RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3′ end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3′ end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome. Nano3P-seq presents a nanopore-based sequencing tool to profile polyA-tailed and non-polyA-tailed transcripts, as well as capture polyA tail length and composition. Polyadenylation of RNA is one such modification, which is known to affect the stability and translation efficiency of the RNA molecule4 and to play an essential role in determining the fate of RNA molecules in a wide range of biological processes6.RNA molecules are subject to multiple co- and post-transcriptional modifications, shaping them to their final mature form6. Indeed, in the first hours after fertilization, vertebrate embryos undergo major cellular reprogramming, a process known as the maternal-to-zygotic transition (MZT)7. During the MZT, maternally inherited RNA and proteins are responsible for activation of the zygotic genome and are later replaced by the zygotic program9. Because the MZT begins in a transcriptionally silent embryo, this transition relies heavily on post-transcriptional regulatory mechanisms7, including modulation of the polyadenylation status of the RNA molecules10. Therefore, characterizing the dynamics of RNA polyadenylation is key to understanding how these modifications regulate the fate and function of RNA molecules.One context in which polyadenylation has been shown to play a major role in determining RNA fate and decay is vertebrate embryogenesis11. While these methods have been successfully employed to characterize the dynamics of polyA tail lengths in various contexts, they have several caveats: (i) they provide a limited perspective on isoform–tail relationships owing to the short-read-length nature of NGS-based technologies; (ii) they do not provide single-molecule resolution; (iii) they are severely affected by PCR amplification biases; and (iv) they can only measure tail lengths that are shorter than the read length.In the past few years, several transcriptome-wide methods have become available for studying the dynamics of polyadenylated tails (polyA tails) based on next-generation sequencing (NGS), such as PAL-seq or TAIL-seq13. To sequence native RNAs using dRNA-seq, polyA-tailed RNA molecules are ligated to a 3′ adapter that contains an oligo(dT) overhang. Consequently, dRNA-seq libraries capture the full-length polyA tail; however, ligation occurs only on RNA molecules that anneal to the oligo(dT) overhang, thus exclusively capturing polyA transcripts with tail lengths greater than 10 nucleotides. A variation of this method consisting of in vitro poly(I)-tailing the transcriptome before library preparation has been proposed for studying nascent RNAs using dRNA-seq15, thus capturing both polyadenylated and non-polyadenylated mRNAs. However, major limitations to this variation include low sequencing yields compared with standard dRNA-seq (10–30%)14 and a lack of tools to distinguish poly(I) and poly(A) signals; therefore, polyA tail length information is lost in these datasets15. An alternative approach for studying the transcriptome using nanopore technologies is direct cDNA sequencing (dcDNA-seq), but this approach cannot sequence the polyA− transcriptome, nor can it capture polyA tail length information. Overall, both dRNA-seq and dcDNA-seq nanopore library preparation protocols are limited to the sequencing of polyadenylated transcripts and thus cannot provide a comprehensive view of both polyadenylated and deadenylated RNA molecules, in addition to being unable to capture RNA molecules with other types of RNA tails .To overcome these limitations, the direct RNA sequencing (dRNA-seq) platform offered by Oxford Nanopore Technologies (ONT) has been proposed as a means to study both the transcriptome and polyA tail lengths simultaneouslyHere we present a novel method that employs nanopore sequencing to simultaneously obtain per-isoform transcriptome abundance and tail lengths in full-length individual reads, with minimal library preparation steps, which we term Nanopore 3′-end-capture sequencing (Nano3P-seq). Notably, Nano3P-seq uses template switching to initiate reverse transcription and, therefore, does not require 3′ end adapter ligation steps, PCR amplification, or second-strand cDNA synthesis. We demonstrate that Nano3P-seq can capture any type of RNA molecule regardless of its 3′ sequence, including polyA-tailed and non-tailed RNA. Moreover, we show that Nano3P-seq can accurately quantify RNA abundances in both the coding and non-coding transcriptome, as well as quantify the polyA tail lengths and tail composition of individual RNA molecules in a highly reproducible manner.+ RNA molecules    and per-isoform (Pearson’s R = 0.97) 93) Fig.  and per-Next, we employed Nano3P-seq to examine the RNA dynamics that occur during the maternal-to-zygotic transition (MZT) at single-molecule resolution Fig. . To this8, with a decay of mRNA genes previously reported to have a ‘maternal decay mode’ . PolyA tail length estimates were highly reproducible across independent biological replicates sequenced in independent flow cells for all three time points studied (R = 0.85–0.95)   Fig. . We obse95) Fig. , in agre21. We observed that the three groups of mRNAs that are known to decay  showed a significant decrease in mRNA abundance , as previously describednce Fig. , as expence Fig. . SpecifiA major feature that distinguishes nanopore sequencing from NGS is its ability to produce long reads, allowing RNA polyadenylation dynamics to be studied at the isoform level. Therefore, we wondered whether Nano3P-seq could identify differentially polyadenylated transcript isoforms during the MZT.P < 0.05) during the MZT. Notably, we observed that analyses at the per-gene level often revealed a different picture compared with analyses at per-isoform level. For example, in khdrbs1a and syncrip, per-isoform analysis revealed opposite tail length dynamics among isoforms during the MZT, with one isoform decreasing and another isoform increasing in polyA tail length as the MZT progressed  of analyzed transcripts varied significantly in polyA tail length  of analyzed genes presented significant differences in their polyA tail lengths across isoforms Fig. . Altoget34. Some modifications occur in base positions that are involved in Watson–Crick (WC) base pairing, causing a disruption during reverse transcription. Consequently, these modifications can be seen as increased ‘errors’ and drop-off rates in RNA-seq datasets38. One example is the hypermodified base m1acp3Ψ, which is present in the eukaryotic small subunit (SSU) rRNA39 crucial for the final processing steps of precursor rRNA (pre-rRNA) into mature SSU rRNA41.RNA molecules are decorated with chemical modifications, which are essential for the stability, maturation, fate, and function of the RNA1acp3Ψ-modified site was very high in mature rRNAs but not present in pre-rRNAs, suggesting that this modification is only present in mature rRNA populations. The presence of the hypermodification was also accompanied by a drop-off in sequencing coverage at the m1acp3Ψ-modified site, in agreement with its ability to disrupt the Watson–Crick base pairing. Analysis of the ‘error’ signatures along the SSU transcripts showed that this position was the only one position to change between precursor and processed rRNA molecules  at this site in the pre-rRNAs, which also causes increased ‘errors’ in dRNA-seq datasets, but not in Nano3P-seq datasets  and analyzed the sequencing error patterns in the two populations Fig. . We obseles Fig. . These r44. However, these methods cannot be used to detect tail modifications among the vast majority of tail nucleotides, as Illumina sequencing quality strongly deteriorates in homopolymeric stretches and with increased read length. By contrast, Pacific BioSciences (PacBio)-based approaches such as FLAM-seq can sequence through the entire tail, however, they cannot unambiguously identify 3′ terminal modifications45.Recent works using TAIL-seq have reported that a number of terminal modifications in polyA tails, such as polyuridine stretches, play a role in mRNA decayTherefore, we explored whether Nano3P-seq could accurately identify nucleotide composition variations  within polyA tails. To this end, we designed synthetic cDNA molecules with polyA tails ending with A (30A), U (1U), UUU (3U), UUUUU (5U), CCCCC (5C), and GGGGG (5G), as well as a polyA tail that contained several internal G nucleotides at fixed positions (IntG) Fig. ; MethodsNext, we analyzed the mRNA tail composition of zebrafish embryos from different developmental stages . Analysis of tail base abundance revealed that G was the most common non-A base in zebrafish mRNAs and that there was a significant decrease in non-A bases with progression of the MZT  Fig. . Our ana46.Finally, we examined the relationship between tail length and the presence of non-A bases in the tails. Although the median tail length increases during the MZT Figs  and 3g, + transcripts using oligo(dT) beads. Although these two approaches are often used interchangeably, their effects on the transcriptome composition are not equal. Nano3P-seq allows us to compare the effects of these two approaches on both the transcriptome composition and polyA tail length distribution. In terms of its effects on transcriptome composition, we found that ribodepletion captures a larger variety of RNA biotypes compared with polyA selection, including several non-polyA-tailed RNA biotypes, as expected  ribodepletion of the sample using biotinylated oligonucleotides that are complementary to rRNAs or (ii) selective enrichment of polyAted Fig. . Howeverted Fig. , suggest12. In terms of sequencing output, the yields of Nano3P-seq runs were similar—or slightly better—to those observed in dRNA-seq runs, producing ~100,000–200,000 reads in Flongle devices and ~500,000–2,000,000 in MinION devices, depending on the RNA input type and quality of the flowcell  and TAIL-seq (Pearson’s R = 0.19) on HeLa cell lines  and to estimate polyA tail lengths . However, a major limitation of NGS-based methods is their inability to assign a given polyA tail length to a specific transcript isoform, which causes a loss of isoform-specific tail length information. In addition, NGS-based methods cannot measure tail lengths greater than the read length, thus biasing our view of polyA tail dynamics to those transcripts that display shorter tail lengths.In the past few years, a variety of NGS-based high-throughput methods have been developed to characterize the 3′ ends of RNA molecules at a transcriptome-wide scale, including methods to reveal polyA tail sites , or molecules with polyA tails shorter than 10 nucleotides, thus biasing the view of the transcriptome toward polyadenylated molecules. Customized dRNA-seq methods involving in vitro poly(G/I)-tailing have been developed to overcome some of these limitations, but a lack of bioinformatic tools to distinguish polyI and polyA signals