C prior to further analyses.Total RNA was extracted from 200 μL of plasma samples with Maxwell RSC miRNA Tissue kit  according to the manufacturer’s protocol. Briefly, samples were thawed on ice and centrifuged at 12,000× The RNA samples isolated from the plasma pool and miRXplore Universal reference were used for small RNA library preparation with four kits: NEXTFLEX Small RNA-Seq kit v3 for Illumina platforms , QIAseq miRNA Library kit , CleanTag small RNA library prep kit  and RealSeq-Biofluids NGS library preparation kit for miRNAs and small RNAs for total RNA samples from biofluids . The maximal possible sample volume of plasma RNA was used as an input for each protocol. One microlitre of either nuclease-free water or miND spike-in were added to each reference sample before the library prep. One picomole of miRXplore Universal Reference was used for each library prep. The input of plasma RNA for library preparation was 9.5 μL for NEXTFLEX, 4 μL for QIAseq, 1 μL for CleanTag and 8.5 μL for RealSeq kits. The adapters were pre-diluted to account for low miRNA abundance in plasma samples.The library preparation workflows were performed for NEXTFLEX, QIAseq, CleanTag and RealSeq kits according to the manufacturer’s protocols. For NEXTFLEX and RealSeq protocols, adapters were pre-diluted 1:4 and PCR amplification was performed for 23 cycles. For QIAseq, PCR adapters were pre-diluted 1:5 and amplification was performed for 23 cycles. For CleanTag, PCR adapters were pre-diluted 1:12 and amplification was performed for 22 cycles.In total, 54 microRNA libraries were prepared and analysed for library fragments distribution on an Agilent DNA 1000 kit  with Agilent DNA1000 reagents .The generated libraries were pooled in equimolar proportion, and the obtained pool was size-selected with the BluePippin system using 3% agarose cassette, 100–250 kb  to remove DNA fragments outside of the target range. The pooled and purified libraries were analysed for fragment distribution with the Agilent High Sensitivity DNA kit  with Agilent High Sensitivity DNA reagents .The library pool was sequenced on an Illumina NextSeq550  according to the manufacturer’s protocol.cDNA was synthesized from total RNA using the miRCURY LNA RT kit . The samples were processed according to the manufacturer’s specifications. In total, 2 μL of total RNA were used per 10 μL reverse transcription (RT) reaction. PCR amplification was performed using a 96-well plate format on a Roche LC96 instrument (Roche Diagnostic) using miRCURY LNA SYBR Green PCR kit  with the following settings: 95 °C for 2 min, 45 cycles of 95 °C for 10 s, and 56 °C for 60 s, followed by melting curve analysis. To calculate the cycle of quantitation values , a combination of the 2nd derivative maximum and the fits point method  was used. Cel-miR-39 was added to the total RNA sample prior to reverse transcription and qPCR to measure the efficiency of cDNA synthesis.The bioinformatic analyses were performed with the software package miND, a data analysis pipeline that generates overall QC data, unsupervised clustering analysis, normalised microRNA count matrices, and differential expression analysis based on raw NGS data. Overall quality of the next-generation sequencing data was evaluated automatically and manually with fastQC v0.11.8  and multThe subsampling was performed using Seqtk v1.3 tool .www.graphpad.com (accessed on 13 December 2021).) [Data processing and analyses were conducted using Microsoft Excel version 16.57  and Grap 2021).) . Data we 2021).) .We established and validated the miND assay that enables one to perform absolute normalisation of small RNA sequencing data based on the concentrations of exogenous spike-ins.A patent was filed for the invention related to novel spike-in oligonucleotides for use in quantitative normalisation of nucleotide sequence data and was granted in the EU under EP3354746B1.


The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Previous structures of the RdRp revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the enzyme and resolve its structure at 5.5 Å resolution. In this structure, the two RdRps contain only one copy of nsp8 each and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs. Jochheim et al report an alternative, dimeric form of the coronavirus SARS-CoV-2 RNA dependent RNA polymerase (RdRp), which is used to replicate and transcribe its genome. They resolve its structure at 5.5 Å resolution and speculate that the dimer facilitates template switching during production of sub-genomic RNAs. Following the structure of the RdRp of SARS-CoV6, structures of the RdRp of SARS-CoV-2 were obtained in free form7 and as a complex with bound RNA template–product duplex11. These structures revealed a monomeric RdRp with a subunit stoichiometry of one copy of the catalytic subunit nsp1212, two copies of the accessory subunit nsp813, and one copy of the accessory subunit nsp714. Two studies additionally observed monomeric RdRp lacking one of the two nsp8 subunits and nsp79. Here we show that the RdRp of SARS-CoV-2 can also adopt a dimeric form with two RdRps arranged in an antiparallel fashion.Replication and transcription of the RNA genome of the coronavirus SARS-CoV-2 rely on the viral RNA-dependent RNA polymerase (RdRp)11), we detected many particles that showed RdRps with a preferred NN distance of 80 Å and a relative orientation of 180° . We calculated nearest-neighbor (NN) distances and relative orientations of neighboring RdRp enzymes. In one of our published data sets  and the sliding pole of the remaining nsp8 subunit  2 heterotetramer17.The structure of the antiparallel RdRp dimer showed that the two polymerases interact via their nsp7 subunits, with the nsp7 helices α1 and α3  contacting each other Fig. . To our 18, coevolved with the globally dominating spike protein mutation D614G19, is often found in severely affected patients20 and is predicted based on the structure to stabilize nsp12 association with nsp8a21. In contrast, the nsp7 mutation S25L is predicted to destabilize nsp7 binding to nsp8a21 and the nsp7 mutation L71F, which is associated with severe COVID-1922, may destabilize binding of the nsp7 C-terminal region to nsp8b. We speculate that mutations in RdRp subunits can influence the relative stabilities of the RdRp monomer and dimer.Frequently occurring mutations in the SARS-CoV-2 genome are predicted to influence formation of the RdRp dimer. The nsp12 mutation P323LNeither the catalytic sites nor the RNA duplexes are involved in RdRp dimer formation. It is therefore likely that the two RdRp enzymes remain functional within the dimer structure. The two RdRp enzymes in the dimer may thus be simultaneously involved in RNA-dependent processes. Unfortunately, we could not test whether the RdRp dimer is functional because we were unable to purify it. In particular, we attempted to reconstitute the RdRp with a nsp12:nsp8:nsp7 stoichiometry of 1:1:1, but obtained preparations showed again an apparent stoichiometry of 1:2:1 that was observed in previous RdRp structures. Therefore, the functional relevance of the RdRp dimer reported here needs to be established.25. In this intricate process, positive-strand genomic RNA (gRNA) is used as a template to synthesize a set of nested, negative-strand sgRNAs that are 5′ and 3′ coterminal with gRNA. The obtained sgRNAs are later used as templates to synthesize viral mRNAs. Production of sgRNAs involves a discontinuous step, a switch of the RdRp from an upstream to a downstream position on the gRNA template26. These positions contain transcription regulatory site (TRS) sequences29, but it is enigmatic how a single RdRp enzyme could ‘jump’ between these.We hypothesize that the RdRp dimer is involved in the production of sub-genomic RNA (sgRNA)31  at the 5′-end of the template. The resulting RNA duplex could then bind to the active center of the second RdRp (RdRp 2) to continue sgRNA synthesis.Our dimer structure suggests a model for sgRNA synthesis that extends a recent proposal31 Fig. . In the 32 can be modeled on our dimer structure without clashes  at 4 °C. Peak fractions corresponding to the RdRp–RNA complex were pooled and diluted to ~2 mg/ml. Three microliters of the concentrated RdRp–RNA complex were mixed with 0.5 µl of octyl ß-d-glucopyranoside  and applied to freshly glow discharged R 2/1 holey carbon grids (Quantifoil). The grids were blotted for 5 s using a Vitrobot MarkIV (Thermo Fischer Scientific) at 4 °C and 100% humidity and plunge frozen in liquid ethane.We reused the already processed data set 3 from our previous publication11. Briefly, data was collected using SerialEM37 on a 300 keV Titan Krios transmission electron microscope (Thermo Fischer Scientific) and a K3 direct electron detector (Gatan). Inelastically scattered electrons were filtered out prior to detection using a GIF quantum energy filter (Gatan) using a slit width of 20 eV. Images were acquired at a nominal magnification of 105,000x and a calibrated pixel size of 0.834 Å/pixel. Due to previously observed preferred orientation when imaging RdRp complexes10, data was collected using a 30° tilt to obtain more particle orientations. 