Patterns of m6A methylation in lncRNAs in developing muscle have not been uncovered. Here, we reveal differentially expressed lncRNAs and report temporal m6A methylation patterns in lncRNAs expressed in mouse myoblasts and myotubes by RNA-seq and methylated RNA immunoprecipitation (MeRIP) sequencing. Many lncRNAs exhibit temporal differential expression, and m6A-lncRNAs harbor the consensus m6A motif “DRACH” along lncRNA transcripts. Interestingly, we found that m6A methylation levels of lncRNAs are positively correlated with the transcript abundance of lncRNAs. Overexpression or knockdown of m6A methyltransferase METTL3 alters the expression levels of these lncRNAs. Furthermore, we highlight that the function of m6A genic lncRNAs might correlate to their nearby mRNAs. Our work reveals a fundamental expression reference of m6A-mediated epitranscriptomic modifications in lncRNAs that are temporally expressed in developing muscle.Proper development of mammalian skeletal muscle relies on precise gene expression regulation. Our previous studies revealed that muscle development is regulated by both mRNA and long non-coding RNAs (lncRNAs). Accumulating evidence has demonstrated that N Benefit tissues .6A modification in gene expression, emerging evidence has revealed the critical role of m6A in skeletal muscle regulation  during proliferative phases . Our results reveal the temporal expression profile and m6A methylation status of lncRNAs during skeletal myogenesis. We found that the m6A methylation levels of lncRNAs were positively correlated with their transcriptional abundance. Our data will provide a fundamental reference for further study on the function of lncRNAs.In this study, we characterized lncRNAs in mouse myoblasts and differentiated myotubes using RNA sequencing (RNA-seq) and further uncovered abundant m2. When C2C12 cells reached about 90% confluency, the GM was replaced with differentiation medium (DM)-DMEM containing 2% horse serum (HyClone).The C2C12 mouse myoblast cell line and HEK-293T cells were purchased from the Cellular Library of the National Collection of Authenticated Cell Cultures . The cells were cultured in growth medium (GM)-Dulbecco’s modified Eagle’s medium  with 10% fetal bovine serum , 100 U/ml penicillin, and 100 μg/ml streptomycin (1 × penicillin–streptomycin) at 37°C in a humidified chamber supplemented with 5% COFor METTL3 overexpression, cDNA of mouse METTL3 was cloned into the pKD-CMV-MCS-EF1-PURO (pKD) vector by Gibson Assembly, and pKD-GFP was used as a negative control. For METTL3 knockdown, the gRNAs downstream of the transcription start sites were used to guide the fusion of inactive Cas9 (dCas9) to the Krüppel-associated box (KRAB) repressor. To generate stable cells, lentiviruses were produced in HEK-293T cells by transfecting vectors together with psPAX.2 and pMD2.G. Lentiviruses were collected and filtered 48 h after transfection and then used to infect target cell lines. Stable cells were selected using the antibiotic puromycin.All sequences of clone primers used in this study are listed in To knock down lncRNAs, custom designed siRNAs targeting selected lncRNAs and control siRNAs were synthesized by Shanghai GenePharma Co., Ltd. C2C12 cells were seeded in 12-well plates and transfected with siRNAs using Lipofectamine 2000 (Invitrogen) after the cells reached 30–40% confluency, according to the manufacturer’s instructions. The transfected cells were maintained in growth medium for two days, and then cells were harvested for analysis.All siRNA sequences used in this study are listed in +, GE) and allowed to air dry for 5 min. Then, RNA was crosslinked to the membrane with UV light . The membrane was washed in TBST , dyed in methylene blue (Sigma–Aldrich) as a quantitative control, and incubated in blocking buffer containing 5% non-fat dry milk in TBST for 2 h at room temperature. Then, the membrane was incubated with m6A antibody  at 4°C overnight. The membrane was washed 3 times for 10 min each in TBST and then incubated with HRP-linked secondary anti-rabbit IgG antibody  for 1 h at room temperature. Signals were detected with WesternBright ECL HRP substrate (Advansta). The dots were quantified using ImageJ.RNA dot blotting was performed as previously described with modifications . Cells wCells were lysed in ice-cold enhanced RIPA lysis buffer  containing phosphatase inhibitor and protease inhibitor cocktail (Roche). Equivalent total protein extracts were separated by SDS–PAGE and transferred to nitrocellulose membranes (Merck Millipore). The membranes were blocked with 5% non-fat dry milk in TBST for 1 h at room temperature. The following antibodies were used in this study: anti-METTL3 , anti-METTL14 , anti-MHC , and anti-GAPDH . Immunoreactivities were determined using WesternBright ECL HRP substrate (Advansta).Total RNA was extracted from cells with TRIzol reagent . First-strand cDNA for PCR analyses was synthesized with HiScript III RT SuperMix for qPCR (+ gDNA wiper) , and quantitative real-time PCR was performed using ChamQ Universal SYBR qPCR Master Mix . The GAPDH gene served as an endogenous control. The qRT-PCR results were analyzed and presented as relative RNA levels of the CT (cycle threshold) values, which were then converted as fold change. The results are presented as the means ± SD.All primers for qPCR are listed in Total RNA was isolated and purified using TRIzol reagent  following the manufacturer’s procedure. The RNA amount and purity were quantified by a NanoDrop ND-1000 (NanoDrop). RNA integrity was assessed using the Agilent 2100 system with RIN number >7.0 and confirmed by electrophoresis with a denaturing agarose gel.TM 6000  following the vendor’s recommended protocol.RNA-seq was performed by BGI Co., Ltd. Briefly, total RNA was used to purify the poly-A containing RNAs using poly-T oligo-attached magnetic beads. Following the purification, the remainder of the RNA was fragmented into small pieces using divalent cations under high temperature. Then, the cleaved RNA fragments were reverse transcribed to create the cDNA library according to the mRNA-Seq sample preparation kit protocol (Illumina). The average insert size for the paired-end libraries was 300 bp (±50 bp). Then, paired-end sequencing was performed on an Illumina NovaSeq6A affinity purified antibody . Then, the IP RNA was reverse transcribed to cDNA by SuperScriptTM II Reverse Transcriptase  followed by U-labeled second-stranded DNA synthesis. Then, after ligation with the adapter to the A-tailed fragmented DNA, the ligated products were amplified with PCR, and 2 × 150 bp paired-end sequencing (PE150) was performed on an Illumina NovaSeqTM 6000  following the vendor’s recommended protocol.RNA extraction and quality control were performed as previously noted. Poly(A) RNA was purified from 50 μg of total RNA using Dynabeads Oligo (dT) 25-61005 (Thermo Fisher) and fragmented into 100-nucleotide-long oligonucleotides using Magnesium RNA Fragmentation Module (NEB). The cleaved RNA fragments were immunoprecipitated using an anti-m6A methylated RNA immunoprecipitation was performed as previously described  for 2 h at 4°C with gentle rotation. Then, 50 μl protein A/G magnetic beads were added and incubated for 2 h at 4°C with gentle rotation. Beads were pelleted at 2,500 rpm for 30 s. Then, the supernatant was removed, and the beads are resuspended in 500 μL RIP buffer. The process is repeated for a total of three RIP washes followed by one wash in PBS. Beads were incubated with DNase I for 30 min at 37°C and were then digested by Proteinase K for 2 h at 37°C with rotation. A MicroElute RNA Clean Up Kit (Omega) was used for RNA purification. Purified RNA was reverse transcribed and quantified by real-time RT-PCR.mescribed . BrieflyP ≤ 0.05. LncRNAs were selected for downstream analysis based on gene type.The sequencing adapters were removed from raw fastq data using cutadapt  softwareThe sequencing adapters were removed from raw fastq data using cutadapt  software6A peaks were identified by HOMER .Significant differential mP ≤ 0.05) were fed into the clusterProfiler  of differentially expressed lncRNAs were obtained using an in-house Python script, and mRNAs with significantly different expression  compared to myoblasts   . To testrker MHC . Howeverrker MHC , and therker MHC . These dP value ≤0.05, In addition to RNA modification, non-coding RNAs, especially long non-coding RNAs (lncRNAs), could represent robust gene expression regulators. To profile the dynamic changes in lncRNAs during C2C12 differentiation, RNA sequencing (RNA-seq) was performed to analyze the expression of lncRNAs in GM and D4. Using bioinformatic analysis, we detected 5,583 lncRNAs expressed in at least one sample. Of these, 679 differentially expressed lncRNAs were identified  in GM and D4 samples to uncover the m6A methylation landscape of lncRNAs. We identified greater than 20 thousand unique peaks from each MeRIP-seq sequencing library  and was similar to a previous report in lincRNAs  and myotubes (D4), respectively  and 14 hypomodified lncRNAs (6 hypo-upregulated and 8 hypo-downregulated) according to the criteria of adjusted P ≤ 0.05 and FDR ≤ 0.05 . As shown in 6A group, indicating that these transcripts were m6A enriched. For example, the enrichment of Xist, Ptgs2os2 and Brip1os was elevated up to hundreds of thousands of fold in the m6A group, which is consistent with the MeRIP-seq data that revealed clear m6A peaks around their RNAs    . However= 0.071) . Intereshylation . Our ana6A modification levels could affect lncRNA expression, we overexpressed METTL3 in C2C12 cells (oe-M3), and GFP-overexpressing cells were used as a negative control (GFP). The real-time PCR and Western blot results validated that METTL3 was successfully overexpressed  to the Krüppel-associated box (KRAB) repressor to inhibit the transcription of METTL3 in C2C12 cells. METTL3 mRNA and protein levels were greatly reduced compared to those with control gRNA . Then, wP ≤ 0.05). GO analysis of biological processes for these mRNAs showed that 94 mRNAs were related to muscle tissue development (P ≤ 0.05). KEGG pathway enrichment analysis showed that the cell cycle and MAPK signaling pathways were significantly enriched (P ≤ 0.05), and such results are consistent with our previous studies showing that the JNK/MAPK and P38/MAPK signaling pathways play essential roles in myogenesis  in the genome  genes play crucial roles in limb development  is expressed at elevated levels among the top ten differentially m6A-enriched lncRNAs in D4 compared to GM. Snhg14 is highly expressed in Parkinson’s disease, and silencing Sngh14 mitigated dopaminergic neuron injury by downregulating a-syn by targeting miR-133b  can be found in the article/The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University.S-JX, Z-DX, QZ, and Y-WP conceived and designed this project. S-JX, ST, and L-TD performed the experiments with the help of Y-XH, Y-RH, HL and, W-YX. S-JX, W-JC, Z-GZ, P-LL, and W-CC analyzed the data. S-JX, Z-DX, ST, L-TD, Y-WP, and QZ wrote the manuscript. All authors contributed to the article and approved the submitted version.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.


