Thus, the shared FinO domain might bear an unusual plasticity enabling it to act either selectively or promiscuously on the same cellular RNA pool. One caveat to this model is that the full suite of in vivo targets of the assumedly highly selective FinO protein is unknown. Here, we have extensively profiled cellular transcripts associated with the virulence plasmid-encoded FinO in Salmonella enterica. While our analysis confirms the FinP sRNA of plasmid pSLT as the primary FinO target, we identify a second major ligand: the RepX sRNA of the unrelated antibiotic resistance plasmid pRSF1010. FinP and RepX are strikingly similar in length and structure, but not in primary sequence, and so may provide clues to understanding the high selectivity of FinO-RNA interactions. Moreover, we observe that the FinO RBP encoded on the Salmonella virulence plasmid controls the replication of a cohabitating antibiotic resistance plasmid, suggesting cross-regulation of plasmids on the RNA level.FinO-domain proteins represent an emerging family of RNA-binding proteins (RBPs) with diverse roles in bacterial post-transcriptional control and physiology. They exhibit an intriguing targeting spectrum, ranging from an assumed single RNA pair (FinP/ Proteins that carry a FinO domain have recently garnered much attention as a new class of widespread RNA-binding proteins (RBPs) in bacteria ,2. To daIn vivo interactome studies of ProQ, a prominent member of this class, found that this RBP recognizes hundreds of small noncoding RNAs (sRNAs) and mRNAs in Escherichia coli, Neisseria meningitidis and Salmonella enterica  and several trans-encoded target mRNAs in the competence regulon  of traJ while the paired RNAs undergo co-degradation by RNase III  the cellular ligands of FinO and ProQ in a transcriptome-wide manner. On the one hand, we confirm that unlike the global RBP ProQ expressed from the Salmonella chromosome, the FinO protein of Salmonella plasmid pSLT has a restricted target suite. On the other hand, we make the surprise discovery of a yet another major sRNA target of FinO. Intriguingly, this sRNA to which we will refer as RepX originates from the replication control locus of an unrelated Salmonella plasmid. While distinct in terms of primary sequence, RepX is similar to FinP in length and RNA structure, which could help to understand the high specificity of FinO-RNA interactions. Most importantly, Salmonella FinO not only regulates the conjugation of its own plasmid, but through the RepX RNA also influences another mobile element in the same cell.As efforts to understand the molecular basis of RNA target recognition and selectivity by FinO domain proteins gain new momentum ,13,29,30 or RocC ,6,10. ToSalmonella enterica serovar Typhimurium SL1344  1:2000 for both FinO-3×FLAG and ProQ-3×FLAG detection, as loading control GroEL was detected with anti-GroEL , anti-mouse  and anti-rabbit  conjugated to horseradish peroxidase were used as secondary antibodies for anti-FLAG and anti-GroEL respectively. For detection, ECL™ Prime Western Blotting Detection Reagent  served as a substrate.Bacterial cultures were grown to the desired conditions. A volume of cells that represents 1 OD600 nm was collected, and total RNA extracted by hot phenol method followed by a DNase treatment. Samples of 10 μg of total RNA were subjected to electrophoretic separation in Tris–borate–EDTA (TBE) 6% acrylamide gels containing 8.3 M urea. RNAs were transferred to Hybond N+  filters. Transcripts of interest were detected by hybridization with 5′ radiolabeled oligos as probes. For oligonucleotide labeling, 10 pmol of the oligonucleotides used as probes were 5′-labeled with 10 μCi of 32P-γ-ATP by PNK  in a 20 μl reaction for 1 h at 37°C. Labeled oligonucleotides were ran over Microspin G-25 columns  to remove unincorporated 32P-γ-ATP. Radioactive signal was imaged with the Typhoon FLA 7000 . Oligonucleotides used as probes are listed in Strains of interest were grown to the desired conditions. The biomass of 4 units of OD600 nm 2.0. At t0, a volume equivalent to 4 ODs of cells was collected for total RNA extraction. Rifampicin to a concentration of 500 μg/ml was added to the cultures to stop transcription. Samples at different time points were collected. A volume of 4 ODs of cells was added to a 14 ml falcon tube containing stop solution (95% ethanol 5% phenol) and snap frozen in liquid nitrogen. Samples were stored at –80°C and when processed, further subjected to total RNA extraction. Extracted RNA was visualized by northern blot. The stability of FinP and RepX was determined. Ribosomal 5S RNA was detected as loading control.Bacterial cultures were grown to ODSalmonella enterica WT and ΔfinO as previously shown with minor modifications (finO was quantified by qPCR for 1 × 105 cells by amplifying a 140 nt DNA site in the chromosome as reference (crp locus) and in pRSF1010 (repA locus) using Power SYBR Green (Applied Biosystems). Average quantification in WT samples was set to 1 to determine fold change in the ΔfinO strain. Oligonucleotides used are listed in The relative plasmid copy number of pRSF1010 was determined in ications ,37. Shor600 nm. A volume of cells equal to 50 ODs was pelleted. Cells were resuspended in 800 μl of lysis buffer  supplemented with 8 μl of DNase I. The suspension was then subjected to mechanical lysis by 0.1 mm glass beads at 30 Hz for 10 min in the Retsch MM200. The lysate was cleared by centrifugation. A volume equivalent to 0.5 OD600 nm was diluted to 90 μl with 1× Laemmli buffer (lysate protein sample) and stored at −20°C. A volume equivalent to 5 OD600 nm was saved for RNA extraction with TRIzol (lysate RNA sample). The remaining lysate was then incubated with 25 μl (1 μl/2 OD600 nm of cells) of monoclonal anti-FLAG M2 (Sigma #F1804) at 4°C with rotation for 30 min. The sample was then added to 75 μl of pre-washed Protein A sepharose beads (Sigma-Aldrich) and further incubated with rotation at 4°C for an additional 30 min. The sample beads were spun down. From the supernatant, representing the flow through, a volume equivalent to 0.5 OD600 nm was diluted to 90 μl with 1× Laemmli buffer (flowthrough protein sample) and stored at −20°C. A volume equivalent to 5 OD600 nm was saved for RNA extraction with TRIzol (flowthrough RNA sample). Next, the beads were washed with lysis buffer for five times and protein and RNA was eluted from the beads. From the last wash, a volume equivalent to 0.5 OD600 nm was diluted to 90 μl with 1× Laemmli buffer (wash protein sample) and stored at −20°C. A volume equivalent to 5 OD600 nm was saved for RNA extraction with TRIzol (wash RNA sample). The beads were resuspended in 532 μl of lysis buffer. To a volume of 32 μl, 8 μl of 5× Laemmli buffer was added and stored at −20°C (elution protein sample). RNA-coimmunoprecipitated was extracted by phenol:chloroform:isoamyl alcohol (P:C:I)  extraction. The purified RNA samples were treated by DNase I and purified by P:C:I extraction. Samples were resuspended in H2O to a final concentration of 1 OD/μl for elution samples and 0.1 OD/μl for lysate, flowthrough and wash. Resulting RNA samples were subjected to visualization via northern blot or quantification via deep sequencing. Protein extracts were further analyzed by western blot.RNA-coimmunoprecipitation was carried out as previously described ,38. BrieLibrary preparation from co-immunoprecipitated RNA samples were carried out with NEBNext Multiplex Small RNA Library Prep Set (New England Biolabs) that allow sequencing in Illumina platforms. The library preparation was carried out in a thermocycler and manufacturer's guidelines were followed with minor modifications. Briefly, 3.5 μl of RNA was mixed with 1 μl of 3′ SR Adaptor (1:10 diluted) and incubated at 70°C for 2 min. The tubes were transferred to ice and then, 6.5 μl of 3′ ligation mix  were added to each tube and incubated at 25°C for 1 h (without lid heated). 2.75 μl of SR mix  was added to the samples and incubated as follows: 75°C for 5 min (lid 85°C), 37°C for 15 min (no lid heated), 25°C for 15 min (no lid heated). For ligation of the 5′ adaptor the 5′ ligation mix was added to each sample ) and incubated at 25°C for 1 hour (no lid heated).To generate the first strand of cDNA 5 μl of the cDNA mix  was added to each reaction and incubated at 50°C for 1 h followed by 15 min at 70°C to inactivate the RT enzyme. 