7043 raw micrographs were acquired in total and preprocessed on-the-fly in Warp for automatic contrast transfer function (CTF) estimation, averaging, motion correction, and particle prediction and extraction. 2.2 million individual RdRp particles were predicted and exported by Warp and imported into cryoSPARC and subjected to a Hetero Refinement job using five ‘Junk‘ classes and one class representing monomeric RdRp as described previously11. The resulting particle set was used for a 3D homogeneous refinement to obtain refined poses and positions for each RdRp monomer.Data collection and preprocessing was the same as previously describedx-axis. We chose to express relative orientation through a single angle by calculating the angle of the rotation around the eigenvector of the rotation matrix. This showed that certain distances and relative orientations between two monomers were highly prevalent  distances and the relative orientations for all neighboring RdRp complexes using the previously refined monomer poses. To account for the tilted data acquisition, we treated the influence on distances observed on the micrograph as a 30° rotation around the 38 using the monomeric RdRp–RNA structure10 filtered to a resolution of 30 Å as a 3D reference to pick further monomers that might have been missed due to their proximity to other particles. Template-based picking of monomeric RdRp however did not introduce any model bias that could influence the dimer structure. Second, we used the previously established NN distance and relative orientation to predict for each RdRp monomer the position where its partner in a dimeric particle should be located on the micrograph. From our NN search we obtained the 3D offset that the second monomer should adopt in a dimeric particle .Because the yield of dimeric particles depended on both halves of a dimer being first detected as monomer, we aimed to detect more RdRp monomers using two strategies. First, we carried out template-based picking and particle extraction in RELION39 (Supplementary Fig. 11, removal of mobile regions and fitting of domains as rigid bodies was done in UCSF Chimera40.Final dimer coordinates were used to extract particles using a box size of 300 Å and a pixel size of 1.201 Å. 2D classification in cryoSPARCFurther information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


GenoLab M is a recently established next-generation sequencing platform from GeneMind Biosciences. Presently, Illumina sequencers are the globally leading sequencing platform in the next-generation sequencing market. Here, we present the first report to compare the transcriptome and LncRNA sequencing data of the GenoLab M sequencer to NovaSeq 6000 platform in various types of analysis.We tested 16 libraries in three species using various library kits from different companies. We compared the data quality, genes expression, alternatively spliced (AS) events, single nucleotide polymorphism (SNP), and insertions–deletions (InDel) between two sequencing platforms. The data suggested that platforms have comparable sensitivity and accuracy in terms of quantification of gene expression levels with technical compatibility.Genolab M is a promising next-generation sequencing platform for transcriptomics and LncRNA studies with high performance at low costs.The online version contains supplementary material available at 10.1186/s12864-021-08150-8. The past dozens of years have witnessed a new era in functional genomics using sequencing technologies . The lauIn the short history of NGS era, many sequencing platforms have emerged: Roche 454, Illumina series  , Ion TorRecently, GeneMind Biosciences Company Limited (GeneMind), launched a new sequencing instrument (GenoLab M™) based on their previous work on GenoCare™ single molecule sequencer . An overNovaSeq 6000, launched in June, 2017, relies on Illumina’s SBS chemistry and two-color reversible terminator-based method. Combined with patterned flow cell technology [Previously, GenoLab M’s performance on transcriptome and LncRNA has not yet been evaluated by the scientific community. Here, we characterized the performance of GenoLab M on transcriptome and LncRNA by parallel comparison with NovaSeq 6000 from Illumina, Inc. on three different species: mouse, bean, and human. The raw data quality, gene expression level, alternatively spliced (AS) events, single nucleotide polymorphism (SNP), and insertions–deletions (InDel) analysis from the two sequencing platforms are compared. The data suggest that the GenoLab M is a promising sequencing platform for transcriptomics and LncRNA studies in animal, plant and human with comparable performance at lower cost.Mouse testicular tissue, human Lieming Xu-2 cells and bean hairy root tissue were collected for RNA extraction. RNAs were extracted by HiPure Universal RNA Mini Kit . Total RNA concentration and purity and integrity were measured via NanoDrop 2000  and RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system , respectively.Transcriptome library construction were performed by Hieff NGS Ultima Dual-mode mRNA Library Prep Kit for Illumina (Yeasen Biotechnology (Shanghai) Co., Ltd., China), Fast RNA-seq Lib Prep Module for Illumina , TIANSeq Stranded RNA-Seq Kit (Illumina) (TIANGEN Biotech (Beijing) Co., Ltd., China) and VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina . These mRNA libraries were marked as Mouse, Human or Bean_mRNA_YS, AB, TG or VZ. LncRNA library were constructed via Hieff NGS Ultima Dual-mode RNA Library Prep Kit for Illumina and Hieff NGS MaxUp rRNA Depletion Kit (human/mouse/rat) (Yeasen Biotechnology (Shanghai) Co., Ltd., China), VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina and Ribo-off rRNA Depletion Kit (Human/Mouse/Rat) , TIANSeq Stranded RNA-Seq Kit (Illumina) and TIANSeq rRNA Depletion Kit (H/M/R) (NR101-TA) (TIANGEN Biotech (Beijing) Co.,Ltd.,China). These libraries were marked as Mouse or Human_LncRNA_YS, VZ or TG. After library QC, they were subjected to NovaSeq 6000 and GenoLab M sequencing in PE150 or PE100 mode.ftp://ftp.ensembl.org/pub/release-101/fasta/homo_sapiens/dna/, ftp://ftp.ensembl.org/pub/release-101/fasta/mus_musculus/dna, and ftp://ftp.ensemblgenomes.org/pub/plants/release-48/fasta/glycine_max/dna/. StringTie [Raw sequencing reads in fastq format were processed through a GeneMind in-house perl pipeline. Reads containing adapter, ploy-N or low-quality reads were filtered out to get clean reads. These clean reads were then mapped to the reference genome sequence with a perfect match or one mismatch method via HISAT2 tools software . The cortringTie  was thentringTie , furthertringTie  was usedtringTie . ExpresstringTie .For LncRNA identification, bioinformatic pipeline was performed according to published methods  with minFollowing RNA extraction, two aliquots of each extract were constructed as Illumina libraries, respectively, using identical amounts of starting material, and then subsequently sequenced to facilitate bioinformatic comparisons on the data. In addition, to verify the compatibility of the library preparation kit for GenoLab M, we used kits from different manufacturers for testing  is gaining more and more attention in recent years. In this context, the ability of each sequencing platform under comparison to detect splicing junctions and corresponding alternative splicing patterns were subsequently analyzed across transcriptomes. In mouse, 53,557, 59,709 and 53,014, 56,741, 64,105 and 48,089 AS events could be detected by GenoLab M and NovaSeq 6000, respectively. Top three AS events in all libraries were TSS: Alternative 5′ first exon (transcription start site), TTS: Alternative 3′ last exon  and AE: Alternative exon ends  cross two platforms  and can capture genes, AS, and SNP at transcriptome and LncRNA levels. The GenoLab M offers a cost-effective alternative to the NovaSeq 6000 platform with similar data quality.Additional file 1.


