Aedes aegypti acts as a vector for several arboviral diseases that impose a major socio-economic burden. Moreover, the absence of a vaccine against these diseases and drug resistance in mosquitoes necessitates the development of new control strategies for vector-borne diseases. ABC transporters that play a vital role in immunity and other cellular processes in different organisms may act as non-canonical immune molecules against arboviruses, however, their role in mosquito immunity remains unexplored. This study comprehensively analyzed various genetic features of putative ABC transporters and classified them into A-H subfamilies based on their evolutionary relationships. Existing RNA-sequencing data analysis indicated higher expression of cytosolic ABC transporter genes (E & F Subfamily) throughout the mosquito development, while members of other subfamilies exhibited tissue and time-specific expression. Furthermore, comparative gene expression analysis from the microarray dataset of mosquito infected with dengue, yellow fever and West Nile viruses revealed 31 commonly expressed ABC transporters suggesting a potentially conserved transcriptomic signature of arboviral infection. Among these, only a few transporters of ABCA, ABCC and ABCF subfamily were upregulated, while most were downregulated. This indicates the possible involvement of ABC transporters in mosquito immunity. Aedes aegypti (Ae. aegypti) mosquito transmits a wide range of arboviruses such as dengue (DENV), Chikungunya (CHIKV), West Nile (WNV), yellow fever (YFV) and Zika virus (ZIKV) that are known to cause severe health problems in human. Among these, DENV is one of the most dangerous infections, which inflicts over 390 million individuals annually with nearly half of the world population at the risk of infection [The nfection ,2. The lAe. aegypti JNK pathway [The interaction of viruses with the mosquito system is initiated just after their ingestion. They spatially and sequentially interact with different mosquito organs; gut, being the first organ of encounter and salivary glands as the last organ of interaction. Although these organs mount an array of immune responses against pathogens, still, the virus somehow manages to escape the mosquito immunity ,5,6. Man pathway . In addi pathway ,13.Drosophila hypersensitive to viral infections [Plasmodium infection in Anopheles gambiae [Identification and characterization of non-canonical or novel immune molecules, which may regulate pathogen development might offer new insights to interrupt the mosquito cycle of blood-borne pathogens . ABC trafections . In addi gambiae ,18.ABC transporters share a typical architectural organization comprising highly conserved hydrophilic nucleotide-binding domains (NBDs) and less conserved hydrophobic transmembrane domains (TMDs) . NBDs, aAe. aegypti genome assembly 5 (AaegL5) [In this study, we identified and comprehensively analyzed the ABC transporters superfamily from (AaegL5)  through (AaegL5) ,27. The Ae. aegypti genome assembly 5 (AaegL5) [Ae. aegypti genome. These reports included long non-coding RNA, uncharacterized protein, and pseudogenes, which is now curated in the recently updated genome assembly (AaegL5) [We have identified a total of 59 putative ABC transporter genes from (AaegL5) , which i(AaegL5) ,28 that (AaegL5) . Our anaAn. gambiae transporters classification [Ae. aegypti ABC transporters were also classified into ABCA to ABCH subfamilies , it wasAe. aegypti ABC transporters into different subfamilies  and AGAP001523 (ABCA7), respectively. Interestingly, the duplication of AaeABCA7 transporter was observed only in Ae. aegypti. These duplicated genes formed a common clade with 100% bootstrap support  and An. gambiae ABCB2 (AGAP005639)  . AaeABCBthropods ,36 and hi larvae . Ae. aegypti ABCC transporters constitute the second-largest ABC subfamily, with 15 full transporters and one-half transporter. All these transporters exhibit 1:1 homology with An. gambiae ABCC members. Interestingly, AaeABCC4 has undergone duplication into AaeABCC4.1 and AaeABCC4.2, and both are located on the opposite strands of the same chromosome. These two duplicates were observed to share high nucleotide similarity (80%) with identical introns-exons architecture. We could not identify ABCC11 and ABCC15 members of this subfamily, which are present in An. gambiae, indicating their absence in Ae. aegypti genome. In the phylogenetic tree, AaeABCC17 formed a cluster with Aedes albopictus and Culex orthologs . All the Ae. aegypti ABCG subfamily members possess a typical reverse domain architecture (NBD-TMD). Among themselves, all the members show high conservation forming a distinct clade upon phylogeny. Most of the AaeABCG transporters show 1:1 orthology with other insects except for An. gambiae ABCG6, which was not identified in Ae. aegypti. AaeABCG19/20 (AAEL026976) is orthologous to both An. gambiae AGAP009471 (ABCG19) and AGAP009472 (ABCG20). We were also able to identify the AaeABCG22 as reported previously [An. gambiae  from embryo to adult stages, as well as in blood-fed adults, formed a separate cluster  and various organs (carcass and gonads) of mosquito based on the transcriptome data from Akbari et al. . The K-merformed  as discuAaeABCA  and AaeABCC  subfamilies. These eight members fall in a distinct cluster . Of these genes, AaeABCH1 showed good expression during larval and pupal development also while AaeABCG3 exhibits minimal expression during these stages. AaeABCA8 and AaeABCC9 showed expression in both male and female carcasses as well as during embryonic development. Few genes that did not show expression in carcass are AaeABCG13 and AaeABCG14, however, they exhibit a high level of expression during embryonic development.Among the 59 ABC transporters, the expression was only detected for 51 transporters , except for four members each from AaeABCC10, AaeABCG2 and AaeABCG11 showed minimal expression in ovaries of blood-fed (BF) females.The genes that showed a decent to high-level expression throughout the mosquito development formed a separate cluster  in different developmental stages of mosquito i.e., eggs, larval stages, pupae and adult males or females, we performed qPCR  which is also evident in the qPCR data. AaeABCG13 expression was minimal during different developmental stages as per the RNA seq data and a similar pattern was observed after qPCR validation.The qPCR data of the selected genes  revealedAe. aegypti ABC transporters, we analyzed datasets available from microarray experiments on three arboviral infections viz. DENV, YFV and WNV [During arboviral infection in the mosquito, the virus must disseminate from the midgut (MG) through the body (CC) to the salivary gland (SG). Therefore, the gene expression is likely to be altered in different organs at various time points. To determine the spatial and sequential expression of  and WNV ,46,47,48Ae. aegypti ABC transporters had a significant differential expression in the whole body of mosquito up to 7 dpi . Our analyses detected the mRNA expression for 54 genes (91.52%) in at least one of the arboviral infections at 1–7 dpi. Out of these 48 genes (81.35%) showed differential expression in at least one of the arboviral infections , AaeABCC  and AaeABCF (AaeABCF2) were upregulated during arboviral infection. AaeABCC14 was continuously upregulated on all-time points of YFV, WNV and DENV infection [Most of the fections . Forty-t in DENV A. AlmostAaeABCG3 gene was downregulated in the carcass at 10 dpi of DENV [AaeABCG11 was upregulated in the carcass and salivary gland on 14 dpi of DENV infection [Further to analyze the DENV infection kinetics we also investigated other available reports for 10 dpi and 14 dpi of DENV ,47. Cont of DENV  and AaeAnfection .AaeABCC13, AaeABCE1, AaeABCF2, AaeABCG11 and AaeABCG13) and assessed their expression levels through qPCR. For the same, RNA was isolated from DENV2 infected Ae. aegypti as well as uninfected blood-fed mosquitoes at different time points. The actin gene was used as an internal control. The qPCR expression pattern of these genes revealed a similar trend as reported for the microarray data  but was observed to be downregulated at 7 dpi, similar to the microarray reports. AaeABCE1 and AaeABCF2 transporters exhibited similar expression patterns post-DENV2 infection; both genes being significantly upregulated at the onset of infection (1 dpi), with subsequent downregulation as the infection progressed. In contrast, a continuous downregulation was observed for AaeABCG11 and AaeABCG13 genes post-DENV2 infection in accordance with the microarray data.To understand the transcriptional regulation of ABC transporter genes having probable roles in mosquito immunity during arboviral infections, the binding sites for transcription factors (TFBS) were predicted in the 5′ UTR sequences of a few representative ABC transporter genes. We could identify the binding sites for STAT, Rel, Cnc, Bgb, Sch and Ets in these ABC transporter genes A, where The involvement of ABC transporters in multiple functions including lipid transport, multidrug resistance (MDR), transport of xenobiotics, chemotherapeutic drugs, and immunity, indicates their accessibility to a wide range of substrates and pathways ,50. HoweAe. aegypti ABC transporters suggested a common evolutionary ancestral origin with other insect ABC transporters. There is a high level of conservation of AaeABC transporters with orthologs from different insects, including the dipterans: secondary vector for DENV , fruit fly (D. melanogaster), malaria vector mosquito (An. gambiae) and Cx. quinquefasciatus, etc. The structural architecture of AaeABC members is also highly conserved with their respective homologues from other insects, plants, and humans. Interestingly, the AaeABC transporters subfamily E remains the smallest like other insects and humans; while, subfamilies C and G have maximum members throughout insect genomes. Gene duplications was observed for AaeABCA2 (AaeABCA2.1 and AaeABCA2.2), AaeABCA7 (AaeABCA7.1 and AaeABCA7.2) and AaeABCC4 (AaeABCC4.1 and AaeABCC4.2) while there were certain gene deletions  and gene additions  when compared to An. gambiae transporters. The ABC transporters of Ae. aegypti still seems less in number when compared to Cx. quinquefasciatus which has the highest number of ABC transporters genes among the reported insect genomes; however, it has many pseudogenes and is still under curation. The Ae. aegypti ABCB subfamily members seem parallel with Anopheles and Drosophila having sequence homology of ~30% with the human TAP proteins, which are involved in protein translocations for antigen processing [DmMdr65 (orthologue of AaeABCB2) has been reported to impart chemical protection to the insect brain and acts as the efflux transporter at the blood-brain barrier [In the current study, the comparative sequence and phylogenetic analysis of 59 putative ocessing . As an e barrier .Ae. aegypti ABC transporters, we performed an in-depth comparative analyses of these genes through mosquito developmental stages and at different time-points post arboviral  infections. A few of the ABC transporters exhibited contrasting expression patterns during developmental stages and post arboviral infections, as observed for AaeABCA2.1 and AaeABCA6 genes. These transporters were upregulated during viral infections through 1 dpi to 7 dpi, but their expression was scanty or minimal during mosquito development stages. AaeABCA4 was also seen upregulated at 7 dpi on DENV infection while it had null expression during development stages [Ae. aegypti ABCA8, ABCB3 and ABCC3 genes were downregulated at 7 dpi, but they had relatively higher expression during development stages. Similarly, Ae. aegypti ABCA9, ABCB2, ABCC1, ABCC9, ABCG8, ABCG10, ABCG11, ABCG13, ABCG15 to ABCG20 transporters had optimum to high expression profile during development to adult stages, while they were mostly downregulated during arboviral infections [Ae. aegypti ABCG11 and ABCG13. These genes showed high expression during adult stages of mosquito development similar to expression patterns observed during data analysis from Akbari et al. [To ensure the functions of t stages . This infections . Our anai et al.  