10 μl of cDNA was PCR amplified with barcoded NEB Index primer (1:10 diluted) and SR primer (1 :10 diluted) in a 50 μl volume reaction . The following PCR program was carried out: 30s at 94°C for initial denaturation, 15s at 94°C, 30s at 62°C and 70°C for 16 or 20 cycles, and a final elongation of 5 min at 70°C. PCR products were purified via AMPure XL beads. The distribution of the library and concentration of library DNA was determined by DNA bioanalyzer and a DNA Qubit measurement respectively. Amplified cDNAs from different libraries were pooled and sequenced on an Illumina NextSeq 500 platform at the Core Unit SysMed at the University of Würzburg.Salmonella enterica serovar Typhimurium strain SL1344 genome: chromosome, NC_016810.1; three plasmids: pCol1B9, NC_017718.1; pRSF1010, NC_017719.1; pSLT, NC_017720.1. Annotation file of pRSF1010 plasmid was amended with the coordinates of the RepX sRNA prior to further data analysis.Approximately 5 million single end 75 bp reads were sequenced per library. Generated FASTQ files were mapped to the P-value and the Benjamini-Hochberg to adjust P-values (P-adj) (2fold 2.0 (4-fold) with P-adj <0.05 were considered significantly enriched for RIP-seq of ProQ-3×FLAG and FinO-3×FLAG at OD600 nm 2.0  the read coverage normalized to TPM of transcripts enriched over 4-fold was used to represent FinO and ProQ occupancy through the growth curve  that takes into account sequencing depth (number of mapped reads) and transcripts length. Enrichment factors were calculated with DESeq2 . RNA coi (P-adj) . Enrichm0 nm 2.0 . For samth curve .6-3C-FinO. Purification of FinO was carried out as previously described for FopA at the recombinant protein expression facility, Rudolf-Virchow-Center Würzburg (The purification of recombinant FinO was carried out using the expression plasmid pMiG007-HisWürzburg .in vitro transcribed in a T7 transcription reaction using the MEGAscript T7 Transcription kit (Thermo Fisher Scientific). As template, 200 ng of the mix of oligonucleotides JVO-18275/ JVO-18276 and JVO-18281/ JVO-18282 was used for FinP and RepX respectively. In vitro transcribed RNA was subjected to electrophoresis in Tris–borate–EDTA (TBE) 6% acrylamide gels containing 8.3 M urea. RNA bands of interest were excised from the gel and eluted in RNA elution buffer  at 4°C overnight. Eluted RNA was further isolated via P:C:I extraction and precipitated in EtOH. Then, 50 pmol of RNA was dephosphorylated with 10 units of calf intestinal phosphatase  in a 50 μl reaction at 37°C for 1 h. Subsequently, CIP-treated RNA was again isolated by P:C:I extraction and EtOH precipitated. In a 20 μl reaction, 20 pmol of the dephosphorylated RNA was 5′-labeled with 20 μCi of 32P-γ-ATP by PNK  for 1 h at 37°C. Labeled RNA was further purified via Microspin G-50 columns  to remove unincorporated 32P-γ-ATP. Labeled RNA was subjected to an additional gel purification step, full length labeled FinP and RepX bands were excised from the gel and eluted in RNA elution buffer  at 8°C overnight. Eluted RNA was further isolated via P:C:I extraction and precipitated in EtOH. Labeled RNA FinP* and RepX* was resuspended in H2O and stored at –20°C. Alternatively, in vitro transcribed RNA was labeled at the 3′ end as previously described  diphosphate (pCp) and T4 RNA ligase I (New England Biolabs). To allow FinO binding, the 3′end of labeled FinP* and RepX* were dephosphorylated via an additional PNK dephosphorylating step as previously described (FinP and RepX sRNA were escribed . Shortlyescribed . Dephosp2) with increasing concentration of FinO protein  in the presence of 1 μg of Yeast RNA/reaction (Ambion) for 1 hour at 37°C. Reactions were stopped by adding 5× RNA native loading buffer  and separated on a native 6% polyacrylamide gels at 4°C in 0.5% TBE at constant current of 40 mA for 3–4 h. For visualization, gels were vacuum dried and signals detected on a Typhoon FLA 7000 phosphoimager . The apparent Kd of FinO binding to FinP or RepX was determined by quantifying the intensity of the bound and unbound fraction by FinO for FinP and RepX on ImageJ. The percentage of RNA bound was plotted as a function of FinO concentration and represented values were fitted to a curve followed by a nonlinear regression. The apparent Kd was determined as the concentration at which 50% of the labeled FinP* or RepX* is bound by FinO in our assay.For the electrophoretic mobility shift assay (EMSA), labeled RNA was denatured by heating at 95°C for 1 min and cooling the samples in ice. For binding, 4 nM of FinP* or RepX* were incubated in binding buffer  in the presence of 1 μg of yeast RNA/reaction (Ambion) for 1 h at 37°C. Reactions were stopped by adding 1× RNA native loading buffer and separated and visualized as described above. Alternatively, the binding of FinP* to FinO was further assessed by classic filter binding assay (2) in the presence of 1 μg of Yeast RNA/reaction (Ambion) for 30 min at 37°C. The binding of the labeled FinP* to FinO (5 μM) was competed with increasing concentration of unlabeled counterpart RNA, i.e. RepX or RepX C-U . Samples were filtered through a 0.2 μm pore size nitrocellulose membrane  and a Hybond+ membrane in a Dot blot apparatus. For washing, 200 μl of binding buffer in absence of yeast RNA was used. FinO bound to RNA is visualized as immobilized RNA in the nitrocellulose membrane while unbound RNA is visualized as bound RNA to the Hybond+ membrane.Where applicable, the binding of the labeled RNA (FinP* or RepX*) to FinO (500 nM) was competed with increasing concentration of non-radiolabeled counterpart RNA (FinP or RepX) . The samples were incubated in binding buffer  and 1 μg of yeast RNA in 10 μl. The reactions were then left untreated (Ctr) or treated with 5 mM lead acetate (PbII)  in a 10 μl reaction. For the T1 ladder, 1 pmol of RNA was denatured in water for 1 min at 95°C followed by addition of RNase T1 enzyme and incubated for 3 min at 37°C. All reactions were stopped by addition of GLII RNA loading buffer . Samples were boiled at 95°C and RNA was phenol:chloroform:isoamyl alcohol (P:C:I)  extracted and ethanol precipitated. Samples were resuspended in H2O and mixed with GLII RNA loading buffer. Samples were then boiled at 95°C and loaded on a 10% polyacrylamide 7 M urea gel and separated for 3 h at 45 W. As for the EMSA, for visualization, gels were vacuum dried and signals detected on a Typhoon FLA 7000 phosphoimager .RNA structure probing of FinP* and RepX* was carried out as previously described ,14. ShorFinP and RepX sequence from pSLT and pRSF1010 respectively was used as input in GLASSgo ,43. Outpin vivo, we chose the Salmonella enterica model strain SL1344, which carries three plasmids: pSLT, pCol1B9 and pRSF1010  protein, thus slightly smaller than the ProQ protein , which is expressed from the Salmonella chromosome , i.e., our standard condition in several previous RBP studies  enrichment of FinP with FinO , but was covered by few reads (0.007%), which is likely due to its overall low cellular abundance.Next, we performed RIP-seq in the early stationary phase of growth  enrichment of another transcript: the 74-nt RepX sRNA expressed from pRSF1010, i.e., the third plasmid in strain SL1344 Figure . RepX cotraJ and included a previously unknown major ligand, the RepX sRNA.Contrasting the enrichment of these two plasmid sRNAs by FinO, the ProQ coIP showed the expected enrichment of chromosomally encoded sRNAs, with SibA, SibD and RyfD being most abundant  Figure . We noteThe above RIP-seq analysis provided a snapshot of the FinO ligands in a single phase of growth. To analyze FinO ligands more comprehensively, we performed RIP-seq at five additional points along the bacterial growth curve, from early exponential growth into deep stationary phase Figure . Using wSalmonella enters late stationary phase. FinP is the most abundant target in all conditions, with 95% of the reads at the two early growth phases to over 80% at later time points. Altogether, FinP and RepX represent over 99% of the enriched sRNAs by FinO in all growth conditions.The coIP time-course showed that FinP and RepX are indeed the major FinO ligands in all tested growth phases Figure . Of noterho mRNA ..repA-repd Figure , and obsE. coli, FinO protects the FinP sRNA from RNase E-mediated degradation and therefore positively regulates its stability , using the ratio between pRSF1010 plasmid DNA and chromosomal DNA as a proxy for specific plasmid abundance in the cell. We observed an increased plasmid/chromosome ratio in the ΔfinO strain as compared to WT, suggesting that absence of FinO alleviates the RepX-mediated repression of pRSF1010 replication   Figure . The Rep of RepX . Thus, iechanism , indicatin vivo  for FinP and RepX in this type of binding assay ), which cleaves single stranded RNA, confirms that FinP forms two stem loops Figure . PromineP Figure . No digeP Figure . In the P Figure . RepX di0 Figure . In cont0 Figure .in vitro structure probing was also carried out with 3′ end-labeled versions of these RNAs. As shown in Figure 70, is mildly protected from Pb(II)-induced cleavage in the presence of FinO. A protection of the base of SLII and the 3′tail, downstream of C61, is also observed for RepX  other transcripts in the cell. FinO, the founding member of this RBP class, was particularly interesting for its previously assumed specialized activity on two transcripts, the in vivo target data for FinO crucially complement available data sets for the Salmonella FopA and ProQ RBPs  and the protein of interest will have to be taken into account as well.As to comparing the RIP-seq results of FinO and ProQ Figures  and 2, tSalmonella enterica strain SL1344 used here, which carries three different plasmids and expresses three distinct FinO domain proteins  plasmid in the same cell. Our present work provides preliminary evidence for a new type of plasmid interdependency, where one plasmid influences the replication rate of another, as suggested by an altered the copy of plasmid pRSF1010 after inactivation of the pRSF1010 . MutatiopRSF1010  or alterpRSF1010 ,60. In oInterestingly, the RepX transcript is strongly conserved among members of the IncQ-1 family of plasmids to which pR1162 and pRSF1010 belong. This raises the intriguing possibility that FinO of pSLT, which belongs to the IncF family of plasmids, would be able to interact with the RepX sRNA of all IncQ-1 plasmids. Of note, IncQ-1 plasmids while not self-transmissible are generally mobilizable thanks to their ability to hijack the type IV conjugation machinery of self-transmissible plasmids . TherefoThe sequencing data have been deposited in NCBI’s Gene Expression Omnibus  and are gkab281_Supplemental_FilesClick here for additional data file.


