Nicotiana benthamiana plants infected by potato spindle tuber viroid (PSTVd). Both hosts develop similar stunting and leaf curling symptoms when infected by PSTVd, thus allowing comparative analyses. About one hundred tomato mRNAs potentially targeted for degradation by vd-sRNAs were initially identified. However, data from biological replicates and comparisons between mock and infected samples reduced the number of bona fide targets—i.e., those identified with high confidence in two infected biological replicates but not in the mock controls—to only eight mRNAs that encode proteins involved in development, transcription or defense. Somewhat surprisingly, results of RT-qPCR assays revealed that the accumulation of only four of these mRNAs was inhibited in the PSTVd-infected tomato. When these analyses were extended to mock inoculated and PSTVd-infected N. benthamiana plants, a completely different set of potential mRNA targets was identified. The failure to identify homologous mRNA(s) targeted by PSTVd-sRNA suggests that different pathways could be involved in the elicitation of similar symptoms in these two species. Moreover, no significant modifications in the accumulation of miRNAs and in the cleavage of their targeted mRNAs were detected in the infected tomato plants with respect to the mock controls. Taken together, these data suggest that stunting and leaf curling symptoms induced by PSTVd are elicited by a complex plant response involving multiple mechanisms, with RNA silencing being only one of the possible components.Viroids are infectious non-coding RNAs that infect plants. During infection, viroid RNAs are targeted by Dicer-like proteins, generating viroid-derived small RNAs (vd-sRNAs) that can guide the sequence specific cleavage of cognate host mRNAs via an RNA silencing mechanism. To assess the involvement of these pathways in pathogenesis associated with nuclear-replicating viroids, high-throughput sequencing of sRNAs and degradome analysis were carried out on tomato and  Pospiviroidae  and Avsunviroidae  that group nuclear and chloroplast replicating viroids, respectively [Viroids are non-protein coding RNAs that infect plants . Accordiectively ,3. Membeectively .Both nuclear and chloroplast replicating viroids are triggers and targets of RNA silencing, a regulatory mechanism of gene expression in most eukaryotes . RNA silEvidence that vd-sRNAs may actually be the initial molecular event of macroscopic symptoms were obtained for sequence variants of peach latent mosaic viroid (PLMVd)  causing The role of vd-sRNAs in pathogenesis of nuclear replicating viroids has been studied in several viroid-host combinations . When stN. benthamiana, two plant species that respond to PSTVd infection by developing similar stunting and leaf curling symptoms.A global view of the mRNAs targeted for degradation by RNA silencing can be achieved by degradome sequencing, a genome-wide approach that allows the identification of most uncapped 5′ phosphate termini of mRNAs in a sample, including those generated by the sequence-specific AGO1-endonucleotidic cleavage guided by miRNA/siRNAs ,25. To eN. benthamiana seedlings inoculated with the PSTVd variant Nb were analyzed 30 days post-inoculation (dpi), when typical symptoms of stunting and leaf curling were clearly observed  genomic RNA  in the three libraries. In agreement with previous studies [N. benthamiana plant infected by PSTVd were mapped on PSTVd genomic and complementary RNAs, an irregular distribution of the reads was found, with very similar profiles observed for all three libraries  PSTVd-sRNAs accumulated at similar levels in both inoculated tomato replicates. The same RNA preparations were then used to generate six libraries of sRNAs and six libraries of degradome RNAs corresponding to the two mock and two infected tomato biological replicates and to the infected and mock  studies ,26,27, Pibraries . These fBetween 38–57 million clean tags, representing the 5′ ends of uncapped, polyadenylated RNAs, were obtained in the degradome libraries prepared from the two mock- and two PSTVd-inoculated tomato replicates. While tags resulting from the cleavage of mRNAs directed by host miRNAs and siRNAs were expected in all samples, the PSTVd-infected replicates should also contain tags corresponding to host mRNAs specifically targeted by PSTVd-sRNAs. In addition, because degradome analysis also identifies random degradation products that are not generated by endonucleolytic cleavage mediated by RNA silencing machinery , false pAs a preliminary test of the quality of the degradome sequencing results, we used the PAREsnip software to identify in the four degradome libraries those mRNAs that were cleaved by the conserved tomato miRNAs. A total of 84 mRNAs were identified as targets of miRNAs belonging to 24 different miRNA families , and mosp < 0.05, Target plots (t-plots) were generated for several selected tomato mRNA targets of miRNAs, including miR-156, miR-167, miR-169, miR-172, mi-R319, miR-396, miR-398, and miR-408 to verify the quality of the sequenced libraries . SimilarThe mRNAs potentially targeted by PSTVd-sRNAs in the viroid-infected tomato plants were identified using the PAREsnip software and a subset of 21-nt PSTVd-sRNAs extracted from the respective sequenced sRNA libraries. Almost 100 potentially targeted mRNAs were identified in each library from infected samples . These mTaking into consideration the fact that vd-sRNAs targeting host mRNAs are expected to be incorporated into AGO1 and that the nucleotide at the 5′ end of the vd-sRNA contributes to its loading into AGO1 , we examThese data indicate that the chance of identifying false targets of PSTVd-sRNAs in the degradome libraries from infected samples is lower when the cleavage sites fall within the category 0. In addition, since the two biological replicates showed identical symptoms, we reasoned that key mRNA(s) targeted by vd-sRNAs and involved in pathogenesis should be found in both replicates. Consequently, category 0 mRNAs that were present in both replicates after the exclusion of potential false positives were further considered as bona fide targets of PSTVd-sRNAs. These correspond to eight tomato mRNAs targeted by eight different PSTVd-sRNAs derived mainly from the plus polarity strand, with only two of them being of negative polarity . IrrespeLINC1 encodes a coiled-coil nuclear protein that acts as a positive regulator of pathogen-associated molecular pattern-triggered immunity (PTI) responses, thereby regulating jasmonic acid signaling and gibberellin biosynthesis [ORC6 is one of the components required for assembly of the pre-replication complex necessary to initiate DNA replication in Arabidopsis [SCN1, CDKF1, PBS-1, and PPI-1) correspond to genes encoding proteins that are not localized in the nucleus. SCN1 encodes a RhoGTPase GDP dissociation inhibitor (RhoGDI), which regulates root hair growth in Arabidopsis [CDKF-1 encodes a cyclin-dependent serine/threonine protein kinase that is involved in activating phosphorylation of cyclin dependent kinases in Arabidopsis [Arabidopsis thaliana PPI1 homolog protein is involved in proton transport through the plasma membrane [The bona fide targeted mRNAs encode eight proteins annotated as CWF19 , Little nuclei 1 , subunit of origin recognition complex protein 6 , NAC domain containing protein 38 , Supercentipede 1 , cyclin-dependent serine/threonine kinase F-1 , putative serine-threonine kinase  and proton pump interactor 1  . Four ofynthesis . This prynthesis . ORC6 isbidopsis , and it bidopsis . CDKF-1 bidopsis . Interesbidopsis . In Arabbidopsis . Arabidomembrane .Analyses of cleavages throughout the sequence of the eight bona fide target mRNAs  showed that, in the PSTVd-infected samples, the frequency of 5′ ends consistent with the specific PSTVd-sRNA-mediated cleavage was always higher than the respective degradation background, with similar results obtained for both biological replicates , Table 1The effect of the mRNA silencing-mediated cleavage on the steady state levels of the eight bona fide targets of PSTVd-sRNAs was tested by RT-qPCR assays on three biological replicates from three independent inoculation experiments. To amplify cDNAs derived exclusively from the uncleaved mRNAs, specific PCR primers were designed to bind upstream and downstream from the cleavage site of each target. Only the mRNAs encoding CWF19, LINC1, ORC6, and SCN1 showed a significantly lower level of accumulation in the PSTVd-infected than in the mock-inoculated tomato plants , supportN. benthamiana plants generated 23 and 28 million of clean reads, respectively. PAREsnip analysis identified a total of 79 mRNAs cleaved by conserved miRNAs belonging to 15 different families. Approximately 70% of the cleavage sites in both the mock-inoculated and PSTVd-infected samples were classified as category 0 or 1 according to the PAREsnip categorization  of the expression of genes involved in defense, hormone signaling, photosynthesis, RNA processing and binding, protein metabolism and modification, RNA silencing and plant innate immune responses ,58,59,60N. benthamiana plants, the direct involvement of these mRNAs in PSTVd-induced stunting and leaf curling symptoms in these hosts appears unlikely. Additional degradome-based studies performed at several infection stages could further clarify the role of RNA silencing in triggering PSTVd pathogenesis.In summary, applying degradome analyses, we found several host mRNAs targeted by PSTVd-sRNAs for a specific cleavage mediated by RNA silencing machinery. However, based on comparative analysis in tomato and Solanum lycopersicum L.) cv. ‘Rutgers’ at the cotyledon/first true leaf stage were mechanically inoculated with nucleic acid extracts from tomato plants infected with PSTVd  in 0.2 M boric acid-NaOH, pH 9 or with the inoculation buffer (mock-inoculated plants). Nicotiana benthamiana seedlings were agroinoculated with Agrobacterium tumefaciens C58 carrying either an empty plant binary expression plasmid (pBIN19Ø) or one containing a head-to-tail dimeric insert of PSTVd-Nb under the control of the 35S promoter of cauliflower mosaic virus [Seedlings of tomato  according to the manufacturer’s instructions. Total RNA preparations were analyzed by polyacrylamide gel electrophoresis under denaturing conditions (8M Urea and 1× TBE buffer) in 5% gels, for detecting genomic PSTVd RNAs, and in 17% gels, for detecting vd-sRNAs. After ethidium bromide staining, the RNAs were electrotransferred to Hybond-N+ membranes  and were hybridized with digoxigenin-labeled full-length riboprobes specific for detecting PSTVd of plus polarity as described previously . HybridicDNA libraries of sRNAs were generated as reported previously  and subjMmeI and purified by PAGE before the Illumina 3′ TruSeq adapter was ligated to the MmeI overhang. After PCR amplification using a modified Gex1 adapter, the library products were sequenced (pair-end 2 × 25 bp) on Illumina NextSeq 500 system  by Vertis Biotechnologie AG . Raw sequences of the sRNAs and degradome libraries have been deposited in the European nucleotide archive (ENA) under the project accession number PRJEB42233.Degradome libraries  were constructed according with the protocol described by German et al. . BrieflyN. benthamiana conserved miRNAs were identified by mapping (Bowtie 1.2.2) against the plant miRNA in miRbase (Release 22.1) and the published tomato and N. benthamiana miRNAs.Raw sRNAs reads generated by HTS were filtered for quality and, after removing the adapters, they were sorted by sequence length. Reads of size between 18 to 26 nt were mapped by Bowtie 1.2.2  on the Pp-value of 0.05, for the identification of PSTVd-sRNA and miRNA targets. The transcripts data set used for mapping