SimilarlAaeABCD2 on 2 dpi and 7 dpi of arbovirus suggested the modulation of peroxisomal machinery by the viruses for the establishment of infection in the host, as reported in the mammalian system [ABCE1 role has been detailed during viral infection and anti-apoptosis in humans [AaeABCE1 showed an increased expression during mosquito development and at 1 dpi of DENV, however, it is downregulated as the infection progresses further, confirming its potential role in viral infection. The reported role of various ABC transporters in different systems motivated us to further investigate their expression patterns. The downregulation of the peroxisomal ABC transporter n system . Similarn system . Furthern humans . AaeABCEAe. aegypti salivary gland transcriptome analysis revealed upregulation of several ABC transporters from A, C, G and H  and 12 h light:dark cycle as described before [ad libitum. For colony propagation, four to five-day-old females were fed on anesthetized mice and their eggs were collected in moist cups. The mice were maintained in the central animal facility and all the procedures were approved by the animal ethics committee (IAEC/RES/28/7). d before ,56. LarvAe. aegypti ABC transporters, protein sequences of already reported fifty-five An. gambiae ABC transporters [Ae. aegypti genome assembly 5 (AaegL5) [Ae. aegypti genome that exhibited blast e-value <10−6 were further screened for ABC transporter specific signature domains through the conserved domain database (CDD) tool at NCBI [Ae. aegypti ABC transporters [To identify putative sporters ,27 were sporters  and subj(AaegL5) . The ret at NCBI . Furthersporters .Ae. aegypti was confirmed through BLASTp search in its genome using already identified ABC transporters from D. melanogaster and Cx. quinquefasciatus as query. In addition, tBLASTn search was also performed using the protein sequences of An. gambiae ABC transporters to rule out the possibility of mis/un-annotated or new ABC transporter genes in Ae. aegypti genome assembly 5 (AaegL5).Finally, the presence of ABC transporter sequences in Ae. aegypti ABC transporters were classified into AaeABCA to AaeABCH subfamilies through multiple sequence alignment , phylogenetic tree reconstruction and orthologous relationship as reported before [Ae. aegypti ABC transporters was based on their homology with An. gambiae ABC transporters [Ae. aegypti ABC transporters that exhibited one to one orthology with An. gambiae ABC transporters were assigned the same nomenclature. However, when more than one Ae. aegypti ABC transporter exhibited orthology with a single ortholog in An. gambiae, the Ae. aegypti ABC transporter with maximum similarity was suffixed with X.1 while the one with less similarity was named X.2 and so on (where X is the gene nomenclature corresponding to An. gambiae). Interestingly, some of the Ae. aegypti ABC transporters exhibited no orthology with An. gambiae transporters and thus, they were named based on homology with their paralogs in Ae. aegypti.The putative d before ,27,60. TAe. aegypti ABC transporters, we performed their phylogenetic analyses with An. gambiae, Cx. quinquefasciatus, D. melanogaster, and Ae. albopictus ABC transporters, using the neighbor-joining (NJ) method in MEGAX program [To understand the evolutionary relationship of putative  program . The evoAe. aegypti AaegL5 assembly was retrieved from VectorBase  and analyzed using R program. First, the raw counts per gene were calculated from RNA-seq data using “featureCounts” [To analyze the expression patterns of ABC transporters during mosquito development, all 42 RNA-seq libraries initially described by Akbari et al.  and alreeCounts” . These reCounts” . DiffereeCounts” ,63.Ae. aegypti ABC transporters during different arboviral infections like WNV, YFV or DENV, we constructed a single non-redundant gene list of these transporters based on prior AaegL1.2 and AaegL3.3 and current AaegL5.1 annotations. We analyzed the corresponding gene expression from the publicly available microarray datasets [p-value <0.05 were considered for expression analysis. The log2 fold change in the expression was used to construct a heatmap using Morpheus software .Furthermore, to analyze the expression of datasets ,46,47,48Ae. aegypti such as eggs, larvae (first to fourth-instar), pupae, adult males or females were collected in RNAlater and stored at −80 °C. Total RNA was isolated from these samples using RNeasy mini kit  with slight modification by adding 30 μL β-mercaptoethanol (2-ME) per 1 mL RLT buffer. First-strand cDNA was synthesized using QuantiTect reverse transcription kit  according to manufacturer’s instructions followed by qPCR using SYBR green supermix in an IQ5 multicolor detection system , where the mRNA for mosquito ribosomal protein subunit S6 gene was used as a normalization control [Different developmental stages of  control . The pri6 pfu/mL. Control mosquitoes were fed with the same blood meal composition except for the virus. Fully engorged females were selected and kept in a cage with 10% sucrose solution ad libitum in incubation at standard rearing conditions. Sham treated control and infected mosquitoes were collected at 1, 2 and 7 dpi.Three-to-five-day old female mosquitoes were starved overnight and offered an infectious blood meal containing rabbit blood washed with 1× PBS and supplemented with 10 mM ATP , 10% fetal bovine serum , 7.5% Sodium bicarbonate and DENV2 virus titer suspended in RPMI media  maintained at 1 × 10ΔΔCt method as described before [Total RNA was extracted from the collected samples using E.Z.N.A. Total RNA kit I  and isolated RNA was treated with DNase using Turbo DNA-free kit (Thermo Fisher Scientific), and reverse transcribed by iScript cDNA synthesis kit . Gene expression was quantified in LightCycler 96 qPCR system  using EvaGreen Master Mix  following standard protocol, using gene-specific primers as detailed in d before . + standard deviation (SD). Statistical significance was analyzed by one-way ANOVA with Tukey’s multiple comparison test for the differential gene expression using GraphPad Prism 8.0 . The data with a p-value < 0.05 were considered significant.All the data were expressed as mean Ae. aegypti ABC transporter gene was analyzed using the online NPP2.2 tool , (organism selected = Eukaryotes and Minimum promoter score = 0.8) [http://jaspar.genereg.net/search?q=&collection=CORE&tax_group=insects, accessed on 31 March 2021) to predict the transcription factor binding (TFB) sites. Here the insect core database was selected, using Drosophila as a reference organism and a similarity threshold of 80% [To identify promoters, the 5′ upstream sequence of each e = 0.8) . The upsd of 80% .


A-to-I RNA editing is a common post transcriptional mechanism, mediated by the Adenosine deaminase that acts on RNA (ADAR) enzymes, that increases transcript and protein diversity. The study of RNA editing is limited by the absence of editing maps for most model organisms, hindering the understanding of its impact on various physiological conditions. Here, we mapped the vertebrate developmental landscape of A-to-I RNA editing, and generated the first comprehensive atlas of editing sites in zebrafish. Tens of thousands unique editing events and 149 coding sites were identified with high-accuracy. Some of these edited sites are conserved between zebrafish and humans. Sequence analysis of RNA over seven developmental stages revealed high levels of editing activity in early stages of embryogenesis, when embryos rely on maternal mRNAs and proteins. In contrast to the other organisms studied so far, the highest levels of editing were detected in the zebrafish ovary and testes. This resource can serve as the basis for understanding of the role of editing during zebrafish development and maturity. Adenosine to inosine (A-to-I) RNA editing is a post-transcriptional modification that diversifies the RNA sequence from the genomic DNA template. This modification, which is the most frequent form of editing in metazoans ,2, is caThe translational machinery recognizes inosine (I) as guanosine (G). As a result, RNA editing in coding regions of the genome can lead to amino acid substitution and alter protein function. For example, a non-synonymous A-to-I editing event inside the pore of an ion channel can affect neuronal excitability . HoweverThe rapid development of high-throughput transcriptome sequencing has enabled a global screen of A-to-I editing levels. Considered naively, detection of specific RNA editing events should simply require screening RNA sequencing (RNAseq) data for A-to-G mismatches between the reference genome and the aligned reads. However, the identification of such events is challenging, since it requires the ability to discriminate between RNA editing events, single nucleotide polymorphisms (SNPs), alignment errors, and somatic mutations . TherefoDanio rerio) has become a pre-eminent model organism and has been widely used to characterize gene expression and genetic pathways, in order to study developmental processes, organ function and complex behavior in vertebrates. The advantages of this transparent vertebrate model result in the accumulation of genomic data on gene expression, mutations, genetic