limits their applicability to study polyA tail length differences across transcripts in these datasets15. In addition, dRNA-seq requires 500 ng RNA as input, whereas Nano3P-seq requires as little as 50 ng, thus decreasing the required input material by tenfold. Nano3P-seq addresses the current limitations by offering a simple and robust solution for studying the coding and non-coding transcriptome simultaneously regardless of the presence or absence of polyA tails or 3′ tail composition, without PCR or ligation biases, and with single-read and single-isoform resolution. Moreover, the use of thermostable group II intron reverse transcriptase (TGIRT) in the Nano3P-seq protocol not only maximizes the production of full-length cDNAs, but also ensures the inclusion of RNA molecules that are highly structured and/or modified, which would often not be captured (or their representation would be significantly biased) using standard viral reverse transcriptases57.Nanopore dRNA-seq has been proposed as an alternative long-read sequencing technology for studying polyA tail lengthsNano3P-seq also provides quantitative measurements of RNA abundances Fig.  and captOverall, our work demonstrates that Nano3P-seq can simultaneously capture both non-polyA-tailed and polyA-tailed transcriptomes, making it possible to accurately quantify the RNA abundances and polyA tail lengths at per-read and per-isoform levels, while minimizing the amount of biases introduced during library preparation. These features set Nano3P-seq as a simple, potent, and robust method that can provide mechanistic insights into the regulation of RNA molecules and improve our understanding of mRNA tailing processes and post-transcriptional control.58  were in vitro transcribed using Ampliscribe T7-Flash Transcription Kit (Lucigen-ASF3507). Curlcake 2 was polyadenylated using Escherichia coli polyA Polymerase (NEB-M0276S). polyA-tailed RNAs were purified using RNAClean XP beads. The quality of the in vitro transcribed (IVT) products as well as the addition of polyA tail to the synthetic constructs was assessed using Agilent 4200 Tapestation  were loaded on a 2.5% agarose gel  and stained with GelRed . Product sizes determined using the GeneRuler 50-base-pair DNA ladder . Subsequently, tail- and gene-specific PCR products of mouse 16S rRNA were purified by gel-elution  and sent for Sanger sequencing with the shared forward primer (5′-GGTCGGTTTCTATCTATTTACGATTTCTC-3′). Resulting chromatograms were analyzed using SnapGene (v.6.0.2). After confirming alignment to the reference sequence, the unclipped chromatograms were used to visualize 3′ ends.Poly(A) Tail-Length Assay Kit  was used according to the manufacturer’s instructions. PCR products corresponding to the tail- and gene-specific primer combinations of mouse 16S rRNA  and human Saccharomyces cerevisiae (strain BY4741) was grown at 30 °C in standard YPD medium . Cells were then quickly transferred into 50-ml pre-chilled falcon tubes, and centrifuged for 5 min at 3,000g in a 4 °C pre-chilled centrifuge. Supernatant was discarded, and cells were flash frozen. Flash frozen pellets were resuspended in 700 µl TRIzol (Life Technologies) with 350 µl acid washed and autoclaved glass beads . The cells were disrupted using a vortex on top speed for 7 cycles of 15 s (the samples were chilled on ice for 30 s between cycles). Afterwards, the samples were incubated at room temperature for 5 min and 200 µl chloroform was added. After briefly vortexing the suspension, the samples were incubated for 5 min at room temperature. Then they were centrifuged at 14,000g for 15 min at 4 °C and the upper aqueous phase was transferred to a new tube. RNA was precipitated with 2× volume molecular grade absolute ethanol and 0.1× volume sodium acetate . The samples were then incubated for 1 h at −20 °C and centrifuged at 14,000g for 15 min at 4 °C. The pellet was then washed with 70% ethanol and resuspended with nuclease-free water after air drying for 5 min on the benchtop. Purity of the total RNA was measured with the NanoDrop 2000 Spectrophotometer. Total RNA was then treated with Turbo DNase  (2 μl enzyme for 50 μl reaction of 200 ng μl−1 RNA) at 37 °C for 15 min, with a subsequent RNAClean XP bead cleanup.2 and tissues were snap frozen in liquid nitrogen. Animals were kept on a 12:12 h light:dark cycle and provided with water and food ad libitum.Experiments were performed with male mice aged between 8 and 10 weeks. All mice were euthanized using COMus musculus) brain, we followed previously published protocols (10.17504/protocols.io.3fkgjkw) with minor changes. A quarter of a C57BL6/J mouse brain was used for this protocol, and all samples and reagents were kept on ice during the protocol. Brain tissue was mined with a razor blade into smaller pieces. Cold Nuclei EZ Lysis Buffer ) was added to the tissue in 1.5-ml eppendorf tube. The sample was homogenized using a 1-ml dounce (stroking ~10–20 times), and the homogenate was transferred into a 2-ml eppendorf tube. One milliliter of cold Nuclei EZ Lysis Buffer was added and mixed, followed by 4 min incubation on ice. During the incubation, the sample was gently mixed a couple of times using a pipette. Homogenate was filtered using a 70-μm strainer mesh, and the flowthrough was collected in a polystyrene round-bottom FACS tube and subsequently transferred into a new 2-ml tube. The sample was centrifuged at 500g for 5 min at 4 °C and the supernatant was removed. The nuclei/mitochondria enriched sample was resuspended in another 1.5 ml EZ Lysis buffer and incubated for 5 min on ice. The sample was centrifuged at 500g for 5 min 4 °C and the supernatant was discarded (cytoplasm). Five-hundred microliters of nuclei wash and resuspension buffer ) was added to the sample and incubated for 5 min without resuspending to allow buffer interchange. After incubation, 1 ml nuclei wash and resuspension buffer was added and the sample was resuspended. The sample was centrifuged at 500g for 5 min at 4 °C. The supernatant was removed and only ~50 μl was left. Using 1.4 ml nuclei wash and resuspension buffer, the sample was resuspended and transferred to a 1.5-ml eppendorf tube. The last washing step was repeated and the pellet was resuspended in 300 μL nuclei wash and resuspension buffer. RNA was extracted using TRIzol (Life Technologies) following the manufacturer’s protocol.To isolate nuclear/mitochondrial-enriched RNA from the mouse (Danio rerio) embryos were obtained through natural mating of the TU-AB strain of mixed ages (5–18 months). Mating pairs were randomly chosen from a pool of 60 males and 60 females allocated for each day of the month. Embryos and adult fish were maintained at 28 °C.Wild-type zebrafish  and total RNA was extracted using the manufacturer’s protocol. Total RNA concentration was calculated by nanodrop.For polyA-selected RNA samples, polyadenylated RNAs were isolated with oligo(dT) magnetic beads  according to the manufacturer’s protocol and eluted in 30 µl before nanodrop quantification.−1) to 37 °C for hybridization. In the meantime, Dynabeads MyOne Streptavidin C1  beads were resuspended by carefully vortexing at medium speed. Eighty microliters of Dynabeads bead resuspension (10 mg ml−1) was transferred into a tube, which then was placed on a magnetic rack. After aspirating the supernatant, 100 μl of bead resuspension buffer  was added to the sample and beads were resuspended in this buffer by agitating the tube. Sample was placed on a magnet and the supernatant was aspirated. This step was performed twice. Beads were then resuspended in 100 μl bead wash buffer (0.1 M NaCl) and placed on magnet to aspirate the supernatant. Beads were then resuspended in a 160 μl depletion buffer . This suspension was then divided into two tubes of 80 μl, which will be used consecutively. Twenty microliters of hybridized riboPOOL and total RNA was briefly centrifuged to spin down droplets and it was pipetted into the tube containing 80 μl of beads in depletion buffer . The tube containing the mix was agitated to resuspend the solution well. Then the mix was incubated at 37 °C for 15 min, followed by a 50 °C incubation for 5 min. Immediately before use, the second tube containing 80 μl of beads was placed on a magnetic rack and the supernatant was aspirated. After the incubation at 50 °C, the first depletion reaction was placed on a magnet and the supernatant was transferred into the tube containing the other set of beads. The mix was incubated again at 37 °C for 15 min, followed by a 50 °C incubation for 5 min. After briefly spinning down the droplets, the mix was placed on a magnet for 2 min. The supernatant was transferred into a different tube and cleaned up using RNA Clean & Concentrator-5 .Ribodepletion was performed on zebrafish total RNA using riboPOOL oligos  following the manufacturer’s protocol. In brief, 5 μg total RNA in 14 μl was mixed with 1 μl resuspended riboPOOL oligos, 5 μl hybridization buffer and 0.5 μl SUPERase•In RNase Inhibitor . The mix was incubated for 10 min at 68 °C, followed by a slow cool down (3 °C minMycoplasma-tested in-house) were cultured in DMEM high glucose  with 10% fetal bovine serum supplement . Pellets were obtained from 6 million cells and 1 ml TRIzol (Life Technologies) was added to each pellet, Sample was incubated at room temperature for a few minutes after adding 200 μl chloroform and vortexing briefly. After the incubation, the solution was centrifuged at 4 °C with 14,000g. Aqueous phase from the previous step was transferred to another eppendorf tube and equal amount of absolute ethanol  was added to the solution. This suspension was then transferred to an RNeasy Mini Spin Column from RNeasy Mini Kit , and total RNA was isolated following the manufacturer’s protocol. Then, polyadenylated RNAs were isolated with Dynabeads Oligo(dT) 25  according to the manufacturer’s protocol and eluted in 20 µL. Concentration and quality of RNA was evaluated using a Nanodrop 2000 Spectrophotometer and an Agilent 4200 TapeStation, respectively.HeLa cell lines  in order to pre-anneal the oligos. Then, 50 ng RNA was mixed with 2 µl pre-annealed R_RNA + D_DNA oligo, 1 μl 100 mM dithiothreitol, 4 µl 5× TGIRT Buffer , 1 µl RNAse Inhibitor Murine , 1 µl TGIRT (InGex) and nuclease-free water up to 19 µl. We should note that if 50 ng are used as input, only 1 µl TGIRT is needed, whereas if 100 ng is used as input, 2 µl TGIRT enzyme is needed. The reverse transcription mix was initially incubated at room temperature for 30 min before adding 1 µl 10 mM dNTP mix. Then the mix was incubated at 60 °C for 60 min and inactivated by heating at 75 °C for 15 min before moving to ice. RNAse Cocktail  was added to the mix to digest the RNA, and the mix was incubated at 37 °C for 10 min. The reaction was then cleaned up using 0.8× AMPure XP Beads . To be able to ligate the sequencing adapters to the first cDNA strand, 1 µl 10 µM CompA_DNA (5′ GAAGATAGAGCGACAGGCAAGTGATCGGAAGA 3′) was annealed to the 15 µl cDNA in a tube with 2.25 µl 0.1 M Tris pH 7.5, 2.25 µl 0.5 M