Kaposi’s sarcoma-associated herpesvirus (KSHV) induces life-long infections and has evolved many ways to exert extensive control over its host’s transcriptional and post-transcriptional machinery to gain better access to resources and dampened immune sensing. The hallmark of this takeover is how KSHV reshapes RNA fate both to control expression of its own gene but also that of its host. From the nucleus to the cytoplasm, control of RNA expression, localization, and decay is a process that is carefully tuned by a multitude of factors and that can adapt or react to rapid changes in the environment. Intriguingly, it appears that KSHV has found ways to co-opt each of these pathways for its own benefit. Here we provide a comprehensive review of recent work in this area and in particular recent advances on the post-transcriptional modifications front. Overall, this review highlights the myriad of ways KSHV uses to control RNA fate and gathers novel insights gained from the past decade of research at the interface of RNA biology and the field of KSHV research. Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8 (HHV-8) is a γ2-herpesvirus that is the causative agent of Kaposi’s sarcoma (KS), KSHV inflammatory cytokine syndrome (KICS), primary effusion lymphoma (PEL), and a plasmablastic form of multicentric Castleman’s disease (MCD) ,2,3. Alt6A) modification, can regulate viral RNA stability and function at a global scale while other more specific features, such as G4 quadruplex motifs, can enforce strict control over viral RNA translation. There are also several virally encoded non-coding RNAs (ncRNA) that indirectly influence viral mRNA fate through a variety of mechanisms that mimic or even outperform their cellular counterparts during infection. We will also examine the regulation of key trans-acting viral proteins like ORF57 that protect viral RNAs from nuclear decay and the KSHV endoribonuclease, SOX, which triggers a massive RNA degradation event during lytic reactivation. KSHV’s capacity to take advantage of cellular RNA regulatory networks with such precision gives researchers a window into the never-ending arms race between viruses and their host. Through the work reviewed here, we wish to reveal research that shines a light on the strategies KSHV uses to enhance its viral gene expression. The studies illustrated here undoubtedly open new avenues in which cellular RNA fate and KSHV exploitation of those systems can be explored in the future. To hijack the host, KSHV triggers an intricate takeover of cellular RNA fate ranging from widespread manipulation of RNA decay to influencing patterns of RNA expression. By seizing control of RNA surveillance pathways, the virus can fine-tune the global gene expression environment throughout both phases of infection. This newly forged pro-viral RNA landscape allows KSHV unprecedented control over viral gene expression, which requires extensive temporal regulation and the capacity to widely remodel gene expression pathways. Many mechanisms are at play to allow the cell to maintain tight control over mRNA fate through mechanisms often associated with physical RNA features such as pre-mRNA splice patterns, 5′ cap, 3′ poly-A tail, and sequences/modifications that recruit trans-acting factors that alter mRNA stability. Post-transcriptional modifications and RNA editing also appear to be particularly important for KSHV-mediated takeover of the host. This review will explore the strategies evolved by KSHV to co-opt or subvert cellular RNA regulatory pathways that influence different stages of mRNA development during viral infection . Many of6A). These chemical additions emerge as crucial for the regulation of many RNA processes, like splicing, translation, stability, and decay. Before RNAs can interact with nuclear export machinery, they must contend with mechanisms that regulate the amount of transcript that gets exported into the cytoplasm or nuclear decay pathways, like the CBCN-mediated RNA decay pathway, which protects the cell from aberrant transcripts. In this section, we will be reviewing how KSHV uses m6A to regulate splicing of its own transcripts, ORF57, to escape nuclear decay pathways and G-Quadruplexes on its mRNA to control nuclear export. Circular RNAs (circRNAs) will also be a new addition to the vast list of KSHV transcript regulator and will be discussed below.As the cell initiates RNA transcription, it also begins the process of regulating the fate of RNA very closely. As transcription occurs, RNA transcripts will undergo a series of maturation processes mainly consisting of co-transcriptional splicing, addition of a 5′ cap, and a 3′ poly-A tail. These processes are mediated by cellular factors that interact with one another and promote maturation of the RNA and ultimately determine their localization and function ,10. Anot6A. The m6A machinery consists of a complex set of proteins that coordinate the regulation of RNA: there is a writer complex that includes METTL3, METTL14, WTAP, METTL16, RBM15, and KIAA1429 [6A addition is a highly dynamic process that also includes a removal mechanism catalyzed by demethylases or erasers like FTO or ALKBH5 [6A modifications both on latent and lytic viral transcripts. Intriguingly, during KSHV lytic reactivation, they also observed a general decrease of m6A deposition on cellular mRNAs [6As possibly allows for enhanced 3′ UTR regulation, which has been known to affect localization, translation initiation, and the stability/decay rate of mRNAs [6A methylation. One of the hypotheses raised by the authors is that the enrichment of methylation in the 3′ UTR on cellular transcripts may help mediate targeting of miRNAs by KSHV own miRNAs that are known to be essential for KSHV-induced cellular transformation [6A modifications during infection and what occurs to the m6A landscape when KSHV switches from the latent to the lytic portion of its life cycle. Recent studies from Ye et al. in vivo and Hesser et al. in vitro have also reported that the amount of m6A-modified KSHV mRNA increases during lytic reactivation while decreasing on cellular mRNAs [6A deposition may be due to viral-induced widespread RNA decay leading to a decrease in available transcripts for m6A deposition and, therefore, an increase in viral transcript availability. Another possibility is KSHV influences m6A deposition directly by increasing viral RNA likelihood of being methylated or by affecting the m6A machinery itself. The counterpart of this shift in m6A deposition towards viral mRNA is that the reduced m6A deposition on cellular RNA could help dampen some host anti-viral processes by affecting host mRNA stability and/or translation. MKIAA1429 ,12,13,14r ALKBH5 ,18. Thesar mRNAs . In latear mRNAs . The disof mRNAs . In factormation . Howeverormation . All thiar mRNAs ,23. The 6A modifications are decoded by a collection of cellular factors that “read” this modification and enact the associated function. Depending on the localization of the modification on the mRNA, different reader proteins will be recruited and the resulting effect on the modified RNA will differ [6A readers that contain an aromatic cage that allows direct binding of the protein to the m6A modification [6A using 3-deazaadenosine (DAA)—an inhibitor of METTL3—a prominent m6A writer—prevented pre-mRNA splicing of the KSHV major lytic switch protein, RTA (ORF50), which resulted in a reduction of viral lytic replication [6A sites found in the RTA pre-mRNA, it was revealed that a single methylation site in exon 2 was responsible for enhancing splicing by using its m6A site to recruit YTHDC1 which in turn recruits SRF3 and SRS10 to aid in splicing. However, RTA pre-mRNA is known to encompass many different splice variants, so it is possible that the other m6A sites found by Ye et al. interact with other m6A nuclear reader proteins such as hnRNP-C1/2 and hnRNP-A2B1 to regulate differential splicing. More recently, a new class of readers from the “Royal” family, specifically Staphylococcal nuclease domain-containing protein 1 (SND1), was shown to bind directly to m6A modifications similarly to YTH domain proteins [6A in aiding the recruitment of splicing factors, uncovering more m6A readers on novel m6A sites will reveal a complex growing regulatory network of co-transcriptional regulators that will help us better understand cellular and KSHV RNA fate. ml differ ,24,25,26fication . One of fication ,29,30. Ylication . Althougproteins . It was proteins . Given tKSHV Circular RNA. Circular RNAs (circRNAs) are a class of 3′ to 5′ cyclized RNAs that are the product of a unique form of precursor RNA processing known as back-splicing ,33. The Recent large-scale screens have uncovered that virally encoded circRNAs are also expressed during latent and lytic KSHV infection. One of the first screens of the KSHV genome for circRNA expression was performed by Toptan et al. in 2018, uncovering several novel viral circRNAs, most abundantly derived from the vIRF4 and PAN/K7.3 gene loci. This same list also found several other low abundance circRNAs potentially derived from multiple lytic ORFs . In para6A methylation could in fact drive the translation of these transcripts [Many questions continue to surround the mechanisms that regulate the expression of KSHV-encoded circRNAs and their impact within the viral RNA pool. Several large-scale screens have already identified numerous small back-splice junctions throughout the KSHV genome, while curiously only a single major junction gives rise to one of the most abundant KSHV circRNAs, circ-vIRF4. For human circRNAs, cis factors such as inverted Alu elements within the nascent pre-RNA between the donor and acceptor sites have been shown to mediate their interaction leading to cyclization . While snscripts . This isnscripts ,45. Furtnscripts . To dateLike circ-PAN, circ-vIRF4 also represents a curious case in both its expression across various KSHV-infected cell types and the current air of ambiguity that surrounds its regulation between latency and lytic reactivation. Previous reports have shown that linear vIRF4 is expressed at low levels during latency and is highly expressed during lytic infection. Conversely, a recent report by Abere et al. found that circ-vIRF4 was expressed at lower levels than its cognate linear form during latency and strangely was only marginally enhanced or even reduced in most PEL cell lines following lytic reactivation . While tThe field of circRNA biology, having matured well over the past decade, has turned much of our understanding of post-transcriptional regulation and the contribution of splicing to control RNA fate on its head. The discovery of KSHV-encoded circRNAs especially calls into question many previously characterized pro- and anti-viral factors that may now have newly defined roles in circRNA biogenesis and regulation or even as circRNAs themselves. One example of this is the protein adenosine deaminase acting on RNA 1 (ADAR1), a recently identified pro-viral inhibitor of RLR-dependent immune signaling, is also inhibitory to circRNA biogenesis, which could in-turn impact KSHV fitness . FurtherG-Quadruplexes. Recently G-quadruplexes (G4s) have gained a lot of attention. These secondary DNA or RNA structures arise from nucleic acids that are rich in guanine residues and result in G-quartets forming from Hoogsteen hydrogen bonding stack upon one another. Multiple proteins are required to help form or unwind these structures . G4s havORF57 and nuclear RNAi-defective 2 (NRDE2). RNA quality control (QC) pathways play a crucial role in the regulation of gene expression and RNA fate. Nuclear RNA QC pathways monitor the presence of improperly matured RNA and prevent their export to the cytoplasm. Among these QC pathways, two play a significant role in controlling the longevity of nuclear transcripts. These pathways are the PABPN1 and PAPα/γ-mediated RNA decay (PPD) pathway and the cap-binding complex (CBC) in association with the nuclear exosome targeting (NEXT) complex, known together as the CBCN-mediated RNA decay pathway. These pathways act on the decay of polyadenylated, short, and intronless transcripts, such as improperly spliced pre-mRNA that do not meet the requirements to become fully