markers, and information networks  and harvested 10 embryos per library after 20 min, 2 h, 4 h, 9 h and 24 h. After 72 h, we removed the head, mainly containing brain tissue, from the body, and stored the samples in Trizol. From the parent fish, we dissected whole brains and muscle tissue from the body and stored them in Trizol. The remaining material of the same adult fish was used to extract DNA.Different brain regions were obtained by dissecting adult wild type fish (AB strain) on ice. Brain regions were manually dissected from the whole brain in RNAlater using forceps and spring scissors , and immWe extracted RNA with Trizol, chloroform and isopropanol according to the manufacturer's protocol, dissolved the pellet in PCR-grade water, checked quantity with qubit and quality with Agilent RNA6000 pico chips on a bioanalyzer 2100 system (Agilent). Libraries for developmental stages and adult whole brain and body were prepared on a robotic system, using Illumina stranded total RNA and mRNA kits. DNA was sheared in TE buffer on a Covaris instrument, then libraries were prepared using the NEBnext Ultra II library prep kit.RNA from adult brain regions was rRNA depleted using NEBnext rRNA depletion kit for human, mouse and rat. Libraries were prepared with the NEBnext Ultra Directional RNA Library prep kit.All libraries were sequenced on an Illumina HiSeq4000 machine  using SnapGene v.5.2.4.The quality of the sequence reads was confirmed using the FastQC  quality Genomes and gene annotations (Ensembl) were downloaded from the USCS genome browser .In order to reduce possible noise and prevent the detection of single-nucleotide polymorphisms as editing events we utilized RNA and matched DNA sequencing data from the same four individuals. Editing events were detected de-novo using REDIToolDnaRna.py . To reduNotably, whole genome duplication occurred during zebrafish evolution, resulting in ∼30% of the genes represented by two copies . To overTo detect RNA editing sites in coding regions, we annotated all sites, using variant effect predictor (VEP) , and theAs editing is very abundant in zebrafish, we expected many reads with dense clusters of hyper-editing events to be ignored by standard alignment tools. In order to resolve this issue, we used the method from our recently published study , which aAlu-specific editing algorithm . The Wilcoxon rank test was used to test statistically significant differences (95% confidence level) between two groups.The statistical analysis was done using R  from Armant et al.  ter STAR . This meter STAR .Together, these analyses have yielded a total of 75 474 putative A-to-I editing events. These editing sites exhibited the known ADAR deamination motif, with a strong depletion of guanosine (G) immediately upstream of the edited site, and some enrichment of G immediately downstream Figure . Next, wgria2 (glur2), gria3 (glur3), gria4 (glur4), cadps and cacna1d  were located in gene regions with 23 774 (69.4%) in exons, and only 608 (2.6%) in coding regions. Out of these 608 A-to-I editing sites, 88 (14.5%) were detected by the hyper-editing algorithm, with 521 sites detected by the approach described here. The false-positive rate in the coding region, estimated by the number of C-to-T mismatches , is rather high, at about 7–47%  with arginine (R) and is known to be essential to normal brain function , and revealed that some of the editing sites show variability in editing levels in different brain regions . Interesfunction  is also Alu elements, which are a primate-specific repeat  structures, required by an ADAR enzyme . In humac repeat . These selements , which aelements   expressx Figure .de novo transcription is initiated  . Out of The RNA editosome has been extensively studied in a variety of human and vertebrate tissues ,77,78. Hs Figure  and surps Figure . All tiss Figure . Moreoves Figure . This suClustering analysis of the editing levels of the 149 detected recoding editing events across tissue types, revealed a clear separation between brain and non-brain tissues , is in aThis study provides a first description of the comprehensive set of editing sites in zebrafish, comprising tens of thousands of unique A-to-I editing events identified in brain RNAseq samples. Our computational approach provides a clean signal, with a minimal rate of false-positives (estimated by the number of A-to-G conversions compared to other mismatches). Most of the editing events are found in transcribed genomic repeats, as long double-strand RNAs, which are the major substrates for ADAR1. Accumulation of these dsRNA structures was recently shown to trigger the innate immune response, with the conclusion that the main role of ADAR1 is to edit these structures and hence block the immune response . Our resAlthough editing is very common across the genome, RNA editing is rare in coding sequences in zebrafish. This is probably due to the presence of an active mechanism for eliminating evolved editing sites from coding regions if they are not beneficial to the organism. Of the 149 A-to-I editing sites identified in coding regions, 117 are non-synonymous, meaning that they may modify the function of the coded protein. Previous studies have shown that editing is critical to the function of nervous system, and essential for neurotransmission . In suppADAR1 and ADAR2b mRNA expression during zebrafish development  database under the accession number PRJNA674002. Other sequencing data used in this study are publicly available.gkab247_Supplemental_FilesClick here for additional data file.


O, a fluorescent tricyclic cytosineanalogue. We demonstrate this by incorporation of tCO inup to 100% of the natural cytosine positions of a 1.2 kb mRNA encodingfor the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterizationof this mRNA shows that the incorporation rate of tCO issimilar to cytosine, which allows for efficient labeling and controlledtuning of labeling ratios for different applications. Using live cellconfocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside humancells. Hence, we not only develop the use of fluorescent base analoguelabeling of nucleic acids in live-cell microscopy but also, importantly,show that the resulting transcript is translated into the correctprotein. Moreover, the spectral properties of our transcripts andtheir translation product allow for their straightforward, simultaneousvisualization in live cells. Finally, we find that chemically transfectedtCO-labeled RNA, unlike a state-of-the-art fluorescentlylabeled RNA, gives rise to expression of a similar amount of proteinas its natural counterpart, hence representing a methodology for studyingnatural, unperturbed processing of mRNA used in RNA therapeutics andin vaccines, like the ones developed against SARS-CoV-2.Methods for trackingRNA inside living cells without perturbingtheir natural interactions and functions are critical within biologyand, in particular, to facilitate studies of therapeutic RNA delivery.We present a stealth labeling approach that can efficiently, and withhigh fidelity, generate RNA transcripts, through enzymatic incorporationof the triphosphate of tC In the context of RNA therapeutics,cellular delivery remains a major challenge, and better understandingof cellular uptake, endosomal release, and cytosolic delivery of RNAsis needed to unleash their full potential.4 A major problemin this regard relates to the challenge of directly visualizing RNAmolecules as they are taken up, processed, and subsequently releasedinto the cytosol.5RNA is a key moleculeof life and a main active player of the centraldogma of molecular biology. RNA is also a crucial regulator of geneexpression via for instance micro (mi)- and small interfering (si)-RNAand through its intrinsic catalytic activity; RNA therefore playsa fundamental role in biology. It has, for these reasons, emergedas a highly promising and versatile drug modality with potential tomodify cellular function at the translational level, opening up entirelynew avenues to address previously undruggable targets.7 eventhough their bulkiness and hydrophobicity significantly impede bothtranscription and translation8  of mRNA. Development of more universallyapplicable RNA labeling schemes and probes that are minimally perturbingto RNA’s functions and compatible with live-cell fluorescenceimaging is therefore needed and may become as crucial for the RNAfield as the discovery and understanding of the green fluorescentprotein (GFP) has been for proteins.11Recent advancesin RNA imaging have generated a broad spectrumof tools and probes by which RNA can be analyzed and quantified, butthey generally rely on the use of heavily modified externally labeledoligonucleotides with chemical and physical properties that are significantlydifferent from their natural counterparts. Cyanine dyes such as Cy3and Cy5 conjugated, for instance, via strain-promoted cycloadditionlinkers remain the most common labeling choice,12 While methods reliant on cell fixation such as fluorescence in situhybridization (FISH) can visualize and quantify endogenous mRNA interactionswith astounding specificity and single-molecule resolution,13 they fall short with respect to capturing thespatiotemporal and conformational dynamics that are important to RNAfunction. The development of FRET-pair-functionalized antisense oligonucleotides(ASOs)15 is highly interesting in this regard, butrequires binding of at least two ASOs to the mRNA target, which maysterically block protein-binding sites, hinder mRNA translation, orinduce tertiary structure formation; in practice the method has alsomainly been used on fixed cells. To visualize RNA in live cells, probesare often conjugated to a vehicle, such as a DNA nanocages16 or gold nanoparticles,17 before delivery. Another emerging strategy is to fuse the RNA ofinterest to an aptamer sequence that binds fluorescent dyes in situ,18 but this can also adversely influence translationefficiency and RNA–protein interactions.19 To reduce the risks of interfering with enzymatic processes,chimeric mRNAs have been developed with multiple stem-loop structuresdownstream of the STOP codon, which serve as binding sites for fluorescentfusion proteins.22 While versatile, all these labeling strategies impactprofoundly on the physicochemical properties and molecular weightsof RNAs and therefore risk significantly perturbing their naturalfunction, spatiotemporal distribution, and transport.A major challengein RNA imaging is to develop new methods andprobes that are functional in living cells.23 3′-polyA tails,24 ornucleotides25 enabling in-cell post-transcriptionallabeling of mRNA. Similarly, Ziemniak et al. have produced a rangeof fluorescent 5′-cap analogues that are