degradome reads was the release ITAGv2.3 of Solanum lycopersicum, and the v1.0.1 N. benthamiana GENOME predicted cDNA . The sRNA data set used in the PAREsnip tool to identify potential cleaved host targets of PSTVd-sRNAs in the degradome data from infected samples was constituted by the 21-nt sRNAs mapped in PSTVd obtained in the corresponding sRNA library (see above). Degradome data from mock controls was analyzed using a sRNA data set generated in silico to contain all possible sRNA derived from PSTVd-Nb. The resulting targets of PSTVd-sRNA in mock samples were considered as false targets.After quality control, adapter trimming, and filtering for 100% overlapping paired reads, high quality degradome reads were analyzed, using PAREsnip software tool  with a cThe putative cleavage sites identified by PAREsnip analysis in each degradome library were assigned to one of five categories (categories 0–4) according to degradome fragment abundance at the cleaved position as described in . This clAliquots (2 μg) of total RNA treated with turbo-DNase  were mixed with 0.25 μg of the RNA adapter (5′-CGACUGGAGCACGAGGACACUGACAUGGACUGAAGGAGUAGAAA-3′) and incubated at 95 °C for 2 min and snap-cooled on ice. Then, the mixture was incubated at 37 °C for one hour with 5U of T4 RNA ligase , 1× buffer T4 RNA ligase , 1 mM ATP and 10 U RNase inhibitor. First, cDNA synthesis of adapter-ligated RNAs was carried out with Superscript II  following the supplier instructions and using a specific primer complementary to the transcript of interest . Then, tΔΔCT method using the ubiquitin conjugating enzyme (UBC) as an internal reference gene for normalization of expression levels. Statistical significance was assessed by a t-test analysis, and the results were considered significant at p < 0.05. Error bars represent the standard error of the mean.Total RNA extracted as described above was treated with Turbo DNase free  and 1 μg of the treated RNA was used for the first strand cDNA synthesis with Superscript II ) and random hexamers according to the manufacturer’s protocol. The cDNA reaction was diluted 5-fold with water and used as template for qPCR amplification. The PCR reaction was carried out in a total volume of 20 μL, containing: 4 μL of 5× PyroTaqEvaGreen qPCR Mix Plus , 0.5 μL of 10 μM of each primer, and 2 μL of the diluted cDNA reaction. Three biological replicates from three independent inoculation experiments were tested, and three technical replicates were performed for each biological replicate. Negative control reactions without cDNA template for each primer pair were included in each amplification experiment. The specificity of amplification was confirmed by melting curve analysis. Gene-specific primer pairs used for qPCR amplification are listed in


TYR, SOX10, CALD1, CD34, DES, and LAMA1, was performed in all cases. TYR, CD34, and CALD1 were the most discriminatory genes in differentiating between OMM and STS, all having 100% specificity and 65, 95, and 60% sensitivity, respectively. While all 20 OMMs were immunohistochemically labeled for SOX-10, two STS were also labeled (100% sensitivity and 90% specificity). MDX IHC labeled all 20 OMMs and no STS. Surprisingly, none of the 20 OSCTs expressed TYR RNA above the cutoff, and 14/20 OSCTs expressed CALD1 or CD34 RNA above the cutoff, thereby confirming them as STS. Four OSCT were suspect STS, and no OSCTs were confirmed as OMMs based on IHC and RNA expression patterns. In conclusion, the RNA levels of TYR, CD34, and CALD1 should be evaluated in suspected amelanotic OMMs that are negative for MDX to accurately differentiate between OMM and STS.Canine oral malignant melanomas (OMMs) exhibit a variety of morphologic phenotypes, including a spindloid variant. The microscopic diagnosis of spindloid OMMs is based on junctional activity and/or the presence of melanin pigment. In the absence of these features, spindloid OMMs are difficult to differentiate from soft tissue sarcomas (STS). An antibody cocktail (MDX) that includes Melan-A, PNL2, and tyrosinase-related proteins 1 and 2 (TRP-1 and TRP-2) is the current gold standard for identifying amelanotic OMMs by immunohistochemistry (IHC). However, MDX is less sensitive for diagnosing spindloid amelanotic OMMs. This raises concern for biopsy specimens that lack overlying epithelium, making it potentially difficult to differentiate OMM from STS by IHC. The goal of this study was to identify additional markers to help differentiate between STS and OMMs that lack pigment and junctional activity. SOX-10 has recently been proposed as a sensitive marker for melanocytes in humans but has not been validated in dogs. Similarly, RNA expression for various genes has been analyzed in humans, but not in the context of diagnosing canine melanocytic neoplasms. For this retrospective study, formalin-fixed, paraffin-embedded tissues from 20 OMMs, 20 STS, and 20 oral spindle cell tumors (OSCTs) that lacked junctional activity and pigmentation were selected. IHC for MDX, SOX-10, and laminin, in parallel with RT-qPCR of  Oral malignant melanomas (OMMs) are the most common malignant oral neoplasm in dogs –3. They TYR mRNA has been detected in 100% of investigated human melanoma cell lines in some studies . The first group (OMM group) included 20 dogs with OMMs  that werTYR, SOX10, CALD1, CD34, DES, and LAMA1 are listed in Regions of high tumor cellularity (at least 80%), away from the overlying epithelium, from inflammation, and from necrosis, were selected  for RNA extraction from each case. Total RNA was isolated from formalin-fixed, paraffin-embedded tissue using RecoverAll™ Total Nucleic Acid Isolation Kit  according to the protocol of the manufacturer. Following deparaffinization with CitroSolv, tumor tissue was incubated in proteinase K containing lysis buffer. RNA was quantified using Qubit . Six hundred nanograms of total RNA was treated with TURBO DNA-free™  to remove contaminating DNA. First-strand cDNA synthesis was performed using SuperScript III Reverse Transcriptase  with random primers . The cDNA was then column-purified by using QIAquick PCR Purification Kit  and eluted with distilled nuclease-free water at 5 ng/μl. For the determination of specific gene expression, each primer was designed with Primer3 software , 14. TheRoutine immunohistochemical labeling was performed as previously described  using MDN), 11–50% (assigned a score of 1), 51–80% (assigned a score of 2), or 81–100% (assigned a score of 3). For MDX, neoplasms were also considered positive if intraepithelial nests, consisting of at least five clustered neoplastic cells, labeled positively or if there were aggregates of at least 20 neoplastic cells that labeled positively anywhere within the neoplasm as previously described  curve. Sensitivity was determined based on the number of cases (OMM or STS) above the established cutoff value; specificity was determined based on the number of (OMM or STS) cases below the established cutoff value. A value of P ≤ 0.05 was considered statistically significant. Analyses were performed using Prism software, v.6.01 (GraphPad Software).Data are presented as means ± SEM. The differential expression of OMM relative to STS genes was analyzed by Student's Based on the selection criteria, MDX IHC correctly labeled all 20 OMMs and did not label any of the 20 STS and none of the 20 OSCT ; Table 1TYR, SOX10, LAMA1, DES, CD34, and CALD1, the cutoff values for relative RNA levels were determined using ROC curves , SOX10 , CD34 , and CALD1 . No significant difference in RNA expression levels was identified between OMMs and STS for LAMA1 (241.8 ± 135.1 vs. 249.3 ± 149.4) and DES (0.1405 ± 0.1327 vs. 0.1040 ± 0.03911). Based on these data, the specificity and the sensitivity of TYR RNA levels for detecting OMM were 100 and 65%, respectively  expressed DES RNA and was positive for laminin and negative for S100, desmin, and MDX by IHC. SOX10 RNA was also expressed above the cutoff level in two OSCTs that had either CD34 or CALD1 RNA levels above the cutoffs (#1 and #20). In total, five OSCT cases expressed DES RNA and seven OSCT expressed LAMA1 RNA above the cutoff. A final diagnosis for each OSCT was made based on the combined RNA expression and immunohistochemical labeling results. Fourteen OSCTs were diagnosed as STS based on RNA levels above the cutoffs for either CD34 or CALD1, no expression of TYR RNA, and lack of labeling for MDX by IHC. Two of these 14 OSCTs labeled positively by IHC for desmin and five for S100, suggesting a differentiation toward pericytes or smooth muscle cells and nerve sheath cells, respectively. Two of the remaining six OSCTs were diagnosed as undifferentiated malignant neoplasms, as they lacked expression and labeling patterns that supported either an OMM or STS. The remaining four OSCTs were diagnosed as suspect STS. While three of those OSCTs had RNA expression levels of SOX10 above the cutoff and immunohistochemically labeled for SOX-10, one OSCT also had RNA expression levels for DES above the cutoff, and all four OSCTs were immunohistochemically positive for laminin and negative for MDX and did not express TYR RNA, making a diagnosis of STS more likely.Following RT-qPCR for C curves . Thirteeectively . None ofe cutoff . The twoe to STS  for TYR ectively . The spes of STS . Of the TYR, CALD1, and CD34 RNA accurately differentiated STSs from OMMs. TYR encodes one of the most studied melanin enzymes that plays a crucial role in the early steps of melanin synthesis and has been detected in 100% of investigated human melanomas in some studies  or if such diverse expression patterns are a reflection of the reactivation of genes that encode different lines of mesenchymal differentiation by malignant mesenchymal cells , and half of these cases were also positive for S100 by IHC, supporting nerve sheath cell differentiation. All five S100 IHC-positive OSCTs also had RNA expression levels for al cells .TYR RNA has been detected in human melanoma cell lines , while seven cases had very low to imperceptible levels of relative RNA expression. An association between TYR RNA expression levels in canine OMM and responsiveness to the ONCEPT® vaccine has not been investigated. In a follow-up study, we intend to investigate a potential correlation between TYR RNA expression levels by OMMs and their responsiveness to the ONCEPT® canine melanoma vaccine.Tyrosinase is a copper-containing membrane glycoprotein that represents a key enzyme in the initiation of melanogenesis . The expll lines . As expe TYR RNA . The lac(TYR RNA . All OMMn humans . The dowanocytes , vaccineanocytes . Similaranocytes . InteresTYR, CD34, and CALD1 discriminate between canine oral melanomas and soft tissue sarcomas. Moreover, RT-qPCR for the RNA expression of TYR, CD34, and CALD may be a useful diagnostic tool following a negative MDX result in suspected spindloid amelanotic OMM cases that lack an overlying epithelium.In conclusion, this study determined that relative RNA expression levels of The original contributions presented in the study are included in the article/MT generated, analyzed, interpreted data, and drafted the manuscript. TT created the study concept and design, generated, analyzed, interpreted data, and revised the manuscript. RS and MK created the study concept and design, interpreted data, and revised the manuscript. EN created the study concept and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The tools of synthetic biology have enormous potential to help us uncover the fundamental mechanisms controlling development and metabolism in plants. However, their effective utilization typically requires transgenesis, which is plagued by long timescales and high costs. In this review we explore how transgenesis can be minimized by delivering foreign