After six rounds of library-vs-library selection, the selected RNAs and LS proteins were analyzed by next-generation sequencing (NGS). Further deconvolution of the enriched RNA and LS protein sequences revealed two synthetic and orthogonal RNA–RBP pairs that exhibit picomolar affinity and >4000-fold selectivity.RNA-binding proteins (RBPs) and their RNA ligands play many critical roles in gene regulation and RNA processing in cells. They are also useful for various applications in cell biology and synthetic biology. However, re-engineering novel and orthogonal RNA–RBP pairs from natural components remains challenging while such synthetic RNA–RBP pairs could significantly expand the RNA–RBP toolbox for various applications. Here, we report a novel library-vs-library in vitro selection strategy based on Phage Display coupled with Systematic Evolution of Ligands by EXponential enrichment (PD-SELEX). Starting with pools of 1.1 × 10  RNA and RNA-binding protein (RBP) pairs, natural or engineered, are used as adaptors to link an RNA of interest with a protein of interest for various applications. For example, the specific interaction between the bacteriophage MS2 coat protein (MS2CP) and MS2 RNA stem–loop  has beenA reasonable strategy to generate novel RNA–RBP pairs is to re-engineer the binding characteristic of an existing RNA–RBP pair. This has typically been done in two steps. (i) Mutate either the parental RNA or RBP to abolish binding ,14. (ii)While these in vitro selection technologies usually involve the selection of an RNA or protein library for binding to a single target, an alternative approach is library-vs-library selection (or screening) which has been employed for identification of protein–protein , proteinEscherichia coli  to develop a method to coevolve RNAs and RBPs through library-vs-library in vitro selection Figure . First, hia coli , and in hia coli ,32,37–45E. coli Rosetta-gami B 5615 strain is not commercially available, because the manufacturer has discontinued production. Accordingly, we generated the strain in the laboratory as follows: pAR5615 plasmid DNA encoding isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible wild-type T7 phage capsid gene was purified from BLT5615 strain (Merck-Millipore) using Zyppy Plasmid Miniprep Kit (Zymo Research). The plasmid DNA was transformed into Rosetta-gami B competent cells (Merck-Millipore), and transformants were selected on an LB plate supplemented with antibiotics (50 μg/ml carbenicillin and 34 μg/ml chloramphenicol). MagStrep ‘type3’ XT beads (2-4090-002) was purchased from IBA Lifesciences. FG HM-NeutrAvidin beads (TAB8848N3171) and magnet stand (TAB4899N12) were purchased from Tamagawa SEIKI.Unmodified oligo DNAs were purchased from Eurofins Genomics, FASMAC, or Integrated DNA Technologies (IDT) Japan. Randomized oligo DNAs containing NNK  codons were purchased from Ajinomoto Bio-Pharma or IDT Japan. DNA oligo pool was purchased from Twist Bioscience. Synthetic genes were purchased from Eurofins Genomics, IDT Japan, or Twist Bioscience. Biotinylated DNA and 2′-OMe RNA were purchased from FASMAC. Q5 High-Fidelity 2× Master Mix and HiScribe T7 High Yield RNA Synthesis Kit were purchased from New England Biolabs (NEB) for polymerase chain reaction (PCR) and in vitro transcription, respectively. SuperScript IV reverse transcriptase was purchased from Thermo Fisher Scientific for reverse transcription (RT). All chemicals used for buffer preparation were purchased from Nacalai Tesque or FUJIFILM Wako Pure Chemical Corporation. Difco LB broth Miller was purchased from BD for bacterial culture. E. coli BL21 (DE3) strain. Transformants were pre-cultured overnight at 37°C in 2 ml of LB medium supplemented with 100 μg/ml carbenicillin and transferred to 100 ml fresh LB medium supplemented with 50 μg/ml carbenicillin. After 1–2 h culture at 30°C, IPTG was added at a final concentration of 0.4 mM, and the cells were further cultured for 20 h at 30°C. The cells were harvested by centrifugation at 6000 × g for 10 min at 4°C and suspended in 20 ml ice-cold His-tag purification buffer A . After cell disruption by ultrasonication, lysozyme , Triton X-100 (0.1% v/v), MgCl2 , DNase I , and RNase A  were added to the crude cell extracts and incubated at 37°C for 30 min. The cell extracts were centrifuged at 8000 × g for 20 min at 4°C to remove cell debris, and the supernatants were filtered. Soluble proteins were loaded onto TALON metal affinity resin (TaKaRa) in a PD-10 (Cytiva) column and washed with 10 ml His-tag purification buffer A. His-tagged proteins were eluted by His-tag elution buffer , and the eluents were incubated with Strep-Tactin XT Superflow high capacity resin (IBA Lifesciences). After 5 min incubation, the resin was washed twice with 0.5 ml His-tag purification buffer A, and Twin-Strep-tagged proteins were eluted twice with 0.5 ml 1× Buffer BXT Strep-Tactin XT Elution Buffer (IBA Lifesciences). The eluents were desalted and concentrated using an ultrafiltration device  and buffer-exchanged into protein storage buffer . Quality of the purified proteins was confirmed by SDS-PAGE  followed by Coomassie blue staining   were subcloned into a pET vector backbone. The bacterial expression vectors  were tra TaKaRa) . The conin vitro transcription. RNA aptamers were synthesized by in vitro transcription at 42°C for 4 h using HiScribe T7 High Yield RNA Synthesis Kit (NEB) according to the manufacturer's manual for in vitro transcription of short templates  was injected into all flow cells at a flow rate of 25 μl/min for 7 min to activate carboxylic acid groups. After injection of ultrapure water for 1 min, 0.3 mg/ml NeutrAvidin in 10 mM sodium acetate (pH 5.2) was injected by setting the immobilization level to 5000 RU. Finally, 1 M ethanolamine–HCl (pH 8.5) was injected for 7 min to quench the activated carboxylic acid. To remove the non-covalently bound NeutrAvidin on the chip surface, 10 mM NaOH was injected three times at a flow rate of 20 μl/min for 20 s. Setting the immobilization level to 400 RU, biotinylated 2′-OMe RNAs (5′-/bio/cguucugugucuuucgucgau-3′) (1 μM) in high salt buffer  was injected and the remaining biotin-binding sites on the chip were quenched by injection of 1 μM free biotin.Binding kinetics were determined using Biacore T200 (Cytiva) at 25°C. CM5 chip surface was washed by three injections of 10 mM NaOH at a flow rate of 20 μl/min for 30 s. Next, a mixture of 200 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 50 mM 2, 0.25 mM TCEP-Na, 0.1 mg/ml BSA, 25 μg/ml yeast tRNA).RNA aptamer solutions (500 nM) were prepared in water and denatured by heating at 80°C for 3 min. After cooling at room temperature for few minutes, the RNA solutions were diluted to 10–50 nM with the high salt buffer. Various concentrations of protein solutions were prepared in SPR running buffer  was determined from the ratio of the association and the dissociation rate constants (KD= koff / kon). The figures were generated using GraphPad Prism software 6.0h (GraphPad Software). The measurements were repeated at least twice using different flow cells (or different SPR chips) in different days to ensure reproducibility.RNAs were injected at a flow rate of 10 μl/min for 30 s, and then the running buffer was injected for 1 min. Proteins were injected at a flow rate of 80 μl/min for 100 s (or 200 s) to monitor association, and then the dissociation kinetics was monitored for 420 s (or 1500 s) in SPR running buffer. The sensor surface was regenerated with 2 μl injection of 10 mM NaOH followed by 6 M urea at a flow rate of 20 μl/min and the running buffer was injected for 1 min. The raw data were analyzed by Biacore T200 Evaluation Software 1.0 (Cytiva) using 1:1 Langmuir interaction model. Background signal from the reference flow cell was subtracted from that of the sample flow cell, and no-protein sample (SPR running buffer only) was injected in each experiment (double referencing). Dissociation constant  before use.MagStrep ‘type3’ XT beads were rinsed three times with ultrapure water and stored in blocking buffer  at 4°C. FG HM-NeutrAvidin beads (100 μl) were rinsed once with immobilization buffer , and mixed with 1.64 nmol of biotinylated capture oligo DNA (5′-/bio/CGTTCTGTGTCTTTCGTCGAT-3′) in the same buffer and incubated at room temperature for 5 min. After washing with the same buffer three times, FG beads were stored in blocking buffer at 4°C. The immobilized oligo DNA was estimated to be ∼6.3 pmol per μl suspended beads. The beads were pre-equilibrated with selection buffer , and the sequence was verified by Sanger sequencing. The partially randomized DNA library encoding L7Ae mutants was prepared by overlap extension PCR (8). The PD library was amplified with logarithmic phase of Rosetta-gami B 5615 strain in 200 ml scale (multiplicity of infection < 0.01). Expression of Gp10A was induced in advance by addition of 1 mM IPTG. Upon lysis , 5 g of sodium chloride was added to the lysate and the E. coli debris was removed by centrifugation at 8000 × g for 20 min at 4°C. After collection of the supernatant, 21 g of polyethylene glycol (PEG) 8000 (Promega) was added and incubated at 4°C overnight. Lysate-PEG mixture was centrifuged at 8000 × g for 40 min at 4°C and the supernatant was discarded. The precipitated phage particles were suspended in T7 phage stock buffer , and subjected to ultrasonication for few minutes. The phage solution was filtered through a 0.45 μm membrane filter , and stored at 4°C until use. Titer of the amplified phage was quantified by plaque assay. Bacteriophage T7-displayed L7Ae or dL7Ae was confirmed by Western blotting  of the purified DNA was used as a template in an in vitro transcription reaction at 37°C for 4 h in 200 μl volume . The transcription product was treated with 2 U TURBO DNase (Thermo Fisher Scientific) at 37°C for 60 min and purified by phenol-chloroform extraction followed by ethanol precipitation. Purified RNAs were dissolved in 1× RNA loading dye (NEB), heat-denatured at 80°C for 3 min, and separated by 8% PAGE. The separated sample was visualized by UV shadowing to excise the gel fragment and the RNA was eluted overnight in TE buffer. The eluents were filtered through a 0.22 μm membrane filter , and concentrated using an ultrafiltration device . RNA concentration was determined from absorbance at 260 nm using NanoDrop One (Thermo Scientific).The template DNA was prepared by primer extension as described previously with modifications . Two olig for 10 min at 4°C. The phage