RNA structures can form functional elements that play crucial roles in the replication of positive-sense RNA viruses. While RNA structures in the untranslated regions (UTRs) of several picornaviruses have been functionally characterized, the roles of putative RNA structures predicted for protein coding sequences (or open reading frames [ORFs]) remain largely undefined. Here, we have undertaken a bioinformatic analysis of the foot-and-mouth disease virus (FMDV) genome to predict 53 conserved RNA structures within the ORF. Forty-six of these structures were located in the regions encoding the nonstructural proteins (nsps). To investigate whether structures located in the regions encoding the nsps are required for FMDV replication, we used a mutagenesis method, CDLR mapping, where sequential coding segments were shuffled to minimize RNA secondary structures while preserving protein coding, native dinucleotide frequencies, and codon usage. To examine the impact of these changes on replicative fitness, mutated sequences were inserted into an FMDV subgenomic replicon. We found that three of the RNA structures, all at the 3′ termini of the FMDV ORF, were critical for replicon replication. In contrast, disruption of the other 43 conserved RNA structures that lie within the regions encoding the nsps had no effect on replicon replication, suggesting that these structures are not required for initiating translation or replication of viral RNA. Conserved RNA structures that are not essential for virus replication could provide ideal targets for the rational attenuation of a wide range of FMDV strains.IMPORTANCE Some RNA structures formed by the genomes of RNA viruses are critical for viral replication. Our study shows that of 46 conserved RNA structures located within the regions of the foot-and-mouth disease virus (FMDV) genome that encode the nonstructural proteins, only three are essential for replication of an FMDV subgenomic replicon. Replicon replication is dependent on RNA translation and synthesis; thus, our results suggest that the three RNA structures are critical for either initiation of viral RNA translation and/or viral RNA synthesis. Although further studies are required to identify whether the remaining 43 RNA structures have other roles in virus replication, they may provide targets for the rational large-scale attenuation of a wide range of FMDV strains. FMDV causes a highly contagious disease, posing a constant threat to global livestock industries. Such weakened FMDV strains could be investigated as live-attenuated vaccines or could enhance biosecurity of conventional inactivated vaccine production. The genomes of RNA viruses not only encode proteins but also contain nontemplated functional elements in both the coding and untranslated regions (UTRs). These elements can be secondary or higher-order RNA structures such as simple stem-loops or more complex structures which include pseudoknots and so-called kissing loops that mediate long-range RNA-RNA interactions –12. The –Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease (FMD), a highly contagious disease of cloven-hoofed animals (including livestock)  which is flanked by 5′ and 3′ UTRs (reviewed in reference pro and 3Cpro) and by a ribosomal skipping event mediated by the 2A peptide into a number of functional precursors and the mature proteins . The P1 region encodes the capsid proteins , while the P2 and P3 regions encode the nonstructural proteins (nsps)  acts as a template for uridylylation of the VPg (3B) protein, which then acts as a primer for synthesis of viral RNA  that are essential for replication of an FMDV subgenomic replicon, suggesting that these structures are required for either initiation of viral RNA translation and/or viral RNA synthesis. In contrast, mutagenesis of the remaining 43 structures had no effect on replicon replication. This approach can aid in the identification of critical viral RNA structures required for viral genome replication and also help identify conserved RNA structures that are not essential for virus replication that could provide ideal targets for the rational attenuation of a wide range of FMDV strains.A number of other secondary RNA structures have been predicted computationally to be present within the FMDV ORF . HoweverWhile previous studies have provided evidence that the FMDV genome is highly structured with conserved RNA base pairing throughout the coding part of the genome , 56, the10.1128/mSphere.00015-21.8TABLE S1Table S1, PDF file, 0.02 MB.FMDV sequences selected from GenBank. Download © Crown copyright 2021.2021Crownhttps://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro folded RNA sequences  of the most thermodynamically stable equences –59. Howequences ––62, in wUsing this approach, all 118 FMDV genomic sequences in their genomic (positive-sense) orientations were scanned using the program Fold Energy Scan in the SSE package  to calcu10.1128/mSphere.00015-21.1FIG S1cre, and IRES) or 3′ UTR (SL1 and SL2) were visualized using the Forna web service. Download FIG S1, PDF file, 0.2 MB.Conserved whole-genome RNA structure was predicted for 118 FMDV field isolates as described in the legend to © Crown copyright 2021.2021Crownhttps://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the x and y axes . Therefore, RNAalifold program, implemented in the ViennaRNA Package , was used y axes . Any paid y axes . The pred y axes .10.1128/mSphere.00015-21.9TABLE S2Table S2, PDF file, 0.04 MB.List of the conserved stem-loops located in the ORF of the FMDV genome. Download © Crown copyright 2021.2021Crownhttps://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the cre, all located in the 5′ UTR, and SL1 and SL2 located in the 3′ UTR  reporter gene that replaced the region encoding the capsid proteins .In order to mutate the conserved RNA structures predicted within the regions encoding the nsps while maintaining codon composition, codon order, and dinucleotide frequencies of the native wild-type (WT) replicon sequence, we applied the CDLR scrambling method , 56. To To identify functional RNA structures, we divided the regions encoding the nsps of the WT replicon (ptGFP replicon) into nine consecutive fragments defined by unique restriction sites and individually permuted each fragment using the CDLR algorithm  and B. T10.1128/mSphere.00015-21.2FIG S2FIG S2, PDF file, 0.4 MB.RNA structure for the whole genome of the WT replicon and a replicon with the 2C-encoding region permuted by the CDLR algorithm was predicted using RNAfold. The predicted structure of the 2C-encoding fragment was visualized using the Forna web service. The lines represent unstructured regions, and the numbers at the beginning and end of each individual stem-loop correspond to the genomic positions of each replicon. Nucleotides forming stems are shown in green, interior loops are shown in yellow, junctions in red, and hairpin loops in blue. Download © Crown copyright 2021.2021Crownhttps://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00015-21.3FIG S33-encoding region permuted by the CDLR algorithm was predicted using RNAfold. The predicted structures of SL1 and SL2 stem-loops located in the 3′ UTR were visualized using the Forna web service. The numbers correspond to the genomic positions of each replicon. Nucleotides forming stems are shown in green, interior loops are shown in yellow, hairpin loops in blue, and the unpaired regions in orange. Download FIG S3, PDF file, 0.3 MB.RNA structure for the whole genome of the WT replicon and a replicon with the Δ1D-3D© Crown copyright 2021.2021Crownhttps://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the polGNN, previously described in reference pol-encoding region -21 cells routinely used to grow FMDV for vaccine production and Madin-Darby bovine kidney (MDBK) cells which originate from a natural host of FMDV and appear to have functional antiviral responses  Fig. 3CFig. 3C. kinetics .10.1128/mSphere.00015-21.4FIG S43]), WT ptGFP (orange), or ptGFP-GNN (gray) was introduced into BHK-21 or MDBK cell monolayers. Results are the means of three independent experiments ± standard errors (error bars). Download FIG S4, PDF file, 0.2 MB.Replication kinetics of FMDV replicon constructs containing the CDLR-permuted regions that were described in the legend to © Crown copyright 2021.2021Crownhttps://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00015-21.5FIG S5FIG S5, PDF file, 0.3 MB.Replication kinetics of FMDV replicons containing larger sections of the region permuted by the CDLR algorithm. (A) Schematic representation of CDLR replicons encoding larger mutated regions. Mutated inserts were cloned directly into the ptGFP replicon using the unique restriction enzymes shown. (B) IncuCyte data represent the average cell (green object) GFP intensity per well over a period of 12 h within BHK-21 cells and MDBK cells. Results are the means of three independent experiments ± standard errors. Download © Crown copyright 2021.2021Crownhttps://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 3 region of the FMDV genome contains conserved secondary RNA structures that may be necessary for replication of the FMDV replicon. Therefore, the RNA structures present in this region were investigated in more detail by visualizing each individual structure and comparing it to the corresponding scrambled region within the CDLR mutant. Analysis of corresponding sequences of FMDV field isolates (over the 3D3 region) revealed five stem-loops (ORF-SL49 to ORF-SL53) with strong nucleotide pairing conservation, with ORF-SL52 being the most conserved structure  and the replicon with the 3D3 region mutated by the CDLR algorithm (mut) demonstrated replication comparable to the ptGFP-3DpolGNN replication-deficient control  was desed above . As preved above . Replicaed above . In conted above . In bothlgorithm . Replicalgorithm . Finallynput RNA ).10.1128/mSphere.00015-21.6FIG S6FIG S6, PDF file, 0.2 MB.Replication kinetics of FMDV replicon constructs containing the individual stem-loop mutants that were described in the legend to © Crown copyright 2021.2021Crownhttps://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the mut), which have not been predicted before. Since mutation of ORF-SL49 and ORF-SL50 had no apparent effect on replicon replication, this replicon showed the effect of combined mutagenesis of the ORF-SL52 and ORF-SL53. While in BHK-21 cells ORF-SL49-50,52-53mut replicon was attenuated at a level similar to that of the replicon containing mutation of the ORF-SL52, in the MDBK cells ORF-SL49-50,52-53mut replicon showed replication levels even lower than the replicon with an inactive polymerase , or all three loops disrupted (ORF-SL51-53mut) were tested (mut), or in combination with ORF-SL52 and ORF-SL53 (ORF-SL51-53mut) resulted in replication levels comparable to that of the ORF-SL49-53mut negative control  resulted in a significant reduction of replicon replication in both cell lines  mutated  to the PV loops α and β using RNAforester. As described in pol do not appear to resemble those found in the equivalent position of the PV genome, while (using the same approach) the cre structures of PV and FMDV showed some structural similarity.Two stem-loops  necessapol . Since PPicornaviridae   were critical for efficient replication of an FMDV replicon, thereby implying that they would provide the same function during virus replication.Many aspects of FMDV replication remain poorly understood, such as the function of RNA structures found within the ORF. Here, we revisited the RNA structural architecture of the FMDV genome and, for the first time, investigated whether the putative stem-loops localized within the ORF are required for viral genome replication. Our results are in line with previous studies showing that FMDV has extensive RNA structure throughout the genome, substantially exceeding that found in viruses of other genera of the family hovirus) , 56, 62.hovirus) , our stuDespite consistently elevated MFED values, the FMDV capsid-encoding region contained only four RNA stem-loops which were conserved in all serotypes. Viral genomes characterized by high MFED values and low conservation of individual RNA structures have been observed before . For ins70–in vitro.To identify functional RNA structures, we applied the CDLR algorithm to permute a genomic FMDV sequence . While tpol-encoding region that were important for replication of the FMDV replicon. Of these structures, ORF-SL52 showed the highest degree of pairing conservation and appeared to be the predominant structure important for replication of the FMDV replicon. Mutation of ORF-SL51, ORF-SL52, or ORF-SL53 showed a much greater reduction of replicon replication in MDBK cells compared to BHK cells. MDBK cells have been shown to secrete high levels of interferon (IFN) upon stimulation  was surprising. The maintenance of extensive conserved internal base pairing and consistently elevated MFED values observed in the relatively diverse set of FMDV isolate sequences analyzed indeed strongly argues that the RNA structures formed by those genomic regions must play some functional role in the FMDV replication cycle. It is possible that at least some of the apparently “nonfunctional” RNA structures are genome-scale ordered RNA structure (GORS) which may play a role in persistence of FMDV in its natural host (Syncerus caffer), which are a natural reservoir of the virus  could be used to enhance biosafety of inactivated vaccine production. The hazards associated with the large-scale production of killed vaccine viruses include both accidental release of virus from high containment production facilities, and the distribution and use of improperly inactivated FMD vaccines –84.–pol that appear critical for FMDV replication. While the function of the other conserved structures remains to be determined, they can be targeted to improve understanding of the FMDV biology. In addition, they have the potential to help develop safer FMDV vaccines, an idea which has been proposed for other viruses  and baby hamster kidney (BHK-21) cells were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle medium (DMEM) containing either 10% fetal bovine serum (FBS) or 10% horse serum (MDBK cells) at 37°C and 5% COFull genome sequences of 105 viruses were selected from GenBank database based on nucleotide distance of their 1D-encoding region, ensuring that they represent sequence variability that is known to be present between all seven FMDV serotypes see .The genomic sequences of the 118 FMDV field isolates were aligned using the MAFFT X-INS-i algorithm which, in addition to nucleotide identity, takes into account RNA secondary structure information , 87. ThePairwise distance and MFED for full genome sequences were prepared using the Sequence Distances and Folding Energy Scan programs implemented in SSE v1.4 package , respectThe average MFED values of the regions encoding the nsps of the FMDV isolates, the ptGFP replicon, and 50 CDLR-permuted ptGFP mutants were calculated as described above.Since there appears to be a lot of ambiguity around the poly(C) tract, that region and its flanking positions were excluded from all analyses.The regions encoding the nsps of the FMDV genome were chosen for mutagenesis by restriction site usage  and B. Tpol-encoding region of the WT replicon which was scrambled by the CDLR algorithm (the 3D3 region) was generated as described above but using RNAfold , nucleotides were changed manually. Individual putative stem-loops and their mutants were predicted using RNAfold implemented in the ViennaRNA package and mfold RNA structure prediction server  were used. The RNA loops α and β were isolated, and their structures were aligned to the 3′-terminal part of the 3Dpol-encoding region containing FMDV stem-loops ORF-SL49 to ORF-SL53 using the RNAforester software and “small-in-large similarity” calculation to determine whether any of the previously described PV stem-loops were similar to any of the FMDV RNA structures identified in this study. For more detailed analysis, each isolated FMDV putative RNA stem-loop (ORF-SL49 to ORF-SL53) was directly compared to PV loop α or β using RNAforester as described above.Computational prediction of two conserved poliovirus (PV) RNA structures located in the 3′-terminal 3Dg et al. ) and desSequences with mutations generated by the CDLR algorithm were synthesized by custom DNA synthesis  and provided within standard cloning vectors. Since the restriction sites flanking nine CDLR regions  were notThe nucleotide fragments containing mutated stem-loops ORF-SL49 to ORF-SL53 were synthesized as described above and directly cloned into the ptGFP replicon.Replicon constructs (5 μg) were linearized with AscI (New England Biolabs) for 1 h at 37°C and purified using the E.Z.N.A. gel extraction kit (Omega Bio-Tek). Linear replicon DNA (500 ng) was added to transcription reaction mixtures at a final volume of 100 μl containing the following: Transcription Optimized buffer (Promega), 10 mM dithiothreitol (DTT) (Promega), 100 U RNasin RNase inhibitor (Promega), 40 U T7 RNA polymerase (Promega), 20 mM ribonucleotide triphosphates (rNTPs) (Promega), and nuclease-free water. Reaction mixtures were incubated at 37°C for 2 h, and the resulting transcript integrity was assessed by agarose gel electrophoresis. RNA yield was quantified using the Quantus fluorometer (Promega), according to the manufacturer’s instructions.Approximately 20 h prior to transfection, cells were seeded into 24- or 12-well plates at the appropriate cell seeding density to achieve ∼80% confluence. The following day, medium was removed and replaced with FluoroBrite DMEM (Gibco) supplemented with 2% FBS and 4 mM glutamine. Replicon transcript RNA (0.5 to 1 μg) was transfected into triplicate or quadruplicate cell monolayers using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) per the manufacturer’s recommendation.Live cell image analysis was performed using the IncuCyte ZOOM kinetic imaging system (Essen BioScience) as described previously . Images P values of <0.05. Error bars represent standard errors of the means (SEM) of multiple independent experiments. Statistical analyses were performed with GraphPad Prism 8.00 .Replicon mutants were compared to WT ptGFP using one-way analysis of variance (ANOVA). Differences between groups were considered to be significant at MW355668 to MW355680.Full genome FMDV sequences generated as a part of this study were submitted to GenBank and are available under the following accession numbers: 10.1128/mSphere.00015-21.7FIG S7FIG S7, PDF file, 0.2 MB.Replication kinetics of FMDV replicon constructs containing the stem-loop mutants that were described in the legend to © Crown copyright 2021.2021Crownhttps://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


The number of neurodegenerative diseases resulting from repeat expansion has increased extraordinarily in recent years. In several of these pathologies, the repeat can be transcribed in RNA from both DNA strands producing, at least, one toxic RNA repeat that causes neurodegeneration by a complex mechanism. Recently, seven diseases have been found caused by a novel intronic pentanucleotide repeat in distinct genes encoding proteins highly expressed in the cerebellum. These disorders are clinically heterogeneous being characterized by impaired motor function, resulting from ataxia or epilepsy. The role that apparently normal proteins from these mutant genes play in these pathologies is not known. However, recent advances in previously known spinocerebellar ataxias originated by abnormal non-coding pentanucleotide repeats point to a gain of a toxic function by the pathogenic repeat-containing RNA that abnormally forms nuclear foci with RNA-binding proteins. In cells, RNA foci have been shown to be formed by phase separation. Moreover, the field of repeat expansions has lately achieved an extraordinary progress with the discovery that RNA repeats, polyglutamine, and polyalanine proteins are crucial for the formation of nuclear membraneless organelles by phase separation, which is perturbed when they are expanded. This review will cover the amazing advances on repeat diseases. The number of hereditary disorders originated by unstable microsatellite repeat tracts has been rapidly increasing. Most of them are neurological, neuromuscular, or neurodegenerative diseases. Recent work has shown that a growing group of these conditions is caused by global RNA dysregulation ,2,3. TheThese repeat tracts can be located in coding or non-coding gene regions. In protein-coding gene regions, only trinucleotide repeat expansions have been found, whereas within introns, promoters, 3’ and 5’ UTRs not only trinucleotide but also tetra-, penta-, hexa-, and dodecanucleotide repeats expanded above a given repeat size threshold have been identified in pathological chromosomes .There is evidence that expanded repeats in protein-coding gene regions could cause pathology by gain-of-function of mislocalized and aggregated polyglutamine  or polyaFXN. In Unverricht-Lundborg progressive myoclonus epilepsy (EPM1), a repeat expansion impairs transcription by altering the spacing of promoter elements. On the other hand, transcribed repeat expansions from non-coding gene sequences cause disease by a complex mechanism involving a toxic RNA gain-of-function through the production of an expanded RNA repeat that leads to the formation of nuclear RNA foci recruiting RBPs and originating splicing misregulation ,33RFC1 a repeats ,107,132.n alleles and