genetic material to plants with systemically mobile and persistent vectors based on RNA viruses. We examine the progress that has been made thus far and highlight the hurdles that need to be overcome and some potential strategies to do so. We conclude with a discussion of biocontainment mechanisms to ensure these vectors can be used safely as well as how these vectors might expand the accessibility of plant synthetic biology techniques. RNA vectors stand poised to revolutionize plant synthetic biology by making genetic manipulation of plants cheaper and easier to deploy, as well as by accelerating experimental timescales from years to weeks. Firstly, by allowing molecular interventions of previously impossible precision and tunability using synthetic signaling systems . This gRNA associates with the protein to guide it to specific region of the genome. While the protein component is too large to load onto most viral vectors, the gRNA is not. Thus, it is possible to create transgenic plant lines that constitutively express large RNA guided enzymes and deliver the gRNAs that direct these enzymes with viral vectors.Nicotiana benthamiana.This strategy has been used by several groups to achieve high efficiency somatic genome editing . Plants In addition to genome editing, site specific recombination via recombinases is another strategy that holds great promise to enable dissection of the links between genotype and phenotype. These recombinase enzymes, such as the commonly used Cre protein, are capable of recognizing a specific sequence of DNA and catalyzing a directional DNA exchange reaction. It has been demonstrated that recombinases can be delivered via RNA viruses, and that the predictable recombination events can be inherited by the progeny of the infected plants . As virain planta via RNA scaffolds (A. thaliana, N. benthamiana, and the crop plant Solanum lycopersicum.The use of RNA viruses has also been successfully applied to somatic transcriptional reprogramming. Plant lines that stably expressed a Cas9-based transcription factor were created, and viral vectors were used to deliver gRNAs to target the transcription factor. This strategy was used to tune the expression of metabolic and developmental master regulator genes and study associated phenotypes in weeks as compared to the months to years it would take with traditional transgenesis. This strategy was also applied to enable targeted methylation in somatic tissue as well as germline tissue to create heritable phenotypes . One limcaffolds  that areThe above approaches, however, are still limited by the inability to load large cargos onto viral vectors, necessitating genomic integration of proteins like Cas9 . Some grFT gene, which are signals that travel up the shoot to trigger flowering, and the BEL5 gene, an RNA that travels to the root and trigger tuber formation in potatoes (Single stranded RNA viruses move through plants in two major ways: in a local fashion through the cell-to-cell connections, the plasmodesmata , and syspotatoes . In addipotatoes . The prepotatoes .While viruses can move systemically through the plant from the initial point of delivery, this movement becomes increasingly patchy and non-uniform at progressively distal points . The virAn important nuance of engineering viral mobility is the impact of virus exclusion, which is when infection of a cell by a particular plant virus prevents the infection by other viruses . CertainAnother exciting avenue of exploration is the potential for engineering viral movement for tissue specific delivery. It has been observed that certain viruses specifically localize to certain tissues . This ma–5 to 10–6 mutations per site per generation (The replication of RNA viral vectors is an important determinant of their capacity to enable stable gene expression. RNA viruses are notorious for having relatively large error rates during replication in the order of 10neration , which wneration . This isneration . Howeverneration . The higReplication speed is also not necessarily in line with an effective gene delivery platform. Viral replication speed has evolved to optimize the propagation of the virus within the host and between hosts . HoweverAn important aspect in designing vectors for effective gene delivery that is often overlooked is engineering the delivered cargo sequence itself. For example, cargos might contain sequence motifs like cryptic splice sites, which can severely inhibit delivery by disrupting the viral genome through splicing. One avenue that has been successfully demonstrated to overcome this challenge is the intentional inclusion of several strong intronic sequences . This apA. thaliana led to higher viral titers and more persistent infections with tobamoviruses and tobraviruses (One of the central challenges to the initial establishment of an infection and maintenance of a consistent viral titer over time is RNA silencing by the host plant. This phenomenon is triggered by the recognition of double stranded viral RNA, which occurs either during replication or before encapsidation due to the high degree of secondary structure in the viral genome . RNA viraviruses . While tThe use of agents capable of self-replication, such as RNA viruses, necessitates a careful consideration of effective biocontainment mechanisms, as well as strategies for virus clearing in case of escape. In laboratory settings, these vectors are used in isolated chambers free of insects or other vectors to prevent potential environmental escapes. For certain viruses, additional layers of containment have been incorporated, such as mutations in the coat proteins to prevent transmission . HoweverHowever, thanks to the low fidelity and rapid replication of RNA viral vectors and the strong selective pressure to mutate the control mechanism, there is a chance of escape from these strategies over time. Thus, it is important to engineer these kinds of control mechanisms to be robust to mutation, for example by layering multiple independent containment mechanisms so that all would have to fail simultaneously to allow escape. Beyond incorporating containment mechanisms, another avenue to engineer effective biocontainment is to fundamentally reengineer the virus and relocate the viral replication machinery into the plant genome . Viral vA. thaliana to resource rich settings. By limiting who is doing this kind of science, we also limit the kinds of questions these tools are used to ask, leaving science poorer for it. RNA vectors, once sufficiently developed, have the potential to dramatically lower both the material cost and person hours required to implement synthetic biology-based strategies to study plants. When coupled with lower cost reporters (Science is founded on the principle that acquired knowledge should be available for all. In practice, however, science is filled with the same inequities that exist across the world. This is, in part, driven by the kinds of enabling tools we develop to move science forward. The focus on expensive and technically challenging methods to deploy plant synthetic biology, such as plant transformation, have restricted its use outside of easily transformed model plants like eporters , they haUsing RNA viral vectors to deploy plant synthetic biology tools can collapse experimental timelines, especially in crop plants. RNA viral vectors can also be easily and cheaply scaled, allowing the interrogation of biology at a scale that was previously unfeasible. For example, they could be used to rapidly tune the expression of every gene in a metabolic pathway and identify expression ratios that optimize metabolic flux to desirable products like vitamins or pharmaceuticals. This approach could also be applied to rapidly interrogate how changes in the expression level or methylation state of developmental regulators translate to whole plant phenotypes. The broad host range of viral vectors means they could expand the use of plant synthetic biology beyond model species. All of these potential benefits highlight the need to invest in the development of these technologies, both through the exploration of some of the engineering strategies we suggest here, as well as through deeper study of plant virome. Combining an expanded understanding of the mechanistic basis of plant virology with the engineering principles of synthetic biology will enable the construction of gene delivery tools that might one day obviate the need for transgenesis. While this next generation of tools is being developed, existing RNA viral vectors will enable the rapid generation of hypotheses about the mechanistic underpinnings of biological phenomena, which can be further interrogated with stable transgenic lines. In this way RNA vectors will accelerate the pace of biological discovery in plants and, once demonstrated to be safe and containable, allow the rapid translation of these insights into crops in the field.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.AK wrote the manuscript and prepared the figures. DV provided the editorial input. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human hepatitis delta virus (HDV) is a small defective RNA satellite virus that requires hepatitis B virus (HBV) envelope proteins to form its own virions. The HDV genome possesses a single coding open reading frame (ORF), located on a replicative intermediate, the antigenome, encoding the small (s) and the large (L) isoforms of the delta antigen (s-HDAg and L-HDAg). The latter is produced following an editing process, changing the amber/stop codon on the s-HDAg-ORF into a tryptophan codon, allowing L-HDAg synthesis by the addition of 19 (or 20) C-terminal amino acids. The two delta proteins play different roles in the viral cell cycle: s-HDAg activates genome replication, while L-HDAg blocks replication and favors virion morphogenesis and propagation. L-HDAg has also been involved in HDV pathogenicity. Understanding the kinetics of viral editing rates in vivo is key to unravel the biology of the virus and understand its spread and natural history. We developed and validated a new assay based on next-generation sequencing and aimed at quantifying HDV RNA editing in plasma. We analyzed plasma samples from 219 patients infected with different HDV genotypes and showed that HDV editing capacity strongly depends on the genotype of the strain. Deltavirus genus. New HDV RNA-like sequences have been recently identified in the animal kingdom [Hepatitis delta virus (HDV) is a small defective RNA virus that infects humans already chronically carrying hepatitis B virus (HBV). Indeed, HDV requires the HBV envelope proteins expressing the HBV surface antigen (HBsAg) for the morphogenesis of its viral particles . HDV has kingdom ,3,4,5. H kingdom ,7. HDV i kingdom .The HDV genus is characterized by a very high genetic diversity. We recently proposed, based on large-scale sequencing of HDV strains for many regions of the world, individualizing eight HDV genotypes, numbered HDV-1 to HDV-8, that differ by ≤20% over the sequence of their full-length genome, with a strong robustness of branch topologies in Bayesian-based tree reconstruction ,10. HDV The HDV virion is approximately 36 nm in diameter. The viral particle is composed of a double lipid layer in which the HBV envelope glycoproteins are anchored surrounding the HDV ribonucleoprotein (RNP). This RNP is composed of the circular single-stranded RNA of negative polarity containing ctively) . The HDVctively) ,13.HDV RNA replication is carried out independently of HBV by the host DNA-dependent RNA polymerase-2 (RNA-Pol-II) . It has As replication proceeds, a fraction of the full-length antigenome RNA undergoes editing by a specific host cellular enzyme, the adenosine deaminase acting on double stranded RNA (ADAR), essentially its short isoform ADAR-1 ,19. As dIn earlier studies ,32 /Non-Edited (NE) RNA + Edited (E) RNA ratio) were measured. The HDV RNA secondary structures of full-length antigenomes from different genotypes were predicted in silico, with special focus on the editing substrate region.Implementation and validation of a new tool to quantify HDV RNA editing rates.