processed mature mRNA. These pathways also oversee the regulation of promoter upstream transcripts (PROMPTs),  short inTo answer this question, Ruiz and colleagues explored the involvement of ORF57 and ALYREF in connection to the CBCN and PPD nuclear decay pathways. The authors knocked down various components of these host RNA QC pathways and observed the effects on mutant of PAN that lacks the cis-acting nuclear retention element (PAN∆ENE) and lytic transcript ORF59 ,55. In tDespite the observation that ORF57 is capable of protecting viral transcripts form nuclear RNA QC decay pathways, it was observed that a small subset of transcripts still underwent PPD and CBCN modulated decay even in the presence of ORF57 ,55. UsinThrough these mechanisms, KSHV can hijack and manipulate host nuclear RNA regulatory pathways to promote infection. Both systems exploit host RNA QC pathways primarily through the manipulation of the mRNA export adaptor ALYREF. While the NRDE2 pathway does not directly require ORF57, interactions surrounding this event may also include ORF57, as it is vital for the recruitment of ALYREF to specific transcripts and plays a major role in splicing within KSHV replication compartments merged with nuclear speckles where it colocalizes with splicing factors . One aspDuring KSHV infection, as mRNAs enter the cytoplasm they are subjected to an even greater repertoire of cellular and viral factors that dictate whether they are translated, stored, or degraded. KSHV co-evolution with its human host has resulted in a wide breadth of viral intrusion into cytoplasmic RNA surveillance and mimicking of RNA features such as RNA interference or RNA structural elements. These processes emerge as crucial to suppress unwanted mRNA translation in favor of both latency maintenance and lytic viral replication .G-quadruplexes. As we previously discussed, G4s within LANA transcript are important to regulate LANA export from the nucleus, but it was also suggested that these G4s also play a role in the cytoplasmic regulation of LANA. It was discovered that when LANA G-quadruplex is stabilized, LANA translation was inhibited and, therefore, protein expression was reduced, despite normal mRNA synthesis. Furthermore, when LANA mRNA was mutated to disrupt the G4 formation, it was revealed that LANA translation was increased, further supporting G4s as a translation regulator of LANA. LANA has been shown to modulate antigen presentation by interacting with components of the antigen presentation ,67. G4s 6A. As previously mentioned, m6A modifications influence many aspects of an mRNA life. In the cytoplasm, m6A readers have been shown to affect the stability, localization, and translation of mRNAs. One such reader that has come up as an important regulator of the cytoplasmic viral mRNA pool is YTHDF2. This reader binds directly to m6A-modified mRNA and promotes their degradation by localizing them to RNA granules known as processing bodies (P-bodies) [6A in promoting lytic reactivation [6A reader [6A in this case would have an antiviral role [6A deposition and/or m6A-mediated functions are highly dynamic. One possibility is that m6A regulation is dependent on temporal factors and that fine-tuning of viral mRNA stability could be a requirement for proper progression of the viral life cycle. Furthermore, mapping of m6A sites within viral mRNA revealed that despite some differences, many of the m6A peaks are consistent across multiple cell types: the same transcripts overall are methylated but the position of this methylation along the transcript can vary. This suggests that the viral mRNAs are not necessarily interfacing with host m6A methyltransferase machinery differently between cell types but instead the contradictory results further enforce the idea that m6A deposition is dynamic and does not always occur on every possible m6A motif. Localization of the m6A mark can be tied to the rate of RNA Pol II elongation where a slow elongation leads to enhanced m6A modification and eventually decrease of translation efficiency [6A deposition and thus be highly context-dependent, possibly explaining this variability. Understanding where and what m6A depositions mean on an mRNA may help in understanding their effects on KSHV replication. M-bodies) ,69. Hesstivation . In starA reader . On the ral role . It is sficiency ,16. It iKSHV miRNA. Having passed an extensive panel of nuclear checkpoints, cytoplasmic mRNAs fall under an equally vast network of regulatory oversight evolved to control the terminal stages of their fate from translation to decay. One form of cytoplasmic RNA surveillance are small non-coding RNAs known as microRNAs (miRNA). miRNAs are short (19–23 nt), single-stranded RNAs that coordinate with cellular RNA silencing factors to dictate the rate of translation and longevity of target transcripts. Mechanistically, miRNAs often target complementary sites within the 3′ UTR or occasionally the coding region of their cognate mRNA. Binding specificity of miRNAs to these sites typically relies on a minimum seed sequence match (2–7 nt), but this requirement has been challenged by several recent reports . Once boStringent control over the switch between the latent and lytic stages of KSHV infection is critical for a successful persistent infection and timely coordination of viral replication. Most KSHV-encoded miRNAs are predominantly expressed during the latent phase of infection except for those derived from the Kaposin locus, which is highly expressed during the lytic phase. These expression patterns are evident across most KS/MCD derived tissues and cell lines albeit with inconsistency between expression levels of the miRNAs themselves . TherefoWhile RTA represents the most direct example of how KSHV-encoded miRNAs regulate the viral life cycle, only a handful of groups have identified other miRNA targets of viral origin. Aside from RTA, these earlier studies identified a few viral gene targets including ORF56, ORF57, and viral interleukin-6 (vIL-6) ,80,83. FWhile miRNA regulation of KSHV genes is typically discussed from the perspective of trans-regulation, pre-miRNAs that give rise to mature viral miRNA can also regulate viral gene expression in cis. In canonical miRNA biogenesis, primary-miRNAs (pri-miRNA) hairpins are transcribed and processed by the nuclear microprocessor complex comprised of DiGeorge Syndrome Critical Region 8 (DGCR8) and the ribonuclease III enzyme, Drosha into long duplex RNA fragments known as pre-miRNAs. These RNA duplexes are then exported into the cytoplasm where they give rise to mature miRNAs. Two pre-miRNAs, miR-K12 and miR-K10, located apart from the remaining 23, are encoded by 3′UTR of the coding gene Kaposin B (KapB). Many KSHV genes are divided into either latency or lytic gene families based on their expression patterns throughout viral infection. However, several KSHV transcripts, including KapB, are constitutively expressed at low levels during latency and highly expressed following lytic reactivation . A curreThis binary fate of the KapB mRNA highlights the interconnectedness between post-transcriptional regulation of viral mRNA and viral miRNA biogenesis. It would be exciting to see if pre-miRNAs of other unrelated DNA tumor viruses, especially those encoded in the 3′ UTRs or exons of coding transcripts, have also been evolved to serve as cis-regulatory factors in the regulation of viral gene expression. Along the same vein, adaptation of the microprocessor machinery by KSHV opens a number of avenues of investigation including studies of viral manipulation of miRNA processing beyond Drosha, the regulation of Drosha in respect to cellular miRNA biogenesis during infection, and the host mRNAs they regulate therein. Much work is still necessary to fully uncover the depth to which miRNAs influence the fate of viral RNAs and the physiological consequences of this activity. Several studies have reported functions for miRNAs far beyond this scope, especially with reports describing their incorporation into extracellular vesicles and even KSHV virions for signaling in a paracrine like manner ,95. WithSOX. Similarly to ORF57 that regulates RNA decay in the cytoplasm, KSHV also has a major RNA decay regulator in the cytoplasm, SOX (ORF37). This protein is expressed as a delayed early gene during lytic reactivation and is responsible for the host shutoff event, which results in decreased host gene expression ,100. Dur6A in the context of KSHV have been elucidated, however, there are conflicting roles depending on cell type and the reason for this remains to be discovered. Similarly, new research has been done into KSHV miRNAs now leaves questions regarding how the host monitors these miRNAs and how the dynamics of P-bodies affect their function as well. KSHV’s use of the G4 structural element is fascinating and makes one wonder if there are more transcripts that use G4s or other structural elements to regulate their translation efficiency. Finally, although a lot of research is done into viral endonucleases like KSHV SOX, there are still mysteries surrounding the mechanisms of targeting and escape of transcripts. Answering these outstanding questions will reveal new avenues of KSHV cytoplasmic regulation of RNA in the cell. The continued research into KSHV’s pervasive use of cellular regulatory pathways has revealed new insights into how KSHV enhances its infection as well as new aspects of cellular RNA fate. Although this research as given more insights into KSHV regulation, more questions remain. New roles for m6A on KSHV biology. The extent of factors that can interact with these structural and chemical modifications is by no means a complete list. Finding and characterizing these factors will greatly expand our knowledge of RNA fate as well as increase our understanding of KSHV host takeover. There are also still over a hundred different RNA modifications and post-transcriptional RNA editing that have been documented like inosine (I), pseudouridine (Y), and 2′-O-methyladenosine (Am). Although m6A has been studied in the context of KSHV, these other RNA editing strategies may be used by the virus in order to regulate cellular and viral RNAs [6A modification could also contribute to the host side of the viral-host battle. By understanding these processes further, we continue to better appreciate how the tenuous and important the balance of RNA fate is.The past two decades have uncovered exciting new ways by which KSHV extensively regulates both its own and its host’s RNA during infection. This emphasizes KSHV large investment in the everlasting battle with its host over the gene expression cascade. Viruses have historically been good models to gain a deeper understanding of basic cellular mechanisms, and by studying how KSHV manipulates RNA fate, we continue to discover novel aspects of the gene expression regulation. It also underlines the critical role of the RNA fate equilibrium and how disrupting that balance can have far-reaching consequences. Currently, there is still much to study and understand about what governs these mechanisms. In particular, we are only starting to appreciate the contribution of G-Quadraplexes or mral RNAs . Given t


In the published article, there was an error in affiliations 2, 3, and 4. Instead of affiliations:2. Department of Integrative Medicine and Neurobiology, School of Basic Medical Sciences, Shanghai, China3. Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, China4. Department of Radiation Oncology, Shanghai Ninth People s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China,it should be:2. Department of Radiation Oncology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China3. Department of Integrative Medicine and Neurobiology, School of Basic Medical Sciences, Shanghai, China4. Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, China.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