NaCl and 2 µl nuclease-free water. The mix was incubated at 94 °C for 1 min and the temperature was ramped down to 25 °C (−0.1 °C s−1) to anneal the complementary to the first-strand cDNA. Then, 22.5 µl first-strand cDNA was mixed with 5 µl Adapter Mix (AMX), 22.5 µl Rnase-free water and 50 µl Blunt/TA Ligase Mix  and incubated in room temperature for 10 min. The reaction was cleaned up using 0.8× AMPure XP beads, using WSB (Washing buffer) buffer for washing. The sample was then eluted in elution buffer and mixed with sequencing buffer and loading beads before loading onto a primed R9.4.1 flowcell. Libraries were run on either Flongle or MinION flow cells with MinKNOW acquisition software version v.3.5.5. A detailed step-by-step Nano3P-seq protocol is provided as a The protocol is based on the direct cDNA Sequencing ONT protocol (DCB_9091_v109_revC_04Feb2019), with several modifications to be able to perform TGIRT template switching. Before starting the library preparation, 1 µl 10µM R_RNA (oligo: 5′ rGrArArGrArUrArGrArGrCrGrArCrArGrGrCrArArGrUrGrArUrCrGrGrArArG/3SpC3/3′) and 1 µl 10 µM D_DNA (5′/5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCN 3′) were mixed with 1 µl 0.1 M Tris pH 7.5, 1 µl 0.5 M NaCl, 0.5 μl RNAse Inhibitor Murine  and 5.5 μl RNase-free water. The mix was incubated at 94 °C for 1 min and the temperature was ramped down to 25 °C (−0.1 °C s−1) to pre-anneal the oligonucleotides. Then, 50 ng polyA-tailed RNA was mixed with 1 µl pre-annealed VNP + CompA, 1 μl 100 mM dithiothreitol, 4 µl 5× TGIRT Buffer , 1 µl RNAse Inhibitor Murine , 1 µl TGIRT and nuclease-free water up to 19 µl. The reverse transcription mix was initially incubated at room temperature for 30 min before adding 1 µl 10 mM dNTP mix. Then the mix was incubated at 60 °C for 60 min and inactivated by heating at 75 °C for 15 min before moving onto ice. Furthermore, RNAse Cocktail  was added to the mix to digest the RNA and the mix was incubated at 37 °C for 10 min. Then the reaction was cleaned up using 0.8× AMPure XP Beads . To be able to ligate the sequencing adapters the the first strand, 1 µl 10 µM CompA was again annealed to the 15 µl cDNA in a tube with 2.25 µl 0.1 M Tris pH 7.5, 2.25 µl 0.5 M NaCl and 2 µl nuclease-free water. The mix was incubated at 94 °C for 1 min and the temperature was ramped down to 25 °C (−0.1 °C s−1) to anneal the complementary to the first-strand cDNA. Furthermore, 22.5 µl first-strand cDNA was mixed with 5 µl Adapter Mix (AMX), 22.5 μl Rnase-free water and 50 µl Blunt/TA Ligase Mix  and incubated at room temperature for 10 min. The reaction was cleaned up using 0.8× AMPure XP beads, using WSB Buffer for washing. The sample was then eluted in elution buffer and mixed with sequencing buffer and loading beads before loading onto a primed R9.4.1 flowcell and run on a MinION sequencer with MinKNOW acquisition software version v.3.5.5.Some adjustments were made to the original Direct cDNA-Sequencing ONT protocol (SQK-DCS109), to be able to use TGIRT (InGex) as reverse transcription enzyme for nanopore sequencing, as this enzyme does not produce CCC overhang, which is typically exploited by the dcDNA-seq library preparation protocol Fig. . In brieA total of 12 synthetic cDNA standards were synthesized as ultramers by IDT (Integrated DNA Technologies) to assess the tail length estimation and tail composition quantification accuracy of Nano3P-seq.Synthetic cDNA standards designed to assess accuracy in tail length estimation:cDNA_pA_standard_0: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGAAAAATGCTTGTTTGCAACCCTATATAGAAcDNA_pA_standard_15: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGAAAAATGCTTGTTTGcDNA_pA_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGcDNA_pA_standard_60: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGcDNA_pA_standard_90: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGcDNA_pA_standard_120: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTSynthetic cDNA standards designed to assess accuracy in tail composition analyses:cDNA_p29A_pU1_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCATTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGcDNA_p27A_pU3_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCAAATTTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGcDNA_p25A_pU5_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCAAAAATTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGcDNA_p25A_pC5_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCGGGGGTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGcDNA_p25A_pG5_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCCCCCCTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGcDNA_pA_internalG_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCTTTTCTTTTCTTTTCTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATG18 with minimap2 with ‘-ax splice -k14 -uf --MD’ parameters59. For zebrafish dRNA-seq samples, reads were base-called with Guppy v.4.0. Base-called reads were first mapped to maternal and somatic zebrafish ribosomal RNA sequences taken from42 and then to the genome (GRCz11) with minimap259 with ‘-ax splice -k14 -uf --MD’ parameters. Mapped reads were intersected with ENSEMBL version 103 annotation (Danio_rerio.GRCz11.103.2.gtf) using bedtools v.2.29.1 intersect option60.dRNA-seq library preparations were prepared following manufacturer’s recommendations, using 450 ng (in the case of polyA-enriched RNA zebrafish run) or 500 ng (in the case of in vitro transcribed ‘sequins’) as input material. Samples were sequenced in an R 9.4.1 MinION flowcell using a GridION sequencing device in the case of ‘sequins’, and in a R 9.4.1 PromethION flowcell using a PromethION sequencing device in the case of zebrafish RNA. For sequins, reads were base-called using stand-alone Guppy v.3.0.3 with default parameters and then the base-called reads were mapped to sequin sequences59 with the following ‘-ax splice -k14 -uf --MD’ parameters when mapping to genome and ‘-ax map-ont -k14 --MD’ when mapping to transcriptome, unless stated otherwise.All the Nano3P-seq runs were base-called and demultiplexed using stand-alone Guppy v.6.0.1 with default parameters. All runs were mapped using minimap258, and mapped reads were then intersected with annotations of Curlcake 1 and 2 sequences to filter out the incomplete reads using bedtools v.2.29.1 intersect option. For yeast total RNA, we first mapped the base-called reads to S. cerevisiae ribosomal RNAs  and then mapped the rest of the reads to the S. cerevisiae genome (SacCer3). Mapped reads were then intersected with SacCer64 annotation exon ends, to filter out incomplete reads. For the analysis of nuclear/mitochondrial-enriched mouse brain RNA spiked in with sequins18, we first mapped the base-called reads to Mus musculus ribosomal RNAs  and then mapped the rest of the rest of the reads to the M. musculus genome (GRCm38), supplemented with sequin chromosome (chrIS). Mapped reads were then intersected with ENSEMBL version 102 annotation (Mus_musculus.GRCm38.102.gtf) and sequin annotation (RNAsequins.v2.2.gtf) exon ends, to filter the incomplete reads. For zebrafish RNA (polyA-selected and ribodepleted), we first mapped the base-called reads to ribosomal RNAs and then mapped the rest of the reads to the genome (GRCz11). Mapped read starts were then intersected with ENSEMBL version 103 annotation (Danio_rerio.GRCz11.103.2.gtf) exon ends, to filter the incomplete reads. For HeLa mRNA, we first mapped the base-called reads to human ribosomal RNAs ; then, the rest of the reads (unmapped) were mapped to the Homo sapiens genome (GRCh38). Mapped read starts were then intersected with ENSEMBL version 107 annotation (Homo_sapiens.GRCh38.107.gtf) exon ends, to filter out incomplete reads . For cDNA standards, base-called reads were mapped to cDNA sequence reference using minimap2, except for the cDNA_pA_standard_120, which was mapped using bwa short-read aligner61 with the following parameters ‘mem -xont2d’. A different aligner was used for cDNA_pA_standard_120 because minimap2, which is a long-read mapping algorithm, did not yield any mapped reads for this standard owing to its short length62 (30 nucleotides once the pA tail length has been soft-clipped). We should note that the use of a different aligner should not affect the polyA tail length estimations of the reads, as these are done at the level of current intensity . We should note that fewer reads were base-called and mapped to cDNA_pA_standard_120, cDNA_pA_standard_90 and cDNA_pA_standard_60, in comparison to the other cDNA standards , base-called reads were mapped to Curlcake 1 and 2 sequenceshttps://github.com/ablab/IsoQuant) was used with Danio_rerio.GRCz11.103.2.gtf annotation using the following parameters ‘--genedb gtf_file --complete_genedb --bam bam_file --data_type nanopore -o output’. A complete step-by-step command line of the bioinformatic analysis done on Nano3P-seq datasets can be found in the GitHub repository . All reference sequences used to map the runs mentioned above are included in the Nano3P-seq GitHub repository.For the assignment of reads to isoforms, IsoQuant package ; rather, they estimate polyA tail lengths by comparing the relative duration of the current signal corresponding to the polyA tail region to the total duration of the sequenced read.For dRNA-seq reads, polyA tail length estimation was performed using NanoTail, a module from Master of Poreshttps://github.com/adnaniazi/tailfindr/tree/nano3p-seq). All code used to estimate polyA tail lengths and post-process Nano3P-seq data can be found at https://github.com/novoalab/Nano3P_Seq.For Nano3P-seq reads, polyA tail length estimation was performed using the Nano3P-seq version of tailfindR (https://github.com/rrwick/Porechop) with the following parameters ‘--extra_end_trim 0 --end_threshold 50’, to remove the adapter sequences. Because Porechop sometimes failed at removal of the adapter sequences, only reads containing more than 80% A bases in their tail composition were kept for downstream analyses, thus ensuring that untrimmed reads are not included in downstream analyses .Base-called reads mapped to the zebrafish genome and cDNA standards for tail composition quantification were first trimmed using the Porechop tool . Mice maintenance was approved by the Garvan/St Vincent’s Hospital Animal Ethics Committee, in accordance with the guidelines of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (Project No. 16/14 and 16/26). All animals were entered into the study in a randomized order and operators were blinded to genotype and treatments.Further information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41592-022-01714-w.Supplementary InformationSupplementary Protocol, Supplementary Tables 1–3, and Supplementary Notes 1–2.Reporting SummaryPeer Review FileSupplementary SoftwareCode corresponding to TailfindR Nano3P-seq branch found in GitHub repository (attached as a separate zip file). This version incorporates the custom adapter sequences that are used in Nano3P-seq library preparation protocols.


