Biochemical assays and computational analyses have discovered RNA structures throughout various transcripts. However, the roles of these structures are mostly unknown. Here we develop folded RNA element profiling with structure library (FOREST), a multiplexed affinity assay system to identify functional interactions from transcriptome-wide RNA structure datasets. We generate an RNA structure library by extracting validated or predicted RNA motifs from gene-annotated RNA regions. The RNA structure library with an affinity enrichment assay allows for the comprehensive identification of target-binding RNA sequences and structures in a high-throughput manner. As a proof-of-concept, FOREST discovers multiple RNA-protein interaction networks with quantitative scores, including translational regulatory elements that function in living cells. Moreover, FOREST reveals different binding landscapes of RNA G-quadruplex (rG4) structures-binding proteins and discovers rG4 structures in the terminal loops of precursor microRNAs. Overall, FOREST serves as a versatile platform to investigate RNA structure-function relationships on a large scale. Structured RNA motifs can be obtained by structure probing, duplex capture, and motif prediction. Here the authors develop a multiplexed affinity assay system to identify functional protein interactors from an RNA structure library with validated or predicted RNA motifs. Structure probing and prediction software have elucidated transcriptome-wide RNA structural architectures, called the RNA structurome, and revealed numerous RNA structures6. Transcriptome-wide double-stranded RNA mapping is also critical for identifying higher-order architectures10. These strategies to determine RNA structures provide the means to investigate the regulatory networks governed by functional RNA elements.Functional RNA elements have specific RNA sequences and structures that work together as a functional unit. Discovering RNA structures from a wide variety of transcripts is a critical step in finding functional RNA elements and understanding their regulatory mechanisms. From the perspective of bioinformatics, homology-based searching for both conserved sequences and secondary structures is effective at finding structured RNA motifs11. However, transcriptome-wide analysis with sequencing  has only revealed footprints of targeted RNA sequences, which are not equal to RNA elements. Thus, subsequent experiments are needed to determine the structural boundaries of continuous RNAs for the discovery of the RNA elements. Furthermore, current sequencing-based methods suffer from technical limitations regarding transcribed RNAs in specific cells and structure-dependent amplification biases in the library preparation.Although RNA structures have been identified, their roles remain mostly unknown. Datasets of RNA structures have been analyzed in combination with large-scale analyses using deep sequencing to elucidate biological phenomenon involving RNA structuresfolded RNA element profiling with structure library) is a system that quantitatively analyzes interactions using a large number of structured RNA motifs extracted from natural RNAs revealed by structure probing, duplex capture, and motif prediction  interactions and demonstrate its potential applications. We first design an RNA structure library that includes 1800 human precursor microRNA (pre-miRNA) loops and structured RNA controls . We then profile RNP interactions between the pre-miRNA loop sublibrary and various RNA-binding proteins (RBPs) to measure the protein-binding intensities of each RNA structure, uncovering both essential RNA sequences and structured motifs for the interactions. Second, we apply FOREST to characterize the structure-binding preferences of RBPs by utilizing affinity-based enrichment. We reveal different binding preferences of rG4-targeting RBPs and identify rG4 structures from the pre-miRNA loops. Finally, we design another RNA structure library by extracting structures from human 5′UTR and the HIV-1 RNA genome to analyze the RNP interactions of a cellular EIF3 complex. We identify EIF3-complex binding elements that regulate translation in living cells.12. We chose these motifs because pre-miRNAs contain stem structures biologically validated by the recognition and processing by Dicer in cells and because the loop regions of some pre-miRNAs are known to regulate cell fate via RNP interactions16. As defined terminal motifs, we extracted single- and multi-terminal motifs consisting of one stem–loop motif with a blunt end without flanking sequences and consisting of two or more single-terminal motifs, respectively , which includes 1800 human pre-miRNA loops, 100 structures of lncRNA (NEAT1), the validated U1A-related aptamer controls, rG4 controls, and triplet repeats Fig. . We conf19, 5′-GCAC-3′, was a vital factor for interacting with U1A  pre-miRNA loops because previous studies have characterized them as LIN28-binding structures, except for hsa-let-7a-320. We confirmed that ten out of twelve let-7 loops interacted with LIN28A with significantly high affinities , cold-inducible RNA binding protein (CIRBP), and DEAH-box helicase 36 (DHX36)28. Fluorescence scanning on the barcode microarray directly quantified the bound RNAs without an RT or PCR step , which affect the stabilization of rG4 16 , and hsa-mir-1234 loop, which also contained a G-rich sequence and high G-score (G-score = 37), but did not show high binding intensities to any of the proteins tested. We analyzed these two pre-miRNA loops using circular dichroism (CD) spectrum analysis and found that the hsa-mir-6850 loop showed a typical CD pattern for rG4 that is dependent on the buffer conditions, whereas the hsa-mir-1234 loop did not  showed higher intensities than the non-rG4 controls, indicating that these pre-miRNA loops form rG4 structures 16) that cross-reactively bound to some rG4-binding proteins did not show enhanced NMM fluorescence intensity , which specifically detects G4 based on the fluorescence intensityres Fig. . In contity Fig. . Togethe36. Using this library, we profiled the RNP interactions of the eukaryotic initiation factor 3 (EIF3) complex, which includes multiple proteins and regulates translation initiation in human cells  from another region (1395–1714 nt) corresponding to upstream of the HIV-1 frameshift region. We validated that the high-ranked structures specifically pulled down EIF3B and an accompanying ribosomal protein from the cell extracts , which contains the terminal motifs from the predicted secondary structure of human conserved 5’UTR regions and SHAPE-MaP datasets of the HIV-1 RNA genome Fig. 5,36. Usills Fig. . We succlls Fig. . In the cts Fig. , demonstTCF25, the longer CU-tract causes a higher intensity than the shorter one  at the first step  . This observation suggested that physiological effects could dynamically alter the RNA structures in the library.In the affinity profiling step of FOREST, multiple reaction conditions can be tested to compare the biophysical and chemical effects on the function and structure of different RNA probes 6) in the gel images under native PAGE conditions  was 230 nt, but eventually extended to 350 nt . Given that the total length of the attachments is 84 nt, the current limitation of the RNA structure length is 266 nt in this condition. Considering the known binding sites, we assume that this maximum length is likely long enough to interact with most ligands. For example, 99.3% of the RNA structures of riboswitches (59404 out of 60290 RNAs) are less than 266 nt, according to RNAcentral, v15In conclusion, we developed FOREST and applied it to the comprehensive analysis of RNP interactions using diverse RNA structures extracted from multiple databases. In the future, FOREST will explore other functional RNA classes in addition to RNP interactions (Supplementary Discussion). These advances will lead to the elucidation of a regulatory layer that is governed by functional RNA elements.The inputs are RNA sequences and the secondary structures represented in dot-bracket format, and the output is the group of RNA motifs extracted from the RNA datasets. The following are procedures for the recognition of terminal motifs  Start and end nucleotides should be in the same column on the secondary structure of the pre-miRNA presented in miRBase. (2) If the pre-miRNA produces one mature miRNA, the start nucleotide of the loop region is set as the fourth nucleotide from the loop-side end of the mature miRNA sequence. (3) If the pre-miRNA produces two mature miRNAs, then the strand closer to the basal end is chosen, and the loop region is set as in (2). If the same loop motifs are confirmed from more than two pre-miRNA genes, the pre-miRNA loop is enrolled in the library with annotation to all original pre-miRNA genes. Following these parameters, we obtained 1805 human pre-miRNA loops. Because of the length limitation (86 nt), we omitted the five pre-miRNA loops from the library .Pre-miRNA loop sublibrary: Human pre-miRNA sequences and their secondary structures were obtained from miRBase version 21  of lncRNA NEAT147. The structures were obtained by RNAcofold48 with default parameters with the input of both the right and left arms of the PARIS duplex groups. Extracted motifs were filtered according to the length (nt). In the case of the RNA structure library, v1, the length limitation was set to 86 nt. The terminal motifs were extracted by removing the flanking regions. Following these procedures, we obtained 48 motifs for RNA structure library, v1.NEAT1 lncRNA sublibrary from PARIS datasets: To design the RNA structure library from NEAT1 lncRNA, we extracted sequence reads from the PARIS data of HeLa cells49. The inputs of ParasoR were the full length of NEAT1 and the three functional regions . The terminal-loop motifs were extracted from the predicted structures. Extracted motifs were filtered according to the length (nt). In the case of the RNA structure library, v1, the length limitation was set to 86 nt. In the case of full-length RNA, we removed the extracted RNA motifs that were not localized at the functional regions. Following these procedures, we obtained 52 motifs for RNA structure library, v1.NEAT1 lncRNA sublibrary from ParasoR datasets: ParasoR software was used to predict the secondary structure of the full-length RNA and functional regions36. To obtain structural information, the RNA secondary structures of the full-length 5′UTR sequences were predicted by CentroidFold v.0.0.1550. To maintain the precision of the prediction, we removed 5′UTR sequences longer than 401 nt from the library. The option gamma was set to 4. The terminal motifs were extracted as mentioned above. To focus on sequences conserved among species, RNA terminal motifs with conserved sequences were preferentially included in the RNA structure library. We used the annotations of the conserved regions registered in UTRdb. Due to the scale of the library synthesis, the library was adjusted to contain 8645 types of terminal motifs. These motifs were loaded into RNA structure library, v2.Human 5′UTR sublibrary: To design the RNA structure library from the human 5′UTR, we extracted RNA sequences from the human 5′UTR