Viroid discovery as well as the economic significance of viroids and biological properties are presented. Next-generation sequencing (NGS) technologies combined with informatics have been applied to viroid research and diagnostics for almost a decade. NGS provides highly efficient, rapid, low-cost high-throughput sequencing of viroid genomes and of the 21–24 nt vd-sRNAs generated by the RNA silencing defense of the host. NGS has been utilized in various viroid studies which are presented. The discovery during the last few years that prokaryotes have heritable adaptive immunity mediated through clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated Cas proteins, have led to transformative advances in molecular biology, notably genome engineering and most recently molecular diagnostics. The potential application of the CRISPR-Cas13a system for engineering viroid interference in plants is suggested by targeting specific motifs of three economically important viroids. The CRISPR-Cas13 system has been utilized recently for the accurate detection of human RNA viruses by visual read out in 90 min or less and by paper-based assay. Multitarget RNA tests by this technology have a good potential for application as a rapid and accurate diagnostic assay for known viroids. The CRISPR/Cas system will work only for known viroids in contrast to NGS, but it should be much faster. Theodor O. Diener discovered the first viroid in 1971, the causal agent of potato spindle tuber disease . He showDiener’s discovery took place 16 years after another virology landmark discovery in which Heinz Fraenkel-Conrat demonstrated that the genetic information controlling viral replication is carried in the nucleic acid core of each virus particle. He showed that he could actually reconstitute a complete infectious tobacco mosaic virus from the protein and RNA .Viroids, the smallest known infectious agents (246–401 nt), comprise a novel class of infectious single-stranded RNA that replicates autonomously and exists as circular and linear forms with a high degree of base pairing. In contrast to viruses, viroids lack capsid proteins, do not code for proteins and completely dependent on host precellular RNA polymerases and processing enzymes for their replication. Pospiviroidae  with CCR and members of its five genera replicate and accumulate in host nuclei; Avsunviroidae  lacks CCR and has hammerhead ribozymes in both genomic and antigenomic RNAs, and members of its three genera replicate and accumulate in host plastids, mainly chloroplasts . Th. Th26]. It is expected in the next few years that the third-generation sequencing (long-read sequencing) platforms will increase sequencing capacity and speed while reducing costs very significantly. Thus, viroid sequencing may be done with costs for less than $ 25–50.Because of the small size of viroid genome, its complete genomic sequence can be determined in a single run by NGS as the sequence of hundreds of thousands to millions of viroid-derived small RNAs (vd-sRNA) of 21–24 nt, can be re-assembled to obtain the viroid genome(s) of interest . The vd-NGS of vd-sRNAs, viroid circular and oligomeric RNAs as well as infected host total RNA, DNase-treated total RNA, rRNA-depleted total RNA and rRNA-depleted dsRNA, combined with informatic and computational tools, have been used for almost a decade in viroid research and diagnostics. In most of these studies, NGS data were confirmed by different methods of RT-PCR to ensure that the viroid putative sequence exist in plants. NGS studies may include, but are not limited to, viroid de novo discovery, identification and detection as well as studies of viroid characterization, profiling, distribution, accumulation, biogenesis, strain differentiation, systemic movement, viroid-host interactions, viroid evolution, pathogenesis, mutation, mRNA targeting, extending host range and others as shown in Methods such as two-dimensional differential gel electrophoresis coupled with mass spectrometry, differential display and microarray have been used to study gene expression in viroid-infected plants . NGS, asA combination of microarray and large-scale RNA sequence analysis have been used to study gene expression in two PSTVd-infected tomato cultivars: the sensitive cultivar “Rutgers” and the dwarf cultivar “MicroTom” . “RutgerOrthologous genes are genes in different species that have similar functions and are similar in their nucleotide sequences which can be traced back to a common ancestral gene. The majority of the identified genes were responsible for diverse metabolic pathways, various stress responses, transcription, translation and transportation as well as genes coding for several cellular components [NGS was used to identify 11, 600 expressed sequence tags (ESTs) from a cDNA library prepared from total RNA extracted from CSVd-infected chrysanthemum leaves which provided good information of the transcriptome in that system . Approximponents .The gene expression changes in CBCVd in infected hop were revealed by transcriptome analyses . CBCVd iFor several years, due to advances in NGS technologies, the complete sequence of ancient genomes from both modern and archaic humans has revolutionized our understanding of human evolution and migration ,64. NGS All known viroids have been sequenced ,8. A priNGS revealed in 2015 that the causal agent of severe stunting and death of hop plants in Slovenia is CBCVd . As a reClustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated Cas proteins comprise the CRISPR-Cas systems, which confer adaptive immunity against foreign genetic elements such as bacteriophages and plasmids in many bacteria and most archaea ,75,76,77The CRISPR-Cas systems are divided into two general classes on the basis of the composition of the interference complex . Class 1Class 2 type VI systems, include a single “effector” protein designated Cas13a, formerly C2c2 , which wNicotiana benthamiana [Classical breeding and different molecular strategies to introduce resistance to viroids have been unsuccessful or partially successful in containing viroid infection . A new mthamiana .PSTVd, PLMVd and ASBVd are economically important and their genomes and replication have been studied extensively . Selecte“A-PSTVd terminal left domain: 1-Initiation site of minus PSTVd RNA synthesis at nt U359 or C1. 2-Three bulges associated with plus PSTVd RNA replication.B-PSTVd center conserved region: 1- Hairpin I, delimited by nt 79 and 110 on the upper strand of the viroid RNA. 2- Hairpin II, delimited by nt 227 and 328 on the lower strand of the viroid RNA. 3-Loop E, located between nt 5’-G97 to C103-3’ and nt 5’-G255 to C262-3’.C-PSTVd terminal right domain: RY motif, composed of two asymmetric internal loops, with sequence elements 5’-ACAGG-3’ (nt 173-177) in the upper strand and 3’-CUCUUCC-5’ (nt 190–184) in the lower strand.D-PLMVd initiation sites: Such sites are located at nt C51 for the plus strand and at nt U286 for the minus strand, both mapping at similar double-stranded motifs of 6–7 bp that also contain the highly conserved GUC triplet preceding the self-cleavage site in both polarity strands. These motifs are located at the base of a similar long hairpin that presumably contains the promoters for a chloroplastic RNA polymerase. Since the in vivo initiation of PLMVd RNAs occurs near the self-cleavage (and self-ligation) sites, CRISPR-Cas13a editing in this region would attenuate both the transcription and processing of PLMVd RNA.E-ASBVd initiation sites: These sites are located at nt U121 and at nt U119 for the plus and minus strands, respectively. The initiation sites are only two nucleotides apart and each site starts with the same sequence, UAAAA, which suggests that the viroid promoters are formed, at least in part, by the sequences flanking the two initiation sites.”The RNA guided nuclease activities of CRISPR-Cas13a, -Cas13b and -Cas12a, which are functionally distinct from -Cas9, have been recently utilized to develop sensitive detection methods for human viruses ,100,101.It has been shown that the Cas13 SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can incorporate pre-amplified input material to develop a paper-based assay with improved sensitivity . SHERLOCCRISPR-Cas editing does not incorporate permanently foreign DNA into the host genome as in transgenic plants, and agricultural applications of this precise technology are already creating valuable products for various markets in the United States. For this reason, the U.S. Department of Agriculture does not regulate foods or plants developed by CRISPR-Cas editing ,108,109.