of (AAGGG)exp/large (AAAGG)n chromosomes . Li. LiSteriSAMD12 antisense strand  gene [390(ATTTC)345 and (ATTTT)340(ATTTC)588 configurations  gene . Oxford urations B. The ATSTARD7 gene encodes a protein involved in lipid transport and metabolism broadly expressed throughout the brain, LCL and fibroblasts. Analysis of the expression levels of STARD7 transcript and protein assessed by quantitative RT-PCR and Western blot, respectively, in affected subject-derived fibroblasts have shown, however, no significant differences compared with controls [The controls . Thus, acontrols ,36, is tn insertions causing FAME [n in intron 1 of the Membrane-associated ring-ch finger protein (MARCH6) gene segregating with the disease in affected individuals with FAME3 [exp(ATTTC)exp tracts n insertion in MARCH6 causes no significant changes in gene expression in LCL or fibroblasts of affected individuals when compared with controls [controls .n in MARCH6 inversely correlates with the age of seizures onset. Moreover, the individuals with the largest expansions present the most severe symptoms. Remarkably, two of these individuals have shown micro-rearrangements of the (ATTTC)n and adjacent ATTTTs, resulting in variable repeat configurations [The size of the (ATTTC)urations . These rYeats domain-containing protein 2 (YEATS2) gene causing FAME4 [819(ATTTC)221 (The gene for FAME4 has been mapped to chromosome 3q in a large Thai pedigree . More reng FAME4 . At thisTTTC)221 B.SAMD12 the search for ATTTC repeats in ATTTT repeat regions of the reference genome has allowed FAME6 gene identification. Expansion of an ATTTC repeat in a 5′ UTR intron of Trinucleotide repeat-containing 6A (TNRC6A) gene, on chromosome 16p, has been identified in one family by searching whole-genome sequencing data followed by Southern blot analysis [18 allele in this repeat location. The ATTTC repeat is flanked by ATTTT repeat tracts (22(ATTTC)exp(ATTTT)exp. In control subjects, there are no ATTTC repeat tracts  gene, on chromosome 4q32.1, in a Japanese family with FAME7 [n flanked by ATTTT repeats [The strategy used to identify the ATTTC repeat insertion in FAME6 has successfully enabled the finding of another ATTTC repeat insertion in intron 14 of th FAME7 . Again,  repeats .RAPGEF2, located in Alu elements  ,166,167.ATXN3 exon 9 and 10 skipping, have shown therapeutic value in SCA3 [ASOs have shown encouraging results for several neurodegenerative and neuromuscular disorders ,182,183. in SCA3 .C9ORF72 FTD/ALS [ATXN2 for the treatment of ALS subjects carrying an ATXN2 intermediate allele (30-33 CAG/CAA repeats) [The most promising ASOs reached clinical trials phase I, being the case of ASOs BIIB105 (NCT04494256) for ALS, and both WVE-004 (NCT04931862) and BIIB078 (NCT03626012) for  FTD/ALS ,179,180.repeats) . Althougrepeats) , ASOs tarepeats)  and in crepeats) , lowerinTo this date, no studies have been reported with ASOs for the treatment of pentanucleotide repeat diseases. Instead, small molecule compounds have successfully targeting the SCA10 RNA repeat  and SCA3Pathogenic pentanucleotide repeats have been identified in three different types of neurological diseases, SCAs, CANVAS, and FAMEs. These pathogenic repeats are all in non-coding gene regions, thus being transcribed in the same orientation of their host gene, but not translated as part of the gene protein. In the host gene antisense strand, they are located in Alu elements, but these Alu transcripts are difficult to grasp and have not been reported. In SCA31 and FAME1 the pathogenic repeat forms RNA foci in the nuclei of cerebellar Purkinje cells that, in SCA31, interact with the ALS protein TDP-43. In SCA10, the nuclear RNA foci sequester hnRNP K causing its loss of function and leading to apoptosis. The AUUUC RNA repeat forms intranuclear RNA foci in transfected human embryonic cells and causes lethal zebrafish developmental malformations in SCA37, whereas FAME1 triggers the formation of nuclear RNA foci in the nucleus of Purkinje and cortical neurons in affected brain tissue. RNA foci are formed by multivalent intermolecular base-pairing of RNAs with repeated nucleotide motifs that recruit RBPs and phase separate in cellular condensates. As all but one pathogenic pentanucleotide repeat is located in Alu elements, differ by only one nucleotide, and, at least, four of them originate the formation of nuclear RNA foci with RBPs that drive nuclear localization, we hypothesized that each one could be toxic through the formation of aberrant nuclear condensates. Thus, in pentanucleotide SCAs and FAME, the data gathered support a role for the pathogenic RNA in neuronal dysfunction by an RNA-gain-of function mechanism. The specificity of the disease alterations likely results from temporal and spatial differences in cellular and tissue gene expression, namely the levels of expression of the affected gene together with free RBP levels for interaction. Furthermore, the abnormal RAN pentapeptides in cell bodies and dendrites of Purkinje cells from SCA31 cerebellar tissue suggest that this mechanism deserves further investigation in the other pentanucleotide repeat disorders. Moreover, since these pentanucleotide repeat diseases present some clinical, genetic, or neuropathological overlap, shared mechanisms underlying the neuronal pathology are expected. The new opportunities for research created by the discovery of pathogenic pentanucleotide repeats will advance knowledge regarding the complexity of mysterious non-coding gene regions in the human genome and their essential role in neurons.


The genomes of RNA viruses present an astonishing source of both sequence and structural diversity. From intracellular viral RNA-host interfaces to interactions between the RNA genome and structural proteins in virus particles themselves, almost the entire viral lifecycle is accompanied by a myriad of RNA–protein interactions that are required to fulfill their replicative potential. It is therefore important to characterize such rich and dynamic collections of viral RNA–protein interactions to understand virus evolution and their adaptation to their hosts and environment. Recent advances in next-generation sequencing technologies have allowed the characterization of viral RNA–protein interactions, including both transient and conserved interactions, where molecular and structural approaches have fallen short. In this review, we will provide a methodological overview of the high-throughput techniques used to study viral RNA–protein interactions, their biochemical mechanisms, and how they evolved from classical methods as well as one another. We will discuss how different techniques have fueled virus research to characterize how viral RNA and proteins interact, both locally and on a global scale. Finally, we will present examples on how these techniques influence the studies of clinically important pathogens such as HIV-1 and SARS-CoV-2. This has broad applications in studying the role and activities of vRNA-binding proteins within the host, as well as in elucidating the structure of macromolecular RNA–protein complexes within virus particles themselves. Furthermore, NGS-based methods can often provide nucleotide or near-nucleotide resolution.in vivo) or the viral genomic material (in virio). This latter point is particularly important in the case of structural viral proteins, such as icosahedral capsid proteins or helical nucleocapsid (NC) proteins that engage in numerous simultaneous contacts with the viral genomic RNA during viral replication, particle assembly, and disassembly. Details of the molecular contacts of these structural proteins with genomic RNA are often lost in crystallographic or electron-microscopy based approaches due to the use of symmetrical averaging during image reconstruction. These techniques may be appropriate to resolve the symmetrical assemblies of the viral capsid proteins, but they can also obscure the inherently asymmetrically arranged genomic RNA within virus particles. Understanding the unique interactions with the numerous capsid proteins is important, as their relative binding positions and affinities with vRNA must be carefully choreographed to successfully guide virus particle assembly , or no crosslinking at all , as well as their methods for RNA–protein complex enrichment (IP or affinity capturing). We will also summarize notable applications in virus research  interactions  due to iIn both classical biochemical and high-throughput NGS assays, the final analyte is an RNA fragment crosslinked to a protein of interest. UV crosslinking is typically followed by RNase digestion and enrichment of the RBP of interest using approaches such as IP or other affinity-capturing methods. Often, the RBP is digested by proteinase K to leave only small peptide adducts crosslinked to the RNA. Finally, the RNA–peptide complex is analyzed either directly or by reverse transcription into cDNA. Coupled with NGS, a number of high-throughput platforms such as high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) , PhotoAcA). CLIP was first applied to investigate RNA targets that are bound and regulated by autoimmune neurologic disease antigens  combines short wavelength UV irradiation and IP to identify RNA sequences interacting with a protein of interest , 26. ShoFig. 