registered in UTRdb5. The datasets were converted to RNA secondary structure information without pseudoknots using RemovePseudoKnots software on the RNA structure web server52. In addition, using ct2dot software on the RNA structure website, the secondary structure information was obtained in a dot-bracket format. Then, the structures were divided into terminal motifs, and all motifs were loaded into RNA structure library, v2.HIV-1 structure sublibrary: To design the RNA structure library from HIV-1 RNA, we obtained RNA structure datasets determined by SHAPE-MaP with structural analysisProtein-binding controls: As controls, selected pre-miRNA loops, protein-binding RNA aptamers, and defective mutants were loaded into RNA structure library, v2.CC + T7 promoter sequence: A sequence for synthesizing RNA from template DNA. The sequences of the following components were transcribed using T7 polymerase.17. In the RNA structure library, multiple barcode sequences were assigned to each RNA structure.Barcode sequence (25 nt): To immobilize each RNA probe on the corresponding microarray spots, the barcode sequences were orthogonally hybridized with the complementary strands of the barcode sequences placed on the DNA microarray. The sequence information of these orthogonal barcodes is listed elsewhereStabilizing stem structure sequence Forward (18 nt): A unique sequence that forms a stem structure to stabilize the RNA structure and insulate the RNA structure and RNA barcode sequence.RNA structure region : Variable RNA motifs extracted from RNA structure datasets.Stabilizing stem structure sequence Reverse (18 nt): A stem structure constructed by base-pairing with the stabilizing stem structure sequence Forward.To generate the RNA structure library, single-stranded DNA sequences were used as templates for RNA probes, including each RNA structure extracted from the datasets. The templates were designed to include five components in the following order from the 3′ end.These template DNAs were synthesized by OLIGONUCLEOTIDE LIBRARY SYNTHESIS . The size of the oligo templates was limited to 170 and 200 nt for RNA structure libraries v1 and v2, respectively. After the assignment of the barcodes to the RNA structure, we converted the barcodes to antisense DNA strands. The listed DNA strands were submitted to the SureDesign server as CGH custom array design services (Agilent Technologies). Probe Replication Factor was set to 5× and 2× for RNA structure library, v1, and v2, respectively. Then, a custom CGH DNA microarray was purchased in the 8× 60 K array format.To produce short RNA probes (less than 300 nt) and the RNA structure libraries, we used the MEGAshortscript T7 Transcription Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Twenty microliters of the reaction solution containing single-stranded DNA templates and single-stranded DNA coding T7 promoter sequence  was prepared and incubated at 37 °C. The reaction time was set to 20 h for the transcription of the RNA structure libraries and 6 h for the others. Then, 2 μL of TURBO DNase (Thermo Fisher Scientific) was added to the reaction solution, mixed by pipetting, and incubated at 37 °C for 15 min. RNA products were purified with Zymo RNA Clean and Concentrator (Zymo Research).For detection and quantification on the microarray, all RNA probes in the library were labeled with fluorescent dye at the 3′ end by mixing 1× T4 Ligase Buffer (Thermo Fisher Scientific), 100 μM pCp-Cy5 (Jena Bioscience), 10 μM RNA structure library, and 0.5 U/μL T4 RNA Ligase (Thermo Fisher Scientific) in a 100 μL reaction mixture. The mixture was incubated at 16 °C for 48 h under light-shielded conditions. The labeled RNA probes in the library were purified with Zymo RNA Clean and Concentrator (Zymo Research). The fluorescently labeled RNA structure library was stored at −28 °C.Eighteen microliters of enriched RNA was mixed with 4.5 μL of 10× Blocking Agent (Agilent Technologies) and 22.5 μL of Hi-RPM Hybridization Buffer (Agilent Technologies). The samples were incubated for 5 min in a heat block set at 104 °C. Then, the samples were immersed in ice water and incubated for 5 min. The samples were applied to an 8× 60 K Agilent microarray gasket slide (Agilent Technologies). The gasket slide and CGH custom array 8× 60 K (Agilent Technologies) were assembled with SureHyb. In a hybridization oven (Robbins Scientific), hybridization was performed for 20 h at a temperature of 55.5 °C at 20 rpm. After hybridization, the microarray slide was washed for 5 min with Gene Expression Wash Buffer 1 (Agilent Technologies) in a glass container at room temperature. Next, the microarray slide was transferred to a glass container containing Gene Expression Wash Buffer 2 (Agilent Technologies), which was immersed in a thermostat bath at 37 °C and washed for 5 min. Finally, we used SureScan (Agilent Technologies) to obtain fluorescence image data on the microarray. The captured images of the microarray slide were converted to numeric fluorescence intensities of each spot by Feature Extraction (Agilent Technologies) and GeneSpringGX (Agilent Technologies).53. His-LIN28A protein was a gift from Dr. Kozo Tomita. His-tagged protein , 20 μL of TALON magnetic beads (Clontech), and 1 μg of the RNA structure library were mixed in 1 mL of protein-binding buffer . To optimize the concentration, we first examined three protein amounts . The optimum concentration was set as the minimum concentration, where the fluorescence intensity was within the dynamic range. A mixture containing no protein was also prepared as a control. The mixture was incubated on a rotator at 4 °C for 30 min and washed with 1× protein-binding buffer three times. Then, 200 μL of elution buffer  was added to the magnetic beads, and the mixture was heated at 95 °C for 3 min. The RNA was collected from the supernatant by removing the magnetic beads. The RNA structure library in the mixture was extracted with phenol and chloroform with ethanol precipitation for purification.His-U1A protein was purified as detailed in our previous paper+ folding buffer  or 1× Li+ folding buffer . For folding, RNA was heated at 95 °C and cooled to 4 °C at −6 °C/s on a ProFlex Thermal Cycler (Thermo Fisher Scientific). For this purpose, a target protein , 20 μL of TALON magnetic beads (Clontech) or 20 μL of the protein G dynabeads  that were preincubated with 10 μg of monoclonal ANTI-FLAG® M2 antibody  and 1 μg of the refolded RNA structure library were mixed in 1 mL of 1× BG4 protein-binding buffer . To optimize the concentration, we first examined three protein amounts . The optimum concentration was set as the minimum concentration, where the fluorescence intensity was almost within the dynamic range. A mixture containing no protein was also prepared as a control. The mixture was incubated on a rotator at 4 °C for 30 min and washed three times with 1× BG4 protein-binding buffer. Then, 200 μL of elution buffer was added to the magnetic beads, and the mixture was heated at 95 °C for 3 min. The RNA was collected from the supernatant by removing the magnetic beads. The RNA structure library in the mixture was extracted with phenol and chloroform with ethanol precipitation for purification.His-tagged BG4 anti-G4 antibody Fab fragment was purchased from Absolute Antibody (Ab00174-1.6). His-tagged CIRBP human recombinant protein was purchased from BioVision (7589-20). C-Myc/DDK-tagged (FLAG-tag compatible) DHX36 human recombinant protein was purchased from OriGene Technologies (TP323929). The RNA structure library was prepared in 1× KThe protein-binding intensities of each RNA probe were calculated by subtracting the fluorescence intensities of the no-protein control samples from those of the enriched protein samples. To alleviate the effect of undesired interactions with the barcode region, we calculated the averaged fluorescence intensities of each RNA structure from the intensities of three RNA probes that had the same RNA structure but different RNA barcodes. In the case of RNA structure library, v1, because there were five barcodes per RNA structure, we excluded the maximum and minimum intensities from a set of five intensities.U1A and LIN28A: First, 1000 nM RNA samples were prepared in 1× folding buffer . For folding, RNA was incubated at 95 °C for 5 min and then immediately placed in tubes on ice. Then, 50 nM refolded RNA and recombinant proteins  were mixed in 10 μL of 1× protein-binding buffer. The mixtures were incubated at 4 °C for 30 min for U1A or 90 min for LIN28A. Then, 2.5 μL of 5× Hi-Density Sample Dye (Thermo Fisher Scientific) was added and mixed. The mixtures were overlaid on a nondenaturing 4-20% polyacrylamide gel (Thermo Fisher Scientific) at 4 °C. After electrophoresis at 10 mA for 50 min, the gel was stained with SYBR Green II. Imaging was performed with a Typhoon FLA7000 . Details of the RNA probes are available in Supplementary Table + folding buffer . For folding, RNA was heated at 95 °C and cooled to 4 °C at −6 °C/s on a ProFlex Thermal Cycler. Refolded RNA was then incubated at 4 °C for at least 30 min. Then, 50 nM refolded RNA and 0 or 750 nM BG4 were mixed in 12 μL of 1× K+ BG4 protein-binding buffer . Next, the mixtures were incubated at 4 °C for 90 min. Then, 3 μL of 5× Hi-Density Sample Dye (Thermo Fisher Scientific) was added and mixed. The mixtures were overlaid on a nondenaturing 6% polyacrylamide gel (Thermo Fisher Scientific) at 4 °C. After electrophoresis at 15 mA for 30 min, the gel was stained with SYBR Green I. Imaging was performed with a Typhoon FLA7000. Details of the RNA probes are available in Supplementary Table BG4 anti-G4 antibody Fab fragment: For this purpose, 500 nM RNA samples were prepared in 1× Khsa-mir-98 loop, we excluded the weak intensity that did not show dose-dependent interaction and the intensity for the 2:1 complex of LIN28A:hsa-mir-98, since the intensities were quite low and difficult to distinguish from the background noise.The band intensities were calculated from the gel images with three biological replicates. The gel images were analyzed using the Gels submenu in ImageJ (NIH). First, the lanes that each enclosed a shifted band and non-shifted band were defined, and the lane profile plots were generated. Then, peak areas were defined based on the bands of the corresponding gel images and measured as band intensities. For EMSA using LIN28A and U1A, the relative intensity of the shifted band compared with that of the non-shifted band in each lane was calculated. For EMSA using BG4, the relative intensity of the non-shifted band in the BG4 (+) lane compared with that in the BG4 (−) lane was calculated for each RNA probe. Relative intensities were normalized to non-shifted bands in the BG4 (−) lanes. In the gel image of http://rna.tbi.univie.ac.at/forna/]54 to generate illustrations of the RNA secondary structures by modifying the colors with Adobe Illustrator.We used the forna website [http://bioinformatics.ramapo.edu/QGRS/help_search.php]33 to calculate G-scores and used the maximum G-score of the inputted RNA sequences for the analysis.We used the QGRS website [http://meme-suite.org/tools/meme]55 and the Weblogo3 website [http://weblogo.threeplusone.com/create.cgi]56 to generate the sequence