Methanosarcina mazei and Sulfolobus acidocaldarius. In this study, only regions directly downstream of annotated genes were analysed, and thus, only part of the genome had been investigated. Here, we developed a novel algorithm  that allows an unbiased, genome-wide identification of RNA 3´ termini independent of annotation. In an RNA fraction enriched for primary transcripts by terminator exonuclease (TEX) treatment we identified 1,543 RNA 3´ termini. Approximately half of these were located in intergenic regions, and the remainder were found in coding regions. A strong sequence signature consistent with known termination events at intergenic loci indicates a clear enrichment for native TTS among them. Using these data we determined distinct putative termination motifs for intergenic (a T stretch) and coding regions (AGATC). In vivo reporter gene tests of selected TTS confirmed termination at these sites, which exemplify the different motifs. For several genes, more than one termination site was detected, resulting in transcripts with different lengths of the 3´ untranslated region (3´ UTR).Archaeal genomes are densely packed; thus, correct transcription termination is an important factor for orchestrated gene expression. A systematic analysis of RNA 3´ termini, to identify transcription termination sites (TTS) using RNAseq data has hitherto only been performed in two archaea,  Archaeal RNA synthesis is generally considered to be more closely related to transcription in eukaryotes than to bacterial transcription. The archaeal RNA polymerase is similar to the eukaryotic RNA polymerase II, and the basal promoter elements in Archaea are similar to their eukaryotic pendants (TATA box and BRE); general transcription factors TBP (TATA binding protein), TFB (transcription factor B) and TFE (transcription factor E) resemble the eukaryotic proteins TBP, TFIIB and TFEII, respectively . Transcriptional regulators, however, seem to be more similar to those in bacteria . Thus, tin vivo reporter assay in the archaeon Thermococcus kodakarensis [Compared to the determination of transcription start sites, the identification of termination sites is more complex. Termination is often leaky and encompasses several consecutive sites, and degradation by exonucleases renders the 3´ ends heterogeneous and less clear. Also, original transcription termination sites are difficult to distinguish from RNA 3´ termini that are due to RNA processing. Data reported on archaeal termination so far show that intrinsic termination occurs at a run of Ts –14 and ikarensis . A recenkarensis .Sulfolobus acidocaldarius and Methanosarcina mazei) were investigated systematically using RNAseq data [S. acidocaldarius and M. mazei, respectively. For more than a third of the genes analysed, multiple consecutive terminators were identified, resulting in 3´ untranslated regions (UTRs) with different lengths [Recently, RNA 3´ ends for two archaea  among the newly identified RNA 3ʹ ends. Therefore, identified RNA 3ʹ ends were considered putative TTS for successive analysis with respect to primary and/or secondary structure motifs as termination signals and 3´ UTR characterization. Selected termination motifs were confirmed using an in vivo reporter gene system.We used a newly developed algorithm to identify RNA 3´ ends from RNAseq data genome-wide in an unbiased manner, independent of annotation and on the basis of reads enriched for primary transcripts. Applying this algorithm to RNAseq data from a terminator exonuclease (TEX) treated library, we found 1,543 RNA 3´ ends for the H. volcanii we applied a self-implemented version of the recently published method from Dar et al. [H. volcanii cells  and cDNA libraries were generated to allow determination of RNA 3´ ends. Libraries were made such that the original RNA 3ʹ end is tagged and can be identified in the resulting sequence. Libraries were subjected to paired-end next-generation sequencing (NGS), resulting in 49 million reads for each library on average. 89–98% of the reads obtained mapped to the Haloferax genome (Supplementary Table 6). Since total RNA was used for library preparation the identified RNA 3´ ends result from either transcription termination or processing. Mapped reads were analysed with DSM, which identifies RNA 3´ ends in a defined region downstream of annotated genes at the position with the highest coverage of mapped read ends. The length of the downstream region is determined by the average insert length of corresponding paired-end reads . A typical RNA 3´ end is shown in Supplementary Figure 2 for the pilA2 gene (HVO_2062).In our dataset, the median length of the analysed region was 126 bp. The resulting 3´ UTRs were mostly shorter than 100 nucleotides, with a median length of 58 nucleotides . The length restriction given in the DSM approach might be too strict for genes with long 3´ UTRs. In addition, the method cannot determine RNA 3´ ends independent of an annotation . Using DSM, we identified 3,155 RNA 3´ ends for the complete DSM analysis only includes sequences downstream of annotated genes and, thus, only a fraction of the genome . To overTo gain further specificity, putative 3´ ends were subsequently censored if the site in question was associated with a sudden decrease in read coverage, indicative of a true RNA 3´ end. This was assessed by a peak calling approach (termed PC), considering the absolute and relative number of fragments ending at the respective position . The twoSince our newly established method IE-PC works independent of any genome annotation, it is able to determine RNA 3´ ends covering the complete genome. Comparison of signals obtained with both methods (DSM and IE-PC) can only be done with the regions that are included in the DSM analysis. The original DSM analysis was based on regions with a median length of 126 nucleotides downstream of an annotated 3´ end, with the RNA 3´ end located at the position with the highest coverage.We compared individual RNA 3´ ends found with DSM and/or IE-PC in downstream regions defined by the DSM analysis in more detail. Examples are shown in Comparing the DSM data with IE-PC results as well as with given coverage data, we see that chosen downstream regions in DSM were frequently too short.This is also clear when comparing the median 3´ UTR length determined by both methods. While DSM found a median 3´ UTR lengthtrmY and tRNAPro, both methods identified one identical RNA 3´ end position and two different RNA 3´ ends positions (Pro gene (upper panel). The corresponding coverage values for DSM (lower panel) showed similar signals, but due to its specific algorithm, DSM assigned one RNA 3´ end downstream of the trmY gene (HVO_1989) and one downstream of the tRNAPro gene (lower panel).The lower panel (DSM) in ositions . The IE-Taken together comparison between the DSM approach and the IE-PC algorithm clearly shows that using the IE-PC approach yields improved and more comprehensive data.To determine RNA 3´ ends the DSM approach used reads from a total cellular RNA fraction that contains RNA 3´ ends derived from transcription termination as well as 3´ ends derived from processing. Thus the 3´ ends identified by DSM are not all TTS but also processing sites (PS). A similar problem exists for the determination of original transcription 5´ ends, where a well established method for the reliable identification of transcription start sites (TSS) has been developed, termed differential RNAseq (dRNAseq). Here, to enrich primary transcripts with original 5´ ends an RNA sample is treated with terminator exonuclease (+TEX) which removes RNAs with a 5´-monophosphate. Primary transcripts are newly synthesized RNA molecules that have not been processed at their 5´ end and also have a higher probability to contain the original 3´ terminus. The majority of bacterial ribonucleases prefer substrates with 5´-monophosphate ends, some even have a specific sensor domain for the 5´-monophosphate , thus prTherefore -similar to the approach for start site determination- we used a + TEX library to enrich original termination ends. To that end we treated a cellular RNA fraction with 5´ terminator exonuclease (TEX) to enrich primary transcripts and thereby original termination ends. After cDNA library generation from the TEX treated RNA, NGS was performed, resulting in an average of 40 million reads for each of the three libraries (Supplementary Table 6). Reads obtained were analysed with our newly established algorithm as described below.To identify transcription termination sites we applied the IE-PC algorithm to the data from the TEX treated samples consisting of enriched original transcription termination sites, and identified 1,543 putative TTS1) and secondary TTS (TTSs): a first TTS is located directly downstream of a 3´ gene end on the same strand  and the remainder in coding regions . DetaileAnalysis of the regions up- and downstream of the TTS were performed separately for TTS located in coding and intergenic regions , and in We next analysed sequences 15 nucleotides up- and five nucleotides downstream of the 807 intergenic termination sites for common sequence motifs. For 748 sites, we found similarities in the sequences, such as a conserved dinucleotide TC as part of the motif as well as a stretch of T’s of variable length upstream of the termination site  and pyrimidines. For 456 TTS in coding regions, we found the downstream motif AGATC  were cloned into a fragment of the reporter gene β-galactosidase (sequences are listed in Supplementary Table 5). Termination activity was monitored with northern blot analyses  [lsm gene can be terminated within the downstream rpl37e gene. A dicistronic transcript and a rpl37e mRNA were identified by northern blot analysis [Sulfolobus islandicus [H. volcanii (Supplementary Table 9) and compared the transcript reads of the first and the last gene in each operon . The majority of genes in an operon has the same amount of transcripts for the first and the last gene however with some clear exceptions that are listed in Supplementary Table 10 and 11. Operons with internal TTS show a slightly stronger tendency to have such outliers.Earlier analyses of TSS data from e operon . Similarigure 5) . In addianalysis . A simillandicus . To analH. volcanii. The dRNAseq method using a +TEX library is an established method to confidently identify transcription start sites by enriching primary transcripts. However, treatement with TEX does not completely remove 5´- monophosphate RNAs, thus only an enrichment of primary transcripts is achieved and the +TEX data set contains to some extent still 5´ processed RNA. Nevertheless, the method is standard and accepted for TSS identification.Applying the new IE-PC algorithm on +TEX RNA sequences, we were able to determine the TTS genome-wide for the archaeon Currently very little is known about archaeal RNases and their preferred substrates, but bacterial ribonucleases clearly prefer substrates with a 5´- monophosphate. Thus an RNA with an 5´-triphosphate is not the preferred substrate for these RNases. Therefore, we used here a +TEX library to enrich transcription termination sites. Altogether, we found 1,543 TTS in the +TEX data set with our new algorithm, representing the first unbiased, truly genome-wide approach.Comparison of both methods for TTS identification, IE-PC and DSM, showed that IE-PC could identify TTS genome-wide, whereas DSM was restricted to regions downstream of annotated genes. Furthermore, algorithmic details of DSM  caused false TTS identifications in some cases.in vivo assays. Motifs in intergenic regions consisted of a stretch of Ts, while termination occurred at a C residue. This motif is similar to those found by in vitro studies in other archaea [S. acidocaldarius and M. mazei using the DSM algorithm [Amongst all TTS, two general sequence motifs for termination were identified. One was predominant in intergenic regions, and the other one in coding regions. Both types of termination