Toroviruses infect cattle, goats, pigs, and horses worldwide and can cause gastrointestinal disease. There is no treatment or vaccine, and their ability to spill over into humans has not been assessed. These viruses are related to important human pathogens, including severe acute respiratory syndrome (SARS) coronavirus, and they share some common features; however, the mechanism that they use to produce sgRNA molecules differs. Here, we performed deep sequencing to determine how equine torovirus produces sgRNAs. In doing so, we also identified two previously unknown open reading frames “hidden” within the genome. Together these results highlight the similarities and differences between this domestic animal virus and related pathogens of humans and livestock. Torovirus  encompasses a range of species that infect domestic ungulates, including cattle, sheep, goats, pigs, and horses, causing an acute self-limiting gastroenteritis. Using the prototype species equine torovirus (EToV), we performed parallel RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) to analyze the relative expression levels of the known torovirus proteins and transcripts, chimeric sequences produced via discontinuous RNA synthesis (a characteristic of the nidovirus replication cycle), and changes in host transcription and translation as a result of EToV infection. RNA sequencing confirmed that EToV utilizes a unique combination of discontinuous and nondiscontinuous RNA synthesis to produce its subgenomic RNAs (sgRNAs); indeed, we identified transcripts arising from both mechanisms that would result in sgRNAs encoding the nucleocapsid. Our ribosome profiling analysis revealed that ribosomes efficiently translate two novel CUG-initiated open reading frames (ORFs), located within the so-called 5′ untranslated region. We have termed the resulting proteins U1 and U2. Comparative genomic analysis confirmed that these ORFs are conserved across all available torovirus sequences, and the inferred amino acid sequences are subject to purifying selection, indicating that U1 and U2 are functionally relevant. This study provides the first high-resolution analysis of transcription and translation in this neglected group of livestock pathogens.The genus IMPORTANCE Toroviruses infect cattle, goats, pigs, and horses worldwide and can cause gastrointestinal disease. There is no treatment or vaccine, and their ability to spill over into humans has not been assessed. These viruses are related to important human pathogens, including severe acute respiratory syndrome (SARS) coronavirus, and they share some common features; however, the mechanism that they use to produce sgRNA molecules differs. Here, we performed deep sequencing to determine how equine torovirus produces sgRNAs. In doing so, we also identified two previously unknown open reading frames “hidden” within the genome. Together these results highlight the similarities and differences between this domestic animal virus and related pathogens of humans and livestock. Nidovirales currently contains four families of positive-sense, single-stranded RNA viruses: the Coronaviridae, Arteriviridae, Roniviridae, and Mesoniviridae  encompasses a range of species with worldwide distribution that infect domestic ungulates, including cattle, goats, sheep, pigs, and horses, causing an acute self-limiting gastroenteritis. Approximately 55% of cattle within the United Kingdom are seropositive for bovine torovirus, and this pathogen represents a significant burden to the industry  1a and 1b  has copied a TRS sequence, a template-switching event may occur during which the anti-TRS at the 3′ end of the nascent strand base pairs with the leader TRS within the 5′ UTR. Transcription reinitiates and continues to the 5′ end of the genomic template. The resulting “anti-leader” sequence that is added ranges from 55 to 92 nucleotides (nt) in coronaviruses to ∼200 nt in arteriviruses. These negative-sense transcripts are therefore 5′- and 3′-coterminal with the full-length negative RNA strand and are identifiable as chimeras with distinct flanking sequences adjacent to the core TRS. The anti-leader sequence in each of the negative-sense templates then functions as a promoter to drive synthesis of a mirror set of positive-sense sgRNAs that are translated to produce the structural proteins.Nidovirales  are thought to produce two major sgRNAs possessing leader sequences of different lengths, indicating that the nidoviral mechanism for discontinuous RNA synthesis can allow two very different leader/body TRS pairs to be utilized in a single viral species  possesses a leader TRS-like sequence (CUUUAGA), but it is only involved in the synthesis of the mRNA used for expression of the spike (S) protein gene . DespiteHere, we describe the first high-resolution analysis of viral transcription during infection by EToV, which is one of the few toroviruses that can be propagated in cell culture , 16. RNARibosome profiling (Ribo-seq) conducted in tandem with the RNA-seq indicated that ribosomes actively translate within the so-called 5′ UTR. Further analysis confirmed the existence of two novel ORFs in this region, which are conserved in all torovirus genome sequences analyzed to date. The specific function(s) of these proteins will be the topic of future work. Together, these results provide an overview of the transcriptional and translational events that accompany infection by this wide-ranging pathogen.We conducted tandem RNA-seq and Ribo-seq of EToV-infected equine dermal (ED) cells. Two biological replicates of virus-infected and mock-infected cells were analyzed, generating 25 to 53 million reads per sample. For RNA-seq, 77 to 92% of reads mapped to the host genome, of which a mean of 1.5% mapped to rRNA, 19% to mRNA, 32% to noncoding RNA (ncRNA), and 47% elsewhere in the genome. For Ribo-seq, 46 to 60% of reads mapped to the host genome, of which a mean of 56% mapped to rRNA, 13% to mRNA, 4.9% to ncRNA, and 26% elsewhere in the genome . Totals of 1.3% and 2.3% of reads mapped to the virus genome in the two EToV-infected RNA-seq replicates and 0.41% and 0.21% in the two EToV-infected Ribo-seq replicates.de novo from RNA-seq reads and confirmed as EToV, Berne isolate. A single 27,694-nt contig was assembled representing almost the entire viral genome. Only 18 nt at the 5′ terminus and 300 nt at the 3′ terminus failed to assemble automatically; however, these regions were clearly covered by reads consistent with the reference sequence on inspection and so were added manually to the consensus sequence. Four single-nucleotide changes were present in all reads, but not the reference sequence compiled from previous sequencing data, at positions 18078 , 21429 , 21814 , and 25596 .The viral genome was assembled The distribution of reads on the virus genome and the phasing of these reads are shown in To calculate the length distributions of host- and virus-mapped RPFs, we used reads mapping within coding regions. After adaptor trimming, the majority (75%) of Ribo-seq reads were 27 to 29 nt in length, which is consistent with the expected size of mammalian ribosome footprints. As expected, the distribution of read lengths for RNA-seq was much broader, peaking between 60 and 70 nt . For quaThe relative RPF density allowed us to estimate the efficiency of ribosomal frameshifting in the context of virus infection. After translating ORF1a, a proportion of ribosomes undergo a −1 ribosomal frameshift to translate ORF1b . This isRNA sequencing reads that did not map to either the viral genome or host databases were analyzed for containing potential viral chimeric junctions, indicative of leader-to-body joining during discontinuous sgRNA synthesis . RelativThree chimeric junctions were identified where the first nucleotide of the corresponding read mapped to the first nucleotide of the viral genome. Of these, one junction was consistent with the previously characterized sgRNA produced via discontinuous RNA synthesis encoding the S gene  . These rA substantial number of additional chimeric reads were identified, indicative of non-TRS-driven cases of discontinuous RNA synthesis, although formally it is possible that some of these are template-switching artifacts introduced during library preparation and/or sequencing. Additionally, a large number of reads spanning from the 5′ UTR to either within the N protein gene or the 3′ UTR were identified. Indeed, the only junction represented by over 1,000 reads spanned nucleotides 673 to 27649; similarly, the second most commonly identified junction, with 642 reads, spanned 687 to 27550. If chimeric reads were predominantly a sequencing artifact, the abundance of any particular chimaera would be approximately proportional to the product of the abundances of the sequences from which the 5′ and 3′ ends of the chimera are derived (with some variation due to sequence-specific biases), thus a high density of chimeras would be expected to fall entirely within the N transcript. In contrast, most of the observed chimeric reads were between N and the 5′ UTR. The relative paucity of reads mapping to generic locations in the ORF1ab region also argues against the majority of chimeras being simply artifactual. The 5′ UTR preference may be due to genome circularization during negative-sense synthesis, as has been proposed for coronaviruses . AlternaThe RNA-seq data were analyzed to identify genes that were differentially expressed between virus-infected and mock-infected ED cells. We identified 61 genes that were upregulated in virus-infected cells, among which eight gene ontology (GO) terms were overrepresented, mostly related to the nucleosome or immune responses . We founOur initial data set indicated an excess density of ribosomes translating within the +2/−1 frame upstream of ORF1a and overlapping the 5′ end of ORF1a . To furtThis Ribo-seq data confirmed translation of two ORFs located within the so-called 5′ UTR and overlapping the 5′ end of ORF1a. We have termed these U1 (80 codons) and U2 (258 codons). We predict that translation of both U1 and U2 is initiated from CUG codons, as a close inspection indicated that ribosomes accumulate at these two sites . It mustRevisiting our first nondrug-treated data set, we calculated the RPF densities and translational efficiencies within the U1 and U2 ORFs . U1 has dN/dS), where dN/dS of <1 indicates selection against nonsynonymous substitutions, which is a strong indicator that a sequence encodes a functional protein. Application of codeml , being more probable than nonconservative substitutions in biologically functional polypeptides. MLOGD 3-frame “sliding window” analysis of a full-genome alignment revealed a strong coding signature in the known protein-coding ORFs (as expected) and also in the U1 ORF  that the observed level of conservation would occur by chance is 6.5 × 10−40.We previously predicted the existence of U2 via an analysis of coding potential and synonymous site conservation across the two torovirus genomes available at that time . Six addof ORF1a . Overlapovo gene . The de sordered . Interessordered  nor HHprsordered  homologuvolution . Consistvolution , synplotvolution , with thBoth U1 and U2 are conserved in all eight torovirus sequences, with no variation in length or initiation or termination position . In all 33–3–4CUUUAGA to reflect this : within the 5′ UTR (leader TRS), the end of U1, central ORF1a, central ORF1b, and immediately before the M, HE, and N ORFs . Consistect this . It is lFor the S gene, our results lend support to the hypothesis suggested previously that a short conserved RNA hairpin, 174 nt upstream of the initiation codon, is involved in stimulating template switching for this sgRNA  Fig. 15Fig. 15. The leader-TRS chimeric reads mapping to the N protein gene initially appear consistent with the coronaviral and arteriviral mechanism of TRS-driven discontinuous RNA synthesis. However, close inspection indicated that the homologous motif mediating copy-choice recombination-like translocation and repairing of RNA strands