logo of enriched motifs from the top 5% of the population in the case of RNA structure library, v2. The k-mer (k = 4) counting was performed with the FOREST datasets of the top 5% (enriched RNA) and the whole population  to identify protein-binding sequence motifs from RNA structure library, v1  = normalized Count (k)Enriched/normalized Count (k)All. Then, we collected the 4-mers whose Z-score for the relative frequency (k) was >2.58. Clustal Omega [https://www.ebi.ac.uk/Tools/msa/clustalo/]57 aligned the collected 4-mers with default parameters. The enriched sequence motif logo was generated from the aligned 4-mers by Weblogo3.We used the MEME website [+ folding buffer. All RNAs were generated by in vitro transcription with a MEGAshortscript T7 Transcription Kit. RNA samples (1000 nM) were prepared in 1× K+ folding buffer. For folding, RNA was incubated at 95 °C and cooled to 4 °C at −6 °C/s on a ProFlex Thermal Cycler. Refolded RNA was then folded at 4 °C for at least 30 min. Then, 15 μL of refolded RNA (1000 nM) was mixed with 15 μL of NMM (100 μM) in 1× K+ folding buffer. The fluorescence was measured on Infinite (TECAN) with an excitation wavelength of 399 nm and an emission wavelength of 610 nm. The fluorescence intensity was corrected by subtracting the intensity of the buffer mixture as a background. Details of the RNA probes are available in Supplementary Table N-Methyl mesoporphyrin IX  was purchased and resuspended in 1× K2 in DMEM with high glucose  containing 10% fetal bovine serum (Japan Bio Serum), 0.1 mM nonessential amino acids (Thermo Fisher Scientific), and 1 mM sodium pyruvate (Sigma-Aldrich).293FT cells (Thermo Fisher Scientific) were cultured at 37 °C in 5% COg for 5 min at 4 °C. The cell pellet was resuspended in chilled PBS and centrifuged again at 300 × g for 5 min at 4 °C. The supernatant was carefully removed. Then, 900 μL or NP40 Cell Lysis Buffer (Thermo Fisher Scientific), 100 μL of 10× Protease Inhibitor Cocktail (Sigma-Aldrich), and 1 μmol of PMSF were added. Next, the mixture was incubated on ice for 30 min. During this time, the mixture was vortexed for 10 s every 10 min. Then, the mixture was centrifuged for 10 min at 13,000 rpm and 4 °C. The proteins in the supernatant were collected and stored at −80 °C in 1.5 mL Protein LoBind Tubes (Eppendorf) until use.293FT cells were seeded in 10 cm dishes and cultured until 80–90% confluency. As a washing step, 10 mL of chilled phosphate-buffered saline (PBS) (4 °C) was added and removed immediately by an aspirator. This washing step was repeated again, and 5 mL of PBS was added. The cells were detached with a cell scraper, and the solution was transferred to a 50 mL tube. The supernatant was removed after centrifugation at 300 × The Dynabeads Protein Immunoprecipitation Kit (Thermo Fisher Scientific) was used following the manufacturer’s instructions with modifications. Twenty microliters of Dynabeads was prepared in 200 μL of Ab Binding buffer (Thermo Fisher Scientific) with 10 μL of rabbit anti-EIF3B antibody  or 10 μL of M2 mouse monoclonal antibodies to FLAG-tag . As a control, we prepared a mixture that did not contain the target antibody. The mixture was incubated at room temperature for 13 min with a tube rotator. The tube was placed on a magnetic rack, and the supernatant was removed. Then, 200 μL of binding/washing buffer (Thermo Fisher Scientific) and 200 μL of the collected 293FT cell extracts (1 μg/μL) were added to the mixture. The mixture was incubated on a tube rotator for 1 h at 4 °C. After the reaction, the tube was placed on a magnetic rack, and the supernatant was removed. A washing step was performed with 1 mL of 1× protein-binding buffer. The tube was placed on a magnetic rack, and the supernatant was removed. Then, 500 μL of 1× protein-binding buffer and 500 ng of Cy5-labeled RNA structure library, v2, were added. The mixture was incubated on a tube rotator at 4 °C for 1 h. Another washing step was performed with 500 μL of the protein-binding buffer, and the supernatant was removed. Washing was repeated three times. Finally, protein-binding RNAs were recovered by phenol–chloroform extraction and ethanol precipitation.Twelve micromolar RNA probes were prepared in 1× hybridization buffer  in the presence of 10 μM biotinylated DNA adapter. Then, RNA and DNA were incubated at 95 °C and cooled to 4 °C at −0.1 °C/s on a ProFlex Thermal Cycler to form RNA–DNA–biotin hybrids and mixed with 50 μL of Streptavidin Mag Sepharose  in 500 μL protein-binding buffer in 1.5 mL tubes. After incubation for 3 h at 4 °C with rotation, the RNA–DNA complexes on the beads were washed with protein-binding buffer. Then, 2 μL of RNase Inhibitor, Murine , 700 μL of 1× protein-binding buffer, and 200 μL of protein extract (1 μg/μL) derived from 293FT was added to the mixture. After incubation for 12 h at 4 °C with rotation, the protein–RNA–DNA complexes on the beads were washed three times with 1× protein-binding buffer. The RBP was detached by heating at 95 °C for 5 min with 5× Sample Buffer . The enriched proteins were collected by removing the magnetic beads. Target proteins were detected by an automated capillary-based immunodetection system  using 20-fold diluted anti-EIF3B , 50-fold diluted anti-RPS19 , and 50-fold diluted anti-ACTB  as primary antibodies. HRP-conjugated anti-rabbit  was used as the secondary antibody. Details of the DNA adapter and the RNA probes are available in Supplementary Table The 5′UTRs of reporter RNA were synthesized by GeneArt (Thermo Fisher Scientific). Templates were prepared by Fusion PCR using the 5′UTR variants, ORFs and common 3′UTR with a poly-A tail. Capped mRNAs were prepared by using the MegaScript Kit (Thermo Fisher Scientific) in the presence of an anti-reverse cap analog (TriLink BioTechnologies). Ten microliters of the reaction solution was prepared and incubated at 37 °C. After 6 h of incubation, 1 μL of TURBO DNase (Thermo Fisher Scientific) was added to the reaction solution, mixed by pipetting, and incubated at 37 °C for 30 min. RNA products were purified with Zymo RNA Clean and Concentrator (Zymo Research). Then, to remove the phosphate at the 5′ terminus, the RNA products were treated with Antarctic Phosphatase (New England Biolabs). The RNA products were purified again, and the concentrations were measured with a NanoDrop 2000 (Thermo Fisher Scientific). Sequence information regarding all DNA materials for constructing the templates of reporter mRNA is available in Supplementary Data 293FT cells were prepared in 24-well plates and transfected with 50 ng of reporter mRNAs encoding Nanoluc Luciferase and 10 ng of inner control mRNA encoding Firefly Luciferase using Lipofectamine messengerMAX (Thermo Fisher Scientific) following the manufacturer’s instructions. To avoid saturation, Nanoluc Luciferase was added to the PEST-tag to promote protein degradation. After 13 h, the plates were placed on ice, and the cells were washed twice with chilled PBS. Then, 200 μL of passive lysis buffer (Promega) was added and mixed by pipetting. Supernatants were collected in 1.5 mL Protein LoBind Tubes and stored at −28 °C until measurement. We used the Nano-Glo Dual-Luciferase Reporter Assay System  following the manufacturer’s instructions to measure the luminescence. The luminescences were acquired on a GloMAX Navigator System (Promega). Translation activity was calculated as a ratio of the luminescence of Nanoluc Luciferase and of Firefly Luciferase Fig. . All samFurther information on research design is available in the Supplementary InformationReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Supplementary Data 12Supplementary Data 13


We explored how tabersonine (Tab) protected against dexamethasone (Dex)-induced osteoporosis. Osteoblasts were treated with Dex (100 µM) with or without Table (5 or 10 µM). We measured cell viability, alkaline phosphatase (ALP) activity, and mitochondrialsuperoxide and reactive oxygen species levels. We used flow cytometry to explore the effects of Tab on mitochondrial membrane potential and osteoblast apoptosis. We used RT-PCR and western blotting to examine the effect of Tab on protein expression. We evaluated the effects of Tab on bone histopathology and bone mineral density in rats with Dex-induced osteoporosis. Tab increased cell viability and ALP activity, and reduced the mitochondrial superoxide, reactive oxygen species and matrix metalloproteinase levels and osteoblast apoptosis. Tab significantly reduced the levels of nuclear factor erythroid 2-related factor 2 (Nrf2), haem oxygenase-1 and NAD(P)H quinone dehydrogenase 1. Moreover, it increased the levels of mRNAs encoding runt-related transcription factor 2, bone morphogenetic protein-2 and osterix. These data suggest that Tab ameliorates Dex-induced osteoporosis by regulating the Nrf2 signalling pathway. Osteoporosis is a metabolic bone disease characterised by the loss of bone mass and bone weakness; it affects about 200 million individuals  worldwide , is a terpene indole alkaloid found in several medicinal plants , originally isolated from Neonatal Sprague-Dawley rats (strain P2) were sacrificed via decapitation and their calvaria were isolated for in vitro analysis. The in vivo study was performed with 8-week-old female rats (mean body weight 250 ± 25 g), held under standard laboratory conditions. All animal procedures were approved by the animal ethics committee of the First Hospital of Jilin University, China .Tab was supplied by the Institute of Botany, Chinese Academy of Sciences, China; Dex was obtained from Tianjin Lisheng Pharmaceutical Co. Ltd., China. Alkaline phosphatase (ALP), Nrf2, and matrix metalloproteinase detection kits were purchased from Abcam, USA. The antibodies used for western blotting and the qRT-PCR primers were obtained from Thermo Fisher Scientific, USA.Calvarial osteoblasts were isolated from neonatal rats as described previously  were seeded into six-well plates, incubated for 24 h, treated with 100 µM Dex with or without the addition of Table (5 or 10 µM) 2 h previously, and cultured for 24 h; CCK-8 was then added, followed by incubation for 2 h. Cell viability was determined by estimating absorbance at 450 nm using a microplate reader.Cell proliferation was estimated using the CCK-8 assay as described previously  were seeded into six-well plates and treated with Dex with or without Tab for various durations. The cells were lysed with lysis buffer, and ALP levels were measured as directed by the kit manufacturer.Osteoblasts  were seeded into six-well plates and the Mitochondrial Membrane Potential Detection Kit was used to estimate MMP according to the manufacturer’s instructions.Mitochondrial membrane potential (MMP) was estimated using the lipophilic, cationic fluorescent dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide. Osteoblasts (2 × 105 cells/well) were seeded into six-well plates and treated with Dex with or without Tab for various durations. The cells were resuspended in 500 µl binding buffer containing PI (5 µl) and Annexin V-APC (5 µl), rinsed with PBS, and placed on ice. Flow cytometry was used to measure apoptosis.Osteoblasts  was plotted against the log of the added cDNA concentration, and relative target gene expression