The innate immune response controls the acute phase of virus infections; critical to this response is the induction of type I interferon (IFN) and resultant IFN-stimulated genes to establish an antiviral environment. One such gene, zinc finger antiviral protein (ZAP), is a potent antiviral factor that inhibits replication of diverse RNA and DNA viruses by binding preferentially to CpG-rich viral RNA. ZAP restricts alphaviruses and the flavivirus Japanese encephalitis virus (JEV) by inhibiting translation of their positive-sense RNA genomes. While ZAP residues important for RNA binding and CpG specificity have been identified by recent structural studies, their role in viral translation inhibition has yet to be characterized. Additionally, the ubiquitin E3 ligase tripartite motif-containing protein 25 (TRIM25) has recently been uncovered as a critical co-factor for ZAP’s suppression of alphavirus translation. While TRIM25 RNA binding is required for efficient TRIM25 ligase activity, its importance in the context of ZAP translation inhibition remains unclear. Here, we characterized the effects of ZAP and TRIM25 RNA binding on translation inhibition in the context of the prototype alphavirus Sindbis virus (SINV) and JEV. To do so, we generated a series of ZAP and TRIM25 RNA binding mutants, characterized loss of their binding to SINV genomic RNA, and assessed their ability to interact with each other and to suppress SINV replication, SINV translation, and JEV translation. We found that mutations compromising general RNA binding of ZAP and TRIM25 impact their ability to restrict SINV replication, but mutations specifically targeting ZAP CpG-mediated RNA binding have a greater effect on SINV and JEV translation inhibition. Interestingly, ZAP-TRIM25 interaction is a critical determinant of JEV translation inhibition. Taken together, these findings illuminate the contribution of RNA binding and co-factor interaction to the synergistic inhibition of viral translation by ZAP and TRIM25. The type I interferon (IFN) response is one of the first lines of cellular defense against invading pathogens. The IFN-induced zinc finger antiviral protein (ZAP) is a potent inhibitor of diverse RNA and DNA viruses . ZAP encEarlier efforts to elucidate ZAP RNA binding activity showed that ZAP binds RNA with its four N-terminal CCCH ZnFs, and mutations of ZnFs 2 and 4 most dramatically reduce ZAP antiviral activity . The firWhile much attention has been given to characterizing ZAP RNA binding, less has been given to its critical co-factors such as TRIM25. TRIM25 was identified as a ZAP co-factor in the context of inhibiting alphavirus translation . HoweverWhile some have attempted to illuminate the contribution of ZAP or TRIM25 RNA binding to the ZAP-TRIM25 interaction, these studies have focused on only a few select mutations. Still, most agree that RNA binding in either ZAP or TRIM25 is not required for the ZAP-TRIM25 interaction. One group demonstrated not only that RNase A treatment has little effect on the ZAPL-TRIM25 interaction, but also that an example ZAPL RNA binding mutant retains and even increases its interaction with TRIM25 . AnotherIn light of the recent novel structural insights, we asked how different ZAP and TRIM25 RNA binding mutations affect the ability of ZAP and TRIM25 to interact with one another and to restrict SINV and JEV translation. We curated a panel of ZAP and TRIM25 RNA binding mutants from prior studies, including mutants with a range of RNA binding and antiviral capabilities . We firsTo investigate the role of ZAP and TRIM25 RNA binding in the context of viral translation, we generated a panel of constructs with mutations previously demonstrated to impact RNA binding. For ZAP, each of the following mutations was introduced in both ZAPS and ZAPL to probe potential isoform differences in RNA binding and antiviral function. These mutations fall into two general categories: 1) ZnF mutants that individually disrupt each CCCH motif and 2) CpG RNA binding cavity mutants. We made four individual mutations to disrupt each N-terminal CCCH ZnF: H86K, C88R, C168R, and H191R, which are located in ZnF 1, 2, 3, and 4, respectively . Two putFor TRIM25, we generated two constructs, one in which we replaced the lysine-rich motif in the L2 linker with alanines (TRIM25 7KA) and one in which we deleted the previously identified RNA binding domain in the PRY/SPRY domain (TRIM25 ΔRBD) .in vitro RNA pull-down assay. We incubated lysates of cells transfected with ZAP or TRIM25 mutants with biotin-labeled SINV (Toto1101 strain) genomic RNA, allowing for RNA and bound protein to be immunoprecipitated using streptavidin beads and probed for the presence of bound ZAP or TRIM25. As a negative control, we also assessed the ability of the WT constructs to bind firefly luciferase (Fluc) RNA. We quantified the resultant ZAP and TRIM25 bound to RNA and normalized to input ZAP and TRIM25 protein levels with ImageJ   . Becausee (Rluc) . We attee (Rluc) .We found that the ZAP and TRIM25 RNA binding mutants inhibit JEV translation to varying degrees Figure 5Upon assaying TRIM25 variants, we found that TRIM25 ΔRBD inhibits JEV translation similarly to TRIM25 WT  and its parental 293T lines . TRIM25 Infections with SINV expressing firefly luciferase (Toto1101/Luc) and temperature-sensitive SINV (Toto1101/Luc:ts6) have been previously described . Each inBamHI and HindIII restriction sites. ZAPS and ZAPL were cloned into pcDNA3.1-3XFLAG from pTRIP-TagRFP-hZAPS and pTRIP-TagRFP-hZAPL ”, to replace wild-type sequence in TRIM25. The 7KA mutated sequence is bolded in the below gene block, and nonessential nucleotides on the 5’ and 3’ ends are written in lowercase.Point mutations in ZAPS and ZAPL were generated using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) and all plasmids were verified by sequencing (Genewiz). Primers for mutations were synthesized by Integrated DNA Technologies  was genealanines , and utiTGTACAGTCAGATCAACGGGGCGTCGAGAGCACTGGATGATGTGAGAAACAGGCAGCAGGATGTGCGGATGACTGCAAACAGAAAGGTGGAGCAGCTACAACAAGAATACACGGAAATGAAGGCTCTCTTGGACGCCTCAGAGACCACCTCGACAAGGAAGATAAAGGAAGAGGAGAAGAGGGTCAACAGCAAGTTTGACACCATTTATCAGATTCTCCTCAAGAAGAAGAGTGAGATCCAGACCTTGAAGGAGGAGATTGAACAGAGCCTGACCAAGAGGGATGAGTTCGAGTTTCTGGAGAAAGCATCAAAACTGCGAGGAATCTCAACAAAGCCAGTCTACATCCCCGAGGTGGAACTGAACCACAAGCTGATAAAAGGCATCCACCAGAGCACCATAGACCTCAAAAACGAGCTGAAGCAGTGCATCGGGCGGCTCCAGGAGCCCACCCCCAGTTCAGGTGACCCTGGAGAGCATGACCCAGCGTCCACACACAAATCCACACGCCCTGTGGCAGCAGTCTCCGCAGAGGAAGCAGCATCCGCAGCACCTCCCCCTGTCCCTGCCTTACCCAGCAAGCTTCCCACGTTTGGAGCCCCGGAACAGTTAGTGGATTTAAAACAAGCTGGCTTGGAGGCTGCAGCCAAAGCCACCAGCTCACATCCGAACTCAACATCTCTCAAGGCCAAGGTGCTGGAGACCTTCCTGGCCAAGTCCAGACCTGAGCTCCTGGAGTATTACATTAAAGTCATCCTGGACTACAACACCGCCCACAACAAAGTGGCTCTGTCAGAGTGCTATACAGTAGCTTCTGTGGCTGAGATGCCTCAGAACTACCGGCCGCATCCCCAGAGGTTCACATACTGCTCTCAGGTGCTGGGCCTGCACTGCTACAAGAAGGGGATCCgttt-3’5’gtttX-tremeGENE9 DNA Transfection Reagent (Roche Life Science) was used to transfect cells at a ratio of 3 μL to 1 μg DNA according to the manufacturer’s instructions. To keep the total plasmid amount in co-transfections constant, empty vectors pcDNA3.1-myc or 3XFLAG were transfected as necessary .XhoI linearization of pToto1101 (in vitro by Sp6 RNA polymerase (New England Biolabs) in the presence of the cap analog [m7G(5’)ppp(5’)G] (New England Biolabs). Fluc DNA templates for transcription were amplified from the pGL3-Control plasmid (Promega). Fluc RNA was transcribed in vitro using the mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen). Biotin-labeled RNAs were generated by adding 10mM biotin-16-UTP (Roche Life Science) to in vitro transcription reactions. JEV replicon DNA templates for transcription were generated by XhoI linearization and transcribed in vitro using the mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen). Transcribed RNAs were purified using the Quick-RNA Miniprep Kit (Zymo Research) and biotinylation was confirmed by streptavidin dot blot  and incubated with RNA probes in a final volume of 100 μL RNA binding buffer supplemented with 1 unit/μL RNAseOUT (Thermo Fisher Scientific), 1 μg/μL heparin (Sigma-Aldrich), and 100 ng/μL yeast tRNA (ThermoFisher) for 30 min at 30°C. Lysate-RNA mixtures were then incubated with 300 μL of Dynabeads M-280 Streptavidin (Invitrogen) for 30 min at room temperature on a shaker. Protein-RNA complexes were washed three times by RNA binding buffer, and proteins were eluted by incubation with 30 μL of 4x Laemmli Sample Buffer (Bio-Rad) for 5 min at 95°C. The proteins were further analyzed by immunoblot and quantified by ImageJ as previously described  and 4-15% precast Mini-PROTEAN TGX Gels (Bio-Rad) before transferring to a PVDF membrane (Bio-Rad). Immunodetection was achieved with 1:2,500 anti-myc , 1:20,000 anti-FLAG (Sigma-Aldrich), and 1:20,000 anti-actin-HRP (Sigma-Aldrich). Primary antibodies were detected with 1:20,000 goat anti-mouse HRP (Jackson ImmunoResearch) or 1:20,000 goat anti-rabbit HRP (Thermo Fisher Scientific). Proteins were visualized on a ChemiDoc (Bio-Rad) using ProSignal Pico ECL Reagent (Genesee Scientific). ImageJ was used to quantify western blots as previously described . BrieflyTo assess ZAP or TRIM25 co-immunoprecipitation (co-IP) with RNA binding mutants of TRIM25 or ZAP, respectively, cells were transfected in 6-well plates, collected, and then lysed by rotating in FLAG IP buffer  supplemented with a complete protease inhibitor cocktail (Roche Life Science) at 4°C for 30 min, before spinning down at 14,000 rpm at 4°C for 15 min. To equilibrate beads prior to use, anti-FLAG beads  or anti-myc beads  were washed 3 times in FLAG IP buffer. Three hundred μL of whole cell lysate (WCL) were incubated with 30 μL of anti-FLAG or -myc beads rotating at 4˚C for 45 minutes. FLAG IP buffer was used to wash immunoprecipitates 3 times before eluting bound proteins with SDS loading buffer, and boiling for 5 minutes for immunoblot analysis. Western blot ImageJ analysis was performed as previously described . BrieflyTransIT-mRNA Transfection Kit (Mirus Bio). Cells were lysed 4 hours post-transfection of replicon RNA and luciferase activity was measured by Dual-Luciferase Reporter Assay (Promega). Each independent experiment included triplicate wells of biological replicates per condition.Following transfection of ZAP or TRIM25 constructs into ZAP or TRIM25 KO 293T for 48 hours, JEV replicon RNA was transfected by Statistical analyses in The original contributions presented in the study are included in the article/EY, LN, and ML conceptualized and designed the study. CW and RK assisted in cloning ZAP and TRIM25 RNA binding mutants. LN performed RNA binding and JEV replicon experiments, while EY performed co-IP and SINV replication and translation experiments. LN performed the correlation analysis. EY and LN co-wrote the first draft of the manuscript. ML provided critical feedback. All authors contributed to manuscript revision, read, and approved the submitted version.This work was supported in part by NIH R01AI158704 (ML), UCLA AIDS Institute and Charity Treks 2019 Seed Grant (ML), Ruth L. Kirschstein Multidisciplinary Training Grant in Microbial Pathogenesis , Ruth L. Kirschstein Cellular and Molecular Biology Training Program , Warsaw Fellowship (EY), and Whitcome Fellowship .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.