The filovirus family includes deadly pathogens such as Ebola virus (EBOV) and Marburg virus (MARV). A substantial portion of filovirus genomes encode 5′ and 3′ untranslated regions (UTRs) of viral mRNAs. Select viral genomic RNA sequences corresponding to 3′ UTRs are prone to editing by adenosine deaminase acting on RNA 1 (ADAR1). A reporter mRNA approach, in which different 5′ or 3′ UTRs were inserted into luciferase-encoding mRNAs, demonstrates that MARV 3′ UTRs yield different levels of reporter gene expression, suggesting modulation of translation. The modulation occurs in cells unable to produce microRNAs (miRNAs) and can be recapitulated in a MARV minigenome assay. Deletion mutants identified negative regulatory regions at the ends of the MARV nucleoprotein (NP) and large protein (L) 3′ UTRs. Apparent ADAR1 editing mutants were previously identified within the MARV NP 3′ UTR. Introduction of these changes into the MARV nucleoprotein (NP) 3′ UTR or deletion of the region targeted for editing enhances translation, as indicated by reporter assays and polysome analysis. In addition, the parental NP 3′ UTR, but not the edited or deletion mutant NP 3′ UTRs, induces a type I interferon (IFN) response upon transfection into cells. Because some EBOV isolates from the West Africa outbreak exhibited ADAR1 editing of the viral protein of 40 kDa (VP40) 3′ UTR, VP40 3′ UTRs with parental and edited sequences were similarly assayed. The EBOV VP40 3′ UTR edits also enhanced translation, but neither the wild-type nor the edited 3′ UTRs induced IFN. These findings implicate filoviral mRNA 3′ UTRs as negative regulators of translation that can be inactivated by innate immune responses that induce ADAR1.IMPORTANCE UTRs comprise a large percentage of filovirus genomes and are apparent targets of editing by ADAR1, an enzyme with pro- and antiviral activities. However, the functional significance of the UTRs and ADAR1 editing has been uncertain. This study demonstrates that MARV and EBOV 3′ UTRs can modulate translation, in some cases negatively. ADAR1 editing or deletion of select regions within the translation suppressing 3′ UTRs relieves the negative effects of the UTRs. These data indicate that filovirus 3′ UTRs contain translation regulatory elements that are modulated by activation of ADAR1, suggesting a complex interplay between filovirus gene expression and innate immunity. Filoviridae, which includes Ebola virus (EBOV) and Marburg virus (MARV), is comprised of filamentous, enveloped, negative-sense RNA viruses  encodes 5′ UTRs ranging from 54 to 108 nucleotides and 3′ UTRs ranging from 302 to 684 nucleotides in length. Overall, these comprise 22.2% of the genome. UTRs occupy a similar percentage of the EBOV genome. This contrasts with vesicular stomatitis virus (VSV), a representative negative-sense RNA virus of the rhabdovirus family, which has 5′ and 3′ UTRs that account for 1.0% and 2.5% of the genome, respectively.The approximately 19-kb-long MARV and EBOV genomic RNAs serve as the templates for viral genome replication, which yields viral antigenomic and genomic RNAs, and for transcription, where seven genes serve as separate transcription units that produce mRNAs encoding viral proteins. MARV and EBOV mRNAs are 5′ capped and 3′ polyadenylated and possess long untranslated regions (UTRs) , 7. The in vitro-transcribed and -transfected model mRNAs  mRNA transcriptional start site makes EBOV transcription dependent on the viral VP30 protein . The impel mRNAs . This imegulated .Marburgvirus, led to the accumulation of 30 A→G changes within sequences corresponding to the 600-nucleotide-long 3′ UTR of the glycoprotein (GP) mRNA (–Sequences within the negative-sense RNA genomes that correspond to 3′ UTRs appear to be targets of editing by adenosine deaminases acting on RNA 1 (ADAR1), enzymes that catalyze the deamination of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA) structures . Such edGP) mRNA . During GP) mRNA –19. ADARP) mRNA –. What fuThe present study addresses the functional significance of filovirus 3′ UTRs and the editing of these sequences by ADAR1. The data demonstrate that the UTRs of MARV Angola regulate translation by microRNA (miRNA)-independent mechanisms. A region that suppresses translation is identified within the MARV NP 3′ UTR, and this corresponds to a region of the viral genome previously implicated as a target for editing by ADAR1. ADAR1-editing mutations or deletions of this region enhance expression from model mRNAs. Whereas the unedited MARV NP 3′ UTR activates the IFN-β promoter, the edited or deleted 3′ UTR does not. The enhanced expression is recapitulated using a minigenome assay and reflects enhanced translation as demonstrated by polysome assays. Finally, the roles of 3′ UTRs and ADAR1 editing are extended to EBOV by studying parental and edited 3′ UTRs corresponding to the VP40 gene of West Africa EBOV-Makona isolates. These data provide functional insight into filoviral UTRs and suggest novel mechanisms regulating filoviral gene expression.Renilla luciferase coding sequence. These were placed adjacent to the T7 promoter sequence (in vitro transcription. Each resulting RNA was capped and poly(A) tailed. Equal amounts of RNAs were then transfected, in parallel and triplicate, into HEK293T cells, and luciferase activity was measured at early (2 h) and late (20 h) times posttransfection. The constructs with the NP and VP35 5′ UTRs yielded modestly higher luciferase signals at both the early and later time points than the other constructs  (MicroRNAs (miRNAs) regulate gene expression, often through the targeting of mRNA 3′ UTRs . To detee (RLuc) , RLuc-3′e (RLuc) , or 5′ Ue (RLuc)  mRNAs. Le (RLuc)  or both e (RLuc) . None ofRenilla luciferase 2 h posttransfection. A substantial increase in expression from the mutated NP, VP30, and L truncation constructs was seen. The L truncation construct (L1-420) exhibited an 18-fold increase in luciferase signal , and A549 cells, which do, yielded similar results at both early and late time points, demonstrating that the enhancing effect of the mutations is not unique to a given cell type and suggesting that the enhancement is not directly related to the IFN response .Stability of mRNAs can dictate the level of translation. To determine whether increased translation from the mutated RNAs was due to altered RNA stability, we measured the ratio of RNA levels at early (3 h) and late (24 h) time points posttransfection by reverse transcription-quantitative PCR (qRT-PCR). Ratios of copy numbers at 3 hours and 24 hours posttransfection were found to be similar for all the transfected mRNAs, suggesting a very similar rate of mRNA decay . We alsoACE, ABHD11, and ACO2, in the context of mRNAs with either the NP wild-type (WT) or edited 3′ UTRs  were taken to identify the polysome-containing fractions. RNA was then extracted from all the fractions and analyzed by qRT-PCR to quantify the amount of transfected RNA present. Compared to the mRNA with WT 3′ UTR mRNA , a largeDuring the 2014 to 2016 West Africa EBOV outbreak, various A→G changes were identified in the negative-sense viral RNAs of different isolates, with sequences encoding the VP40 3′ UTR being a hot spot for such changes –19. A co–To directly assess effects of 3′-UTR mutations on translation, polysome analysis was performed. A higher abundance of the edited 3′-UTR mRNA was present in the polysome fractions than the WT 3′-UTR mRNA . This suRenilla luciferase were flanked by MARV 5′ UTRs, 3′ UTRs, or both 5′ and 3′ UTRs. After in vitro transcription, capping, and polyadenylation, the mRNAs were purified and transfected into cells. Using luciferase activity as a measure of gene expression at different time points posttransfection, we characterized the impact of both the 5′ and 3′ UTRs on expression. Comparing the translation efficiency of the model MARV mRNAs with both 5′ and 3′ UTRs identified differences in expression for each model mRNA. Expression increased in the following order: L, GP, VP40, NP, VP30, VP35, and VP24. When we compared the reporter mRNAs containing only the 5′ UTR, we observed that the expression levels for all mRNAs were comparable, although the NP and the VP35 5′-UTR mRNAs yielded slightly higher levels than the rest. This suggested that the variation in translation efficiency in the MARV mRNAs was mostly due to the 3′ UTRs. When constructs with only the MARV 3′ UTRs were compared, various effects on translation were obtained. The NP, GP, VP24, and L 3′ UTR-containing mRNAs showed lower expression than the rest. Interestingly, the L 3′ UTR yielded the least signal, suggesting limited expression of the L protein in infected cells. Low expression of protein translated from mRNAs with MARV L UTRs parallels the observation that EBOV L mRNA translation is maintained at low levels by cis-acting elements present in the L mRNA 5′ UTR  present in 3′ UTRs are well documented to regulate mRNA stability in the eukaryotic cells . StructuA major mechanism regulating mRNA translation in eukaryotic cells is the action of miRNAs. While coding sequences and both the 5′ and 3′ UTRs in host and viral mRNAs are known to harbor miRNA target sites, it is the 3′ UTRs where such target sites are most abundant –31. The –32–The negative regulatory elements in the NP 3′ UTR correspond to sites in the MARV genome that appear to be edited by ADAR1 . ADAR1 e36–42–Marburgvirus, led to the accumulation of 30 A→G changes within sequences corresponding to the 600-nucleotide-long 3′ UTR of the GP mRNA  promoter or an IFN-inducible ISRE promoter. Our data demonstrate that 5′-capped, 3′-polyadenylated, and phosphatase-treated mRNAs possessing the MARV WT NP 3′ UTR, but not the edited 3′ UTR, induced IFN responses, suggesting this UTR may contribute to IFN responses. Interestingly, a similar effect was not detected with the EBOV VP40 3′ UTR. Why the MARV NP and EBOV VP40 3′ UTRs differ in this respect is unclear. It could reflect stability or structure of any stem-loop structures but this will require further study. There is precedent for the activation of IFN responses by capped RNAs, as 3′ UTRs from influenza virus and some RNA aptamers have been described as 5′ triphosphate-independent RIG-I activators , 47. TheThe finding that filovirus 3′ UTRs include elements that decrease translation efficiency is intriguing. That such elements can also activate innate immune responses, as in the case of the NP 3′ UTR-containing mRNAs, and correspond to genome RNA sequences susceptible to modification by ADAR1 is also notable. These findings suggest that possible costs of these features are offset by other, yet to be identified, benefits. A previous study identified an upstream open reading frame in the EBOV L 5′ UTR as a suppressor of L translation, but the suppression could be relieved when phosphorylation of translation initiation factor eIF2a was induced. This was proposed as a mechanism by which L protein levels could be maintained at a low but consistent level, even when innate immune responses are activated . A simil2. Dulbecco’s modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) was used as growth medium.A549 cells, human embryonic kidney 293T (HEK293T) cells, and Vero cells were obtained from ATCC. RNase III knockout cells were a kind gift from Benjamin tenOever . Stable cell lines with IFN-β and ISRE reporters have been described previously . All celKJ660346). Wild-type 3′-UTR and 3′-UTR coding sequences containing all 16 VP40 editing mutations were synthesized commercially (GenScript). Forward primers were designed to add the T7 promoter gactcgtaatacgactcactataggggaagag