was actually a short AGAA sequence, not the true TRS  is tolerant to modifications; indeed, even when this spacer is extended to six nucleotides, transcripts are still detectable at 20% of wild-type levels  across all torovirus genomes  suggestse levels . Again, e levels . Initiate levels . It may Coronaviridae, subfamily Torovirinae, genus Bafinivirus) has an extended TRS sequence (CA[G/A]CACUAC) which is not conserved with the mammalian toroviruses analyzed in this study. Bafinivirus replication produces three sgRNAs which share an identical 42-nt leader also found at the far 5′ terminus of the genome, indicating this species utilizes discontinuous RNA synthesis in a manner similar to that of the corona- and arteriviruses  homology-driven reinitiation , (ii) structure-driven discontinuous transcription (EToV S gene), and (iii) transcription termination . These mechanisms require an RdRp which is prone to template switching when even relatively short homologous sequences are present, potentially leading to a large number of irrelevant transcripts being produced  andThe differential transcription analysis indicated that infection with EToV induces increased transcription of multiple genes, the products of which are significantly more likely than random to be involved in (i) nucleosome function and DNA binding and (ii) immune responses to infection than genes which were not differentially transcribed. Some of the identified GO categories, including cytokine signaling, innate immune responses, and ribosome biogenesis, have been identified in previous RNA-seq analyses of various coronaviruses , 44. SimEquus caballus) genome is not highly annotated, and thus many Ensembl gene identifications do not possess an annotated orthologue, a limiting factor in our analysis.To the best of our knowledge, this is the first analysis of differential gene expression following infection with a torovirus. It would be of interest to repeat this analysis at later time points, as a previous study found that EToV-mediated global inhibition of host protein synthesis was only detectable at 16 h postinfection (h.p.i.). The same study found induction of both the intrinsic and extrinsic apoptotic pathways was evident only by 24 h.p.i. . It is cThe current lack of a published reverse genetics system to study torovirus replication means we are unable to perform targeted mutagenesis. This would enable definitive experimental confirmation that U1 and U2 are translated from their respective CUG codons, followed by phenotypic analysis of knockout mutants. However, the comparative genomic analysis together with the accumulation of ribosomes on both CUG codons is highly suggestive of this being the site of initiation; CUG has previously been reported as the most commonly utilized non-AUG initiation codon in mammalian systems . In the cis-replication elements.The ORFs of both U1 and U2 are intact in all torovirus genomic sequences that we have analyzed to date, including bovine , 52, capTorovirinae  of the proteins encoded by both U1 and U2 remain to be elucidated. Despite the relatively large size of the U2 protein (∼30 kDa), after extensive database searches no structural homologs were identified. By comparison, the U1 protein is small (∼10 kDa) and highly basic (pI of 10.4), and it possesses many of the predicted features of a double-spanning transmembrane protein, including two hydrophobic stretches separated by a hinge and a predicted coiled-coil tertiary topology. Based on structural similarity to known proteins, one potential function is a virally encoded ion channel (viroporin) embedded in either intracellular or plasma membranes. It is possible that U1 plays a role in toroviruses similar to that of the coronaviral and arteriviral E proteins, which have no known toroviral homologue. The coronavirus E protein is a small transmembrane protein (∼10 kDa) which possesses ion channel activity and is required for virion assembly, forming a pentamer that traverses the viral envelope , 55. E pAlternatively, viroporin activity may be mediated by a small, basic double-transmembrane protein, the ORF of which is embedded within the EToV N gene in the +1 frame (with respect to N). An analogous “N + 1” protein has been identified in some group II coronaviruses and is postulated to play a structural role; however, it is not essential for replication , 57. NeiNidovirales.Our data have revealed that the transcriptional landscape of a prototypic torovirus is complex and driven by many factors beyond the canonical multilocus TRS model of coronaviruses. The development of a torovirus reverse genetics system would allow manipulation of potential template switch-inducing sequences and allow us to elucidate which features of the toroviral TRS cause them to act as terminators of RNA synthesis rather than consistently inducing homology-assisted recombination. Our accompanying translational analysis has revealed two conserved novel ORFs and suggests the EToV 5′ UTR is only 523 nt. Together these data provide insight into the molecular biology of the replication cycle of this neglected pathogen and highlight the disparities between the families of the l-glutamine in a humidified incubator at 37°C with 5% CO2.A plaque-purified isolate of equine torovirus, Berne strain (isolate P138/72) (EToV), was kindly provided by Raoul de Groot (Utrecht University) and cultured in equine dermis (ED) cells. This virus was initially isolated from a symptomatic horse in 1972 . ED cellED cells were infected with EToV for 1 h in serum-free medium (MOI of 0.1) and flash-frozen in liquid nitrogen at 8 h.p.i. prior to either RNA isolation or ribosome purification for profiling. Cells were either not pretreated or, where stated, were treated with a final concentration of 100 μg/ml cycloheximide (CHX) for 2 min (Sigma-Aldrich) or 2 μg/ml of harringtonine for 3 min (LKT Laboratories), followed by CHX for 2 min before flash-freezing. RNA and ribosomes were harvested according to previously published protocols , 58, withttp://hannonlab.cshl.edu/fastx_toolkit/). Reads shorter than 25 nt after trimming were discarded. Bowtie (version 1.2.1.1) databases were generated as follows. Horse rRNA sequences were downloaded from the National Center for Biotechnology Information (NCBI) Entrez Nucleotide database  (DQ310701.1 (positions 1 to 14531), X52374.1 (13475 to 21394), X52506.1 (21250 to 26086), X52505.1 (26054 to 26850), X52375.1 (26784 to 27316), and D00563.1 (27264 to 279923). Horse mRNA sequences from EquCab2.0 (GCF_000002305.2) were downloaded from NCBI RefSeq (Both Ribo-seq and RNA-seq reads were demultiplexed and adaptor sequences trimmed using the FASTX-Toolkit (02126.1) . As the I RefSeq  and combvice versa. Reads mapping to the first five codons at the 5′ end of each region or the last six codons at the 3′ end of each region were excluded. For sgRNAs, RNA-seq density was calculated for the same regions as those described for Ribo-seq. For the genomic RNA the regions for ORF1a and ORF1b were combined into the interval from the start codon of ORF1a (position 882) to the 5′ end of the S gene hairpin (position 21118). Ribo-seq and RNA-seq densities were calculated as the RPM values for which the 5′ end maps to each region, divided by the length of the region in nucleotides and then multiplied by 1,000 . For RNA-seq, a decumulation strategy was used to subtract the estimated RNA-seq density for longer overlapping genomic and subgenomic transcripts that would contribute to the RNA-seq density measured for each of the 3′ ORFs: the genomic RNA-seq density was subtracted from all subgenomic densities, and then the RNA-seq densities of overlapping upstream subgenomic transcripts were iteratively subtracted from downstream regions . Translation efficiency for each gene was calculated as Ribo-seq density/decumulated RNA-seq density. Translational efficiencies for HE could not be accurately estimated, as the low expression of the HE transcript made the decumulation procedure for HE susceptible to noise.Ribo-seq density and RNA-seq density were calculated for each gene in the EToV genome  and 12. Read length distributions were calculated for Ribo-seq and RNA-seq reads mapping to positive-sense host mRNA annotated coding DNA sequences (CDSs) or to the positive-sense coding sequence of the EToV genome . Histogrde novo assembly of unmapped and vRNA reads from the infected RNA-seq samples. Assembly was performed using Trinity (The EToV (Berne isolate) genome sequence was confirmed by  Trinity  with the Trinity  against  Trinity .Chimeric reads were classified as reads for which the entire read mapped uniquely to the viral genome, with no mismatches, after adding a single breakpoint, with a minimum of 12 nt mapping on either side of the breakpoint, at least 5 nt apart. To identify such reads, all unmapped reads were split into two subreads at every possible position ≥12 nt from either end, and these subreads were mapped to the viral genome using bowtie with no mismatches and no multimapping permitted. Transcription junctions were defined as donor/acceptor pairs that were either supported by at least 10 chimeric reads or contained the entire 5′ leader and TRS sequence in the 5′ segment of the read. At some positions single-nucleotide resolution for the chimeric breakpoint could not be established. Where reads were found to break at adjacent possible positions, these positions were merged to give a short region containing the breakpoint. The number of nonchimeric reads spanning each donor and acceptor site was calculated as the number of reads which overlapped the site by at least 12 nt in either direction (as chimeric reads overlapping the site by <12 nt are not detectable). The proportion of chimeric reads at each donor or acceptor site is therefore the number of chimeric reads with a breakpoint at the site divided by this number plus the number of nonchimeric reads spanning the site .To visualize TRS conservation, multiple-sequence alignments were generated using Clustal Omega with default parameters . RNA str2 fold change of >1 and an FDR of less than 0.1 were considered to be differentially expressed. Gene ontology (GO) term enrichment analysis  using STAR , with a analysis  was perfanalysis . Differeanalysis  by folloDQ310701.1 (Berne virus), AY427798.1 (Breda virus) (JQ860350.1 (porcine torovirus) (LT900503.1 (porcine torovirus), LC088094.1 (bovine torovirus), and LC088095.1 (bovine torovirus) ; 100 randomized alignments were generated, and their dN/dS values were calculated with codeml.The GenBank accession numbers utilized for comparative genomic analysis were a virus) , KR52715rovirus) , KM40339rovirus) , LT90050 package . The eig package . AlignmeAY427798) was used as a reference, since unlike Berne virus it contains an intact HE gene. Genomes were then mapped to reference sequence coordinates by removing alignment positions that contained a gap character in the reference sequence, and these pairwise alignments were combined to give the multiple-sequence alignment. This was analyzed with MLOGD using a 40-codon sliding window and a 5-codon step size. For each of the three reading frames, within each window the null model is that the sequence is noncoding, whereas the alternative model is that the sequence is coding in the given reading frame. Positive/negative values indicate that the sequences in the alignment are likely/unlikely to be coding in the given reading frame. To assess conservation at synonymous sites, the concatenated coding regions were extracted from the alignment and analyzed with synplot2.Coding potential within each reading frame was analyzed using MLOGD , and synhttp://www.ebi.ac.uk/arrayexpress) under the accession number E-MTAB-6656. The full-length virus sequence has been deposited in GenBank (accession no. MG996765).The sequencing data reported in this paper have been deposited in ArrayExpress (