XRN1-knockdown HeLa cell lines, followed by a comparison of steady state and degraded mRNA levels from RNA-seq and PARE-seq data, respectively. The results revealed 1103 and 1347 transcripts classified as stable and unstable candidates, respectively. Of the unstable candidates, we found that a subset of the replication-dependent histone transcripts was polyadenylated and rapidly degraded. Additionally, we identified 380 endonucleolytically cleaved candidates by analyzing the most abundant PARE sequence on a transcript. Of these, 41.4% of genes were classified as unstable genes, which implied that their endonucleolytic cleavage might affect their mRNA stability. Furthermore, we identified 1877 decapped candidates, including HSP90B1 and SWI5, having the most abundant PARE sequences at the 5′-end positions of the transcripts. These results provide a useful resource for further analysis of RNA decay patterns in human cells.RNA decay is an important regulatory mechanism for gene expression at the posttranscriptional level. Although the main pathways and major enzymes that facilitate this process are well defined, global analysis of RNA turnover remains under-investigated. Recent advances in the application of next-generation sequencing technology enable its use in order to examine various RNA decay patterns at the genome-wide scale. In this study, we investigated human RNA decay patterns using parallel analysis of RNA end-sequencing (PARE-seq) data from  The gene expression levels within a cell are determined by the interplay of tightly regulated processes for RNA synthesis and decay. Although transcription-mediated mRNA synthesis plays a major role, mRNA decay pathways at the posttranscriptional level also significantly contribute to regulating gene expression. Cytoplasmic mRNAs are internally degraded by endoribonucleases or from one of the extremities by exoribonucleases. These ribonucleolytic activities include decapping, 5′ to 3′ exonucleolytic decay, deadenylation, 3′ to 5′ exonucleolytic decay, and endonucleolytic cleavage ,2,3,4,5.RNA decay on a transcriptome-wide scale has been studied while using several methods. One well-established method takes advantage of inhibitor-mediated global transcriptional arrest. In this method, global transcription is blocked by transcription inhibitors, such as actinomycin D, 5,6-dichloro-1–D-ribofuranosyl-benzimidazole and α-amanitin , with su6-methyladenosine-containing RNAs by the RNase P/MRP complex [Parallel analysis of RNA ends (PARE) or RNA degradome sequencing is another method adopted for RNA decay analysis ,21,22. P complex ,28.XRN1-knockdown HeLa cell lines to investigate global mRNA stability. Using genome-wide bioinformatics analysis, we classified stable and unstable genes and identified endonucleolytically cleaved and decapped candidate transcripts.In this study, we analyzed PARE-seq and RNA-seq data that were established from XRN1-knockdown HeLa cells in order to investigate global RNA stability in human cells [2 = 0.999; and PARE-seq R2 = 0.704), indicating the high quality and reproducibility of the data (2 = 0.484)  C. We ado= 0.484) A to iden= 0.484) .The set of genes was subjected to Gene Ontology (GO) analysis in order to understand the biological processes associated with these stable and unstable transcripts. We observed that the stable gene set showed a high enrichment for genes that were involved in nucleic acid metabolism, RNA metabolic process, and gene expression A. By conLARP1, PABPN1, BMI1, and PTEN, with their RNA-seq read coverages and PARE-seq D-plots being shown in LARP1 and PABPN1 both encode RNA-binding proteins and they are involved in gene expression by promoting translation or protecting mRNA poly(A) tails from degradation, respectively. BMI1 is a proto-oncogene encoding a ring protein that is a major component of polycomb repressive complex 1, and PTEN encodes a phosphatase that acts as a tumor suppressor. As unstable genes, we selected CIRBP, PDRG1, IER3, and HIST2H4A , and HIST2H4A encodes a histone H4 protein that is responsible for nucleosome structure.We then examined the decay plots (D-plots) and RNA-seq read coverage of the representative stable and unstable transcripts . D-plotsHIST2H4A B. CIRBP HIST2H4A, which encodes a replication-dependent histone (RDH), was identified as an unstable gene (RDH mRNAs have conserved stem-loop structures instead of poly(A) tails, their expression could not be detected in the poly(A)-selected RNA-seq and PARE-seq data. However, we noticed that polyadenylated and degraded HIST2H4A mRNA was well represented in PARE-seq data, but barely detected by RNA-seq. Recent reports indicated that a subset of histone genes produces poly(A) mRNAs under a variety of cellular conditions and stress conditions [RDH mRNAs was polyadenylated and rapidly degraded in XRN1-knockdown HeLa cells. Indeed, many RDH transcripts were classified as unstable genes and enriched in the chromatin-silencing GO term.Interestingly, ble gene B. Given nditions ,31,32. ORDH and 13 replication-independent histone (RIH) transcripts, in order to globally investigate the RNA stability of the histone transcripts. As expected, most RDH transcripts were detected at lower levels in poly(A)-selected RNA-seq data, whereas RIH transcripts were well represented  were classified as unstable according to our analysis. We found that no stable genes contained endoribonucleolytic cleavage sites, implying that endonucleolytic cleavage of the 77 genes might affect mRNA stability.We identified the most abundant PARE sequence on a transcript, designated as a Max PARE sequence (Max-seq), for all annotated human transcripts to identify endonucleolytically cleaved transcripts. For better identification, we adopted four stringent filters: reproducible Max-seq position, reliable decay abundance, Max-seq abundance, and the more prominent Max-seq value relative to that of other decay byproducts A. By anaNuclear speckle splicing regulatory protein 1 encodes an RNA-binding protein that mediates pre-mRNA alternative-splicing regulation   B and ence 3′ UTR C, with tanalysis D. Moreovequences E, whereaanscript F.XRN1-knockdown cell lines and PARE-seq profiles at the 5′ ends of monophosphorylated mRNAs. A prerequisite to identifying decapped transcripts involves the determination of the precise 5′ end positions of mRNAs. To this end, we took advantage of C-PARE-seq, which profiles the 5′ ends of the capped transcript. C-PARE-seq data were established from the same RNAs used in PARE-seq library construction [The process of mRNA decapping involves the hydrolysis of the 5′ cap structure and exposure of the 5′ monophosphate by the decapping enzyme DCL2. These decapped mRNAs are further processed by XRN1 and quickly decayed. We assumed that our PARE-seq data could be used to identify decapped transcripts, because the PARE-seq libraries were constructed from truction .C-PARE sequences were mapped to 200-nt regions upstream and downstream of the annotated cDNAs (+/− 200-nt windows) in order to identify precise 5′-end positions of mRNAs, resulting in the identification of 41,718 transcripts with at least one C-PARE sequence. Empirical cap positions were defined using three filters A. First,PARE sequences were mapped to +/− 200-nt windows to identify decapped transcripts, obtaining 85,178 transcripts with at least one PARE sequence. These were further filtered using the same criteria used for C-PARE analysis. The results identified Max-seqs within these windows for 3852 transcripts. We then compared the positions of C-PARE Max-seq and PARE Max-seq in each transcript, identifying 1877 transcripts as decapped candidates based on their C-PARE Max-seqs and PARE Max-seqs being at the same positions . These 1We then performed GO analysis of the 1877 decapped candidate transcripts. The set was highly enriched for genes that were involved in the mRNA metabolic process, nuclear-transcribed mRNA catabolic process, viral gene expression, and NMD A. In theHSP90B1-001 and RPL11-001 are examples of transcripts categorized as stable genes  were categorized as unstable genes -tailed canonical RDH transcripts. RDH mRNAs are non-polyadenylated in metazoans and instead contain their characteristic 3′ UTR with highly conserved stem-loop structures [RDH mRNAs are inappropriately processed at the 3′ ends and contain polyadenylated tails, they are rapidly degraded by AU-rich element-mediated mRNA decay via BRF1 [RDH mRNAs increases [RDH mRNAs. In the present study, we found that most polyadenylated RDH mRNAs were unstable along with high accumulation of their decay byproducts. Although we were unable to identify the conserved decay pattern of polyadenylated RDHs, we did identify potential endoribonucleolytic cleavage patterns in various positions of RDH cDNAs  were adjusted to <0.01 and >|2.5|, respectively.DPKM was calculated by summing the abundance of all PARE sequences matching a cDNA and normalizing the summed abundance by cDNA length and total read number. The EdgeR program  was usedhttps://www.gencodegenes.org/) protein-coding transcripts. Four filters were adopted (Endonucleolytic cleavage sites and decapped transcripts were identified while using custom Python scripts. The SOA and the abundance of PARE Max-seqs were first obtained after mapping the PARE sequences to the annotated transcript in order to identify endonucleolytic cleavage sites. Annotated transcripts were downloaded from the Gencode V11 ( adopted . SOA andGO analysis of a set of genes was performed using the DAVID bioinformatics analysis platform ,52. A fuXRN1-knockdown HeLa cells, additional data from wild-type and mutated cells defective in other RNA decay machinery will provide a better understanding of the roles of RNA decay mechanisms in regulating gene expression. Additionally, our analysis can be applied in order to understand the role of mRNA stability in various conditions or among the different tissues.In this study, we analyzed RNA decay byproducts from PARE-seq data and identified the diverse RNA-decay patterns of human genes, including endonucleolytically cleaved and decapped candidates. The findings revealed various RNA decay patterns not only with quantitative information, but also the exact positions of the cleaved sites at single-nucleotide resolution. The list of candidate genes identified in this study will be useful resources for further analysis of their expression. Although the data were limited to those from


However, the regulatory mechanism of mRNA structure in miRNA cleavage remains elusive. We investigated the role of in vivo RNA secondary structure in miRNA cleavage by developing the new CAP-STRUCTURE-seq method to capture the intact mRNA structurome in Arabidopsis thaliana. This approach revealed that miRNA target sites were not structurally accessible for miRISC binding prior to cleavage in vivo. Instead, we found that the unfolding of the target site structure plays a key role in miRISC activity in vivo. We found that the single-strandedness of the two nucleotides immediately downstream of the target site, named Target Adjacent nucleotide Motif, can promote miRNA cleavage but not miRNA binding, thus decoupling target site binding from cleavage. Our findings demonstrate that mRNA structure in vivo can modulate miRNA cleavage, providing evidence of mRNA structure-dependent regulation of biological processes.MicroRNA (miRNA)-mediated cleavage is involved in numerous essential cellular pathways. miRNAs recognize target RNAs via sequence complementarity. In addition to complementarity,  Howevng cells . Second,ng cells . This coin vivo, a method is required that obtains RNA structures before cleavage. Recently, several transcriptome-wide in vivo structure probing methods for RNA have been established -based methods utilize a variety of chemicals, e.g. 2-methylnicotinic acid (NAI), to probe the single-strandedness of all four RNA nucleotides , can promote miRNA cleavage but not miRNA binding. Thus, TAM decouples target site binding from cleavage. Our study revealed the role of in vivo mRNA structure in the regulation of miRNA cleavage, providing evidence of mRNA structure-dependent regulation of biological processes.To decipher the Arabidopsis thaliana seeds of the Columbia (Col-0) and the xrn4 mutant accession . The whole procedure was performed according to Ding et al.  and 0.5 mM MgCl2) in a 50 ml Falcon tube. NAI was added to a final concentration of 150 mM and the tube swirled on a shaker (1000 rpm) for 15 min at room temperature (22°C). This NAI concentration and reaction time had been optimized to allow NAI to penetrate plant cells and modify the RNA in vivo under single-hit kinetics conditions (We modified the protocol  by changprotocol . Brieflynditions . After qCAP-enriched and 3′ poly(A)-enriched RNA structure sequencing.To capture the structure information around the cleavage site of miRNA target genes, we adopted the feature of 5PSeq . The whoet al. (iP is the (+)SHAPE RT count and iM is the (−)SHAPE RT count at nucleotide i. The factor, α  stop counts to obtain a single (−)SHAPE library and a single (+)SHAPE library. We calculated the SHAPE reactivity using a slightly modified version of the formula in Ding et al. ,\documeng et al. . The reag et al. . For theA. thaliana etiolated seedlings were grown as described above. They were collected and immediately frozen in liquid nitrogen and stored at −80°C. The seedlings were ground into powder. Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions. On-column DNAaseI treatment was carried out according to RNase-Free DNase Set (Qiagen). To construct the Illumina library for degradome analysis, polyA+ selection was carried out using the Poly(A)Purist Kit (Ambion™). Selectively captured polyadenylated RNAs (1 μg) were ligated directly to an DNA/RNA hybrid adapter (5′-CTACAC GACGCTrCrUrUrCrCrGrArUrCrUrNrNrN-3′) using T4 RNA ligase (NEB) at 37°C for 30 min. The ligated RNAs were subjected to RT by SuperScript III First-Strand Synthesis System (Invitrogen) with random hexamers fused with Illumina TruSeq adapters (5′-CAGACGTGTGCTCTTCCGATCTNNNNNN-3′). Polymerase chain reaction (PCR) amplification was performed on the ligated cDNA using Illumina TruSeq Primers. Two different barcode indices were used for two degradome biological replicates. The final dsDNA degradome libraries were subjected to next-generation sequencing on Illumina HiSeq 4000 (BGI).Five-day-old Raw reads were processed to remove 5′and 3′ adapter sequences. Degradome reads were mapped to the TAIR10 transcript reference and a degradome density file was generated. The degradation level of target genes was normalized by reads per kilobase per million mapped reads (RPKM).The same seedling samples stored at −80°C, as described above, were ground into powder using liquid nitrogen. Total RNA was extracted using mirVana miRNA Isolation Kit  following the manufacturer's instructions. The integrity analysis was performed on a Bioanalyzer by the Beijing Genomics Institute (BGI), Shenzhen, China, which also performed the library construction according to standard protocols.A. thaliana mature miRNAs  can be estimated by,A. thaliana by assigning a sequence complementarity penalty score .ΔGunpaired ,26. By eunpaired , we chos package  together‡cutting measures the energy required to raise the initial substrate target RNA to the transition catalysis-compatible state  (Kluyveromyces polysporus Argonaute (KpAGO)  ,29, huma (TtAgo)  and yeas (KpAGO)  suggest  (KpAGO) ,29. Theral mol−1 . TherefoFor CE validation, the miRNA156 target sites, followed by 0 or 2 Adenines (As) and ending with a G-quadruplex (GQS) or a stem-loop (SL) were synthesized and inserted into AflII and PacI of Firefly 3′UTR in vector inter2. We labeled the GQS constructs as 0A_GQS and 2A_GQS, and the stem-loop constructs as 0A_SL and 2A_SL, with the prefix indicating the number of Adenines. Antisense of miRNA156 target site constructs with the same flanking sequence were also synthesized as the control for each construct.in vivo binding assay, the MIR156B genomic sequence was inserted into AscI and SacI of vector pMDC32. Primers are listed in For the miRNA156 overexpression vector construction in AGO1 MIR156 over-expression line were prepared and transformed according to the Tape-Arabidopsis Sandwich method  by Invitrogen SuperScript III reverse transcriptase. The resulting cDNA pellet was dissolved in RNase-free water and mixed with 1 μl 50 μm Mly1-HBLPCR-5′ssDNA linker modified by a 5′-phosphate and a 3′-3-Carbon spacer group (5′P-AGATCGACTCAGCGTCGTGTAGCTGAGTCGATCTNNNNNN-C3-3′), 10 μl Quick Ligase Reaction Buffer (2×), 1U Quick Ligase (New England Biolab) in a 20 μl system. The ligation was performed at 25°C for 1 h, followed by Phenol:Chloroform:Isoamyl Alcohol  and Chloroform:Isoamyl  purification.Four-week-old tobacco leaves were co-infiltrated with agrobacterium strains harboring plasmids of 0A_SL, 2A_SL, 0A_GQS or 2A_GQS. Two days after infiltration, the leaves were treated with 150 mM of the SHAPE reagent (NAI). The control group was treated with DMSO. Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen) and then by on-column DNaseI treatment (following the respective manufacturer's protocol). Gene-specific reverse transcription was performed as previously described by Kwok et al. , with a The ligated cDNA samples were dissolved in 10 μl of water and used for the PCR reaction. The PCR reaction contained final concentrations of 0.5 mM VIC-labeled DNA gene-specific primers (the same as that used in the reverse transcription primers except the 5′ end was labeled with Vic), 0.5 mM of linker reverse primer (AGATCGACTCAGCTACACGACGC), 200 mM dNTPs, 1× ThermoPol reaction buffer and 1.25U of NEB Taq DNA polymerase in 25 μl. The solution was then extracted with Phenol:Chloroform:Isoamyl  and incubated with Mly1 restriction enzyme, according to the manufacturer's protocol. Finally, the reaction pellets were dried and resuspended in Hi-Di formamide (Applied Biosystems/Life Technologies).The Ned-labeled gene-specific primer (the same as that used in reverse transcription primer except the 5′ end was labeled with NED) was used to make sequencing ladders using linear DNA and 1 μl 5 mM ddTTP by Klenow DNA Polymerase I (New England Biolab) . Then, tWT was immuno-purified from Arabidopsis seedlings  as substrates. To perform the slice assay, cleavage buffer  was added to 20 μl beads in extraction buffer (1:1) bearing freshly purified HA-AGO1 from 3 g seedling on the beads’ surface. A total of 50 cps of labeled substrate was added to the reaction and incubated at 25°C. A total of 10μl of the resultant liquid was added to 10 μl 2× RNA loading buffer , 0.02%, Bromothymol Blue, 0.01% Xylene Cyanol), denatured for 5 min at 95°C and loaded into a 1 mm polyacrylamide gel electrophoresis gel . Then the gel was dried and exposed to a phosphor screen for image analysis.HA-tagged AGO1eedlings . The 0A_DAH and 0A_GQS or 2A_GQS. Two days after infiltration, the leaves were collected and ground in liquid nitrogen. The protein/RNA complexes were extracted using two volumes of IP buffer . After removing insoluble debris by centrifugation, cell extracts were incubated with anti-HA antibody (Abcam ab9110) for 1h at 4°C with gentle mixing. The anti-HA-decorated extracts were then incubated with pre-washed protein G magnetic beads for 1 h. After incubation, the beads were washed 6 times with the IP buffer. The RNA produced after co-immunoprecipitation was precipitated with ethanol and glycogen, and analysed by RT-PCR. The miRNA156 expression levels were analysed by miRNA RT-PCR  and andin vis Figure ,39. We ls Figure  with thes Figure . We checs Figure . We thens Figure  and the s Figure . We founTURE-seq   activity . We thenAP2 targeted by miRNA172, which has been shown to act through translational repression rather than mRNA cleavage   , had higasi-RNA) . These oasi-RNA) . Since sasi-RNA)  we then asi-RNA) . In addiasi-RNA) . Our resin vivo. Since our CAP-STRUCTURE-seq directly measured the in vivo structural accessibility via SHAPE reactivity  between the inaccessible and accessible structural states (i.e. the unfolding of the target site) is lower than the barrier for catalytic cleavage , and would be less affected by the RNA structure of the target site. In the structure-limiting scenario, the energy barrier (ΔG‡open) between the inaccessible and accessible structural states is higher than the barrier for cleavage   Figure . Howeverg Figure , contrartivation , where sDAH) (‡open anti-correlated with the enrichment ratio of target RNAs from previous AGO1DAH-RIP-seq results (P = 0.05). In contrast, both the free energy of binding of the miRNA-target duplex (ΔGduplex) and ΔG‡cutting show no correlation with the enrichment . These observations suggest that the target sites are not structurally accessible in vivo, but rather the unfolding of the target site structure plays a key role in AGO processing of target RNAs.Our analysis favors the structure-limiting scenario rather than the enzyme-limiting scenario, implying that unwinding the miRNA target may be the limiting step in miRISC action, but once the miRNA is bound cleavage occurs quickly. This is consistent with AGO RIP-seq results, where few target transcripts have been captured in wild-type (WT) with less than 1-fold enrichment, while target mRNAs were enriched 7-fold in the catalytic mutant of (AGO1DAH) . Further results   and cleaved (positive CE) groups. We found higher SHAPE reactivity at the +1 and +2 nt immediately downstream of target sites in the cleaved group relative to the non-cleaved group .Having revealed that the target site structure affects cleavage p Figure , suggest utility  to calcu utility , indicat utility  or AT co utility  because in vivo by using capillary electrophoresis  than those without TAM  involving the 20 nt miRNA156 target site and a designed stable structure module. We concatenated the target site with either a G-quadruplex structure or a stem-loop structure with the aim of mimicking the base-pairing state of the 2 nt immediately downstream of the target site Figure . To mainphoresis  to resolM Figure . This suin vitro AGO cleavage assay using HA immuno-affinity-purified WT AGO protein. We found that the target RNA was cleaved only when TAM was present  by using the slicing-defective AGO1 mutant, AGO1D762A. We found that AGO1 was able to bind the target RNAs with the same binding affinity irrespective of whether the TAM was present or absent  is a well-characterized RNA-structure probing chemical which can detect accessibility of all four RNA nucleotides invivo . We applg method  , to find and bind its target site . Thus, the lower the energy barrier the easier miRISC can bind to the target sites  show tho Figure . Insteado Figure  from an e Figure . Thus, te Figure . This eqs Figure .‡open) can contribute 23% to the final miRNA-mediated cleavage in vivo , the mio Figure . ConsideThermus thermophilus Argonaute (TtAgo) complexes has been solved (2+) with the AGO nuclease activation site, the phosphate oxygens and in-line water, in order to facilitate the attack on the cleavable phosphate. The single-stranded TAM may promote this conformation transition and trigger the nuclease activity of AGO. Since the TAM is located at the 3′ end of the target site on the RNA, which is in parallel with 5′ end position of the miRNA and the 5′ end of the miRNA interacts with the MID domain of AGO, we suspect that single-stranded TAM may engage or ‘touch’ amino acids in the MID domain  ternary structures of n solved , providin Figure , therebyn Figure .in vivo  function rather than be regulated by RBPs (Our work indicates that the accessibility of the mRNA target site may be the primary determinant for RISC endonuclease efficacy. And we determined that a motif comprising two unpaired nucleotides immediately downstream of the pre-cleaved mRNA target site is required to direct RISC cleavage. Adaptation of this motif within the  by RBPs . Furtherin vivo through CAP-STRUCTURE-seq, we found that miRNA target sites were not structurally accessible in vivo and we demonstrated that the unfolding of the miRNA target site structure predominantly affected miRISC activity in vivo. Furthermore, we discovered that the native RNA structure motif, TAM, was sufficient to regulate miRNA cleavage in vivo. The TAM mechanism provides evidence of mRNA structure-dependent regulation of biological processes in vivo. Our study reveals that in vivo mRNA structure serves as an additional regulator of miRISC activity, which could also facilitate the biotechnological engineering of gene silencing, and possibly provide an additional avenue toward crop improvement.In summary, by deciphering intact mRNA structures gkaa577_Supplemental_FileClick here for additional data file.