motifs (T stretch and AGATC) were confirmed using  archaea ,12–14,32lgorithm .in vivo termination test confirmed that AGATC is used as termination motif but revealed that it is not very efficient (11–27%). This lower termination efficiency is important for TTS in coding regions since it allows full length transcription of the gene in which the termination site is located. A termination event as strong as T5C would be detrimental for the expression of the terminator containing gene. In addition, it would be very difficult to place such a T stretch in a coding region since the coding region is governed by the requirement to contain the specific triplets for the encoded protein, and especially a G/C rich genome like the one from Haloferax can rarely contain a T stretch in a coding region. Thus, to be able to terminate in coding regions other signals have to be used and these motifs should allow for sufficient read-through.The presence of TTS in coding regions, terminating transcription of an upstream located gene, has been observed previously in archaea including the RNASeq analysis by Dar et al. ,33. Our The prevailing motif we have found in coding regions was AGATC, located downstream of the termination site. The DNA sequence downstream of the TTS has been shown to influence termination efficiency in bacteria and eukaryotes . Furtherin vivo termination experiments and indeed terminated transcription, however not as efficiently as the T stretch. A certain amount of folding potential upstream of TTS was also found in M. mazei but was considered not to be essential, whereas no structures were identified near TTS in S. acidocaldarius [The search for secondary structure motifs located close to the TTS was successful for a subset of the TTS and revealed hairpin structures upstream of the TTS, suggesting that these structures might influence transcription termination. One of the identified structures was tested in the aldarius . PossiblH. volcanii. The median 3´ UTR length for first TTS was 97 nucleotides, which is longer than the median 3´ UTR length recently reported for two archaea  and the bacterium B. subtilis (40 nucleotides) [With our genome-wide TTS determination approach, we can present the first comprehensive determination of 3´ UTR lengths in eotides) . The preM. mazei and S. acidocaldarius [In many cases, we found several TTS downstream of one gene, showing that mRNAs with different 3´ UTR lengths are generated. The lengths of the UTRs as well as the fact that some genes have several 3´ UTRs provide a good platform for interactions with regulatory molecules, such as sRNAs. This is similar to the situation in eukaryotes, in which termination at different sites generates different 3´ UTRs, that can interact with different regulatory proteins or RNAs such as miRNAs. Different UTR lengths were also found in aldarius .S. acidocaldarius, 301 gene pairs with convergent orientations were investigated, and in 52% of them, the terminator of one gene was located in the coding region of a gene on the opposite strand [M. mazei, only 8% of the convergent genes showed such an overlap. The potential functions and the physiological relevance of these antisense transcripts must be uncovered in the future.If a gene is located on the opposite strand of a TTS  [leuB, ∆pyrE2, ∆trpA, ΔbgaH)  were grown aerobically at 45°C in Hv-YPC or Hv-Ca medium [600 of 0.8–0.9 (for a list of strains used see Supplementary Table 5). Total RNA was isolated from three biological replicates using TRIzol (ThermoFisher scientific). RNA fractions were sent to vertis  for further treatment, cDNA library preparation and high throughput sequencing. RNA preparations were made for each of the three different RNAseq approaches: (1) To obtain an RNA fraction enriched in primary transcripts RNA was treated with terminator exonuclease (this fraction was termed +TEX), (2) RNA from one preparation was left untreated representing the complete RNA pool (this fraction was termed -TEX). RNA preparations (1) and (2) were performed such that the original RNA 3´ end was maintained (for details see Supplementary methods). For RNA preparation (3) RNA was isolated for obtaining transcriptome data (this fraction was termed RNAseq data). In all sequencing approaches primers were designed such that the identification of the RNA strand was possible. Details for library construction and sequencing are reported in Supplementary methods., ∆trpA)  and HV55a medium  to an ODE. coli strains DH5α (Invitrogen) and GM121 [nd GM121  were groRaw reads were adapter clipped and quality trimmed using cutadapt version 1.10  based onhttp://www.bioinf.uni-leipzig.de/publications/supplements/18-059).Based on the Dar-Sorek-Method (DSM) ,47, RNA To detect sites with a significant enrichment of sequenced and mapped fragment ends a sound background without enrichment is desired. In the current setting, we used the intrinsic properties of a paired-end sequencing run to directly deduce the following information . We alsoi denotes the number of fragment ends at position i, Cj the number of fragment starts at position j, and j as a background distribution. Based on the distributions, native fragment end scores can be assigned an empirical p-value, evaluating its likelihood to occur by chance alone. The above detailed software, named Internal-Enrichment (IE) was implemented in Perl and is available at Bioinformatics Leipzig (http://www.bioinf.uni-leipzig.de/publications/supplements/18-059). We ran IE using all position showing signals for starts and ends (-mr 1), we omitted read fragments with length more than 100 nt (-mf 100) to reduce outliers, and the geometric mean as a method to calculate a score for the given signals (-mode GeomMean). We only included signals that were present in all three replica such that each showed a maximal empirical p-value of 0.05. The geometric mean of all empirical p-values for one position from all replica is used as the final score for the given position.Thereby, CA complementary approach to find RNA 3´ ends is identifying peaks in read stops . In ordeWe then used a sliding window approach with a window size of 150 nt and an overlap of 50 nt to find positions in the respective windows with a significantly higher number of read stops than the rest of the window. This was done by computing the mean number of stops as well as the standard deviation by window and then calculating the z-score for the number of read ends at every position of the window. Subsequently, we required a minimum number of 5 reads stopping, at least 10% of all reads covering position i-1 must end at position i as well as a minimum z-score of +2 at a site to report it as a putative RNA 3´ end. As a consequence of the overlapping of the windows, a site is evaluated multiple times and must fulfil the criteria in at least one of the contexts evaluated.To be able to differentiate between 3´ end processing sites and termination sites we isolated RNA and enriched this RNA for primary transcripts by digestion with the terminator exonuclease (TEX) (for details see Supplementary methods). Primary transcripts contain 5´ triphosphates and are not digested by TEX. Bacterial ribonucleases preferentially use 5´-monophosphate RNA as substrates , thus thWe report results for the sample enriched in primary transcripts (+TEX), in which IE and PC were both computed for the +TEX sample, followed by intersection analysis, resulting in the identification of TTS. In most cases, both methods (IE and PC) reported the same sites. For a few cases, more than one position was reported within a distance of 10 nt. In this case, the position with the highest coverage was chosen as TTS.http://www.bioinf.uni-leipzig.de/publications/supplements/18-059).More information is available at Bioinformatics Leipzig  (Supplementary Table 4).http://www.bioinf.uni-leipzig.de/publications/supplements/18-059 .The code is available at: Haloferax alicante and Haloferax volcanii)(bgaHa) [H. volcanii ß-galactosidase gene (bgaH) was deleted in strain H119. H119 was transformed with the bgaH deletion plasmid pTA617 (for a list of plasmids used see Supplementary Table 5) [SalI digested genomic DNA and probe bgaHaDO (primers: bgaHKODO-for and bgaHKODO-rev (for a list of primers used see Supplementary Table 5); template genomic DNA of H. volcanii). Probe labelling and detection of the blot were carried out using the DIG-DNA labelling mix and detection reagents (Anti-Digoxigenin-AP) (Roche) according to the manufacturer´s protocol. The resulting deletion strain was termed HV55.To allow the use of a plasmid carrying the ß-galactosidase reporter gene (bgaHa) ,51, the Table 5) ,52 and gTable 5) . Homozygsyn expression cassette  synthesized by lifetechnologiesTM (Thermofisher Scientific) was introduced via NotI/EcoRI into pTA231 [BamHI site by digestion with BamHI, blunting by Klenow fragment and religation. Then a C-terminal fragment of the reporter gene bgaHa was inserted at the NdeI site. After NdeI digestion, the vector was treated with Pfu polymerase to fill-in the NdeI site. The inserted bgaHa fragment was generated by PCR using primers bgaHatermifw and bgaHatermirev and pTA599 as template [BamHI site that is present in the newly inserted fragment. Terminator fragments were generated with PCR using primers as listed in Supplementary Table 5 and templates pTA599 (for the control construct) or genomic DNA of H. volcanii . HV55 cells were transformed with pTA231termtest constructs, and grown in Hv-Ca medium with uracil . Cells were grown to an OD650 of 0.6–1.0 and RNA was isolated using NucleoZOL (Machery-Nagel) according to manufacturer´s instructions.To construct the terminator-test plasmid a p.o pTA231 , resultitemplate . The corH. volcanii as described above. Ten µg RNA was separated on a 1.5% agarose gel and subsequently transferred to a nylon membrane . After UV-crosslinking the membrane was hybridized with a radioactively labelled probe against the 5ʹ-part of the bgaHa mRNA to detect termination events. The probe was generated by PCR  and the purified PCR fragment was labelled using α-32P-dCTP and the random primed DNA labelling kit DECAprimeTMII (Invitrogen). Experiments were done in triplicate. For detection of a potential processing fragment downstream of the termination site, a radioactively labelled probe against the 3ʹ-part of the bgaHa mRNA was used. The probe was generated and labelled as described above using primers BetaHinten1 and BetaHinten2 and pTA231-termtestA1 as template. For quantification of northern blot signals, membranes were exposed to phosphorimaging plates (FujiFilm) and the resulting signals detected using a Typhoon imager (GE). Analysis of three replicates was carried out using the ImageQuant TL software (GE).To analyse the RNA levels, total RNA was isolated from http://www.bioinf.uni-leipzig.de/publications/supplements/18-059), RNAseq data are available at the European Nucleotide Archive  under the project accession number PRJEB30349, with the following assigned experiment accession numbers.The data supporting our findings are available in the Supplementary Information, in addition the code is available under (