In most Sub-Saharan African countries, little is known about young adolescents’ sexual and reproductive health (SRH). Though some efforts have been made to understand and improve SRH of older adolescents, very young adolescents (VYAs) are often overlooked, and little is known about their sexual knowledge and behaviors. The goal of this study was to describe SRH knowledge, information-seeking, and sexual behavior of VYAs in Uganda.A cross-sectional survey was administered in 33 primary schools in June and July of 2016. Trained interviewers administered surveys to adolescents ages 10–14 regarding SRH knowledge, information-seeking, sexual behavior, and relevant covariates. Continuous variables were summarized as means (SD) or medians (IQR) whereas categorical variables were summarized as proportions (percentages).A total of 1096 adolescents were included in this analysis, 81.8% of which were from rural areas, with a median age of 12. Regarding sexually transmitted infections (STIs) knowledge; 95% knew HIV while 37% knew other STIs apart from HIV. Although 47% knew at least one way in which HIV is acquired only 8% knew at least four ways. Regarding contraceptive knowledge, 56% mentioned at least one modern method of preventing pregnancy . The majority (85%) of VYAs reported accessing SRH information in the media with 35% reporting accessing media with sexual content while 10% vs 22% consulted their father or mother respectively and 31% a school source. At least 7.6% of VYAs had ever had sexual intercourse, 90% of which were not using any protection.Comprehensive SRH knowledge was low among VYAs in this study. Media remains an important source of information for SRH for this age group though it may be misused as some adolescents reported accessing sexual content that may be inappropriate. A large proportion of sexually active VYAs reported sexual risky behaviors. This study highlights the need for an accurate and more comprehensive SRH education approach for VYAs in Uganda at an opportune age before the majority engage in sexual behavior. Uganda has the second youngest population in the world, with high rates of sexually transmitted diseases and adolescent pregnancy. Access to sexual and reproductive health (SRH) information and resources is a challenge, especially for very young adolescents (VYAs) aged 10–14. The goal of this study is to assess levels of SRH knowledge, SRH information seeking and sexual behavior among VYAs. We found that most students are aware of HIV, but few have detailed knowledge of how HIV is acquired, what other STIs exist, and what methods of pregnancy prevention are available. Seven percent of VYAs were already sexually active with most of them not using any form of protection such as condoms. The majority of VYAs had been exposed to SRH information in the media  with at least one third of them accessing media with sexual content, while far fewer had received such information in schools or from their parents. This study highlights the importance of implementing school-based SRH education at a young age. Because the majority of VYAs are not yet sexually active, SRH interventions in this age group may be more effective than those in older adolescents in delaying sexual onset and decreasing risky sexual behavior. Furthermore, because these students are already being exposed to sexual information in the media, it is important for schools and parents to provide accurate information that helps students to reflect more critically on SRH information in media sources.With a median age of just 15, Uganda has the second youngest population in the world . This yoAccess to SRH information and prevention services remains a challenge in most developing countries, including Uganda. The 2016 DHS found that only 40% of Ugandan adolescents ages 15–19 years have comprehensive HIV prevention knowledge . A studyDespite these challenges, there has been a lot of pushback on SRH education in Uganda from parents, teachers, and even Members of Parliament (MPs), who feel that teaching these topics in schools will erode Uganda’s culture and morality . In 2016Following evidence on the proven benefits of comprehensive sexuality education in diverse settings , UNESCO The goal of this study is to investigate the current status of sexual behavior, SRH knowledge, and information-seeking behaviors of adolescents ages 10–14 in Uganda. It aims to fill the gap in the literature regarding the SRH of VYAs in Uganda. This information should guide the development and implementation of educational SRH interventions across the country.This paper presents the results of a cross-sectional survey of very young adolescents in primary schools in Mbarara district of South Western Uganda. This data was collected as the baseline of a cluster randomized trial to evaluate the implementation and effectiveness of a comprehensive sexuality education program in primary schools. According to the 2015 District Education reports, Mbarara has a total of 248 primary schools with an estimated primary school enrolment of 80,924 .Participant eligibility criteria included being 10–14 years old and in primary level 5 or 6 of education from rural and urban schools within Mbarara district. The final sample size included 1096 adolescents from 33 schools in both urban and rural settings. A list of primary schools in Mbarara was available in district records. We eliminated schools that had no upper classes (primary 5 to 7), and schools that offer unique services, including one school for children with hearing impairments and one school for children with visual impairments. The remaining schools were entered into an Excel spreadsheet and a formula was developed to randomly select the required 33 schools for the study. A second Excel formula was used to randomly assign each of the 33 schools to the treatment or control arm. Research assistants (RAs) selected the final sample of students in each school from class registration lists using systematic sampling methods and substituting pupils who were absent on the day that data was collected.The data were collected between June and July 2016. Standardized closed surveys were administered by trained RAs and were crosschecked by the onsite investigator and research monitors and cleaned for any errors. All RAs were fluent in both English and Runyankole  and surveys were administered based on each students’ preference. Responses to all questions were recorded and coded either using scales or binary yes/no questions, with the option to refrain from responding. Additionally, questions that assessed pupils’ knowledge included the option to respond with “I do not know”. Anonymity was maintained throughout the study and participants’ responses were deidentified.Sexual behavior was measured starting from a screening question: “have you ever been to a secret place alone with a friend of the opposite sex?” If the answer to this question was yes, further questions were asked regarding sexual experiences. These included “Did you ever kiss on the lips?”, “Did you or your friend ever touch your private parts?”, “Have you ever had sex?” “How old were you when you first had sex?”, “What method did you use to prevent HIV/AIDS or STIs?” and “Were you willing to have sex the first time you did?”Sexual and reproductive health knowledge was measured based on knowledge of puberty, HIV/STIs and pregnancy prevention. This included knowledge on how HIV/AIDS can be acquired, types of common Sexually Transmitted Infections, knowledge of pubertal changes in boys  and in girls , and knowledge about ways to prevent pregnancy .Sources of Information on SRH were assessed. We asked students if they had sought SRH information from their mother, father, a school source, a community health facility and/or mass media sources. Media was further categorized into seeking information on SRH topics or sexual content.Data was collected on socio-demographic variables such as age in years, pubertal stage, gender, religion, socio-economic status , whether the school was located in rural or urban area and whether one, both or neither parents were alive.p-value of less than 0.05 was considered a significant association.Data was double entered into Epi-data 3.1 . The data utilized in this analysis represent the baseline data of a cluster randomized control trial evaluating the effectiveness of a Comprehensive Sexuality Education intervention. Based on an intra-class correlation calculated in a pilot test among 105 VYAs and an estimated drop-out rate of 20%, we determined that a minimum sample size of 1100 students was needed to measure a 10% score difference in outcomes between baseline and endline among the intervention vs control groups with a power of 90%. Data analysis was done using Stata® . Continuous variables were summarized as means (SD) or medians (IQR) whereas categorical variables were summarized as proportions (percentages). In addition, a bivariate analysis among categorical variables was made using chi square. All analyses were stratified by gender. A A total of 1096 pupils were interviewed between June and July 2016. The median age was 12  years and 58% were female. Over 80% of the study population were from rural areas and over 80% of the pupils had both parents alive. Twenty-nine percent were ranked as having a high socio-economic score, 47% ranked medium and 24% ranked low  knew HIV is an STI and 37% knew other STIs apart from HIV. Although 47% knew at least one way in which HIV may be acquired, only 8% knew at least 4 methods by which HIV is acquired. Regarding contraceptive knowledge, 59% mentioned abstinence, 56% mentioned any of the more effective methods of preventing pregnancy , and 3% knew natural methods for contraception. More boys than girls knew of any effective ways to prevent pregnancy and the difference was significant . More boys than girls watched visual media with sexual content. A total of 31.7% reported seeking SRH information from a source at school. Only 22.3% and 9.9% had discussions about love or sex with their mother or father respectively. Pupils were more likely to communicate with a parent of their same gender, meaning more boys communicated with fathers and more girls communicated with mothers  of VYAs were aware of HIV, few were aware of the four most common ways that HIV is transmitted and over a third said that it is not possible for a person who looks healthy to have HIV. Similar findings about SRH knowledge were earlier described by Bankole et al., 2007 . This shIn this study, 7.6% of VYAs were sexually active, which is lower than previous findings among the same age group in Sub-Saharan Africa of 11–15% , 37. MorFurthermore, because the majority of these students are still not sexually active, this is an opportune age to implement behavioral SRH interventions. A plethora of studies in low and middle income countries have shown that comprehensive SRH education is associated with delayed sexual onset , 43, butSexual behavior was self-reported and this being a sensitive subject, under reporting could have occurred among some participants. During data collection, we ensured privacy and tried as much as possible to have interviewers of the same sex as respondents conduct the interviews in an effort to limit reporting bias, a form of information bias. While dichotomizing certain variables was helpful in demonstrating statistical results, this may have led to a loss of relevant data. Furthermore, because this was a cross sectional survey, we could not determine the direction of causality. Finally, due to cultural and legal restrictions around homosexuality in Uganda, we were only able to ask students about heterosexual behavior, and did not ask about homosexual activity.Very young adolescents in Uganda face great challenges in SRH, having low SRH knowledge, early sexual debut and risky sexual practices. SRH education should start during early adolescence, before the majority of these students are sexually active and while most of them are still in school. Comprehensive, accurate, school-based SRH education is crucial. Further research should be conducted and used by policy makers in the development of nation-wide policies regarding SRH education for VYAs.