levels were determined using the 2C (1:200) and β-actin (1:1000), and then at room temperature for 60 min with appropriate secondary antibodies. The enhanced chemiluminescence assay was used to detect protein bands, and the ImageLab software was employed for densitometric analysis.Total osteoblast proteins were extracted into ice-cold radioimmunoprecipitation lysis buffer, and the DC Protein Assay was performed to estimate protein levels. Sodium dodecyl sulphate-polyacrylamide gel (10% w/v) electrophoresis was used to separate the proteins, which were then transferred to polyvinylidene difluoride membranes. The membranes were incubated with a 5% (w/v) fresh nonfat dry milk solution to block non-specific reactions. They were incubated at 4 ºC overnight with primary antibodies against Nrf2 (1:500), Ho-1 (1:1000), NAD(P)H quinone dehydrogenase 1 , caspase-3 (1:500), Bcl-2 (1:1000), Bax (1:1000), cytochrome-Rats were separated into four groups: control, Dex , and Tables 20 and 40 . The animals were then sacrificed, and the bilateral femora were collected.Dual energy X-ray absorptiometry was used to determine the bone mineral density (BMD). Rats were anesthetised with Zoletil/Rompun, and the femora, lumbar vertebrae and whole body were scanned for the evaluation of BMD.The isolated tibiae were fixed in 10% (v/v) formalin at 40 °C for 2 days. Ethylenediaminetetraacetic acid (10% v/v) was used to decalcify the bones, which were then placed in liquid paraffin to create wax blocks. Sections (4 µm thickness) were prepared and stained with haematoxylin and eosin. Histopathological changes were identified under a tri-ocular microscope at 100× magnification.n = 10/group). One-way analysis of variance, followed by the Dunnett test, was performed using the GraphPad Prism software . p values < 0.05 were considered to reflect statistical significance.All data are expressed as means ± standard errors of the means (Dex reduced osteoblast viability (compared with the control) and Tab rescued viability, as revealed by the CCK-8 assay Fig. .p < 0.01; Fig. Dex significantly reduced the osteoblast ALP level compared with the control , bone morphogenetic protein-2 (BMP-2) and osterix were measured in osteoblasts treated with Dex alone or Dex and Tab Fig. . The mRNThe effect of Tab on Nrf2 signalling was evaluated by measuring the levels of Nrf2, Ho-1 and NQO-1 in osteoblasts via western blotting; Nrf2 activity was also determined using the TransAM Nrf2 kit Fig. a, b. Thec and reduced that of Bcl-2 compared with controls. Tab neutralised these adverse effects of Dex.Tab affected the levels of pre- and pro-apoptotic proteins in Dex-treated osteoblasts Fig. . Dex sigTab affected bone histopathology and BMD in rats with Dex-induced osteoporosis Fig. . The conBone homeostasis is affected by osteoporosis, a disease characterised by the dysregulation of bone formation and resorption. Osteoblasts regulate bone formation and osteoclasts control bone resorption . In conclusion, our findings suggest that Tab ameliorates Dex-induced osteoporosis by regulating Nrf2/ROS/Bax signalling. Thus, Tab may be clinically valuable for osteoporosis management.Nrf2 signalling protects osteoblasts (Sena and Chandel


Nearly all retroviruses selectively package two copies of their unspliced RNA genomes from a cellular milieu that contains a substantial excess of non-viral and spliced viral RNAs. Over the past four decades, combinations of genetic experiments, phylogenetic analyses, nucleotide accessibility mapping, in silico RNA structure predictions, and biophysical experiments were employed to understand how retroviral genomes are selected for packaging. Genetic studies provided early clues regarding the protein and RNA elements required for packaging, and nucleotide accessibility mapping experiments provided insights into the secondary structures of functionally important elements in the genome. Three-dimensional structural determinants of packaging were primarily derived by nuclear magnetic resonance (NMR) spectroscopy. A key advantage of NMR, relative to other methods for determining biomolecular structure , is that it is well suited for studies of conformationally dynamic and heterogeneous systems—a hallmark of the retrovirus packaging machinery. Here, we review advances in understanding of the structures, dynamics, and interactions of the proteins and RNA elements involved in retroviral genome selection and packaging that are facilitated by NMR. During the late phase of the retroviral replication cycle , two copies of the unspliced, 5′-capped, and 3′-polyadenylated RNA transcript are specifically selected for packaging into assembling virions. Genomes are selected against a background that includes a substantial excess of non-genomic RNA, including cellular mRNAs , monomerRetroviridae family includes two subfamilies encompassing eleven genera, categorized primarily by sequence similarity within the pol gene [Spumaretrovirinae subfamily  diverged early and is evolutionarily distant from the Orthoretrovirinae subfamily . Evolutionarily related retroviruses within a given genus can preferentially package each other’s viral genomes [lentiviruses (HIV and simian immunodeficiency virus (SIV)) [gammaretroviruses (Moloney murine sarcoma virus and spleen necrosis virus) [The pol gene . The Spu genomes ,25,26, as (SIV)) , as wells virus) , whereass virus) , indicats virus) . Althougs virus) ) to the s virus) , which ls virus) ,31 and cs virus) ,33. Alths virus) ,37,38,39s virus) ,41, as ls virus) .http://www.hiv.lanl.gov/). Although some studies with mutant genomes suggested that residues in the gag open reading frame might also be important for packaging, it now appears that packaging defects were caused by misfolding of the 5′-leader due to interactions with non-native downstream sequences [Retroviruses contain two copies of their genomes , both ofequences . Studiesequences , MoMuLV equences ,58, and equences ,62,63,64equences ,66,67,68equences ,70,71, requences ,73,74,75equences ,73,74,75http://www.rcsb.org). However, new 2H-edited NMR methods recently enabled structural studies of RNA elements within the intact, dimeric 5′-leaders of two strains of HIV-1 (HIV-1NL4-3 and HIV-1MAL)  [Solution-state nuclear magnetic resonance (NMR) contributed substantially to current knowledge of the structures and mechanisms that contribute to retroviral genome selection and packaging. NMR offers a number of advantages for structural characterization of biomolecules, including the ability to characterize structures and dynamics of conformationally heterogeneous samples, and to obtain data without the need to prepare crystalline samples. Due to these advantages, seven of the first twelve HIV-1 protein domain structures that were solved during the first decade of HIV-1 structural biology were determined by NMR . However230 kDa) ,75,81. HXenopus protein transcription factors and proposals that these arrays function as nucleic acid-binding “zinc fingers” [2+ and Zn2+ with high affinity, likely via 3S/1N coordination [1H-113Cd heteronuclear spin echo difference NMR applied to a 113Cd-substituted HIV-1 CCHC peptide, revealed that the cysteine-S and histidine-Nε atoms were coordinated to the metal [The NC domain of the HIV-1 Gag protein was the first retroviral constituent studied by NMR. Except for the spumaviruses, all retroviral NC proteins contain one or two copies of a conserved cysteine/histidine rich sequence   that wasfingers” , Berg etfingers” ; Figure fingers” ,87, mutafingers” . Furtherfingers” . Berg etdination . 1H-113Che metal , and NOEhe metal . Subsequhe metal ; Figure he metal ; Figure NMR structures were reported for several isolated retroviral zinc knuckle domains ,97,98,99β-sheet arranged such that several basic residues were available on the protein surface to interact with plasma membrane assembly sites [γ led to the suggestion that the MA protein might operate as a dimer [10 helical stretch, and a three-strand mixed β-sheet [10 helix participating in the trimer interface and ultimately led to the proposal that MA undergoes this structural rearrangement during assembly [NMR is a powerful tool in structural characterization of Gag’s MA domain ,120,121,ly sites . Structu a dimer . However a dimer . Matthew a dimer . Aside f β-sheet . NMR stu β-sheet , also re β-sheet . Compariassembly . Recent assembly ,135,136;The MA–plasma membrane interaction is dependent not only on the region of basic residues located on MA’s surface but also on N-terminal myristoylation ,137,138,2) [2 lipids [4-PIP2 is buried in MA’s hydrophobic cleft in an “extended lipid” conformation, promoting myristoyl exposure [2 promotes MA–liposome interactions, no PIP2-dependent chemical shift perturbation are seen in the hydrophobic cleft, calling into question the validity of the extended lipid binding model; 2 head groups but the acyl chains of PIP2 do not extend from the membrane; 2 discrimination and membrane binding, NMR studies also played a key role in characterizing the targeting of MA to the plasma membrane during assembly. MA specifically targets phosphatidylinositol--bisphosphate P2) ,142,143 2) ,145,146.2 lipids , in whicexposure . A varieexposure . Developexposure . NMR titLys3 isoform that is present in virions and serves as the primer for reverse transcription [2 [Lys3 interacts with residues in the basic patch region of MA. Interestingly, the affinity of MA for tRNALys3 was roughly one order of magnitude stronger than PIP2-containing liposomes, and the NMR-based liposome binding assays revealed that tRNALys3 inhibited MA–PIP2 interactions [Recent studies demonstrated that MA interacts with cytosolic tRNAs , includicription ,151,152.ption [2 . Gaines ractions . The binractions , which cractions . Taken tractions . Thus, iractions .CTD) has a relatively weak propensity to form dimers in solution (dimerization Kd ~10 µM) [CTD through NC (CACTD-SP1-NC) [CTD and SP1 domains. An even larger HIV-1 Gag construct (∆Gag) comprising domains spanning unmyristoylated MA through NC (myr(-)MA-CA-SP1-NC; ~100 kDa) was studied by Clore et al. using protein perdeuteration and transverse relaxation optimized spectroscopy (TROSY) NMR [1H-15N chemical shift perturbation mapping was used to explore the structural changes that occur as ∆Gag interacts with two different DNA oligonucleotides—∆P(-)PBS and d(TG)15 [1H-15N chemical shift perturbations are observed within the MA domain, following saturation of the NC binding site, suggesting weak secondary interactions between the MA and the DNA oligonucleotides [The CA domain of Gag promotes assembly via formation of intermolecular Gag–Gag interactions. The C-terminal domain of CA (CA ~10 µM) , and bot ~10 µM) . To dete-SP1-NC) . These sOSY) NMR , to optiOSY) NMR . 