particles were dissolved in 500 μl selection buffer and was incubated with 10 μl pre-equilibrated MagStrep ‘type3’ XT beads at room temperature for 30 min. The phage-immobilized beads were washed once with 500 μl selection buffer and re-suspended in 295 μl of the same buffer. RNA solution of an appropriate concentration was prepared in 200 μl volume, and the RNAs were folded by heating at 80°C for 3 min followed by incubation at room temperature. The folded RNAs were mixed with 4 μl yeast tRNA , 40 U Murine RNase Inhibitor (NEB), and 395 μl of the phage-immobilized magnetic beads. After washing with selection buffer, phage particles were eluted with 50 μl 1× Buffer BXT Strep-Tactin XT Elution Buffer  (IBA Lifesciences).A detailed summary of the selection parameters is shown in 2, 5 mM dithiothreitol) were added to another aliquot of the beads and incubated at 30°C for 15 min. The beads were separated by a magnet and the supernatant containing phage particles was collected.In the 2nd purification, the eluent from the 1st purification was mixed with 10 μl of capture oligo DNA-immobilized NeutrAvidin beads and 440 μl selection buffer and incubated. After washing with the same buffer, the beads were re-suspended in 20 μl of TE buffer. The bead suspension was split into two aliquots for separate RNA and phage recovery. To recover RNAs, 20 pmol of reverse primer N20L_Rv,  was addeThe recovered RNAs were reverse transcribed at 60°C for 15 min in 20 μl volume using SuperScript IV, and the reaction was quenched by incubation at 85°C for 5 min. The cDNAs were amplified by PCR  with appropriate primers  or two-step PCR (for PD library). The RNAs were mixed with a barcoded reverse transcription primer  according to manufacturer's instructions. PhiX control v3 DNA  was spiked into the libraries as an internal control, and sequencing was performed on Illumina MiSeq sequencer using MiSeq Reagent Kit v3 (150 cycle single read or 300 cycle paired-end read). Raw sequence data (fastq files) were analyzed and visualized by custom Python scripts.The oligo pool (426 fmol) containing 2000 distinct sequences was amplified by PCR  using N20L_Fw and N20L_Rev  as primeE. coli BL21 (DE3) strain. The transformants were cultured overnight at 30°C in 2 ml of MagicMedia Medium (Thermo Fisher Scientific) supplemented with 50 μg/ml carbenicillin. The cells were harvested and suspended in ice-cold His-tag purification buffer B . After cell disruption by ultrasonication, Triton X-100 (0.05% v/v) was added to the crude cell extract. The extract was centrifuged at 18 000 × g for 10 min at 4°C to remove cell debris. The supernatant was incubated with TALON metal affinity resin  for 10 min at 4°C. After washing several times with the same buffer, His-tagged protein was eluted with His-tag elution buffer. His-tag purified proteins were confirmed by Western blotting with Strep-Tactin-HRP conjugate   .MagStrep ‘type3’ XT beads  were rinsed three times with ultrapure water and then incubated with blocking buffer  at 4°C overnight. His-tag purified proteins were incubated with 10 μl of MagStrep ‘type3’ XT beads at 4°C for 30 min. After washing with 500 μl each of high salt buffer  and tTBS  , the beads were stored in blocking buffer at 4°C.RNA solution (0.2 μM) was prepared in 500 μl selection buffer supplemented with 0.5 M urea, and the RNAs were folded by heating to 80°C for 3 min followed by incubation at room temperature. The folded RNAs were mixed with 1 μl yeast tRNA  and 5 μl recombinant protein-immobilized beads, and incubated at 37°C for 30 min. After washing eight times with 500 μl sample buffer, RNAs were eluted with 50 μl 1× Buffer BXT Strep-Tactin XT Elution Buffer (IBA Lifesciences). The eluents were purified and concentrated by ethanol precipitation and then dissolved in 10 μl TE.We designed the following strategy to enrich complexes of RNA and phage-displayed protein from unbound species via two-step affinity purification Figure . First  which isE. coli to be amplified. The amplified RNA and phage pools are then used for the subsequent round of selection. After several rounds of selection, the enriched RNA and phage-displayed RBP sequences are analyzed by next-generation sequencing (NGS).Through this two-step affinity purification protocol, complexes of RNA and phage-displayed protein are enriched. The beads are split into two aliquots to recover the enriched RNAs and phage particles separately. RNAs and phage particles are recovered by heating and ribonuclease H (RNase H) treatment, respectively. As RNase H cleaves the RNA strand in DNA–RNA hybrid, it helps to reduce phage particles that non-specifically bind to the capture DNA, NeutrAvidin, or magnetic beads. The recovered RNAs are amplified by reverse transcription (RT) followed by polymerase chain reaction (PCR) and in vitro transcription (IVT). The recovered phage particles are used to infect A. fulgidus L7Ae that lacks affinity for box C/D Kt as a negative control protein  between L7Ae and box C/D Kt was 2.13 × 10–11 M in physiological salt and magnesium concentrations at pH 7.4  measured by stopped-flow kinetic analysis  of a mock PD library was immobilized on StrepTactin magnetic beads. Next, the beads were incubated with 0.2 μM mock RNA library in 500 μl buffer for 10 min, and the beads were washed three times with the same buffer. The phage particles and the RNAs were eluted with D-biotin and then incubated with DNA-immobilized NeutrAvidin magnetic beads in 500 μl of buffer for 10 min to capture RNA-phage complexes. After washing three times, RNAs were eluted, reverse transcribed, and amplified by PCR. Since dKt was designed to be 5-nt longer than box C/D Kt, relative abundance of the two RNA species could be evaluated by gel electrophoresis of the PCR products  and five residues  in the loop connecting α4 helix and β4 sheet were randomized. We did not randomize R41 because it is considered to form a universal hydrogen bond with a phosphate group in Kt RNA (10 ( = 208), diversity of the actual library is limited by the efficiency of in vitro packaging reaction of the phage genomic DNA which was estimated to be approximately 4.0 × 108 based on plaque assay. The PD library size is comparable to those of the previously reported T7 PD libraries , 5 and 6 were analyzed by Illumina sequencing. As expected, almost all sequences in the initial RNA and PD pools (99.8% of RNA and 99.2% of PD) were sequenced only once Figure  and C. RSequencing of the enriched RNA and LS protein sequences does not indicate which RNA interacts with which LS protein. To discover specific RNA–LS protein pairs, we prepared recombinant LS1–LS20 and L7Ae proteins and immobilized them individually on magnetic beads. We also synthesized the 1991 most abundant RNA sequences after the 6th round and 9 control RNAs  ,42 by inA simple inspection of the RNA sequences after PD-SELEX revealed two highly enriched consensus sequences. Consensus sequence 1  was found in 57% of the population, and consensus sequence 2 (UCCAUGACGC) was found in 25% of the population after round 6. The 2000-sequence RNA library also contained 59% and 33% of the consensus sequence 1 and 2, respectively. In contrast, the unselected pool contained 0.0109% (consensus sequence 1) and 0.0011% (consensus sequence 2) of the two sequences. Consequently, we focused on binding of these consensus sequences to the individual LS proteins.Enrichment of the RNAs possessing consensus sequence 1 or 2 was statistically analyzed and visualized by box plots Figure  and C. JFurther inspection revealed that LS4 has low EF values (<0.5) for consensus sequence 2, and LS12 and L7Ae have low EF values (<0.5) for consensus sequence 1 Figure  and C. TKD of 7.36 × 10–11 M, and LS12-1 RNA bound LS12 with a KD of 1.05 × 10–11 M  of CS1 RNA–LS4 and CS2 RNA–LS12 using 0.5 nM Cy5-labeled RNA showed binding profiles characteristic of titration regime  and measured their binding affinities using SPR Figure . RNAs weM Figure  suggesteM Figure , a minimA Figure  which coA Figure . AccordiA Figure  whose KDn regime  which in 10–10 M .–8 M, and that of CS1 RNA and L7Ae was 1.49 × 10–9 M •A (Hoogsteen) type base pairs (KD = 2.21 × 10–9 M) for LS4 but its affinity for LS12 and L7Ae was not measurable (KD > ∼1 μM). This prompted us to characterize additional mutants at these positions . CS1-M2b RNA exhibited respectable binding selectivity for LS4 (1273-fold over LS12) with a lower KD (8.65 × 10–11 M). The possibility of tuning the binding affinity while preserving selectivity by mutating CS1 RNA may be advantageous for adapting the RNA–RBP pair for specific applications.It is known that G•A mismatches in the core of Kt RNAs form se pairs . We founse pairs . To exam–7 M KD which is 15800-fold greater than that for LS12 (7.24 × 10–12 M). On the other hand, the KD value of CS2 RNA and L7Ae was 5.71 × 10–12 M (We next examined the binding affinities of CS2 RNA with LS4 and L7Ae. CS2 RNA bound LS4 at 1.14 × 10 10–12 M  which wa 10–12 M . Therefo 10–12 M .KD except for M3. Impact of the M3 mutation on binding affinity was more pronounced for L7Ae (KD = 2.00 × 10–7 M) than for LS12 (KD = 6.02 × 10–9 M)  in the asymmetric internal loop of CS2 RNA  Figure . LS12 an 10–9 M) . This ob 10–9 M) . It was  10–9 M)  which coKD = 5.99 × 10–7 M and 1.45 × 10–8 M, respectively) compared to the box C/D Kt – L7Ae interaction (KD = 2.13 × 10–11 M). (The affinities of LS4 and LS12 for the box C/D Kt RNA were significantly impaired (0–11 M). . Therefo0–11 M).  and B.KD value of 2.13 × 10–11 M  based on Förster resonance energy transfer (FRET) measurement using a stopped-flow mixer   ,42. We a 2.5 nM) ,63. More 2.5 nM) . The RNA nM) (KD . While t nM)   . Moreoved Lilley , suggestd Lilley . While iE. coli or yeast cell-based in vivo library-vs-library selections have been reported to identify protein-protein  that are represented more frequently in the randomized RNA library (relative to larger and more complex RNA motifs) may have contributed to the successful enrichment the RNA–RBP pairs. In principle, RNA–RBP pairs with lower affinity (e.g. nanomolar KD) should also be selectable by PD-SELEX. In Bowley et al., an antigen-antibody pair with a KD of 50 nM was selected from phage-displayed antibody and yeast-displayed antigen libraries  show low picomolar affinities which are among the strongest RNA-protein interactions observed in natural or synthetic systems . We expeAll data and materials are available upon request.gkab527_Supplemental_FileClick here for additional data file.