compatible with bothtranscription and translation.26 However,the resulting mRNA products only carry one single label and have thereforenot been possible to visualize inside cells, limiting the applicabilityof this method.Additionalless perturbing strategies for labeling mRNA have thereforebeen developed, relying for example on click chemistry approacheswith azido-functionalized 5′-cap analogues,29 significantchallenges have remained regarding development of FBAs that are sufficientlyenzyme-compatible to be effectively processed during transcriptionand translation.30 FBAs have the advantageof being internal fluorophores, with relatively small chemical modificationsto the natural base that they replace. Furthermore, their design enablesnormal base-pairing and -stacking of the target nucleic acid. FBAsare therefore considered to be native-like fluorescent labels andhave been extensively used in vitro to probe nucleic acid structureand behavior. We have, for example, designed FBA interbase FRET pairsto obtain detailed information on the structure and base orientationin DNA31 and RNA,33 and others have used FBAs to study biophysically ribosome-mediatedcodon:anticodon base-pair formations.34 A handful of studies indicate that FBAs can be incorporated intoRNA via cell-free transcription, resulting in for example ca. 800nucleotide (nt) RNA strands with a modified cystosine35 or short transcripts with fluorescent isomorphic guanine37 and uridine.39 However, none of these studieshave proven that FBA transcripts can be translated, and FBA-labeledRNAs have not yet been used in biological applications or to visualizeRNA molecules inside living cells. This progress is needed to translateFBAs from useful in vitro probes to functional tools for chemicaland medical biology.30Fluorescent nucleobase analogues (FBAs) haveemerged as attractivelabels for DNA and RNA. However, even though we and others have significantlyimproved FBAs with respect to brightness, excitation, and emissionto facilitate their use in fluorescence microscopy,O; Absmax = 369 nm; Emmax = 457 nm; ε369 = 9370M–1 cm–1; ⟨ΦF⟩ = 0.24)32 can be enzymaticallyincorporated in high numbers into RNA via end-labeling reactions aswell as cell-free transcription. We furthermore show that it is possibleto exchange all natural cytosines in a 1.2 kb long mRNA for tCO  in equally good yields, and we envision it could thereforebecome a generic and convenient route toward any modified nucleosidetriphosphate.To overcome this“hit-and-miss” aspect of triphosphate synthesis, wedeveloped a new synthesis scheme that requires no preliminary protectionof the 2′- and 3′-positions and that facilitates purificationof the final product 1. In ourO ribonucleoside triphosphatewas synthesizedfrom a solid-supported ribonucleoside, a strategy that has never beenreported for modified nucleobases, but attempted with some successfor unmodified nucleobases with 2′-OMe backbone protection.Our synthetic scheme relies on phosphoramidite chemistry, which involvesthe use of cycloSal-phosphoramidite 5 and bis(tetrabutylammonium) dihydrogen pyrophosphate 6  for 2 h at roomtemperature. Subsequent reverse phase or ion-exchange chromatographyallowed the desired triphosphate 7 in a triphosphorylationyield of 60% and with a high UV purity of 99%. Importantly, up to85% of the unreacted nucleoside 1 could convenientlybe recovered by precipitation from the first reaction crude, compensatingfor the low loading achieved. Considering this, our triphosphorylationmethod gives an overall yield of up to 30% of the tCO ribonucleosidetriphosphate, which is higher than most solution-based alternatives.Briefly, a long-chain alkylamino controlled-porosityglass (CPG)support was functionalized with a succinyl moiety. Protected ribonucleoside ormation 1. The reO ribonucleoside triphosphate (tCOTP)was used to produce fluorescently labeled RNA via two different enzymaticmethods.The tC49 Starting from a 17-mer ssRNA on nearly all RNA primers  resulted in slightlyfewer aborted transcripts and hence more full-length transcripts withtCOTP compared to native CTP. This suggests a minor .35 As a further comparisonto this, we tested the fidelity of incorporation of tCOTP by reverse transcription for the tricyclic cytosine used by Stengel et al., would representa better cytosine analogue that would minimize perturbations to biologicalprocesses. We used a DNA template encoding for histone protein H2Bfused to GFP (H2B:GFP) to produce mRNA via cell-free transcription.The template was codon optimized (O moieties56 (videinfra). This optimization could also have the additional positiveeffect that consecutive incorporations of tCOs would belimited; previous work indeed suggested difficulties for DNA polymerasesto incorporate consecutive dtCOTPs.57 However, we also observed in vitro transcription of a nonoptimizedcalmodulin mRNA (vide infra) even at 100% tCOTP (0% CTP)despite this transcript containing three CCCC repeats. We observedefficient transcription and tCO incorporation with bothT7 and SP6 RNA polymerases , as demonstratedby agarose bleach gel electrophoresis of the resulting transcripts confirmsthat the degree of tCO incorporation is concentration-dependentand allowed us to determine the relative rate constants for the incorporationof CTP and tCOTP . The ratios between the rate constants kC/ktCO were close to or slightlyabove unity for all transcripts  for d(tCOTP) incorporation into DNAand analogues d(tCTP) into DNA and tCTP into RNA.58 This suggests that tCO is a goodnature-mimic with minimal perturbing effects on the transcriptionalprocess. Furthermore, a trivial but noteworthy consequence of thedetermined rate constants is that the tCO incorporationdegree in our mRNA matches extremely well the percentage of tCOTP added to the transcription reaction (We used absorption and fluorescencespectroscopy to quantify the incorporation efficiency of tC spectra 4a of tCOreaction 4b.O in themRNA transcripts as a function of its degree of incorporation. Wereport a minor red-shift of the emission spectrum (ca. 4 nm) withincreasing tCO incorporation .In quantitative fluorescence-based cell analyses,it is importantthat fluorescence intensity is proportional to probe concentration.We therefore examined the emissive behavior of tCporation 4c, whereporation 4d. The ftitution . Howeverled mRNA6c, indicO-labeled mRNA is functionalin translation, providing the first evidence that this is possibleusing an FBA-modified mRNA. We used a commercial transcription–translationkit to produce calmodulin-3 under cell-free conditions in bacteriallysates (OTP/CTP ratios asused above (0 to 100% of tCOTP), was produced using thecalmodulin-3 DNA template plasmid provided with the kit, i.e., withoutany codon optimization. The expression of calmodulin-3 protein wasfirst confirmed by SDS PAGE followed by Coomassie staining  to avoiddegradation; we found this to be necessary also for tCO-containing mRNA, suggesting that the FBA does not alter its stability(see further below).We used two different approaches  tointroduce tCO-labeled mRNAs and folded (fluorescent) protein product following both electroporationand chemical transfection of the tCed mRNAs , suggestO content ofthe mRNA, an effect that rendered an effective lowering of the meanfluorescence intensity of the cell populations electroporated withmRNA containing tCO. Although this could indicate thattCO impedes in-cell translation, our results using chemicaltransfection (see below) suggest this is rather an effect of how themRNA was introduced. It is possible that the increased hydrophobicityof tCO-labeled mRNAs in combination with the high invasivenessand electric fields of electroporation caused some tCO mRNAdegradation and/or imposed secondary structures that prevented translation.A slightly higher propensity to adopt secondary structures in vitrois indicated from the gel analysis in O content, suggesting that the tCO-labeled mRNA that reaches the cytosol in a functional form is processedas the corresponding nonlabeled mRNA.We thereafter usedflow cytometry to quantify the translation ofprotein, based on mean cellular GFP fluorescence intensities . ElectroO-dependent effectson either transfection efficiency or mean cellular GFP fluorescencewere observed   6d. We ob orange)  of the tO-labeledH2B-GFP encoding mRNA to that of the nonlabeled variant  template. In addition, both in vitrotranslation experiments and chemical transfection of cells indicatethat tCO-labeled mRNAs can translate equally well as thecorresponding nonlabeled control, suggesting that this type of internallabel can indeed be less disrupting to mRNA function compared to aCy5-label. This points to the flexibility and versatility of the hereindiscovered tCO-labeling method, which offers opportunityto tune labeling density to optimize brightness and distribution offluorophores along the mRNA sequence. Moreover, when using Cy5-labeledmRNA to study delivery, the modus in the field is to mix in a fractionof the labeled mRNA with the corresponding unlabeled mRNA, a procedurethat increases the risk of incorrectly reported rates and levels ofdelivery and translation even further, something that now can be avoidedwith our nature-mimicking labeling technique.Astoundingly, we also found that tCO’s brightness and absorption at 405 nm are sufficientto overcome previous limitations with FBA probes in biological applications.30 Moreover, we demonstrate how this convenientlyallows for spatiotemporal monitoring of uptake, trafficking, and organellecolocalization of chemically transfected mRNA in a live cell modelwith simultaneous detection of its translation into H2B:GFP protein.Finally, we presentthe first example of an FBA-labeled nucleicacid that can be directly visualized in live cells, showing that tCWe envision that our straightforward approach for introducing nonperturbingfluorescent labels into RNA will be an excellent addition to existingimaging tools, applicable for elucidating trafficking mechanisms suchas endosomal escape and exosome formation, both of which are of fundamentalimportance for pharmaceutical development. Applying both TdT end-labelingand T7 transcription strategies also holds significant combined potential,as it would enable selective and site-specific incorporation of duallabels to allow for, for example, FRET applications. We believe thatthe development reported here will benefit pharmaceutical industry,clinical laboratories, and academic partners aiming at furtheringtheir understanding of uptake and endosomal escape mechanisms andallow them to take vital steps toward new and improved delivery strategiesfor next-generation nucleic acid-based drugs as well as further developmentof the recently successful mRNA-based vaccines.