at the 5′ end. Renilla luciferase (RLuc) coding sequence was flanked by either the 5′ UTR, 3′ UTR, or both the UTRs to create templates for transcribing test mRNAs. Assembly of the RNA coding sequence and template was accomplished by PCR using Q5 High-Fidelity DNA polymerase (NEB). Finally, template DNA was amplified with the forward primer gactcgtaatacgactcactataggggaag and a 3′ end-specific reverse primer. The amplified template was gel purified, in vitro transcribed using HiScribe T7 Quick high-yield RNA synthesis kit (NEB) using the manufacturer’s protocol, purified by LiCl4 precipitation, and capped using the Vaccinia capping system (NEB). Capped mRNAs were again purified by LiCl4 precipitation followed by the addition of poly(A) tails using Escherichia coli poly(A) polymerase (NEB) as per the manufacturer’s protocol. After a final round of LiCl4 purification, mRNA was resuspended in nuclease-free water, analyzed by formaldehyde agarose gel electrophoresis, aliquoted, and stored at −20°C. Truncated mRNAs were generated in a similar fashion by using the full-length template for amplification. Reverse primers specific to internal sites were designed with a conserved terminal sequence, ATTAAGAAAAA, at the 3′ end.Both wild-type and edited MARV NP 3′-UTR coding sequences were synthesized by GenScript . For amplifying the remaining MARV UTRs, purified total RNA from MARV-infected THP1 cells at 24 hours postinfection was used for cDNA synthesis. The 5′- and 3′-UTR coding sequences were amplified with UTR-specific primers. Appearance of mutations in the 3′-UTR coding region of Ebola virus VP40 gene during the course of infection was reported from patients in Sierra Leone during the 2014 to 2016 West Africa Ebola virus outbreak . A totalIn vitro-transcribed RNA was transfected into cells using Lipofectamine 2000 reagent (Invitrogen). Briefly, cells were seeded in a T75 flask 24 hours prior to transfection such that they would be ∼80% confluent on the day of transfection. These cells were reverse transfected. The transfection used 10,000 cells and 100 ng of RNA per well of a 96-well plate or scaled accordingly. At the specified times, levels of luciferase expression were measured using the Renilla-Glo luciferase assay system (Promega) per the manufacturer’s protocol.Time points for the luciferase assays were based on pilot studies. In pilot studies, expression from transfected RNA was monitored over a time course of 1 to 48 hours posttransfection. It was observed that more transfectable cells, such as Vero cells and HEK293T cells, exhibited high levels of expression within an hour of transfection, whereas, for A549 cells, the highest levels of expression were observed after 2 hours posttransfection. In all the cell types, reporter activity was very high, with little change up to ∼6 to 8 hours. Therefore, early time points in the 2- to 6-hour time frame were used. Experiments that included A549 cells used the 6-hour time point. After 24 hours, pilot studies indicated that signal declined and that this corresponded to lower levels of the reporter mRNA in cells, as determined by RT-PCR. Therefore,14 to 24 hours were chosen as intermediate time points, and 48 hours was used as a late time point.CACCCACTCCTCCTACTTT; reverse, CCCTGTTGCTGTAGCCAAAT). Comparisons were made by the threshold cycle (ΔΔCT) method. For rate of decay assays, total RNA was extracted at early (2-hour to 4-hour) and late (18-hour to 24-hour) time points. Following cDNA synthesis using oligo(dT) primers, reporter mRNA was quantified was by using Renilla luciferase-specific primers . A ratio of RNA levels at early and later time points was taken as a measure of input RNA stability.Total RNA was prepared from transfected samples using TRIzol (Invitrogen). cDNA synthesis was performed using oligo(dT) primers for mRNA or random hexamers for total RNA using the SuperScript IV reverse transcriptase kit (Thermo Fisher Scientific) as per the manufacturer’s protocol. Quantitative PCR (qPCR) was carried out following the manufacturer’s protocol using SYBR Green master mix (Thermo Fisher) on a Bio-Rad CFX real-time PCR system . GAPDH  following the manufacturer’s protocol. Student’s t tests were carried out with GraphPad Prism to assess statistical significance.Stable cell lines with an IFN-β promoter reporter gene and an ISRE promoter reporter gene have been described previously . Both cein vitro-transcribed and purified minigenome RNA was reverse transfected into the cells and reseeded in 24-well or 96-well plates, and the cells were grown for the indicated times. Minigenome assays were evaluated by measuring Renilla luciferase activity and by quantitative RT-PCR. Strand-specific primers were used for cDNA synthesis from genomic sense (GGACACACAAAAAAGATGAAGAATG) or antisense (TGGACACACTAAAAAGATGAAGAATG) minigenome RNA strands. Following cDNA synthesis, standard qPCR was done using the Renilla luciferase primers.HEK293T cells were grown in T75 flasks until they were ∼80% confluent on the day of transfection. Helper plasmids that express the MARV L, NP, VP30, and VP35 proteins have been previously described . These pRenilla luciferase coding sequences flanked by the MARV NP 5′ and 3′ UTRs, and the second gene possessed firefly luciferase coding sequences flanked by the MARV L 5′ and 3′ UTRs. The region encoding wild-type or edited MARV NP 3′ UTRs was PCR amplified from constructs used in the comparative expression assays. cDNA obtained from infected THP1 cells was used to amplify the MARV L 5′ UTR. A “middle fragment” was assembled by PCR and was comprised of the MARV NP 3′ UTR (the intergenic sequence TCAAT), the L 5′ UTR, and firefly luciferase. Similarly, a “front fragment” comprised of T7 terminator, ribozyme, MARV leader, NP 5′ UTR, and RLuc and an “end fragment” comprised of L 3′ UTR, trailer, and T7 promoter were PCR amplified from a monocistronic MARV minigenome template. Next, the front, middle, and end fragments were assembled by overlapping PCR amplification. All PCRs were carried out using Q5 high-fidelity polymerase (NEB). The resulting construct was designed to produce a genomic-sense RNA upon in vitro transcription. The PCR amplicons were gel purified, sequenced, and used as a template for in vitro transcription. Transcribed bicistronic minigenome RNA was purified by LiCl4 precipitation and size verified by running on a formaldehyde agarose gel.A bicistronic minigenome was designed to contain two genes. The first gene possessed To confirm transcription of mRNAs containing the wild-type and edited NP 3′ UTRs in cells transfected with the bicistronic minigenome and helper plasmids, total RNA was harvested from transfected cells. cDNA was generated by using oligo(dT) primers and quantitative PCR, and sequencing was performed.Conserved sequences at the 3′ ends of the MARV UTRs were identified with WebLogo .2, 100 μg/ml cycloheximide, and 1 mM dithiothreitol [DTT] in RNase-free H2O). One milliliter each of decreasing concentrations of sucrose buffers, starting with 50% and topping off with 15% for a total of 11 ml, was added to Thinwall polypropylene ultracentrifuge tubes (Beckman Coulter). The tubes were immediately frozen and stored at −80°C. On the day of use, the tubes were thawed at room temperature, resulting in the formation of a continuous gradient, and immediately placed on ice. Formation of a continuous gradient was confirmed by adding different color dyes to the 50% and 15% starter sucrose solutions in a test preparation.Continuous sucrose gradients were manually prepared. Fifty percent and 15% sucrose solutions were prepared in RNase-free buffer  containing 100 μg/ml cycloheximide. Cells were lysed on the plates and harvested by adding 1 ml ice-cold lysis buffer . Lysates were centrifuged at 13,000 rpm for 10 min at 4°C to remove cellular debris. We loaded 800 μl of the lysate onto the sucrose gradient. The gradient tubes were ultracentrifuged in a Beckman Coulter SW41 Ti swinging-bucket rotor at 40,000 rpm for 2 hours at 4°C. Samples were fractionated by pumping into the tubes a chase solution of 60% sucrose in RNase-free water using a BR-188 density gradient fractionation system (Brandel). Real-time concentration of RNA in the flow was measured by reading absorbance at 260 nm. One-milliliter fractions were collected and set aside for RNA extraction. RNA was extracted from 200 μl of each fraction with TRIzol reagent, and the quantity of transfected RNA in each fraction was determined by qRT-PCR. Absorbance data were analyzed by PEAK CHART data acquisition software (Brandel) and overlaid with qRT-PCR data using Prism software (GraphPad).Polysome fractionation was performed as outlined previously . Briefly


Bombyx mori Vasa, which unwinds truncated target transposon RNAs from the piRNA-induced silencing complex piRISC. In this study, we used single-molecule techniques to visualise how Vasa dynamically interacts with piRISC and investigate how Vasa oligomerization is involved in the process of piRNA amplification, named the ping-pong pathway. We found that Vasa’s oligomerization is required during these processes in vitro and in vivo, and that Vasa triggers the dissociation of truncated RNA in heterogeneous pathways. Our single-molecule results suggest that oligomerized Vasa guides the timing of the process regulating overall dissociation efficiency.RNA helicases are enzymes that generally unwind double-stranded RNA using ATP hydrolysis energy, mainly involved in RNA metabolism, transcription, translation, and mRNA splicing. While the helicase core is crucial for RNA unwinding activity, N- and C-terminal extensions of specific helicases may contain an intrinsically disordered region for electrostatic interaction, resulting in the formation of droplets in the cytoplasm. However, how the disordered region of the RNA helicase contributes to RNA unwinding and dissociation remains unclear. Here, we focused on  Kinoshita et al. use single molecule imaging to show that the N-terminal extension of BmVasa, a germ-specific DEAD box RNA helicase, is necessary for protein oligomerization and the dissociation of target RNA from the Siwi-piRISC complex. The authors conclude that oligomerized Vasa guides the timing of the regulation of overall dissociation efficiency. These RNA helicases are enzymes that typically unwind double-stranded RNA using the hydrolysis energy of ATP in highly conserved residues, including the D-E-A-D motif3. RNA helicases disrupt the conformations of elongated RNAs with specific secondary structures and reconstruct the RNA-protein complex by the ATP-driven facilitation of the helicase core to the closed conformation, consisting of two RecA-like domains with flanking linkers5. While the helicase core is crucial for RNA unwinding, the N- and C-terminal extensions of specific helicases have a unique function. For example, the N-terminus of the human homolog of DEAD-box helicase 4 (DDX4) may contain an intrinsically disordered region (IDR) for electrostatic interaction with other DDX4 molecules, resulting in the formation of droplets in the cytoplasm6. These results suggest that the N-terminus region of DDX4 plays an important role for aggregation in cells. Thus, the amino-acid sequence of Vasa, a homolog of human DDX4, suggests that its N-terminal IDR undergoes oligomerization. However, the mechanism by which Vasa oligomerization links to the unwinding function of RNA is yet unknown.DEAD-box RNA helicases are the largest helicase family involved in all aspects of RNA metabolism, including transcription, translation, and mRNA splicingBombyx mori Vasa, an ATP-dependent RNA helicase that unwinds truncated