® Illumina sequencer  to quantify the proportion of edited versus non-edited genome sequences in the plasma of HDV-infected patients. To analyze the large amount of sequence data generated, we took advantage of the patent-protected “in-house” software recently developed for HIV sequence analyses, including the Pyromute, Pyrotrop and Pyrolink modules, which were further developed to create the PyroEdit software.To implement a new assay to measure HDV RNA editing rates, we used an NGS ‘method using the MiSeq3, 5 × 105 and 105 sequences; (II) consensus sequences from all HDV genotypes (HDV-1 to -8); (III) the presence of the W codon on all sequences of each genotype; (IV) sequence variability up to 30%; (V) Phred Quality Score ≥30, and (VI) a lower limit of detection of the W codon of 1%.Initial validation of the software was performed using computer-generated edited and non-edited sequences from reference sequences. Briefly, we created a program that generated several synthetic fastq files with the following characteristics: (I) fastq files containing 10Then, we generated clones (see below) expressing edited and non-edited genome sequences that were mixed at different ratios, respectively: 0%/100%, 5%/95%, 10%/90%, 25%/75%, 50%/50%, 75%/25%, 90%/10%, 95%/5%, 100%/0%. Each different mixture was analyzed 8 times in the same experiment.® flow cell and 3 times on 3 different MiSeq® flow cells .Finally, we analyzed the plasma samples from 219 HDV-infected patients described below. Repeatability and reproducibility experiments were conducted from 8 samples, including 3 low (10%), 3 median (32%) and 2 high (52%) RNA editing rates, that were tested 10 times using the same MiSeqWe generated non-edited and edited clones from a well-characterized HDV-1 strain (dFr663d unpublished), which exhibited a double population by Sanger sequencing, ATC (for non-edited) and ACC (for edited) at the 1012 position of the genome corresponding to amber (UAG)/W (UGG) codon for the antigenome. E. Coli bacteria. The final non-edited and edited purified clones were collected, further amplified using the KAPA high fidelity PCR kit and sequenced by the Sanger method using the ABI Prism Big Dye Terminator Cycle Sequencing Kit v3.1 . DNA plasmid purification was carried out with the GeneJET plasmid miniprep kit . DNA was normalized using a Qubit Fluorometer , and new PCRs were performed to characterize each non-edited and edited clones by means of Sanger sequencing. Quantification of each clone by NGS was performed through new PCR amplifications using a pair of primers containing the 5′ NGS-adaptor sequences P5 and P7 . PCR products were further purified using the NXp Station . DNA quantity and quality were assessed using Qubit and BioAnalyser .Briefly, amplicons of the so-called R0 genome region, spanning nt 920 to 1289 and covering the editing region [Clinical samples from 219 untreated patients infected with different HDV genotypes were selected from the collection of the French HDV National Reference Laboratory according to the HDV genotype and a HDV viral load > 1000 UI/mL. HDV RNA levels ranged between 2.7 and 10.2 Log IU/mL, with a median of 6.3 ± 1.62 Log IU/mL, as quantified by the commercial Eurobioplex HDV kit  . HBV DNA® System  according to the manufacturer’s protocol.For the quantification of HDV RNA editing rate, RNAs were extracted from plasma samples with the m2000sp system  and reverse-transcribed with the SuperScript III First-Strand Synthesis System  according to the manufacturer’s instructions. The cDNAs were amplified as previously described ,37 usingNGS data analysis was performed with our new dedicated tool PyroEdit. After a specific quality filter step, only complete sequences (up to 151 pb) with a Phred Quality Scores >30 were recorded and aligned  with a genotype-specific consensus reference sequence. The percentage of sequences bearing the W at the amber/W codon was calculated for each patient sample and recorded in the final report. http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi accessed on 1 November 2019) and UNAfold WebServer (http://www.unafold.org/mfold/applications/rna-folding-form.php accessed on 1 June 2021). We then compared the patterns of the putative minimum editing substrate for the ADAR-1 enzyme according to the genotype and the editing rate.To analyze secondary HDV RNA structures, especially in the region surrounding the amber/W editing site, we determined the full-length genome sequence of 32 out of the 219 strains from the initial cohort , as previously described [p values of <0.05 denoted statistical significance. Figures were carried out under ggplot2.Statistical analyses were carried out using the R software . The non-parametric Kruskal–Wallis test was used to compare population distributions. The Wilcoxon test was used to analyze paired samples of non-categorical data, while Pearson’s test was used for statistical correlations. We developed a new tool to measure the editing capacity of HDV strains in samples from infected patients. The workflow is based on NGS and quantifies editing of the amber/stop codon at positions 1010–1013 of the antigenomic RNA.Previously created in-house software was adapted to develop the PyroEdit application, that was subsequently validated to be used regardless of the number of sequences to analyze, threshold values, virus variability and HDV genotype. Firstly, the validation process was conducted by using computer-generated fastq files, as described in the “Methods” section. The mean difference between the expected values of edited genome rates (input) and the values calculated by PyroEdit (output) was less than 0.1%, whatever the HDV genotype is (data not shown). Secondly, eight mixtures containing clones expressing either edited or non-edited genomes in various proportions were prepared . A totaln = 115), HDV-5 (n = 55), HDV-6 (n = 12), HDV-7 (n = 17) and HDV-8 (n = 20). The editing rates ranged from <5% to 69% (median: 32%). In three samples, the editing rate was repeatedly comprised between the LLOD (1%) and the LLOQ (5%). Higher and lower values were controlled twice, and an identical result was found. The median editing rate values were different across the different HDV genotypes: 31%, 41%, 27%, 39% and 26% for HDV-1, HDV-5, HDV-6, HDV-7 and HDV-8, respectively. The difference was significant between HDV-5 and HDV-7 compared to that between HDV-1, -6 and -8, as assessed by multivariate analyses . No significant difference was observed between non-African and African-HDV-1 strains . Although there was no correlation between the initial HDV viral loads and the editing rate values (p < 0.01).Editing rates were quantified in plasma samples from 219 patients infected with different HDV genotypes, including HDV-1  on the viral RNA by histone mimicry, favors DNA-dependent RNA Pol 2 hijacking, and allows the Pol2 RNA elongation process to occur ,40. At l® Illumina device, together with a new in-house software, PyroEdit, which allowed us to measure the viral editing rate in plasma samples from treatment-naïve patients infected with different HDV genotypes. We also analyzed the predicted RNA structural features of the editing region of several strains from this cohort according to editing rate and HDV genotype.In this study, we report the development and validation of a new NGS-based assay using the MiSeqOur results show that our assay was able to: (I) be robust in spite of the high variability of the HDV genome, characterized by a nucleotide dissimilarity rate ranging from 20% to up to 35% over the entire genome sequence between the different HDV genotypes; (II) individualize the sequence of interest of 251 nt in length with a Phred Quality Score > 20% and with 99% accuracy; (III) correctly align each consensus sequence; (IV) check which nucleotide was present at the amber/W codon position and characterize the codon; and (V) calculate the editing rate and provide a final report. With this quantitative method, the editing rate values in clinical samples ranged from 7% to 69% in patients infected with HDV genotypes HDV-1, -5, -6, -7 and -8. Previous in vitro (cell culture) and in vivo (plasma) studies using HDV-1 strains found editing rates around 40% ,35,43. Rp < 0.001). In addition, HDV-1 strains exhibited significantly higher editing capacities than HDV-6 and HDV-8 (p < 0.05). Interestingly, in the HDV-1 group, no difference in editing efficiency was seen between African and non-African strains. Because HDV-1 is the most frequent, ubiquitously distributed genotype worldwide, this suggests that the in vivo HDV-1 editing rate, around 30%, could be optimal for a most efficient spreading fitness, with an optimal early-to-late phase of HDAg production ratio.Another important result of our study is that the editing rate values differed significantly between different HDV genotypes. Indeed, the African HDV-5 and HDV-7 strains showed significant higher in vivo rates of editing than HDV-1, HDV-6 and HDV-8  were more at risk of developing cirrhosis than those infected with other African genotypes, such as HDV-1 [L-HDAg synthesis and its accumulation in several cell compartments has been shown to interfere with multiple cellular signaling pathways and alter the pathogenesis of the infection ,28,29,30It has been reported that HDV proteins (s- and L-HDAg) are involved in immune-mediated liver damage . Data frIt has been demonstrated in cell culture experiments that L-HDAg is involved in the inhibition of HBV replication observed in HBV-HDV co- or super-infected individuals . ActivatAnother known consequence of HDV RNA editing is the existence of HDV particles containing edited genomes. Such particles are unable to be at the origin of full viral replication cycles. In the event of a co-infection with virions containing genomes capable of coding for both proteins, L-HDAg would likely inhibit the replication of otherwise competent genomes. With our assay, we found that up to 69% of viral particles contained such edited genomes. These edited particles could behave as defective interfering particles (DIPs), as described for HBV and several other viruses . DIPs arThe s-HDAg/L-HDAg ratio and its regulation govern the switch from genome replication to packaging, which seems to be unrelated to the HDV viral levels, as we found no clear relationship between viral loads and editing rates. New treatments against HDV will be available soon in addition to pegylated interferon alpha, including Myrcludex, Lonafarnib, Replicor and others in clinical development. Longitudinal studies assessing the kinetics of HDV editing rates on and after treatment could be particularly interesting to better understand their mechanisms of success and failure. The NGS-based method we have developed appears to be particularly well-suited to this endeavor. In addition, the assay will be very useful to finely characterize the editing process control during the natural history of the infection, both in in vitro and in in vivo studies.In conclusion, our study provides a new specific tool dedicated to the in vitro or in vivo quantification of HDV editing rates and novel insights into the genotype dependency of this important step of the HDV life cycle. This tool will be useful in large-scale clinical studies in the future to assess the role of editing rates as a predictive marker of the natural history and treatment response of the infection.