RNA sequencing is an indispensable research tool used in a broad range of transcriptome analysis studies. The most common application of RNA Sequencing is differential expression analysis and it is used to determine genetic loci with distinct expression across different conditions. An emerging field called single-cell RNA sequencing is used for transcriptome profiling at the individual cell level. The standard protocols for both of these approaches include the processing of sequencing libraries and result in the generation of count matrices. An obstacle to these analyses and the acquisition of meaningful results is that they require programing expertise. Although some effort has been directed toward the development of user-friendly RNA-Seq analysis analysis tools, few have the flexibility to explore both Bulk and single-cell RNA sequencing.BingleSeq was developed as an intuitive application that provides a user-friendly solution for the analysis of count matrices produced by both Bulk and Single-cell RNA-Seq experiments. This was achieved by building an interactive dashboard-like user interface which incorporates three state-of-the-art software packages for each type of the aforementioned analyses. Furthermore, BingleSeq includes additional features such as visualization techniques, extensive functional annotation analysis and rank-based consensus for differential gene analysis results. As a result, BingleSeq puts some of the best reviewed and most widely used packages and tools for RNA-Seq analyses at the fingertips of biologists with no programing experience.https://github.com/dbdimitrov/BingleSeq/.BingleSeq is as an easy-to-install R package available on GitHub at  About a decade ago, a transcriptome profiling approach, known as RNA Sequencing (RNA-Seq), was predicted to revolutionize transcriptome analyses . Today, Single-cell RNA Sequencing (scRNA-Seq) is an emerging field that enables the gene expression profiling at the individual cell level and it is believed to lead to the reconstruction of an entire human cell lineage tree . scRNA-SAlthough there is a wide range of software tools available for both Bulk RNA-Seq and scRNA-Seq analyses, most require some proficiency in programing languages such as R. This creates a challenge for the analysis of RNA-seq data for a large portion of biologists lacking programing experience. Here we present an application, called BingleSeq, the primary goal of which is to enable the user-friendly analysis of count tables obtained by both Bulk RNA-Seq and scRNA-Seq protocols.BingleSeq is based on shiny  and it iUser experience is complemented with customizable and interactive tables and plots as well as analysis-related tips and pop-up messages to guide the correct execution of the pipelines. Plot interactivity was achieved via conversion from ggplot to ggplotly .To begin the DE analysis of Bulk RNA-Seq data, a count table and a metadata table must be loaded in the appropriate formats . Genes cSubsequent to quality control, users can investigate differentially expressed genes (DEGs) using three state-of-the-art packages: DESeq2 , edgeR , and limUpon obtaining DE results, users can visualize them using interactive plotting techniques –2E. Thesp-value threshold and fold-change threshold, among others. Due to their versatility, heatmaps were designed as BingleSeq’s most customizable plotting component —see https://support.10xgenomics.com/single-cell-gene-expression/datasets/1.1.0/pbmc3k.An evaluation of BingleSeq’s scRNA-Seq pipeline was performed by reproducing and extending the results of Seurat’s online tutorial  method from the viper package  and togeT test.BingleSeq supplies an option to assess the agreement between the different DE analysis methods in the form of a Venn diagram . MoreovePRJEB27501. The single-cell RNA-Seq example is a Cell Ranger 10× Genomics dataset looking at filtered data of 2,700 human peripheral blood mononuclear cells.BingleSeq also provides inbuilt Bulk RNA-Seq and scRNA-Seq test data. Bulk RNA-Seq data is represented by a 3-sample contrast between HSV-1 infected control and interferon B treatment . The datSome effort has already been directed towards lowering the entry requirements to RNA-Seq analyses as there are some software tools which implement UI components. However, many of these applications are limited to only some key features or particular parts of RNA-Seq analysis .From the available software packages providing comprehensive solutions for Bulk RNA-Seq, NetworkAnalyst , DEapp , DEBrowsWhen looking at similar applications providing solutions to scRNA-Seq analysis, BingleSeq is most analogous to SeuratWizard  as both In terms of providing a solution to both Bulk and single-cell RNA-Seq analyses, BingleSeq’s features and comprehensiveness are only contested by those of Omics Playground. However, what sets BingleSeq apart is that it provides multiple clustering packages and algorithms.As solutions to Bulk RNA-Seq, BingleSeq implements DESeq2, edgeR, and limma. These packages are well-tested and regarded as being among the best performing ones . DespiteBingleSeq’s scRNA-Seq pipeline includes three unsupervised clustering solutions provided by monocle, Seurat, and SC3 packages. The latter two packages are regarded as having the best overall clustering performance . HoweverThus, by implementing multiple DE and clustering packages BingleSeq enables users to explore and pick the method that is most suitable for their experiment.BingleSeq is a comprehensive and intuitive solution that enables users to choose from multiple state-of-the-art DE analysis and unsupervised clustering packages according to their preferences or the dataset in question. In terms of Bulk RNA-Seq analyses, BingleSeq implements functionality that puts it close to, what are to our understanding, the best available similar applications. In terms of scRNA-Seq, BingleSeq could be argued to be among the most exhaustive applications, such as ASAP  and singFuture work will focus on including more functional annotation options and on extending user control over the packages implemented in BingleSeq. Moreover, the implementation of both Bulk RNA-Seq and scRNA-Seq pipelines puts BingleSeq in a particularly good position to implement user-friendly deconvolution of Bulk RNA-Seq results using scRNA-Seq data. Hence, an excellent and practical conclusion to the development of BingleSeq would be to include state-of-the-art deconvolution methods such as Cell Population Mapping (CPM)  or MUltihttps://github.com/dbdimitrov/BingleSeq/. The application’s GitHub page provides an easy guide on how to install the application as well as examples of its general applicability and an extensive description of typical workflows when working with Bulk RNA-Seq and scRNA-Seq data.BingleSeq is as an easy-to-install R package available on GitHub at 10.7717/peerj.10469/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.10469/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.10469/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.10469/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.10469/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.10469/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.10469/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.10469/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.10469/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.10469/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.10469/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.10469/supp-12Supplemental Information 12Click here for additional data file.


Here, we report a crystal structure of EV71 3Dpol elongation complex (EC) at 1.8 Å resolution. The structure reveals that the 5′-end guanosine of the downstream RNA template interacts with a fingers domain pocket, with the base sandwiched by H44 and R277 side chains through hydrophobic stacking interactions, and these interactions are still maintained after one in-crystal translocation event induced by nucleotide incorporation, implying that the pocket could regulate the functional properties of the polymerase by interacting with RNA. When mutated, residue R277 showed an impact on virus proliferation in virological studies with residue H44 having a synergistic effect. In vitro biochemical data further suggest that mutations at these two sites affect RNA binding, EC stability, but not polymerase catalytic rate (kcat) and apparent NTP affinity . We propose that, although rarely captured by crystallography, similar surface pocket interaction with nucleobase may commonly exist in nucleic acid motor enzymes to facilitate their processivity. Potential applications in antiviral drug and vaccine development are also discussed.The enterovirus 71 (EV71) 3D Enterovirus A (EV_A) species belonging to the Enterovirus genus of the Picornaviridae family, is one of the major causative agents of hand, foot and mouth disease (HFMD), and can sometimes cause severe neurological complications such as encephalitis, aseptic meningitis, and acute flaccid paralysis (pol) protein is an RNA-dependent RNA polymerase (RdRP) that plays the central role in viral RNA genome replication, and has become a major target for antiviral studies. To date, numerous crystal structures of picornavirus RdRP proteins have been solved. Along with RdRPs from other RNA viruses, the polymerase catalytic core adopts an encircled right hand architecture with palm, fingers and thumb domains surrounding the active site (Enterovirus C species) RdRP (Human enterovirus 71 (EV71), a member of the aralysis . The genaralysis . The 3D ive site . The fines) RdRP . The indes) RdRP ,8. A coles) RdRP .pol by viral protein 3CD and host SUMOylation  machineries  and the N-terminal domain  regulating the corresponding polymerases, respectively, through intra-molecular interactions  that are part of the RdRP molecule but are beyond the RdRP catalytic core. Examples of the first type include the regulation of flavivirus NS5 by viral protein NS3 and viral RNA elements within the 5′-untranslated region (UTR) , picornahineries ,18, and hineries . Represeractions .In this work, we identified and characterized a type of regulatory interaction that is quite unique if compared with the aforementioned interaction types. These interactions only involve a small surface pocket within the RdRP fingers domain and a nucleobase of the RNA that is part of RdRP–RNA catalytic complex. We unintentionally identified these interactions when solving an EV71 RdRP elongation complex (EC) crystal structure. Through a set of biochemical characterizations, we further demonstrated that this surface pocket facilitates EC stability, likely through its interactions with the nucleobase within the RdRP–RNA complex, while the elongation catalytic rates are not affected. Cell-based virological data comparing the wild type (WT) and mutant viruses further suggest that this pocket is important to virus proliferation. To the best of our knowledge, regulatory sites within the RdRP catalytic core that interact with the downstream RNA have not been reported with support of structural data. Our study provides an example of evolution and utilization of nucleobase anchoring sites by viral RdRPs to facilitate or maintain their essential catalytic properties, and the identified surface pocket may also serve as an intervention target for developing antiviral applications.pol gene within the DNA clone of strains HeN09-17/HeN/CHN2009  and BrCr-ts  was cloned into a pET26b-Ub vector (Escherichia coli strain BL21(DE3)pCG1  for the production of the full-length 3Dpol protein with a native glycine at its N-terminus according to previously described methods and a C-terminal hexa-histidine tag for affinity purification phosphine (TCEP). 