Mucor irregularis is a causative agent of mucormycosis. The transcriptome analysis of the isolated M. irregularis strain C3B revealed the presence of an RNA polymerase domain of a negative-polarity RNA virus. In this work, we describe the gene ontology-based annotation of the Mucor irregularis transcriptome, which includes a putative RNA mycovirus.The fungus  Mucor irregularis is a causative agent of mucormycosis. The transcriptome analysis of the isolated M. irregularis strain C3B revealed the presence of an RNA polymerase domain of a negative-polarity RNA virus. In this work, we describe the gene ontology-based annotation of the Mucor irregularis transcriptome, which includes a putative RNA mycovirus.The fungus  Mucor irregularis is a causative agent of mucormycosis, especially in people with an impaired immune system; however, it can also affect immunocompetent hosts, causing primary cutaneous mycosis , a region of the Amazon forest, located south of the city of Belém, Pará State, Brazil. The soil sample was collected, serially diluted to 1 mg/ml, and inoculated in potato dextrose agar.For total RNA extraction, the fungal mycelium was transferred to a microtube containing Tris-EDTA buffer and 5-mm beads. Then, thermal lysis was carried out by three successive immersion cycles of the microtube in water at 100°C for 1 min and liquid nitrogen for 30 s. Subsequently, the tube was vigorously shaken in the TissueLyser II (Qiagen) for mechanical lysis. Total RNA purification was performed using the TRIzol reagent and the QIAamp viral RNA minikit (Qiagen).Furthermore, the cDNA was synthesized through the cDNA synthesis system kit (Roche), as described by the manufacturer. The library was constructed using the Illumina Nextera DNA library preparation kit, with 100-bp paired-end reads. Sequencing of the library was performed on the HiSeq 2500 platform (Illumina).De novo assembly was performed using Trinity v2.8.4  and present at 30.19% identity  to the polymerase sequence of Beihai sesarmid crab virus 5 (GenBank accession number KX884794), a crustacean-associated virus, according to the CD-Search  long containing a partial viral polymerase gene was obtained from the assembly of the transcriptome of the fungus N50, 11,037 nt; GC content, 38.13%), of which 21,313 presented a BLAST hit result. The gene ontology (GO) distribution of the sequences for molecular function, biological processes, and cellular components is presented in The assembly transcriptome generated 30,195 contigs (MN082151. This transcriptome shotgun assembly project has been deposited at GenBank under the accession number GHMU00000000. The primary sequence data were deposited in the Sequence Read Archive under the accession number SRR8992276.The partial viral sequence was deposited at GenBank under accession number


Genetic, transcriptional, and post-transcriptional variations shape the transcriptome of individual cells, rendering establishing an exhaustive set of reference RNAs a complicated matter. Current reference transcriptomes, which are based on carefully curated transcripts, are lagging behind the extensive RNA variation revealed by massively parallel sequencing. Much may be missed by ignoring this unreferenced RNA diversity. There is plentiful evidence for non-reference transcripts with important phenotypic effects. Although reference transcriptomes are inestimable for gene expression analysis, they may turn limiting in important medical applications. We discuss computational strategies for retrieving hidden transcript diversity.The online version of this article (10.1186/s13059-019-1710-7) contains supplementary material, which is available to authorized users. Caenorhabditis elegans ModENCODE sets combined RNA-seq, RACE, and expressed sequence tag (EST) sequencing [Reference transcriptomes (RefTs) aim to provide a comprehensive picture of transcripts produced by an organism. Early RefTs were produced at the turn of the century based on sanger sequencing of full-length cDNAs (flcDNA) –3. Laterquencing , 6. In yquencing –10 . A tquencing , 12 and quencing , 14 and C. elegans [Drosophila [Arabidopsis [Major genome databases integrate sequence data from the above sources into non-redundant, curated transcript datasets Fig. . RefSeq  elegans , Flybaseosophila , 23, andbidopsis , are probidopsis  does notThe most striking lessons drawn from large-scale transcript sequencing have been the widespread expression of long non-coding RNA genes and the abundance of alternative transcripts. This is well reflected in the number of genes and transcripts in current genome annotations Fig. . For insWhile current transcript counts in RefTs may seem impressive, these datasets have actually grown relatively slowly, constrained by their rigorous curation process. For instance, Gencode has grown from 161,000 human transcripts in 2012 to 207,000 now, i.e., a 29% growth in 7 years. In the meantime, projects generating raw RNA-seq data have exploded. Three projects alone, The Cancer Genome Atlas (TCGA) , GTEX 2, and HumWe describe below the different types of transcript variations that may be missing from RefTs. We contend that the information gap between RefTs and high-throughput data is not going to be closed. Based on multiple evidence gathered from medical transcriptome studies, we argue that non-reference transcript information is highly significant and its neglect limits our understanding of genotype–phenotype relationships. This underlines the need for computational methods that can extract non-reference events from RNA-seq data., including a wide array of RNA processing, editing, base modification, and cleavage/degradation mechanisms, are specific to cell type, cell compartment , and environmental conditions. It is worthy to note that transcriptomic complexity is not limited to higher eukaryotes, as illustrated by the discovery of bidirectional promoters [Each cell of an organism produces a distinct set of transcripts. Transcriptome differences between cells stem from three mechanisms that are potentially cumulative Fig. . First, romoters , 32 and romoters  in yeastMost individual RNA variations do not find their way into RefTs. An analysis of splice junctions in approximately 21,500 human RNA-seq libraries from SRA  identifiOne last important factor limiting RefT completeness stems from the nature of RNA libraries analyzed  RNA sequencing will help improving RefTs faster than current technologies that require capture of cDNA ends in complement to short reads. However, the combinatorial nature of transcript variation, the higher yield of short-read sequencing, and the huge diversity of tissues, diseases, and transcript classes probed by short-read sequencing make it unlikely that RefTs will ever match the level of diversity observed in short read data.Of note, limitations of RefTs are in a large part intentional. Indeed, these databases are manually curated to exclude a majority of pervasive transcripts resulting from expressed repeats, pseudogenes, or erroneous splicing. Transcript catalogues computationally generated from thousands of RNA-seq libraries apply less stringent inclusion criteria and are poised to include a large fraction of non-functional and pathological products, as well as incorrect boundaries and exon structures , 77.Well-curated RefTs are essential resources for measuring gene expression. RefT-based gene expression analyzes are now highly efficient , 89, proThere is no consensus on the most efficient RNA-seq analysis protocols for characterizing and quantifying non-reference transcripts. Strategies focusing on local or regional transcript variations are a powerful way to circumvent limitations related to full-length assembly. Such methods can be combined to conventional RefT-based analysis to achieve a complete description of normal and aberrant transcript forms present in a set of RNA-seq libraries.Additional file 1:Table S1. Overview of major eukaryotic transcriptome databases. Table S2. Large-scale RNA-seq projects (human). Table S3. Sequencing methods providing insight on specific events shown in Fig Table S4. Transcript variations related to cancer and other diseases; and software for retrieving these variations from RNA-seq data. (XLSX 18 kb)


In this study, we report that KSHV stimulates the RIG-I signaling pathway in a RNA polymerase (Pol) III-independent manner and subsequently induces type I interferon (IFN) responses. Knockdown or inhibition of RNA Pol III had no effect on beta interferon (IFN-β) induction by KSHV. By using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) approach, we identified multiple KSHV regions that give rise to RNA fragments binding to RIG-I, such as ORF810420-10496, Repeat region (LIR1)119059-119204, and ORF2543561-43650. The sequence dissimilarity between these fragments suggests that RIG-I detects a particular structure rather than a specific sequence motif. Synthesized ORF810420-10496 RNA stimulated RIG-I-dependent but RNA Pol III-independent IFN-β signaling. In summary, several KSHV RNAs are sensed by RIG-I in a RNA Pol III-independent manner.Retinoic acid-inducible gene I (RIG-I) is a cytosolic pathogen recognition receptor that initiates the innate immune response against many RNA viruses. We previously showed that RIG-I restricts Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation  is the causative agent of Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. Innate immune responses against viral infections, especially the induction of type I interferon, are critical for limiting the replication of viruses. Retinoic acid-inducible gene I (RIG-I), a cytosolic RNA helicase sensor, plays a significant role in the induction of type I interferon responses following viral infection. Here, we identified multiple RNA regions in KSHV as potential virus ligands that bind to RIG-I and stimulate RIG-I-dependent but RNA Pol III-independent IFN-β signaling. Our results expand the role of RIG-I by providing an example of a DNA virus activating a