This method converts s4U base‐pairing with guanine through the nucleophilic addition of s4U to acrylonitrile. The high reaction efficiency permits AMUC‐seq directly and efficiently to recover nascent RNA information from total RNAs. AMUC‐seq is validated by being used to detect mRNA half‐lives and investigating the direct gene targets of a G‐quadruplex stabilizer, which can be regarded as potential anticancer drug, in human cells. Thousands of direct gene targets of this drug are verified (these genes are significantly enriched in cancer such as SRC and HRAS). AMUC‐seq also confirms G‐quadruplex stabilization that impacts RNA polyadenylation. These results show AMUC‐seq is qualified for the study of temporal RNA dynamics, and it can be a promising strategy to study the therapeutic mechanism of transcription‐modulating drugs.RNA sequencing has greatly facilitated gene expression studies but is weak in studying temporal RNA dynamics; this issue can be addressed by analyzing nascent RNAs. A famous method for nascent RNA analysis is metabolic labeling with noncanonic nucleoside followed by affinity purification, however, purification processes can always introduce biases into data analysis. Here, a chemical method for nascent RNA sequencing that avoids affinity purification based on acrylonitrile‐mediated uridine‐to‐cytidine (U‐to‐C) conversion (AMUC‐seq) via 4‐thiouridine (s 4U) cyanoethylation converts in the nascent RNA to cytidine in a base‐pairing manner, representing a promising strategy for enrichment‐free nascent RNA detection that further benefits transcriptome‐wide analysis of temporal RNA dynamics.4‐thiouridine (s However, traditional RNA‐seq technology is generally performed at the steady‐state level of cellular RNAs, changes in RNA transcription and decay rates cannot be easily distinguished. In addition, it provides poor resolution of the temporal information of RNA kinetics. Cells always require relatively long hours of stimuli to achieve detectable differential expression upon RNA‐seq, and multiple secondary signaling events could be promoted and contribute to the observed changes within that time, which confuses the underlying regulatory mechanisms.RNA sequencing (RNA‐seq) is a revolutionary tool for transcriptome profiling in a given biological sample that allows us to look at the dynamic changes of gene expression against different conditions or stimuli.1 Unde Nascent RNA profiling can reveal the temporal information of gene expression changes. Metabolic labeling and downstream purification of metabolically labeled nascent RNA followed by RNA sequencing has become a famous approach to study nascent RNA behaviors. For example, global run‐on sequencing (GRO‐seq) uses 5Br‐UTP to label nascent RNA and then nascent RNAs containing 5Br‐U are immune‐precipitated with an anti‐BrU antibody. 4‐thiouridine (s4U) and 5‐ethynyl uridine (5EU) can also be used for nascent RNA labeling and then act as chemical handles for the separation of labeled RNA via thio/ethynyl‐specific biotinylation. However, these techniques require large amounts of input sample. And the conventional purification assay for isolating metabolically labeled nascent RNAs is laborious, a certain amount of contamination can be induced during purification processes due to the non‐specific binding. Sometimes RNA purification suffers from limited efficiency and tends to enrich transcripts that contain high uridine content . These issues can result in experimental bias unless additional experiments and/or computational processing is employed for data normalization.To address these issues, recently, new strategies for analyzing newly synthesized (nascent) RNA instead of total cellular RNA have been developed.3 Nasc This strategy directly distinguished nascent RNA from total RNA population in single‐base resolution by marking the mapping reads of nascent RNA with introduced base mutations. Briefly, cells are exposed to a thiol‐labeled nucleoside (s4U or s6G), resulting in rapid uptake and incorporation into the newly synthesized RNA, which can be then isolated and treated with specific chemical reagents, that lead to the change in the base‐pairing manner of the metabolically incorporated nucleoside. In this way, nascent RNA information can be directly extracted from the sequencing data of total RNA by tracking and segregating the sequencing fragments, which contain anticipated base mutations.Recently, an innovative enrichment‐free strategy for nascent RNA detection has been presented, adding a new dimension for the study of RNA dynamics.9 This SLAM seq employs a nucleophilic substitution chemistry to alkylate s4U, inducing s4U‐to‐C mutation in a reverse transcription (RT)‐dependent manner. TimeLapse‐seq transformed s4U into cytidine derivative underlying an oxidative nucleophilic aromatic substitution reaction, that has also been successfully extended to recode s6G to A analogue. This oxidation condition caused certain oxidative damage to guanine, and TUC‐seq directly converted s4U to C by osmium tetroxide and ammonia, while not been applied to transcriptome‐wide sequencing. Herein, we employed a novel method based on a nucleophilic addition chemistry to realize s4U‐to‐C conversion in NGS‐compatible manner. This strategy is highly efficient and reliable, and can quantitatively analyze nascent RNA at transcriptome scale with little influence on the base‐paring manner of other nucleosides.Thus far, only limited methods have been developed for enrichment‐free nascent RNA detection.13 SLA which functions by binding and stabilizing G‐quadruplex (G4) structures, a non‐canonical nucleic acid secondary structure formed by guanine‐rich sequences. G4 motifs have been identified overrepresented in promoters, particularly in genes driving cancer and associating with cell proliferation, that offers G4 targeting a therapeutic advantage since the stabilization of these G4s result in inhibition of the expression of these genes. To pursue G4s efficiently as therapeutic targets, it is necessary to uncover the direct gene targets of G4 ligands in vivo in order to understand how G4s involved in cancer intervention and therapeutics.By applying our sequencing strategy, we successfully detected the transcriptome‐wide direct gene targets of one potential anticancer drug PDP,14 whi22.1Figure4U in the newly made RNA is caused by the electrophile acrylonitrile to form the S‐cyanoethylated 4‐thiouridine (ces4U) via a type of Michael addition under alkaline condition. The cyanoethyl group has been used for thiocarbonyl protection in the synthesis of thiopyrimidine containing oligonucleotide. We predicted that the original N3 of s4U is no longer a competent proton donor after cyanoethylation, leading to s4U inhibiting Watson–Crick base pairing with A. Instead, it forms non‐canonical base pair with G. Taking advantage of this, cyanoethylated s4U were replaced by C in cDNA via RT and PCR during sequencing.The principle of our method is outlined in 4U‐to‐C conversions are the identification signals of nascent RNAs, an adequate conversion efficiency is necessary for the recovery of nascent RNAs from total RNAs as complete as it can be. Thus, we first tested the reactivity of the s4U cyanoethylation reaction in vitro. We found that the s4U‐to‐ces4U conversion rate could reach ≈90% under very gentle conditions. By adjusting the reaction conditions, the reaction could be completed within hours . We compared the selectivity and reactivity of our method with the reported ones (SLAM‐seq and TimeLapse‐seq) all these three methods can achieve high reaction selectivity and efficiency . Next, we validated the hydrogen bonding patterns of s4U and ces4U. We prepared s4U and ces4U‐containing RNA oligos as templates and performed RT‐directed single‐nucleotide incorporation reactions by using RevertAid reverse transcriptase. Undoubtedly, s4U preferred to pair with T, yet also has a weak base‐pairing ability toward G, which is probably induced by the tautomeric form of s4U. In contrast, only dGTP incorporation was observed opposite to the ces4U‐RNA template even after a long reaction time . Thus, s4U cyanoethylation resulted in a thoroughly s4U‐to‐C conversion in the base‐pairing manner.Because s. The isolated total RNAs, treated RNAs with acrylonitrile, amplified specific mRNA , and then subjected it to TA cloning and Sanger sequencing. Ten clones were selected for mutation analysis. As expected, distinct U‐to‐C transitions appeared only in the acrylonitrile‐treated s4U‐RNA populations .23 The2.34U cyanoethylation reaction for s4U‐RNA detection on a transcriptome‐wide scale. s4U‐labeled mRNAs of HEK293T cells cultured with 50 µm s4U for 24 h were prepared for the following tests. First, we performed LC‐MS to measure the reaction efficiency of s4U cyanoethylation in total mRNAs. To alleviate the suppression to the s4U cyanoethylation reaction imposed by the intricate RNA secondary structures, the purified full‐length mRNAs were fragmented into ≈150 nt RNA fragments prior to acrylonitrile treatment by treating RNA with zinc (II) and heat (70 °C for 10 min), which catalyze the cleavage of RNA by attacking the 2′ hydroxyl group of ribose to produce a mix of 2′ or 3′ phosphate ends with no sequence and structure specificity. LC‐MS analysis identified that nearly 90% of s4U in total mRNAs converted to ces4U , consisting with the reaction efficiency we tested in vitro on the nucleoside. Then, acrylonitrile‐treated total mRNAs were subjected to library construction and next‐generation sequencing  were kept at low background levels. What is more, all the non‐U‐to‐C mutations in four samples were kept at background levels. These results identified s4U or acrylonitrile had little impact on the base‐pairing patterns of all the other nucleotides. In addition, the RNA expression patterns displayed a good correlation between the acrylonitrile‐treated and untreated RNAs , suggesting acrylonitrile treatment has an inappreciable influence on the analysis of gene expression.Inspired by these results, we therefore implemented scificity.24 LC‐g Figure . The fras Figure. However2.44U labeled RNA information from total mRNA sequencing reads by tracking the U‐to‐C transition signals and retracting SNPs, and analyzed the s4U labeled RNA recovery efficiency. After 24 h s4U labeling, 75% of the mapping fragments have been marked by the desired T‐to‐C mutations  of human mRNAs has been suggested to be a few hours. We then checked the mapping reads of several fast‐decayed mRNAs in BCL‐2 family whose t1/2 have been estimated to be 1–4 h in human B‐cells. In theory, the pre‐existing fraction of short‐lived transcripts ought to be completely decayed within the 24 h s4U‐incubation time. Indeed, 88.5–95.6% of their mapping fragments contained T‐to‐C conversions . The same analysis was also applied to the extremely stable transcripts APLP2, MYH9, FLNA, MSN, CRTAP and PRDX5. These transcripts have been examined to be long‐lived, with t1/2 in excess of 12 h, in human cells. A large fraction of pre‐existing mRNAs was still detected in the total mapping reads . These results indicate our method can efficiently recover the s4U labeled RNA information from total RNAs.We then dissected the ss Figure , indicat Figure t1/2 of hu Figure t1/2 of hu Figure t1/2 of hu2.5 RNA stability is determined by the interplay of RNA synthesis, processing, and decay. Results showed that the median mRNA t1/2 in HEK293T cells was calculated to be 5.5 h, which correlated with previous observations in human B‐cells ‐interacting drug PDP Figure5A, a py we defined, the genes whose temporal transcription level downregulated were the direct targets of PDP. A short‐term PDP treatment and s4U labeling  revealed 684 downregulated transcripts in the nascent mRNA outputs  , so PDP has a wide range of gene targets in cells. No apparent changes were observed in the total mRNA abundance , suggesting nascent RNA detection provides high resolution of RNA dynamics. Gene ontology analysis revealed that those downregulated genes were enriched in genes involved in transcription regulation and kinase activity and development, while lacked in genes involved in olfactory receptor activity, and G‐protein coupled receptor activity .The experimental approach is outlined in Figure 20 we  We also have compared the transcription levels of all detected G4‐containing genes to that of detected genes containing no G4s. On the whole, the transcription levels of genes without G4s have no obvious changes, while genes containing G4 sites  were globally downregulated, the downregulation levels were correlated with the number of G4 sites , such as TERT, PIM2, and VEGFA, which have been evidenced to be regulated via G‐quadruplex and involved in the anticancer therapy of G4 stabilizers, and BAD, AKT2, and NOTCH3 which have not yet been studied to be