Paramyxoviridae family share a common and complex molecular machinery for transcribing and replicating their genomes. Their non-segmented, negative-strand RNA genome is encased in a tight homopolymer of viral nucleoproteins (N). This ribonucleoprotein complex, termed a nucleocapsid, is the template of the viral polymerase complex made of the large protein (L) and its co-factor, the phosphoprotein (P). This review summarizes the current knowledge on several aspects of paramyxovirus transcription and replication, including structural and functional data on (1) the architecture of the nucleocapsid , (2) the encapsidation of the genomic RNAs (structure of the nucleoprotein in complex with its chaperon P and kinetics of RNA encapsidation in vitro), and (3) the use of the nucleocapsid as a template for the polymerase complex . Finally, this review presents models of paramyxovirus transcription and replication.Viruses of the  Paramyxoviridae family are enveloped viruses with non-segmented negative-strand RNA genomes. These viruses share similar gene expression and genome replication mechanisms to the members of the other ten families of the Mononegavirales order. Paramyxoviruses are currently divided into 4 subfamilies, 17 genera, and 78 species . Paramyxoviruses are found in a broad range of animals, including mammals, fishes, reptiles, or birds and include several human and animal pathogens such as measles virus (MeV), mumps virus (MuV), canine distemper virus (CDV), and Newcastle disease virus (NDV). Some, such as Nipah virus (NiV) and Hendra virus (HeV), regularly emerge in human or animal populations where they cause deadly epidemics. Although vaccines exist against some paramyxoviruses, effective antiviral treatments for post-exposure prophylaxis are urgently needed [Members of the  species  1]  and its co-factor the phosphoprotein (P) ,5,6,7. TThe mechanisms used by the polymerase complex to access the encased RNA, progress along the nucleocapsid, and generate new encapsidated products with soluble nucleoprotein monomers are yet to be fully deciphered. However, in the past few years, the atomic structures of the nucleoprotein of a number of paramyxoviruses from different genera have been solved in either assembled ,21,22,23The genomes of paramyxoviruses contain a single, bipartite, transcription promoter at the 3′ end, followed by at least six conserved, adjacent genes separated by non-transcribed intergenic regions A 29,30,,30,31. Tcore) followed by a long intrinsically disordered tail (Ntail) [core is responsible for the oligomerization on the RNA and constitutes the main body of the nucleocapsid [tail mediates the recruitment of the polymerase complex [The nucleoproteins of paramyxoviruses are made of a folded domain (N (Ntail)  .tail [OD [Finally, viral transcription and replication are also affected by the phosphorylation status of N and P. Indeed, both Ntail ,84,85 antail [OD ,92,93,94tail [OD ,100,101.tail [OD  or upregtail [OD ,84,91, mtail [OD , alter ttail [OD , particitail [OD , tune thtail [OD .Nucleocapsids extracted from disrupted virions or infected cells form left-handed, rod-like helical structures with a herringbone appearance under an electron-microscope ,109,110 Expression of the nucleoprotein in avian cells , mammalicore are well conserved across the Paramyxoviridae family, while the sequence and length of Ntail vary greatly  was solved for NiV [The sequence and the organization of N greatly A. The st for NiV , MeV [25 for NiV , and hPI for NiV   and C-terminal domains (NCTD), which form a positively charged cavity to accommodate the RNA (NTDarm and NCTDarm) connected by flexible linkers; these arms are responsible for the self-assembly of N protomers onto the RNA. The superposition of the structures of the N proteins of paramyxoviruses shows high conservation of the tertiary and secondary structures  [N the RNA B,C. Thesructures D. The glus (RSV) ,130 and us (RSV)  (two pneus (RSV) ,135,136 us (RSV)  (two filus (RSV)  (a bornaus (RSV)  and rabius (RSV)  (two rhaus (RSV) )  and all the tight inter-protomer connections are hidden inside the helix C 17,18,,18,117. NTD and the NCTD  RNA or an RNA corresponding to the first six nucleotides of the viral genome [Using recombinant N proteins fused to a cleavable Pl genome ,23. For l genome .Paramyxoviruses have bipartite promoters ,147,148 The side chain of the residue Q202 of the nucleoproteins of PIV5 and MeV interacts with the base of the nucleotide in position six, and thus the first nucleotide of the promoter ,20 FiguA. In a cIn addition to its impact on the promoters, the phase of the RNA bases also affects the transcription signals. Indeed, during transcription, the P messenger RNA of paramyxoviruses can be edited by the viral polymerase which adds one or several extra G residues by stuttering along a short stretch of C residues . Incremetail belongs to the group of pre-molten globules within the class of intrinsically disordered proteins [core is sufficient to generate the helical core of the nucleocapsid [tail influences the conformation of the helix. Indeed, treatment of the nucleocapsid or NCLC by trypsin cleaves off Ntail, reduces the pitch, and tightens the helix [tail generates helical structures [Nproteins ,57,59. Aeocapsid ,36,38, Nhe helix ,117,121 ructures .tail is not visible by electron microscopy [tail escapes from the nucleocapsid core toward the exterior between two helical turns [tail [tail are spatially constrained by the helical turns of the nucleocapsid core, the C-terminal residues have high angular freedom [tail, the nucleocapsids were found to become more rigid and allowed a resolution of 2.9 Å. The atomic model was reconstituted up to residue 414, thus including the first thirteen residues of the tail (aa 402-414). The residues bind the surface of NCTD and point toward the exterior of the nucleocapsid core  which folds upon binding to the C-terminal domain of the phosphoprotein (PXD) and thus participates in the recruitment of the polymerase complex on its template [XD, Ntail also binds the matrix protein, thus promoting the packaging of the nucleocapsids into viral particles [tail also mediates the interaction with several cellular factors [Nynthesis ,154. Indtemplate ,156,157 articles ,161,162. factors ,164 and  factors .0), thus forming the so-called N0-P complex [0-P complexes are used as the substrate for the encapsidation of genomes and antigenomes [The N-terminal domain of the phosphoprotein interacts with the monomeric, RNA-free form of the N protein  of paramyxoviruses contain a conserved α-MoRE [core (0-PNTD complex of NiV [NTDarm and NCTDarm were removed (with the exception of MeV in which the NCTDarm was included). For all three viruses, PNTD binds to NCTD in the groove occupied by the NNTDarm of the adjacent Ni+1 protomer in the nucleocapsid conformation  have been solved. The overall architecture of Ncore is conserved between the N0 conformation (or “open” conformation) and the NNUC conformation (or “closed” conformation)  [NTD and NCTD most likely participate in the stabilization of the NNUC conformation, even in the absence of adjacent protomers.For MeV, NiV, and PIV5, the structures of both the RNA-free Nrmation) D. Althou differs D–F. Indethe NCTD F. This rtructure , modelin or RNA) . The intNTD, another ultra-weak interaction was detected between Ncore and a fragment of the long, disordered region of P located between PNTD and POD [NTD (0 state [tail (Nα-MoRE) and PXD may also further stabilize the N0-P complex [In addition to P and POD . This frPOD [NTD G. The se(0 state . The int complex .core) to a cleavable PNTD allows the production of soluble N0-P complexes [The fusion of full-length N protein , although dispensable [OD enhances genome replication possibly by increasing the local concentration of N0 at the site of encapsidation [Finally, the local concentration of Nin vitro . For VSIpensable , POD enhsidation .Articulavirales and Bunyavirales orders for which the polymerase can directly bind the extremity of the viral genomes, the viral polymerase of mononegaviruses cannot interact with its nucleocapsid template without its cofactor P [Unlike the distantly related members of the factor P . MoreoveXD mediates the interaction of L with the nucleocapsid and the polymerase complex is made of the L protein and a tetramer of P proteins, the polymerase complex possesses four potential nucleocapsid-binding sites. This architecture is the basis of the so-called “cartwheeling” model in which the polymerase complex progresses on its template due to cycles of association/dissociation between PXD and the nucleocapsid [Since Peocapsid ,185.XD of paramyxoviruses adopts a three-helix bundle structure [XD binds to the α-MoRE of Ntail [core [XD has a prism-like shape with three faces: α1 + α2 form face 1 (F1), α2 + α3 form face 2 (F2), and α3 + α1 form face 3 (F3)  A. Each i with F2 , the L pXD with Nα-MoRE is weak, with a constant of dissociation (KD) in the µM range [α-MoRE folds into an alpha-helix upon binding to PXD, the flanking regions of Ntail remain disordered [tail and its partners. Indeed, the long-disordered region upstream of Nα-MoRE slows the disorder-to-order transition of Nα-MoRE upon binding to PXD [α-MoRE increases the affinity for PXD  [tail upstream the Nα-MoRE affects the interaction with PXD [The interaction of PµM range ,157,193.sordered ,196,197.sordered , affectsg to PXD ,200. Accfor MeV) . The damwith PXD ,200,201.XD and Nα-MoRE permits efficient progression of the polymerase complex on the nucleocapsid. Indeed, modifications of the binding strength between MeV PXD and Nα-MoRE alter the activity of the polymerase [XD/Nα-MoRE interaction strength and the efficiency of the re-initiation of the transcription at gene junctions [XD/Nα-MoRE interaction keeps the polymerase in an active state when crossing the gap between transcription units.The low affinity between Plymerase ,203,204.XD/Nα-MoRE interaction regulates the activity of the polymerase, it is not strictly required for RNA synthesis. Indeed, while the truncation of MeV N from Nα-MoRE to its C-terminus abrogates gene expression in a minigenome assay, further deletion of the 43 upstream residues restores activity [CDR) upstream of the Nα-MoRE has a negative effect on the activity of the polymerase and the binding of PXD to Nα-MoRE counteracts this effect. Accordingly, deletions in the NCDR of CDV and NiV increase activity [α-MoRE is relocated to a variable region of NNTD, removing NCDR enhances gene expression [α-MoRE on Ncore and devoid of NCDR produces more polycistronic mRNAs. Similarly, two recombinant CDV with deletions in NCDR produce more polycistronic mRNA and show an altered RNA synthesis balance with enhanced transcription over replication [tail inhibits RNA synthesis, and PXD counterbalances this negative effect. This interplay would finely tune the polymerase’s activity and ensure the quality of the generated mRNAs by improving the recognition of the signals at the intergenic junctions.Although the Pactivity   and requires the presence of Nα-MoRE in complex with PXD  of the NNTD of the other protomer  also binds the nucleocapsid on the same location on NNTD, suggesting an ancestral mode of interaction between the polymerase complex and its template [In addition to interacting with the α-MoRE, Pth Ncore . This inwith PXD C–E. Accoeraction . After sprotomer D,E. The of Ntail ,190. Thiechanism ,190. Thed to PXD . The P ptemplate ,208,209.OD and the third face of the PXD of one P protomer [XD of the tetramer must have the ability to bind the L protein and one different PXD must be able to bind Nα-MoRE [XD that binds L does not have to be able to bind Nα-MoRE and it is unclear whether PXD can simultaneously bind L and Nα-MoRE. It is also unknown whether the interaction between L and PXD is stable or undergoes rounds of association/dissociation during RNA synthesis.P binds the L protein via the C-terminal part of the Pprotomer ,188,189 XD for the binding to Ntail and enhance RNA synthesis.Of note, some cellular factors influence the progression of the polymerase complex. For instance, hsp70  and peroXD is at the core of an interaction network, which is more complex than initially anticipated. PXD participates in a finely regulated dance allowing the progression of the polymerase complex onto the nucleocapsid and the optimal recognition of the transcription signals.Taken together, these results show that PNTD and NCTD of each N protomers, with only half of its bases facing the solvent. Thus, copying of the genome requires significant conformational rearrangements to allow the RNA to reach the catalytic site of the polymerase.The viral genome is encased between the Ntail affects the rigidity and the pitch of the helical nucleocapsids [XD to intact nucleocapsids of HeV or MuV does not affect the coiling of the helix [XD does not make the RNA more accessible to nucleases either. Similarly, the binding of the C-terminal domain of VSIV P to NCLC does not significantly modify the structure of the N proteins [XD does not participate in the uncoiling of the nucleocapsid, or the oligomerization of PXD is required. Some cellular factors may also contribute to the uncoiling of the nucleocapsid, such as hsp70, which binds MeV Ntail [NTD of MuV can uncoil the helical nucleocapsid and increase gene expression in a minigenome assay [XD to Ncore, the atypical behavior of MuV PNTD could reflect a possibly common uncoiling mechanism.First, in the straight helical conformation of purified nucleocapsids, the RNA is covered by the N protomers of the adjacent helical turn, leaving an insufficient empty space above the RNA to accommodate the large polymerase complex C. Therefocapsids ,117,121,he helix ,114. Forproteins . This sueV Ntail ,163, stieV Ntail ,211, andeV Ntail ,213. Conme assay ,114. SimNUC to the N0 conformation [NTD), the N protein of PIV5, initially in the NNUC conformation, adopts a conformation similar to the N0 conformation. This suggests that the N0 conformation is the most stable in the absence of RNA and adjacent N proteins [NTD and NCTD [i+1 and Ni protomers slightly change toward the N0 conformation, with the conformation of the Ni protomer being modified to a lower extent. Thus, the conformational rearrangement from the NNUC to the N0 conformation may partially take place only at the 3′ end of the genome to facilitate the release of the RNA terminus and the