target transposon RNAs from the piRNA-induced silencing complex (piRISC) during piRNA metabolic processes8. This cleavage reaction is induced by the formation of piRISC by piRNA (PIWI-interacting RNA), a noncoding RNA generated specifically for germ cells, and Siwi (Silkworm Piwi), one of the PIWI (P-element induced wimpy testis) proteins of the Argonaute subfamily (AGO and PIWI proteins). Furthermore, germ cells have an ingenious piRNA amplification mechanism for efficient transposon RNA cleavage, known as the ping-pong pathway9. In this pathway, Siwi, Vasa, Ago3, and DDX43 are orchestrate a droplet called Nuage near the nuclear membrane in silkworm germ cells BmN410.Therefore, in this study, we focused on the function of the N-terminal region of Although dynamic changes in truncated RNA dissociation triggered by Vasa in piRISCs are intimately tied to the ping-pong pathway, the mechanism by which Vasa interacts with piRISC and triggers truncated RNA dissociation remains unknown. In this study, we used single-molecule techniques to visualise how Vasa dynamically interacts with piRISC and evaluate how Vasa’s oligomerization is involved in the ping-pong pathway. As the result, we found that Vasa triggers the dissociation of truncated RNA in heterogeneous pathways, and that Vasa oligomerization is required during these processes both in vitro and in vivo.Bombyx mori (silkworm) ovary  and a mutant with N-IDR deletion (“dN-Vasa”) were prepared using anti-FLAG gel column by culturing germ cell line BmN4 derived from ary Fig. . Both saary Fig. , S1a–b, Next, we visualised the oligomerized Vasa in vivo. BmN4 cell imaging showed that the transfected WT-Vasa formed Nuage, whereas dN-Vasa did not form Nuage with dispersion in the cytoplasm Fig. , suggest7, Vasa has been found to aggregate more in the presence of exogenous RNA using TMR-labelled Vasa. To observe the effect of exogeneous RNA on Vasa’s oligomerization, biotinylated TMR-labelled Vasa was immobilised on a glass substrate at a single-molecule density in the presence of yeast RNA  microscopy at the single-molecule level. We first confirmed the cleavage and unwinding activities of the reconstructed system in the bulk. The results showed that in the presence of WT-Vasa, target RNA complementary to endogenous piRNA was dissociated from the beads bound to Siwi-piRISC Fig. . On the The 3′ end of target RNA in Siwi-piRISC was immobilised on the glass surface via a biotin-avidin bond at higher densities. The average fluorescence intensity of target RNA in the field of view was monitored in real time immediately after the introduction of non-fluorescent WT-Vasa and dN-Vasa Fig. . In the We subsequently monitored the individual dissociation processes that were not obtained from the averaged signals. By reducing the concentrations of immobilised target RNA to 10–100 pM, fluorescent signals could be successfully identified at single-molecule resolution Fig. . Since tFurthermore, to visualise how Vasa directly interacts with and dissociates from Siwi-piRISC in the presence of exogenous piRNA-4 (see “Methods”), we simultaneously measured the behaviour of Vasa and target RNA fluorescently labelled with TMR and ATTO647N, respectively. The results showed that Vasa colocalized well in the presence of Siwi-piRISC, but not in its absence in TIRF experiments Figs. , S3c. ThIn addition, we also visualised Siwi via Siwi antibody fluorescently labelled with Alexa 488 and confirmed the simultaneous localisation of the three factors including Vasa and target RNA Fig. . This inN = 14 out of 72 traces in total)   Figs.  and S5a,s) Figs.  and S5b.In the case of dissociation at different times, Vasa progressively dissociated from the oligomerized Vasa. Furthermore, even after the RNA dissociated, a part of Vasa remained bound to the truncated RNA immobilised at the 3′ end on the surface. This suggests that a part of the Vasa remains bound to the truncated RNA even after the dissociation of the 5′-terminal truncated RNA attached to ATTO647N.N = 27)  Figs.  and S5c.In all cases, oligomerized Vasa progressively dissociated independently of the dissociation event of the target RNA Figs. , S5a, b.In summary, the obtained results suggest that these heterogeneous dissociations may contribute to the overall RNA silencing activity of the ping-pong cycle.11. The RNA-binding and unwinding activity and IDRs of the Drosophila ortholog Belle are also required for viability and fertility13. Furthermore, in addition to DEAD-box RNA helicase, one of the prokaryotic DNA helicases, Escherichia coli UvrD forms oligomers to unwind duplex DNA, as indicated by not only single-molecule imaging approaches14 but also biochemical approaches15. Therefore, the Vasa oligomerization observed in this study is consistent with previously reported experimental results. Our further coarse-grained MD simulation supported the importance of IDRs for Vasa oligomerization.  was designed. The 5′ end of the target RNA was fluorescently labelled with Alexa647 via the SSH amino linker, and the 3′ end was labelled with biotin.The endogenous target RNA, 5′-CGA CCU CGA GGG GWe also designed an exogenous target RNA (“piRNA-4 target”) complementary to piRNA-4.8. The designed sequence of target RNA  was 5′-CCC CCG GGA GUA ACU UCG GUU UCU GGG AAU UUA AUC AAG CUU AUC AAU ACC GUC GAC CUC GUG UAC UGG C-3′ . The 5′ end was fluorescently labelled with ATTO647N via an SSH amino linker, and the 3′ end was labelled with biotin.The sequence of piRNA-4 (28 nt) was 5′-UAA AUU CCC AGA AAC CGA AGU UAC UCC C-3′, where the 5′ end was phosphorylated and the 3′ end base (C) was methylatedBombyx mori (silkworm) ovary, and purified using an anti-FLAG gel column . In the case of Vasa, we also prepared deleted constructs of the N-terminal disordered region (K133-W601 referred to as dN-Vasa). FLAG-tag and HaloTag (Promega) were fused at the N-terminus of Siwi and Vasa  as the primary antibody while Siwi was treated with anti-Siwi antibodies  kindly gifted from Siomi lab at the University of Tokyo, followed by treatment with a secondary antibody, IgG1/IgG2a labelled with Alexa Fluor 488/555 , for fluorescent detection. The nuclei were stained with DAPI. Fluorescent images were obtained using an LSM800 with AiryScan (Zeiss).BmN4 lysates were prepared and incubated with Dynabeads Protein G (Invitrogen) and anti-Siwi antibody (2 µg) for 1 h at 4 °C. Guide RNA  was first added to the beads and incubated for 30 min at 26 °C, as necessary. Fluorescently labelled target RNA (50 nM) was then added to the beads and incubated for 3 h at 26 °C in cleavage buffer . Then, 500 nM Vasa and 1 mM ATP were added to the beads and incubated in cleavage buffer for 2 h at 26 °C. After the supernatant and beads were collected separately, RNAs in these fractions were extracted by phenol-chloroform treatment and precipitated with ethanol. Electrophoresis was performed using denaturing gel in 15% urea, and the fluorescence of RNA in the gel was scanned with Typhoon FLA 9500 .19. The observed glass surface of the microchamber was coated with 0.01% poly-L-lysine for 5 min. The carboxylic acid group in Vasa was strongly adsorbed onto the glass surface in the assay buffer containing the oxygen scavengers: 30 mM HEPES, pH 7.3, 150 mM potassium acetate, 5 mM magnesium acetate 0.1% NP-40, 500 µM TCEP, 10 mM TSY, 10 mM PCA, 12-fold diluted PCD (Pacific Biosciences), and 200-fold diluted RNasin® Plus RNase Inhibitor (Promega). 5 µg/ml yeast RNA was included for the contribution test of exogenous RNA to the oligomer formation of Vasa. The fluorescent images illuminated by a 532-nm laser  were acquired at a frame rate of 100 ms using an EMCCD camera  equipped with a Nikon Ti-E TIRF microscope19.Cover glasses were cleaned using 1 N KOH sonication and a plasma cleaner, and then the micro-chamber was built using adhesive tape19. Briefly, 1 mg/ml Neutralised Avidin (Wako) was adsorbed onto the glass surface, and then the excess avidin was removed by washing three times with buffer after 2 min incubation. Biotinylated TMR-labelled Vasa, labelled with Biotin-XX-NHS (Sigma-Aldrich), was fixed on the substrate. After introducing 5 µg/ml yeast RNA and removing the excess, 50 nM TMR-Vasa was applied during the recording, and a series of 1000 fluorescent images were acquired at a time interval of 600 ms in total using TIRF microscopy.In the Vasa assembly experiment in the presence of RNA, the plasma-cleaned glass surface of the microchamber was coated with amino-silane and PEG-biotinThe fluorescence intensities of TMR-labelled Vasa were analysed, and the size of the optimal region of interest (ROI) was 8 × 8 pixels  and the noise tolerance was ~1500 using the plugin of spot intensity analysis .20. The intensity of a single TMR was determined from the averaged stepwise intensity   Fig. . We deteFor the measurement of red fluorescence intensity only, we mixed 20 nM target RNA with 760 nM Siwi for 20 min (endogenous target RNA) or 3 h (exogenous piRNA4 target RNA) at room temperature, and then fixed Siwi-piRISC on the glass surface via biotinylated target RNA by avidin-biotin binding. We monitored the fluorescence intensity of target RNA illuminated by a 642-nm laser  soon after adding 25 nM non-fluorescent Vasa and ATP in the assay buffer, including oxygen scavengers. We monitored 1000 successive images each in the time interval of 400 ms. The image analysis of single molecular tracking of red fluorescence spots was the same as explained above.For the simultaneous measurement of cleaved target RNA and vasa, a premix of 10 nM biotinylated target RNA (labelled with ATTO647N), 625 nM vasa (labelled with TMR), and 380 nM Siwi  and was fixed on the surface by avidin-biotin binding in the assay buffer containing the oxygen scavenger. To measure the dissociation of cleaved target RNA and Vasa, 1 mM ATP was introduced into the chamber during recording. We acquired 1000 successive images each in a time interval of 400 ms by shutter control. The image analysis of single molecular tracking was the same as that explained above.Statistical analyses were all performed using MATLAB 2020b and Curve Fitting Toolbox. The averaged intensity of a single fluorescent dye was analysed by fitting data with a single Gaussian curve Figs. , S3d. ThFurther information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Circularization of RNA mediated by association of translation factors and RNA elements in 5′ and 3′ UTRs is a common feature for translation control in eukaryotes. There is no information about translation in plant rhabdoviruses and little information is known in animal rhabdoviruses.The role of 5′ and 3′ UTRs in two genes of EMDV in translation were studied using luciferase constructs and RNA structures of these sequences were analyzed by SHAPE and Inline probing.carmovirus Turnip crinkle virus, which contains a well-characterized cap-independent translation enhancer, was unable to complement the lack of EMDV 3′ UTR. Addition of cap analog to luciferase constructs containing the UTRs of the N gene did not restore translation, and translation of the reporter construct in the absence of the 5′ cap was higher than the capped construct. No RNA-RNA interactions between 5′ and 3′ UTRs were detected by EMSA or in-line cleavage structural assays. Deletion of 11 nucleotides from the 3′ terminus negated the synergistic activity of the 3′UTR.We have found that efficient translation of N and X mRNAs of nucleorhabdovirus Eggplant mottled dwarf virus (EMDV) requires elements present in both 5′ and 3′ UTRs. Luciferase reporter constructs containing precise 5′ and 3′ UTRs of the N and X genes had substantially higher translational activity compared with constructs containing only the 5′ or 3′ UTR. The 3′UTR of The results with RNA-RNA interaction suggesting that translational synergy between the UTRs may utilize alternative means. Mutation analysis in 3′UTR suggesting that the polyadenylation signal sequence contained in this location may play a critical role in translation. The Rhabdoviridae family, in the order of Mononegavirales, contains viruses with negative-sense RNA genomes that include plant-infecting and animal-infecting viruses. Rhabdovirus genes contain the following five canonical genes (beginning at the 5′ end of the complementary plus-strand) that encode: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and large protein (L). The conservation of genes and gene order among all rhabdoviruses suggests that these viruses likely share common mechanisms of replication, transcription and translation. However, the low sequence similarity among orthologues of these proteins suggests that considerable host adaptation has occurred –4. mRNAsInhibition of canonical cap-dependent translation by depletion of translation factors eIF4E and eIF4G or rapamycin treatment had no effect on translation of the Vesicular stomatitis virus (VSV), suggesting that rhabdoviruses are translated by a cap-independent mechanism , 13. VSVPlant-infecting rhabdoviruses are now 6 genera of unsegmented and segmented genomes that nucleorhabdoviruses composed of viruses that replicate in the nucleus , 19, 20.SspI and NruI restriction sites. 