regulated via G4s in these genes, these cancer genes may also be the candidates that involved in the anticancer therapy of PDP. We also have conducted the same experiment in MCF7 cells, a breast cancer cell line, and achieved quite similar results .To assess the PDP effects in cancer, we focused on the cancer genes in these downregulated genes by searching downregulated genes in a cancer gene database summarized by Bushman.37 We 2.7 A large fraction of these genes acts in ribosome biogenesis, translation initiation, mRNA splicing, and transcription factor activity , suggesting PDP can disrupt mRNA transcription and protein translation by disturbing the normal polyadenylation processes of these mRNAs.The annotated PASs of these affected mRNAs were remarkably enriched with RNA G4 , indicating PDP disrupted their normal polyadenylation processes by interacting these RNA G4s. So these mRNAs are also the direct targets of PDP. The same results have also been confirmed in the MCF7 cell line .Except for inhibiting gene transcription, we found that PDP can also interfere with the normal 3′‐end polyadenylation process of the transcribed pre‐mRNAs. After PDP treatment, 724 genes (involving 124 cancer genes) displayed distinctly enhanced transcription levels downstream their annotated polyadenylation sites (PASs) . This result indicated that PDP hindered the pre‐mRNA polyadenylation at their annotated PASs, instead, they skipped these PASs and may be terminated at another PAS behind, that led these aberrant mRNAs had longer 3′ UTRs. The 3′ ends of mRNAs are closely related to mRNA stability, localization, and translation, disruption in the normal polyadenylation processes of mRNAs can lead to mRNA dysfunctions.40 A l34U‐to‐C mutation permits the recognition of metabolically labeled RNA at 1 bp resolution without additional RNA‐purification processes. The high reactivity of this method allowed for efficient recovery of the nascent RNA information. It is noteworthy that acrylonitrile treatment caused negligible non‐s4U‐to‐C mutations and undesired U‐to‐C mutations, such as the resulted G‐to‐T mutation rising from the oxidation of guanine to 8‐oxo‐guanine by using TimeLapse‐seq. This enrichment‐free nascent‐RNA analysis method is qualified to transcriptome‐wide study of the temporal RNA kinetics. Except for evaluating the stability of protein‐coding RNAs in human cells, we also have successfully investigated the direct gene targets of a G4‐interactive small molecule. In addition to the well‐known transcriptional regulation effects, we evidenced the blocking of G4 structures also affected the 3′ end polyadenylation of mRNAs that may disrupt gene expression. Both regulatory roles of G‐quadruplex‐interacting drug could be involved in their anticancer therapy. These results could be helpful for the future understanding of the anticancer mechanisms of this kind of potential antitumor drugs.Taken together, we presented a new chemical method for enrichment‐free nascent RNA sequencing. Our experiments demonstrated that acrylonitrile‐mediated sTo the best of our knowledge, this is the first use of nascent RNA sequencing to investigate the active targets of transcription‐modulating drugs. Compared to the conventional analysis methods, enrichment‐free nascent RNA sequencing provides much higher resolution of RNA dynamics that is adequate for defining the primary therapeutic mechanisms of transcription‐modulating drugs, because it can precisely define their direct gene targets and simultaneously identify their transcriptional regulatory effects. We believe our method can be applied to a broader range of applications in biological research, biomedical research, and chemical genomics and has great potential to facilitate the progresses of these fields.4−1 s4U were reacted in the presence of 306, 764, or 1146 mm acrylonitrile under mild reaction condition  at indicated temperature for different time periods. The samples were separated on Agilent HPLC system (Thermo Fisher Scientific), employing a Thermo Scientific Hypersil ODS Column  with a flow rate of 1 mL min−1 at 35 °C. eluent A: 0.1 m TEAA (pH 7.0), eluent B: acetonitrile; gradient: 5−40% B in A within 20 min and UV detection at 260 nm.10 µg mL4U) was treated with acrylonitrile and purified by HPLC. Purified chemically treated RNA  was then annealed to a FAM 5′ end‐labeled primer, and reverse transcription was performed using RevertAid First Strand cDNA Synthesis Kit (42 °C). The reaction was then stopped and subjected to 20% denaturing PAGE.300 ng RNA oligo  were prepared under the exactly same conditions. After the incubation period, cell survival was evaluated using MTT assay. 10 µL aliquot of MTT solution (5 mg mL−1 in PBS) was added to each well. After 4 h of incubation, the medium was replaced and supplemented with 100 µL of DMSO. The absorbance at 492 nm was measured for each well. Data were expressed as mean values of three individual experiments conducted in duplicate.12 000 HEK293T cells were seeded per 96 well and grown at 37 °C in DMEM containing 10% FBS and 1% P/S for 24 h. Then, indicated concentrations of s4U/PDP were added to the media for indicated time periods. Control cells (treated with only solvents) were prepared under the exactly same conditions. After the incubation period, the culture media was removed, rinsed the cells with PBS and added trypsin to dissociate cells. The culture media and PBS rinse were pooled and saved. Once cells were fully trypsinized, the media was quenched with the saved media and PBS wash from the previous step. Cell cycle profile and apoptosis analysis were then performed.100 000 cells were seeded per 24 well and grown at 37 °C in DMEM containing 10% FBS and 1% P/S for 24 h. Then, indicated concentrations of sm laser excitation was collected.Cells were fixed in 66% ethanol and stored at +4 °C for 2 h. 50 000–100 000 resuspended cells were taken, centrifuged at 1000 g for 5 min, discarded the supernatant, and rehydrated cells in PBS. Cells were the stained with propidium iodide + RNase for 30 min (Immediately prior to use prepare the Propidium Iodide + RNase staining solution in PBS). For flow cytometry detection, propidium iodide fluorescence intensity on FL2 (660 nm) of a flow cytometer and 488 nm laser excitation were collected.Apoptosis analysis: 50 000–100 000 resuspended cells were taken, centrifuged at 1000 g for 5 min, discarded the supernatant, and added 195 µL of Annexin V‐FITC binding solution to gently resuspend the cells. 5 µL Annexin V‐FITC was added and mixed gently then 10 µL of propidium iodide staining solution was added and mixed gently. Incubated at room temperature in the dark for 10–20 min, then placed in an ice bath. For flow cytometry detection Annexin FITC fluorescence intensity on FL1 (530 nm) and propidium iodide fluorescence intensity on FL2 (660 nm) of a flow cytometer and 488 n6 HEK293T cells were seeded per 10 cm cell dish and grown for 24 h. Then, s4U was added to the medium at a final concertation of 50 µm and grown for another 24 h. Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacture's protocol and the RNA concentration was determined by Nanodrop (Thermo Fisher). gDNA was removed by TURBO DNase (Invitrogen), then RNA was purified by RNA Clean & Concentrator Kits (Zymo Research). Isolated RNA was then subjected to s4U cyanoethylation reaction. After reacted with acrylonitrile, RNA was purified by RNA Clean & Concentrator Kits. 500 ng purified RNA was reverse transcribed using PrimeScript RT Master Mix (TaKaRa). The resulting cDNA was then amplified with Taq DNA polymerase (Thermo Fisher) using CCND1‐specific primers . PCR products were purified by gel extraction. Isolated PCR products were further applied to TA cloning analysis by using pEASY‐T1 Simple Cloning Kit (TransGen Biotech). For each sample, 30 clones were selected for Sanger sequencing.3 × 106 HEK293T cells were seeded per 10 cm cell dish and grown for 24 h. Total RNA was extracted after that 50 µm s4U was added to the medium and grown for another 24 h. Polyadenylated RNA was isolated by subjecting total RNA to oligo(dT) enrichment using Dynabeads Oligo(dT)25 (NEB) following manufacturer's instructions and then gDNA was removed. Purified polyadenylated RNA was fragmented using RNA fragmentation reagents (Invitrogen) at 70 °C for 10 min, then stopped by stop solution and purified by RNA Clean & Concentrator Kits. 2 µg RNA fragments were subjected to s4U cyanoethylation reaction at 45, 50, and 55 °C for different time periods. To enzymatically degrade RNA to monomeric ribonucleosides, Nuclease P1  was added to a 50 µL solution containing 2 mm ZnCl2, 10 mm NaCl, 100 µm DTT, and 600 ng purified RNA and incubated at 37 °C for 2 h. Then, 6 µL 200 mm Tris‐HCl (pH 7.9), 1 µL 1 mm DTT, 3 µL 100 mm MgCl2, and 2 µL Shrimp Alkaline Phosphatase  were added to the reaction solution and incubated for another 2 h at 37 °C. Each sample was centrifuged at 12 000 g at 4 °C for 20 min, 50 µL supernatant was collected and directly subjected to HPLC‐MS analysis. s4U and ces4U ribonucleosides which diluted in enzymatic buffer containing a mixture of four common bases were used to make a standard curve for quantitative MS analysis. Standards were prepared as Table S2, Supporting Information.3 × 10−1. Nucleosides were on‐line analyzed using a triple quadrupole mass spectrometer after electrospray ionization with the following multiple reaction monitoring: s4U m/z 260→129, and ces4U m/z 318→129.The samples were separated on a Shimadzu HPLC system (LCMS‐8050), employing a ZORBAX Rapid Resolution High Definition column  with a flow rate of 200 µL min6 HEK293T cells were seeded per 10 cm cell dish and grown for 24 h. For T > C conversion identification, 50 µm s4U was added to the medium and grown for another 24 h. For PDP‐mediated RNA dynamic analysis, PDP was added to the medium for 20 min prior to 1 h 500 µm s4U incubation. After that, RNA samples for library construction were prepared by total RNA extraction, polyadenylated RNA isolation, gDNA removal, RNA fragmentation, and RNA cyanoethylation . Standard RNA seq libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) according to manufacturer's instructions. Sequencing was performed on Illumina HiSeq X Ten.3 × 10 Then, only one read from reads group with identical sequence is kept to remove potential PCR amplification bias. Next, the remaining reads were mapped to human ribosome RNA sequences  using bowtie2. The paired‐end reads that failed to map to rRNA were finally mapped to human genome (GRCh37) using hisat2 with parameter “–mp 2,2 –rna‐strandness FR –no‐mixed –no‐discordant –seed 1234” (index of hisat2 was download from hisat2 office website). Only the proper pair and uniquely mapped alignments was persisted for the downstream pipelines.All library samples shared the same processing methods in the initial few steps. First, all the paired‐end reads from NGS technology were trimmed using cutadapt to remove Illumina adapter sequences with parameters “‐m 20 –max‐n 3 ‐q 30”.41 Theref/alt represents the mismatch ratio of “ref‐to‐alt” , Nref/alt represents the number of “ref‐to‐alt” mismatch event, and Nref represents the expected sequencing number of base “ref”.Mismatch events were extracted from alignment results by custom script. For two mismatch events with the same position and mismatch type extracted from read 1 and read 2 in the same fragment, only the one with the best sequencing quality was retained. Mismatch events induced by SNP or with lower sequencing quality than 41 were excluded. For specific mismatch site, if the count and ratio of correspond mismatch event was larger than or equal to 5 and 5% in control samples, respectively, it was treated as a new SNP, and the mismatch events induced by new SNPs were finally excluded from all samples. Counting the number of mismatch events for all mismatch type and the number of reference base, the mismatch ratio was calculated as following formula3+N1‐7 G3+N1‐7G3+N1‐7G3+”. G4 