canonical RNA-sensing pathway. The inflammatory response triggered by the cytosolic sensors known as RIG-I-like receptors (RLRs) is one of the first and most important lines of defense against infection. There are three members of the RLR family: retinoic acid-inducible gene I (RIG-I), melanoma differentiation gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). During RNA virus infection, RIG-I recognizes viral RNA fragments, and RNA binding induces conformational changes and oligomerization of RIG-I . Activat2–RIG-I signaling is essential for the control of infection by many RNA viruses, such as hepatitis C virus (HCV) , vesicul8–11–RIG-I has previously been shown to be involved in sensing DNA viruses in a RNA polymerase (Pol) III-dependent manner. RNA polymerase III transcribes cytosolic AT-rich DNA from DNA viruses into RNA that can then serve as a pathogen-associated molecular pattern (PAMP) for RIG-I-mediated immune responses , 21. Forde novo infection, while beta interferon (IFN-β) induction was attenuated, suggesting that RIG-I was indeed activated and had an inhibitory role during primary infection and during reactivation from latency (KSHV is a large double-stranded DNA virus that is the etiologic agent of Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD) , 27. An 28– latency . The mecRNA polymerase (Pol) III is an intracellular DNA sensor that has been shown to transcribe viral DNA into a RNA intermediate that stimulates RIG-I . To deteEscherichia coli-produced recombinant RIG-I incubated in vitro with iSLK.219 total RNA. Endogenous RIG-I/RNA complexes were immunoprecipitated with a monoclonal antibody against RIG-I. For a control, we used an anti-IgG antibody in parallel throughout the immunoprecipitation (IP) procedure  and reactivated (RFP) iSLK.219 cells. Virus reactivation in iSLK.219 cells was induced for 48 h by 0.2 µg/ml doxycycline treatment, and the extent of reactivation was measured by detection of RFP-positive cells using high-content fluorescence imaging Download Copyright © 2018 Zhang et al.2018Zhang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the −/−) mouse embryo fibroblasts (MEF). Poly(I·C) transfection and VSV infection served as positive controls. Twenty-four hours after RNA transfection or virus infection, total RNA was purified, and mouse IFN-β mRNA levels were determined by qRT-PCR. As seen in −/− MEF, demonstrating that the IFN-β induction was dependent on RIG-I. Poly(I⋅C) and VSV induced IFN-β mRNA independent of RIG-I status presumably via TLR3-TRIF .To establish the biological activity of the RIG-I copurifying RNA fragments, the bound RNA was tested for immunostimulatory activity. RNAs derived from the following samples were compared: (i) IgG antibody immunoprecipitated from latent iSLK.219 cells with no doxycycline (Dox) ; (ii) IgG antibody immunoprecipitated from lytic iSLK.219 cells reactivated with doxycycline ; (iii) RIG-I antibody immunoprecipitated from latent iSLK.219 cells ; and (iv) RIG-I antibody immunoprecipitated from lytic iSLK.219 cells . Purified RNA was transfected into wild-type (WT) and RIG-I knockout  and reactivated iSLK.219 cells . There was significant induction of IFN-β in response to RNA from the RIG-I IP but only a moderate amount of IFN-β induction from Input RNA . Individual peaks on the graph correspond to a sequencing read that starts at that particular position mapped to the KSHV genome, and the x axis corresponds to all possible positions in the KSHV genome. The distribution of sequences in the RIG-I IP; Dox sample displayed a large number of discrete peaks across the KSHV genome, while IgG; Dox IP samples had very few peaks across the KSHV genome , since RIG-I is known to bind small RNAs. Reads were mapped to the KSHV genome, and relative abundances between RIG-I IP and GFP control IP, as well as total RNA from latent iSLK.219 cells (No Dox) and lytic iSLK.219 cells (Dox), were compared. V genome , indicatV genome . A compaThe positions of some KSHV sequences identified to bind RIG-I in at least three independent biological replicates are shown in 10420-10496, Repeat region (LIR1)119059-119204, and ORF2543561-43650 were selected for qRT-PCR assay, because multiple discrete peaks were concentrated in these regions across multiple experiments. The locations of these genes in the KSHV genome are shown in 10420-10496, Repeat region (LIR1)119059-119204, and ORF2543561-43650 were found in the endogenous RIG-IP from lytic iSLK.219 cells, but not from latent iSLK.219 cells or IgG IP samples 117812-117957, and ORF2543265-43354 regions in RIG-IP samples from reactivated BCBL-1 cells, but not in samples from RIG-IP latent cells or IgG IP samples (Since B cells are the primary reservoirs of KSHV and KSHV is tightly associated with primary effusion lymphoma (PEL), we repeated the RIG-I IP and qRT-PCR amplification in biologically relevant, BCBL-1 PEL cells. We used the BCBL1-TRex-RTA cell line since in these cells, similar to iSLK.219 cells, RTA  is expressed under the control of a doxycycline-inducible promoter, and viral reactivation can be 100% induced in the majority of cells without simultaneously affecting other cellular signaling pathways and increasing cellular transcription as is the case with phorbol esters and sodium butyrate. The qRT-PCR procedure is illustrated in  samples .10420-10496, Repeat region (LIR1)119059-119204, and ORF2543561-43650 119059-119204, and ORF2543561-43650], we aligned their sequences , and a circular tree of the alignment was constructed for the purpose of visualization. Tree building used the neighbor-joining algorithm with Jukes-Cantor distance measure and 100 bootstrap replicates. Branches are indicated by lines, and terminal leaves are indicated by blue circles. The scale bar indicates relative size for each of the panels. In this representation, a smaller diameter circle and fewer branches indicate a less diverse sequence population. Note that the tree for RIG-I plus Dox (A) is bigger, i.e., representing a more diverse sequence set, not only by visual comparisons with the other conditions (B to D) but also that the scale bars are different. Hence, the set of sequences that bound RIG-I under Dox-induced conditions is very large and diverse. Download FIG S3, TIF file, 0.7 MB.The circular tree shows the diversity of the sequences obtained from RIG-I IP of doxycycline-treated iSLK.219 cells. To visualize the complexity of the RNA population that was immunoprecipitated in each experiment, all small RNA fragment sequences were Copyright © 2018 Zhang et al.2018Zhang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00823-18.4FIG S4 x axis for each of the four conditions listed is indicated by four different colors. The y axis shows the relative fraction  for each of the four conditions. Sequences bound to RIG-I under Dox-induced conditions dominate composition at >75% AU, whereas sequences bound to IgG under Dox-induced conditions dominate the composition at <25% AU. Download FIG S4, TIF file, 16.1 MB.RIG-I preferentially binds AU-rich sequences in reactivated cells. Another way to visualize the different sequences bound to RIG-I under Dox-induced conditions is to determine the AU content (as a percentage) for each sequence. The percent AU on the Copyright © 2018 Zhang et al.2018Zhang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10420-10496 RNA to activate RIG-I. Different amounts of ORF810420-10496 RNA were transfected into HEK293 cells. Poly(I·C) and 5′ triphosphate double-stranded RNA (5′-ppp-dsRNA), a RIG-I synthetic ligand, were transfected into HEK293 cells as controls. As seen in 10420-10496 RNA induced IFN-β transcription in a dose-dependent manner. Interestingly, ORF810420-10496 RNA also induced high levels of RIG-I mRNA in a dose-dependent manner  and 5′-ppp-dsRNA transfection served as positive controls. As seen in 10420-10496 RNA was dependent on RIG-I. Next, we tested IFN-β induction in WT RIG-I and RIG-I−/− MEF that were transfected with different amounts of ORF810420-10496 RNA or 5′-ppp-dsRNA as a positive control. As seen in 10420-10496 RNA in WT RIG-I cells but not in RIG-I−/− cells. Finally, RNA Pol III and RIG-I were knocked down in HEK293 cells by siRNA transfection followed by transfection of different amounts of ORF810420-10496 RNA. Poly(dA·dT) and 5′-ppp-dsRNA transfection served as positive controls. IFN-β induction was similar in Pol III siRNA- and nontargeting control siRNA-treated cells, but reduced in cells transfected with RIG-I siRNA prior to ORF810420-10496 RNA transfection , short dsRNA, full-length genomes, and poly(U/UC) motifs in the genomes of RNA viruses  activate RIG-I , 42, 43.46–in vitro. We validated three RNA fragments of KSHV  that bind to RIG-I. We further showed that synthesized ORF810420-10496 was sufficient to induce RIG-I-dependent IFN-β signaling. These RNA fragments represent a minimal set of potential RIG-I activators and should not be taken as evidence that these are the only regions in the KSHV genome that give rise to RIG-I binding RNAs or that these represent RNA structures with the highest affinity for RIG-I. As far as RIG-I/RNA interactions are concerned, the amount of potential viral versus cellular RNAs bound to RIG-I may be a determining factor for RIG-I activation. This was borne out for KSHV, as the total number of KSHV RNAs associated with RIG-I increased dramatically during viral reactivation.We have identified multiple regions in the KSHV genome that encode RNA molecules that interact with RIG-I during the course of KSHV reactivation as well as 10420-10496, did not induce any IFN-β when transfected into HEK293 or iSLK cells (data not shown).Alignment results showed that these three fragments do not share any significant sequence similarity, suggesting that RIG-I senses either RNA structures within the KSHV genome or an as yet unidentified stimulatory motif. The KSHV RNA fragments can internally base pair and form RNA secondary structures. Indeed, secondary structural predictions with the RNA Mfold program indicate that the identified stimulatory regions within the KSHV genome are highly structured see , 50–52. 