45 additional nucleotides (including the 6 nt restriction site) were present between the Fluc stop codon and the 3′ UTR. All fragments were generated by PCR using primers designed for digestion with BamHI/SacI sites for the 5′ fragments and NruI/SspI for 3′ fragments and then inserted into the appropriate vector sites. Reporter constructs were linearized with PmlI and SspI before transcription using T7 RNA polymerase to generate both capped and uncapped RNAs according to the manufacturer's instruction. Deletion of 11 nt from the 3′ terminus of 5′N80-Fluc-N3′UTR was generated using oligonucleotide-mediated site-directed mutagenesis  methionine and an amino acid mixture in the absence of unlabeled methionine. Transcripts were synthesized at 37 °C for 2.5 h and then subjected to phenol–chloroform extraction and ethanol precipitation. RNA integrity was verified on 1% agarose gels. Products were subjected to electrophoresis through 5% polyacrylamide gels.Fragments containing the N gene 5′ UTR  or 3′ UTR  were amplified by PCR using a primer containing a T7 promoter sequence at the 5′ end, followed by synthesis of transcripts using T7 RNA polymerase. In-line probing was performed as previously described –28. 5′ UTo investigate if two RNA fragments were interacting thereby reducing in-line cleavage at interacting residues, tenfold excess unlabeled 3′ or 5′ UTR N-gene fragments were combined with the labeled fragment and cleavages allowed to take place as described above.SHAPE was carried out on a full-length EMDV N gene as described previously , 30. BriEMSA was performed based on a previously described procedure . EMDV N Both 5′ and 3′ UTRs are required for efficient translation of reporter constructs.Since little is known about animal rhabdovirus translation, and nothing is known about plant rhabdovirus translation, we were interested in investigating whether sequences and/or RNA structures in rhabdovirus UTRs might help to facilitate translation of the capped genome. EMDV has a genome organization with the order 3′-N-X-P-Y-M-G-L-5′ (negative strand) Fig. a, which To determine if the N gene 5′ and 3′ UTRs contain elements that promote efficient translation, luciferase reporter constructs were generated containing either the precise 5′ and 3′ UTRs (N5′80 + N3′UTR), or the individual UTRs  Fig. b. Compar5′ and 3′ sequence comparisons in different rhabdoviruses genes and also between different genes of EMDV (like most plant rhabdoviruses) showed conservation of 5′-AACA-3′ at the 5′ end and 5′-UUUAAUAAAAA-3′ at the 3′ end of all genes. When 5′-UUUAAUAAAAA-3′ was deleted in 5′N80-Fluc-N3′UTR, translation was reduced by 12.5-fold  , 36, 37.The role of host proteins in translation of rhabdoviruses mRNAs has been suggested in different studies. PCBP2 protein in interaction with the 3′UTR of G mRNA in rabies virus can help to circularization and RNA stability of viral RNA , 44–46. VSV mRNA, a type member of rhabdoviridae, has a 5′ cap and 3′ poly-A tail. If, however, translation factors such as eIF4E and eIF4G are depleted or cleaved, VSV proteins synthesis prevails, suggesting that VSV has alternative translation mechanisms in association with cell proteins and translate by use of several protein like rpl40, eIf3a and eIf3d , 48. EleIn the present study, we found that the 5′ and 3′ UTRs of N and X genes in EMDV are de-void of pronounced RNA secondary structure. Our results showed that 5′ and 3′ UTRs are required for efficient translation on N and X mRNAs in EMDV. It is also possible that EMDV uses protein-mediated translation when there is no detectable RNA-RNA interaction between 5′ and 3′ UTRs of N mRNA. VSV and EMDV both replicate in vector insects suggests both viruses possibly share their translation strategy. Our results of efficient translation of N and X transcript genes and also non-capped luciferase mRNA construct together with previous results for protein-mediated alternative translation in VSV, suggesting possible similar translation mechanism in plant and animal rhabdoviruses. Identification of host protein interacting with EMDV transcripts during viral infection of different hosts may provide novel insights into translation and host infection mechanisms of EMDV and other plant rhabdoviruses. Further studies are aimed to develop protein-mediated translation control in EMDV and other plant rhabdoviruses.Analysis of 5′ and 3′ UTRs sequences in plant and animal rhabdoviruses didn’t show any conserved sequence. The presence of both UTRs of n and X genes in the luciferase construct showed a higher translation level compare to one of them. Both 5′ and 3′ N gene UTR’s showed low structure in SHAPE and inline probing but didn’t show any interaction. Mutation in 11 bp of 3′UTR end decrease translation level suggests polyadenylation signal sequence in this region may play a critical role in interaction for translation. UTRs may utilize alternative ways for translation using protein factors.


However, the underlying mechanism of RNA m5C methylation in cell mobility and metastasis remains unclear. The functions of m5C writers and readers are believed to regulate gene expression at the post-transcription level and are involved in cellular metabolism and movement. In this review, we emphatically summarize the recent updates on m5C components and related regulatory networks. The content will be focused on writers and readers of the RNA m5C modification and potential mechanisms in diseases. We will discuss relevant upstream and downstream interacting molecules and their associations with cell migration and metastasis.Epigenetic modification plays a crucial regulatory role in the biological processes of eukaryotic cells. The recent characterization of DNA and RNA methylation is still ongoing. Tumor metastasis has long been an unconquerable feature in the fight against cancer. As an inevitable component of the epigenetic regulatory network, 5-methylcytosine is associated with multifarious cellular processes and systemic diseases, including cell migration and cancer metastasis. Recently, gratifying progress has been achieved in determining the molecular interactions between m The biological roles of this modification in RNA, including RNA export, translation, RNA fragmentation and ribosome composition, were not fully comprehended until recently 5C have been improved and employed to further elucidate the functions of the m5C modification 5C modification, extensive target sites were discovered in multiple RNA classes and diverse organisms 5C modification on eukaryotes.In eukaryotes, a wide range of nucleotide modifications, including acetylation, methylation and glycosylation, are essential for cellular biological processes and carcinogenesis. Among the hundreds of chemical modifications, 5-methylcytosine , demethylases and readers  have been identified. Earlier studies have detected DNA methylation induced by the human homologous DNA methyltransferase (DNMT) family, including DNMT1, DNMT2 and DNMT3A/3B. Exclusively, a multiomics study revealed that human DNMT2 not only methylates DNA but also a small RNA (aspartic acid transfer RNA) 5C demethylation, ten-eleven translocation (TET) genes were initially implicated as tumor suppressors 5C, f5C and ca5C), followed by the excision of f5C or ca5C which is probably induced by thymine DNA glycosidase (TDG) in DNA 5C readers and subsequent physiological functions further indicate the complicacy of cascading regulation at the molecular level. The RNA m5C modification is involved in a large number of human cancers, including leukemia, lung cancer, gastric cancer, squamous cell carcinomas, hepatocellular carcinoma and gynecologic cancers 5C modification and its elimination are closely associated with molecular and cytological processes in diseases and various cancer types, and thus research on the mechanism and clinical application are hotspots.An integrated m5C modifications involved in metastasis, cell migration and invasion, discussing their functions in cellular biological processes and tumor development. Classified by enzyme family, we review the recent progress in studies of m5C writers, demethylases and readers to summarize the current knowledge on methylation associated molecular regulation and pathway activation, as well as the reversion of demethylation. We then discuss the clinical advances in the evaluation and prognosis of human cancers.In this review, we focus on the pivotal mechanisms of RNA m6A) modification was established as a pivotal modification in prokaryotes but was not detectable in eukaryotes. Importantly, m6A modifications have now been identified as closely related to many biological processes and diseases, such as RNA metabolism related gene expression regulation, DNA damage repair, cell development and differentiation, cell cycle, immune response and even cancer progression 6A in the human genome indicated that specific enzymes induce methylation and demethylation-mediated gene transcription by targeting the AGG motif 6A methyltransferase and demethylase, methyltransferase-like 3 (METTL3) and fat mass and obesity-associated protein (FTO), respectively, are involved in the DNA damage response to ultraviolet irradiation 6A plays a crucial role in epigenetic regulation at the transcriptional level. To date, various RNA classes have been verified to be regulated by m6A modification, including precursor mRNAs, long noncoding RNAs, ribosomal RNA and microRNAs 6A. Similarly, METTL3-induced m6A promotes the mRNA translation of several oncogenes in human acute myeloid leukemia cells, and then mediates cell proliferation, apoptosis and leukemia progression In earlier studies, the DNA N6-methyladenosine  modification, related functional research has become more common in the last five years. The biological effect of the reversible m1A modification is to meditate RNA stability and RNA processing, and this modification has been verified to have a transcriptome-wide distribution in mammalian cells and tissues 1A is distributed in mRNAs and enriched at translation start sites (5' UTRs), which may promote translation 1A also occurs in several