intervals from G4‐seq were also used to perform analysis. The distribution of G4 around TSSs and PASs of each transcripts were calculated.The potential G4 intervals were extracted from the genome sequences by using custom script. The sequences in these intervals conform to the regular expression “GN1‐7G3+”.34 G4 4U and C2H3CN treatment. According to this, the total number of fragments and the number of labeled‐fragments (with T‐C mismatch event) of each transcript were calculated respectively. According to previous reports, use the following formula to calculate the half‐life of the transcriptNe represents the fragment number of unlabeled‐RNA, Nt represents the fragment number of total‐RNA, Nn represents the fragment number of labeled‐RNA, and T represents the treatment time.For a transcript, a shorter half‐life corresponded to a higher proportion of labeled‐RNA over total‐RNA after sranscript27(2)N represents the fragment count, L represents the transcript length, and S represents the count of all fragment. The calculation of labeled‐FPKM is similar to the calculation of FPKM, except that C represents the number of labeled fragments.The expression level of each transcripts was represented by fragment count per kilobase per million fragment (FPKM), which were calculated using below formula2(labeled‐FPKM of PDP treatment/labeled‐FPKM of control) ≤ ‐log2(1.5).In order to identify the target genes of PDP, the fold‐change of FPKM and labeled‐FPKM were calculated for PDP treatment sample versus control sample respectively. The canonical protein coding transcripts that met following criteria at all replicates and comparison group were treated as PDP target genes: a) FPKM of PDP treatment and control ≥1; b) labeled‐FPKM of control ≥1; c) log2(1.2) ≤ log2(FPKM of PDP treatment/FPKM of control) ≤ log2(1.2); d) log2(downstream‐FPKM of PDP treatment/downstream‐FPKM of control) ≥ log2(1.5).The impaired mRNA maturation led to transcriptional read‐through, which resulted in increment of the fragment count of downstream of PAS. Downstream‐FPKM  was used to measure the fragment count of PAS downstream and eliminate deviation in library size. Transcripts whose downstream regions overlap in the same direction as other transcripts are not considered. The canonical protein coding transcripts that met following criterions at all replications and comparison group were treated as impairs mRNA maturation: a) FPKM of PDP treatment and control ≥1; b) downstream‐FPKM of PDP treatment ≥0.2; c) ‐logGO‐term enrichment analysis was performed using the PANTHER database.45The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Neuroendocrine carcinoma of the cervix (NECC) accounts for 2% of all cervical cancers. Brain metastasis is rare, with few cases described in the literature, and is usually associated with preceding pulmonary metastasis. We describe an additional case of isolated brain metastasis without pulmonary metastasis from NECC and reflect on unique management.A 37-year-old woman with a history of NECC presented with severe headache post-total hysterectomy with pelvic lymph node dissection. The computed tomography (CT) scan demonstrated obstructive hydrocephalus with several intra-axial lesions located in the pineal region, left cerebellar hemisphere, and left frontal operculum. A right frontal ventriculostomy was initially placed to relieve the hydrocephalus. CSF was sent for cytology but was unrevealing. Due to the degree of brainstem compression and the need to obtain a pathologic diagnosis, a posterior fossa craniotomy for the removal of the lesion was performed. Histopathology demonstrated small blue cell tumors positive for neuroendocrine markers consistent with neuroendocrine carcinoma of the cervix. Resection of additional metastasis was not recommended. An endoscopic third ventriculostomy (ETV) was then performed in order to remove the ventriculostomy with success. The patient was then referred to radiation oncology and received whole-brain radiotherapy (WBRT) for a total of 30 Grays (3000 cGy) over 10 fractions. Interval imaging demonstrated complete resolution of the pineal and left frontal lesions. The patient was symptom-free for approximately three months. She then presented with paraplegia consistent with follow-up imaging of her neuraxis, demonstrating drop metastasis in her cervical, thoracic, and lumbar spine. Spinal radiation was given with partial recovery in upper extremity function, however, lower extremity function did not recover. The patient was then transferred to palliative care.There are no guidelines on NECC brain metastasis management. Brain metastasis is associated with reduced longevity. NECC has a propensity for early dissemination and treatment failure. ETV is preferred over ventriculoperitoneal shunting in cases with obstructive hydrocephalus, as it may reduce the risk of tumor seeding. Retrospectively, our patient may have benefitted from upfront craniospinal radiation. Neuroendocrine carcinomas are derived from neuroendocrine cells, which originate from the embryonic neuroectoderm. Neuroendocrine carcinomas are divided into well-differentiated  and poorly differentiated , with small cell being the most common subtype . NeuroenA 37-year-old female with a known history of neuroendocrine carcinoma of the cervix (NECC) for the past 11 months who had undergone a partial hysterectomy presented to the neurosurgical service with a severe headache unresponsive to medical treatment after recently undergoing a follow-up completion hysterectomy with pelvic lymph node dissection. Figure Brain metastasis is rare in NECC. There are no guidelines on how to manage brain metastasis when it reaches this stage. Per the Society of Gynecologic Oncology (SGO), treatment, especially chemotherapy, is generally translated from cervical cancer and small cell lung cancer (SCLC) management, given the histological and clinical similarities . For SCLOur patient had a survival of 13 months from diagnosis. Given that her neurologic status declined due to spinal metastasis after the complete resolution of her cranial lesions, our patient may have benefited from prophylactic craniospinal radiation upfront and maintained her ambulatory status longer. This is apparently in contrast with current guidelines that do not recommend prophylactic craniospinal radiation in patients with early NECC.While brain metastases are typically observed concurrently with lung metastases , our casThe development of guidelines for the management of NECC and metastasis are needed to better guide treatment. Further research looking at the management and outcomes of NECC brain metastasis and the use of craniospinal radiation is needed. Research studying other management, including stereotactic radiosurgery and multimodal approaches, would be beneficial. There are potential opportunities for investigations into determining what mutations are responsible in NECC to allow for CNS penetration through the blood-brain barrier. It would also be of interest to study the incidence of microscopic CNS disease.It is important to acknowledge some limitations. The use of prophylactic craniospinal radiation is often at the discretion of the treating physician or radiation oncologist given that there are no established guidelines for the management of NECC. There is limited research on survival rates with the use of prophylactic craniospinal radiation. Whether the type of NECC plays a role in increasing longevity with the use of prophylactic craniospinal radiation is not fully understood.Brain metastasis of NECC is associated with reduced longevity and has a propensity for early dissemination and treatment failure. Our patient with isolated CNS metastasis from NECC may have had longer ambulatory status with craniospinal radiation. In our patient with obstructive hydrocephalus, ETV was preferred over ventriculoperitoneal shunting to reduce the risk of tumor seeding. The development of guidelines on NECC brain metastasis management is needed.


APOBEC1 (APO1), a member of AID/APOBEC nucleic acid cytosine deaminase family, can edit apolipoprotein B mRNA to regulate cholesterol metabolism. This APO1 RNA editing activity requires a cellular cofactor to achieve tight regulation. However, no cofactors are required for deamination on DNA by APO1 and other AID/APOBEC members, and aberrant deamination on genomic DNA by AID/APOBEC deaminases has been linked to cancer. Here, we present the crystal structure of APO1, which reveals a typical APOBEC deaminase core structure, plus a unique well-folded C-terminal domain that is highly hydrophobic. This APO1 C-terminal hydrophobic domain (A1HD) interacts to form a stable dimer mainly through hydrophobic interactions within the dimer interface to create a four-stranded β-sheet positively charged surface. Structure-guided mutagenesis within this and other regions of APO1 clarified the importance of the A1HD in directing RNA and cofactor interactions, providing insights into the structural basis of selectivity on DNA or RNA substrates. Nucleic acid base editing on DNA or RNA is a versatile tool that serves a variety of biological roles within living systems. The APOBEC family of proteins deaminates cytosine (C) residues on DNA or RNA into uracil, resulting in a range of biological effects such as antiviral restriction , immune APOB) responsible for producing a stop codon and shorter version of the protein in order to regulate lipid uptake and the level of low-density lipoproteins (LDLs)  structure that is composed of a five-β-stranded (β1–β5) sheet adjoined by six helices (h1–h6), with a Zn atom coordinated at the active center ,22. Mosts (LDLs) . APO1 has (LDLs) ,27 and ws (LDLs) ,29. In as (LDLs) . The clas (LDLs)  and the s (LDLs)  are the s (LDLs) ,33. In as (LDLs) ,32,34,35s (LDLs) .Like other members of its family, APO1 is also capable of deamination on single-stranded DNA (ssDNA) ,39, and Despite many years of studies investigating RNA and DNA cytosine deamination by APO1, there are still many questions remaining with regard to the molecular mechanisms behind its activity and regulation, mainly due to the lack of high-resolution structural information of APO1. In order to better understand the structural basis of APO1 activity and function, we have determined a crystal structure of APO1 that reveals dimerization occurs through its uniquely folded C-terminal domain. Subsequent structure-guided mutational and functional studies using direct biochemical and cell-based assays provide new insights into the molecular basis for APO1 activity on RNA and DNA substrates as well as the functional role of its unique C-terminal domain. The findings should contribute to the further understanding of cellular functions of APO1, as well as the potential application of APO1 in developing base editors with greatly improved substrate specificity for therapeutic purposes.de novo via the ab initio function of RosettaScripts  APO1 construct was cloned into a pMAL-C5X vector. Mutations were made using Primestar Max (Takara Biosciences) high-fidelity DNA polymerase, with primer design done via Snapgene Viewer. MBP–A1CF was generated by purchasing a dsDNA gene string from Thermo Fisher (GeneArt) that contained the first 582 residues of A1CF and two phosphoserine analog mutations  thought to confer additional APO1 complementation activity , followeGenerally, 4 l of LB–ampicillin media were inoculated with starter culture and grown at 37°C until log phase; induction of protein expression was done with 100 μM final concentration of IPTG overnight at 16°C. Cells were lysed using a shear force fluid homogenizer  in a lysis buffer containing 20 mM HEPES, pH 7.5, 500 mM NaCl and 0.5 mM TCEP, with additional 100 μg/ml of RNAse A (Qiagen) added. Centrifuged cell lysates were passed over 10 ml of amylose affinity resin and washed once with lysis buffer, once with high-salt buffer  and again with lysis buffer. Lysis buffer with 40 mM maltose was used to elute the MBP fusion proteins, and the protein