engagement of the polymerase.Second, the RNA cavity of the N proteins must open to release the RNA upon the passage of the polymerase. A first model proposes that the N protomers undergo a large conformational rearrangement reverting from the Normation ,214. Molproteins . Howeverand NCTD ,215. MorNTD of MeV NNUC and N0 are superimposed, the loop-α7 region is the only fragment that does not properly align  is not occupied and may therefore be engaged by PNTD to help to release the RNA. Due to the binding register of the first six nucleotides of the genome and the rule of six, the terminal 3′ nucleotides are positioned such that they are exposed to the solvent and are therefore more accessible [NTD only to the terminal N protomer may be sufficient for the polymerase to access the RNA. However, for SeV and hPIV3, the deletion of the PNTD abolishes genome replication but not transcription in vitro, thus suggesting that the potential role of PNTD in releasing the RNA is not essential (at least for members of the Respirovirus genus) [Pcessible . Therefos genus) ,217.XD binds Ntail and recruits the polymerase complex on the nucleocapsid , thus stabilizing the Ntail, decreasing the fuzziness, and facilitating the progression of the polymerase . In this alternative model, the anchoring of the Ntail to the previous helical turn would further remove it from the path of the polymerase  A. Indeed0-P complex, plays a key role in this process. In the replicase mode, the polymerase complex encapsidates the nascent RNA and does not recognize any transcription signals. As the N protein has a higher affinity for an RNA with the sequence of the 5′ end of the genome, the first six nucleotides of the nascent genome outcompete PNTD for the binding to N , the molecular machinery and the mechanisms at play during RNA synthesis appear to be well conserved among paramyxoviruses. The exposure of the RNA on the outside of the helical nucleocapsid, the binding site of the C-terminal domain of P to the NNTD, the coiled-coil structure of POD, and the interaction sites between P and L suggest these mechanisms are shared with pneumoviruses and filoviruses but are quite different for rhabdoviruses.Except for MuV and its atypical features (antiparallel coiled-coil for Ptail, Ncore, and L? How do other viral proteins such as the V, C, and M proteins interact with the ribonucleoprotein complex and regulate RNA synthesis?Despite these new insights, several questions remain unanswered. The helical nucleocapsid must uncoil to allow the progression of the polymerase. What factors induce this conformational change? It is also still unclear how the nucleoproteins release the RNA and what signals trigger this release. Although structural information is available on the individual components, there is little insight into the organization or the structure of the polymerase complex on the nucleocapsid while synthesizing RNA. In addition, how does the second element of the bipartite promoter enhance initiation? How does the polymerase complex add nucleoproteins to the nascent RNA? How does the phosphoprotein orchestrate its interactions with NAnswering these questions should bring significant insight into the mechanisms at play and assist the development of safe and potent vaccine vectors, oncolytic viruses, and antiviral therapeutics. With the momentum provided by the recent advances and the constant improvement of powerful tools such as cryo-electron microscopy, there is no doubt our understanding of the viral molecular machinery of paramyxoviruses will continue to rapidly improve.


Recent outbreak of deadly Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) urges the scientist to identify the potential vaccine or drug to control the disease. SARS-CoV-2 with its single stranded RNA genome (length ~ 30 kb) is enveloped with active spike proteins. The genome is non-segmental with 5’-cap and 3’-poly tail and acts as a mRNA for the synthesis of replicase polyproteins. The replicase gene lying downstream to 5’-end encodes for non-structural protein, which in turn pose multiple functions ranging from envelope to nucleocapsid development. This study aims to identify the highly stable, effective and less toxic single strand RNA-based aptamers against non-structural protein 10 (NSP10). NSP10 is the significant activator of methyltransferase enzymes (NSP14 and NSP16) in SARS-CoV-2. Inhibiting the activation of methyltransferase leads to partial viral RNA capping or lack of capping, which makes the virus particles susceptible to host defence system.In this study, we focused on designing RNA aptamers through computational approach, docking of protein-aptamer followed by molecular dynamics simulation to perceive the binding stability of complex. Docking study reveals the high binding affinity of three aptamers namely RNA-053, 001, 010 to NSP10 with the HADDOCK score of − 88.5 ± 7.0, − 87.7 ± 11.5, − 86.1 ± 12 respectively. Molecular Dynamics suggests high conformational stability between the aptamer and the protein. Among the screened aptamers two aptamers maintained at least 3-4 intermolecular H-bonds throughout the simulation period.The study identifies the potential aptamer candidate against less investigated but significant antiviral target i.e., NSP10/NSP16 interface complex.The online version contains supplementary material available at 10.1186/s43088-021-00152-5. At the end of 2019, the viral pneumonia similar to pneumonia was reported in Wuhan, Hubei, People’s Republic of China. After sample analysis the clinical features revealed that the pneumonia was caused by the novel coronavirus. In the early 2020, World Health Organization (WHO) and International Committee on Taxonomy of Viruses (ICTV) have named the disease as COVID-19 and the virus as Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) , 4. The The SARS-CoV-2 genome is organized into core genome and accessory genome. The core genome encodes for structural proteins and non-structural proteins are considered essential in viral genome production, replication, viral particles formation and its assembly , 7 whereAs known, mRNA of eukaryotic cells undergoes post-transcriptional modifications like 5′-RNA capping, splicing and polyadenylation before being exported to cytoplasm. Similarly, these viruses utilize the RNA capping mechanism as a weapon to avoid the encounter by host defence mechanism, to improve RNA stability and efficient translation using its own RNA capping machinery . The hosDNA/RNA aptamers are single stranded oligonucleotide with defined structure which binds to target specifically. They can be targeted against nucleic acids and protein molecules. The main advantages of aptamer are less immunogenic, easily synthesized or modified, long term storage and stable in various conditions like temperature or pH . RNA aptCurrent study focuses on emerging field of aptamer technology for the identification of specific RNA based aptamers to confront SARS-CoV-2 by targeting the NSP10. This work provides the structural insights of RNA aptamer and NSP10 interaction which are studied through molecular docking and molecular dynamics approach.The 3D structure of most significant protein in SARS-CoV-2 viral capping mechanism i.e., NSP10 (PDB id: 6ZCT with resolution of 2.55 Å) was retrieved from RCSB Protein Data Bank (PDB) . The cryThe 30-mer RNA pool of 100 sequences were generated randomly using shuffleseq program of EMBOSS package . The RNAPrior to docking, the water molecules and/or ions or small molecules found in retrieved structure of protein were eliminated and saved for further studies. The 3D form of protein and predicted RNA aptamers were energy minimised to obtain its stable conformation. The energy minimisation was carried out by GROMACS v2020.4  using AMProtein- RNA aptamer docking was performed by High Ambiguity Driven biomolecular DOCKing (HADDOCK v2.2) web server . This da+ and Cl− ions while maintaining the buffer concentration at 0.1 M NaCl. Steepest Descent Algorithm was used to energy minimize the built systems with maximum of 50,000 steps and with energy tolerance of 1000 kJmol−1 nm−1. Bond lengths in the system were constrained using LINCS algorithm [To determine the binding efficacy and stability of the protein-RNA binding, molecular dynamics was performed using GROningen MAchine for Chemical Simulations (GROMACS 2020.4) . Simulatlgorithm  and perilgorithm . The V-rlgorithm .The equilibrated systems were set up for final production run for 200 ns. The structural coordinates were saved for every 10 ps and resultant trajectories were analyzed using GROMACS v2020.4 modules. Molecular Dynamics simulation were executed on an Intel i7 workstation powered by Nvidia GTX 2080Ti GPU system and analysis were carried out on Dell PowerEdge T20 workstation with 32 GB RAM.The gmx rms, gmx rmsf, gmx gyrate and other important modules were used to study about the conformational stability, flexibility of residues and compactness of the protein and docked complexes. MD analysis was carried out as mentioned in our previous study .The solvent accessible surface area (SASA) was calculated using gmx sasa of GROMACS package. SASA addresses the surface hydropathic property of the protein. SASA calculation is performed by defining the radius of a probe sphere  radius of water) which is ‘rolled’ along the surface of the protein. If the probe overlaps with the vdW radii of the atoms of amino acids, the area is not accessible by the solvent and therefore accounted to the solvent excluded surface . IntermoThe secondary structure of NSP10 consists of 4 long α-helices and 3 β-sheets in which 2 sheets being antiparallel to each other  is displayed in Fig. In case of NSP10-010 complex Fig. , the steFrom the molecular docking simulation, it is hypothesized that the binding of aptamers to NSP10 may inhibit the obligatory function of protein i.e., activation of methyltransferase enzymes in SARS-CoV-2. Although these aptamers were believed to retard NSP10, in order to check the stability of the docked complexes, they were subjected for molecular dynamics simulation studies over 200 ns.–4–0.45 nm. The protein was stable after 40 ns of the simulation and there was a sudden spike at 114.5 ns which might be due to conformational change in Gln65 and Pro100-Thr102 of B2 and B3 into loop structure  plot. From the Fig. 2. SASA of the protein decreases with increment in compactness of the protein. From Fig. The change in compactness of unbound NSP10 and the aptamer-bound NSP10 was evaluated by Rg as a function of simulation time. The intermediate Rg values of NSP10, NSP10-001, NSP10-010 and NSP10-053 was found in between 1.4 and 1.5 nm. From the Fig. The interactions between protein-RNA aptamer complexes were assessed by g_hbond module. The hydrogen bond interaction stabilizes the protein-aptamer complex. Throughout the simulation period, at least 3–4 H-bonds were found between protein and the aptamer in both the systems Fig. . AdditioThe collective motion of protein was studied by essential dynamics or Principal Component Analysis (PCA). The essential dynamics study was carried out to have deeper knowledge about conformational stability and flexibility of residues. In general, the protein is required to have certain degree of rigidity, flexibility to perform its functions freely. The sturdy interaction between protein and the ligand would restrict the motion of protein, hence making the protein not to attain the required specific conformation essential for its activity. The PCA Fig. A plot coTo examine the minimum free energy conformation ensemble of protein, the free energy landscape analysis was performed using first two PC’s i.e., PC1 and PC2. FES analysis provides Gibbs free energy difference of protein conformation in docked complexes with high accuracy. From Fig. In this study, Non-Structural Protein 10 (NSP10) has been suggested as the potential drug target in SARS-CoV-2. To date, several lead molecules have been proposed and being investigated against various targets of coronavirus. With the high exponential mutational rate of virus, the search for potent drug/lead molecules has been complicated. In this work, we have designed RNA based aptamers to act as an inhibitor against NSP10/ NSP14 and/or NSP10/NSP16 interface site. The screened aptamers were docked to NSP10. Although, RNA-073 aptamer shows better minimum free energy i.e., − 11.70 kcal/mol (2nd best) but it failed to show considerable amount of binding with the target NSP10. The work was further moved forward to study about the dynamics of best protein-aptamer complex. Considering that the protein-aptamers complex exhibits a very dynamic nature, in this study we have implemented molecular dynamics study for a time of 200 ns to mimic the natural environment. The dynamics study provided more in-depth insights into the mechanism of binding. Initial docking results revealed that the RNA-053 aptamers pose as a significant molecule among screened aptamers. Surprisingly, during the MD analysis, the above said RNA-053 aptamer moved out of the binding pocket at the end of MD simulation, signifying the best inhibitor should have optimum size thus it fits inside the pocket perfectly . On compAmidst the COVID-19 pandemic situation, there is a much demand of less toxic and highly potential drugs in the market. The distinguished non-structural protein 10 is found only in coronaviruses, shares ~ 99% of sequence identity with SARS-CoV and involved in mRNA cap methylation. Binding of this protein with NSP14 and NSP16 is essential as it increases the methyl transferring activity to certain folds in SARS-CoV-2. Computationally designed RNA aptamers were employed to disrupt the NSP10/NSP14 and NSP10/NSP16 interface, a significant factor of RNA capping mechanism. Docking studies identified the potential 3 RNA-based aptamers with high binding affinity. Atomistic model simulation revealed that among 3 chosen aptamers, 2 aptamers-001 and 010 binds with NSP10 at crucial residues Lys43, His80, Lys93, Tyr96. This interaction encourages us to believe in high chances of inhibitory action by aptamers against NSP10. Our research work greatly contributes for the discovery of potential candidate against highly infectious SARS-CoV-2. The efficacy and inhibitory concentration of designed RNA aptamers must be evaluated using in-vitro and in-vivo studies.Additional file 1. The secondary hairpin loop conformation of screened aptamers.