Currently, a total of 81 corticalis group species are known worldwide, amongst them 67 were recorded from China. However, the diversity of this group in China is still insufficiently known.The Clubionaxianningsp. nov. is described as a new species of the C.corticalis species-group collected from Hubei Province, China. Clubiona Latreille, 1804 currently contains 518 catalogued species that are found worldwide, except for the Polar Regions and South America .OP675437; YHCLU0273, ♀, GenBank OP675436.A DNA barcode was also obtained for species matching. A partial fragment of the mitochondrial cytochrome oxidase subunit I (CO1) gene was amplified and sequenced for two specimens, using the primers LCOI1490 (5’-GGTCAACAAATCATAAAGATATTG-3’) and HCOI2198 (5’-TAAACTTCAGGGTGACCAAAAAAT-3’) . For addAll measurements were obtained using an Olympus SZX7 stereomicroscope and given in millimetres. Eye diameters are taken at the widest point. The total body length does not include chelicerae or spinnerets length. Leg lengths are given as total length . Most of the terminologies used in text and figure legends follows All specimens are deposited Museum of Guizhou Normal University, Guiyang, Guizhou, China.Zhong & Yusp. n.B0CC6F23-8DFB-5B4A-BAD0-E8AC5114D6878DFF1CC4-1C1A-46A6-8300-FF36FFFA2644Type status:Holotype. Occurrence: recordedBy: Yang Zhong, Xusheng Gong, Qianle Lu; individualID: YHCLU0272; individualCount: 1; sex: male; lifeStage: adult; behavior: foraging; preparations: whole animal (ETOH); associatedSequences: OP675437GenBank: ; occurrenceID: 1312422A-3320-5AE6-9BA9-5DCB3013B14F; Taxon: order: Araneae; family: Clubionidae; genus: Clubiona; specificEpithet: xianning; scientificNameAuthorship: Zhong & Yu; Location: continent: Asian; country: China; countryCode: CHN; stateProvince: Hubei; county: Tongshan; municipality: Xianning; locality: Jiugongshan Nature Reserve; decimalLatitude: 29.39; decimalLongitude: 114.65; Identification: identifiedBy: Hao Yu; dateIdentified: 2022-05; Event: samplingProtocol: by hand; samplingEffort: 10 km by foot; year: 2020; month: 7; day: 4; Record Level: basisOfRecord: PreservedSpecimenType status:Holotype. Occurrence: recordedBy: Yang Zhong, Xusheng Gong, Qianle Lu; individualID: YHCLU0273; individualCount: 1; sex: female; lifeStage: adult; behavior: foraging; preparations: whole animal (ETOH); associatedSequences: OP675436GenBank: ; occurrenceID: 085B9104-F139-5EC1-9913-5A5DB9C7C9C6; Taxon: order: Araneae; family: Clubionidae; genus: Clubiona; specificEpithet: xianning; scientificNameAuthorship: Zhong & Yu; Location: continent: Asian; country: China; countryCode: CHN; stateProvince: Hubei; county: Tongshan; municipality: Xianning; locality: Jiugongshan Nature Reserve; decimalLatitude: 29.39; decimalLongitude: 114.65; Identification: identifiedBy: Hao Yu; dateIdentified: 2022-05; Event: samplingProtocol: by hand; samplingEffort: 10 km by foot; year: 2020; month: 7; day: 4; Record Level: basisOfRecord: PreservedSpecimenMale 5'TTCTGGTCAGCTATAGTTGGTACAGCTATAAGAGTTATAATTCGTATAGAATTAGGTCAATCTGGAGCTTTTTTAGGTGATGATCATTTGTATAATGTAGTAGTTACTGCTCATGCTTTTGTTATAATTTTTTTTATAGTAATACCTATTATAATTGGGGGGTTTGGAAATTGATTAGTTCCATTAATATTAGGGGCAGGTGATATAGCTTTTCCTCGTATAAATAATTTAAGTTTTTGACTTTTACCACCTTCATTAATATTATTAGTTATATCATCTATGGCTGAGATGGGAGTTGGGGCTGGATGAACAGTTTATCCCCCTCTTGCTTCTTTAGTAGGTCATACGGGAAGAGCAATGGATTTTGCTATTTTTTCATTACATTTAGCTGGGGCTTCTTCTATTATAGGAGCTGTTAATTTTATTACTACTATTATGAATATACGATCTTTTGGAATAATAATGGAAAAGATTTCATTATTTGTTTGGTCTGTTTTAATTACAGCTATTTTATTATTATTATCTTTGCCAGTTTTAGCCGGGGCTATTACTATATTATTAACTGATCGTAATTTTAATACGTCTTTTTTTGACCCTGCTGGGGGAGGTGATCCTATTTTATTTCAACATTTATTTTGATTTTTTGGTCACCC3' .5'TCTGGTCAGCTATAGTTGGTACAGCTATAAGAGTTATAATTCGTATAGAATTAGGTCAATCTGGAGCTTTTTTAGGTGATGATCATTTGTATAATGTAGTAGTTACTGCTCATGCTTTTGTTATAATTTTTTTTATAGTAATACCTATTATAATTGGGGGGTTTGGAAATTGATTAGTTCCATTAATATTAGGGGCAGGTGATATAGCTTTTCCTCGTATAAATAATTTAAGTTTTTGACTTTTACCACCTTCATTAATATTATTAGTTATATCATCTATGGCTGAGATGGGAGTTGGGGCTGGATGAACAGTTTATCCCCCTCTTGCTTCTTTAGTAGGTCATACGGGAAGAGCAATGGATTTTGCTATTTTTTCATTACATTTAGCTGGGGCTTCTTCTATTATAGGAGCTGTTAATTTTATTACTACTATTATGAATATACGATCTTTTGGAATAATAATGGAAAAGATTTCATTATTTGTTTGGTCTGTTTTAATTACAGCTATTTTATTATTATTATCTTTGCCAGTTTTAGCCGGGGCTATTACTATATTATTAACTGATCGTAATTTTAATACGTCTTTTTTTGACCCTGCTGGGGGAGGTGATCCTATTTTATTTCAACATTTATTTTGATTTTTTGGTCACCC3' , differ from C.altissimus by: (1) atrium large, nearly as wide as epigynal plate  (cf. Fig. C.xianningsp. nov. also can by separated from C.caohai and C.altissimus by their habitus: abdomen without distinct colour pattern in C.xianningsp. nov. (Fig. C.caohai and C.altissimus (Male of the new species resembles that of Yu, 2020 : fig. 2DYu, 2020 : fig. 2Bov. Fig. E–H, but The specific name refers to the type locality and is a noun in apposition.Known from the Mt. Jiugong, Hubei Province, China Fig. .


P < 0.05). The relative expression of circHIPK3 in patients with TNM stage II/III was dramatically inferior to that in patients with tumor, node, and metastasis (TNM) stage I (P < 0.05). Expression of circHIPK3 in patients with lymph node metastasis was dramatically inferior to that in patients without lymph node metastasis (P < 0.05). Of the lung cancer tissues of patients with different TNM stages, only six patients had high expression, and the remaining 104 patients had low expression. Moreover, electrophoresis revealed that circHIPK3 can only be amplified in cDNA, but not in gDNA. Gefitinib-mediated apoptosis rate of lung cancer drug-resistant cell lines decreased notably. In summary, the circular RNA circHIPK3 may have a notably low expression in lung cancer tissues, whose low expression had a certain enhancement effect on the drug resistance of lung adenocarcinoma cells to gefitinib.To study the mechanism of circular ribonucleic acid (RNA) circHIPK3 involved in the resistance of lung cancer cells to gefitinib, 110 patients with lung cancer were recruited as the research objects, and the tumor tissue and para-cancerous tissue of each patient's surgical specimens were collected and paraffinized to detect the expression of circHIPK3 in different tissues. Gefitinib drug-resistant cell line of lung cancer was constructed with gefitinib to detect cell apoptosis under different conditions. As a result, the relative expression of circHIPK3 in patients with tumor diameter no less than 3 cm was dramatically inferior to that in patients with tumor diameter less than 3 cm ( Lung cancer is a malignant tumor, mainly originated from the mucosal epithelium of the bronchus, which is classified into small cell carcinoma and nonsmall cell carcinoma according to the histological changes. In recent years, with the serious air pollution and the gradual deterioration of the environment, the global mortality rate of lung cancer is on the rise, seriously threatening people's health . AccordicircRNA is a new type of RNA molecule that is characterized by a covalent closed loop and widely exists in eukaryotes . circRNAIn recent years, related studies found that using siRNA to knockdown circHIPK3 can inhibit cell proliferation. In addition, circHIPK3 can adsorb miR-124 in liver cancer and promote the proliferation of liver cancer cells by regulating the expression of target genes. However, its regulation and expression mechanism in lung cancer is still unclear. Therefore, this study was developed to explore the mechanism of the circular RNA circHIPK3 involved in the resistance of lung cancer cells to gefitinib.In this study, 110 lung cancer patients admitted to X Hospital from September 2019 to September 2020 were collected as the research objects. Paraffin specimens were collected from 58 male patients and 52 female patients. The study has been approved by the Medical Ethics Committee. Patients and their families understood the study content and methods of specimen acquisition and agreed to sign corresponding informed consent forms.Inclusion criteria: (i) patients diagnosed with lung cancer by pathology and imaging; (ii) patients aged between 45 and 75 years; (iii) there was no metastasis of lung lesions, mediastinal lymph node enlargement, or pleural hypertrophy; (iv) patients who were not recently treated with other drugs or antibiotics in the study; (v) patients with normal coagulation function and platelets.Exclusion criteria: (i) patients with diseases of other systems or organs; (ii) patients who had not received cooperative treatment due to personal or other factors; (iii) patients with incomplete clinical data and history information , etc.).μm/slice) and stored in an enzyme-free centrifuge tube  containing xylene .Paraffin sections of tumor tissue and para-cancer tissue  from surgical specimens of each included subject were collected. The collected paraffin samples were sliced with uniform thickness  were added. After the tissue was dissolved, it was bathed in water at 95°C for 10 min and centrifuged at 8,600 rpm at room temperature for 5 min. The supernatant was absorbed and transferred to a new Rnase-free centrifuge tube. 220 μL buffer RB was added, shaken, and mixed, then added with anhydrous ethanol, and mixed again.Sectioning: the collected paraffin specimens were sectionalized with a thickness of 10 μL solution and precipitate were absorbed and added to the adsorption column, centrifuged at high speed for 1 min. The waste solution was filtered and repeated several times until the solution and precipitate passed through the adsorption column. 80 μL DNase I working solution was added to the center of the adsorption column and left for 15 min at room temperature. 