regions of tRNAs and mitochondrial genes and regulates corresponding biological processes 1A remain ambiguous. The m1A level in mitochondrial tRNAs is significantly altered in various human cancers, and is associated with the clinical prognosis 1A in tRNA, alkB homolog 1 (ALKBH1) facilitates cancer progression and migration in vivo, resulting in demethylation-induced tRNA fragmentation 1A modification remains unclear and requires further identification.For the N1-methyladenosine , shares a conserved residue at the hydrophobic pocket for binding m5C-modified RNA 5C-modified mRNAs across the cytoplasm and nucleus, which was confirmed by ALYREF protein sequencing 5C modified mRNAs by residue Trp45 in the cold shock domain of YBX1 5C regulatory network mainly consists of functional readers and downstream effector, which needs further investigation of novel specific binding proteins.After the ms Figure . We name5C modification is widely distributed in RNA involved in various biological processes. As a fundamental function of the RNA m5C modification, the regulation of RNA function and metabolism is crucial to the development of biological processes  and NSUN5 (yeast homologue Rcm1) regulate ribosome synthesis and processing 5C methylation in mRNAs and noncoding RNAs (ncRNAs). For example, DNMT2 is responsible for RNA processing by relocating and interacting with mRNA as a component of the processing complex 5C modification at C69, which maintains the cell cycle in progenitor cells 5C modification promotes RNA stabilization, transportation and translation, but not translation termination induced by NSUN6 mediated mRNA methylation to allow RNAs to perform their regular functions 5C of the anticodon stem-loop 5C modification is involved in epigenetic regulation by affecting RNA metabolism. RNA fragmentation is a common metabolic pattern of tRNA that is related to epigenetic regulation and cancer progression The ms Figure . With im5C modification in diseases and cancer is noteworthy. The disorder of tRNA fragmentation induced by DNMT2 leads to high-fat-diet-induced metabolic disturbance in sperm cells 5C leads to reduced protein translation rates, which activates stress pathways and promotes neuronal apoptosis 5C modification is strongly correlated with dysplasia and dysfunction of the nervous system. Diseases related to energy metabolism disorder are also associated with the mitochondrial RNA m5C modification. For example, NSUN3 may be associated with multisystem mitochondrial disease associated with a combined oxidative phosphorylation deficiency 5C that leads to myelopoiesis in infections, such as sepsis and parasitosis, in the mammalian system Since RNA function and epigenetic regulation are strongly associated with the occurrence and development of diseases, the role of the RNA m5C modification in malignant solid tumors has been a focus of cancer research at the epigenetic level. Various m5C writers and readers play crucial roles in cancer progression, including cell proliferation, differentiation, migration, invasion and drug sensitivity. Taking the typical m5C methylase NSUN2 as an example, NSUN2 has been shown to participate in multiple pan-cancer pathways: promoting cell progression, migration and invasion in breast cancer 5C modification in cancer, DNMT1 overexpression directly leads to hypermethylation of tumor suppressor genes, which results in lung tumorigenesis and a poor prognosis 5C readers, ALYREF is associated with tumor progression in patients with glioblastoma and HCC, but regulates glucose metabolism and tumorigenesis in individuals with bladder cancer 5C readers are associated with cancer development in collaboration with methylases 5C level was reported in cancerous tissues compared to adjacent normal tissues in human colorectal carcinoma and hepatocellular carcinoma, suggesting possible anticancer mechanisms 5C components and high levels of the m5C modification are significantly correlated with malignancy and a poor prognosis in patients with the cancers mentioned above and some other cancer types, such as head and neck squamous cell carcinoma (HSNCC) and ovarian cancer The RNA m+ T cells suggests a possible treatment for acquired immune deficiency syndrome.In addition to eukaryotes, in RNA virus HIV-1, the transcription and replication of TAR RNA are restricted by NSUN1 induced RNA methylation 6A in RNA possesses multiple epigenetic regulatory activities in various biological processes, diseases and especially cancer 5C can mediates the activation of oncogenic pathways and forms a microenvironment suitable for the migration and metastasis of various cancer cell lines . The activation of the NSUN2/YBX1/HDGF axis was proven to promote cell growth, tumor progression and metastasis 5C modification, which specifically binds to oncoprotein G3BP1. The mechanism was verified to be associated with tumorigenesis, malignancy and cell migration and invasion. Poor differentiation of cancer cells was observed in H19-overexpressing hepatocellular carcinoma 5C modifications play a pivotal role in tumor metastasis and cell motility -induced endogenous p53 inactivation in lung cancer, while p53 is now better known for its anticancer effect than as an oncoprotein in various tumors, as it is associated with cell survival, invasion, tumor growth, metastasis, the EMT and multiple pro-oncogenic signaling pathways 5C modification at the transcriptional level and related oncogenic pathways is needed to improve our knowledge of the epigenetic regulation of tumor metastasis.As an immature RNA methylase family, the relationship between the DNMT-induced m5C modification in tumor cells and other diseases is inevitably deficient and has the potential for further exploration. The m5C modification may not be as popular as sibling methylation m6A, but it is still an integral part of epigenetic modification. Abundant writers and readers for RNA m5C have been identified, leading to a variety of interactions and regulation of biological processes. Typically, DNMT2 recognizes and methylates tRNA through a highly conserved region that was previously linked to DNA methylation. Along with NSUN2, NSUN3 and NSUN6, these enzymes target tRNAs and participate in tRNA fragmentation and energy metabolism. NSUN1, NSUN4 and NSUN5 preferentially bind rRNA and induce the process of ribosome metabolism. For mRNA, DNMT2 and NSUN2 are the main writers, since mRNA m5C is critical for numerous cellular biological processes, such as cell growth, differentiation, movement and RNA modification. Furthermore, NSUN2 and NSUN7 bind to noncoding RNAs that regulate cell metabolism 5C usually work in partnership with readers. YBX1 and ALYREF tend to recognize NSUN-induced methylation, which promotes the transportation and improves the stability of target mRNAs. YTHDF2, on the other hand, binds to m5C-modified rRNA at the same conserved residue for the m6A modification. Unfortunately, erasers for RNA m5C have not been comprehensively identified. Members of the well-known DNA demethylase family TETs trigger cascade oxidations, which may activate the underlying mechanism of RNA demethylation in the m5C region. Since a duality of methylation in DNA and RNA was found in m6A, m5C, hm5C and m1A modifications, studies aiming to identify whether a co-function of same methylation component induces RNA and DNA methylation or demethylation exists and the related mechanisms and outcomes are needed. Nevertheless, different types of RNA methylation are able to collaborate to regulate downstream targets and modulate the development of diseases. For example, NSUN2-mediated m5C collaborates with METTL-mediated m6A promote p21 expression, and various types of RNA methylation participate in glioma genesis and progression Epigenetic modification is increasing in significance in biological processes. The elucidation of nucleic acid methylation has been reported in recent years. Our review focused on the RNA m5C is relevant to numerous diseases, including energy metabolism disorders, hematopoietic system diseases, viral infection and the most remarkable pathological process, the development of malignancies. The expression of RNA m5C writers and readers is significantly correlated with a poor prognosis and the TNM classification of neoplasms. These writers and readers are mainly involved in pathways of tumorigenesis and metastasis in the brain, lung, breast, prostate and others tissue. Many cytokines, such as HDGF, TGF-β, FGF2 and G3BP1, which are validated tumor promoters, participate in cell migration and metastasis induced by the m5C modification, indicating a strong correlation between the RNA m5C level and mobility of cancer cells. Moreover, some studies discovered high RNA m5C levels in circulating tumor cells 5C in blood DNA was identified as a promising marker for cancer prognosis, increased general RNA m5C levels may be a novel indicator of metastasis. Nevertheless, a more comprehensive regulatory network for cancer cell transfer requires many mechanistic investigations of epigenetic modifications. RNA m5C has the potential to be a unique target for clinical cancer assessment and treatment.RNA m5C modification, gene therapy is a feasible treatment for RNA m5C-induced diseases and cancer development. By modifying the gene sequence of key RNA m5C methylases, such as NSUN2, reduced RNA methylation may prevent the occurrence of disease or reverse cancer progression. However, considering the non-specificity of gene therapy, a well-designed inhibitor based on molecular research may effectively reduce the function of a specific RNA m5C writer and reverse the development of diseases since inhibitors of m6A components are being used in cancer therapy. In addition, the upstream molecules that regulate the functional activity of the RNA m5C component may represent an optional treatment for diseases related to specific molecules. For example, aurora kinase B phosphorylates NSUN2 at Ser-139 and reduces RNA methylation levels, suggesting that regulation at the protein level is feasible 5C modification and exploit it in clinical applications.Regarding the existing targets of RNA m5C research when the final goal is to fully understand the RNA m5C modification and translate the findings into the clinic. Regarding the detection and research trends of RNA m6A modification, detection methods based on homogeneous techniques are still evolving to obtain better resolution or sensitivity 5C sites are inefficient, computer-based learning techniques that quickly and accurately predict RNA m5C modifications are urgently needed. For example, the novel m5C detection method, direct RNA sequencing revealed a more complex transcriptome characterization than expected in Arabidopsis5C site detection with good predictive performance will be helpful in structural and functional research on the RNA m5C modification 5C research. Database websites such as “RMBase” integrate a large number of RNA methylation data for review and verification while “RMDisease” and “RMVar” have been applied in RNA modification-related disease research 5C modification sites for functional research However, barriers still exist to further RNA mBXZ and ZGZ conceived this study and provided financial and administrative support. QFZ and FRL did most of the data generalization and summarization. WC, HRM and HFL help collecting supplement information. All authors further discussed about data and prepared the panels for tables and figures. QFZ wrote the manuscript. All authors read and approved the final manuscript.The researches that support the conclusion of this review have been referenced within this article.