N7-methylguanosine (m7G), 5-hydroxymethylcytosine (hm5C), 5-methylcytosine (m5C), N1-methyladenosine (m1A), N6-methyladenosine (m6A), and others. In this study, we focus on non-coding RNAs (ncRNAs) to summarize the epigenetic consequences of RNA modifications, and the pathogenesis of cancer, as diagnostic markers and therapeutic targets for cancer, as well as the mechanisms affecting the immune environment of cancer. In addition, we summarize the current status of epigenetic drugs for tumor therapy based on ncRNA modifications and the progress of bioinformatics methods in elucidating RNA modifications in recent years.As next-generation sequencing (NGS) is leaping forward, more than 160 covalent RNA modification processes have been reported, and they are widely present in every organism and overall RNA type. Many modification processes of RNA introduce a new layer to the gene regulation process, resulting in novel RNA epigenetics. The commonest RNA modification includes pseudouridine (Ψ),   In this review, Rong et al. focus on ncRNAs to summarize the epigenetic consequences of RNA modification, and the pathogenesis of cancer, as diagnostic markers and therapeutic targets for cancer, as well as the mechanisms affecting the immune environment of cancer. The mentioned changes indicate “epigenetically related” changing processes, which extensively cover changing processes inside the methylating process of DNA cytosines, changing processes inside chromatin and histone configurations, as well as changing processes inside the expressing state of non-coding RNAs (ncRNAs), controlling the stable characteristic of numerous messenger RNAs (mRNAs), and becoming “master regulating elements” for the gene-expressing state.,,N7-methylguanosine (m7G), 5-hydroxymethylcytosine (hm5C), N1-methyladenosine (m1A), 5-methylcytosine (m5C), N6-methyladenosine (m6A), and others.,N4-acetylcytidine (ac4C) inside the gene-expressing state and regulating process and proved ac4C’s correlation with different diseases in humans, in particular carcinoma. Investigations of RNA-modifying process functions and the relevant molecular systems attention, boosting tool development for detecting particular modifying processes in transcriptomes and genomes. Developing novel technologies promotes knowledge regarding RNA-modifying processes.,Overall life modes require systems regulating RNA conditions as well as activity. One vital proportion of the mentioned RNA control bands appears under the transcribing process condition, whereas an important proportion pertaining to RNA homeostasis is increasingly dependent on the RNA stable characteristic and degrading process. It is especially true in terms of normal diseases facing humans, e.g., carcinoma, in which abnormal RNA transcriptomes constitute markers of cells under transformation that fail to comprehensively result from changes in the transcription initiation sites related to DNA.,Current studies have pointed out that there are two different ways of RNA modifications: one is to change the structure of modified RNA, and then regulate the interaction between RNA and proteins, blocking or inducing; the other is that modified RNA-binding proteins (RBPs) directly recognize and induce follow-up reactions. Dynamic and reversible methylation on RNA is mediated by RNA-modifying proteins called “writers” (methyltransferases) and “erasers” (demethylases). “Readers” (modified RNA-binding proteins) recognize and bind to RNA methylation sites, which influence the splicing, stability, or translation of modified RNAs.Herein, the roles of epigenetics and the therapeutic implications of ncRNAs are reviewed, including transfer RNA (tRNAs), microRNAs (miRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and ribosomal RNAs (rRNAs) in the regulation in human cancers. We mainly summarize the epigenetic consequences of RNA modification, and the pathogenesis of cancer, as diagnostic markers and therapeutic targets for cancer, as well as the mechanisms affecting the immune environment of cancer. We also summarize the current status of epigenetic drugs for tumor therapy based on ncRNA modifications and bioinformatics methods for RNA-modifying processes, as well as questions to be addressed.,,Homo sapiens YTH domain containing 1 [YTHDC1]).,in vivo and in vitro, bound to m6A-assuming RNAs; its biochemically related footprint complies with the m6A consensus motif. HNRNPA2B1 directly binds to several nuclear transcripts and can elicit basically the same selective splicing influence as that of the m6A writing element METTL3. In addition, HNRNPA2B1 can promote primary miRNA processing, interact with DGCR8 (the miRNA microprocessor complex protein), and bind to m6A signs inside one subdivided primary miRNA transcript set. As proven by an m6A map analysis, lncRNAs undergo extensive m6A modification as well. The m6A methylating process is an RNA structure-related switching and also engages in the competing endogenous RNA (ceRNA) model mediated by lncRNA.,m6A modification regulates mRNA at different levels, including structure, maturation, stability, splicing, export, translation, and decay.,m5C is also reported to be involved in the regulation of RNA metabolism. m5C is not only detected in ribosomal RNA and metastatic RNA. It was also found that m5C modification in mRNAs was catalyzed by members of the NOP2/Sun RNA methyltransferase family and accumulated significantly near the translation initiation 3′ untranslated region (UTR) site. ALY/REF output factor (ALYREF) was further identified as the m5C-binding protein (reader) mRNA, which in the nucleus promotes the output of m5C-modified mRNAs.,,,m1A is found in mitochondrial (mt) transcripts, mRNA, rRNA, and tRNA .48, 49, ,Escherichia coli ribosomes, which are m7G modified on both SSU and large ribosomal subunit (LSU) rRNA.,m7G exists in the internal sites of tRNA and rRNA. In yeast, small ribosomal subunit (SSU) rRNA, the 1575 site, is m7G-modified by the Bud23-Trm112 heterodimer.O-methylated adenosine (Am) refers to the first adenosine proximal to the 5′ cap, capable of undergoing in-depth methylation based on methyltransferase PDX1 C-terminal inhibiting factor 1 (PCIF1) for forming m6Am. Consistent with m6A, the N6-methyl group of m6Am is also capable of undergoing demethylation by FTO  under an AAG motif in vitro and in vivo. Their study indicates that the RNA inner m6Am-modifying process undergoes mediation by METTL4; however, whether METTL4 exhibits additional substrates other than U2 snRNA should be studied in depth.2′-n by FTO .67 m6Am ,RNA m5C can receive the deeper oxidization from ten-eleven translocating (TET) class enzymes for forming hm5C.,,ψ, the largest amount of RNA modification process, appears in various RNAs .+ T cells of systemic lupus erythematosus are associated with disease activity.As mentioned above, we have introduced some types of ncRNA modifications, mainly including m6A, m5C, m1A, and m7G, all of which may lead to some epigenetic consequences. For example, METTL3 promotes tumorigenesis of breast cancer (BC), acute myeloid leukemia (AML), and hepatocellular carcinoma (HCC) by enhancing m6A modification.The effect of ncRNA modifications on carcinoma has a reflection inside the variations of tumor-associated genes. System-based investigation on the cross-link of post-modification regulation, ncRNA modifications, and substrate genes can help indicate the system pertaining to ncRNA modifications inside carcinoma in a comprehensive manner. The present review elucidates the effects exerted by ncRNA modifications in carcinoma pathogenic mechanisms, as shown in 124ncRNAs plays a key role in the occurrence and development of many cancers. Studies have shown that peptides and proteins encoded by ncRNA, such as HOXB-AS3, FBXW7-185aa, PINT-87aa, and SHPRH-146aa, miPEP-200a, and miPEP-200b, have been proven to play an important role in tumorigenesis and the development of cancer though regulating glucose metabolism, the epithelial-to-mesenchymal transition (EMT), and the ubiquitination pathway.in vitro and in vivo aggravation of HCC malignant phenotypes. In a mechanistic manner, LINC00958 could sponge miR-3619-5p for increasing the hepatoma-originating growing element (HDGF)-expressing state, helping the HCC lipogenic process and progressing process. The N-methyladenosine-modifying process under the mediation of METTL3 generated the LINC00958 upregulating process by the stabilization of its RNA transcript.In HCC, Zuo et al.Specific to squamous cell cancer of the head and neck (HNSCC), Ban et al.Specific to bladder carcinoma, according to Han et al.,ncRNA modifications not only plays an important role in promoting tumor progression, but also plays an important role in tumor inhibition. It has been reported that YAP, ALKBH5, IGF2 and other tumor suppressor elements can interact with ncRNA to inhibit tumor progression.Specific to colon cancer (CC), according to Liu et al.,in vitro and in vivo inhibiting CRC progressing process. It is noteworthy that they demonstrated the mA reading element YTHDF3 as an emerging YAP target and one vital YAP signaling player through the facilitation on lncRNA GAS5 degradation under the modification of mA, presenting novel insights for CRC progression.In colorectal carcinoma (CRC), Ni et al.Epigenetics has turned out to be a hotspot in existing scientific research. The RNA-modifying process engages in carcinoma malignancy phenotype regulation through control of the expressing state of carcinoma-associated genes. The uncommon level of the RNA-modifying process facilitates the tumor pathogenic mechanism and progressing process. Although m6A was highlighted extensively, it remains unclear. How RNA modification readers’ expressing state and process receive regulation inside cancer require in-depth studies.It has been confirmed that ncRNA modifications have become a universal biomarker of carcinoma. By microarray analysis, Zhang et al.ncRNA modification has been identified to act on carcinogenic driving factors and tumor suppressor factors in a variety of major cancer types. Therefore, a deeper understanding of the complex interaction network coordinated by ncRNA modifications will provide a unique opportunity to design better treatment interventions. Previous studies have reported the presence of m6A, m5C, and pseudo-uracil (ψ) in coding RNA and ncRNA and described their physiological and pathological roles in cancer. These post-transcriptional modifications affect the development, maintenance, and progression of tumors. m6A, m5C, or ψ regulates cell survival in many environments to cope with stress and stem cell function, and targeting them represents a very promising opportunity to specifically target these cell populations and reduce chemotherapy resistance and recurrence.In CRC cells, the m6A process is suggested to mediate the cytoplasmic export of a crucial oncogenic circNSun2. Chen et al.in vitro and in vivo HCC metastasis. Furthermore, miR-29a silenced anti-metastatic SOCS1 through direct TET family targeting, resulting in SOCS1 promoter demethylation inhibition. As proved by the chromatin immunoprecipitation analysis processes, TET1 could change the SOCS1 expressing state via binding to the promoting element area pertaining to SOCS1. Lastly, the miR-29a overexpressing state displayed a relationship to weak clinical outcomes and TET-SOCS1-matrix metalloproteinase (MMP)9 axis silencing inside HCC cases. According to their findings, 5-hmC loss is an epigenetic hallmark of HCC, and miR-29a is an important epigenetic modifier, promoting HCC metastasis through TET-SOCS1-MMP9 axis silencing. The results offer a new strategy for epigenetic carcinoma therapy.In HCC, Wang et al.in vitro and tumor growth in vivo. This study suggests that ALKBH5-mediated mA modification of PVT1 contributes to OS tumorigenesis.In osteosarcoma (OS), it was found that lncRNA PVT1 expression was upregulated in OS tissues and cells and was significantly related with clinical stage, tumor size, and prognosis of patients with OS. Further investigation revealed that mA demethylase ALKBH5 could associate with PVT1 and suppress its degradation. ALKBH5 decreased the mA modification of PVT1, thus inhibiting the binding of reader protein YTHDF2 in PVT1. Functionally, ALKBH5-mediated PVT1 upregulation promoted the OS cell proliferation The RNA-modifying process and tumor immune microenvironment (TME) critically impact carcinoma development. Although immune checkpoint blockade (ICB) therapy has revolutionized carcinoma treatment, many patients do not respond to, or develop, resistance to ICB. m6A in RNA regulates many pathophysiological processes. Li et al.