was concentrated using centrifugal spin concentrators (Millipore). Concentrated MBP–APO1 proteins were further purified by size exclusion chromatography (SEC) in lysis buffer to purify the different oligomeric species. For MBP–A1CF fusion, the concentrated protein was first cleaved overnight with HRV protease at 4°C and then diluted at least 10-fold into CEX buffer A . Cation-exchange chromatography was performed to separate A1CF from cleaved MBP on a 6-ml Resource S column (GE) using a gradient from buffer A to CEX buffer B . Another SEC run was performed to further purify A1CF in a storage buffer containing 20 mM HEPES, pH 7.5, 250 mM NaCl and 0.5 mM TCEP. For more precise measurement of molecular weights (MWs), multi-angle light scattering (MALS) was performed.P21212 space group via HKL2000  was performed in Phaser within the Phenix software suite  by searc−ung variant of NR9404 Escherichia coli cells, and the cells were grown on non-inducing minimal media plate overnight. Individual colonies were picked and grown in 5 ml cultures of LB media in the presence of 1 mM IPTG and 100 μg/ml carbenicillin overnight for at least 20 h, and the OD600 was measured and normalized to a value of ∼1.50. One hundred microliters of cells were then plated on either an LB plate containing 100 μg/ml rifampicin or an antibiotic-free equivalent after diluting the cells by 107-fold. Plates were incubated at 37°C for 20 h before cells were counted. For statistical tests, data were assessed for outliers using the ROUT nonlinear regression-based methodology at a 1% Q value, which resulted in the removal of no more than two outliers per assessment. The cleaned dataset was then analyzed using ANOVA with a Tukey post-test to assess the significance of different results.pMAL vectors encoding MBP fusions of each of the tested mutants were transformed into a All DNA reactions were completed using an abasic hydrolysis technique ,50 on a APOB-derived RNA substrate for additional biochemistry. ssDNA oligos were ordered and annealed together that contained the T7 promoter and desired final APOB RNA sequence of GGAUAUAUGAUACAAUUUGAUCAGUAUAUUAAAGAUAGUUAUGAUUUACAUGAUU. Reactions were 10–15 ml in a buffer of 40 mM Tris–HCl, pH 7.9, 6 mM MgCl2, 20 mM DTT, 2 mM spermidine, 500 μM dNTPs, 1 U/μl T7 RNA polymerase and 0.2 U/μl RNAseOUT (Thermo Fisher), and allowed to react overnight at 37°C. Resulting RNA was concentrated by ethanol precipitation followed by purification using TRIzol (Thermo Fisher) with the standard technique.Recombinant T7 RNA polymerase (McLAB) was used to generate a 55-nt in vitro transcribed APOB RNA described earlier. The reaction buffer consisted of 20 mM HEPES, pH 7.5, 50 mM NaCl, 5 mM DTT, 1 U/μl of RNAseOUT (Thermo Fisher) and 1 μM RNA. Ten-microliter reactions were allowed to incubate at 37°C for 1 h followed by the addition of 1.2 μM final of a 5′-FAM-labeled primer that binds downstream of the editing site with the sequence 5′-AAT CAT GTA AAT CAT AAC TAT CTT TAA TAT ACT GA-3′ and a denaturation step of 95°C for 10 min and subsequent step down to room temperature that stops the reaction, denatures the RNA strand and allows the primer to anneal. A reverse transcription buffer was then added that results in a final concentration of 2.5 U/μl Protoscript II (NEB), 1× manufacturer-recommended reverse transcription buffer, 5 mM DTT, 250 μM dTTP, dCTP and dATP, and 250 mM ddGTP (Tri-link Bio), and 0.1 U/μl of RNAseOUT (Thermo Fisher). After reverse transcription, the resulting products were run on a 20% acrylamide urea denaturing gel similar to DNA deamination.An assay for RNA deamination was adapted for use with recombinant proteins from the poisoned primer extension design previously proposed . PurifieAPOB RNA in the presence of 20 mM HEPES, pH 7.5, 5 mM DTT, 10% glycerol, 50 mM NaCl and, if RNA, 0.4 U of RNAse inhibitor. The same FAM-labeled ssDNA substrate and the FAM-labeled APOB RNA used for deamination were used for this binding assay. Samples were incubated on ice for 10 min prior to running EMSA at 4°C in 1% TBE. Resulting quantification was done by determining the ratio of remaining unshifted DNA to the blank for each protein concentration and plotting the resulting amount of shifted DNA to the concentrations used. Nonlinear regression based on a modified Hill saturation curve (Kd estimates.Electrophoresis mobility shift assay (EMSA) was used to estimate nucleic acid binding by APO1 mutants. All EMSA gels were run on 8% acrylamide TBE native gels. The listed protein concentrations were allowed to incubate with 50 nM of either ssDNA or the on curve  was dete2, 1.0 mM MgCl2, 20 mM HEPES, pH 7.4, and 5 mM glucose. Visualization of fluorescence was done on a Zeiss LSM-700 inverted confocal microscope using a 40× water immersion objective with a laser intensity of 15–20% and gain set to around 500 units. Excitation wavelengths for Hoechst 33342, eGFP and mCherry were set to 405, 488 and 555 nm, respectively; emission bandpass filters were set to 400–480, 490–555 and 555–700 nm. Images were captured as multichannel 16-bit grayscale intensity images 1012 × 1012 pixels across using two-line averaging and pixel dwell time of 0.8 μs. Resulting fluorescence images were quantified using the LSMtoolbox plugin of FIJI , mouse monoclonal IgG1 anti-HA antibody for A1CF or mouse monoclonal IgG2a anti-α-Tubulin antibody (Genetex) as a loading control. The secondary antibody used was a cy3-labeled ECL plex goat anti-mouse IgG . All antibodies were diluted 1:3000 into PBST + 5% milk for usage. Co-immunoprecipitation on RNase A (100 μg/ml) treated cell lysates expressing only the tested APO1/A1CF co-expression vectors was completed using a classic magnetic bead co-IP kit (Pierce) as per manufacturer’s instructions, using the anti-FLAG antibody as the pull-down target. The subsequent pull-down samples were then stained with the same western antibodies as described.E. coli expression system  showed increased solubility compared to the WT, with the locations of these residues in these six constructs mapped on the structure model and the linear sequence shown in Figure P21212 with eight molecules per ASU  and three small helices . Within an APO1 molecule, the A1HD uses its β-hairpin and h7 and h8 to form hydrophobic interactions with its own CDD, positioning its β-hairpin across the top side of the Zn active center and in close proximity to loops 1 and 7 of the CDD Figure . The welThe A1HD mediates dimerization of APO1 primarily through the pairing of the two β-hairpins via β7 of the two monomers, forming a four-stranded β-sheet connecting the two subunits. The interior face of this β-sheet interacts extensively with helices h6 and h8 of A1HD through hydrophobic residues to create a hydrophobic core between the two APO1 subunits Figure . There aE. coli (ecCDA)   and yeas (ecCDA) . These m (ecCDA) , and the (ecCDA) . However (ecCDA)  were sho (ecCDA) , which l (ecCDA) , consist (ecCDA) .The dimeric structure of APO1 reveals a large positively charged surface spanning across the two paired β-hairpins of A1HDs and branching out to the two active centers near the N-termini Figure . MutatioR/109 viable cells, while the WT APO1 showed a median of 63 RifR/109 viable cells. These observations were also consistent with a previously reported mutagenicity of ∼50 RifR/109 viable cells for untagged human APO1, which is notably far less than the mutagenicity of APO1 from other mammals such as rat and rabbit , WT APO1 alone showed obvious binding to APOB RNA   Figure . For the) Figure . As for ) Figure . W121 is) Figure . The loo) Figure ,72,74. W) Figure , where Y) Figure  seen at ) Figure . Recent ) Figure ,76, A3BC) Figure  and A3GC) Figure ; all for) Figure . As suchin vitro. The result showed that F120A had near-complete abolishment of deaminase activity for both RNA and DNA substrates, while W121A only showed near-complete abolishment of activity on RNA but retained ∼60% of WT activity for deamination on DNA  to interact with RNA in a specific fashion needed for deamination. A more mechanistic understanding of the role of W121 in RNA deamination may require a co-crystal structure of APO1/cofactor binding to substrate RNA in the future.DNA and RNA binding assays showed that the four mutants  had estimated  0.75 μM  and did While the dimer form of APO1 is consistent with the prior biochemistry evidence ,42–44, tAPOB RNA. One caveat is that the activity of L173Q could come from the residual dimer, even though such possibility is low as only the fractions centered around the monomeric peak were used for the activity assay. This result is consistent with a recent report where GFP fused to the C-terminus (APO1–GFP) but not to the N-terminus (GFP–APO1) significantly lowered dimerization, and this APO1–GFP fusion defective in dimerization is still active , on the mSOL construct . Both thl active . To testF Figure . This moF Figure . Residuein vitro poisoned primer extension and further characterize the RNA editing activity of APO1 mutations in a cellular environment, we used an in-cell fluorescence assay to detect and quantify RNA editing activity as previously described  in previous reports , W199A and F205A all had RNA editing activities comparable to WT Figure , consistn Figure . Howevern Figure , implyinn Figure . L173A an Figure . The difThe APO1 molecular surface is very basic and contains a continuous patch of positively charged and polar residues Figure , left, sThe RNA editing result showed that mutants R30A and R197A, like the inactive control F120A mutant and the mCherry only negative control, showed no RNA editing activity Figure . The wesTo investigate whether the reduced RNA editing of these mutations was due to reduced APO1–A1CF binding, co-immunoprecipitation was performed using the FLAG-tagged APO1 that is co-expressed with A1CF in HEK 293T cells. The result showed that, except for L173A, which had slightly reduced A1CF binding, all other tested mutants had WT levels of A1CF binding, including the F120A and W121A Figure . This reHere, we have described the crystal structure of APO1 and its structure-based functional characterization. The APO1 structure reveals an N-terminal CDD that is similar to the canonical APOBEC deaminase domain and an extra well-structured C-terminal A1HD domain with a unique fold Figure . The A1HAPOB-derived RNA that is known to be specifically deaminated by WT APO1  during RNA editing are mutually exclusive events. A previous model proposed for the mechanism of RNA editing by APO1 and its cofactor A1CF is that APO1 cofactor recognizes a targeted RNA through specific structural features of the RNA and then melts the structured RNA to present an unfolded RNA region to APO1 active site for deamination . The detde cells . It is wde cells . Thus, iIn summary, we have determined the crystal structure of APO1 that reveals a canonical deaminase core structure and an extra C-terminal A1HD domain fold unique to APO1. The structure shows how APO1 dimerizes through its A1HD domain through hydrophobic interactions. The stable dimer formation can be disrupted by mutating the interface, but dimerization does not appear to be required for APO1 cofactor binding or deamination on RNA or DNA substrates. The results from the subsequent structure-guided mutational and functional studies using biochemical and cell-based assays provide new insights into the deaminase activity on both RNA and ssDNA substrates and the importance of this C-terminal domain in regulating these enzymatic functions. There are still many interesting questions with regard to how APO1 interacts with its cofactors and recognizes specific target RNA for deamination and other potential biological functions of APO1. The results reported here provide a strong structural foundation for addressing these questions in the future.Coordinates and structure factors have been deposited in the Protein Data Bank with PDB accession code 6X91.zcaa027_Supplemental_FileClick here for additional data file.