50–We focused on the specific, testable hypothesis that KSHV RNAs bound to RIG-I. Beyond viral transcripts, our analysis also identified cellular RNAs of multiple biotypes that were enriched with either RIG-I or control IgG under conditions of KSHV reactivation. Future studies are needed to understand the mechanism of cellular RNAs bound to RIG-I in the context of KSHV reactivation. It should be noted that in the case of viral infections, where pathogen-derived RNAs take over the total RNA pool during viral replication, there is enough overrepresentation of viral sequences to make individual genome locus assignments on the basis of next-generation sequencing (NGS). The differential is much less pronounced for cellular RNAs.We also investigated the role of RNA Pol III in the KSHV-mediated induction of the RIG-I pathway. Knockdown or inhibition of RNA Pol III had no effect on IFN-β induction by KSHV infection , suggest10420-10496, Repeat region (LIR1)119059-119204, and ORF2543561-43650] were present only in the doxycycline-induced reactivated iSLK.219 cells but not in latent iSLK.219 cells  was added to the BCBL1-TRex-RTA cell medium and incubated for 24 h for reactivation. Wild-type (WT) RIG-I and RIG-I−/− mouse embryo fibroblast (MEF) cell lines were kindly provided by Mark Heise . Cells were maintained at 37°C in 5% carbon dioxide.HEK293 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (Cellgro) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Pen-Str). iSLK.219 cells harboring latent rKSHV.219 (r stands for recombinant) were maintained and reactivated as previously described . BCBL1-T10420-10496 RNA (5′-UCCUUAUACACCAGAGUCUCGUUGCGGGUGAUGAAGUAGUGUUCGCAGGUGUCUUUGCAGGUUUCCACCUGGUUGUUGGU-3′) was synthesized and purified by Eurofins Genomics . RNAs were delivered into the cells with Lipofectamine RNAiMAX  according to the manufacturer’s instructions. Vesicular stomatitis virus (VSV) was provided by Douglas Lyles . For virus infection, cells were washed with phosphate-buffered saline (PBS) and treated with the culture medium (“mock treated”) or infected with VSV in serum-free and antibiotic-free medium. KSHV was prepared and titrated as previously described (High-molecular-weight polyinosinic-polycytidylic acid [poly(I·C)] (1 mg/ml) and the RIG-I synthetic ligand 5′-triphosphate double-stranded RNA (5′-ppp-dsRNA) (100 µg/ml) were purchased from InvivoGen. Poly(dAT-dTA), and poly(I·C) were transfected at a final concentration of 4 µg/ml, and 5′-ppp-dsRNA was transfected at a final concentration of 1 µg/ml. Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF8escribed . The proescribed .2 to 365-nm UV light using an XL-1000 UV cross-linker . Cells were harvested and incubated in NP-40 lysis buffer , and 100 U/ml RNaseOUT (Invitrogen) for 15 min on ice. The lysate was cleared by centrifugation to remove cell debris. The resulting supernatants containing endogenous RIG-I were divided equally and incubated with anti-RIG-I (D14G6) rabbit monoclonal antibody (MAb)  or anti-IgG (DA1E) rabbit MAb  overnight at 4°C on a rotating shaker before adding protein G magnetic beads . Two hours later, beads were separated using a magnetic separation rack and were washed based on published techniques for immunoprecipitation (IP) with KSHV specifically (The iSLK.219 cells were reactivated with doxycycline (Dox), and the unreactivated control iSLK.219 cells were washed with PBS  and exposed at 250 ml/cmifically  and as fE. coli BL21(DE3) RIPL (Agilent). Conditions for expression of RIG-I were taken from a previous published protocol  was cloned into pE-SUMOSTAR (Life Sensors), and the plasmid DNA was transformed into protocol . FLAG-RI2), complete protease inhibitor cocktail (Roche), and 100 U/ml RNaseOUT (Invitrogen) for 2 h at 4°C on a rotating shaker. The reaction mixtures were further incubated with anti-FLAG M2 antibody (Sigma-Aldrich) or control mouse IgG1 (mIgG1) (Sigma-Aldrich) overnight at 4°C on a rotating shaker before adding Gamma Bind Plus Sepharose beads . The rest of the IP was performed the same way as the endogenous RIG-I IP.For IP using purified RIG-I, 10 µg of purified FLAG-tagged RIG-I protein was incubated with RNAs extracted from reactivated or unreactivated iSLK.219 cells in ice-cold buffer C  according to the manufacturer’s protocol. Total RNA from RNA-transfected or virus-infected RIG-I MEF was isolated according to the manufacturer’s protocol . The same amount of RNA from each sample was processed for reverse transcription and quantitative PCR using SuperScript III reverse transcriptase (Invitrogen), SYBR green PCR master mix (Bio-Rad), and ABI 7300, as described previously . Primer http://hannonlab.cshl.edu/fastx_toolkit/) in order to remove adaptor sequences and reads with PHRED scores below 30. Alignment to the KSHV strain JSC-1 BAC16 genome (GenBank accession no. GQ994935.1) or the BCBL-1-BAC36 genome (GenBank accession no. HQ404500) (https://bitbucket.org/ddittmer/rig-i-ip/src/master/. The raw reads are available from SRA archives under BioProject accession no. PRJNA451459 and SRA accession no. SRP145129.Next-generation sequencing and library preparation were performed on the Illumina HiSeq2500 system (rapid-run mode) in the High Throughput Sequencing Facility (HTSF) at UNC-Chapel Hill. RNA was purified and treated with RNase-free DNase to remove any genomic DNA contamination (Zymo Research). The RNA concentration was estimated using Qubit methodology (Invitrogen). Then, the RNA was prepared for Illumina sequencing using a modified small RNA sequencing kit  according to the manufacturer’s protocol (Perkin-Elmer). The ligated cDNA was amplified using 24 PCR cycles. Prepared libraries were pooled for DNA sequencing. DNA concentration of prepared libraries and pool was performed using a broad-range Qubit (Invitrogen) kit, and a profile of DNA size was established by Bioanalyzer QC DNA 1000 chip (Agilent). A single-end 100-cycle sequencing run was used for the analyzed pool on HiSeq2500 rapid mode. The obtained sequences were processed with the FASTX toolkit (Q404500)  was perfThe RNA Mfold program  was usedThe small interfering RNA (siRNA) negative control (D-001810-10-05), siRNAs targeting human RIG-I (Ddx58) (L-012511-00-0005), and the two subunits of RNA polymerase (Pol) III (Polr3A and Polr3D) (L-019741-01-0005 and L-011594-00-005) were purchased from Dharmacon. For knockdown of target genes, siRNAs were transfected into the cells with Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s recommendations. At 48 h or 72 h posttransfection, cells were harvested or infected with viruses for further experiments. For immunoblotting, cells were washed with PBS and lysed in 0.1% NP-40 buffer plus protease inhibitor complete tablets (Roche). Equal amounts of lysate were separated using SDS-PAGE and transferred onto Hybond ECL nitrocellulose membranes . The antibodies used included anti-RIG-I (D14GG) , anti-RNA Pol III (POLR3D) (Sigma), anti-IRF3 (D83B9) , anti-phospho-IRF3 (S396) , anti-IκB-alpha (H-4) , anti-phospho-IκB-alpha (Ser32) (14D4) , horseradish peroxidase (HRP)-labeled antitubulin , and antiactin (Santa Cruz).t test was used to determine statistically significant differences. P values of less than 0.01 or 0.001 were considered statistically significant.An unpaired two-tailed Student’s 10.1128/mBio.00823-18.5FIG S5 FIG S5, TIF file, 6.7 MB.Summary of the mapping results of two additional independent repeats of Copyright © 2018 Zhang et al.2018Zhang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


RNA is an essential player in almost all biological processes, and has an ever-growing number of roles in regulating cellular growth and organization. RNA functions extend far beyond just coding for proteins and RNA has been shown to function in signaling events, chromatin organization and transcriptional regulation. Dissecting how the complex network of RNA-binding proteins (RBPs) and regulatory RNAs interact with their substrates within the cell is a real, but exciting, challenge for the RNA community. Investigating these biological questions has fueled the development of new quantitative technologies to measure how RNA and RBPs interact both locally and on a global scale. In this review, we provide an assessment of available approaches to enable researchers to select the protocol most applicable for their experimental question. To identify RBP–RNA interactions in vivo, a number of experimental strategies are available. One of the most widely used approaches for detecting direct RNA–protein interactions, crosslinking and immunoprecipitation (CLIP), utilizes ultraviolet (UV) light to induce zero-length covalent bonds between RNA and the directly attached protein. Specific antibodies against the RBP of interest are then used to immunoprecipitate the RBP–RNA complexes. The purified RNA fragments can then be used to identify the position of the direct RBP–RNA interaction  have important functions in controlling the fate of RNAs including the modulation of pre-mRNA splicing, RNA modification, translation, stability and localization. RBPs are a diverse group of proteins that interact with RNA using an array of strategies from well-defined RNA-binding domains to disordered regions that recognize RNA sequence and/or secondary structures. This fluidity in RBP structure and target identification has made the eraction . Many ofEvolution of the CLIP protocol has generated a number of variants that have improved the efficiency of multiple steps including RNA fragmentation, RBP purification and cDNA library preparation. The addition of high-throughput sequencing (HITS-CLIP) to the CLIP protocol has enabled a genome-wide view of RBP–RNA interactions and nucleotide resolution Figure . One of These protocols have formed the foundation for additional CLIP strategies that are designed to purify specialized RBP–RNA complexes. These include the