Picornaviridae. In addition to being the first example of internal ribosome entry site (IRES) utilization, cardioviruses also employ a series of alternative translation strategies, such as Stop-Go translation and programmed ribosome frameshifting. Here, we focus on cardiovirus 2A protein, which is not only a primary virulence factor, but also exerts crucial regulatory functions during translation, including activation of viral ribosome frameshifting and inhibition of host cap-dependent translation. Only recently, biochemical and structural studies have allowed us to close the gaps in our knowledge of how cardiovirus 2A is able to act in diverse translation-related processes as a novel RNA-binding protein. This review will summarize these findings, which ultimately may lead to the discovery of other RNA-mediated gene expression strategies across a broad range of RNA viruses.Cardioviruses are single-stranded RNA viruses of the family  Viruses are obligate intracellular pathogens that depend on the host translation machinery to translate their own genome and replicate within the cell . In the Picornaviridae [Cardiovirus A is the encephalomyocarditis virus (EMCV), a well-established model for studying non-canonical translation [Cardiovirus B or Theilovirus, includes rat theilovirus (RTV), Vilyuisk human encephalomyelitis virus (VHEV) and Theiler’s murine encephalomyelitis virus (TMEV), all of which are genetically divergent from Cardiovirus A [Cardiovirus B before re-classification as new species Cardiovirus D in 2019 [Cardiovirus C, E and F each comprise only one or two isolates [Cardioviruses comprise a diverse group of viruses within the family aviridae . They haaviridae ,14. The aviridae . The arcnslation . The secovirus A . Within ovirus A . Human S in 2019 . To dateisolates ,18.Like other picornaviruses, all viral processes following viral entry take place in the cytoplasm, including replication, translation and viral assembly ,15,19. TDespite sharing a similar genomic organization to other picornaviruses, cardioviruses possess several unique features that facilitate key regulatory events during genome translation, thus allowing the virus to regulate its own replication and interfere with host cellular processes. Similar to other picornaviruses, the polyprotein encoding ORF is flanked at both ends by lengthy untranslated regions (UTRs) with significant secondary and tertiary structures ,33. The Other conserved RNA structures both at the 5′ UTR and within the coding region of the viral genome are also important for regulating viral gene expression. A region of ⁓450 nucleotides within the 5′ UTR comprises a type II internal ribosome entry site (IRES), which directs cap-independent internal initiation of protein synthesis ,45. In ctrans-frame product. In EMCV this is a 128-amino acid protein, whereas in TMEV it is a short 14–15-amino acid peptide [Although the 5′ UTR of cardioviruses has been a hotspot of research, other translational regulatory regions within the coding sequences are also crucial for viral pathogenicity. The coding region of the cardioviruses contains a conserved RNA element that directs programmed–1 ribosomal frameshifting (PRF) at the junction of 2A–2B genes . Ribosom peptide . 2B enco peptide . On the  peptide . Further peptide . This le peptide . This ev peptide . OverallEscherichia coli a simple hairpin can lead to 50% frameshifting on the dnaX coding sequence and is largely defined by the thermodynamics of codon–anticodon base pairing rather than the kinetics of RNA unfolding [Before we discuss protein-mediated frameshifting, it is useful to briefly consider the general principles of conventional PRF, applicable to hundreds of RNA viruses. Frameshifting ensures the production of proteins in optimal ratios for efficient virus assembly and enables viruses to expand their coding capacity through the utilization of overlapping ORFs . Typicalnfolding ,71. Framnfolding ,73.Arteriviridae), a −2 frameshift event occurs during translation of the ORF1a on a G_GUU_UUU slippery sequence. Unlike typical frameshift motifs, instead of the usual downstream structured RNA element, a C-rich sequence (CCCANCUCC) is present [trans-activator for −1 frameshifting [Recently, it was discovered that arteriviruses and cardioviruses utilize a new mechanism of PRF in which the stimulatory element is not limited to mRNA structures, but also involves an RNA-binding protein ,74,75. I present . Frameshshifting ,55,58. Tshifting .Cardiovirus species, with only ∼14% pairwise amino acid sequence identity  [EMCV 2A protein has also been shown to interact directly with 40S subunits in infected cells  and in v(∼10 nM) . This ma(∼10 nM)   . Early w(∼10 nM) . Given t(∼10 nM) . The funDisruption of nucleocytoplasmic trafficking is another hallmark of host cell shut-off in cardiovirus infection . The leaFinally, 2A has been implicated in the inhibition of apoptosis during EMCV infection . Virus rThe arginine loop A was fir


FT2a and Dt1. Taken together, we demonstrated the adoption of CB RNA-seq in soybean, which may shed light on gene expression regulation of important agronomic traits in leguminous crops.Transcription is the first step of central dogma, in which the genetic information stored in DNA is copied into RNA. In addition to mature RNA sequencing (RNA-seq), high-throughput nascent RNA assays have been established and applied to provide detailed transcriptional information. Here, we present the profiling of nascent RNA from trifoliate leaves and shoot apices of soybean. In combination with nascent RNA  and RNA-seq, we found that introns were largely spliced cotranscriptionally. Although alternative splicing (AS) was mainly determined at nascent RNA biogenesis, differential AS between the leaf and shoot apex at the mature RNA level did not correlate well with cotranscriptional differential AS. Overall, RNA abundance was moderately correlated between nascent RNA and mature RNA within each tissue, but the fold changes between the leaf and shoot apex were highly correlated. Thousands of novel transcripts (mainly non-coding RNA) were detected by CB RNA-seq, including the overlap of natural antisense RNA with two important genes controlling soybean reproductive development,  Drosophila melanogaster and Chironomus tentans  is involved in protein-coding gene transcription and some non-coding gene transcription. Before maturation, messenger RNA precursors (pre-mRNAs) are subjected to multiple processing steps, including 5′ capping, splicing of introns, 3′ cleavage and polyadenylation, and editing . These s tentans . In addi tentans . Studies tentans .Arabidopsis using genome-wide nascent RNA-seq approaches, plant native elongating transcript sequencing (pNET-seq), and plaNET-seq , with an average gene length of 2,000 bp and an average intron length of 180 bp  Merr.] is a paleopolyploid derived from two whole genome duplication events approximately 59 and 13 million years ago. It has a relatively complicated and large genome, with a size of approximately 1.1 gigabases  to measure the extent of cotranscriptional splicing . In shorbidopsis . The PIRDrosophila and Arabidopsis have indicated that multiple factors, such as intron characteristics, gene expression level, and number of introns, are related to cotranscriptional splicing efficiency , as an important regulatory step of gene expression, plays a critical role in the development and stress response of organisms . Previoutionally . Thus, wtionally  to descrtionally . The PSItionally . This wationally . Howevertionally , insets.tionally . Second,p-value < 0.05. A small number of the different splicing events between the leaf and shoot apex tissues were detected by both CB RNA and polyA RNA (mRNA and ΔPSICB were barely correlated (Spearman correlation ranged from 0.22 to 0.35)  . Furtherto 0.35) . Althoug7G206100 .Genes associated with intertissue differential splicing events detected by CB RNA and polyA RNA were also different . To explChromatin-bound RNA-seq is applied to detect transcribed RNAs, which are subject to multiple steps of mRNA processing, including cotranscriptional and posttranscriptional processes prior to maturation. Thus, there might be discordance in the abundance at the nascent RNA and mRNA levels. To test this hypothesis, we compared the TPM values of nascent RNA and mature RNA. Overall, the levels of nascent RNA and mature RNA were moderately correlated (Spearman correlation = 0.71–0.73) . There aTo select unstable and stable RNA transcripts, we first established a linear regression model of the log2 values of TPM genes obtained with CB RNA-seq and polyA RNA-seq. Then, the predicted TPM values of genes in polyA RNA were calculated based on the linear regression model. If the actual TPM of a gene was threefold higher (or lower) than the predicted TPM, the gene was considered to be stable (or unstable) . To inveWe then identified differentially expressed genes (DEGs) between 15-day apex and 15-day leaf tissues at both nascent and mature RNA levels. More than 10,000 genes were expressed more in the apex than in the leaf, and vice versa . Most ofGene Ontology enrichment analysis was performed to determine the biological functions of the DEGs. Genes with higher expression in the apex were mainly associated with RNA methylation, histone methylation, translation, DNA replication, and meristem initiation and maintenance. Genes with higher expression levels in the leaves were mainly related to photosynthesis and plastid organization .In addition, only a small number of genes were called DEGs between the 15-day apex and 10-day apex , and theFT2a, the essential gene involved in flowering timing, was identified in 15-day leaves by CB RNA-seq. Dt1, the key gene controlling growth habit, overlapped with another strong antisense transcript at the nascent RNA level in the apex . Only intergenic transcripts were included for further analysis. In total, there were 5,927 and 1,515 active intergenic transcripts from CB RNA-seq and polyA RNA-seq, respectively, with 1,326 transcripts overlapping . These tWe then applied two tools, CNCI and FEELnc, to evaluate the protein-coding potential of these new transcripts. In total, 4,001 and 974 active new transcripts of CB RNA and polyA RNA were considered non-coding transcripts by both methods, respectively , and morNon-coding RNA detected only at the nascent RNA level might be unstable or unpolyadenylated. ncRNAs detected only at the polyA RNA level might be very stable and accumulate by slow transcription. Different types of ncRNAs may be regulated differently at the transcriptional level. To gain insight into the effects of histone modifications on ncRNA expression, we compared the metaprofiles of histone modifications for three groups of ncRNAs from the leaf tissue  . Group IArabidopsis. Here, we describe the soybean transcriptome using CB RNA-seq. As expected, CB RNA isolation greatly enriched the nascent RNA by removing the abundant cytosolic mRNAs and nucleoplasmic RNAs. We demonstrated that CB RNA-seq successfully detected nascent RNA biogenesis and cotranscriptional processing of pre-mRNA from the leaves and growing apex tissues. This method can be applied to other tissues at various developmental stages and/or under different environmental conditions, which may further shed light on the transcriptional regulation of the soybean genome.Although nascent RNA-seq has been extensively used to detect cotranscriptional regulation in yeast, fly, and mammalian cells, its application in plants is still lagging behind. Recently, several methods have been developed to detect nascent RNA and reveal plant-specific transcriptional features . HoweverArabidopsis cells, indicating a conserved mechanism that controls cotranscriptional splicing in eukaryotic cells  was observed between CB RNA and polyA RNA, suggesting that most AS events are determined cotranscriptionally. This agrees with a previous study in bidopsis . HoweverArabidopsis, genes induced highly and quickly by short-term heat shock usually exhibit basic transcription under normal temperature  with a constant 25°C temperature in a growth chamber. Shoot apexes from 10- to 15-day seedlings were collected in three biological replicates, with each replicate collected from approximately 20 plants. For the leaves, the first trifoliolate leaves of two 15-day-old plants were collected as one biological replicate. All samples were frozen with liquid nitrogen immediately after collection.2, 0.75% Triton X-100, 0.5 mM EDTA, 1 mM DTT, 8 mM β-mercaptoethanol, 1 μg/ml pepstatin A, 1 μg/ml aprotinin, and 1 mM PMSF). The crude nuclei were precipitated at 3,500 rpm and washed with resuspension buffer  once, followed by washing buffer  twice. Chromatin RNA was extracted from washed nuclei using TRIzol reagent (Life Technologies).The chromatin RNA extraction protocol was modified from a previously published method . BrieflyAfter degrading genomic DNA by TURBO DNase (Life Technologies), CB RNA was subjected to rRNA depletion using a riboPOOL kit  and polyA RNA removal by oligo(dT) beads . Poly(A) RNA was enriched from total RNA by oligo(dT) beads. Both CB RNA and polyA RNA were transformed into cDNA libraries using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7765) and sequenced on an Illumina NovaSeq platform.1, and then Cutadapt was used to remove adapters and low-quality reads .For detecting genes with differential expression, mapped reads in each gene were quantified using featureCounts. Then, differential gene expression was evaluated by the R package DESeq2 . DEGs wep-value to control the FDR. All analysis was done in R software.Gene Ontology annotation of genes was extracted from the annotation file for Wm82.a2.v1. A hypergeometric test was explored for the statistical test, and the Benjamini and Hochberg method (1995) was used to adjust the To detect new ncRNA genes at the nascent RNA and polyA RNA levels, transcripts were assembled in CB RNA and polyA RNA data separately and merged by TACO as described above in the AS event analysis. Then, annotation GTF files of transcripts were compared with reference annotation GTF files using GffCompare (with the -r option). For each putative transcript, its relationship to the closest reference transcript was described by a “class code” value. For example, the code “=” indicates that the introns of a transcript completely match the introns of the reference transcript. We chose only unknown, intergenic transcripts that were assigned the code “u” and estimated their protein-coding potential by two software programs, CNCI and FEELnc .ChIP-seq raw data of histone modifications were downloaded from NCBI . The rawPRJNA689321.The original contributions presented in the study are publicly available. This data can be found here: NCBI website under Bioproject accession ZD, ML, and JZ designed the research. JZ and ML performed the research. ML, HZ, FK, and BL analyzed the data. ML and ZD wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.