Retroviruses package two copies of their genomic RNA (gRNA) as non-covalently linked dimers. Many studies suggest that the retroviral nucleocapsid protein (NC) plays an important role in gRNA dimerization. The upper part of the L3 RNA stem-loop in the 5′ leader of the avian leukosis virus (ALV) is converted to the extended dimer by ALV NC. The L3 hairpin contains three stems and two internal loops. To investigate the roles of internal loops and stems in the NC-mediated extended dimer formation, we performed site-directed mutagenesis, gel electrophoresis, and analysis of thermostability of dimeric RNAs. We showed that the internal loops are necessary for efficient extended dimer formation. Destabilization of the lower stem of L3 is necessary for RNA dimerization, although it is not involved in the linkage structure of the extended dimer. We found that NCs from ALV, human immunodeficiency virus type 1 (HIV-1), and Moloney murine leukemia virus (M-MuLV) cannot promote the formation of the extended dimer when the apical stem contains ten consecutive base pairs. Five base pairs correspond to the maximum length for efficient L3 dimerization induced by the three NCs. L3 dimerization was less efficient with M-MuLV NC than with ALV NC and HIV-1 NC. A hallmark of retroviruses is that they package two copies of their genomic RNA (gRNA) to form an infectious virion. The two packaged gRNA molecules interact to generate an RNA dimer in which the subunits are held together by noncovalent bonds. Conservation of the dimeric gRNA among retroviruses is required for several stages of the retroviral life cycle (reviewed in Ref. ). IntereAfter virus budding, the non-infectious immature form of the virus particle is reorganized into the infectious mature form . The dimThere are numerous studies on dimerization of human immunodeficiency virus type 1 (HIV-1) gRNA  is necessary for gRNA dimer formation in the context of the virion . NCp12 p is necesWe showed that the dimerization properties defined for our ALV strain (type Schmidt-Ruppin A) differ from other alpharetrovirus strains . More prIn a previous study , we inveNCp7 (NC(1–55)) of the HIV-1 MAL isolate, NCp10 of M-MuLV, and NCp12 of ALV (type Schmidt-Ruppin A) were synthesized by the Fmoc/opfp chemical method and purified to homogeneity by HPLC, as described previously ,36,37,38DNA oligonucleotides were purchased from Eurogentec . Nucleotides of the avian leukosis virus of subgroup A (ALV-RSA termed ALV in the text) are numbered according to the Pr-C strain of RSV . In the Standard molecular biology methods were used for plasmid construction. Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs . The expand high-fidelity PCR system was from Roche Diagnostics . Cloned sequences and mutations were verified by DNA sequencing. Plasmid pEP241 contains the 241–629 sequence of the ALV genome  with EcoTM large-scale RNA production system  and five micrograms of plasmids cleaved by the endonuclease restriction enzyme DdeI. The 5′- and 3′-ends of L RNAs correspond only to the ALV sequence, i.e., they do not possess additional sequences derived from the plasmid sequence. Each L RNA was purified by denaturing polyacrylamide gel electrophoresis as described previously [32P] ATP  were used to label the 5′-end of L RNAs. Each L RNA labeled at its 5′-end was purified by denaturing polyacrylamide gel electrophoresis.L RNAs were synthesized by in vitro transcription using the T7 RiboMAXeviously . Calf inw/v glycerol, 0.05% w/v bromophenol blue, 0.05% w/v xylene cyanol). The dimerization assay was performed in the final volume of ten microliters and the final concentrations of 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.2 mM MgCl2, and 5 mM DTT [4 cpm/pmol) in 6 µL of water was incubated at 90 °C for 2 min and placed on ice for 2 min. The NC buffer was added, and each sample was incubated at 37 °C for 15 min in the absence or presence of the nucleocapsid protein  at various concentrations. At the end of incubation, 2 µL of SDS-EDTA solution (7.1% SDS and 39.6 mM EDTA) was added to the sample. Then, RNA was phenol–chloroform extracted, and the aqueous phase was mixed with 3.5 µL of loading buffer. The samples were analyzed by electrophoresis on a 12% poylyacrylamide gel (19:1 (w/v), acrylamide/bisacrylamide) at 25 °C in the TBE buffer (89 mM Tris-borate (pH 8.3), 2 mM EDTA). After electrophoresis, the gel was fixed, dried, and autoradiographed. The monomeric (m) and dimeric (d) forms of L RNAs were quantified using a TyphoonTM TRIO  and ImageQuant software . The percent of dimer was determined as 100 × (d/(m + d)).To prepare the heat-denatured L RNAs (lanes D in the gels), 1.9 pmol of each L RNA in 10 µL of double-distilled water was incubated at 90 °C for 2 min and placed on ice for 2 min and mixed with 3.5 µL of loading buffer  was dissolved in 48 µL of water, heated at 90 °C for 5 min, and placed on ice for 5 min. The NC buffer was added, and the sample was incubated with NCp12 (866 pmol) at 37 °C for 15 min. Sixteen microliters of the SDS-EDTA solution  were added to the sample before the phenol–chloroform extraction. Aliquots (10 µL) of the aqueous phase were heated for 5 min at temperatures ranging from 30 to 75 °C before the addition of 3.5 µL of loading buffer. Aliquots were analyzed by 12% polyacrylamide gel electrophoresis as described above.Dimerization was performed in the final volume of 80 µL and the final concentrations of 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.2 mM MgClNine RNAs corresponding to the wild-type and mutant conformations of the L3 domain were generated by in vitro transcription . The mfoSpecific interactions between NCp12 and the loop B sequence could play a role in NCp12-mediated L3 dimerization. Lwt RNA and the loop B mutants without NCp12 mainly remained monomeric after the treatments that removed the protein , lanes CDeletion of loop B leads to the formation of the long stem containing two bulges , LdLB. LNCp12 was unable to promote dimerization of LdLA RNA . This reThe results obtained with the LdLB and LdLA mutants suggest that the stability of stems A, B, and C modulates the efficiency of NCp12-mediated L3 RNA dimerization. Phylogenetic analysis of 28 alpharetrovirus sequences predicted that stem C contained 6 base pairs in one strain and 5 base pairs in 27 strains [Annealing of mini-TAR RNA to mini-TAR DNA hairpin in the presence of NCp7, NCp10, and NCp12 has been studied by gel shift assays . The annThe dimerization yields of Lwt and LAU+1 RNAs were similar in the presence of NCp7 and NCp12, whereas they were lower in the presence of NCp10 . The dimThe L3 stem-loop is part of the highly structured 5′ leader sequence that has critical functions in virus replication, such as reverse transcription, translation, dimerization, and packaging . The L3 We first sought to evaluate the role of loop B in extended dimer formation. Interestingly, the m1LB and m2LB mutations did not lead to reductions in L3 RNA dimerization . These rStructural rearrangements leading to extended dimer formation require destabilization of stem C by NCp12. Loop B appears to be an important destabilizing element in L3. Consistent with this view, the deletion of loop B dramatically reduced L3 RNA dimerization. However, this deletion did not abolish L3 RNA dimerization in the presence of NCp12 at high concentrations  at the 3′-end of gRNA with the complementary r sequence at the 3′-end of minus-strand strong-stop DNA (ssDNA) . Each coTaken together, these observations indicate that there are notable differences in the mechanism of NC-mediated annealing of DNA–RNA and RNA–RNA duplexes. A fully base-paired region of 10 base pairs cannot be an RNA dimerization site, whereas it can form a DNA–RNA duplex.