Here we describe a new integrative approach for accurate annotation and quantification of circRNAs named Short Read circRNA Pipeline (SRCP). Our strategy involves two steps: annotation of validated circRNAs followed by a quantification step. We show that SRCP is more sensitive than other individual pipelines and allows for more comprehensive quantification of a larger number of differentially expressed circRNAs. To facilitate the use of SRCP, we generate a comprehensive collection of validated circRNAs in five different organisms, including humans. We then utilize our approach and identify a subset of circRNAs bound to the miRNA-effector protein AGO2 in human brain samples.The online version contains supplementary material available at 10.1186/s13059-021-02497-7. Circular RNAs (circRNAs) are abundant RNAs generated through circularization of specific exons by a process called back splicing –4. circRMany computational pipelines exist for de novo discovery and quantification of circRNAs from RNA-seq data –25. ThesGenerally, circRNA detection relies on the identification of a splicing junction that is unique for a given circRNA . Importantly, other biological processes and technical artifacts can generate splicing junctions that can be confused with those characteristic of circRNAs . These iHere, we developed an alternative approach for both identifying and quantifying circRNAs. We first generate a reliable database of circRNAs by analyzing RNA-seq data from RNaseR and mock-treated samples through several existing pipelines. In the second step, we use this database to create a reference to which the RNA-seq data is aligned. We showed that our approach outperforms the use of single pipelines with regard to both annotation and quantification. Then, we generated, sequenced, and analyzed mock and RNaseR-treated samples from mouse, rat, monkey, and human tissues. This allowed us to create a catalog of bona fide circRNAs for utilizing our new pipeline in those species. Finally, we used this catalog to identify circRNAs bound to the miRNA-effector protein AGO2 in human brain samples.To accurately annotate and quantify circRNAs, we utilize a two-step approach that we call the Short Read CircRNA Pipeline  splicing junctions of the first and last exon contained in each circRNAs Fig. D; the laDrosophila melanogaster RNA-seq dataset obtained on samples with and without RNaseR treatment [GSE55872]. As previously observed for other datasets [We began by assessing circRNA levels detected by five commonly utilized circRNA pipelines  have lower RNaseR/mock RNA ratios than circRNA junctions Fig. B. For liA given circRNA would be consider real if the ratio of the signals obtained between the RNaseR and mock samples is higher than a given threshold/cutoff value. The choice of this threshold is somehow arbitrary, and it is initially based on the distributions of these ratios for the circRNA candidates and their hosting mRNAs in a given tissue/species. Moreover, this threshold can be accommodated by the user at a later point. Previous work showed that circRNA junctions found by more pipelines are more likely to be generated from bona fide circRNAs , 35. TheBy utilizing the SCRP strategy with a cutoff of 0.9, the proportion of circRNAs identified by all pipelines increased from about 35% to almost 60% . Indeed, validated circRNAs had very low expression levels in polyA+-selected RNA-seq libraries . Neither intron length nor GC content clearly discriminated between true and false-positive circRNA junctions, although exons included within true- and false-positive circRNA junctions were flanked by much longer introns than randomly selected exons  using one read of the paired-end RNAseq sample (SRR1197359) which we computationally downsized in length and depth. As expected for each library depth, we detected more circular reads and a larger repertoire of circRNAs as we shortened the seed . Each set was designed to contain half of the number of circRNA read counts as the one before . To test this hypothesis, we compared the performance of SRCP with the different pipelines in two of the samples in which the reads were maintained 100 bases long or computationally truncated using fastx trimmer to 50 or 70 bases. SRCP consistently outperformed the other pipelines at all the assayed lengths  rather than paired end (PE) RNAseq. To do so, we utilized the same dataset but ran the pipelines utilizing the data as single end reads. We compared their power to detect circRNAs from the intact or computationally truncated reads and determined how they compare with the PE assessments. Interestingly, while utilizing SE reads, the number of total R1 + R2) circRNA reads identified by CIRI2 is slightly higher than SRCP  were similar in young and aged flies  by SRCP between young and aged flies. For the validation, we chose one circRNA (circWbp2) that we found DE by all pipelines, four that were found only by SRCP , and three that were found to be DE by SRCP and one or two additional pipelines . As expected, the levels of the control mRNA  and run SRCP to identify the circRNA and linear junctions for each tissue/specie. We then proceeded to determine the abundance of all circRNAs in the RNaseR-treated and mock samples and plot them according their RNaseR resistance. As for D. melanogaster, we observed that circRNAs presented higher RNaseR/mock ratios than their linear counterparts , generated the circRNA index as we did for the Previous work has shown that at least two circRNAs (CDR1as and circSry) strongly bind to the miRNA-effector protein AGO2 and that this binding might result on the regulation of specific miRNAs , quantifies circRNAs with high sensitivity and with a low number of false negatives. This method is general and is not limited to a specific circRNA detection pipelines. The quantification step is simpler than all other pipelines since it does not search de novo for circular RNA junctions.A number of circRNA identification and quantification tools have been described including acfs , DCC 2727, segemAnother strength of our approach is that the user can modify the annotation of the false-positive circRNAs identified by different circRNA detection pipelines. As the annotation is based on the RNaseR-treated library to the total (rRNA depleted) library (referred as mock) ratio, the user can control the false-positive rate. While the RNaseR/mock ratio is the gold standard for validating the circularity of RNA molecules, some linear highly structured RNAs are also resistant to this RNAse . In prinDrosophila circRNAs identified and quantified using SRCP, we found that bona fide circRNAs share some genomic features and expression characteristics. These include their very low abundance in polyA-selected RNA-seq libraries, slightly lower expression levels, larger exon size, hosted by genes with more introns, and better genomic annotation compared to randomly selected exons. Although none of these features individually allowed us to computationally discriminate between “true” and “false” circRNAs, machine learning approaches might be trained to take these features into account to improve de novo identification of circRNAs to diminish false negatives.Interestingly, in our analysis of Importantly, SRCP requires the presence of a validated list of circRNAs which can only be generated by running several pipelines in mock and RNaseR-treated samples. Here, we provide a comprehensive list of validated circRNAs for human brain and blood as well as for mouse, rat, and monkey brain. Nevertheless, new datasets will have to be generated and analyzed for using SRCP in other tissues and/or organisms. Once generated, those lists can be utilized in further experiments and the computational time and framework for analyzing circRNA expression will diminish considerably.In sum, SRCP is a novel method that combines circRNA annotation and an efficient algorithm for their quantification. To identify false positives from the several different circRNA-identification pipelines, we compared the expression of the putative circRNAs in mock and RNaseR-treated samples. We adjusted the threshold/cutoff of the RNaseR/mock ratio for minimizing the number of false negatives by taking into consideration the detectability by several pipelines and the expression level of the circRNA. By comparing the results obtained using SRCP with the ones obtained using five circRNA identification and quantification pipelines on multiple simulated and real RNA-seq datasets, we found that SRCP identifies more differentially expressed circRNAs than any of the other methods. Finally, SRCP allows quantification of circRNAs that were identified by different pipelines.Using available genome annotation, we extract for every putative circRNA all its potential transcripts. We do this using bedtools intersect, we intersect the circRNA coordinates with the genome transcriptome annotation. Next, we score the transcripts as follows: (i) If the start coordinate and the end coordinate of the circle are both exactly on a 5′ and 3′ boundaries of the transcript’s exons, the score is maximal. (ii) If only one coordinate is exactly on an exon boundary and the other is not, the score is 1. (iii) If neither coordinate is on any exon boundary, the score is 0. Next, we choose for each circRNA in the database, a transcript that best fits the circle. This is the transcript with the highest score or, if a few transcripts all have the same highest score, one is randomly chosen. Next, using bedtools getfasta , we extrTo create linear references, for each circRNA junction, we select the closest annotated exons upstream and downstream of the circular junction. We then extract the FASTA sequences for those exons using bedtools get_fasta . We concj) bases upstream or downstream or the putative junction have to match the exon with no mismatches.RNA-seq reads are aligned to the circular index with bowtie2 . The reaTo quantify circular reads, the number of reads that align to the circular index for each circRNA is calculated. For linear reads, the number of reads that align to the linear index is calculated for all transcripts once for the downstream of the circle and once to the upstream side of the circle.To select an appropriate cutoff value, we compared the number of circular and linear RNA reads classified as RnaseR resistant for an array of different cutoff values of the RNaseR/mock ratio  and used that as a reference to exclude “false” circRNAs. If a “false” circRNA was found in the collapsed “true” circ list, we changed its annotation to “true.” The final “true” circRNA list per tissue consists of circRNAs that passed the cutoff plus some circRNAs that passed the cutoff in a different tissue.Drosophila melanogaster heads and extracted the fasta sequences for the junction area (70 bases upstream and downstream of the circular junction) plus the fasta sequences for the upstream and downstream exons. We then randomly selected start positions on the circRNA and generated 70-base circular reads (from the circle sequences) and linear reads from the upstream and downstream exons.In order to create a simulated dataset, we took all the circular junctions found in Drosophila melanogaster annotation from UCSC. For each gene, we selected the starting exon in the transcript. We then randomly selected the desired number of exons. Using the gene annotation from UCSC, we extracted the annotation for each of these random made transcripts. We then use bedtools getfasta to extract the corresponding fasta sequences for each of these random transcripts.To select random exons, we used the We ran circRNA detection pipelines Acfs, CircExplorer, circRNA_finder, CIRI2, and find_circ with default parameters with the dm3 genome and genome annotation from the UCSC genome browser as input. In all cases, we utilized the default parameters.We downloaded the data ran SRCP on these data, using the circ lists we had established from the previous analysis of the atlas. We used the same parameters as we used for the datasets in the atlas with hg38(human), mm10(mouse), and rheMac10(monkey) genomes. The corresponding genome annotations were downloaded from the UCSC genome browser and used as input. In all cases, we utilized the default parameters.Similar to the rescue we performed by collapsing the tissues, we collected the “true circRNAs” found in these datasets while in our data did not pass the cutoff and changed their annotation to “true.”We ran circRNA detection pipelines CircExplorer, circRNA_finder, CIRI2, and find_circ with default parameters with hg38(human), mm10(mouse), rheMac10(monkey), and rn5(rat) genomes. The corresponding genome annotations were downloaded from the UCSC genome browser and used as input. In all cases, we utilized the default parameters.To determine with certainty that the SRCP does not contain double counted reads, we ran SRCP on each of the sides: R1 and R2. Next, we extracted the SRCP reported reads and filtered to look at the true circs only. We compare the list of read names which are associated to R1 as well as to R2. None of the reads appear in both lists.p value < 0.05 as significantly differently expressed.We normalized the circular reads to the number of aligned reads in the sample. We used DESeq2  to detecWe downloaded the data from GEO (GSE52082) and realigned it to human genome version Hg38 after trimming linker and adapter. SRCP was run using 5 base pair seeds and the brain circRNA list used as a reference. We retrieved miRNA abundance analysis from . BigWig Homo Sapiens miRNA family information.We expanded the miRNA binding site prediction in the detected circRNA backsplice junction reads using TargetScan7.0. For this, we extracted the reads supporting splice junctions from SAM files generated by SRCP. Then we run TragetScan 7.0 using Total RNA was prepared from female fly heads  using TRI Reagent  according to the manufacturer’s protocol. RNA was DNase treated  and cDNA derived from this RNA  was utilized as a template for quantitative real-time PCR performed with the C1000 Thermal Cycler Bio-Rad. The PCR mixture contained Taq polymerase (SYBR green Bio-Rad). Cycling parameters were as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s, 55 °C for 10 s, and 72 °C for 30 s. Fluorescence intensities were plotted versus the number of cycles by using an algorithm provided by the manufacturer. All the primers used in this assay are listed in Additional file Human Adult Normal Brain Total RNA (R1234035-50), Human Adult Normal Cerebellum Total RNA (R1234039-50), Rat Brain Total RNA (R1434035-50), Monkey (Rh) Brain (R1534035-50) Total RNA, and Human Blood Total RNA (custom made) were obtained by BioChain.One C57BL/6 male and one female were sacrificed at p21, different brain regions were harvested  by the Nelson’s lab at Brandeis University. The RNA was extracted with TRI reagent , followed by Zymo RNA cleaning and concentrator -25 Kit (Zymo), according to the manufacturers’ instruction. The RNA was treated with DNase .D. melanogaster (Canton-S) heads and brains. Total RNA was extracted with TRI reagent , followed by DNase  treatment.We collected and dissected 10-day-old D. melanogaster samples, 2 μg of total RNA was rRNA-depleted with a homemade protocol and then treated with RNaseR as indicated above. Recovered RNA was used to prepare a total RNA library, adapting the protocol from [D. melanogaster samples, libraries were prepared using the NEXTFLEX® Rapid Directional RNA-Seq Kit 2.0 by PerkinElmer, following the manufacturer’s instructions. The samples were sequenced by Novogene  with Hiseq-4000.Ribodepletion was performed on 5 μg of total RNA with Ribo-Zero Gold rRNA Removal Kit according to the manufacturer’s instruction. Degradation of rRNA was checked by Tape Station. Ribodepleted RNA was treated with 3 units of RNaseR (Lucigen) per 1 μg of total RNA or with buffer only (Mock) for 15 min at 37 °C. The RNA was immediately extracted with TRI reagent  according to the manufacturer’s instruction. For Human Blood Total RNA, only 2 μg of total RNA were rRNA-depleted and then treated with RNaseR as indicated above. For col from . SpecifiAdditional file 1: Figures S1-S8.Additional file 2: Supplementary tables. 1-5.Additional file 3. Correlations between Biological Replicas.Additional file 4. List of validated circRNAs in the different species.Additional file 5. AGO-HITS CLIP clusters and miRNA seed across backsplicing junctions of circRNAs found to be bound to AGO2.Additional file 6. Oligonucleotides utilized in this study.Additional file 7. Review history.