+ T cells. Overall, these findings suggest a novel anticarcinoma strategy for restoring balanced RNA methylation in tumor cells and guide clinical physicians in developing a new practical approach for considering the impact of related genes on prognosis. Zhang et al.Additionally, Tang et al.S-adenosylmethionine (SAM) is a universal methyl donor for S-adenosylhomocysteine (SAH), receiving the hydrolyzation into homocysteine and adenosine via SAH hydrolase. SAH hydrolase is capable of regulating SAH, and SAH inside cells acts as a competitive inhibiting element for several methyltransferases dependent on adenosylmethionine.in vitro. Furthermore, FB23-2 noticeably suppresses human AML cell lines from progressing and primary cells inside xeno-transplanted rats. On the whole, as revealed from the information here, FTO refers to one druggable target, and the process to target FTO by small-molecule-inhibiting elements is promising for treating AML.Given that the RNA-modifying process plays an important role in carcinomas, a chemical that inhibits the RNA-modifying process editing could serve as an effective drug. ,,,6A peaks in different subcellular components and gene regions. Among those human m6A-modifying process, 190,050 and 150,900 peaks were identified in carcinoma and non-carcinoma cells, respectively. Lastly, all results were integrated and imported into a visualized cell-dependent m6A database CVm6A. CVm6A specificity is considered to noticeably promote the function and regulation study of the cell-dependent m6A-modifying process in disease and developing process. Xuan et al.http://rna.sysu.edu.cn/rmbase/), integrating epitranscriptome sequencing data to investigate post-transcriptional-modifying processes of RNAs, as well as how they are correlated to RNA-binding proteins, disease-related SNPs and miRNA-binding events. From 47 studies among 13 species, RMBase v2.0 expanded with ∼600 datasets and ∼1,397,000 modifying process sites, representing 10-fold expansion nearly in comparison with the existing release. It covers ∼1,373,000 m6A, ∼5,400 m1A, ∼9,600 Ψ-modifying processes, ∼1,000 m5C-modifying processes, ∼5,100 2′-O-methylating (2′-O-Me) processes, and ∼2,800 modifying processes of other modifying process types. Moreover, they developed a novel web-based tool termed as “modMetagene” for plotting RNA-modifying process metagenes with the use of a transcript mode. Such a database is conducive to investigating the potential functions and mechanisms of RNA-modifying processes.Information with respect to the positions of RNA-modifying process sites is critical to the characterization of mechanisms and functions of those modifying processes. As impacted by recent advances in genomics and molecular biology, biologists can identify a variety of RNA-modifying processes in an experimental manner. Most currently available high-throughput experimental techniques exhibit limitations due to the following reasons: (1) although certain technology, e.g., methylated RNA immunoprecipitation sequencing (MeRIP-seq), m6A sequencing (m6A-seq), and photo-crosslinking-assisted m6A sequencing (PA-m6A-seq), can be used to identify m6A sites and detect 10s of thousands of m6A-containing sequence fragments in the transcriptome, they cannot locate the exact positions of the m6A sites;As revealed in the collective evidence, RNA modification is progressively critical to the occurrence and development of cancer. Some writers, erasers, and even RNA-modified readers have potential as biomarkers that could serve as drug targets to diagnose and treat cancer. However, existing research on ncRNA and RNA modification is only the tip of the iceberg. Whether ncRNA and RNA modification are normal or specific in cancer needs to be clarified. In particular, we look forward to the relationship between RNA modification and tumor immunity. Therefore, the modification process of ncRNA and its specific targets need to be studied further.Although many sequencing technologies and databases are essential to predict and study RNA modification, more accurate and faster detection and prediction methods are needed. In addition, we need more information prediction software for ncRNAs and RNA modification sites. After solving the above problems, we expect that scientists can study the correlation between RNA modification and cancer pathogenesis, prognosis, and diagnosis, further explore the mechanism behind its function, design specific small molecules targeting RNA modification, and detect targeted drugs that can penetrate blood to treat cancer by brain barrier.


The single-stranded, negative-sense, viral genomic RNA (vRNA) of influenza A virus is encapsidated by viral nucleoproteins (NPs) and an RNA polymerase to form a ribonucleoprotein complex (vRNP) with a helical, rod-shaped structure. The vRNP is responsible for transcription and replication of the vRNA. However, the vRNP conformation during RNA synthesis is not well understood. Here, using high-speed atomic force microscopy and cryo-electron microscopy, we investigated the native structure of influenza A vRNPs during RNA synthesis in vitro. Two distinct types of vRNPs were observed in association with newly synthesized RNAs: an intact, helical rod-shaped vRNP connected with a folded RNA and a deformed vRNP associated with a looped RNA. Interestingly, the looped RNA was a double-stranded RNA, which likely comprises a nascent RNA and the template RNA detached from NPs of the vRNP. These results suggest that while some vRNPs keep their helical structures during RNA synthesis, for the repeated cycle of RNA synthesis, others accidentally become structurally deformed, which likely results in failure to commence or continue RNA synthesis. Thus, our findings provide the ultrastructural feature of vRNPs during RNA synthesis. Nakano et al. use high-speed AFM and cryo-EM to study influenza virus RNA synthesis in vitro. They observe the synthesis of two types of RNA: folded single stranded RNA associated to an intact viral ribonucleoprotein (vRNP) and looped, double stranded RNA associated with a partially rearranged vRNP structure. Orthomyxoviridae, has eight single-stranded, negative-sense RNA (vRNA) segments as its genome. Transcription and replication of influenza A virus are carried out by ribonucleoprotein complexes called vRNPs in the nucleus of infected cells. A vRNP comprises a vRNA, multiple copies of nucleoprotein (NP) and a heterotrimeric, RNA-dependent RNA polymerase complex comprising PB2, PB1 and PA subunits4. Each vRNP adopts a helical, rod-shaped structure, in which a single strand of a multiple NP–RNA complex is folded back on itself and coiled, forming a double-stranded helix with a loop structure at one end6. The heterotrimeric RNA polymerase is located opposite to the loop end of the helical rod-shaped vRNP9.Influenza A virus, a member of the 7GpppXm) of host pre-mRNAs11, and the PA subunit cleaves the pre-mRNA 10–13 nucleotides downstream from the cap with its endonuclease activity14. The resultant capped RNA fragment is directed to the PB1 active site where it is used as a primer15. After elongation, a poly(A) tail is added to the 3′ end of the transcript by stuttering of the polymerase on the oligo-U stretch of the template vRNA18. Hence, the 5′-capped and 3′-polyadenylated viral mRNAs are synthesized in a primer-dependent manner. In contrast, genome replication is thought to be primer-independent19. Replication involves generation of positive-sense complementary RNAs (cRNAs), which are replication intermediates that act as templates for vRNA synthesis. Elongation of a nascent cRNA by cis-acting RNA polymerase proceeds concomitantly with sequential binding of free NPs, forming a rod-shaped, double-helical cRNP complex22. Afterward, trans-acting or trans-activating RNA polymerase generates vRNAs from intermediate cRNAs24.Although influenza virus vRNPs conduct both transcription and replication of the vRNAs, mechanisms of the two processes are quite different. During transcription, the PB2 subunit binds to the 5′-terminal methylated cap structure  and concluded that the vRNPs maintain their double-helical structures during transcription31. However, it remains unclear whether the helical vRNPs they observed are the only conformation of vRNPs producing nascent RNA. Here, to further characterize RNA synthesis from an ultrastructural perspective, we analysed virion-derived vRNPs producing nascent RNAs during in vitro RNA synthesis, using high-speed atomic force microscopy (HS-AFM) and cryo-EM. The combination of these two techniques enabled us to clearly visualize and characterize the native structures of vRNPs producing nascent RNAs.Great progress has been made in delineating the molecular mechanisms by which the RNA polymerase conducts transcription and replication based on its atomic structure32. To investigate the structure of vRNP during RNA synthesis, we purified vRNPs from influenza A/Puerto Rico/8/34 (PR8) virions and performed in vitro RNA synthesis using primers in the presence of nucleoside triphosphates. Autoradiography of the RNA products after electrophoresis showed bands corresponding to the eight vRNAs of influenza A virus in a 15-min incubation after the reactions  for the NP and NA genes35. After in vitro RNA synthesis using primers in the presence of nucleoside triphosphates, vRNPs were adsorbed onto a mica substrate and were visualized in solution. Without the primer, after in vitro RNA synthesis, vRNPs appeared as rod-shaped structures with helical grooves .Although vRNPs are helical rod-shaped structures in a static state, it is possible that they may change their conformations during RNA synthesis. To investigate near-native vRNP structures producing nascent RNA, we employed HS-AFM, which can provide topographic images in solution by scanning sample surfaces with a probe tip, without fixation or staining31. The tomographic reconstruction of a deformed vRNP was able to resolve the continuous RNA loop associated with the deformed vRNP , dsRNA was not detected in mock-infected cells , for in vitro RNA synthesis. When Br-UTP was used for in vitro RNA synthesis instead of UTP, both folded and looped RNAs associated with vRNPs were similarly observed Fig. , indicatlls Fig. , suggestNext, to determine whether double-stranded, looped RNA contains nascent RNA, we used 5-ethynyl-UTP (EUTP) for in vitro RNA synthesis and evaluated its incorporation into looped RNAs using Click chemistry. A Click reaction with biotin-azide was followed by incubation with streptavidin, and specific binding of streptavidin to looped RNAs was confirmed with HS-AFM Fig. . However36, vRNPs treated with a low concentration of RNase A were prepared as control vRNPs lacking intact residential vRNA  were lysed in 50 mM Tris-HCl buffer (pH 8.0) containing 100 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol (DTT), 2% Triton X-100, 5% glycerol, 2% lysolecithin and 1 U μL−1 RNasin Plus RNase inhibitor  for 1 h at 30 °C. The sample was ultracentrifuged through a 30–70% (w/v) glycerol gradient in Tris-NaCl buffer [50 mM Tris-HCl (pH 8.0) and 150 mM NaCl] at 45,000 rpm for 3 h at 4 °C in a SW55Ti rotor . Collected fractions were mixed with 2× Tris-glycine SDS sample buffer  and then subjected to SDS-PAGE using a 4–15% Mini Protean TGX precast gel .Influenza A virus, A/Puerto Rico/8/34 (H1N1) (PR8), was prepared, as previously reported−1 for cryo-EM and 0.01 mg mL−1 for other experiments) was incubated in 50 mM Tris-HCl buffer (pH 7.9) containing 5 mM MgCl2, 40 mM KCl, 1 mM DTT, 10 μg mL−1 actinomycin D, 1 mM each of ATP, CTP, GTP and UTP, 1 U μL−1 RNasin Plus RNase inhibitor with a primer, and 1 mM ApG . In some experiments, 10 μg mL−1 rabbit globin mRNA  was used as a primer instead of ApG. The reaction was performed at 30 °C for 15 min (for ApG primer) or 30 min (for globin mRNA primer) unless otherwise noted. For detection of newly synthesized RNA by radioisotope, the same reaction mixture was used with the exception that 0.25 μCi μL−1 [α-32P] UTP and 0.05 mM UTP were added. After in vitro RNA synthesis, RNA was purified with an RNeasy Mini kit , mixed with an equal volume of 2× RNA loading dye , heated at 90 °C for 2 min, and immediately chilled on ice. The sample was electrophoresed on a 4% polyacrylamide gel containing 7 M urea in 0.5× TBE buffer  at 120 V for 5 h. The gel was dried at 80 °C for 2 h, exposed to an imaging plate  for 12 h to 24 h, and scanned with a Typhoon 3000 Phosphorimager . Labelling of nascent RNA with a nucleotide analogue was also performed in the same reaction mixture using 1 mM Br-UTP (Sigma–Aldrich) or 1 mM EUTP  instead of UTP. Inhibition of RNA synthesis was evaluated by adding 0.1–100 μM of 