1H-15N  d(TG)15 . Upon adleotides ; Figure Lys3 primer binding site for initiation of reverse transcription [Considerable effort was made over the past 30 years to understand the structure of the HIV-1 5′-leader and its role in genome packaging (reviewed in ). A combcription ,169,170 cription . The SD cription , thus alcription . The Ψ hcription .gag start codon  were prackaging ,162, or ackaging ; Figure ackaging ,164,165.Packaging of the HIV-1 genome is intrinsically dependent on RNA dimerization ,186,187.The loop–loop kissing interactions were structurally characterized by both NMR ,208,209 Oligonucleotides that adopt the kissing dimer structure can be converted to a more thermodynamically stable extended dimer ,211,212 Residues of the internal loops (bulges) adopt similar structures in the kissing and extended duplex forms of the DIS dimers . NMR stu1H-15N heteronuclear single quantum coherence (HSQC) studies of wild type and mutant NC constructs identified the N-terminal residues and residues flanking the zinc-knuckle domains as critical to this conversion [2+, suggesting a mechanism through which Mg2+ regulates the kissing loop unfolding, to prevent premature conversion to the extended duplex structure, in the absence of NC. Further work also established this mechanism of interconversion to be pH-dependent, upon protonation of a flanking purine residue [NMR methods are used to monitor the mechanism of DIS conversion from the kissing to the extended duplex structure, as well as the temporal formation and discrimination of intermolecular contacts ,221,222.nversion ,225. RDCnversion . These m residue .15N-labeled RNAs, to distinguish between kissing and extended duplex dimer interfaces in DIS fragments [15N splitting patterns observed for Watson–Crick imino protons confirmed that “weak dimers” contain a kissing interface, whereas the dimers that are more thermally stable contain an extended duplex intermolecular interface.Takahashi et al. utilized mixed samples containing unlabeled and ragments . 15N splgag initiation codon, demonstrated that these residues are essential for wild-type packaging efficiency [d ~40 nM) and that structural rearrangements induced by NC binding are required for in vivo RNA packaging [Early mutagenesis studies that deleted residues spanning from the major splice donor site to the ficiency ,228,229.ficiency . A conseficiency ,157,159;ficiency ,232,233.ficiency ,235. Altficiency  .13C-labeled 3′-fragment (residues 328–356) were enzymatically ligated to facilitate the direct probing of the AUG conformations in the context of the large, intact, leader RNA. When the RNA sample was incubated in physiological-like buffers that promote dimerization, NMR spectral changes consistent with an AUG structural conversion from a hairpin to a U5:AUG duplex were observed. The U5:AUG base pairing was then probed directly, using an NMR method for probing long-range interactions through adenosine–interaction detection  [Long-range U5:AUG base pairing was later supported by NMR studies. 2D NMR analysis showed that an 11-mer U5 RNA can disrupt the AUG stem-loop structure, to form intermolecular U5:AUG duplex . More ree below) . An upfie below) . The thre below) .The TAR element is a 59-nt sequence located at the 5′-end of all HIV-1 nascent viral transcripts and plays an essential role in Tat-mediated transcriptional activation ,247,248.HIV-1 TAR adopts a hairpin structure that consists of two A-form helical domains connected by a three-pyrimidine residue bulge that binds to the Tat protein ,263,264 Lys3—the packaged host transfer RNA (see section on matrix) that serves as the primer for viral reverse transcriptase [Lys3 and a complementary 18-nt PBS fragment (see section on NC–RNA interactions) [The function of the 5′ PBS is well characterized as the annealing site for tRNAcriptase . NC mediactions) . An addiactions) . NMR stuactions) ,280. Theactions) .Lys3 annealing. The recognition mechanism of RHA by the 5′-leader of HIV-1NL4-3 during viral assembly was recently characterized via a combination of NMR and ITC studies, and it was found that RHA and NC noncompetitively interact with the leader RNA [The PBS might also play a role in packaging cellular RNA helicase A (RHA) into virus particles ,284,285.ader RNA . Chemicaader RNA .CES) [CES were facilitated by substituting the dimer-promoting GC-rich loop of the DIS hairpin by a GAGA tetraloop (CESm) was five times larger than the average size of RNA structures previously solved by NMR. Additionally, nucleotide-specific deuterium labeling did not provide sufficient resolution to enable complete assignment of the ΨCESm spectra. Resolution was further improved using a novel approach involving the non-covalent annealing of differentially 2H-labeled RNA fragments [CESm [CESm structure also explains how HIV-1 selectively packages its unspliced genome [NMR was used to help identify a minimal region of the 5′-leader capable of directing RNA packaging. Deletions and mutations that did not significantly affect NC binding or RNA misfolding in vitro were employed in RNA packaging experiments. These studies revealed that the TAR, polyA, and PBS elements are not required for packaging, and identified a minimal ~260 nt region of the 5′-leader that is capable of efficiently promoting the packaging of heterologous vector RNAs    conformation in the dimer.The 5′-leader of the NL4-3 strain of HIV-1  enabled the direct detection of U5:AUG base pairing in the 230 kDa dimeric HIV-1NL4-3 leader [MAL leader (see below) [2H-edited NOESY NMR spectra were also obtained for HIV-1NL4-3 leader RNAs containing deletions or loop substitutions and compared with spectra of the intact, dimeric leader [CES, see above).The 3 leader ; Figure e below) . 2H-editc leader . Mutatioc leader    and a Gr-labeling scheme (sample 2) were mixed, and 2D NOE spectroscopy (NOESY) data were obtained. A268-H2-to-G251-H1′ NOE signals were detected for a sample containing an equimolar mixture of A2-leader and CrGrUr-leader RNAs, which is only possible if the leader adopts the extended duplex conformation; r labeling schemes showed similar results. In contrast, experiments probing long-range interactions in the TAR, polyA, PBS, and Ψ-hairpins gave results consistent with intramolecular base pairing in these regions of the leader; 3 leader . SeparatNL4-3 5′-leader. Unfortunately, the high propensity of this leader to form dimers made it difficult to obtain more extensive NMR data for the monomeric form of the RNA, due to the high sample concentrations required for NMR studies . In studies of in vitro transcribed 5′-leader RNAs from different strains of HIV and SIV, it was discovered that the dimerization propensity of the simian immunodeficiency virus (SIVcpzUS strain) leader, which contains a GUGCAC palindrome in the dimer promoting DIS loop, is substantially lower than that of the HIV-1NL4-3 leader [MAL, which also contains a GUGCAC palindrome and exhibits a dimerization propensity similar to that of SIVcpzUS [MAL 5′-leader could be studied by NMR in its monomeric (low ionic strength) and dimeric (PI buffer) states [The above studies provided insights into the secondary structures adopted by selected regions within the HIV-13 leader . TherefoSIVcpzUS . Thus, t) states . In addi) states , it was ) states ,296,297;) states .A review of published retroviral DNA sequences  revealedMAL 1G, 2G, and 3G 5′-leader RNAs were probed by 2H-edited NMR [NL4-3 leader; MAL transcript, unlike the HIV-1NL4-3 transcript that required mutagenesis  for U5:AUG detection. It is also noteworthy that signals were not resolved for the H1 helix observed in the HIV-1NL4-3 ΨCES structure. It is possible these signals were obscured by spectral overlap, and future studies to test for the presence of a tandem three-way junction structure similar to that observed for the HIV-1NL4-3 ΨCES are warranted. The NMR spectra for the intact 5′-capped 1G HIV-1MAL leader also exhibited evidence that the cap residue is sandwiched between the TAR and polyA helices. NMR studies of a leader fragment comprising the capped TAR, polyA, and U5:AUG helices confirmed that the TAR and polyA helices are stacked in an “end-to-end” manner; To understand how transcriptional addition of as few as one or two 5′-guanosines influence the dimerization properties and function of ~9 kilobase HIV-1 transcripts, the secondary structures of 5′-capped HIV-1ited NMR . The dim2H-edited NMR studies performed for the monomeric forms of the capped 2G/3G MAL leader RNAs, revealed extensive structural remodeling, compared to the capped 1G transcript [MAL data were obtained for the native RNA whereas the HIV-1NL4-3 studies were conducted with mutants using the lr-AID NMR method [anscript . ResidueR method ). The reR method . BiochemR method ; Figure env splice site and gag start codon abrogated the MoMuLV genome packaging [+) containing the Ψ site and an additional 474 nt of the gag coding region [NL4-3 vectors with non-native residues downstream of the leader were also poorly packaged. This was originally attributed to a role of coding residues in packaging; however, more recent studies indicate that the non-native residues might disrupt the structure of the leader and thereby inhibit packaging [MoMuLV is an evolutionarily distant C-type retrovirus that was extensively studied as a model for retroviral genome encapsidation, and as a potential vector for therapeutic gene delivery . A 350-nackaging . Althougackaging  and downackaging  residuesg region . We noteackaging . It is, d ~132 nM), but interacts weakly with constructs comprising only one or two stem-loops [CES).Mutagenesis, chemical accessibility probing, and computational analysis indicated that the Ψ region folds into several stem-loops, which are further condensed by long-range interactions ,309,310.em-loops ,314. ThuCES RNA readily forms dimers under physiological-like ionic strength conditions (as does the entire leader) and undergoes conformational rearrangement upon dimerization [The MoMuLV Ψrization . Specifirization ,315,317 rization ,319,320;rization  . However, isolated stem-loops and multi-stem-loop-fragments bind weakly to NC [The O3 stem was found to be essential for genome packaging ,327,345.ly to NC . These fly to NC   and RNA binding-induced contacts between the two zinc knuckle domains [The three-dimensional structure of HIV-1 NC bound to a 20-residue RNA, containing the apical loop of the Ψ-hairpin , was determined by heteronuclear NMR spectroscopy . The spe domains , which a10 helix, upon binding to SD-hairpin, but form different protein-RNA interactions, compared to that of the NC-Ψ-hairpin structure [The 3D structure of HIV-1 NC bound to a 19-residue SD-hairpin was also determined by solution NMR . Similartructure ,110. NMRd ~40 nM, compared with Kd ~ 300 nM for the Ψ-hairpin apical loop, under physiologic-like ionic strength) [In addition to the loop region of RNA hairpins, the three-dimensional structure of NC bound to a single-stranded U5 derivative (5′ -UGUGCCCUUCU-3′) was also determined using solution NMR . The twotrength) . The strtrength) . However1H-NMR studies of the complex identified a cluster of hydrophobic residues on the surface of the MoMuLV zinc knuckle that form a binding pocket for a guanosine base . 