3Dpol concentrations were measured by absorbance at 280 nm using an extinction coefficient of 71 280 M−1cm−1 calculated by the ExPASy ProtParam program (http://web.expasy.org/protparam/). The typical protein yield was 10–15 mg per liter of bacteria culture.The EV71 3Db vector . The resfication . All 3Dptemplate . Cell grtemplate ,28, excein vitro T7 RNA polymerase transcription and subsequent glmS ribozyme cleavage according to protocols described previously  was prepared by eviously ,31. T31 eviously , except 2. Crystals were directly cooled and stored in liquid nitrogen prior to data collection.The EC crystals were grown by sitting-drop vapor diffusion at 16°C using a 12.5 mg/ml EC sample. Rhombohedron-shape crystals grew to their final size within 1–2 weeks in a precipitant solution containing 0.17 M sodium acetate, 0.085 M Tris (pH 8.5), 25.5% (wt./vol.) PEG4000 and 15% (vol./vol.) glycerol. Crystal soaking trials were done for 10 h using the precipitant solution supplemented with 5 mM ddCTP and 10 mM MgClo-Fc electron density maps were generated using CNS (X-ray diffraction data was collected at Shanghai Synchrotron Radiation Facility (SSRF) beamline BL17U1 . Data of at least 180° were typically collected in 1° oscillation steps. Reflections were integrated, merged and scaled using D*Trek . The inising CNS . Unless sing CNS .pol or its variant to the template/primer RNA was assessed using a fluorescence polarization (FP)-based assay. A T31/P8F (with a 1:1.1 molar ratio) construct was used as the RNA substrate. Except for bearing a 6-FAM (6-carboxyfluorescein) at the 5′-end of P8, it is otherwise identical to the P31/P8 construct used in the EC assembly. For each 3Dpol construct, a set of 14 mixtures were prepared, each with 25 nM T31/P8F construct (according to T31 concentration) and concentrations of 3Dpol in the range of 3.2 nM to 5.6 μM in a FP buffer containing 50 mM potassium glutamate (pH 7.5), 50 mM arginine, 5 mM dithiolthreitol (DTT), 5 mM MgCl2, 30 mM NaCl and 5% (vol./vol.) glycerol. The mixtures were loaded into the wells of a black 384-well plate . After at least 60-min of incubation at room temperature (r.t.), the fluorescent signals were measured in the 2102 EnVision Multilabel reader (PerkinElmer) using an excitation wavelength of 485 nm (monochromator) and a 535-nm high band pass emission filter. The FP values were calculated based on the fluorescent signals parallel (F∥) and perpendicular (F⊥) to the excitation light and the equation FP = (F∥-F⊥)/(F∥+F⊥). The FP values at different 3Dpol concentrations were fitted to the one-site binding model for determination of the dissociation constant (Kd) values following the equation FP = offset+amplitude•[3Dpol]/([3Dpol]+dK). Five replicates were obtained for each 3Dpol concentration. The FP values were decreased at high-concentration data points, possibly resulting from protein aggregation and therefore these data points were omitted in fitting routines.The binding affinity of the WT EV71 3Dpol and its variants, the EC assembly reactions were carried out as described above with a total volume of 20 μl except that the 3Dpol and RNA concentrations were 6 and 4 μM, respectively, the KCl concentration was 55 mM, and the NaCl concentration was 20 mM. At each indicated reaction time point, an aliquot of the reaction mixture was withdrawn and mixed with an equal volume of stop solution containing 95% (vol./vol.) formamide, 20 mM EDTA (pH 8.0), and 0.02% (wt./vol.) xylene cyano. The RNA species were analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) followed by Stains-All staining and band intensity quantification as previously described  were annealed at a 1:1 molar ratio to yield the T35Fterm/P11L construct before being used in a stopped-flow assay for measuring the overall 3Dpol elongation rates over a stretch of 17 nt. The LI-COR function was not used in this study due to the lack of an appropriate imaging instrument. By modifying the sequence at the 5′ region of the RNA template from previously described methods , 20 mM NaCl, 55 mM KCl, 5.5 mM MgCl2 and 4 mM TCEP. The reaction mixture was then 20-fold diluted using the SF buffer to a reach a final RNA concentration of 50 nM. The fluorescence signal change was measured at 22.5°C after rapid-mixing, producing a final solution with 25 nM RNA, 200 nM 3Dpol, 150 μM ATP/GTP/CTP and 2.5–160 μM UTP in SF buffer.Two types of stopped-flow fluorescence assays were established in measuring the EC elongation rates. The first assay was modified from protocols described previously ,40. The  methods , the flukpol and apparent KM values by fitting the data to the Michaelis–Menten type equation, rate = kpol•[UTP]/([UTP]+KM).Data analysis and fitting were done as described previously . The datint template (Integrated DNA technologies) were annealed into duplex mimicking T31/P8 RNA duplex to determine the incorporation rates of a 2′-deoxy-CMP (2dCMP) directed by a templating guanosine 5 nt upstream of the fluorescein  Figure . Hydrogee Figure . Sequence Figure . We therSome picornavirus RdRP EC crystal lattices allow in-crystal catalysis and translocation ,43. As tin vitro transcription, equal amount of the WT and mutant viral RNAs were transfected into Vero cells. Viral protein expression was compared between the WT and mutant viruses. An immunofluorescence assay (IFA) was used to monitor virus replication by detecting expression of the viral VP1 protein. As show in Figure pol residue R277 is critical for the EV71 viral replication and viability, while residue H44 may play a synergistic role with R277.To assess the biological relevance of H44 and R277 in viral replication, we engineered a set of point mutations at these two sites into an infectious cDNA clone of EV71. Besides alanine mutations, a threonine mutation at residue 44 was also tested because threonine is the other naturally occurring amino acid at this position in EV71 Figure . After iKd). The results showed that WT, H44A, R277A and H44A-R277A RdRP had Kd values of 0.18, 0.17, 0.40 and 0.60 μM, respectively  of EC was estimated as 0.031 h−1, corresponding to an EC half life (t1/2) of about 22.4 h   at 30°C over time Figure  and B. Wh Figure . These dRdRP EC) .While the RdRP from the genotype-C EV71 (EV71-C) has a histidine at residue 44, those from genotype-A EV71 (EV71-A) and CV A16 have a threonine at this position . We intrterm/P11L  RNA construct was used to measure the average elongation rates on a 17-nt stretch of the template  duplex with the P11L primer, and the EC was assembled after incorporating a GAGA tetra-nucleotide sequence by providing GTP and ATP as the only NTP substrates. The EC was then mixed in the stopped-flow instrument with all four NTPs, and the fluorescence signal only started to increase when the polymerase reached the fifth nucleotide to the 5′-end of the template (kpol) and the Michaelis constant (KM) for UTP  and KM (10.0–15.4 μM) values  and KM (99.5–132.4 μM) values for single nucleotide addition . Although structurally conserved, the residues responsible for nucleobase stacking and purine base recognition are only conserved in enteroviruses   Figure . Here we) Figure . In a pr) Figure , suggest) Figure . With th) Figure . It is a) Figure .The regu) Figure . Structu) Figure . Though ) Figure , not adj) Figure .NTP-driven nucleic acid motor enzymes, such as polymerase, helicase and translocases, utilize the energy provided by phosphoryl transfer or NTP hydrolysis reactions to move along nucleic acids. Typically in these proteins, the NTP binding pocket is the active site while the surface of the protein is accessible to the interactions with the nucleic acids up- or down-stream of the active site. Although not always essential to the enzymatic activities, the sequence-non-specific interactions between these proteins and the nucleic acids beyond the active site could contribute to the processivity of these enzymes, because they provide extra anchoring point(s) to the nucleic acids Figure . In thisEnterovirus genus include causative agents of HFMD, poliomyelitis and common cold. The identification of the functionally conserved nucleobase-binding pocket in this study may provide opportunities for developing inhibitors or live attenuated vaccines. As discussed above, the assembly of this surface pocket is analogous to nucleotide binding pocket observed in other enzymes. Therefore, it may have the potential to bind not only nucleobases within a nucleic acid strand, but also nucleotide analogs commonly studied in antiviral research for RNA viruses. With respect to the development of live-attenuated vaccine, this pocket has two advantages. First, the two key residues are far apart in the primary structure, and therefore it is less susceptible to reversion if both sites are chosen for mutation in the vaccine strain. Second, the residue type for both positions is not strictly conserved as long as it can provide suitable hydrophobic interactions to the nucleobase, and allows a variety of amino acid combinations to be tested for viable viruses with attenuated features.Members of the in vitro biochemical characterization and cell-based virological studies. Taken together, this pocket contributes to RdRP EC stability and virus proliferation, likely through its nucleobase binding capability. We propose that similar regulatory mechanisms, although rarely reported, may commonly exist in nucleic acid motor enzymes. Our work could serve as a general reference for future studies to dissect the mechanism behind nucleobase anchoring sites identified in these enzymes through non-crystallographic approaches.In summary, we identified a nucleobase-binding pocket in enterovirus RdRP by crystallography and analyzed its relevance to polymerase function through Atomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data bank under accession numbers 6KWQ and 6KWR.gkz1170_Supplemental_FilesClick here for additional data file.