Surgery for metastatic pancreatic cancer remains controversial as the survival benefit is questionable. The aim of the present study was to analyze the survival of these patients using data extracted from the surveillance, epidemiology, and end results (SEER) program database. Further, studies on resection for metastatic disease to the lung were systematically reviewed. A total of 11,541 cases with synchronous distant metastasis were analyzed. The median survival of single-organ metastasis was better than of multi-organ metastasis . Single organ lung metastasis had longer median survival times compared to the other sites . Resection of the primary tumor was associated with longer survival in synchronous single-organ metastasis to the lung compared to no resection . A systematic literature review identified 79 cases of metachronous lung metastasis with a survival of 120.0 ± 6.32 months and 83.0 ± 24.84 months following resection of the primary tumor and metastasis, respectively. Lower TNM staging, longer interval to metastasis, and single metastatic lesion correlated with better survival. Resection in highly selected pancreatic cancer patients with synchronous and metachronous lung only metastasis might confer a survival benefit and should be considered on an individual basis. It is the fourth leading cause of cancer related deaths in the US, and it is estimated to be the second leading cause in the 2030s2. The majority of the PDAC patients is diagnosed at an advanced stage, and only about 20% of the patients are potential candidates for oncological resection5. Most of the patients die from peritoneal spread or distant metastasis. In consideration of the relevant risk of postoperative complications and the poor survival, it has generally been accepted that for PDAC patients with metastatic disease, resection of the primary tumor or metastatic lesions is contraindicated. However, with the rapid development of new surgical techniques, more precise and minimally invasive operations, and better perioperative care, morbidity and mortality of pancreatic surgery has decreased in experienced centers6. New chemotherapeutic regimens also significantly prolong the survival of PDAC patients with metastatic tumors8. Thus, especially in the context of multimodal therapies, surgery for PDAC patients with metastasis, especially those with oligometastatic and metachronous disease, has gained interest10. Several studies have reported that for selected cases with distant metastasis, resection might relevantly prolong survival, compared to palliative treatment11.Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignant tumors with a rising incidence in both economically developed and developing countriesIn this study, we utilized the surveillance, epidemiology, and end results (SEER) program database and relevant reported clinical studies and case series to comprehensively generate evidence of the impact of surgery for PDAC patients with distant metastasis.A total of 11,541 patients with synchronous distant metastasis with definitive information of the site of metastasis in the liver, lung, bone and/or brain were included. A total of 9372 cases (81.2%) had single organ metastasis, 1911 cases (16.6%) had two organ metastasis, 250 cases (2.2%) had metastasis in three organs and 8 cases had metastasis in all four analyzed organs. Of the 9372 cases of single-organ metastasis, 8222 cases (87.7%) were liver, 941 cases lung (10.1%), 191 cases (2.0%) bone, and 18 cases (0.15%) brain metastasis. In 7156 out of the 9372 cases of synchronous single-organ metastasis, information was available whether the patients were operated/resected or not  of the 11,541 patients was 4.0 ± 0.07 months . The 1-year, 2-year, and 3-year survival rates were 13.9%, 2.6% and 0.6%, respectively Fig. . After mp = 0.015). Well or moderate differentiation predicted better survival compared to poorly/undifferentiated tumors . Lung metastasis had better survival than other metastasis   Table . Tumor l3) Table , older a3) Table , metasta3) Table  and poor3) Table  correlat3) Table .Table 1Up < 0.0001; liver metastasis, 10.0 ± 1.16 months vs 4.0 ± 0.09 months, p < 0.0001) . (ii): metachronous metastasis from unresectable PDAC, with non-surgical treatment (1 study). Kruger et al.12 reported 5 cases with a median survival after diagnosis of metastasis of 10.7 months . (iii): metachronous metastasis after radical resection of the primary tumor, with non-surgical treatment of the metastasis (8 studies); These 8 studies reported on 212 cases. The median survival after initial resection and diagnosis of lung metastasis ranged from 23.0–50.5 months and 7.5–31.3 months, respectively. (iv): metachronous metastasis after resection of the primary tumor, with surgical treatment of the metastasis (17 studies). These 17 studies (including 154 cases) reported a median survival after the initial resection and resection of metastasis of 19.0–92.0 months and 16.0–37.0 months, respectively.Four scenarios of single-organ lung metastasis were initially analyzed Table . (i): syOf those 154 cases from 17 studies, only 79 cases from 11 studies were available with complete survival information for each case and were extracted for further quantitative synthesis Tables  and S3. 14. For pancreatic cancer with metastasis, resection of either the primary tumor or metastasis is generally not indicated; however, for cases with limited (oligo-) metastatic lesions, which could potentially be resected, surgery has been advocated on an individual basis in some experienced pancreatic cancer centers15.Radical resection of both primary and metastatic lesions has been carried for an increasing number of tumor types with promising results18. Obviously, the most important point is to select those patients that might benefit from this aggressive surgical approach17.One reason being that during the last years, more effective chemotherapeutic regimens, such as Folfirinox or gemcitabine plus nab-paclitaxel, have significantly prolonged the survival of metastatic pancreatic cancer patients. In case of initially unresectable cases or limited metastatic spread, these effective therapies have given some patients the chance for resection of the primary tumor or resection of both primary tumor and metastasis21.Although the liver is the most common site of distant metastasis of pancreatic cancer, the lung ranks second. Single-organ metastasis to the lung are not common, but several studies of small cohorts have reported a better prognosis than for other types of metastasis, including distant lymph node metastasis, liver metastasis, and peritoneal metastasis, which indicates that the metastatic site is another important aspect which should be taken into account when considering surgical treatment for metastatic pancreatic cancer patients22. We have also shown that for synchronous single-organ metastasis, older age, worse differentiation, and non-resection of the primary tumor were independent risk factors for worse survival and that survival was better for resected versus non resected synchronous single-organ metastasis to the lung and liver.According to our SEER database analysis, synchronous multi-organ metastasis had worse survival compared to single-organ metastasis, and synchronous single-organ metastasis to the lung had better survival compared to other metastatic sites. This is in line with a recent analysis that also demonstrated better survival for isolated lung metastasis compared to isolated liver metastasisMany factors, such as the general physical status, tumor burden, any additive/palliative chemotherapy/radiotherapy among others, could affect patient survival. Information regarding these items are either absent or incomplete in the SEER database. Importantly, information regarding chemo-/radiotherapy is insufficient and it is therefore officially suggested not to analyze its impact on survival. Together, the extent of selection and treatment bias is difficult to estimate. Obviously, only few patients with synchronous single-organ metastasis to the lung or the liver would benefit from resection. The present study provides some information on how to select potential candidates for resection. For both multi-organ and single-organ metastasis, younger age and better tumor differentiation were independent factors for increased survival in the SEER database analysis.A systematic review of the available clinical reports on resection for metachronous single-organ lung metastasis revealed a striking median survival of 83.0 months (95% CI 34.31–131.69 months) after resection of metastasis. Single metastatic lesions, initial lower TNM staging, and longer interval from primary resection to recurrence were associated with increased survival, although this was not statistically significant.Since resection of pulmonary metastasis can be carried out safely with low morbidity and mortality rates, resection could be considered for those patients with a longer interval between resection of the primary tumor and detection of lung-only metastasis, better differentiated tumor, younger age, and single metastatic lesions.23. Thus, pancreatic cancer with Her-2 amplifications were noted to have less frequent liver metastases and an increased frequency of lung and brain metastasis24. SMAD4 loss was more common in patients with widespread metastatic disease than locally advanced tumors25. Molecular analysis has helped to identify those patients that may benefit from resection of metastasis of colorectal cancer25. In the future, molecular subtyping might also help to identify key signaling pathways and potential surrogates to select metastatic pancreatic cancer patients for whom surgical resection might be beneficial28. Since there are no prospective, randomized trials, solid evidence of criteria for patient selection is lacking.It has been reported that the occurrence of pulmonary metastasis is generally later than that of liver metastasis, and it has also been proposed that the biological behavior of PDAC presenting with a first site of metastasis other than the liver may be related to its molecular phenotypeAlthough this is a comprehensive retrospective study based on the SEER database and a systematic analysis of published reports, there are limitations. (1) Data of systemic treatment of the patients included in the SEER database is incomplete. Therefore, important prognostic factors could not be analyzing in this context. (2) There is a substantial selection bias regarding patients that were resected and those that were not resected in the SEER database. (3) There are no randomized clinical trials and all the published reports were either case series or case reports, and (4) published cases were highly selected, resulting in a significant selection bias.In conclusion, most pancreatic cancer patients with distant metastasis have a poor prognosis. In contrast to other metastatic sites, resection of synchronous and metachronous single-organ lung metastasis shows promising results in terms of safety and survival in highly selected patients. Younger age, better tumor differentiation, longer interval between the initial resection and diagnosis of metastasis, and single metastatic lesions predict better outcome, and all patients should be treated with chemotherapy first. Patient selection is important as expansion of this practice may offer little advantage or even harm patients.Further, it is currently not clear whether there is a true benefit for resection, or whether the data merely reflect a selection bias of patients with a better prognosis independent of resection. Clinical trials or at least multi-center, prospective data are needed to evaluate the outcome of surgical treatment for both synchronous and metachronous single-organ lung metastasis in pancreatic cancer patients.The clinicopathological, surgical and survival information of PDAC patients with distant metastasis were extracted from the SEER database (2010–2014) by using the SEER* Stat 8.3.4 software. The inclusion criteria were as follows: (1) Patients had to be 20 years or older, be pathologically diagnosed with PDAC from January 1, 2010 to December 31, 2014; (2) patient had active follow-up, and did not suffer any other kind of malignant tumor; (3) the information of metastasis in liver, lung, bone, and brain was available. The exclusion criteria were as follows: (1) Patient died from any other cause which was not related to PDAC; (2) metastasis at any other site, besides liver, lung, bone and brain; (3) the information of surgery for the primary tumor or metastatic lesion was missing.The setting of mining strategies of the SEER database was as follows: “cases in research database”, “known age”,“age at diagnosis = 20–85 + ”, “year of diagnosis = 2010–2014”,“site recode ICD-3/WHO 2008 = pancreas”, “malignant behavior”, “diagnostic confirmation = microscopically confirmed”, “ICD-0-3His/behav, malignant = 8140/3 and 8500/3”. Details of the data extraction are presented in Fig. The following variables were extracted: Patient ID, age, sex, year of diagnosis, vital status recode, survival months (>0), COD to site recode, SEER cause-specific death classification, type of follow-up expected, survival months flag, CS mets DX-bone (2010+) (bone metastasis), CS mets at DX-brain (2010+) (lung metastasis), CS mets at DX-liver (2010+) (liver metastasis) and CS mets at DX-lung (2010+) (lung metastasis) and RX Summ–Surg Prim Site (1998+)(surgery for primary site), RX Summ–Scope Reg LN Sur (2003+) (surgery for lymph node) and RX Summ–Surg Oth Reg/Dis (2003+) (surgery for metastatic lesion or the others). All patients were initially diagnosed as stage IV, thus all patients had synchronous metastasis. Since the information of adjuvant treatment is largely incomplete and sometime it could be misleading, it is officially suggested not to analyze the survival impact of adjuvant treatment by using data from SEER database. Therefore, in this study, the information of adjuvant treatment was not analyzed.st, January 1990 and 1st, October 2019) and Science Citation Index Expanded database . The Cochrane Central Register of Controlled Trials (CENTRAL) and ClinicalTrial.org  were also searched to identify randomized controlled trials and non-randomized studies following the Preferred Reporting Items for Systematic reviews and Meta-analysis (PRISMA) guidelines. Both non-randomized  and randomized studies were collected for further analysis. Conference abstracts were excluded.We comprehensively searched the published English literature for cases of PDAC with lung metastasis in the Medline database .Disease specific survival (DSS) was defined as duration from the date of diagnosis to the date of death and recorded as median value ± standard error (SE) with 95% confidence intervals (CI). Survival time was defined as censored if the patients were alive at the last follow-up. The DSS were analyzed by using Kaplan-Meier curves and log-rank tests. Multivariate analysis was performed with a logistic regression model to identify factors predicting survival. All tests were two-tailed and a Supplemental Information.