500 μL protein-free RW1 was added and centrifuged, then added with rinse solution, centrifuged again, and the waste solution was discarded. After drying, the adsorption column was transferred to a new RNase-free centrifuge tube. 35 μL RNase-free ddH2O was added and centrifuged at 8,600 rpm for 5 min to obtain RNA solution. RNase R was used to process the extracted total RNA to avoid artifacts. RNase R processing reaction system: 2 μg RNA, 1 μL RiboLock, 2 μL 10x RNase R reaction buffer, and 1 μL RNase R, with sterile water supplementing the system to 20 μL [μL 5 × PrimeScript Buffer, 0.5 μL enzyme mix I, 0.5 μL random 6 mers, and 7 μL total RNA were added to a 0.2 mL enzyme-free EP tube to configure a 10 μL system. The EP tube was closed and shaken for 15 s to mix, centrifuged, and put in the reverse transcription machine. The reverse transcription conditions: 37°C for 15 min, 85°C for 5 sec. After the reaction, the synthesized cDNA was transferred to -20°C refrigerator for storage. The length of circHIPK3 searched on NCBI was 1,099 bp, and the specific base sequence was as follows: GTATGGCCTCACAAGTCTTGGTCTACCCACCATATGTTTATCAAACTCAGTCAAGTGCCTTTTGTAGTGTGAAGAAACTCAAAGTAGAGCCAAGCAGTTGTGTATTCCAGGAAAGAAACTATCCACGGACCTATGTGAATGGTAGAAACTTTGGAAATTCTCATCCTCCCACTAAGGGTAGTGCTTTTCAGACAAAGATACCATTTAATAGACCTCGAGGACACAACTTTTCATTGCGACAAGTGCTGTTGTTTTGAAAAACACTGCAGGTGCTACAAAGGTCATAGCAGCTCAGGCACAGCAAGCTCACGTGCAGGCACCTCAGATTGGGGCGTGGCGAAACAGATTGCATTTCCTAGAAGGCCCCCAGCGATGTGGATTGAAGCGCAAGAGTGAGGAGTTGGATAATCATAGCAGCGCAATGCAGATTGTCGATGAATTGTCCATACTTCCTGCAATGTTGCAAACCAACATGGGAAATCCAGTGACAGTTGTGACAGCTACCACAGGATCAAAACAGAATTGTACCACTGGAGAAGGTGACTATCAGTTAGTACAGCATGAAGTCTTATGCTCCATGAAAAATACTTACGAAGTCCTTGATTTTCTTGGTCGAGGCACGTTTGGCCAGGTAGTTAAATGCTGGAAAAGAGGGACAAATGAAATTGTAGCAATCAAAATTTTGAAGAATCATCCTTCTTATGCCCGTCAAGGTCAAATAGAAGTGAGCATATTAGCAAGGCTCAGTACTGAAAATGCTGATGAATATAACTTTGTACGAGCTTATGAATGCTTTCAGCACCGTAACCATACTTGTTTAGTCTTTGAGATGCTGGAACAAAACTTGTATGACTTTCTGAAACAAAATAAATTTAGTCCCCTGCCACTAAAAGTGATTCGGCCCATTCTTCAACAAGTGGCCACTGCACTGAAAAAATTGAAAAGTCTTGGTTTAATTCATGCTGATCTCAAGCCAGAGAATATTATGTTGGTGGATCCTGTTCGGCAGCCTTACAGGGTTAAAGTAATAGACTTTGGGTCGGCCAGTCATGTATCAAAGACTGTTTGT TCAACATATCTACAATCTCGGTACTACAG.700 to 20 μL . The insThe designed upstream and downstream primers were as follows: circHIPK3-F: TAGACTTTGGGTCGGCCAGT; circHIPK3-R: TGGAATACACAACTGCTTGGC.The expression of circHIPK3 was detected by real-time fluorescence quantitative polymerase chain reaction (PCR). circHIPK3 is derived from the HIPK3 gene and consists of exon 2  head-to-tail splicing. Head-to-tail splicing can be generated by trans-splicing or genome rearrangement. The sequence is a circular sequence, which is assembled from the first position of the corresponding linear RNA, and a new unique sequence is generated at the reverse splicing site. The circRNA specific primers were designed with this as the target sequence, so as to specifically recognize circRNA and distinguish circular sequence from linear RNA. The identification and location of circHIPK3 was shown in Serum-free cell freezing medium (RPMI) 1640 cell culture medium consisted of 10% fetal bovine serum (FBS) , 100 U/mL penicillin  and 100 mg/mL streptomycin . The cell cryopreservation tube was taken out of the liquid nitrogen and quickly melted in a constant temperature water bath at 37°C. The thawed cell cryopreservation solution was transferred to a centrifuge tube containing cell culture medium and centrifuged at high speed for 5 min. Then, after cell culture medium was added to the lower sediment to resuspend the cells, they were transferred to RPMI medium and incubated at constant temperature. The medium was changed once the cells grew adherently.After the cell coverage in the culture dish reached more than 80%, passaging began. After the cell culture solution was aspirated, trypsin digestion solution was added, and cells were observed under a microscope. After culturing until the cells became round, culture medium was added to stop the digestion, and the cells were resuspended and passed to a culture dish.50) of gefitinib on sensitive cells was 1.78 mol/L. Then, the culture medium containing 17.5 μmol/L gefitinib was used for 24 h, and the medium containing half of the inhibitory concentration gefitinib was immediately replaced for 5-10 d, until the cells grew steadily and were subcultured for 3 times. IC50 was determined again. The medium containing 17.5 μmol/L gefitinib was cultured for 24 h, and the medium with gradually increased inhibitory concentration of gefitinib was replaced until the cells grew statically in the medium containing 17.5 μmol/L gefitinib and were continuously passed for 3 times. Then, the IC50 of gefitinib was 34.5 μmol/L and was named A549/GR.Gefitinib was dissolved in dimethyl sulfoxide (DMSO) (Abis (Shanghai) Biotechnology Co., Ltd.). The methyl tetrazolium (MTT) assay was used to determine the median lethal concentration of gefitinib on lung cancer cells . In this study, human lung adenocarcinoma cell line A549 was used to induce gefitinib-resistant cell lines. Resistant cells were induced by a combination of high-dose shocks and gradually increased doses. MTT assay was used to determine the 50% inhibitory concentration  was added to each well for 4 h, and the culture was terminated. The supernatant cultured in the hole should be carefully sucked and discarded. For suspended cells, the supernatant should be discarded after centrifugation. 150 μL dimethyl sulfoxide (DMSO) was added to each well and shaken to dissolve completely. At 490 nm, the light absorption value (OD) of each hole was measured by enzyme-linked immunosorbent assay (ELISA) and recorded. The cell growth curve was plotted with time as abscissa and light absorption value as ordinate.Single cell suspension was prepared with culture medium containing 10% fetal bovine serum and inoculated into 96-well plates with 1,000-10,000 cells per well, and volume of each well was 200 P < 0.05.SPSS 19.0 was employed for data statistics and analysis. Mean ± standard deviation (P > 0.05). There was also no considerable difference in the relative expression of circHIPK3 between patients less than 60 years old and patients no less than 60 years old (P > 0.05).P < 0.05). The relative expression of circHIPK3 in patients with T stage II/III tumors was dramatically inferior to that in patients with T stage I (P < 0.05).P < 0.05). The relative expression of circHIPK3 in patients with lymph node metastasis was dramatically inferior to that in patients without lymph node metastasis (P < 0.05).The circular RNA circHIPK3 is widely and highly expressed in lung cancer cells, mainly located in the cytoplasm, but also in the nucleus. The expression of circHIPK3 in cancer tissues and normal lung tissues adjacent to cancer is notably different, so it is used for the diagnosis of lung cancer. With pathological and imaging findings as diagnostic criteria, The 110 lung cancer patients included in this study were graded into stage I, stage II, and stage III according to the TNM staging. Among them, 39 patients were in TNM stage I, 36 were in stage II, and 35 were in stage III. The circular RNA circHIPK3 was amplified by designing primers and then electrophoresed on agarose gel plate to get the electrophoresis, as shown in The surface of normal cells is composed of lipids distributed asymmetrically on the inner and outer lobes of the plasma membrane. Phosphatidylserine is mostly distributed in the inner lobes of the plasma membrane and exposed to the cytoplasm . HoweverGefitinib can specifically block a certain pathway of lung cancer cells, block the conduction, infiltration, and metastasis of lung cancer cells, locate and kill cancer cells, and cause little damage to normal human cells , 26. It P < 0.05). The relative expression of circHIPK3 in patients with TNM stage II/III was dramatically inferior to that in patients with TNM stage I (P < 0.05). In addition, the relative expression of circHIPK3 in patients with lymph node metastasis was dramatically inferior to that in patients without lymph node metastasis (P < 0.05). The results indicated that the relative expression of circHIPK3 in the tumor tissues of patients with lung cancer was closely related to the diameter of the patient's tumor, the TNM stage, and the occurrence of lymph node metastasis. However, there was no high correlation with the patient's age, gender, and other information. Among lung cancer tissues of patients with different TNM stages, only 6 patients had high expression, and the remaining 104 patients had low expression. Therefore, circHIPK3 may have a notably low expression in lung cancer tissues and related cell lines, which had a high correlation with the tumor stage of patients. Flow cytometry apoptosis detection results found that gefitinib-mediated apoptosis rate of lung cancer drug-resistant cell lines decreased notably, which was similar to Zhou et al.'s (2020) [In this study, the tumor tissues and para-cancerous tissues from surgical specimens of 110 study subjects were collected to detect differences in the expression of circHIPK3 in different tissues. Moreover, a lung cancer gefitinib drug-resistant cell line was constructed, and cell apoptosis was detected under different conditions, to study the mechanism of circRNA involved in the resistance of lung cancer cells to gefitinib. As a result, the relative expression of circHIPK3 in patients with tumor diameter no less than 3 cm was dramatically inferior to that in patients with tumor diameter less than 3 cm (s (2020)  researchIn this study, tumor tissues and adjacent tissues were collected from surgical specimens of lung cancer patients to detect differences in the expression of circHIPK3 in different patients and different tumor tissues. In addition, a lung cancer gefitinib drug-resistant cell line was constructed to detect cell apoptosis under different conditions. The results showed that the circular RNA circHIPK3 may have a considerably low expression in lung cancer tissues, and its low expression had a certain enhancement effect on the drug resistance of lung adenocarcinoma cells to gefitinib. However, the sample size selected in this study is small, and the representativeness is low. Therefore, the selection of test sample size will be increased in subsequent experiments. Further study is required to clarify the mechanism of circRNA involved in the resistance of lung cancer cells to gefitinib. In short, this study provides a certain theoretical basis and data support for the drug treatment of lung cancer.