6-fluoro-3-hydroxy-2-pyrazinecarboxamide-4-ribofuranosyl-5′-triphosphate  to the reaction mixture.Purified vRNP (1–2 mg mL34. Templates containing a T7 phage promoter sequence (TAATACGACTCACTATAGGG) were amplified by PCR using sets of primers listed in Supplementary Table 41. PCR products were purified with a Min Elute gel extraction kit (Qiagen) and transcribed in vitro with RiboMAX large-scale RNA production sysytem-T7 (Promega) according to manufacturer instructions. After RQ1 DNase I (Promega) treatment for 30 min at 37 °C, transcripts were purified using an RNeasy mini kit. The concentration of purified RNA was determined by spectrophotometry, and the copy number was calculated from the molecular weight of each RNA. RNA (1 × 1010 copies) was then subjected to electrophoresis on a 4% polyacrylamide gel containing 7 M urea and visualized with silver staining using a Silver Staining II kit . Additionally, all eight vRNA segments of influenza A virus (A/WSN/33 strain) were similarly prepared and used as markers for electrophoresis. Template DNAs were amplified with the primers listed in Supplementary Table −1 [α-32P] UTP. Transcribed RNAs were purified and mixed before electrophoresis.RNA standards for NP and NA segments of PR8 virus were prepared, as described previously34. The RNA standard or in vitro transcribed RNA was mixed with a quarter volume of 10 μM tagged primer (sequence is provided in Supplementary Table −1 Superscript III reverse transcriptase (Invitrogen), and 1 U μL−1 RNasin Plus RNase inhibitor prepared with saturated trehalose and pre-heated at 60 °C for 5 min] were added to the RNA solution at 60 °C, and the mixture was further incubated for 1 h at 60 °C. The reaction was stopped by heating at 85 °C for 5 min, and the cDNA solution was stored on ice until use. cDNA solution (diluted 1:50) was mixed with forward and reverse qPCR primers  of synthetic RNA standards prepared as described were used to generate a standard curve. The copy number was calculated from the standard curve with a strong linear correlation (R2 > 0.99) and amplification efficiency between 95 and 105%. The detection limit was 1 × 104 copies μL−1.RT-qPCR was performed as described by Kawakami et al.2, 40 mM KCl and 1 mM DTT] and immersed in a liquid chamber filled with 80 µL of the imaging buffer for observation at room temperature using an HS-AFM system . We performed HS-AFM in tapping mode, in which the cantilever was excited to oscillate at its resonant frequency in the vertical direction during lateral and vertical scanning of the cantilever chip in order to allow the tip to intermittently tap the sample surface. Images were collected at two images s−1 using cantilevers with a 0.1 N m−1 spring constant and a resonance frequency in water of 0.6 MHz . To acquire high-resolution images, the electron-beam-deposited tips were fabricated using phenol or ferrocene powder, as described by Uchihashi et al.42. Incorporation of Br-UTP into nascent RNA was confirmed by incubating in vitro transcripts with 0.1 mg mL−1 monoclonal anti-5-bromodeoxyuridine antibody  for 2 h at 4 °C in a microcentrifuge tube and imaging the sample with HS-AFM. Production of the dsRNA was examined by adding 1 μg mL−1 J2 antibody  to the reaction mixture in a microcentrifuge tube, incubating for 2 h at 4 °C, and observing it by HS-AFM. In situ observation of RNA digestion by each RNase was performed by adding a 10% volume of the indicated concentration of RNase A  or ShortCut RNase III (New England Biolabs) to the AFM liquid cell during imaging. At least five independent experiments were performed for each RNase, and all HS-AFM images were viewed and analysed with Kodec 4.4.7.3943. A low-pass filter and a flattening filter were applied to individual images to remove spike noise and flatten xy-plane, respectively.In vitro RNA synthesis was performed in a microcentrifuge tube, with 2 µL of sample dropped onto freshly cleaved mica without surface modification. After incubation for the desired time (~1–5 min) at room temperature, the mica surface was then washed sufficiently with imaging buffer  and incubated for 30 min under light shielding. The mica surface was then washed with imaging buffer, and the buffer was replaced with 0.1 mg mL−1 of streptavidin solution . After 15 min of incubation at room temperature, the mica surface was washed again with imaging buffer for the HS-AFM observation.A Click-iT RNA imaging kit was purchased from Invitrogen. After in vitro RNA synthesis with EUTP, the sample was deposited on mica and incubated for 3 min at room temperature. The mica surface was then washed with imaging buffer, and the following reactions were all performed on mica without drying the surface. Imaging buffer on the mica surface was replaced with Click-iT reaction cocktail  of the deflection signal of a cantilever, we determined the spring constant (kc) [pN nm−1] and quality factor (Qc) of the cantilever, as described previously47. Determined kc values were in good agreement with nominal values reported by the manufacturer. During observation, the vRNP was destroyed by gradually lowering the set point. Destruction of the vRNP was determined from the apparent change in the height of the vRNP. After imaging, the amplitude value [V] at the frame in which the vRNP was destroyed was measured from the amplitude image, and the obtained value was converted into Asp [nm] using the InvOLS value. The free cantilever-oscillation amplitude A0 was measured by releasing the cantilever from the sample surface. The average tip–sample-interaction force <Ft> was calculated using the following equation: <Fts> = kc (A02 − Asp2)1/2/ Qc48. For preparation of control vRNP, we treated 0.01 mg mL−1 of vRNP with 0.05 μg mL−1 of RNase A for 10 min at 37 °C. Degradation of the vRNA was confirmed by RT-PCR using primers used to detect the full-length NP segment.To measure the force applied to the sample surface, two types of HS-AFM images were obtained simultaneously: topographic images and amplitude images. By measuring the thermal noise and the inverse optical lever sensitivity (InvOLS) value [nm Vhttps://www.r-project.org/). P-value of <0.05 was considered statistically significant. All experiments performed on the paper were successfully replicated more than three times.All statistical analyses were performed using available R packages (Further information on research design is available in the Peer Review FileSupplementary informationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Data 1Reporting Summary


Metastasis is the leading cause of death of patients with esophageal squamous cell carcinoma (ESCC). Although an increasing number of studies have demonstrated the involvement of G3BP2 in several human cancers, how G3BP2 interacts with long noncoding RNAs and regulates mRNA transcripts in mediating ESCC metastasis remains unclear. In this study, we uncovered that G3BP2 was upregulated in ESCC. Further analysis revealed that upregulation of G3BP2 was significantly correlated with lymph node metastasis, depth of tumor invasion and unfavorable outcomes in ESCC patients. Both in vitro and in vivo functional assays demonstrated that G3BP2 dramatically enhanced ESCC cell migration and invasion. Mechanistically, LINC01554 maintained the high G3BP2 expression in ESCC by protecting G3BP2 from degradation through ubiquitination and the interaction domains within LINC01554 and G3BP2 were identified. In addition, RNA-seq revealed that HDGF was regulated by G3BP2. G3BP2 bound to HDGF mRNA transcript to stabilize its expression. Ectopic expression of HDGF effectively abolished the G3BP2 depletion-mediated inhibitory effect on tumor cell migration. Intriguingly, introduction of compound C108 which can inhibit G3BP2 remarkedly suppressed ESCC cell metastasis in vitro and in vivo. Collectively, this study describes a newly discovered regulatory axis, LINC01554/G3BP2/HDGF, that facilitates ESCC metastasis and will provide novel therapeutic strategies for ESCC. Esophageal carcinoma (EC) is an exceedingly aggressive cancer with a high mortality rate and poor prognosis worldwide , and squRas-GTPase-activating protein (SH3 domain)-binding protein 2 (G3BP2) is located on the long arm of chromosome 4 (4q21), which has been reported to harbor genetic variant risk factors for ESCC in the Chinese population . G3BP2 iG3BP2 belongs to the G3BP protein family, which has four distinct motifs in its protein structure, including a nuclear transport factor 2-like domain, acidic and proline-rich regions, an RNA recognition motif (RRM) and an arginine and glycine rich box (RGG box). Among the motifs, the RRM domain is the most common specific basic domain of RNA binding proteins (RBPs) . Mountinhttp://service.tartaglialab.com) and RPI-seq (http://pridb.gdcb.iastate.edu) suggest that G3BP2 may interact with the lncRNA LINC01554. LINC01554 is a long intergenic noncoding RNA and is mainly localized in the cytoplasm. It has been demonstrated to attenuate cell growth by inhibiting glycolytic effects [Long noncoding RNAs (lncRNAs), transcribed RNAs of more than 200 nucleotides in length, engage in diverse biological processes involved in tumor initiation, growth and metastasis at the epigenetic, transcriptional, posttranscriptional, and translational levels . LncRNAs effects  and acti effects . HoweverIn this study, our data revealed that G3BP2 was frequently upregulated in ESCC tissues and predicted unfavorable patient outcomes. Mechanistically, LINC01554 maintained the high expression of G3BP2 by inhibiting ubiquitin mediated proteasome degradation of G3BP2. RNA-seq further identified hepatoma-derived growth factor (HDGF) as the objective mRNA that regulated by G3BP2. G3BP2 stabilized HDGF mRNA transcript to maintain its high expression level and then increased ESCC metastasis. Notably, the introduction of compound C108, a small molecule that inhibits G3BP2, markedly attenuated ESCC cell metastasis in vitro and in vivo. In conclusion, our findings describe a new regulatory signaling axis, LINC01554/G3BP2/HDGF, that facilitates ESCC metastasis, and highlight that G3BP2 could be a promising therapeutic target for treatment of ESCC.https://www.oncomine.org) and TCGA databases (https://www.cancer.gov) Both databases indicated that G3BP2 was highly expressed in ESCC  and high endogenous G3BP2 expression cells (KYSE30 and KYSE150 cells), respectively. The overexpressed and knockdown efficiency was examined by Western blot Fig. . EctopicP < 0.05 and log2 (fold change) > 1, 387 and 263 differentially downregulated genes were identified in KYSE30 cells and KYSE150 cells, respectively, among which 25 genes were shared in both cell lines  and the result revealed that HDGF was upregulated in ESCC  and head and neck squamous cell carcinoma (HNSCC). We observed that G3BP2 was also highly expressed in both LUSC and HNSCC  tumor invasion and metastasis in breast cancer . In lineSo far, some mechanisms for regulating G3BP2 expression have been described. At the RNA level, G3BP2 is upregulated by androgen , miR-117In contrast to G3BP1, which has been reported to degrade c-Myc BART, and CTNNB1 mRNA transcripts, thereby promoting cancer cell growth, metastasis and invasion –33, and To better understand the pivotal role of the LINC01554/G3BP2/HDGF signaling axis in facilitating ESCC metastasis, we designed and conducted a series of rescue experiments. The results showed that disrupting the interaction between G3BP2 and LINC01554 significantly downregulated HDGF expression and subsequently attenuated ESCC cell migration and invasion. In addition, both ectopic overexpression of LINC01554 and HDGF effectively restored the inhibitory effect of G3BP2 silencing on ESCC cell metastasis. Moreover, we found that the staining pattern of LINC01554, G3BP2, and HDGF was the same in ESCC and matched nontumor samples, which was in line with the results of the functional assays observed in vitro.To explore whether G3BP2 is a promising therapeutic target for metastatic ESCC, a small molecule that inhibits G3BP2, compound C108, was exploited in this study. Compound C108 has been proven to inhibit tumor initiation in breast cancer in vitro and in vivo . It mainIn conclusion, our findings Fig.  identifyThe materials and methods in detail were provided in the Supplementary information file