The genome is pervasively transcribed across various species, yielding numerous non-coding RNAs. As a counterbalance for pervasive transcription, various organisms have a nuclear RNA exosome complex, whose structure is well conserved between yeast and mammalian cells. The RNA exosome not only regulates the processing of stable RNA species, such as rRNAs, tRNAs, small nucleolar RNAs, and small nuclear RNAs, but also plays a central role in RNA surveillance by degrading many unstable RNAs and misprocessed pre-mRNAs. In addition, associated cofactors of RNA exosome direct the exosome to distinct classes of RNA substrates, suggesting divergent and/or multi-layer control of RNA quality in the cell. While the RNA exosome is essential for cell viability and influences various cellular processes, mutations and alterations in the RNA exosome components are linked to the collection of rare diseases and various diseases including cancer, respectively. The present review summarizes the relationships between pervasive transcription and RNA exosome, including evolutionary crosstalk, mechanisms of RNA exosome-mediated RNA surveillance, and physiopathological effects of perturbation of RNA exosome. Precise spatio-temporal control of gene expression is critical for converting genomic information into biological functions of life systems. Transcription forms an initial foundation of the gene expression programs; eukaryotic genomes, including those of yeasts, are pervasively transcribed beyond the canonical protein-coding genes and representative non-coding RNA genes, such as rRNAs, yielding numerous RNA species such as long non-coding RNAs (lncRNAs), upstream antisense RNAs  in mammals or cryptic unstable transcripts (CUTs) in yeasts), transcription start site (TSS)-associated RNAs (tssRNAs), and enhancer RNAs (eRNAs) ,2. TypicThe structure and composition of RNA exosome are well conserved among eukaryotes and archaea, and a similar complex has been identified in bacteria . The humA recent proteomic analysis of cell cycle-dependent protein interactions suggests that the dynamics of EXOSC components can be divided into three groups: (1) PH ring components (EXOSC4–9) and EXOSC2, (2) EXOSC1 and EXOSC3, and (3) EXOSC10 . EXOSC1,Saccharomyces cerevisiae. In humans, the TRAMP complex consists of MTR4  and human homologs of the yeast Trf4/5 and Air1/2, non-canonical poly(A) polymerase PAPD5  and ZCCHC7, respectively [While the RNA exosome can degrade most RNA per se, multiple distinct adaptor complexes have been shown to direct the RNA exosome to distinct classes of RNA substrates . In mostectively . In contectively .Schizosaccharomyces pombe, especially PAXT. The NEXT complex consists of MTR4, the RNA-binding protein RBM7, the zinc-finger protein ZCCHC8, and degrades snRNA, snoRNA, replication-dependent histone mRNAs, uaRNAs, eRNAs, and LINE1 RNAs [In addition to the TRAMP complex, additional nucleoplasmic adaptor complexes have been identified in mammals. The nuclear exosome targeting complex (NEXT) and the poly(A) tail exosome targeting complex (PAXT) remove various types of nucleoplasmic lncRNAs, and many components of these complexes are conserved in NE1 RNAs ,21,22. TNE1 RNAs . Three aNE1 RNAs . The PAXNE1 RNAs ,25. WhilNE1 RNAs . PromiscNE1 RNAs . In addiNE1 RNAs ,23,26,27A recent report has shown that NEXT targets become polyadenylated upon NEXT inhibition and are handed over to PAXT-mediated decay, indicating fail-safe RNA decay via PAXT . This maCBCA  plays an important role in determining whether target RNAs are subjected to productive processing and export or to nuclear retention and decay . CBCA-boRecent studies have revealed that PAXT plays an important role in balancing RNA degradation and export. Depletion of MTR4 and ZFC3H1 has been shown to cause accumulation of uaRNAs and ptRNAs not only in the nucleoplasm but also in the cytoplasm, resulting in cytoplasmic accumulation of abnormally ribosome-bound RNAs ,33. One An additional important factor is the MTR4-binding partner NRDE2. NRDE2 interacts with MTR4 in a 1:1 stoichiometry, but the complex is devoid of the core exosome complex and is independent of the other adaptor complexes ,38. NRDEHow many of the unstable transcripts targeted by RNA exosome have biological functions? An ostensibly wasteful process of pervasive transcription and subsequent degradation by the RNA exosome may be reconciled with their evolutionary interplay. A recent review of lncRNAs provides insights into this issue . In contFurthermore, the introduction of the “constructive neutral evolution (CNE)” mechanism may help to understand step-by-step evolution from pervasive transcription to the acquisition of functionality of lncRNAs . In the RNA exosome complexes are thought to be cell-essential genes required for cell viability. Reflecting that the RNA exosome targets numerous types of RNAs, experimental depletion of RNA exosome causes complex phenotypes, including alterations in translation, chromatin regulation, genome structure, R-loop formation, DNA damage response, telomere regulation, cell viability, differentiation, and cellular senescence ,4,55. HoDepletion of the RNA exosome can drastically change the localization and accumulation of multiple RNA species. As described earlier, depletion of MTR4 and ZFC3H1 induces cytoplasmic accumulation of nuclear RNAs and perturbs translation ,33. In tC9orf72 gene in patients with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) [A recent analysis further demonstrated that the RNA exosome was necessary for DNA repair for homologous recombination (HR) . In the is (ALS) . Conversis (ALS) .Gene expression programs consist of multiple layers of feedback and feedforward regulation. This is also the case for RNA processing and RNA degradation. PABPN1 autoregulates itself by binding to an A-rich region in the 3′ UTRs of PABPN1 pre-mRNA, preventing splicing, and inducing nuclear decay . Such poA study in mESCs has revealed that the transcripts that are differentially expressed between distinct differentiation states tend to be more exosome-sensitive in either state where the expression is lower than the other . Thus, iIn addition, a recent report has shown that the RNA exosome-mediated RNA turnover has an important role in cellular senescence . SenesceEXOSC3, EXOSC8, and EXOSC9 cause several forms of autosomal-recessive neurodegenerative disease, pontocerebellar hypoplasia (PCH), namely, PCH type 1b (PCH1b), PCH1c, and PCH1d, respectively [EXOSC3 mutations is characterized by atrophy of the cerebellum and pons [EXOSC8 mutations is characterized by psychomotor deficits, cerebellar and corpus callosum hypoplasia, hypomyelination, and spinal muscular atrophy (SMA) [EXOSC9 mutations are associated with PCH1d that exhibit spinal motor neuropathy and cerebellar atrophy, while the abnormalities of the pons are modest [EXOSC5 are associated with a novel form of exosomepathy, characterized by developmental delays, short stature, cerebellar hypoplasia, and motor weakness [EXOSC1 was recently linked to the disease condition of PCH [Mutations in distinct components of the RNA exosome have been linked to distinct, tissue-specific disease phenotypes, while some partly overlap ,84,85; sectively ,84,85. Pand pons . PCH1c why (SMA) . EXOSC9 e modest . A recenweakness . In addin of PCH . Althougn of PCH .EXOSC2 are linked to a distinct syndrome, characterized by short stature, hearing loss, retinitis pigmentosa, and distinctive facies (SHRF), where the cerebellar atrophy is mild [EXOSC2-associated SHRF may be associated with misprocessing of pre-mRNAs and snRNAs [PABPN1 have been linked to oculopharyngeal muscular dystrophy (OPMD) [RBM7 and ZCCHC8, have been linked to spinal motor neuropathy and familial pulmonary fibrosis with short telomere syndrome features (including bone marrow failure), respectively [SKIV2L and TTC37  have been linked to syndromic diarrhea/trichohepatoenteric syndromes (SD/THES2 and SD/THES1), respectively [In contrast to these mutations, mutations in  is mild . Given td snRNAs . As othey (OPMD) . Mutatioectively ,96. Althectively . In addiectively ,99.EXOSC2 mutations may only mildly reduce the overall level of the RNA exosome, the degrees of disruption of the functional complex may partly explain the presence of the PCH phenotype. It is also likely that mutations in distinct core exosome genes differentially affect distinct entry paths for the RNA exosome, distinct RNA target classes, and distinct cofactors, finally leading to diverse mutation-dependent and cell type-dependent phenotypes. Furthermore, an interplay between known complex configurations and cell cycle-dependent interaction dynamics may distinguish the effects of each component on the overall behaviors of the RNA exosome [The mechanistic consequences of mutations in core exosome genes remain unclear. However, mutations in the exosome subunit appear to generally impair the assembly of the RNA exosome complex and reduce overall levels of the functional complex ,84,85. A exosome .EXOSC4 and ZC3H3 genes are relatively frequent in certain cancer types, and clearly exhibit the co-occurrence  are essential in most cancer cell lines a 100,1001. PABPNcurrence b. Howeve cancers ,105,106;er types ,108,109. myeloma . DIS3 muin yeast . In addiin yeast . AlthougA recent integrative survey of rRNA metabolism-related genes using TCGA database showed that specific upregulation of EXOSC8 expression is driven by alterations in copy number variations (CNV) and correlates with worse prognosis in colorectal cancer . In addiCDK1 mRNA, respectively [With respect to functional consequences, EXOSC9 depletion inhibits growth and survival under various stress conditions in several cancer cell lines . Along wectively ,119. A cectively ,120. FutThe present review summarizes the relationships between pervasive transcription and RNA exosome, including evolutionary crosstalk, mechanisms of RNA exosome-mediated RNA surveillance, and physiopathological effects of perturbation of RNA exosome. Co-occurrence of pervasive transcription and RNA exosome system appears to fit well with a weak selection regime, conferring evolvability to organisms based on individually weak mutations, which are promiscuous but rich sources for transcription. In addition, various phenotypes observed upon perturbation of the RNA exosome and cofactors may be viewed as functions of the RNA exosome, independent of the counterbalance system for pervasive transcription. As the RNA exosome influences genome stability, it is important to understand how the RNA exosome controls the dynamics of nuclear meso-scale structures and controls compartmentation of biochemical reactions in the nuclei. Given that various RNA molecules targeted by the RNA exosome play important roles in intracellular liquid-liquid phase separation (LLPS), the biology of biomolecular condensate and LLPS may be intimately associated with the functions of the RNA exosome ,122. Fro