2roteins, ) in mousroteins, . After Uroteins, . These Nhodology .Figure 2via short wavelength UV irradiation  in cell culture; (2) cell lysate is treated with RNase to digest unprotected RNA; (3) IP with specific antibody of the RBP of interest enriches the targeted RNA–protein complex; (4) subsequent SDS-PAGE is used to remove noncrosslinked RNA  pronked RNA ; (6) theNGS-coupled HITS-CLIP libraries can readily reveal hundreds of thousands of CLIP sequence-tags , which cvia their interactions with numerous classes of small noncoding RNAs (such as miRNAs), AGO proteins cleave mRNA and repress translation  or pre- . This s , Guo et al. used AGO HITS-CLIP to identify the enriched mRNA targets of miR-27 in T-cell receptor signaling pathway  by Lee et al. . Infecte  can efficiently stabilize RNA–protein complexes , UV radiation may also lead to mutagenesis in the target of interest , 57, 58.B). These crosslink-specific mutations (U-C or G-A transitions) enable high-throughput screening for the precise nucleotide at the RNA-protein crosslink and therefore engaged in RNA–protein interactions.PAR-CLIP provides a different path to high-throughput discovery of RNA–protein interactions . Based oFig. 2PAR-CLIP takes advantage of thionucleobase-enhanced crosslinking with the following basic scheme  protein, pumilio homolog 2 (PUM2), insulin-like growth factor proteins , 83, as et al. . Thet al. , 90 usedet al. . Motif eet al.  uncoveren others . Togetheet al. identified IN-binding RNA targets in virions  is a multi-domain enzyme and one of the cleaved products of Pol polyprotein . IN medi virions . IN show virions  and RRE, virions . In vitrprinting  were conprinting . In one  . For exread’ by the cytoplasmic YTH domain family (YTHDF) proteins . Although it is possible to supplement with a different analog in parallel experiment , they each exhibit different crosslink efficiencies and signal intensities (8 and 109 cells)  . Additioin virio RNA–protein interactions of an RNA virus . In return, vPAR-CL obviates the time- and resource-consuming IP process but instead relies on the standard purification of the given virus  is supplemented to cell culture during virus infection to label newly synthesized vRNA genomes. After cell lysis, the virus particles are purified  and RNase treated to remove any copurified unpackaged viral or host RNAs. The intact, 4SU-containing viruses are crosslinked with 365 nm UV and then are subjected to proteinase K digestion, RNA extraction, reverse transcription, and NGS library construction. In comparison to PAR-CLIP   . An impo3 cells)  from int3 cells)  Fig. 3)in virio vPAR-CL has been demonstrated with FHV to reveal highly conserved RNA-capsid interactions across the entire encapsidated viral genome. This suggests a structural tropism of the RNA inside virion  that likin virio RNA–protein interaction sites. Both vPAR-CL and PAR-CLIP utilize photo-activatable nucleoside analogs to achieve efficient crosslinking and investigate nucleotide-resolution interactions sites. Therefore, vPAR-CL shares the same biochemical limitations as PAR-CLIP related to thio-based photo-crosslinking. Additionally, due to the lack of enrichment of crosslinked 4SU-containing RNA–protein complexes, the incorporation rate of 4SU is critical in vPAR-CL experiments, which determines whether the vPAR-CL signal can stand out above the background. This requires optimization on 4SU dosage and virus harvest timing. vPAR-CL is demonstrated to be reliable to resolve the protein-interaction sites in icosahedral particles such as FHV, whose RNA genome is in direct contact with single protein component-the capsid. It remains an open question whether vPAR-CL can be adapted to other enveloped viruses such as flavivirus, in which the vRNA genome may be in contact with multiple protein components . Nvia cDNA chain termination at the introduced covalent bonds during reverse transcription, which is expected to occur in more than 80% cases  , crossli0% cases . This coInfrared-CLIP  utilizesvia streptavidin-horseradish peroxidase to avoid radiolabeling. This is followed by proteinase K digestion and polyadenylation of RNA to allow reverse transcription with a modified oligo-d(T) primer. This obviates the ineffective RNA ligation of sequencing adapters, allows for low input materials during reverse transcription, and omits size-selection of cDNA products. Therefore, as the name suggests, simplified CLIP presents a simplified and efficient procedure compared to traditional HITS-CLIP.In simplified CLIP , after IEnhanced CLIP  builds uIn terms of virus research, we list examples of these CLIP-derived methods that have been used to study protein–RNA interactions in response to viral infections .in vitro, in virion, and cell culture studies. When it comes to a whole tissue or whole organism scale, UV-light often falls short in providing a viable crosslinking solution, due to its deficiency in evenly penetrating dense and complex tissues. Instead, crosslinking methods using chemicals such as formaldehyde are widely used due to their ability to permeate different tissues.Although photo- (UV-) crosslinking methods offer reliable solutions to characterize direct RNA-protein contacts with nucleotide or near-nucleotide resolution, methods such as HITS-CLIP, PAR-CLIP, vPAR-CL, and other similar technologies are only effective for C) , whose subsequent interaction with amino groups of DNA/RNA bases results in covalent crosslinking C (reviewiewed in ). In comiewed in  . To address this, ‘RNA:protein immunoprecipitation in tandem (RIPiT)-Seq’ can be performed . After formaldehyde crosslinking, two sequential IP steps  are involved and separated by RNase treatment to generate RNP footprints. This is followed by crosslink reversal, RNA extraction, dephosphorylation, size selection, and NGS library construction , which therefore improves signal-to-noise ratio in the downstream NGS assays.PIP-seq is not accompanied with any conventional IP methods to allow specific enrichment of RBPs of interest. Unlike vPAR-CL which is only applied in virions with highly selected RNA/protein components, PIP-seq instead searches the entire transcriptomic length in a complex cellular or erformed . RIPiT-Struction . As a maAlthough formaldehyde is commonly regarded as a more efficient and powerful crosslinker to UV irradiation, formaldehyde readily induces protein-protein crosslinking, which can interfere with the specificity of identification of desired RNA–protein interaction as well as yielding indirect signal . It is avia mass spectrometry) and interacting RNAs (via RNA-seq)  . A similRNA-seq) , 159, isaldehyde , 158. Inaldehyde .As a recent development, VIR-CLASP  also presents a method that is designed to investigate the protein components of the vRNA-cellular RBP interactions . In VIR-There are also numerous RNA-centric methods that utilize affinity-capture to investigate RNA–protein interactions. For example, RNA Bind-n-Seq , 168 is Native RNA immunoprecipitation (RIP) was first applied to isolate and purify proteins interacting with the XIST RNA that controls X chromosome inactivation , 171. RIet al. combined m6A IP and RIP-seq (MeRIP-seq) to study the HIV-1 induced m6A increase in both host and viral mRNAs and uncovered the connection between HIV-1 RRE methylation and nuclear export efficiency of RNA (6A-RIP-seq and m6A-PAR-CLIP (miCLIP) , Nie et al. conducted AGO2 RIP-seq and identified numerous small noncoding RNAs, including highly enriched rRNA-derived fragments   to revea2 genome . Importaragments . With EBragments  and lympragments . Anotherragments . EBNA1-Rragments . Interesragments .In RIP-seq, the obviation of crosslinking largely shortens the experimental procedure and reduces the requirement of starting material. However, the exclusion of crosslinking also limits the application of RIP-seq. The requirement for strong interactions within an RNA–RBP complex that can withstand purification procedures is unguaranteed and the IP of noncrosslinked RNA inevitably precludes stringent washing during purification that would remove contaminants. Similarly, RNase digestion must be controlled carefully in RIP-seq. RIP-based techniques are typically suitable for stable RNP, such as those interacting with noncoding RNAs . RIP thee.g., base pairing) and functional restrictions  can preclude methylation and allow unveiling of the structural/functional relations. Indeed, Smola et al.  and dimethyl sulfate (DMS) chemistries have been extensively used as RNA secondary structural probing methods, due to their extraordinary abilities to methylate flexible nucleotides in biological samples (SHAPE methylates 2′-OH of RNA ribose while DMS methylates N1 of adenine and N3 of cytosine) , 193. Moa et al.  used SHA PIP-seq , SHAPE-Mh worlds . XL-SHAP Tat/TAR  complex  Tat/TAR . Similar Tat/TAR .Virus research has been fueled by rapidly emerging high-throughput and quantitative tools to study RNA–protein interactions. Since the development of HITS-CLIP, RNA research as well as the RNA virus field has evolved to transcriptomic and genomic scale studies. It is apparent that the NGS-enabled RNA–protein interaction technologies are an ever-growing field and that more and more such techniques will continue to be applied in an expanding range of viral and biological systems. It is inevitable that many of these technologies have been adapted or developed to study pressing issues such as the ongoing SARS-CoV-2 pandemic.e.g., whether antibody is available for IP). This flowchart is intended for brief guidance, while many other factors should be considered during experimental design.With such a great availability of NGS-enhanced technologies to investigate RNA–protein interactions, it could be overwhelming to determine the most appropriate scenarios to apply each technique. We hereby provide a quick reference flowchart , in whicCertainly, many challenges remain. Although novel methods such as vPAR-CL reduce experimental burden, many techniques are still labor-intensive and require handling of hazardous materials such as radioisotopes. Both technological and computational advances are necessary to improve RNA-binding site discovery to have a higher sensitivity, lower bias, and better specificity. It is also challenging to adapt these methods to applications in more complex scenarios such as in animal models or higher plants. Finally, as many techniques only focus on end-point analysis, it is important but remains difficult to understand how the RNA–protein interactions and the associated regulatory networks dynamically change at different timescales. This is especially important to virus research as vRNA genomes encounter a myriad of protein partners throughout their replication cycles.e.g., HITS-CLIP focuses on a wide range of binding sites, while PAR-CLIP focuses on higher specificity of the binding sites); each will discover different sets of RBP-binding sites on a given transcriptome or genome. It also demonstrates that the NGS and bioinformatic discoveries resulting from any of these approaches should be considered as a highly efficient hypothesis-generating approach, which ultimately necessitate experimental validation.It is also tempting to cross-compare different platforms for their reliability, sensitivity, and the biological relevance of the discovered sites. However, it remains challenging to draw such conclusions, as different experimentalists will inevitably employ different cell lines, culturing conditions, virus strains, infection methods to address their own research need. Numerous crosslinking methods, enzymes , sequencLooking ahead, several rapidly growing new technologies also help depict a bright picture of virus RNA–protein interaction research. For example, single-cell RNA sequencing allows for comprehensive understanding of an individual cell in the context of its original microenvironment. Additionally, Oxford Nanopore Technologies’ long-read nanopore sequencing is advantageous in generating long and continuous single-molecule sequencing reads, which can effectively cover the entire virus genome to understand the coevolution of distant virus mutations and other features . FurtherThe authors declare that they have no conflicts of interest with the contents of this article.