Tetranychus urticae, is prime candidate for developing RNAi approaches due to frequent occurrence of conventional pesticide resistance. Using a sequencing-based approach, the fate of ingested RNAs was explored to identify features and conditions that affect small RNA biogenesis from external sources to better inform RNAi design.RNA interference (RNAi) regulates gene expression in most multicellular organisms through binding of small RNA effectors to target transcripts. Exploiting this process is a popular strategy for genetic manipulation and has applications that includes arthropod pest control. RNAi technologies are dependent on delivery method with the most convenient likely being feeding, which is effective in some animals while others are insensitive. The two-spotted spider mite, T. urticae. This chelicerae arthropod shows only modest response to oral RNAi and has biogenesis pathways distinct from model organisms. Processing of synthetic and plant host RNAs ingested during feeding were evaluated to identify active substrates for spider mite RNAi pathways. Through cataloging characteristics of biochemically purified RNA from these sources, trans-acting small RNAs could be distinguished from degradation fragments and their origins documented.Biochemical and sequencing approaches in conjunction with extensive computational assessment were used to evaluate metabolism of ingested RNAs in trans-acting RNAs appear very unstable and rare. This suggests potential RNAi pathway substrates from ingested materials are mostly degraded and infrequently converted into regulators of gene expression. Spider mites infest a variety of plants, and it would be maladaptive to generate diverse gene regulators from dietary RNAs. This study provides a framework for assessing RNAi technology in organisms where genetic and biochemical tools are absent and benefit rationale design of RNAi triggers for T.urticae.Using a strategy that delineates small RNA processing, we found many transcripts have the potential to enter spider mite RNAi pathways, however,  D. melanogaster segregates miRNA biogenesis from siRNAs [Drosophila show distinct small RNA biology, which is most apparent in chelicerates such as the two-spotted spider mite, Tetranychus urticae, the subject of this study [RNAi is a mode of post transcriptional gene silencing where small 18–30 nucleotide (nt) RNAs associated with Argonaute (Ago) proteins bind to complementary transcripts . While am siRNAs . A potenm siRNAs . Organisis study –8. Hundris study .A consequence of the rapidly evolving nature of siRNAs is inconsistency in RNAi efficacy across arthropods. For example, long dsRNA feeding is effective in beetles, but not in moths and butterflies . ExploitThe small RNA effectors of RNAi are particularly amenable to high-throughput sequencing approaches due to their short length. Fragmentation is not part of sequencing library creation, so RNA ends created by RNases like Dicer are preserved. Also due to sequential ligation of adapters strand is preserved. By analyzing read sizes, nucleotide content, and overlaps between complementary, alternate-strand mapping reads; class and biogenesis patterns can be elucidated. In arthropods, Dicer products (siRNAs and miRNAs) are typically 19–23 nt long. The RNase III domain of Dicer causes staggered cleavage of dsRNAs that leaves 2 nt 3′ overhangs between strands of siRNA duplexes. In contrast, piRNAs are represented by longer 25-30 nt reads. Biogenesis of piRNAs involves cleavage by piwi proteins guided by the activity of pre-existing piRNAs. Ago and Piwi proteins may exhibit slicer activity which cuts the base of target transcript 10 bases from the 5′ end of the bound small RNA. After piwi cleavage, the fragmented transcript is converted into a new piRNA directly or by alternative nucleases. Each of these biogenesis modes can be determined through sequencing data analysis independent of genetic tools.Tetranychus urticae, an approach was used that combines a purification method alongside detailed analysis of small RNA sequencing libraries. Several studies have tested T. urticae for sensitivity to dsRNA, finding saturating exposure like soaking in dsRNA solution is required for robust phenotypes [T.urticae could lead to insights that could lead to superior RNAi techniques. Furthermore, mites as with other chelicerae arthropods, harbor RNA-dependent RNA polymerases (Rdrps), a factor in nematodes and plants that potentiates RNAi through amplifying dsRNA. However, there is little functional evidence that animal Rdrps, other than those found in nematodes, have a role in RNAi pathways that interact with exogenous RNAs [D. melanogaster have a central role for siRNAs in genome surveillance [To evaluate efficiency of feeding-based RNAi in the two-spotted spider mite, enotypes , 14. Thious RNAs –17. Addieillance , 18.By applying this strategy, we find a variety of exogenous RNAs are processed into small RNAs following ingestion by spider mites. Interestingly, the most active configuration is not the expected long dsRNA. We find various plant produced-RNAs are converted into small RNAs after ingestion. However, very few seem to associate with regulatory complexes, something that is affected by feeding behavior and prior exposure to dsRNA. This suggests a scenario where foreign RNAs appear to transit the spider mite RNAi pathway but are diverted from regulatory complexes, which is modulated by the status of the mite physiology.trans-acting is complicated by fragmented transcripts in samples. Immunopurification of Ago complexes followed by isolation and sequencing of associated RNAs can be used to validate small RNA identity [T.urticae miRbase miRNAs . In sRNA sequencing libraries created from total RNA extracted from bean-raised animals, ~ 3.13% map perfectly to bean sequences. Of these reads, 85% align exclusively while 15% also map to the mite genome. When Hi Trap purified libraries are aligned to plant sequences there is a 10-fold decrease in mapping rates, and a substantial shift in the proportion of reads that align to both mites and the plant (67%). Hi Trap mite purified RNA was also extracted from animals raised on Arabidopsis thaliana, which is not a preferred host of spider mites [P. vulgaris and A. thaliana) showed shifts in the dominant read sizes . Thus, the RNAs in the mite library are unlikely to be mature plant sRNA contaminants.Next, we investigated another source of exogenous RNAs–those acquired from plant hosts to assess the universe of RNA species that infiltrate RNAi pathways. Spider mites infest many plants but are readily reared on bean plants  followed by rinsing in PBS (pH 7.4), and 0.1% Tween 20 solution. Afterwards, mites were collected on a paper towel and allowed to dry before being placed onto a P. vulgaris leaf inside of a large petri dish at room temperature. After five days, mites were collected, flash frozen and stored at -80 °C.Mites were reared on two plants: T. urticae actin and of Green Fluorescent Protein (GFP) from Aequorea victoria . Actin long dsRNA and GFP long dsRNA, were both approximately 350 nt long and created using the MEGAscript™ in vitro transcription kit (Ambion). Templates for dsRNA synthesis were PCR products amplified with primers encoding T7 RNA polymerase promoter sequences on their 5′ ends. Following in vitro transcription, lithium chloride precipitation was performed to purify products, and the resulting RNAs were annealed through heating and gradual cooling.For the initial experiment, two dsRNAs were generated from cloned fragments of Four synthetic structures were designed based on RNAs shown to enter miRNA pathways. Templates for these structures were created by annealing long, ~ 100 nt, oligonucleotides (supplement). The resultant dsDNAs encoded a T7 promoter at the 5′ end followed by the structured RNA sequence. RNAs were synthesized using the MEGAshortscript™ in vitro transcription kit (Ambion). The synthesis products were purified by phenol chloroform extraction, and ethanol precipitation.2, 10% glycerol, 0.2% PMSF, 1 mM DTT, 1X Roche EDTA-free protease inhibitor cocktail) was mixed with the pulverized animals. The lysate was then clarified by centrifugation. Cleared lysate was applied to the column and passed at a speed of 1 mL/minute. The column was washed with binding buffer followed by elution buffer (binding buffer with 300 mM KOAc). An equal volume of acid phenol-chloroform was added to the tube, followed by rocking at room temperature. Following phase separation, RNAs were isopropanol precipitated and resuspended in 30 μL of ddH2O. Total RNAs extracted from mites used TRI reagent and followed manufacturer protocols.Column enrichment of small RNA containing complexes used 1 mL HiTrap Q FF columns (GE Lifesciences) as previously described . BrieflyTotal RNA from spider mite samples was used for cDNA synthesis (Thermo Fisher T-7 kit) using random hexamer primers. cDNAs were used in qPCR assays with SYBR Green real-time PCR master mix (Thermo Fisher), following manufacturer protocols. In the qPCR assay, actin transcripts were normalized by assessing levels of 18S ribosomal RNA. Primer sequences for actin and were previously published .RNA obtained from both column extraction and total RNA extraction were sequenced from an Illumina TrueSeq Small RNA Library Prep Kit library. Sequencing occurred on an Illumina Nextseq500 or Miseq machine using a single-read 50 base pair protocol.Small RNA TruSeq libraries were initially processed using Fastx toolkit to remove adapter sequences . LibrariAlignments were then processed with Samtools and Bedtools to find regions of expression based on read density and merging of juxtaposed features , 36. BedP.vulgaris), and SRR7947145 .A python-based algorithm was used to find overlapping read pairs that represent Dicer cleavage produced small RNA duplexes . Small RAdditional file 1.