31P NMR experiments revealed significant chemical shift changes for phosphodiester groups between G3 and C4, and between A1 and C2. These results revealed that the MoMuLV zinc knuckle binds exposed guanosines bases in a manner similar to that observed for HIV-1 zinc knuckles.Initial NMR studies of MoMuLV NC-nucleic acid interactions were conducted with a pentanucleotide, d(ACGCC), which was previously shown to bind to a HIV-1 zinc knuckle peptide  and to HCES hairpins , nor to the SL-CD tandem stem-loop RNA [CES), and the binding required (and was shown to induce) RNA dimerization [CES) [Unexpectedly, MoMuLV NC did not exhibit significant affinity for any isolated Ψloop RNA . Insteadrization . These don [CES) .CES construct, with loop residue mutations designed to prevent dimerization, while retaining the internal base pairing of the dimer  was examined [To facilitate 3D structural studies, a Ψexamined ,314. Theexamined . The G30examined . These hi gave rise to intermolecular NOEs between Gi-H8 and Trp35 aromatic, Ala27 methyl, Ala36 methyl, and Leu21 side-chain protons, and between Gi-H1′ and Leu21, Trp35, and Arg23 side-chain protons [i-1, i-2, and i-3, and the side-chain protons of Ala27, Leu21, Arg18, Tyr28, Ala36, Lys42, and Lys30 [Chemical accessibility experiments conducted with a larger portion of the MoMuLV 5′-leader indicated that multiple UCUG sequences become exposed and accessible, following RNA dimerization, suggesting the presence of additional NC-binding sites for genome recognition and packaging ,310. Sho protons . Additiond Lys30 . These fThe structure of the RSV NC bound to the minimal packaging signal, µΨ, is also solved by NMR. Unlike other retroviruses, the 5′ UTR of alpha-retroviruses contain three distinct open reading frames or AUG motifs—AUG-1, AUG-2, and AUG-3, and there is controversy over their functional differences ,330,331.NMR analysis showed that the SL-C tetraloop gave rise to distinct NOEs with the aromatic residues in the N-terminal zinc knuckle . SimilarThe C-terminal zinc knuckle structure lacked a hydrophobic pocket and did not give rise to NOEs to guanosine residues, suggesting that it functions differently than other characterized zinc knuckle domains. However, there were NOEs to the linker residues adjacent to the 5′ and 3′ bases of SL-A . On the Lys3 primer to the primer binding site (PBS) [In addition to its role in genome packaging, the NC domain of Gag (or the mature NC protein) functions as an RNA chaperone to catalyze conformational rearrangements . NC stimte (PBS) ,360, prote (PBS) ,362, andte (PBS) ,369,370.te (PBS) ,371,372.te (PBS) ,377,378.Efforts to develop inhibitors of HIV-1 genome packaging involved approaches that targeted both the NC domain of Gag and the RNA packaging signal. Small molecules that bound to both viral targets and interfered with NC-RNA binding in vitro, were identified. Although these compounds exhibited antiviral activity in cell culture assays, none exhibited the appropriate efficacy and toxicity required for advancement to the clinic. The current progress is summarized below.Current anti-retroviral therapies involve a combination of drugs that suppress HIV-1 replication, but resistance can still occur, and this life-long treatment regimen can have adverse side effects. Therefore, there is a need to develop antivirals that target different components of HIV that are essential for replication. The zinc knuckles of NC are essential for replication and, therefore, are attractive potential targets. One class of antiviral agents was shown by NMR to function by a novel “zinc ejection” mechanism, in which the cysteine sulfur atoms that are required for binding zinc and maintaining proper folding are selectively oxidized ,382,383.More recently, a series of compounds were identified that could block interactions between NC and TG-rich deoxynucleotides. NMR-based structural studies revealed that two inhibitor molecules interact with the N-terminal and C-terminal zinc knuckles by mimicking exposed guanosine residues  Figure , which a1H NMR spectroscopy showed that NSC specifically bound to the GGAG tetraloop  small-angle scattering profiles do not require sample crystallization, (ii) there are no size limitations, (ii) the electron-rich backbone of RNA scatters X-rays strongly, (iii) the 3D RNA structures are typically non-spherical, and (iv) procedures for implementing sparse NMR restraints and SAXS data could be accomplished with existing NMR structure refinement software packages ,395,396.Cryo-EM is another complementary technique traditionally used to determine the tertiary structures of large protein complexes ,404. UnlThe combination of NMR and cryo-EM was further utilized in the structural characterization of the extended duplex form of the 30 kDa HIV-1 DIS ; Figure During the late 1980’s and early 90’s, NMR was used to study relatively small retroviral proteins and RNAs were believed to be important for packaging. These studies provided insights into the role of zinc in viral replication, characterized the structure of NC, and its intrinsic CCHC zinc knuckle domains, and compared and contrasted the NC domains of different retroviruses. These studies were complemented by studies with small nucleic acids, which identified sequences that bind the NC zinc knuckles with high affinity, established the mechanism of DIS-promoted RNA dimerization, and led to the structural characterization of nucleic acid complexes with isolated zinc knuckles and intact NC proteins. NMR studies also provided insights into the structures of other regions of the 5′-leader that are important for viral replication, and led to the identification of novel reagents and mechanisms for inhibiting viral replication by promoting zinc ejection from the CCHC zinc knuckles or binding competitively to NC or to RNA elements within the 5′-leader. In addition, NMR studies of a minimal packaging signal from the MoMuLV 5′-leader, led to the proposal for a dimerization-dependent RNA structural switch packaging mechanism, in which nucleotides important for NC binding are sequestered by base pairing in the monomeric form of the RNA and become exposed to promote NC-binding upon dimerization. It remains to be determined if MoMuLV or other retroviruses utilizes heterogeneous transcriptional start site usage as a means of controlling transcript dimerization, as appears to be the case for HIV-1. Interestingly, more than 40 years ago, Levin and Rosenak proposed that MuLV-producing cells likely contain two non-interconverting pools of viral transcripts that function separately as viral mRNA and gRNA, based on the sensitivity of gRNA incorporation into virions produced in the presence of the transcription inhibitor, actinomycin D , and mor13C-labeled and un-labeled) fragments and nucleotide specific 2H editing. Both strategies aimed to enhance spectral sensitivity and resolution and enable detection of NMR chemical shifts and NOE signals for the specific nucleotides of interest [1H-13C signals were detectable for the regions of the leader undergoing rapid rotational motions , they could not be detected for residues in larger, more globular regions of the RNA undergoing more restricted rotational motions . Thus, although the 1H-13C correlated NMR methods that are widely used to study protein structure were able to characterize smaller, structurally independent regions of the leader, they had a limited utility for the characterization of large globular regions. For example, 1H-13C correlated NMR methods showed that the AUG residues adopt a hairpin in the monomer and an alternate (undetermined) structure in the dimer, likely due to their participation in a larger, globular structure in the dimer.RNA dimerization-dependent packaging mechanisms were proposed for HIV, based primarily on biochemical and nucleotide accessibility mapping studies of in vitro transcribed RNAs, but applications of NMR to test structural and mechanistic hypotheses were not performed until the past decade or so, due to the large size of the HIV 5′-leader (~350 nucleotides). The first reported NMR studies of the intact HIV-1 5′-leader were performed using both chemical ligation of differentially labeled . Initial studies focused on the non-capped 2G HIV-1NL4-3 leader were confounded by the inability to obtain homogeneous samples of monomeric species, at the high concentrations required for NMR measurements. Nevertheless, studies of samples with measurable populations of the monomer revealed that residues of AUG form a hairpin, and residues of the DIS palindrome form base pairs with U5, in the monomeric form of the HIV-1NL4-3 leader. Based on the observation of A-H2 signals that are unambiguously diagnostic of the DIS hairpin, those studies suggested that the DIS retains its hairpin structure in the monomer. Subsequent studies shifted focus to the leader of HIV-1MAL because the samples containing higher percentages of the monomer could be obtained. These studies clearly showed that the DIS does not adopt a hairpin structure in the monomer, and it now appears that the DIS hairpin signals observed in the HIV-1NL4-3 spectra were associated with an equilibrium presence of the dimeric species. The NMR data clearly showed that the monomeric form of the HIV-1MAL leader adopted a structure in which the DIS palindrome formed base pairs with U5, as observed by NMR for the NL4-3 leader, and the remaining residues form long-range base pairs with residues of the upstream polyA and U5 elements. This topology has similarities to the “Long Distance Interactive” (LDI) model proposed by Abbink and Berkhout . NMR also helped shed light on the factors that modulate RNA dimerization and thereby modulate the transcript function and fate. 5′-Capped transcripts predominantly begin with either one or three guanosines, resulting from “twinned” start site usage. Transcripts that begin with a single guanosine, adopt a dimeric structure that sequesters the 5′-cap and exposes high-affinity NC binding sites, whereas 5′-capped transcripts that begin with two (less common) or three guanosines, form monomers that are substantially remodeled, compared to the dimeric RNA. The energetics are exquisitely balanced, with remodeling occurring at the energetic cost of a single G-C base pair .Attempts to study the monomeric form of the 2G HIV-11H-15N correlated NMR methods for detecting the H2 protons of 15N-labeled adenosines [Future studies of retroviral packaging mechanisms will benefit from studies of ever larger RNAs and protein–RNA complexes, including spliced RNA transcripts and unspliced transcripts bound to Gag, both within the cytoplasm and associated with the plasma membrane; enosines  (e.g., fIn summary, NMR contributed considerably to our understanding of the atomic-level structural determinants of retroviral genome selection and packaging, more so than any other structural methodology. Since the first reported NMR structure of a retroviral zinc knuckle 30 years ago, all 3D structural information for retroviral zinc knuckle domains, NC proteins, and NC–RNA complexes were provided by NMR. Challenges associated with the crystallization of structurally flexible polycationic proteins such as NC, or structurally heterogeneous RNA polyanions, makes NMR an ideal methodology for studying retroviral RNA packaging. The NMR techniques developed over the course of these studies should be broadly applicable to the rapidly expanding field of non-coding cellular RNAs. As questions about genome packaging shift to larger systems, new approaches involving hybrid methodologies, advancements in labeling techniques, and possibly solid-state NMR will likely make important future contributions.