N6,2′-O-dimethyladenosine (m6A) reader, YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), in regulating the malignant progression of gliomas.The prognosis for diffuse gliomas is very poor and the mechanism underlying their malignant progression remains unclear. Here, we aimed to elucidate the role and mechanism of the RNA YTHDF2 mRNA levels and functions were assessed using several independent datasets. Western blotting, quantitative polymerase chain reaction, and immunohistochemistry were used to evaluate the expression levels of YTHDF2 and other molecules in human and mouse tumor tissues and cells. Knockdown and overexpression were used to evaluate the effects of YTHDF2, methyltransferase-like 3 (METTL3), and UBX domain protein 1 (UBXN1) on glioma malignancy in cell and orthotopic xenograft models. RNA immunoprecipitation (RIP), methylated RIP, and RNA stability experiments were performed to study the mechanisms underlying the oncogenic role of YTHDF2.UBXN1 mRNA degradation via METTL3-mediated m6A, which, in turn, promoted NF-κB activation. We further revealed that UBXN1 overexpression attenuated the oncogenic effect of YTHDF2 overexpression and was associated with better survival in patients with elevated YTHDF2 expression.YTHDF2 expression was positively associated with a higher malignant grade and molecular subtype of glioma and poorer prognosis. YTHDF2 promoted the malignant progression of gliomas in both in vitro and in vivo models. Mechanistically, YTHDF2 accelerated 6A modification.Our findings confirmed that YTHDF2 promotes the malignant progression of gliomas and revealed important insight into the upstream regulatory mechanism of NF-κB activation via UBXN1 with a primary focus on mThe online version contains supplementary material available at 10.1186/s13045-021-01124-z. IDH)-mutant and 1p/19q co-deletion status according to the latest World Health Organization classification of central nervous system tumors   modification have provided new insights into the molecular mechanism of malignant progression of diffuse gliomas [6A is dynamically regulated by methyltransferases (‘writers’) and demethylases (‘erasers’) [6A can influence the entire RNA life cycle, including alternative polyadenylation, pre-RNA processing, RNA export from the nucleus to the cytoplasm, RNA translation, and RNA decay, through binding with different ‘readers’ of m6A [6A writers , erasers (FTO and ALKBH5), and readers  in diffuse gliomas and have revealed that the expression level of YTHDF2 (YTH N6-methyladenosine RNA binding protein 2) is significantly correlated with increased malignancy [Recent advances in understanding RNA  gliomas –9. Similrasers’) , 11. m6A’ of m6A –14. We h6A-modified RNA via serval different mechanisms, including de-adenylation by the CCR4–NOT complex, and endoribonucleolytic cleavage by HRSP12-RNase P/MRP [6A modification remains unclear.YTHDF2 destabilizes mse P/MRP –17. YTHDse P/MRP  and is ise P/MRP . These fse P/MRP , 20, 21.UBXN1 mRNA degradation by recognizing m6A modification mediated by the m6A writer, METTL3. Finally, we also validate the role of YTHDF2 in promoting tumor progression and activating NF-κB signaling in orthotopic xenograft models.Here, we investigated the expression levels of YTHDF2 mRNA and protein in gliomas and observed that YTHDF2 expression was positively correlated with increased glioma malignancy. Elevated YTHDF2 expression was associated with decreased survival of all diffuse glioma and GBM patients in both the Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA) datasets. We demonstrate that YTHDF2 can promote cell proliferation and migration in several glioma models. Additionally, we show that elevated YTHDF2 can promote nuclear factor kappa B (NF-κB) activation by suppressing UBX domain protein 1 (UBXN1) expression. Mechanistically, we show that YTHDF2 can accelerate http://www.cgga.org.cn/) and the TCGA database (https://tcga-data.nci.nih.gov/tcga/tcgaDownload.jsp) [We obtained transcriptional and clinical data from the CGGA official website (oad.jsp) , 23. The2.All cells were cultured as previously reported , 24. Bri3 cells/well were seeded into six pairs of duplicate wells of a 96-well plate and incubated at 37 °C for 1–6 days. At the predesigned time point, the medium for six-repeated wells was changed, and 10 μl CCK-8 reagent was added to the 100 μl fresh culture medium. Absorbance was detected daily at 450 nm for 1–6 days.Cell proliferation was measured using the Cell Counting Kit-8 . Specifically, 1.5 × 105) in serum-free DMEM were added to the Transwell upper chamber, with the lower chamber containing DMEM with 10% FBS. After incubation for 6 h at 37 °C, the migrated cells present on the underside of the Transwell membrane were stained with crystal violet and counted under a microscope .For migration assays, GBM cells  following the manufacturer’s protocol and quantified samples with a NanoDrop 2000. cDNA was generated from 1 µg RNA and real-time qPCR was performed on a 7500 Fast Real-Time PCR system to determine target RNA levels. The primer sequences used are listed in Additional file Western blotting was performed as previously described . After wScrambled siRNA and YTHDF2 siRNA were purchased from GenePharma . The siRNAs were transfected into cells using PolyPlus-transfection according to the manufacturer’s instructions . The growth medium was changed 8 h after transfection. Target RNAs and proteins were measured by qRT-PCR and Western blotting, respectively, 48 h after transfection. Two non-overlapping siRNAs that effectively suppressed target gene expression were selected for subsequent experiments. The sequences of siRNAs used are presented in Additional file Lentiviruses expressing YTHDF2, METTL3, UBXN1, METTL3-shRNA (target sequence: gc CTT AAC ATT GCC CAC TGA T), YTHDF2-shRNA , scrambled shRNA, or empty vector were purchased from Beijing Syngentech Company and Shanghai Genechem Company.4 cells were plated into a 6-well plate and transfected with the indicated lentivirus using HitransG P (Genechem) in accordance with the manufacturer’s instructions. Infected cells were selected using 2 μg/ml puromycin  for ≥ 1 week, and the transfection efficiency was determined by RT-qPCR and Western blotting analysis.U87, LN229, and N33 cells were selected to establish stable YTHDF2 overexpression cell lines. U87 cells were used to establish METTL3 knockdown and overexpression cell lines. UBXN1 was overexpressed in YTHDF2-overexpressing U87 cells. Transfection was performed as previously reported . Briefly6A antibody (Abcam) was incubated with RNA for 1 h at 4 °C. Prewashed Pierce™ Protein A/G Magnetic Beads  were mixed with the antibody-treated RNA in immunoprecipitation buffer at 4 °C overnight. Finally, the methylated RNA was purified for sequencing or PCR.After extracting total RNA (200 µg) using TRIzol reagent, ribosomal RNA was removed using a RiboMinus™ Eukaryote Kit v2 . Then, the RNA was sheared into approximately 100-nucleotide fragments using RNA Fragmentation Reagents . Next, a portion of the RNA solution was stored at − 80 °C as input for RNA sequencing or PCR. An anti-mRIP assays were performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit  according to the manufacturer’s instructions. IP lysis buffer was used to lyse U87 cells and the cell lysate was divided into anti-SNRNP70, anti-METTL3, anti-YTHDF2, anti-IgG, and input samples. The cell lysates were incubated with magnetic beads coated with 5 µg of specific antibodies overnight at 4 °C. After washing, the lysates were digested with Proteinase K, and the RNA bound to immunoprecipitated proteins was purified. qPCR was performed to measure the target RNA levels.Cells were seeded in 6-well plates, and siRNAs were used to knockdown specific expression for 2 days if necessary. Then, cells were treated with Actinomycin D  for 0, 1, 2, and 4 h or longer. RNA with added bacterial 16 s RNA was then extracted by TRIzol. The target RNA was measured by qPCR, and the bacterial 16 s RNA was used as a reference.5) transfected with an empty vector, YTHDF2 overexpression, or METTL3 overexpression vectors were suspended in PBS and injected into the right frontal node of nude mice. The inoculation position was 2 mm lateral and 2 mm posterior to the anterior fontanel. Tumor size was estimated from luciferase volume measurements and MRI. The mice were sacrificed when they exhibited disturbed activity or convulsion. The brain was then harvested and embedded in paraffin.All experiments were approved by Beijing Neurosurgical Institute following the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines. Five-week-old female BALB/c nude mice  were selected for the experiments. U87 cells  and Prism 7 . A value of P < 0.05 was considered statistically significant.All statistical analyses were conducted using R v3.4.1  of CGGA. YTHDF2 mRNA levels were also increased in molecular subgroups of primary gliomas with higher malignancy   and TCGA RNA-seq (n = 595) cohorts used in our previous study [The above findings suggest that the elevated expression of YTHDF2 may play an oncogenic role in glioma. To explore potential effects of YTHDF2 expression on glioma functions, we identified the genes that positively correlated with YTHDF2 expression in all primary gliomas of four different datasets, including the CGGA RNA-seq  enriched pathway  and TCGA RNA-seq (n = 595) cohorts. METTL3 expression was significantly negatively correlated  with UBXN1 expression in CGGA cohort  to UBXN1 expression in TCGA cohort  and TCGA  datasets  in several datasets  [6A modification, it is still required to carefully verify the expression, prognostic value, potential biological functions, and underlying mechanisms of m6A ‘writers,’ ‘erasers,’ and ‘readers’ in different cohorts of gliomas. Here, based