Biomechanical and molecular processes of premature cervical remodeling preceding spontaneous preterm birth (sPTB) likely result from interactions between the cervicovaginal microbiota and host immune responses. A non-optimal cervicovaginal microbiota confers increased risk of sPTB. The cervicovaginal space is metabolically active in pregancy; microbiota can produce, modify, and degrade metabolites within this ecosystem. We establish that cervicovaginal metabolomic output clusters by microbial community in pregnancy among Black individuals, revealing increased metabolism within the amino acid and dipeptide pathways as hallmarks of a non-optimal microbiota. Few differences were detected in metabolomic profiles when stratified by birth outcome. The study raises the possibility that metabolites could distinguish women with greater risk of sPTB among those with similar cervicovaginal microbiota, and that metabolites within the amino acid and carbohydrate pathways may play a role in this distinction. Contemporary studies of reproductive health and disease commonly utilize hierarchical clustering of cervicovaginal microbiota into community state types (CST) based on microbiota composition10. CST I, II, III, and V are enriched in Lactobacillus species, while CST IV is relatively poor in Lactobacillus and comprised of a wide range of strict and facultative anaerobes. Communities lacking Lactobacillus are considered non-optimal11 and have been associated with adverse reproductive health outcomes, including increased risk of urogenital disease and sexually transmitted infections18.Spontaneous preterm birth (sPTB) is a major cause of neonatal morbidity and mortality, and worldwide, sequelae of prematurity account for 1.1 million deaths annually. Premature cervical remodeling is thought to represent a precursory event on the pathway to sPTB. Recent evidence suggests that the biomechanical and molecular processes of premature cervical remodeling likely result from interactions between the cervicovaginal microbiota and host immune responses24. We reported outcomes from a 2000-women cohort revealing that CST IV, specific bacterial taxa, and local immune factors were significantly associated with sPTB9. We determined that Lactobacillus species and antimicrobial peptide β-defensin mitigate risk associated with certain bacterial taxa. This study further demonstrated that the prevalence of a non-optimal microbiota differed by race, as 40% of non-Hispanic Black women were colonized by CST IV early in pregnancy. While these findings are compelling in the context of known racial disparities, not all women with CST IV, regardless of race, will deliver preterm. Similarly, not all cases of sPTB occur in the setting of a non-optimal microbiota. These collective observations raise questions about other undefined factors in the cervicovaginal space that might mediate or distinguish clinical outcome in the presence of the same microbiota.Recent studies examining cervicovaginal microbiota in pregnancy have established an association between a non-optimal microbiota and sPTB28. Metabolomics is the study of sub-kilodalton biochemicals, many of which have been implicated in cellular function and structure. The cervicovaginal space is metabolically active in pregnancy, and microbiota in this ecosystem can produce, modify, and degrade metabolites31. Our group previously identified a cervicovaginal metabolomic signature in pregnancy, including distinct biochemicals associated with sPTB30. While these early studies suggest that metabolites may provide a lens into mechanisms underlying premature cervical remodeling, they did not incorporate data on microbiota composition. Published literature on the gastrointestinal system has established that the metabolome differs within the same microbial community, in part due to genetic and dietary factors32. As such, it is not just the microbial composition but the functional output of the collective microbiota that drives host response. Applied to the cervicovaginal space in pregnancy, it is plausible that obstetrical outcomes and risk may be governed by the effect of microbial metabolites on host function. However, no study in pregnancy to date has examined the cervicovaginal metabolome within the same ethnic group and microbial community to determine microbiome-metabolome patterns that may identify women at greatest risk for sPTB.Observations within the reproductive tract, as well as those in other biological niches, support the premise that microbiota affect host organ systems through regulation of microbial metabolitesGiven this background, we posit that the cervicovaginal metabolome may play a critical role in modulating microbiota-immune interactions, and that specific metabolites may function as biomarkers of sPTB. We therefore sought to examine cervicovaginal metabolomic output among women within specific CSTs to identify novel metabolites and related functional pathways associated with cervical remodeling and sPTB.Obstetric and clinical data are presented in Table Analysis across groups identified 304 biochemicals present in cervicovaginal fluid. Metabolite profiles clustered primarily by CST Fig. . Among tth component in IPCA  was significantly associated with birth outcome (t-test p < 0.05) rather than with CST . We found that the 4We next evaluated whether specific metabolites were associated with birth outcome. Five metabolites differed among women with sPTB versus term birth, among which four were increased in abundance in cases of sPTB Fig. , althougWe performed analyses to address the possibility that the metabolomic profile differs by birth outcome within cervicovaginal CST. We first compared metabolites abundances in CST I women who delivered spontaneously preterm or at term. We identified 10 metabolites that differed by birth outcome among women with CST I, four of which were increased in abundance in cases of sPTB Fig. . Among tTo determine whether within non-optimal cervicovaginal microbiota (CST IV), a known risk factor for sPTB, metabolites could identify women at greatest risk of early delivery, we compared metabolite abundances in CST IV women who delivered spontaneously preterm or at term. There were 13 differentially abundant metabolites, the top six of which based on absolute fold change are presented in Fig. Our analyses establish that cervicovaginal metabolomic output clusters by microbial community in pregnancy among individuals self-identifying as Black, and reveal increased metabolism within the amino acid and dipeptide superpathways as hallmarks of CST IV compared to CST I. To our knowledge, this study is examining for the first time the cervicovaginal metabolome in Black individuals and stratified by cervicovaginal microbiota. Relatively few differences were detected in metabolomic profiles when stratified by birth outcome. After correction for multiple comparisons, we did not detect significant differences in metabolomic profiles by birth outcome within the same CST though the small sample size limit our ability to draw conclusions from these subanalyses. Nonetheless, our study raises the possibility that metabolites, in particular those within the amino acid and carbohydrate superpathways could potentially distinguish women with greater risk of sPTB among those with similar cervicovaginal CSTs.Lactobacillus species, thereby leading to increased abundance of amino acid catabolites. Alternatively, anaerobes may produce greater amounts of amino acids, which they subsequently convert to biogenic amines  and non-optimal (CST IV) microbiota. Our observations support the concept that anaerobic organisms preferentially utilize amino acids as carbon and nitrogen energy sources, in contrast to Lactobacillus abundance35. Tryptamine also appears to mediate key physiologic functions of the gastrointestinal tract including epithelial secretion, which is important for host functions such as regulating immune response to pathogenic organisms and maintaining luminal fluid concentrations37. Among women with a non-optimal microbiota who go on to sPTB, only kynurenate (and not tryptamine) is significantly increased in abundance prior to FDR correction, raising the possibility that a relative lower abundance of tryptamine in a CST IV community may compromise host immune surveillance or function of the cervicovaginal epithelium. It is plausible that tryptamine confers some degree of host protection in the setting of high-risk cervicovaginal microbiota, and that this defense is lacking in women with CST IV who delivery spontaneously preterm. Notably, perturbations in the kynurenine pathway of tryptophan metabolism have been linked to inflammatory conditions, cardiovascular disorders, and neurodegenerative disease38. Our observations raise the possibility that tryptophan metabolites, including tryptamine and kynurenate, may regulate host immune response and potentially serve as mediators of cervical remodeling. As these metabolites have yet to be explored in the cervicovaginal space, this hypothesis warrants mechanistic investigation in future studies.It is worth noting that metabolites in the amino acid superpathway were both significantly upregulated and downregulated when comparing optimal to non-optimal cervicovaginal microbiota, and this observation may reflect physiological shifts to maintain homeostasis in response to distinct microenvironments. Metabolites that are increased may counteract those that are decreased in abundance, thereby providing metabolomic equipoise. An alternative and perhaps more biologically plausible explanation, especially in light of our understanding that select cervicovaginal microbiota confer differential risk of adverse pregnancy outcomes, is that these observed alterations in the relative abundance of metabolites in the amino acid subpathway generate relatively unopposed bioactive metabolites. These metabolites may induce host inflammation or barrier disruption in the absence of protective metabolites or factors. For example, we identified a 3.8-fold decrease in tryptophan among participants with CST IV compared to CST I. Conversely, breakdown products of tryptophan, including tryptamine and kynurenate, were among those significantly increased metabolites when comparing CST IV to CST I. Tryptamine is a biogenic amine that has been associated with CST IV cervicovaginal microbiota and negatively correlates with 39. Spermine is commonly associated with Lactobacillus-dominated cervicovaginal microbial communities and has been shown to increase production of lactic acid by Lactobacillus40. As lactic acid can function in an anti-inflammatory capacity in this space44, it is possible that spermine plays a major role at the cervicovaginal epithelium. In other systems, polyamines have been shown to regulate epithelial barrier function, specifically through modulation of cell adhesion proteins, including E-cadherin and occludin, as well select signaling pathways regulating the expression of these proteins47. In intestinal epithelial cells, spermine has been shown to prevent disassembly of focal adhesions required for cell migration by binding to the SH2 domain of Src48. These observations collectively raise the possibility that decreased abundance of spermine in the cervicovaginal space impedes epithelial barrier stabilization and/or promotes barrier permeability, thus contributing to cervical remodeling.With respect to stabilization of epithelial barriers, another interesting finding is the decrease in the polyamine spermine among women with a non-optimal microbiota and sPTB prior to FDR correction. Notably, our group has established that microbial supernatants from select high-risk vaginal bacteria associated with a non-optimal microbiota can disrupt the cervical epithelial barrier in vitro, and that this process involves cleavage of cell adhesion protein E-cadherinLactobacillus grows under anaerobic conditions and can convert pyruvate to lactic acid. Under aerobic conditions, however, Lactobacillus utilizes lactic acid to generate pyruvate, thereby increasing vaginal pH. This physiology is consistent with our observation that pyruvate abundance is significantly higher among women with CST IV. Though not statistically significant in our analyses post FDR correction, select glycolytic intermediates, including glucose 6-phosphate and pyruvate, may be further increased among women with CST IV who delivered spontaneously preterm, suggesting that cervicovaginal carbohydrate metabolism is potentially amplified in this subset of women. The role of glycolysis and microbiota has been explored in other mucosal surfaces, including the gastrointestinal tract, where increased glucose metabolism has been implicated in the breakdown of intestinal epithelial cell barriers, thereby leading to transcriptional reprogramming, inflammation, and enteric infection49. Our laboratory has demonstrated that specific bacterial species present in CST IV compromise the integrity of cervical epithelial cell barriers, alter immune response, and induce epigenetic changes31. Future studies investigating