In chromatin, linker histone H1 binds to nucleosomes, forming chromatosomes, and changes the transcription status. However, the mechanism by which RNA polymerase II (RNAPII) transcribes the DNA in the chromatosome has remained enigmatic. Here we report the cryo-electron microscopy (cryo-EM) structures of transcribing RNAPII-chromatosome complexes (forms I and II), in which RNAPII is paused at the entry linker DNA region of the chromatosome due to H1 binding. In the form I complex, the H1 bound to the nucleosome restricts the linker DNA orientation, and the exit linker DNA is captured by the RNAPII DNA binding cleft. In the form II complex, the RNAPII progresses a few bases ahead by releasing the exit linker DNA from the RNAPII cleft, and directly clashes with the H1 bound to the nucleosome. The transcription elongation factor Spt4/5 masks the RNAPII DNA binding region, and drastically reduces the H1-mediated RNAPII pausing. The mechanism by which RNAPII transcribes the DNA in the chromatosome with H1 has remained enigmatic. Here the authors present the cryo-EM structures of the RNAPII-chromatosome complexes, and explain how RNAPII is regulated by H1 in chromatin. NCPs are connected by linker DNA segments, and appear as a beads-on-a-string fiber2.Chromatin compacts genomic DNA in eukaryotes, and the nucleosome is an elemental architecture. The nucleosome is composed of linker DNAs and the nucleosome core particle (NCP), which folds an approximately 150 base-pair DNA segment into a histone octamer, comprising the four core histones H2A, H2B, H3, and H4. In the NCP, the DNA is symmetrically wrapped 1.7-turns around the histone octamer, and the central region of the nucleosomal DNA is located on the dyad axis3, and promote further compaction of chromatin7. H1 consists of three domains, the N-terminal disordered region (∼35 residues), the central globular domain (∼80 residues), and the C-terminal disordered region (∼100 residues), and in the chromatosome, these domains mainly bind to a linker DNA, the DNA around the dyad axis, and another linker DNA, respectively11. This tripartite nucleosome binding by H1 is termed “on-dyad” binding, and it compacts the nucleosome architecture by restricting the linker DNA orientation and flexibility14. H1 also binds the nucleosome in an “off-dyad” mode, in which the central globular domain mainly binds to a linker DNA16.Linker histones, such as H1, are major chromatin components that specifically bind to the nucleosomal DNA forming the chromatosome17. H1 binding to the nucleosome induces structural changes in chromatin, thus regulating transcription processes catalyzed by RNA polymerase II (RNAPII)5. For instance, a biochemical study with Drosophila egg extracts indicated that H1 binding to reconstituted chromatin represses RNAPII transcription18. A mouse knock-out study revealed that embryos with an H1 ratio reduced to approximately 50% cease development at mid-gestation19. H1 reportedly functions in gene regulation through chromatin compaction and 3D genome organization20. Consistently, acute depletion of H1 leads to perturbations of gene expression in the constitutive heterochromatin in mouse embryonic stem cells21. H1 alleles have been identified as genes with driver mutations in lymphoma, revealing that H1 is a bona fide tumor suppressor22. These facts indicate that H1 plays pivotal roles in development, differentiation, and tumorigenesis by appropriately reducing transcription efficiency according to the cellular status.H1 is the most abundant nucleosome binding protein, and exists in a 50–130% amount relative to nucleosomes5. H1.2 is the major somatic linker histone. Genome-wide studies revealed that the genomic H1.2 binding loci negatively correlate with the RNAPII transcription activation, suggesting the repressor function of H1.225. In contrast, genetic and biochemical studies demonstrated that H1.2 potentially upregulates gene expression30. Therefore, H1.2 may function to both repress and activate RNAPII transcription5, probably depending on cell types and genomic loci. These different outcomes may be the results of complex transcription regulation at the levels of transcription initiation and elongation, but the direct effects of H1 on transcription are not well understood.Eleven non-allelic human H1 isoforms have been described, and they may have specific functions in altering the cellular transcription status, primarily by repressing transcription by RNAPII32. H1 causes RNAPII pausing at the 3′ splice site, and regulates alternative splicing32. These observations suggested the specific involvement of linker histones in the regulation of transcription elongation, but the molecular details have remained elusive. In the present study, we investigate the basic properties of the chromatosome in transcription elongation by performing in vitro transcription experiments and cryo-EM analyses, which reveal how H1 accomplishes nucleosome repression in transcription elongation.Linker histones are associated with the gene-body regions of actively transcribed genesKomagataella pastoris was loaded onto the bubble, and the transcription reaction was conducted in the presence of the transcription elongation factor TFIIS , lacking half of the C-terminal region (amino acid residues 151-212)  Fig. . Interes12) Fig. . The RNA12) Fig. , probablSus scrofa domesticus36  analysis  of the RNAPII transcribing the entry linker DNA , the exit linker DNA of the chromatosome sterically conflicts with the transcribing RNAPII. In the natural chromatin context, this exit linker DNA is followed by another nucleosome (or chromatosome). This led us to test whether a second, neighboring nucleosome/chromatosome affects the RNAPII pausing. To do so, we performed the chromatosome transcription assay with the di-nucleosome template in the presence of H1.2 Fig. . In thisner Fig. . The strner Fig. . Therefo40. It binds and seals the RNAPII DNA-binding cleft41, and enhances the transcription elongation efficiency in the nucleosome43. Interestingly, when the Spt4/5-bound RNAPII structure was superimposed on the current RNAPII-chromatosome complex, Spt4/5 overlapped with the exit linker DNA in the form I complex  is one of the transcription elongation factors that is tightly associated with transcribing RNAPIIlex Fig. . This filex Fig. . This sulex Fig. , lane 4.This study has revealed that a chromatosome poses a higher transcriptional barrier than an H1-free nucleosome in vitro. H1 causes RNAPII pausing before it enters the chromatosome. This is because H1 brings the entry and exit linker DNAs close together, and the RNAPII on the entry linker DNA sterically conflicts with the exit linker DNA, hindering its progression. As shown in the form I complex, the exit linker DNA directly interacts with the RNAPII DNA-binding cleft, thus inducing the RNAPII pausing Fig. . The for14. Therefore, the bound H1 may persist on the nucleosome while the RNAPII invades the nucleosomal DNA region  strain  and the JM109(DE3) strain (for H4) as N-terminally hexa-histidine (His6)-tagged proteins, and isolated under denaturing conditions. The His6-tagged proteins were purified by chromatography with nickel-nitrilotriacetic acid agarose (Ni-NTA) resin (QIAGEN). The His6-tag portion was then removed by thrombin protease, and the histones were purified by cation exchange chromatography on a MonoS column under denaturing conditions. The purified histones were dialyzed against water and lyophilized.Human histones H2A, H2B, H3.1, and H4 were expressed in E. coli cells and purified by the method described previously49. Briefly, H1.2 and H1.2 deltaC were produced in the BL21-CodonPlus(DE3)-RIL strain, as C-terminally His6-SUMO-tagged proteins. The His6-SUMO-tagged proteins were purified by Ni-NTA agarose (QIAGEN) chromatography. The His6-SUMO tag portion was then removed by PreScission protease treatment, and H1.2 and H1.2 deltaC were purified by MonoS cation exchange column chromatography. Purified H1.2 and H1.2 deltaC were stored at −80 °C. K. pastoris RNAPII, composed of 12 subunits, was purified as described previously50. Briefly, K. pastoris encoding a TAP-tagged Rpb2 was cultured, and the tagged-RNAPII was purified by affinity column chromatography on anti-FLAG M2 affinity gel (Sigma-Aldrich), and anion-exchange column chromatography. K. pastoris TFIIS and Spt4/5 were purified as described previously40. These proteins were produced as His6-tagged proteins in the E. coli KRX strain. The His6-tagged proteins were then purified by chromatography on a COSMOGEL His-Accept column , and eluted by cleavage of the His6-tag portion by HRV-3C protease treatment. The eluted proteins were purified by Resource S cation-exchange column chromatography.Human H1.2 and H1.2 deltaC (H1.2(1-150)) were expressed in S. scrofa RNAPII preparation, fresh S. scrofa thymus was homogenized using a 2L blender (Waring) in buffer A (50 mM Tris-HCl (pH 7.9), 1 mM EDTA, 10 μM ZnCl2, 10% glycerol, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.6 µM leupeptin, 2 µM pepstatin A). The homogenized material was centrifuged at 12,200 × g for 20 min at 4 °C, and the supernatant was filtered through Miracloth (Millipore). A 5% solution of polyethyleneimine (adjusted to pH 7.9 at 25 °C) was added to a final concentration of 0.1%. After mixing for 10 min at 4 °C, the precipitate was collected by centrifugation at 15,750 xg for 20 min. The pellet was washed twice with buffer A, and the RNAP II fraction was extracted from the pellet with buffer A containing 0.15–0.2 M ammonium sulfate. A Q Sepharose (Cytiva) slurry was added to the eluted fraction, and the ammonium sulfate concentration was adjusted to 0.15 M. After rotation at 4 °C for 30 min, the resin was washed with 6 volumes of buffer A containing 0.15 M ammonium sulfate, followed by 3 volumes of buffer A containing 0.2 M ammonium sulfate. RNAPII was eluted with buffer A containing 0.4 M ammonium sulfate. The eluate was precipitated by 50% ammonium sulfate overnight. The precipitate was collected by centrifugation at 22,900 × g for 20 min at 4 °C, resuspended in buffer A, and then dialyzed against 1 L of buffer B (50 mM Tris-HCl (pH 7.9), 150 mM NaCl, 1 mM EDTA, 10 µM ZnCl2, 2 mM DTT). The dialyzed sample was subjected to anion-exchange chromatography on a Mono Q (Cytiva) column. The RNAPII-containing fraction was concentrated with an Amicon Ultra-15 centrifugal filter unit (Merck) and purified by size exclusion chromatography on a Superose 6 column (Cytiva), equilibrated with buffer C (5 mM HEPES-NaOH (pH 7.3), 150 mM NaCl, 10 µM ZnCl2, 10 mM DTT). Peak fractions containing RNAPII were collected and stored as a 10% glycerol stock at −80 °C. Judging from the electrophoretic band intensities, the purity of RNAPII in this fraction is about 30%  buffer, containing 150 mM NaCl, 1 mM DTT, 5% glycerol, and 10 μM zinc chloride, flash-frozen in liquid nitrogen, and stored at −80 °C.For the E. coli DH5 strain. The 193 bp Widom 601 fragment was excised from the plasmid DNA by EcoRV (Takara), and purified by polyethylene glycol precipitation. The ends of the DNA fragments were dephosphorylated by calf intestinal alkaline phosphatase (Takara). The 193 bp Widom 601 DNA fragment was also produced by polymerase chain reaction. The purified 193 bp Widom 601 DNA fragment was cleaved with HinfI, resulting in the 161 base-pair DNA with a 3 bp overhang at the 5′ end (Takara). This DNA fragment was further purified by DEAE-5PW anion-exchange column chromatography (TOSOH) or native polyacrylamide gel electrophoresis purification using a Prep Cell model 491 apparatus (Bio-Rad).For the preparation of the DNA fragment used in the mono-chromatosome transcription, the plasmid DNA containing the 193 bp Widom 601 sequence was prepared from the The sequences of both strands of the resulting DNA fragment are as follows:non-template strand: 