African swine fever virus (ASFV) causes incurable and often lethal hemorrhagic fever in domestic pigs. In 2020, ASF presents an acute and global animal health emergency that has the potential to devastate entire national economies as effective vaccines or antiviral drugs are not currently available (according to the Food and Agriculture Organization of the United Nations). With major outbreaks ongoing in Eastern Europe and Asia, urgent action is needed to advance our knowledge about the fundamental biology of ASFV, including the mechanisms and temporal control of gene expression. A thorough understanding of RNAP and transcription factor function, and of the sequence context of their promoter motifs, as well as accurate knowledge of which genes are expressed when and the amino acid sequence of the encoded proteins, is direly needed for the development of antiviral drugs and vaccines. African swine fever virus (ASFV) causes hemorrhagic fever in domestic pigs, presenting the biggest global threat to animal farming in recorded history. Despite the importance of ASFV, little is known about the mechanisms and regulation of ASFV transcription. Using RNA sequencing methods, we have determined total RNA abundance, transcription start sites, and transcription termination sites at single-nucleotide resolution. This allowed us to characterize DNA consensus motifs of early and late ASFV core promoters, as well as a polythymidylate sequence determinant for transcription termination. Our results demonstrate that ASFV utilizes alternative transcription start sites between early and late stages of infection and that ASFV RNA polymerase (RNAP) undergoes promoter-proximal transcript slippage at 5′ ends of transcription units, adding quasitemplated AU- and AUAU-5′ extensions to mRNAs. Here, we present the first much-needed genome-wide transcriptome study that provides unique insight into ASFV transcription and serves as a resource to aid future functional analyses of ASFV genes which are essential to combat this devastating disease.IMPORTANCE African swine fever virus (ASFV) causes incurable and often lethal hemorrhagic fever in domestic pigs. In 2020, ASF presents an acute and global animal health emergency that has the potential to devastate entire national economies as effective vaccines or antiviral drugs are not currently available (according to the Food and Agriculture Organization of the United Nations). With major outbreaks ongoing in Eastern Europe and Asia, urgent action is needed to advance our knowledge about the fundamental biology of ASFV, including the mechanisms and temporal control of gene expression. A thorough understanding of RNAP and transcription factor function, and of the sequence context of their promoter motifs, as well as accurate knowledge of which genes are expressed when and the amino acid sequence of the encoded proteins, is direly needed for the development of antiviral drugs and vaccines. Asfarviridae , while the faustoviruses show similarity to ASFV but have larger genomes and infect amoeba (Vermamoeba vermiformis) (8Poxviridae family member (African swine fever virus (ASFV) is the sole characterized member of rviridae , a familes order , 3. Asfaformis) 810, a disy member .We have focused our analysis on the BA71V strain  , 13 beca15–ASFV inhabits the eukaryotic cytoplasm and appears to be self-sufficient in terms of transcription and modification of viral mRNA. It encodes an RNAP, a poly(A) polymerase, and an mRNA capping enzyme; importantly, extracts obtained from mature virus particles are fully transcription competent , 20, 21.cap analysis gene expression sequencing (CAGE-seq), and RNA 3′-end sequencing (3′ RNA-seq). We report (i) the ASFV transcriptome map showing differences in gene expression levels between early and late infection, (ii) a genome-wide TSS map that has allowed us to define early and late ASFV promoter consensus motifs as well as 5′ mRNA leaders, and (iii) a genome-wide TTS map that provides novel insights into the mechanism of transcription termination in ASFV. We have applied a combination of next-generation sequencing (NGS) techniques including transcriptome sequencing (RNA-seq), RNA 5′-end https://bit.ly/2TazQxK).A transcriptome is defined by the overall expression levels of transcripts and their 5′ and 3′ termini. We carried out RNA-seq, CAGE-seq, and 3′ RNA-seq in order to characterize these parameters during early and late ASFV infection; when the methods are combined, they provide information about the ASFV transcriptome and DNA sequence signatures associated with transcription initiation and termination. The processed data are compiled in an assembly hub and can be publicly accessed in the UCSC Genome Browser (available at r ≥ 0.9), with the exception of RNA-seq data from 16 h (r of 0.74 and 0.84 for two strands [data not shown]). 28–Vero cells were infected with BA71V, and viral RNA was extracted at 5 h and 16 h postinfection (p.i.). These time points were chosen based on a previous report of a small subset of genes that were experimentally characterized using nuclease S1 mapping and primer extension analysis , 26. BowFollowing mapping of CAGE-seq reads to the ASFV BA71V genome, we located regions with an enrichment of reads corresponding to the 5′ ends of transcripts and thereby the TSS. We detected 779 clusters of CAGE-seq signals, and CAGE-seq clusters upstream of annotated ORFs were manually investigated to confirm that they represent primary TSSs (pTSSs) based on peak height, proximity to the ORF initiation codon, and coverage from our complementing RNA-seq data. We identified pTSSs fulfilling these criteria upstream of 151 BA71V ORFs; thus, only two genes, E66L and C62L, were not found associated with a pTSS. Overall, our data showed good agreement with previously individually mapped TSSs of 44 ORFs (Table S2). Not all of the ∼780 clusters were located within 500 bp upstream of ASFV ORFs but were within, or in the antisense orientation relative to, ORF coding sequences . We reanSeveral genes have a bona fide pTSS upstream of the annotated start codon and an alternative TSS residing within the included J64R  and B169Twenty-eight TSSs in our CAGE-seq data set were not associated with annotated ORFs (Table S3), and seven of these pTSSs were associated with transcripts that encode short ORFs, which we call putative novel genes (pNGs). These encode polypeptides 25 to 56 amino acids (aa) long that were missed in the initial BA71V ORF prediction as only ORFs of ≥60 aa were annotated . Five pNIn order to gain insights into expression of individual genes, we quantified mRNA levels obtained by CAGE-seq and compared the most abundant mRNAs at early and late time points . Table SP value of <0.05), with 47 genes downregulated and 56 genes upregulated during the progression from early to late infection. Henceforth, we focused on the CAGE-seq data set because the reads are associated with the nascent transcription start sites and thus cannot arise from transcription readthrough from upstream genes (unlike mRNA quantification using RNA-seq), which would complicate the analyses. RNA-seq also has the disadvantage of a lower sequencing depth and thus lower apparent sensitivity than that of CAGE-seq. Indeed, CAGE-seq identified 149 genes as significantly differentially expressed, with 65 downregulated genes and 84 upregulated genes . They remain poorly characterized beyond a domain prediction, and 9 proteins (out of 12) of these ORFs could be detected in BA71V virions by mass spectrometry  . Only a trometry .Kluyveromyces lactis virus-like element (VLE) promoters  and the initiator (Inr). The first two interact with initiation factors TBP and TFIIB, while the last interacts with RNA Pol II . Alignmeromoters . Howeverromoters  motif seromoters . Figure ly genes , we searly genes  and i, wP value of 2.85e−03; E value of 5.16e+00)  late genes, which we called the late promoter motif (LPM) . The spa.16e+00) . HoweverP value of <0.05) (Early and late genes in ASFV vary with regard to the length of 5′ untranslated regions (UTRs), i.e., the distance between the 5′ mRNA end and the translation start codon. The 5′ UTRs of late genes are significantly shorter and have a higher AT content than early genes (f <0.05)  and f. Sf <0.05) , and thef <0.05) . The mosf <0.05) . In ordef <0.05) . This suf <0.05) .Previous mapping of mRNA 3′ ends has revealed a conserved sequence motif consisting of ≥7 thymidylate residues in the template, which is consistent with 3′ end formation via transcription termination, similar to that of the RNA polymerase III paradigm , 47. To Here, we report the first comprehensive ASFV transcriptome study at single-nucleotide resolution. The mapping of 158 TSS and 114 TTS for 159 ASFV genes allowed us to reannotate the BA71V genome. Our results provide detailed information about differential gene expression during early and late infection, the sequence motifs for early and late gene promoters (EPM and LPM and Inr elements) and terminators [poly(T) motif], and evidence of quasitemplated AU RNA-5′ tailing by the ASFV-RNAP.We have discovered seven novel putative genes, some of which are highly conserved with the aggressively virulent strains (Georgia 2007/1 and Belgium 2018/1) that have caused the current outbreak in Europe . This suOur results demonstrate that the majority of ASFV genes show some degree of differential expression from early to late infection . InteresWe have shown that MGFs show distinct downregulation from early to late infection, while genes annotated as transmembrane region or