Lilium davidii var. unicolor is a great challenge due to its polysaccharides, polyphenols and other secondary metabolites. In this study, different RNA extraction methods, namely TRIzol method, the modified TRIzol method, Kit method and cetyltrimethylammonium bromide (CTAB) method were employed to obtain total RNA from different tissues in L. davidii var. unicolor. A Nano drop spectrophotometer and 1% agarose gel electrophoresis were used to detect the RNA quality and integrity. Compared with TRIzol, Kit and CTAB methods, the modified TRIzol method obtained higher RNA concentrations from different tissues and the A260/A280 ratios of RNA samples were ranged from 1.97 to 2.27. Thus, the modified TRIzol method was shown to be the most effective RNA extraction protocol in acquiring RNA with high concentrations. Furthermore, the RNA samples isolated by the modified TRIzol and Kit methods were intact, whereas different degrees of degradation happened within RNA samples isolated by the TRIzol and CTAB methods. In addition, the modified TRIzol method could also isolate high-quality RNA from other edible lily bulbs. Taken together, the modified TRIzol method is an efficient method for total RNA isolation from L. davidii var. unicolor.The high quality, yield and purity total RNA samples are essential for molecular experiments. However, harvesting high quality RNA in  Lilium davidii var. unicolor), an important economic crop, is the only edible sweet lily in China. It is an endemic species that is only suitable at the altitude of 2000–2600 m, resulting in a narrow distribution in Gansu Province, western China1. Bulbs of the L. davidii var. unicolor are one of the main traditional Chinese medicines for several centuries, and its flowers also have ornamental value3. In recent years, more and more studies focused on bulbs of L. davidii var. unicolor, such as bulblets formation and development, and dormancy of bulbs. High levels of starches, proteins, fats, celluloses, saponins, colchicines, and polysaccharides in scales of the bulb can be used as potential biomarkers for bulb quality5. The starch and sucrose metabolism is the major pathway in the scales of this Lilium species7. In addition, the content of polysaccharides in Lilium roots is also very high. During the study concerning growth and development of Lilium, works on the physiological and molecular aspects is necessary, whereas it is difficult to obtain high quality RNA to support molecular biology techniques. Polysaccharides and polyphenols limit nucleotide purification and precipitation due to their co-precipitation with the endogenous RNA9. Thus, obtaining high-quality total RNA for many molecular analyses from scales and other tissues of Lilium is difficult due to the high level of various metabolites. Therefore, the optimized method to isolate total RNA effectively from different tissues of Lilium should be explored.Lanzhou lily  method was reported to be more suitable for RNA extraction in the bulb18. In addition, TRIzol method and RNA extraction Kit were employed to separate RNA from leaf and bulb of Lanzhou lily21. Nevertheless, few protocols are suitable for isolating total RNA from different tissues of Lilium, especially from different parts of the scales. Despite the rapid advances in genetic engineering and transgenic technology of model plants, the genomic database of Lilium is needed to be further improved. Therefore, obtaining integrity, high quality and yield total RNA from Lilium is an essential condition for functional genomics research.Over the past several decades, commercial plant kits play an important role in plant molecular studies, but extracting RNA with high quality and quantity from plants rich in polysaccharides remains a challengeL. davidii var. unicolor . In addition, the quality and yield of RNA obtained by TRIzol method and the modified TRIzol method were compared with each other within different tissues of L. davidii var. unicolor, and within different edible bulbs. A Nano drop spectrophotometer and 1% agarose gel electrophoresis were used to assess the quality of the extracted RNA samples. Here, we concluded that the modified TRIzol method could be applicable to gain high quality and quantity total RNA in different tissues of L. davidii var. unicolor containing polyphenols, polysaccharides, proteins and lipids.In this study, different RNA extraction methods, including the modified TRIzol method, TaKaRa MiniBEST Plant RNA Extraction Kit (Kit) method and CTAB method, were used to extract total RNA from different tissues of L. davidii var. unicolor, including the modified TRIzol method, Kit method and CTAB method. In Lilium root, RNA samples obtained from the modified TRIzol method showed that the A260/A230 and A260/A280 ratios were closed to 1.5 and 2.1, and RNA samples gained from the CTAB method showed higher ratios  (Table −1) (Table Lilium as shown in Table −1). Therefore, high-quality total RNA could be isolated from Lilium stem by using the modified TRIzol method. When RNA was extracted from Lilium leaf using the Kit and CTAB methods, the spectrophotometric results showed that the A260/A230 ratios of RNA ranged from 0.91 to 1.07, and the A260/A280 ratios ranged from 1.69 to 2.01, indicating that the RNA samples were contaminated by proteins, polyphenols and polysaccharides. However, when using the modified TRIzol method, the A260/A230 and A260/A280 ratios and the RNA concentrations were increased significantly  were significantly lower than that from the modified TRIzol method  from stem and leaf were quite shallow  of RNA samples isolated by the modified TRIzol method were lower than that by the Kit (ranged from 1.96 to 2.06) and CTAB (ranged from 1.74 to 1.92) methods, but were higher than that by the TRIzol method (ranged from 0.19 to 0.22) (Table −1) compared to the other methods, and quality of RNA gained by the TRIzol method (ranged from 131.31 to 401.52 ng μL−1) were the worst   was higher than that from the TRIzol method (53.11 ng μL−1), while the yields of RNA of top-inner scales and basal-inner scales from the modified TRIzol method were relatively low  were significantly higher than that by the TRIzol method (ranged from 9.24 to 35.90 ng μL−1)   Figs. , 6. The 31. Also, the CTAB method could obtain higher quality and quantity total RNA from leaf, root and bulb of Lanzhou lily29. In this study, while isolating RNA samples with the CTAB method from root, stem and leaf, one of the main obstacles is the existence of DNA contamination  of Gansu Agricultural University, Lanzhou, Gansu Province, China . Firstly, fresh plants were collected and washed with deionized sterile water and dissected leaf, stem, root and bulb separately. Secondly, scales were removed carefully from mother bulb and sorted into three groups: external scales (1–3 layers), middle scales (4–6 layers) and inner scales (7–9 layers). Then different scales were sorted into three equal parts, named as top part, middle part and basal part  and harvested after the aerial part of the plants withered in October 2021. Lily bulbs of Lilium brownii var. viridulum Baker were produced in Longhui, Hunan Province, China  and harvested after the aboveground part of the plants withered in August 2021. Different layers of the tested inner scales of these two edible bulbs were collected by the same method used in L. davidii var. unicolor . Other reagents included chloroform-isoamylalcohol (C: I) at a ratio of 24:1, 8 M LiCl and 75% ethanol.Plastic materials (tubes and pipette tips), mortars and pestles used in the current paper were soaked overnight in 0.01% DEPC-treated distilled water and then autoclaved at 121 °C for 1 h. In our methods, TRIzol® reagent  and TaKaRa MiniBEST Plant RNA Extraction Kit  were applied. Buffers and solutions were prepared as follows. CTAB extraction buffer: 2% CTAB (w/v); 2% soluble polyvinylpyrrolidone ; 100 mM Tris–HCl (pH 8.0); 2 M NaCl; 25 mM ethylenediamine tetraacetic acid (EDTA); 0.5 g L33. In order to obtain higher concentration of total RNA, RNA purification adsorption column were used after Steps 4. Our modified procedure is described as follows: (1) About 300 mg of each tissue samples was ground into a fine powder using liquid nitrogen and transferred into a 1.5 mL centrifuge tubes. Then 1 mL of TRIzol reagent was added, and mixed by a micropipettor. Incubated the samples for 10 min at 4 °C, and centrifuged at 12,000 g for 5 min at 4 °C. (2) The supernatant was transferred to a new 1.5 mL Eppendorf tube. 200 µL of cold chloroform was added and the mixture was hand-shaken and then incubated at room temperature for 5 min. Then it was centrifuged at 12,000 g for 15 min at 4 °C. (3) The supernatant (~ 700 μL) was transferred to a new 1.5 mL chilled Eppendorf tube and then an equal volume of chilled isopropanol was added and mixed by inverting the tube for 6–10 times. The final mixture was incubated at − 20 °C overnight to precipitate RNA. (4) The obtained RNA pellet from two repeats above was added to one RNA purification adsorption column (700 μL for each time) followed by a centrifugation of 15 min at 12,000 g at 4 °C to obtain a visible precipitate, and the supernatant was discarded. (5) 700 µL pre-cooled 75% ethanol were added and mixed by inverting the tube for 4–6 times, and centrifuged at 12,000 g for 1 min at 4 °C, then the supernatant were discarded. The ethanol washing step was repeated twice, then centrifuged at 12,000 g for 2 min at 4 °C and the redundant ethanol was discarded. (6) After centrifugation, the tubes were carefully transferred to the ice, and the washed RNA pellet was air-dried for 10 min in super clean bench. (7) 40 μL RNase free H2O was added onto the membrane of the RNA purification adsorption column to dissolve RNA. RNA samples were incubated at 4 °C for 3 min, and then centrifuged at 12,000 g for 2 min at 4 °C to elute RNA. To obtain high concentration of RNA, the first eluent was added back into the RNA purification adsorption column, incubated and centrifuged again as above.TRIzol-based RNA extraction was done according to Ahmad et al. (2017), and the modified TRIzol method was done according to this TRIzol method with some modifications10, the RNase-free DNase I and RNA purification adsorption column were used in Steps 10 and 11, respectively. Our modified procedure is described as follows: (1) The extraction buffer was preheated in a 50 mL Eppendorf tube (with 3% β-mercaptoethanol added) and bathed at 65 °C for 30 min. (2) About 300 mg of each sample was grounded into a fine powder by using liquid nitrogen, then the sample was transferred to 1.5 mL chilled Eppendorf tube and 1.2 mL pre-heated extraction buffer was added. (3) The tube was vortexed immediately and then incubated at 65 °C in water bath for at least 30 min with vortexing once every 5 min. (4) The tube was centrifuged at 12,000 g for 10 min at 4 °C and the supernatant was transferred to a new 1.5 mL Eppendorf tube. (5) An equal volume (650–850 µL) of C: I (24:1) was added to the tube, with vortexing for 30 s and then centrifuged at 12,000 g for 15 min at 4 °C. (6) The aqueous phase was transferred to a new 1.5 mL Eppendorf tube without disruption of the white interphase. (7) Repeated C: I abstraction as in the previous steps (steps 5 and 6). (8) 1/3 volumes of 8 M pre-cooled LiCl was added to the supernatant in 1.5 mL Eppendorf tube and was mixed upside down, then the mixture was stored at 4 °C overnight for nucleic acid precipitation. (9) The obtained RNA pellet from two repeats above was combined and added to one RNA purification adsorption column (700 μL for each time) followed by a centrifugation of 15 min at 12, 000 g at 4 °C, then discarded the supernatant. (10) 5 µL 10 × DNase I Buffer, 4 µL Recombinant DNase I  and 41 µL RNase free H2O were pipetted onto the membrane of the RNA purification adsorption column, followed by an incubation of 15 min at room temperature (25 °C) to eliminate possible genomic DNA contamination. (11) The RNA was washed, air-dried and eluted as described in steps 5–7 of the modified TRIzol protocol.CTAB method was constructed according to White et al. (2008) with some modificationsPlant RNA Extraction Kit, which contained two types of cracking buffer named PE and RL, was used according to the manufacturer’s instructions (Cat. No. 9769). The method was modified when using RNA purification adsorption column to collecting RNA. In order to ensure high quality total RNA, the same volume of the treatment mixture were collected to the same adsorption column as in method 1 and 2. Importantly, all of the operating steps were performed at 4 °C to prevent RNA degradation during the experiment process.32. The gels were stained with ethidium bromide (0.5 µg mL−1) and then visualized and documented by a Gel Imager System .The A230/260 and A260/280 ratios were measured by a Nano drop spectrophotometer to determine the purity of the extracted total RNA. The concentration of the RNA sample was detected simultaneously by the spectrophotometer to determine the RNA yield. RNA integrity was evaluated by the clarity of ribosomal RNA bands in 1% agarose gel electrophoresisSupplementary Information.


M = 21.06; SD = 2.62). Turkish young adults completed an online survey, including measures of coronavirus stress, subjective well-being, meaning-based coping, and spiritual well-being. The results indicate that greater meaning-based coping and spiritual well-being mediated decreases in the adverse impacts of coronavirus stress on subjective well-being. These results suggest that the importance of a combination of meaning-based coping and spirituality processes mitigate the adverse effects of stress on well-being during the coronavirus pandemic. Interventions focusing on meaning-based coping and spirituality in those experiencing high coronavirus stress are urgently needed to improve the mental health and well-being of young adults.The coronavirus pandemic has significantly affected the Turkish population. During the pandemic, people with high coronavirus stress are at risk of experiencing poor subjective well-being. There is no research investigating the role of meaning-based coping and spirituality in explaining the link between coronavirus stress and subjective well-being. This study examined the mediating roles of meaning-based coping and spiritual well-being in the link between coronavirus stress and subjective well-being in young adults during the COVID-19 pandemic. The sample included 427 young adults , ranging in age between 18 and 48 years ( The coronavirus disease 2019 (COVID-19) pandemic has become a major public health problem in many countries in the world, including Turkey. It has expeditiously spread over the world, originating from Wuhan city of China in early December 2019. By March 3, 2021, the One well-known psychological aspect of the COVID-19 pandemic is coronavirus stress. The uncertain atmosphere related to COVID-19 may aggravate stress among people coping with the pandemic. For example, data from China shows that nearly 25% of the population has experienced moderate-to-severe levels of anxiety- or stress-related symptoms during COVID-19 . SimilarThe influence of the COVID-19 pandemic on well-being and mental health outcomes has been investigated in various studies. Taken together, the results of these studies reveal a positive association between coronavirus stress and depression, anxiety, fear, burnout, trauma, and other variables related to well-being and mental health outcomes . For exaPerceived risk of COVID-19 is another important psychological aspect of the COVID-19 pandemic. Perceived risk plays a fundamental role in forming one’s health-related behaviors . The resThe COVID-19 pandemic has exacerbated psychosocial challenges . SubjectAlthough the pioneering body of research has mainly examined the influence of coronavirus stress on well-being and mental health outcomes, little is known about the underlying psychological mechanism that explains how the detrimental effect of coronavirus stress on well-being can be mitigated during the COVID-19 pandemic. Researchers highlight the importance of finding effective ways to protect and improve well-being of individuals in this unprecedented period . As suchResearch findings highlight that the absence of stress or distress does not necessarily mean that a person is optimally functioning, and instead, optimal positive psychosocial human functioning can be achieved with greater use of meaning-based coping strategies . FurtherAnother important mechanism that can explain the relationship between coronavirus stress and subjective well-being is that of spiritual well-being, which refers to spiritual maturity and the integral experience of an individual who is functioning as God intended . ResearcIn light of the sketched literature, there are enough reasons to believe that coronavirus risk is a positive predictor of coronavirus stress, which is a negative predictor of subjective well-being, and meaning-based coping and spirituality are positive predictors of subjective well-being. Understanding the mechanisms underlying the relationships between coronavirus stress and subjective well-being would improve our understanding of these associations. In this study, we focus on meaning-based coping and spirituality and investigate the possible roles of these two variables for the associations between coronavirus stress and subjective well-being. To this end, we conducted a cross-sectional survey to examine the relationship between coronavirus risk, coronavirus stress, and satisfaction with life as a measure of subjective well-being by focusing on meaning-based coping and spirituality as possible mediators. More specifically, we tested whether (i) coronavirus risk would predict coronavirus stress; (ii) coronavirus stress would predict meaning-based coping, spirituality, and subjective well-being; (iii) meaning-based coping and spirituality would predict subjective well-being; and (iv) meaning-based coping and spirituality would mediate the relationship between coronavirus stress and subjective well-being.M = 21.06; SD = 2.62), and the majority of the sample was female (71%). With regard to coronavirus characteristics, the majority of the sample was not infected (95%). The data were collected during the COVID-19 pandemic throughout November 2020. A web-based survey was created using data-collection measures and demographic items. Social media was used to contact the participants. Turkish young adults completed an online survey, including measures of coronavirus stress, subjective well-being, meaning-based coping, and spiritual well-being. Before the survey items, informed consent was presented to the participants. Participation in the study was voluntary, and the survey was completed anonymously. The study was also approved by the Ağrı İbrahim Çeçen University Institutional Review Board.The sample of this study comprised 427 undergraduate students from two different universities in Turkey. Participants ranged in age between 18 and 48 years (A single item was used to assess perceived coronavirus risk of young adults (“Do you feel yourself at risk due to coronavirus pandemic?”). The item was scored using a 5-point Likert-type scale, ranging from 1 (negligible) to 5 (very high).Coronavirus stress was measured using the coronavirus stress measure (CSM), which is a 5-item self-report scale  developed to assess COVID-19-related stress as a stressful event . All iteSubjective well-being was assessed using the satisfaction with life scale (SWLS), which was developed to assess people’s cognitive assessments and judgments of life . The SWLχ2 = 497.76, df = 10, p < 0.001) and adequate sample size (Kaiser-Meyer-Olkin measure of sampling adequacy = 0.86). The SWS had strong factor loadings, ranging between 0.56 and 0.85. Next, confirmatory factor analysis affirmed the unidimensional structure of the measure with the second subsample. Results from this analysis indicate a close data-model fit , characterized by strong factor loadings (λ range = 0.55–0.85) and a latent construct reliability estimate (H = 0.89). These indicate that the SWS has psychometrically adequate properties for use in measuring the spiritual well-being of Turkish people. The internal reliability estimate with the total sample of the study was strong; see Spiritual well-being was measured using the spiritual well-being scale (SWS) adapted from the 12-item functional assessment of chronic illness therapy-spiritual well-being (FACIT-Sp-12), which was originally developed to assess the spiritual well-being of people with cancer and chronic illnesses . The SWSα = 0.86; Eisenbeck et al., unpublished). The internal reliability estimate in this study was strong; see The meaning-centered coping scale  was used to assess people’s meaning-based coping strategies,. It is a 9-item self-report measure , scored based on a 7-point Likert-type scale between 1  and 7 (I completely agree). Previous research indicates that the scale is psychometrically adequate for use in many cultures, including Turkey  and kurtosis scores (range = −0.88 to −0.37) as shown in χ2 = 262.20, df = 111 p < 0.001, CFI = 0.96, TLI = 0.95, RMSEA [95% CI] = 0.057 , SRMR = 0.035).Prior to testing the structural equation model, a measurement model was suggested to examine the association between latent constructs and their observed variables . Coronavχ2 = 152.02, df = 52, p < 0.001, CFI = 0.96, TLI = 0.96, RMSEA [95% CI] = 0.067 , SRMR = 0.032). The results also showed that perceived coronavirus risk was a significant predictor of coronavirus stress  and accounted for 15% of the variance in this variable. Coronavirus stress had a significant predictive effect on meaning-based coping  and subjective well-being . We also found a significant mediating effect of meaning-based coping  on the association between coronavirus stress and subjective well-being. All variables together explained 23% of the variance in subjective