5′:AATCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACATCCAGGCCTTGTGTCGCGAAATTCATAGATtemplate strand: 5′:ATCTATGAATTTCGCGACACAAGGCCTGGATGTATATATCTGACACGTGCCTGGAGACTAGGGAGTAATCCCCTTGGCGGTTAAAACGCGGGGGACAGCGCGTACGTGCGTTTAAGCGGTGCTAGAGCTGTCTACGACCAATTGAGCGGCCTCGGCACCGG.The purified DNA fragment was ligated with the double-stranded oligonucleotide 50-mer (purchased from Eurofins) by T4 DNA ligase (NIPPON GENE). The sequences of both strands of the double-stranded oligonucleotide 50-mer are as follows:non-template strand: 5′:CCTTTAAAGCAATAGGAGCTTACGGTCCACTTGTGTTTGGTGTGTTTGGGtemplate strand: 5′:ATTCCCAAACACACCAAACACAAGTGGACCGTAAGCTCCTATTGCTTTAA.For the preparation of the DNA fragment used in the di-chromatosome transcription, the 368 bp DNA fragment containing the Widom 601 and 603 sequences connected with 48 bp linker DNA was produced by polymerase chain reaction. The purified DNA fragment was cleaved with HinfI, resulting in the 354 bp DNA with a 3 bp overhang at the 5′ end (Takara). This DNA fragment was further purified by native polyacrylamide gel electrophoresis, using a Prep Cell model 491 apparatus (Bio-Rad).The sequences of both strands of the resulting DNA fragment are as follows:non-template strand: 5′:AATCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACATCCAGGTCATTCCGGACGTGTTTGTCCTCTGCCTTTAAAGCAATAGGAGCTTACCCCCAGGGACTTGAAGTAATAAGGACGGAGGGCCTCTTTCAACATCGATGCACGGTGGTTAGCCTTGGATTGCGCTCTACCGTGCGCTAAGCGTACTTAGAAGCCCGAGTGACGACTTCACACGGTAGGTGGGCGCGCGAACTGAGCCTTGTGTCGCGAAATTCATGATtemplate strand: 5′:ATCATGAATTTCGCGACACAAGGCTCAGTTCGCGCGCCCACCTACCGTGTGAAGTCGTCACTCGGGCTTCTAAGTACGCTTAGCGCACGGTAGAGCGCAATCCAAGGCTAACCACCGTGCATCGATGTTGAAAGAGGCCCTCCGTCCTTATTACTTCAAGTCCCTGGGGGTAAGCTCCTATTGCTTTAAAGGCAGAGGACAAACACGTCCGGAATGACCTGGATGTATATATCTGACACGTGCCTGGAGACTAGGGAGTAATCCCCTTGGCGGTTAAAACGCGGGGGACAGCGCGTACGTGCGTTTAAGCGGTGCTAGAGCTGTCTACGACCAATTGAGCGGCCTCGGCACCGG.The purified DNA fragment was ligated with the same double-stranded oligonucleotide 50-mer (purchased from Eurofins) as described above by T4 DNA ligase (NIPPON GENE).48. Briefly, the freeze-dried histones were mixed at an equal molar ratio under denaturing conditions. The histone octamer was refolded by dialysis against refolding buffer, containing 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 M NaCl, and 5 mM 2-mercaptoethanol. The histone octamer was purified by chromatography on a Superdex 200 gel filtration column . The resulting octamer was flash-frozen in liquid nitrogen, and stored at −80 °C. The histone octamer and the 161 or 354 bp DNA fragment were mixed, and the nucleosome was reconstituted by the salt dialysis method. The double-stranded oligonucleotide 47-mer containing a 3 bp overhang at the 5′ end was ligated to the cohesive DNA end of the reconstituted nucleosome. The oligonucleotide also contained a 9 base mismatched region (bubble) at the region 14–22 bps from the 3′ end. The resulting nucleosome with a bubble region was purified by non-denaturing gel electrophoresis, using a Prep Cell apparatus (Bio-Rad).The histone octamer was reconstituted as described previouslyThe sequences of both strands of the oligonucleotide 47-mer containing a bubble region are as follows:non-template strand: 5′TTCTTAAATACCATGGCCATCTTCATTCCGGACGTGTTTGTCCTCTGtemplate strand: 5′:AGGCAGAGGACAAACACGTCCGGAATGAGAGCTAATTTGGTATTTAAGAA.The complete DNA sequences of both strands for the mono-chromatosome transcription experiments are as follows:non-template strand: 5′TTCTTAAATACCATGGCCATCTTCATTCCGGACGTGTTTGTCCTCTGCCTTTAAAGCAATAGGAGCTTACGGTCCACTTGTGTTTGGTGTGTTTGGGAATCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACATCCAGGCCTTGTGTCGCGAAATTCATAGATtemplate strand: 5′ATCTATGAATTTCGCGACACAAGGCCTGGATGTATATATCTGACACGTGCCTGGAGACTAGGGAGTAATCCCCTTGGCGGTTAAAACGCGGGGGACAGCGCGTACGTGCGTTTAAGCGGTGCTAGAGCTGTCTACGACCAATTGAGCGGCCTCGGCACCGGATTCCCAAACACACCAAACACAAGTGGACCGTAAGCTCCTATTGCTTTAAAGGCAGAGGACAAACACGTCCGGAATGAGAGCTAATTTGGTATTTAAGAA.The complete DNA sequences of both strands for the di-chromatosome transcription experiments are as follows:non-template strand: 5′TTCTTAAATACCATGGCCATCTTCATTCCGGACGTGTTTGTCCTCTGCCTTTAAAGCAATAGGAGCTTACGGTCCACTTGTGTTTGGTGTGTTTGGGAATCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACATCCAGGTCATTCCGGACGTGTTTGTCCTCTGCCTTTAAAGCAATAGGAGCTTACCCCCAGGGACTTGAAGTAATAAGGACGGAGGGCCTCTTTCAACATCGATGCACGGTGGTTAGCCTTGGATTGCGCTCTACCGTGCGCTAAGCGTACTTAGAAGCCCGAGTGACGACTTCACACGGTAGGTGGGCGCGCGAACTGAGCCTTGTGTCGCGAAATTCATGATtemplate strand: 5′ATCATGAATTTCGCGACACAAGGCTCAGTTCGCGCGCCCACCTACCGTGTGAAGTCGTCACTCGGGCTTCTAAGTACGCTTAGCGCACGGTAGAGCGCAATCCAAGGCTAACCACCGTGCATCGATGTTGAAAGAGGCCCTCCGTCCTTATTACTTCAAGTCCCTGGGGGTAAGCTCCTATTGCTTTAAAGGCAGAGGACAAACACGTCCGGAATGACCTGGATGTATATATCTGACACGTGCCTGGAGACTAGGGAGTAATCCCCTTGGCGGTTAAAACGCGGGGGACAGCGCGTACGTGCGTTTAAGCGGTGCTAGAGCTGTCTACGACCAATTGAGCGGCCTCGGCACCGGATTCCCAAACACACCAAACACAAGTGGACCGTAAGCTCCTATTGCTTTAAAGGCAGAGGACAAACACGTCCGGAATGAGAGCTAATTTGGTATTTAAGAAK. pastoris RNAPII or approximately the corresponding amount of the partially purified S. scrofa RNAPII), TFIIS (0.14 μM of K. pastoris TFIIS or 0.11 μM of H. sapiens TFIIS), and the DY647-labeled primer RNA (5′-DY647- AUAAUUAGCUC-3′) (0.57 μM) (Dharmacon), in the presence or absence of Spt4/5 (0.57 μM), in a 7 μL reaction at 30 °C for 30 min. For the experiment with K. pastoris RNAPII, the reaction solution contained 37 mM HEPES-KOH (pH 7.5), 7.1 mM MgCl2, 43 mM potassium acetate, 0.29 μM zinc acetate , 0.37 mM HEPES-NaOH (pH 7.5), 7.1 mM MgCl2, 37 mM potassium acetate, 0.29 μM zinc acetate, 29 μM Tris(2-carboxyethyl)phosphine, 0.46 mM DTT, 2.0% glycerol, 6.14 mM NaCl, 0.29 μM zinc chloride, 0.57 mM UTP, 0.57 mM GTP, and 0.57 mM ATP. The reactions were then incubated for 15 min at 4 °C for transcription with K. pastoris RNAPII or 30 °C for transcription with S. scrofa RNAPII. H1.2 was added to the reaction mixture in nucleosome:H1.2 ratios of 1:0, 1:3, 1:4.5, 1:6, and 1:9, and was bound to the nucleosome for 30 min by an incubation at 4 °C for transcription with K. pastoris RNAPII or at 30 °C for transcription with S. scrofa RNAPII. H1.2 was dissolved in 10 mM Tris-HCl buffer (pH 7.5), 0.05 mM phenylmethylsulfonyl fluoride (PMSF), 0.25 mM EDTA, 75 mM NaCl, 10% glycerol, 10 mM HEPES-KOH (pH 7.5), 0.5 mM dithiothreitol, 50 mM KCl, and 1 mM 2-mercaptoethanol. CTP  was then added to re-start the RNAPII progression. After the reaction  was incubated with RNAPII phosphine, 0.14 mM DTT, 0.7% glycerol, 0.57 mM UTP, 0.57 mM GTP, and 0.57 mM ATP, at 30 °C for 30 min, followed by an incubation at 4 °C for 15 min. Afterwards, H1.2 was added to the reaction mixture at a nucleosome:H1.2 ratio of 1:6, and was bound to the nucleosome by an incubation at 4 °C for 30 min. CTP  was then added to re-start the RNAPII progression. After a 5 min reaction, 216 μL of 0.5 M EDTA was added to the reaction mixture. Under these conditions, a ~60-nt RNA product predominantly accumulated in the presence of H1.2. The resulting RNAPII-chromatosome complexes were fractionated by the GraFix method51. A gradient was prepared with low buffer (20 mM HEPES-KOH (pH 7.5), 50 mM potassium acetate, 0.2 μM zinc acetate, 0.1 mM Tris(2-carboxyethyl)phosphine, and 10% (w/v) sucrose) and high buffer (20 mM HEPES-KOH (pH 7.5), 50 mM potassium acetate, 0.2 μM zinc acetate, 0.1 mM Tris(2-carboxyethyl)phosphine, 25% (w/v) sucrose, and 0.1% glutaraldehyde). The sample was applied to the top of the gradient and centrifuged at 4 °C for 16 h at 124,779 × g, using an SW41 rotor (Beckman Coulter). After centrifugation, the fractions containing the RNAPII-chromatosome complexes were collected and dialyzed twice against 20 mM HEPES-NaOH (pH 7.5) buffer, containing 0.2 μM zinc acetate and 0.1 mM Tris(2-carboxyethyl)phosphine. The resulting RNAPII-chromatosome complexes were then concentrated with an Amicon Ultra 100 K centrifugal filter unit (Millipore), until the DNA concentration of the sample reached 78.4 μg/mL. The samples were applied to glow-discharged Quantifoil grids . Each grid was blotted at 4 °C and 100% humidity in a Vitrobot Mark IV (Thermo Fisher Scientific) , and then immediately plunged into liquid ethane.The 6-FAM-labeled nucleosome (0.14 μM) was mixed with RNAPII (0.14 μM), TFIIS (0.14 μM), and the DY647-labeled primer RNA (5′-DY647- AUAAUUAGCUC-3′) (0.57 μM) (Dharmacon) in 1.5 mL of reaction solution, containing 37 mM HEPES-KOH (pH 7.5), 7.1 mM MgCl−/Å, using the SerialEM software52.Cryo-EM images of the RNAPII-chromatosome complexes were collected by a Krios G3i electron microscope (Thermo Fisher Scientific), equipped with a K3 direct electron detector with a 25 eV slit width of the BioQuantum energy filter (Gatan), and operated at 300 kV at a pixel size of 1.07 Å. Each image of the RNAPII-chromatosome complex was recorded with a 7 s exposure time, and then fractionated into 40 frames with a total dose of 56.2 e53 and Gctf54, respectively. Preliminary RNAPII-chromatosome maps were then obtained after particle picking with gautomatch (http://www.mrc-lmb.cam.ac.uk/kzhang/) and quick 2D and 3D classifications with Relion3. Using the particle coordinates from these preliminary processing results, the neural networks for Topaz55 were trained, and then another round of particle picking was performed with Topaz. Finally, the coordinates from the Topaz picking and the initial gautomatch picking were merged, and served as the starting set for further image processing with Relion3.First, the motion correction and CTF estimation were performed with Relion3During the initial stage of image processing, the dataset was split in two batches that were processed independently. As the dataset was highly heterogeneous, refinements were mostly focused on the RNAPII region of the molecule, and to remove bad particles and particles centered on the nucleosome, 2D and 3D classifications were performed occasionally. After the initial 3D refinement using the RNAPII mask, the Bayesian polishing and CTF refinement were performed. After another 3D refinement, 3D classification was performed to remove RNAPII particles lacking downstream DNA. A 2.8 Å resolution RNAPII map was obtained at this stage, and then another Bayesian polishing was performed to downscale particles to 1.49 Å/pix. Finally, 3D classifications with the mask enveloping RNAPII and nucleosome were done to select particles containing both RNAPII and nucleosome.56.Next, the particles from the two batches were merged and subjected to 3D classification, which revealed two different complexes of RNAPII transcribing a chromatosome (forms I and II). For each complex, RNAPII subtraction and 3D classification around the nucleosome were performed to select classes with high quality chromatosome density. A significant amount of continuous motion remained between the RNAPII and chromatosome, so local refinements for the RNAPII region and the chromatosome region were performed, in addition to the overall reconstruction. Composite maps were also calculated for each complex structure, using the phenix.combine_focused_maps tool in the Phenix package40, modeled manually with WinCoot (COOT57 for windows), and then refined against the consensus RNAPII reconstruction using phenix.real_space_refine tool in the Phenix package56. The atomic model for the chromatosome was based on previous crystal structures (PDB: 3LZ0 and 4QLC)58, modeled manually, and then refined against the nucleosome reconstruction of the form I complex. The RNAPII and nucleosome models were then fit into their respective overall reconstructions, and the interfaces between the two bodies and the connecting DNA were manually edited with WinCoot and ISOLDE59. Figures were prepared using UCSF Chimera60, ChimeraX61, and PyMOL.First, the RNAPII and the chromatosome were independently modeled. The atomic model for RNAPII was based on the previous crystal structure (PDB: 5XOG)Further information on research design is available in the Supplementary informationPeer Review FileReporting Summary