putative signal peptide genes (though poorly characterized beyond this), along with structural or viral morphology genes, are overrepresented in late infection . Our CAGWhile eukaryotic Pol II and archaeal RNAP critically rely on initiation factors TBP and TFIIB for transcription initiation on all mRNA genes, bacterial RNAP obtains specificity for subsets of gene promoters by associating with distinct sigma factors . ASFV-RNASFV-TBP (B263R) is an early gene, and ASFV-TFIIB (C315R) is expressed throughout infection. We propose that the LPM is utilized by ASFV-TBP and -TFIIB homologues, neither of which was detected in virions . A functAn in-depth characterization of the global gene regulation in ASFV with a higher temporal resolution is essential to assess how closely ASFV follows the cascade-like patterns of VACV . While tWe found several examples of alternative, gene-internal, TSS utilization with the potential to increase the complexity of the viral proteome; protein variants may provide the means to generate distinct functionalities, which has also been described in VACV by Yang et al. . Our TSScis-acting motif without any RNA secondary structures characteristic of bacterial intrinsic terminators . The rational design of drugs that target the gene expression machinery is crucially reliant on our knowledge about the ASFV-RNAP, the basal factors that govern its function, and the DNA sequences they interact with, while vaccine development benefits from the intricate knowledge about gene expression patterns. Our results directly contribute to these burning issues for animal husbandry.Understanding the molecular mechanisms of the ASFV transcription system is not only of academic interest. Unless effective vaccines in conjunction with antiviral treatments against ASFV are developed, a large proportion of the global pig population is projected to die in the context of this terrible disease  were grown in six-well plates, infected in two replicate wells for 5 h or 16 h at a multiplicity of infection of 5 with the ASFV BA71V strain, and collected in TRIzol lysis reagent (Thermo Fisher Scientific) separately after growth medium was removed. Infected cells were collected at 5 h postinfection  and at 16 h postinfection . RNA was extracted according to the manufacturer’s instructions for TRIzol extraction, and the subsequent RNA-pellets were resuspended in 50 μl of RNase-free water and DNase treated . RNA quality was assessed via a Bioanalyzer (Agilent 2100) before ethanol precipitation. For CAGE-seq and 3′ RNA-seq, samples were sent to CAGE-seq  and Cambridge Genomic Services , respectively.For RNA-seq, samples were resuspended in 100 μl of RNase-free water and poly(A) enriched using a NEXTflex poly(A) beads kit (Bioo Scientific) according to the manufacturer’s instructions, and quality was assessed via a Bioanalyzer. A NEXTflex Rapid Directional qRNA-Seq kit was utilized to produce paired-end indexed cDNA libraries from the poly(A)-enriched RNA samples, according to the manufacturer’s instructions. Per-sample cDNA library concentrations were calculated via a Bioanalyzer and Qubit fluorometric quantitation (Thermo Fisher Scientific). Sample S3-5h, S4-5h, S5-16h, and S6-16h cDNA libraries were twice separately sequenced on an Illumina MiSeq instrument, generating 75-bp reads  and 12 FASTQ files.Library preparation and CAGE-seq of RNA samples S1-5h, S2-5h, S3-16h, and S4-16h was carried out by CAGE-seq . Library preparation produced single-end indexed cDNA libraries for sequencing; in brief, this included reverse transcription with random primers and oxidation and biotinylation of 5′ mRNA cap, followed by RNase One treatment removing RNA not protected in a cDNA-RNA hybrid. Two rounds of cap-trapping were performed using streptavidin beads, washing away uncapped RNA-cDNA hybrids. Next, RNase One and RNase H treatment degraded any remaining RNA, and cDNA strands were subsequently released from the streptavidin beads and quality assessed via Bioanalyzer. Single-strand index linker and 3′ linker were ligated to released cDNA strands, and primer containing an Illumina sequencer priming site was used for second-strand synthesis. Samples were sequenced using an Illumina NextSeq 500 platform producing 76-bp reads (Table S1).3′ RNA-seq was carried out with samples E-5h_1, E-5h_2, L-16h_1, and L-16h_2 using a Lexogen QuantSeq 3′ mRNA-Seq Library Prep kit FWD for Illumina according to the manufacturer’s instructions. Library preparation and sequencing were carried out at Cambridge Genomic Services  on a single NextSeq flow cell producing 150 bp reads (Table S1).https://usegalaxy.org) (NC_001659.2) and Vero cell  genomes using Bowtie 2 directly after trimming (U18466.2) and Vero cell (NCBI RefSeq GCF_000409795.2) genomes.FastQC  analysisaxy.org) , 69, andaxy.org) . After rtrimming , with altrimming ; therefoU18466.2) and Vero cell (NCBI RefSeq GCF_000409795.2) genomes.FASTQC-analyzed 3′ RNA-seq reads showed relatively varying and poorer quality after nucleotide 65. Cutadapt  was utilU18466.2 genome gene feature file (GFF3), modified to include C44L (U18466.2 ORFs (if clusters were between 300 bp upstream and 200 bp downstream of an ORF). Clusters were classified tssUpstream if located within 300 bp upstream of an ORF, proximal if located within 500 bp of an ORF, coding DNA sequence (CDS) if within the ORF, not available (NA) if no annotated ORF was within these regions (excepting pNG), and antisense if within these regions but antisense relative to the ORF.CAGE-seq-mapped sample BAM files were converted to BigWig (BW) format with BEDtools genomecov  to produude C44L . CAGEfigCluster classification was not successful in all cases; therefore, manual adjustment was necessary. Integrative Genomics Viewer (IGV)  was usedIf the strongest CTSS location was intra-ORF and corroborated with RNA-seq coverage, then the ORF was redefined as starting from the next ATG downstream. For the 28 intergenic CTSSs, IGV was used to visualize if CAGE BW peaks were followed by RNA-seq coverage downstream and whether the transcribed region encoded a putative ORF by using NCBI Open Reading Frame Finder .TTSs were mapped in a similar manner to TSSs, and CAGEfightR was utilized as described above to locate clusters of 3′ RNA-seq peaks though the method differed in some respects: input BigWig files contained the 3′ read-end coverage extracted from BAM files using BEDtools genomecov. Clusters were detected for the 3′ RNA-seq peaks in the same manner as before, except that clusters < 25 nt apart were merged, giving a total of detected 567 clusters. BEDtools was used to check whether the highest point of each cluster (TTS) was within 500 bp or 1,000 bp downstream of annotated ORFs and pNGs. TTSs were then filtered out if 10 nt downstream of the 3′ end had ≥ 50% As to exclude clusters potentially originated from miss-priming. TTS clusters for pNG3 and pNG4 were initially filtered out but included in the final 212 TTSs due to their strong RNA-seq agreement. In cases of multiple TTS clusters per gene, we defined the highest CAGEfightR-scored one within 1,000 bp downstream of ORFs as the primary (pTTS) unless no clear RNA-seq coverage was shown or manually annotated from the literature for O61R .U18466.2. For all 151 ASFV ORFs which had identified pTSSs, we defined their transcription units as beginning from the pTSS coordinate to the ORF end. Since no pTSS was identified for ORFs E66L and C62L, these entries were left as ORFs within the GFF, while the seven putative pNGs were defined as their pTSSs down to the genome coordinate at which the RNA-seq coverage ends. In eight cases in which genes had alternative pTSSs for the different time points, the TUs were defined as the most upstream pTSS down to the ORF end. For analyzing differential expression with the CAGE-seq data set, a GFF was created with BEDtools extending from the pTSS coordinate 25 bp upstream and 75 bp downstream; however, in cases of alternating pTSSs, this TU was defined as 25 bp upstream of the most upstream pTSS and 75 bp downstream of the most downstream pTSS. HTSeq-count  of <0.05 were considered significant. Further analysis of ASFV genes used their characterized or predicted functions as found in the VOCS tool database (https://4virology.net/)  , 81 or Agy.net/)  entries http://meme-suite.org)  of 60 nt upstream of the total 166 pTSS sequences , and TomTom software , sequences 30 bp upstream and 5 bp downstream were extracted from the ASFV-BA71V genome in FASTA format using BEDtools. The 36 early and 55 late genes and all 166 pTSSs  at once were analyzed using MEME software (ite.org) , searchiite.org)  search , raw coverage of 5′ ends (CAGE-seq) and 3′ ends (3′-RNA-seq), and reads per kilobase per million (RPKM) values for the RNA-seq data. Coverage for the forward and reverse strands are shown in blue and red, respectively.Sequencing data from RNA-seq, CAGE-seq, and 3′ RNA-seq are available in the NCBI Sequence Read Archive (SRA) under BioProject accession number  Browser  and can Results from differential gene expression analysis with DESeq2 of CAGE-seq and RNA-seq are found in Tables S4 and S5, respectively. The 91 genes showing the same patterns of differential expression according to both of these NGS techniques are found in Table S6. Details of nontemplated extensions detected from CAGE-seq are in Table S7. CAGEfightR-detected cluster peaks from 3′ RNA-seq after removal of those arriving from poly(A) miss-priming are described in Table S8. All 779 CAGEfightR-detected cluster peaks from CAGE-seq are listed in Table S9.