on our previous work, we investigated the expression levels and prognostic values of m6A reader YTHDF2 in additional two large cohorts of CGGA. We also verified the protein expression of YTHDF2 in gliomas with different histological grade and IDH-mutant status. We showed that both the mRNA and protein levels of YTHDF2 are positively correlated with increased grade of glioma malignancy and poor prognosis. Based on this, we showed that the elevated expression of YTHDF2 can promote the proliferation and migration of differentiated GBM cells by promoting NF-κB activation both in vitro and in vivo. Mechanistically, YTHDF2 accelerated m6A-dependent degradation of UBXN1 mRNA, which is a negative regulator of NF-κB activation.RNA mf glioma , 27, 32. cohorts . Howevers (GSCs) , 33, 34.IDH-mutant status can also dramatically impact the expression levels, prognostic values, and functions of m6A ‘writers,’ ‘erasers,’ and ‘readers’ in gliomas. IDH mutation can inhibit the activity of m6A ‘eraser’ FTO in leukemia and glioma cells by producing R-2-hydroxyglutarate [IDH-wildtype glioma, and METTL3 expression is positively associated with a higher malignant grade and poorer prognosis of IDH-wildtype gliomas but not IDH-mutant gliomas [IDH-mutant status, WHO 2016 classifications, and primary/recurrent status in this study. Unlike METTL3, which only increases with grade in IDH-wildtype gliomas, we demonstrated that YTHDF2 increased along with increasing WHO grades in both IDH-mutant and IDH-wildtype gliomas. Furthermore, we revealed that the expression of YTHDF2 significantly increased in recurrent gliomas compared with primary gliomas. We also showed that the expression of YTHDF2 has prognostic value in GBM, excluding the potential impact of histological grade on survival analysis in previous studies. These findings provide more detailed and reliable information to further understand the roles of YTHDF2 in gliomas.Consistent with our previous report, a recent study also showed that YTHDF2 expression is increased in GBM and has prognostic value in pan-gliomas , 19. Howlutarate , 36. Our gliomas . Thus, wThe specific biological functions and their mechanisms of YTHDF2 in glioma have not been reported until recently, mainly focusing on the studies of GSCs , 19. YTH6A-dependent mRNA clearance, which is required for GSC growth, invasion, and tumor formation in vivo [6A-dependent mRNA decay. UBXN1, a UBX domain-containing protein, suppresses NF-κB activation by maintaining the expression of IκB [YTHDF2 has been shown to accelerate the degradation of more than 3000 RNA targets, most of which are mRNAs, but which also include non-coding RNAs . In glio in vivo . Here, wn of IκB , 31. Addn of IκB , 40. HIVn of IκB . CollectRecently, downregulation of UBXN1 expression was also reported to be involved in the mechanism by which EGFR promotes NF-κB activation and the mechanism through which the long non-coding RNA, PRADX, activates NF-κB , 21. Mea6A writer, METTL3, plays a key role in the maintenance of GBM stem cell characteristics by regulating m6A enrichment in ADAM19, SOX2, and SRSF genes [6A enrichment in UBXN1 mRNA, and is therefore involved in modulating NF-κB activation. Overall, these findings indicate that m6A has the potential to modulate NF-κB in multiple ways, which should be clarified by further studies.Activated NF-κB can mediate the activation of STAT3, CEBPB, and TAZ by upregulating the expression of CD44, vimentin, and N-cadherin, thereby promoting the invasion, angiogenesis, and therapeutic resistance of glioma –45. The SF genes . RecentlSF genes . Here, oUBXN1 mRNA decay in gliomas by recognizing the m6A modification mediated by METTL3. This, in turn, enhances NF-κB activation (Fig. 6A modification.In conclusion, our findings demonstrate that YTHDF2 expression in diffuse gliomas increases with greater histological or molecular features of malignancy. Elevated YTHDF2 promotes malignancy of gliomas in both in vitro and in vivo models. Mechanistically, YTHDF2 accelerates ion Fig. E. Taken Additional file 1. The primers and supplementary figures for this manuscript.Additional file 2. Identified genes that were negatively correlated with YTHDF2 expression.Additional file 3. The raw gel figures used in this manuscript.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus, a member of the coronavirus family of respiratory viruses that includes severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and the Middle East respiratory syndrome (MERS). It has had an acute and dramatic impact on health care systems, economies, and societies of affected countries during the past 8 months. Widespread testing and tracing efforts are being employed in many countries in attempts to contain and mitigate this pandemic. Recent data has indicated that fecal shedding of SARS-CoV-2 is common and that the virus RNA can be detected in wastewater. This indicates that wastewater monitoring may provide a potentially efficient tool for the epidemiological surveillance of SARS-CoV-2 infection in large populations at relevant scales. In particular, this provides important means of (i) estimating the extent of outbreaks and their spatial distributions, based primarily on in-sewer measurements, (ii) managing the early-warning system quantitatively and efficiently, and (iii) verifying disease elimination. Here we report different virus concentration methods using polyethylene glycol (PEG), alum, or filtration techniques as well as different RNA extraction methodologies, providing important insights regarding the detection of SARS-CoV-2 RNA in sewage. Virus RNA particles were detected in wastewater in several geographic locations in Israel. In addition, a correlation of virus RNA concentration to morbidity was detected in Bnei-Barak city during April 2020. This study presents a proof of concept for the use of direct raw sewage-associated virus data, during the pandemic in the country as a potential epidemiological tool. SARS-CoV-2 is one of more than 37 coronaviruses in the ronments –10. Interonments , 12. Recronments –16. Despronments  and its The presence and prevalence of SARS-CoV-2 in wastewater provide a valuable epidemiological data source . WastewaTable 3). For the examination of virus concentration methods, samples of raw sewage from Dan-Panaroma hotel, Tel Aviv (termed here TLV1-10), and from Zfat (termed here Z1-5) were collected. The sampling equipment was sanitized and properly sterilized . In addition, for sampling different geographic locations  were used to ascertain a preferable kit for SARS-CoV-2 RNA extraction. Four raw sewage samples from areas with numerous COVID-19 populations were spiked with MS2 and human coronavirus CO43 and were analyzed by different kits to evaluate which kit works best with wastewater. The spiked sewage samples were stirred for 30 min, divided into aliquots, and kept at −80 degrees. Several RNA extraction kits were tested to optimize the removal of impurities from the raw sewage samples. The kits used were; AccuPrep® Viral RNA Extraction Kit (Bioneer); NucleoSpin RNA, NucleoSpin RNA Stool and NucleoSpin RNA Virus , Quick-DNA/RNA Viral MagBead , and Monarch® Total RNA Miniprep Kit .Sewage is composed mostly of water and may include detergents and organic matter, which may have a great impact on inhibition when using molecular methods. Therefore, to decrease the effect of inhibitors, several extraction kits (A few concentration methods were used to achieve improved Ct (Threshold detection cycle in qPCR) values. Viral particles from ~0.25 to 1 liter of sewage samples were first centrifuged to remove sediment and large particles. Virus precipitation of the supernatant was performed using different methodologies: Polyethylene glycol (PEG) 8,000 or 6,000, alum , skim milk solution (0.01% w/v in sewage), or ultrafiltration. The sewage mixtures were mixed at 4°C with 100-rpm agitation for 12 h, followed by centrifugation at 4,500–14,000 g for 45 min at 4°C to pellet the virus particles. The virus particles were then resuspended in phosphate-buffered saline (PBS). The aqueous phase (containing virus particles) was collected and filtered through a 0.22 μm filter. The TLV2 sample was mixed with glycine buffer  at 1:4 volume ratios before the precipitation. The mixture was incubated at 4°C with 100-rpm agitation for 2 h, to detach virions bound to organic material. To continue in optimizing precipitation methods, different precipitant weight percentages were tested using different salt (NaCl) concentrations . In somehttps://www.bgi.com/global/sars-cov-2-real-time-fluorescent-rt-pcr-kit-ivd). Quantitative PCR amplification was performed in a Step One Plus real-time PCR system . Both positive-control and negative-control assays were performed for quality control. In CVL, positive controls for the SARS-2 E and N genes were generated, by synthesizing RNA fragments corresponding to the amplified regions in the qPCR test. Serial dilutions of these synthetic RNA fragments were used to generate the standard curves of plasmid log copy number vs. Ct value (see https://www.bioline.com/us/), thereby shortening the time of the reaction, while maintaining its sensitivity. The test was successfully performed with both ABI7500 Bio-Rad CFX-96 instruments. The analytical sensitivity was tested and was found to be comparable to that described by Corman et al. The analytical limit of detection (LOD) of the E gene target was <10 target copies per reaction for the E gene.In the BGU laboratory, the extracted viral RNA samples were tested using the BGI commercial kit  and funding from the Israeli ministry of Health.We would like to thank funding from the Ben Gurion University, the Corona Challenge Covid-19 (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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.