miRNA machinery (Ago2 HITS-CLP)  and RNA One of the long-standing issues with CLIP approaches has been the high level of non-specific RNA that is purified. In an attempt to minimize this issue, a number of groups have shifted to using epitopes tags and denaturing wash conditions. The crosslinking and analysis of cDNAs (CRAC) protocol employs a two-step affinity purification of tagged proteins under denaturing conditions . More reAll polymerase chain reaction (PCR)-based library preparations have the potential to contain products generated by PCR and primer errors. To account for this during CLIP library construction, the use of unique molecular identifiers (UMIs) within the reverse transcription primer has become standard. This makes it possible to identify the effects of PCR amplification biases . SeveralThe standardization of the CLIP approach has facilated an ever-growing number of RBP–RNA interactomes to be identified. The availability of these datasets has enabled the ENCODE consortium to integrate RBP–RNA maps to generate predictive regulatory networks of RBP and RNA interactions. Using this resource, new pre-published work has used 233 eCLIP datasets to construct these networks. They then depleted (shRNA) or deleted (CRISPR) RBPs and used RNA Bind-N-seq and CLIP-seq platforms to map RBP-controlled biological processes. This unique approach builds on existing datasets and has expanded the catalog of functional elements encoded in the human genome  uses two affinity tags, PP7 and tobramycin, to capture RNA–RBP interactions under stringent conditions . RBP purThe limitation of aptamer tagging of target RNA is that these techniques require the production of an exogenous RNA or RBP that may be prone to aggregation and/or mis-localization. Emerging nucleic acid hybridization-related strategies that circumvent some of these potential issues are now available including RNA antisense purification sequencers (RAP RNA-seq)  and RNA in trans and binds upstream of the target ssRNA sequence. This allows the sgRNA to recognize the specific RNA target instead of the encoding DNA locus  (MS2-TRAP)  Figure . This ex) Figure . A modif) Figure .in vivo RNA duplexes  was one of the initial tools that enabled the identification of s Figure  44). CL. CLin vis Figure ,46 and ss Figure . To overs Figure   have a remarkable number of biological functions. NcRNAs operate as RNA–protein complexes and also act on other nucleic acids that utilize base pairing interactions to selectively bind. Identifying the proteins and nucleic acids that interact with ncRNAs is likely to further expand our understanding of their function. To detect LNC–RNA–chromatin interactions, multiple methods are available. These include capture hybridization analysis of RNA targets (CHART) , chromatin vivo RNA duplexes recognized by RBPs in a high-throughput manner. It is however limited in its capacity to determine the structure of unbound RNA regions. To globally map RNA structures, several protocols leverage enzymes or chemicals that specifically react with local RNA features and/or structures. Enzyme-based methods include: sequencing of psoralen-cross-linked, ligated and selected hybrids (SPLASH)  can identify ) Figure  52), ps, psin vi) Figure  53), an, anin vi) Figure  54). Th. Thin viAlternative experimental strategies use chemical-based approaches to map RNA structure. These protocols include selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE)  Figure , selectiin vitro binding assay that identifies RBP-binding motifs by quantifying the relative affinity of an RBP for a pre-defined set of RNAs  is an alternative method that enables the identification of protein-assisted inter- and intra-molecular RNA interactions as well as RNA structures  rather than oligo-dT to purify polyadenylated RNA may provide the solution for this issue  Figure . LNAs haMany technologies investigate interacting partners of one RBP at a time. This, however, limits our ability to study the global landscape of RNPs and to determine the combinatorial binding and regulation of RBPs. Recent advanced methods now enable the global mapping of RBP–RNA interactions, including global PAR-CLIP (gPAR-CLIP) and protein occupancy profiling ,73. ThesThe RPBS-Cap capture protein and binding RNA protocol leverages a chemical reagent, Sulfo-SMCC , rather than an antibody to covalently conjugate the amine groups within the RBPs into protein G beads  Figure . This is2 on a nanosecond timescale, and this approach may significantly increase crosslinking efficiencies to levels approaching 50% (One of the benefits of UV crosslinking is that it requires direct contact between an amino acid and a nucleotide for the reaction. There are however some limitations in this process including UV-crosslinking has low efficiency (0.1–5%), a bias in the crosslinking levels of different RBPs at varying wavelengths and difficulty in crosslinking RBPs that interact with double-stranded RNA . One pothing 50% . PrelimiPoor UV-crosslinking efficiency is a problem for capturing RBPs that interact with dsRNA. One potential reagent that may circumvent many of the technical issues of RBP–dsRNA capture is methylene blue . MethyleLow UV cross-linking efficiencies and sequence biases have led to the development of experimental strategies that ‘label’ the RNA rather than purify the RBP and its substrates. The target of RNA-binding proteins identified by editing (TRIBE) approach is based on a fusion protein consisting of the deaminase domain from the ADAR family of RNA-editing enzymes and an RBP that marks target RNAs near the RBP-binding sites by an A-to-I change . The advThis is an exciting time to be investigating the roles of RNA and their complex relationships with RBPs. An evergrowing body of work has found important functions for these interactions in almost every biological system. The protocols and technological advances/possibilities discussed within this review will continue to grow, expanding our capacity to quantitate how different RNA species fold, function and complex with RBPs in different cellular conditions.


In higher plants, roots have important functions, such as the acquisition of water and ions, as well as transportation into the aerial parts of the plant via the xylem vessels. Radial ion transport in the root is strongly regulated in the endodermis, which is characterized by the presence of the Casparian strip (CS) and suberin lamellae. Although tremendous progress has been made with regard to the ion transporters and endodermal cells, little is known about the relationship between the ion transporters in the endodermis and ion homeostasis in aboveground tissues. This review summarizes the current knowledge about the location of the ion transporters (or channels) in the endodermis. We mainly discuss the effects of mutants related to the CS and/or suberin formation on the role of endodermal ion transporters in ion homeostasis. We also wish to emphasize the critical role of endodermal ion transporters in the regulation of radial ion transport in the root. The root system plays an indispensable role during the uptake of mineral ions. In the roots, various cell layers are organized in concentric rings around the vascular tissues, which are, from the outside to the inside, the epidermis, exodermis (if exists), cortex, and endodermis . To reacArabidopsis thaliana have also been uncovered by membrane proteomic approaches , barley (Hordeum vulgare), and maize (Zea mays) are summarized in Substantial progress has been made in the identification of various types of ion transporters in the root, in addition to the understanding of ion absorption and long-distance transport . Many trproaches . Among tThe movement of water and solutes in the root has been classified into three pathways: the apoplastic pathway, symplastic pathway, and coupled transcellular pathways . The apoArabidopsis, endodermis differentiation can be divided into two stages: the CS formation at the first stage, and “patchy” appearance of suberin lamellae at the second stage that gradually enclose the whole cell in an upward direction along the longitudinal axis   and manganese (Mn) contents  and natural-resistance-associated macrophage proteins1 (nramp1), consistent with the effects of Fe and Mn deficiency on suberization  . Howeverdodermis . Hence, contents . Notablycontents . In ricecontents . The spa in rice . Howeverrization . One posnditions . In contnditions . The autns  and MGT6 are two plasma membrane transporters involved in the uptake of Mg in is roots . Althougis roots . Interesis roots . AdditioThe research described above lends strong support to the notion that the endodermis is a checkpoint for ion accumulation in the shoots.Arabidopsis roots, the nitrate transporter2.1 (AtNRT2.1), ammonium transporter1;2 , chlorate resistant 1 (CHL1/AtNRT1.1), and inward-rectifier K+ channel (AKT1) are all highly expressed in the mature region of the root. However, the degree of suberization in the mature region remains unclear. CHL1 is mainly expressed in the epidermal cells, whereas in the older part, CHL1 is expressed in the cortex and endodermis, and its expression domain becomes restricted to the endodermis  technique, which has been used to measure the net fluxes of different ions within the root surface. Another issue yet to be addressed is the contribution of endodermal ion transporters to radial ion transport from the roots to shoots. Many ion transporters, such as CHL1, AMT1.1, and AKT1, can serve as sensors of nitrate, ammonium, and potassium, respectively . AccordiAs the gene expression atlas at a single cell resolution has become available, cell type specific ion transporters have been identified . For insesb1, casp1casp3, myb36, and sgn3 mutants (The formation of the CS and suberin lamellae is closely correlated with the leaf ionome in the  mutants . More im mutants . NeverthZB drafted and revised the manuscript. HC, CG, and JB revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.