the functional and immune effects of select glycolytic intermediates on the cervicovaginal epithelium are warranted.Our findings raise questions as to the role of increased glycolytic intermediates, including glucose, glucose 6-phosphate, pyruvate, and 2-phosphoglycerate in women with a non-optimal compared to optimal microbiota. In the vagina, Another notable finding includes the predominance of lipid metabolites comprising the group of 11 metabolites associated with sPTB in the ICPA. Nine of the 11 metabolites belonged to the lipid superpathway, and all were decreased in abundance in cases of sPTB compared to term birth. Several of these metabolites are components of linolenic and arachidonic acid metabolism pathways, raising the possibility that these eicosanoid precursors are being consumed in the production of prostaglandins, which play a known role in cervical ripening and inflammation. Among the decreased metabolites are also sphingolipids, a class of lipid metabolites that stabilize plasma membranes and protect cells against harmful environmental exposures, as well as function in cell signaling and immune regulation. It is biologically plausible that this relative decrease in abundance of sphingolipids renders the cervicovaginal epithelium more vulnerable to environmental factors while potentially contributing to a maladaptive host immune response.30. We demonstrated previously that low-risk symptomatic women  between 22 to 34 weeks of gestation who go on to sPTB have distinct cervicovaginal biochemical output compared to controls with threatened preterm labor who go on to term deliveries30. Though none of the individual metabolites were overlapping between this current study and those identified in that cohort, common differentially detected superpathways were identified among women who delivered spontaneously preterm, including superpathways associated with amino acid and carbohydrate metabolism. When our current findings are compared to patterns of cervicovaginal metabolomic output identified in a cohort of asymptomatic women between 20 to 28 weeks of gestation at high risk of early delivery29, metabolic changes in the carbohydrate superpathway were detected, including increased abundance of glucose-6-phosphate, in women who delivered preterm. That study also identified differential detection of metabolites in the amino acid superpathway in cases of sPTB, consistent with our current study.Our findings expand upon the published literature from our group characterizing cervicovaginal metabolomic signatures in pregnancy associated with sPTBMetabolomic variance between symptomatic and asymptomatic women may be explained, at least in part, by differences in host immune response, including local and systemic inflammation. Differences between this current study and those two previously published cohorts from our laboratory may result from differences in the timing of cervicovaginal fluid sampling, as well as racial differences. The observation that increased glucose and amino acid metabolism is associated with sPTB across all three studies, however, provides compelling biologic plausibility that glycolytic intermediates and amino acid catabolites may play a key role in cervical remodeling and sPTB.Our data suggest a critical role for the cervicovaginal metabolome in modulating microbiota-immune interactions. Identifying bioactive metabolites and defining their mechanisms of action will likely facilitate recognition of novel biomarkers and pathways through which targeted interventions can successfully block premature cervical remodeling. Cervicovaginal metabolomic profiling in pregnancy may offer potential as a tool for sPTB risk stratification, thereby enhancing existing clinical strategies such as cervical length measurement. When considered in the context of a non-optimal microbiota, metabolites may be useful to identify women at greatest risk clinically. While certain high-risk bacterial species in combination with select metabolites may induce similar biologic effecs, and therefore represent the same hit to the system, it is possible that the presence of both is additive or even synergistic. Our observations also carry significant translational implications; metabolites offer potential as therapeutics in maintaining or restoring cervicovaginal homeostasis, thereby blocking the cascade of events that lead to aberrant cervical remodeling and sPTB. Spermine may represent one such example given our observations and its known role in epithelial barrier stabilization in other microenvironments.Strengths of this study include our ability to capture a cohort of non-Hispanic Black women with well-characterized cervicovaginal microbiota early in the second trimester. Focus on a single racial group and stratification by CST provides insight into metabolomic changes that may confer increased risk of early delivery whether the vaginal microbiota is considered optimal or non-optimal. Many published studies examining risk factors associated with sPTB have been limited by poorly phenotyped birth outcomes, resulting in clinical and biologic heterogeneity. Our pregnancy cohort is comprised of women with clear preterm birth phenotyping. Limitations of this study include the sample size of 40 women with cervicovaginal metabolomic analysis at a single time point in pregnancy; these data underscore the need for larger prospective studies characterizing metabolomic output across gestation associated with both optimal and non-optimal cervicovaginal microbial communities and birth outcome. The study is also limited by potential confounding of vaginal exposures predecing swab collection, which could impact metabolite quantification. This cohort, however, has a low prevalence of these exposures with only two participants reporting vaginal intercourse, douching, or pelvic exam within the 24 h preceding swab collection.50, it is likely that both host- and microbiota-derived metabolites play a biologically active role in the cervicovaginal space. It is reasonable to conclude that biochemicals belonging to the xenobiotic superpathway arise from microbiota or dietary intake rather than eukaryotic cells. While metabolites of bacterial origin may induce pathologic changes in mucosal immunity and epithelial barrier integrity, these small molecules may also be inhibitory to other bacterial species and alter microbial homeostasis within the cervicovaginal space40.The cellular origin of differentially detected biochemicals remains a major unanswered question; it is unclear whether metabolites of interest are derived from host cells, specific microbial species, or both. Based on our understanding of the microbiome-metabolome interface in other biological systems in the context of health and diseaseFuture mechanistic studies involving in vitro models may unveil pathways through which select metabolites contribute to premature cervical remodeling and sPTB. The ability of such biochemicals to inhibit or promote growth of commensal microbiota is of particular interest. Conversely, the potential for healthy microbial species to modify metabolomic signatures associated with adverse outcomes, as well as the role of host immune response in mediating these processes, warrant further investigation.Motherhood and Microbiome (M&M) in which 2,000 women enrolled from December 2013 through February 2017 as previously described9. The Institutional Review Board at the University of Pennsylvania approved this study. All methods were performed in accordance with the relevant guidelines and regulations. Informed consent was obtained from all subjects. Exclusion criteria included a major fetal anomaly, HIV seropositive status, history of organ transplant, chronic steroid use, enrollment into the study during a previous pregnancy, or multiple gestations. Particiants were asked whether they had vaginal intercourse, douched, or had a pelvic exam in the 24 h preceding cervical swab collection. Cervical swabs were collected at 16 to 20 weeks of gestation and cervicovaginal microbiota were analyzed by 16S rRNA gene sequencing9. Microbial communities were classified into CST as previously reported using hierarchical clustering with Jensen-Shannon divergence and Ward linkage10. CST I is predominated with L. crispatus, CST II with L. gasseri, CST III with L. iners and CST V with L. jensenii. CST IV is defined by a paucity of Lactobacillus species and a diverse set of strict and facultative anaerobes. Cases of preterm birth (PTB) were individually adjudicated by a maternal–fetal medicine physician (MAE) to distinguish spontaneous from medically indicated. PTB was considered spontaneous when a woman presented with either cervical dilation and/or premature rupture of membranes and delivered prior to 37 weeks of gestation.A nested case–control study was performed from a prospective cohort study entitled Inclusion criteria for this nested case–control study was self-reported non-Hispanic Black race and 16S rRNA gene data from cervicovaginal samples obtained at 16–20 weeks of gestation. Twenty sPTB and term birth control pairs were frequency-matched by CST. Four groups of 10 women were analyzed: CST I/sPTB; CST I/term birth; CST IV/sPTB; and CST IV/term birth. Only women who delivered between 38 to 40 weeks of gestation were included in the term birth group to avoid misclassification.Cervicovaginal fluid was collected following insertion of a sterile speculum using a sterile cotton-tipped swab. Samples were collected in 0.5 mL phosphate-buffered saline, immediately placed in liquid nitrogen, and stored at − 80 °C for future use.51, and were analyzed by both Ultra-Performance Liquid Chromatography/tandem mass spectrometry  and Gas Chromatography/Mass Spectrometry .Metabolomics analysis was performed by Metabolon®, Inc.  as previously described52. Compounds were identified by comparison to library entries of purified standards or recurrent unknown entities and assigned to key metabolic pathway groups, including amino acids, carbohydrates, cofactors and vitamins, energy, lipids, nucleotides, peptides, and xenobiotic metabolism.Raw data was extracted, peak-identified and quality control processed using Metabolon's hardware and softwareBivariate comparisons of maternal characteristics were performed between cases of sPTB and term birth controls using Chi-square (χ2) or Fisher's exact test, where appropriate. Continuous variables were compared by the Student t-test or the Mann–Whitney U test, depending on the distribution of the data.10 transformed values . Metabolite measurements of zero were imputed using the minimum transformed value across all samples. Similar transformations have previously yielded excellent agreement with measurements performed by an independent certified medical laboratory53. IPCA was run using the ipca function of the R mixOmics package33. Permutational multivariate analysis of variance (PERMANOVA)54 was employed to test whether the centroids of samples as grouped by CST or birth outcome are equivalent based on Bray–Curtis distance, using the adonis function of the R vegan package. Area under the receiver operating characteristics (auROC) for the prediction of CST and birth outcome using the five IPCs identified in IPCA was measured. Independent t-tests were employed to test the difference of IPCs by birth outcome and by CST. Sparse IPCA (sIPCA)33 was further conducted to select features for IPCs using the sipca function of the R mixOmics package. We assessed the relationship between the number of features selected and the difference of IPC4 between sPTB and term birth measured by the p values of Mann–Whitney U tests. The optimal features selected would be a set of minimum features which yield the most significant difference in IPC4 components between sPTB and term birth. The optimal number of selected features was determined as 11  and by birth outcome (sPTB versus term). Sensitivity analyses were conducted comparing median metabolite abundance by birth outcome among CSTs. Two-sided Student t-tests were used for comparison, and resulting p-values were corrected by false discovery rates calculated using the Benjamini–Hochberg (BH) method with a threshold of ≤ 0.1. Fold change was calculated to quantify the difference in log2 change between groups for each metabolite. Only metabolites with a p-value ≤ 0.05 and absolute fold changes ≥ 1.5 were considered significant. Clinical data were analyzed using STATA . An estimate of the false discovery rate  was calculated to account for multiple comparisons55.The primary metabolomics analysis was conducted on metabolite abundance data that was volume-normalized to protein content. All data were logSupplementary Information.