Negative strand RNA viruses (NSVs) include many important human pathogens, such as influenza virus, Ebola virus, and rabies virus. One of the unique characteristics that NSVs share is the assembly of the nucleocapsid and its role in viral RNA synthesis. In NSVs, the single strand RNA genome is encapsidated in the linear nucleocapsid throughout the viral replication cycle. Subunits of the nucleocapsid protein are parallelly aligned along the RNA genome that is sandwiched between two domains composed of conserved helix motifs. The viral RNA-dependent-RNA polymerase (vRdRp) must recognize the protein–RNA complex of the nucleocapsid and unveil the protected genomic RNA in order to initiate viral RNA synthesis. In addition, vRdRp must continuously translocate along the protein–RNA complex during elongation in viral RNA synthesis. This unique mechanism of viral RNA synthesis suggests that the nucleocapsid may play a regulatory role during NSV replication. The primary function of the capsid  is to carry and protect the viral genome during transmission between cells. The term “nucleocapsid” refers to the capsid protein–nucleotide complex. For efficiency, the viral nucleocapsid is assembled by organization of capsid protein subunits following a geometric symmetry, including the icosahedral symmetry for spherical viruses and the helical symmetry for filamentous viruses (read Chapter 3 in Fields Virology for details) [All viruses assemble a nucleocapsid. The capsid consists of viral proteins and encloses the nucleotide genome of the virus. The word “capsid” is originated from the Latin details) . For negNegarnaviricota, Realm: Riboviria [Negarnaviricota , NSVs belong to Phylum iboviria ,3. Sinceviricota . The detNegarnaviricota has been divided into two Subphyla; Haploviricotina and Polyploviricotina. The distinction between the two Subphyla is the nonsegmented or segmented nature of the viral genome. The NSVs are then further classified phylogenetically ultimately yielding a separation by virion morphology. Although these viruses can differ greatly in their global structures, structural and functional similarities are observed when comparing their capsid proteins.The Phylum The capsid proteins (N) of NSVs share a common fold. As shown for vesicular stomatitis virus (VSV) in Mononegavirales and Bunyavirales. Beyond these, there are a small number of structures from Articulavirales represented by the segmented Orthomyxoviruses. Structures from the order Alphaflexiviridae are included in this review because of structural similarities, even though these are positive single strand RNA viruses.The two largest NSV orders contain the majority of known capsid protein structures are Haploviricotina capsid proteins from 11 members in the order Mononegavirales were superimposed using the program Fr-TM-align [The structures of TM-align   . The strFirst, the subunits of the nucleocapsid protein are parallelly aligned along the linear RNA genome. Unlike the icosahedral symmetry in the spherical viral nucleocapsid, this one dimensional linear symmetry allows the genomic RNA to be encapsidated at any length and to be fully protected . AlthougSecond, the encapsidated genomic RNA is accommodated in the cleft formed between the N- and C-terminal lobes. The genomic RNA assumes a fixed three dimensional structure in the protein subunit with bases stacked similarly as one strand in an A-form RNA duplex. However, it should be noted that the bases are stacked as separated fragments with bases in one fragment facing the interior of the cleft and bases in the next fragment facing the opening of the cleft. The nucleotides situated between subunits may possess a more flexible conformation. Some of the backbone phosphate groups in the genomic RNA may be coordinated with positively charged residues in the cleft, but there are no defined motifs as found in specific RNA binding proteins .Third, the assembly of the nucleocapsid is stabilized mainly by interactions between the subunits of nucleocapsid protein. This is proved by the fact that a similar capsid could be assembled in the absence of RNA . There aMononegavirales with limited variations. In RSV and hMPV, there are seven nucleotides associated with each subunit rather than the nine seen in VSV. In PIV5, MeV, NDV, EBOV, and MARV, there are six nucleotides associated with each subunit. The nucleocapsid structure of NiV and BoDV was determined in the absence of RNA. A notable difference in these viruses is that the C-terminus instead of a large loop in the C-lobe is involved in cross subunit interactions.This assembly scheme of linear nucleocapsid is maintained in all members in the order Orthobunyaviruses, there are 11 nucleotides associated with each subunit  or Orthotospovirus (TSWV) was solved as a protein–RNA complex. However, these structures are not NLPs and the protein–RNA interactions observed in these structures reflect portions of those interactions in the nucleocapsid. For members in Nairovirida, the structures were protein monomers.For members in truction ,46. Its truction . The strParamyxoviridae, Pneumoviridae, and Rhabdoviridae, there is a virus-coded chaperone protein, called phosphoprotein (P) because of its heavy phosphorylation, that binds the N protein to keep it monomeric and RNA free  and complimentary RNA (+ sense) genomes. Before the nucleocapsid subunit is incorporated in the nucleocapsid, it has to remain as a monomer and RNA free. Several mechanisms may be employed by different NSVs. For members in RNA free A. For meeocapsid . Second,eocapsid . For memeocapsid ,11,49. Tormation ,51. It iormation .Peribunyaviruses, the two termini of the N protein are highly flexible in the monomer2. They become well ordered through cross subunit interactions in the nucleocapsid. In Phenuiviruses, on the other hand, the N-terminus folds back in the cleft when the N protein is monomeric and becomes extended to interact with the neighboring subunit in the nucleocapsid [In eocapsid ,52  showed that there are direct interactions between dimers of the P protein [The encapsidation of genomic RNA in the nucleocapsid occurs concomitantly with viral RNA replication. This requires that monomeric N subunits are delivered at the replication site to encapsidate the genomic RNA as it emerges from the polymerase. For  protein . It is p protein .One of the unique features in viral RNA synthesis of NSVs is that the template is the nucleocapsid, not the naked genomic RNA . This reIt has been long recognized that the nucleocapsid protein plays an essential role in the viral RNA synthesis of NSVs. In paramyxoviruses such as Sendai virus, there is so called “rule of six” that stipulates that the viral genome is only replicated efficiently when the genome length is 6n nucleotides  . This inOther features also suggest that the nucleocapsid protein plays a role in viral RNA synthesis. It has been observed that RNA editing occurs at conserved sites during mRNA transcription of certain paramyxoviruses. In Sendai virus, this is 3′ UAA UUUUUU CC*C in which additional Gs may be inserted at C* during mRNA transcription . During Mononegavirales species in that the level of gene expression, i.e., viral transcription, is the key determinant of gene compositions [Since the viral genome is encapsidated in the nucleocapsid during viral RNA synthesis, interactions of the RNA sequence with the nucleocapsid protein may be involved in regulation of polymerase activities. One piece of evidence could be related to the unusual codon usage bias by the genome of NSV. For instance, the pattern of relative synonymous codon usage is highly conserved throughout the influenza A virus subtypes, suggesting the integrity of viral RNA structures must be maintained for efficient viral replication . In straositions . In a muositions . The ratositions .To define which step in viral RNA synthesis is mostly affected by the genomic RNA sequence, the codon usage preference of VSV genome was mutated to that of the mammalian host . The mosUltimately, the viral synthesis during NSV replication may proceed only when a productive complex is formed between the nucleocapsid and the viral polymerase. The structure of the polymerase in complex with the promoter of influenza virus has been reported ,69,70,71Mononegavirales, the viral polymerase is composed of the large subunit (L) that contains the active site, and a cofactor. In most families, the cofactor is named the phosphoprotein (P) due to its heavy phosphorylation. In Filoviridae, the cofactors are VP35, a homolog of the P protein, and VP30 required for viral transcription [For members in cription ,77. The cription  (Figure cription ,80,81. Dcription , similarcription ,83.There are two types of viral RNA synthesis: transcription and replication. In an NSV genome that encodes more than one viral protein, the promotor for transcription is located at the internal gene junction. The structure of the L-P complexes ,84,85,86Mononegavirales, the promoter for viral replication is at the 3′ end and the promoters for viral transcription are internal at the gene junctions. How the viral polymerase may recognize the different promoters is not entirely clear. Unlike in influenza virus, multiple copies of the viral polymerase are packaged in the virion [For members in e virion , but note virion . It was e virion . Howevere virion . The actSince the template RNA is encapsidated in the nucleocapsid, it must be made available to the active site of the polymerase during viral RNA synthesis. Two elements are essential to this process: First, a conformational change must be induced in the N protein subunits to release the encapsidated RNA, and second, the integrity of the nucleocapsid must be restored after RNA synthesis is completed. Several structures showed that the P protein chaperoned N monomer has an open conformation in comparison with that in the nucleocapsid ,49, but The nucleocapsid structure of NSV has many common features as found in spherical viruses . In sphe


Viral RNA genomes change shape as virus particles disassemble, form replication complexes, attach to ribosomes for translation, evade host defense mechanisms, and assemble new virus particles. These structurally dynamic RNA shapeshifters present a challenging RNA folding problem, because the RNA sequence adopts multiple structures and may sometimes contain regions of partial disorder. Recent advances in high resolution asymmetric cryoelectron microscopy and chemical probing provide new ways to probe the degree of structure and disorder, and have identified more than one conformation in dynamic equilibrium in viral RNA. Chemical probing and the Detection of RNA Folding Ensembles using Expectation Maximization (DREEM) algorithm has been applied to studies of the dynamic equilibrium conformations in HIV RNA in vitro, in virio, and in vivo. This new type of data provides insight into important questions about virus assembly mechanisms and the fundamental physical forces driving virus particle assembly. Michael Rossman’s lab’s diffraction images of Southern Bean Mosaic Virus particles revealed beautiful icosahedral symmetry for the protein shell , but theThe current models for encapsidated viral RNA range from completely disordered to a single, well-defined minimum energy structure. The different models for encapsidated viral RNA genomes in several viruses have different assumptions about the degree of order underlying the approach to structure elucidation. For example, three models have been proposed for the RNA genome of Human Immunodeficiency Virus: a model focused on a structured 5′UTR , an enseThe models for encapsidated viral RNA may also incorporate hypotheses about the virus assembly mechanism. Many models include a series of hairpin loops which can serve as packaging signals that bind proteins and direct virus particle assembly, for example MS2 , STMV 1, and STNAll of these models use a variety of information from crystallography, cryoelectron microscopy, phylogenetic sequence alignment, RNA nucleotide chemical probing experiments, and thermodynamic-based RNA prediction tools. RNA crosslinking and immunoprecipitation strategies have also been employed to identify encapsidated viral RNA-coat protein interactions ,22,23. HThe recent advances in cryoelectron microscopy techniques have opened a new window to look at the ways that viral RNA genomes can be partially ordered and/or dynamically structured inside a viral capsid. The improvements in direct electron detectors, image contrast, and single particle tracking have made high resolution asymmetric reconstructions of virus particles possible . ImportaMost recently, the high-resolution asymmetric reconstruction of BMV shows a partially ordered ensemble of conformations for the viral RNA genome . BMV hasOn the other hand, the asymmetric reconstruction of MS2 bacteriophage supports a model of virus assembly that nucleates at the RNA interactions with the maturation protein, followed by capsid protein interactions with RNA hairpin packaging signals . When thDetection of RNA folding Ensembles using Expectation Maximization (DREEM) presents a new approach to studying nucleotide chemical modification with Illumina sequencing . Dimethyhttps://codeocean.com/capsule/0380995/tree.The DREEM clustering approach uses a Bernoulli mixture model that assumes each sequencing reads is a random draw from models that include different subpopulations. The expectation maximization algorithm first assigns the sequencing reads to clusters, then calculates an expected likelihood of observing the data according to the model parameters, and then re-estimates the model parameters to maximize the expected value likelihood. After the iterations of the algorithm converge, the data are normalized and formatted for inclusion as chemical probing constraints in RNAStructure software. The final weight and distribution of each cluster provides an estimate of the relative abundance of each structural conformation. The details of the DREEM algorithms are described in the supporting information of Tomeszko et al. , and the2 at pH 7 and 37 °C after reannealing from 95 °C and equilibrating at 37 °C to generate a thermodynamically-determined conformational ensemble. Surprisingly, these in vitro chemical probing conditions yielded a similar conformational population distribution as the in virio conditions (phosphate buffered solution with 10 mM Tris pH 7 and 3 mM MgCl2) and the in vivo cell culture conditions with standard media. The similar ratios of the two RRE conformations supports the hypothesis that the RRE conformational equilibrium is thermodynamically driven and does not depend on specific cellular conditions. RNA folds cotranscriptionally in vivo, and the viral replication process may be kinetically controlled. However, these DREEM results suggest that a locally-folded, thermodynamically-stable conformational ensemble may form during kinetically controlled cotranscriptional in vivo folding.DREEM was applied to the study of HIV RNA in vitro, in virio, and in vivo in HEK293T and CD4+T cells . DREEM wDREEM has also recently been applied to the study of SARS-CoV-2 RNA in vivo in Vero cells . DREEM iThus, advances in cryoelectron microscopy and chemical probing techniques are providing more insight into the way that a virus folds an RNA genome inside a virus particle. Methods to combine both cryoelectron microscopy and chemical probing data will further refine models for encapsidated viral RNA. Although DREEM uses short 150-nucletoide reads in Illumina sequencing methods, the use of long-read, single molecule sequencing techniques, such as Pac Bio or Oxford nanopore technology, will further enable the identification of chemically accessible nucleotides that are far apart in the sequence but still may change chemical accessibility in a coordinated way, and thus indicate more than one conformation of an RNA molecule. More accurate and detailed models for encapsidated viral RNA will provide a foundation for further testing hypotheses of kinetic or thermodynamic control of virus assembly and the determination of virus assembly mechanisms.