Partitiviridae sequences found in this study were classified into two genera, which have both plant- and fungi-infecting partitiviruses. This observation suggests that the lichen provides an opportunity for horizontal transfer of these partitiviruses among microbes that form the lichen consortium.The lichen is a microbial consortium that mainly consists of fungi and either algae (Viridiplantae) or cyanobacteria. This structure also contains other bacteria, fungi, and viruses. However, RNA virus diversity associated with lichens is still unknown. Here, we analyzed RNA virus diversity in a lichen dominated by fungi and algae using dsRNA-seq technology and revealed that partitiviruses were dominant and active in the microbial consortium. The  The lichen is a symbiotic microbial consortium that is mainly composed of a fungus (mycobiont) and photosynthetic partner (photobiont), either green algae (Viridiplantae) or cyanobacteria, or both, that harbor chlorophyll. The phototrophic partner feeds organic compounds to the fungus. In contrast, the fungus protects minute photosynthetic cells from environmental stresses, such as drought and nutrient deficiencies, and provides a suitable environment for photosynthesis and gas exchange . In geneRhabdoviridae) and Apple mosaic virus (family Bromoviridae) were detected by RT-PCR from lichens (Chrysothrix chrysovirus 1 (family Chrysoviridae) and Lepraria chrysovirus 1 (family Chrysoviridae) were identified from Chrysothrix chlorina and Lepraria incana lichens, respectively, and the former was observed in accompanying endolichenic fungus in the lichen by in situ hybridization  have been established in this family. Among them, host of Gammapartitivirus and Deltapartitivirus are identified as fungi and plant, respectively, and some of the species in Alphapartitivirus and Betapartitivirus infect fungi or plant  was sampled in February 2019 . Sample The lichen sample was disrupted in liquid nitrogen in a mortar. For dsRNA extraction and purification, the ISOVIRUS  kit was used. In brief, total RNA was extracted in the extraction buffer, and dsRNA was purified with cellulose resin and eluted by nuclease-free water after DNase I treatment according to the manufacturer’s protocol. To obtain total RNA, the TRIzol Plus RNA Purification Kit  was used according to the manufacturer’s protocol. Total RNA was treated with DNase I (Invitrogen) and further purified using the RNA Clean and Concentrator-5 Kit .In this study, we applied FLDS to obtain RNA viral sequences. dsRNA, which is a molecular marker of RNA virus infection, is used as a template for cDNA synthesis because cellular long dsRNA is a replicative intermediate of ssRNA virus as well as genome of dsRNA virus. Simultaneously, full-length cDNA is synthesized by applying template-switching activity of the reverse transcriptase with an oligonucleotide primer against adapter-ligated dsRNA fragments, which enabled us to obtain complete genome sequences of non-retro RNA viruses.Sequencing libraries were constructed as described previously , 2018b. Illumina sequence libraries were constructed from the double-stranded cDNAs. Double-stranded cDNAs were fragmented using Covaris S220 , and fragmented cDNAs were applied to KAPA Hyper Prep Kit Illumina platforms . The quality and quantity of the Illumina libraries were evaluated using the KAPA library quantification kit (Kapa Biosystems) and applied to the Illumina MiSeq platform  according to the manufacturer’s protocol (600-cycle kit to perform 300-bp paired-end sequencing).e-value ≤ 1 × 10–5. Sequences that matched a known RNA-dependent RNA polymerase (RdRp) gene by BLASTX with an e-value ≤ 1 × 10–5 were collected from RNA virus contigs and segments. In addition, a conserved domain database (CDD) search was also used. Nucleotide sequences encoding the RdRp gene were clustered at 90% identity using VSEARCH  and Short Read Archive database (accession no. DDBJ: DRA009807).Sequences obtained in this study are available in the GenBank database repository   using phde novo assembly and reconstruction of the full-length genome segments, a total of 65 RdRp-encoding operational taxonomic units (OTUs)  were identified from the dsRNA-seq library  suggested that 17 of 65 OTUs were related to Partitiviridae. In addition, viruses related to seven dsRNA virus families , three ssRNA virus families , and one unclassified RNA virus family (Polymycoviridae) were also identified , and additional unknown proteins. The genome structures of LpaRV1–6 resemble those of partitiviruses, which are known to have a bisegmented genome encoding RdRp and CP in each segment . Phylogeiviridae . To dateiviridae . The phyor fungi . It is nor fungi , it was Partitiviridae but not classified into the family , and the LRV1-FgV4 cluster was not clarified. Therefore, we could not provide family-level classification of LRV1 as in the case of FgV4.The published top hit sequence of LRV1 in the BLASTX search was Fusarium graminearum virus 4 (FgV4), which was shown to be closely related to e family . The phyPartitiviridae or its relative group. Among non-retro RNA viruses, only Partitiviridae and Endornaviridae harbor the genera including plant and fungal viruses. Although a single partitivirus that can infect both plant and fungal hosts has never been reported, recent studies revealed that a few viruses belonging to another RNA virus family can infect both plant and fungal hosts. For instance, cucumber mosaic virus, a positive-sense ssRNA virus belonging to the genus Cucumovirus in the family Bromoviridae, was discovered to infect both plants and fungi  and Short Read Archive database (accession no. DDBJ: DRA009807).Sequences obtained in this study are available in the GenBank database repository (accession nos. DDBJ: SU, TN, ND, and YM designed the research. FK and MH performed the research. ND and YM collected samples. YT, YC, and SU analyzed the data. All the authors listed wrote the manuscript. All authors contributed to the article and approved the submitted version.This research is a joint project funded by Nippon Gene Co., Ltd. In this research, we used ISOVIRUS, a product sold by Nippon Gene Co., Ltd. YM and ND are employed by Nippon Gene Co., Ltd. The remaining 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.


S haplotypes previously determined in Japanese citrus accessions, sharing characteristics such as isoelectric point, extracellular localization, molecular weight, intron number and position, and tissue-specific expression with S-RNases. One T2 RNase gene in self-incompatible Hyuganatsu was significantly down-regulated in the styles of a self-compatible mutant of Hyuganatsu in RNA-seq and qPCR analyses. In addition, the inheritance pattern of some T2 RNase genes was consistent with the pattern of the S haplotype in the progeny population of Hyuganatsu and Tosabuntan. As all results supported citrus self-incompatibility being based on S-RNase, we believe that six T2 RNase genes were S-RNases. The homology comparison between the six T2 RNases and S-RNases recently reported in Chinese citrus revealed that three out of six T2 RNases were identical to S-RNases from Chinese citrus. Thus, the other three T2 RNases were finally concluded to be novel citrus S-RNases involved in self-incompatibility.Several citrus varieties show gametophytic self-incompatibility (GSI), which can contribute to seedless fruit production in several cultivars. This study investigated the genes regulating this trait through RNA-seq performed using styles collected from the flowers of Japanese citrus cultivars ‘Hyuganatsu,' ‘Tosabuntan,' ‘Hassaku,' ‘Banpeiyu,' and ‘Sweet Spring'. We screened the transcripts of putative T2 RNases, i.e., the protein family including all S-RNases from S-RNase-based GSI plants, and constructed a phylogenetic tree using the screened T2 RNases and S-RNases retrieved from citrus genome databases and a public database. Three major clusters (class I–III) were formed, among which, the class III cluster contained family specific subclusters formed by S-RNase and a citrus-specific cluster monophyletic to the S-RNase clusters. From the citrus class III cluster, six transcripts were consistent with the  S phenotype determination: gametophytic self-incompatibility (GSI) and sporophytic self-incompatibility (SSI), in which incompatible pollen is determined by its own haploid genome or the diploid genome of the plant (sporophyte) that produced it, respectively  is a genetic mechanism that causes pistils to reject self-pollen or pollen from close relatives, avoiding a decrease in genetic variability and adaptive potential by self-fertilization  allele  and SC of mandarin (C. reticulata) by the transition of SI to SC through S-RNase mutation , ‘Hassaku' (C. hassaku hort. ex Tanaka), ‘Banpeiyu' (C. maxima), and ‘Tosabuntan' (C. maxima or C. ootachibana hort. ex Tanaka) showed SI  in Rosaceae, which are S-RNase-based GSI plants  in a recent genome analysis , locus structures, and diversifying selection  had common features to S-RNases, such as close phylogenetic position, pistil-specific expression, pI > 8, and a single intron in the genomic structure  and its SC mutant (HYSC), ‘Tosabuntan' , ‘Banpeiyu' , ‘Hassaku'  and ‘Sweet Spring' .Plant materials used in this study were grown in the field of the University of Miyazaki : SI ‘HyuDuring the flowering season of 2016, styles from TBN, BAN, HSK, and SWS were collected from flowers at the balloon stage. In 2017, additional styles were collected from HYSI and HYSC. Sample collection was duplicated in 2016 and triplicated in 2017 from the same tree on different dates. Young leaves were collected from all the cultivars. Floral and fruit tissues were separately collected from HYSI, i.e., stigma, style, ovary, anther, filament, petal, sepal and pedicel, albedo, flavedo, and juice sac. All samples were quickly frozen in liquid nitrogen immediately after collection and stored at −80°C until further use.In 2018, HYSI × TBN and reciprocal outcross TBN × HYSI were performed. Seeds were collected and sown in soil to germinate seedlings. Leaves of the parents (HYSI and TBN) were collected for DNA extraction.Total RNA was extracted from approximately 100 mg of style tissue using NucleoSpin RNA Plus  from all the cultivars according to the manufacturer's protocol. RNA was quantified using a NanoDrop 2000 (ThermoFisher) and Qubit 3.0 Fluorometer .For the style RNA samples collected in 2016, eight Illumina sequence libraries were constructed for each sample using the KAPA Stranded mRNA-Seq Kit (KAPA Biosystems) and sequenced using Illumina HiSeq 4000 to obtain 10M PE100 reads. For the HYSI and HYSC samples obtained in 2017, RNA-seq was performed for six libraries by Novogene to obtain 40M PE150 reads. Samples collected in 2016 and 2017 had two and three replications, respectively.de novo assembly. Reads from different replicates of a single cultivar were pooled for de novo assembly of the transcriptome. The pooled reads of each cultivar were used as input for a Galaxy-based version of Trinity  and Shiikwasha (C. depressa Hayata) are the only indigenous citrus species in Japan  and LC575202–LC575209 (https://getentry.ddbj.nig.ac.jp/).The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: CH conceived the project, conducted experiments, and wrote the manuscript. KU performed RNA-seq library construction and data acquisition. MA and SI conducted PCR and electrophoresis in inheritance analysis. QY helped with qPCR analysis and revised the manuscript. FG supervised the project and revised the manuscript. TT supervised the project. All authors contributed to the article and approved the manuscript before submission.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.