well-being.We next performed several structural equation models to test the role of mediators in the association between coronavirus experiences and subjective well-being of young adults. Findings from the first model, which was carried out to examine the mediating role of meaning-based coping on the effect of coronavirus experiences on subjective well-being, provided good data-model fit statistics . Coronavirus stress significantly predicted spiritual well-being  but was not a significant predictor of subjective well-being . Spiritual well-being significantly mediated the effect of coronavirus stress on subjective well-being  as seen in We next tested the mediating effect of spiritual well-being on the relationship between coronavirus stress and subjective well-being, indicating good data-model fit statistics . Coronavirus stress had a significant and direct effect on spiritual well-being , and meaning based coping mitigated the negative effect of stress on spiritual well-being  as shown in Findings from the third model, which was carried out to examine the mediating role of meaning-based coping on the effect of coronavirus experiences on spiritual well-being, yielded good data-model fit statistics . Coronavirus stress significantly predicted meaning-based coping  and spiritual well-being , yet the predictive effect of it on subjective well-being was nonsignificant . Coronavirus stress indirectly predicted subjective well-being through meaning-based coping  and spiritual well-being  as shown in Spiritual well-being and meaning-based coping together were finally included in the model and examined the mediating effect of these variables together in the association between coronavirus stress and subjective well-being; see The present study aimed to develop our understanding of the associations between coronavirus risk, coronavirus stress, and subjective well-being by focusing on meaning-based coping and spirituality as underlying mechanisms. Results support the study hypotheses. The coronavirus pandemic has become an adverse worldwide public health concern associated with various negative mental health and well-being outcomes . TherefoMost importantly, findings from the study show that meaning-based coping and spirituality explain the relationship between coronavirus stress and subjective well-being. The findings extend coronavirus research by showing that meaning-based coping and spirituality play important roles in the association between coronavirus stress and subjective well-being. The coronavirus pandemic has exacerbated psychological and social challenges , which iDespite efforts to understand the impacts of the pandemic , little We also find that spiritual well-being is another important factor that mitigates the negative effect of coronavirus stress on the subjective well-being of participants. These results suggest that people with high levels of spiritual maturity  have greThe present study is not without limitations. First, the present research was a cross-sectional design. Longitudinal studies would advance our understanding of the underlying mechanisms that relate coronavirus risk and stress to subjective well-being by identifying the potential causal and temporal relationships. Second, the data were collected using self-report measures, which are considered another limitation of the study. Future studies could be conducted using various data-collection methods  to provide additional understanding of the associations between the variables in the study. Furthermore, the convenience sampling method is also a clear limitation of this study, and therefore, future research is warranted to perform with randomly collected diverse samples, including equal genders and individuals living larger cities.In addition to these limitations, results from this study provide important implications for research and practice that aim to improve individuals’ mental health and well-being against coronavirus experiences. The results indicate how people cope with the coronavirus pandemic that has detrimental consequences for their mental health and well-being and report that meaning-based coping mitigates the adverse impact of coronavirus stress on subjective well-being. Additionally, the findings suggest that spiritual well-being is an essential source to cultivate people’s subjective well-being by mitigating the effect of coronavirus stress. Based on these results, mental health providers could design preventions and interventions that help to improve meaning-based coping strategies, which, in turn, alleviate the effect of coronavirus stress on subjective well-being. Spiritual well-being could also be promoted through these programs, and spiritual resources might be integrated with meaning-based strategies to provide more effective coping with pandemic experiences.In conclusion, we find that meaning-based coping and spirituality mediate the associations between coronavirus stress and subjective well-being. In addition, coronavirus risk appears to be a significant positive predictor of coronavirus stress. The present study serves to further develop our understanding of the underlying mechanisms by which coronavirus stress impacts subjective well-being. More research in this area is needed and will facilitate fully understanding the associations between coronavirus risk, coronavirus stress, meaning-based coping, spirituality, and subjective well-being.The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.The studies involving human participants were reviewed and approved by Ağrı İbrahim Çeçen University. The patients/participants provided their written informed consent to participate in this study.GA and MY contributed to the design of the study. GA analyzed the data and wrote the materials and methods, results, and discussion sections. MY wrote the introduction. Both the authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The complete genome sequence of novel polerovirus-associated RNA infecting pepper in South Africa was determined. The nucleotide sequence identity of 78.3% with closely related species suggested that this associated RNA was novel, and the name Pepper Vein Yellows Virus-associated RNA is proposed for this RNA fragment. The complete genome sequence of a novel polerovirus-associated RNA infecting pepper in South Africa was determined. The nucleotide sequence identity of 78.3% with closely related species suggested that this associated RNA was novel, and the name pepper vein yellows virus-associated RNA is proposed for this RNA fragment. In February 2020, previously unknown disease symptoms were observed in field-cultivated sweet peppers in Limpopo, South Africa. Symptoms included yellowing of the leaf veins at the top of the plant. As the infection progressed, interveinal yellowing with eventual stunting of the growing tips was observed .Polerovirus. Symptomatic leaf material was collected, from which total RNA was isolated using the Qiagen RNeasy plant minikit. The RNA was used as the template for double-stranded (ds) cDNA synthesis using the Maxima H Minus ds cDNA kit (Thermo Scientific) using the random hexamers provided by the manufacturer. A sequencing library was constructed following the manufacturers protocol  and sequenced on the R9.4 MinION flow cells for 72 hours. In short, the double-stranded cDNA was end prepped with use of NEBNext formalin-fixed, paraffin-embedded (FFPE) DNA repair mix (New England BioLabs) and Ultra II end prep mix (New England BioLabs). The DNA was washed with use of AMPure XP beads (Beckman Coulter), and Oxford Nanopore Technologies (ONT) AMX adapters were ligated with use of the NEBNext quick T4 ligase (New England BioLabs). The washing step was repeated, and the library was loaded onto a MinION flow cell. After 72 hours of sequencing, the data were base called with use of Guppy v. 3.6 (Oxford Nanopore Technology) and trimmed/filtered based on length and quality scores  with use of Porechop v. 0.2.4 (https://github.com/rrwick/porechop) and Nanopolish v. 0.13.2 (https://github.com/jts/nanopolish). The average read length was 1,441 bp, the total number of bases was 7.39 Gbp, and the number of reads was 5.1 million. All tools were run with default parameters unless otherwise specified. The processed reads were assembled using CLC Genomics Workbench v. 12.0 (Qiagen) using the de novo assembly option in the long-read module (beta). No polishing steps were executed prior to assembly. Reads originating from pepper were subtracted by mapping the reads to a reference genome (GenBank accession number AYRZ00000000.2). The remaining reads were used for de novo assembly. The assembled contigs were used to search the NCBI nucleotide (nt) collection database using BLASTn.The observed symptoms led us to screen for begomoviruses, potyviruses, and tospoviruses, with use of Pathogen Immunostrips (Agdia) and enzyme-linked immunosorbent assay (ELISA), which returned a negative result. We therefore started screening for a putative infection with a virus from the genus MK931185.1). An additional contig of about 2.9 kb  was found in the sweet pepper sample, which shares a 78.3% nucleotide identity with the complete sequence of Tobacco bushy top disease-associated RNA (GenBank accession number EF529625.1)  showed 94.7% nucleotide similarity to pepper vein yellows virus  amplicons along the genomic RNA. For the amplification of the Sanger sequencing fragments, the following reaction setup was used: 4 µl 5× Phusion high-fidelity (HF) buffer, 0.4 µl 10 mM deoxynucleoside triphosphates (dNTPs), 1 µl 10 mM forward and reverse primer, 0.2 µl Phusion DNA polymerase (New England BioLabs), 50 ng of template cDNA, and nuclease-free water, added to a total volume of 20 µl. The 5′ and 3′ termini of the viral RNA were obtained using rapid amplification of cDNA ends (RACE) technology. The 5′-terminal end was amplified following the manufacturer’s protocol using the 5/3′ RACE kit, 2nd generation (Roche). For the amplification of the 3′ terminus, the molecules were tailed with dATP and TdT. These tailed molecules were used as input material for 3′ RACE using the manufacturers protocol (see EF529625.1 as RefSeq in the toolbox Whole Genome Alignment (beta) using default settings in CLC Genomics Workbench v. 12.0 (Qiagen). The genome contains the following two ORFs: an ORF coding for a protein of unknown function of 259 amino acids (aa) and an ORF coding for a putative RNA-dependent RNA polymerase of 786 aa. We propose the name pepper vein yellows virus-associated RNA (PeVYVaRNA) for this RNA fragment.The complete genome of the polerovirus-associated RNA with a size of 2,973 nt and a GC content of 50.6% was obtained and further analyzed and annotated by using the Tobacco bushy top disease-associated RNA reference genome under GenBank accession number MT321510. Sequencing data are available under SRA accession number SRR12835201.The full sequence of PeVYVaRNA isolate PRO54353 can be found in NCBI GenBank under accession number