Advances in high throughput, next generation sequencing technologies have allowed an in-depth examination of biological environments and phenomena, and are particularly useful for culture-independent microbial community studies. Recently the use of RNA for metatranscriptomic studies has been used to elucidate the role of active microbes in the environment. Extraction of RNA of appropriate quality is critical in these experiments and TRIzol reagent is often used for maintaining stability of RNA molecules during extraction. However, for studies using rumen content there is no consensus on (1) the amount of rumen digesta to use or (2) the amount of TRIzol reagent to be used in RNA extraction procedures. This study evaluated the effect of using various quantities of ground rumen digesta and of TRIzol reagent on the yield and quality of extracted RNA. It also investigated the possibility of using lower masses of solid-phase rumen digesta and lower amounts of TRIzol reagent than is used currently, for extraction of RNA for metatranscriptomic studies. We found that high quality RNA could be isolated from 2 g of ground rumen digesta sample, whilst using 0.6 g of ground matter for RNA extraction and using 3 mL (a 5:1 TRIzol : extraction mass ratio) of TRIzol reagent. This represents a significant savings in the cost of RNA isolation. These lower masses and volumes were then applied in the RNA-Seq analysis of solid-phase rumen samples obtained from 6 Angus X Hereford beef heifers which had been fed a high forage diet (comprised of barley straw in a forage-to-concentrate ratio of 70:30) for 102 days. A bioinformatics analysis pipeline was developed in-house that generated relative abundance values of archaea, protozoa, fungi and bacteria in the rumen and also allowed the extraction of individual rRNA variable regions that could be analyzed in downstream molecular ecology programs. The average relative abundances of rRNA transcripts of archaea, bacteria, protozoa and fungi in our samples were 1.4 ± 0.06, 44.16 ± 1.55, 35.38 ± 1.64, and 16.37 ± 0.65% respectively. This represents the first study to define the relative active contributions of these populations to the rumen ecosystem and is especially important in defining the role of the anaerobic fungi and protozoa. Current day advances in high throughput next generation sequencing technologies has revolutionized and enhanced the molecular biology and life sciences research landscape. Increases in speed and reductions in cost of sequencing, advances in the development of computational resources and capabilities has resulted in a cost effective ability to study a wide array of biological systems, phenomena and environments at a depth that was hitherto impossible. Culture-independent microbial community studies have particularly benefited from this revolution. Driven primarily by improvements in the ability to study and analyze the well conserved small subunit (SSU) 16S rRNA gene, it is now possible to explore complex heterogeneous communities and ecosystems . This has provided insights into the diversity and functions of microbial life, and the relationships between microbial community dynamics and the physiological states of related biological systems. These technologies have been broadly applied in the study of a wide range of environments and ecosystems. It is becoming more and more usual to find studies which extend beyond elucidating taxonomic profiles, to actually inferring metabolic and functional profiles from such taxonomic distributions and highlighting metabolic and biochemical pathways that might be implicated in such systems , therebyMany recent microbiome studies have relied on 16S rRNA amplicon sequencing. This approach involves the extraction, amplification and sequencing of a targeted phylogenetic marker region which must (1) be flanked by highly conserved and ‘known’ sequences, (2) be ubiquitous in the required taxonomic region of interest, and (3) be variable enough to discriminate between variant species. This characteristic (of focusing on targeted regions of the genome) makes statistical comparisons and analysis of qualitative/quantitative differential expression information, allele-specific expression measurement and transcript/gene fusion verification, possible. It also allows high throughput, simultaneous measurement of thousands of targets and provides greater specificity and accuracy in the measurement of transcripts of interest . SequencTotal RNA sequencing (RNA-Seq) on the other hand provides a snapshot of entire transcriptomes. It thus enables measurement of transcript/gene abundances, detection of coding and non-coding RNA, identifying both known and novel features of the transcriptome, and allowing the prediction of potential functions of the collective organisms in a sample (by studying microbiota associated genes), thus leading to a more comprehensive understanding of habitats, ecosystems and biological systems . CompariThe analysis of rRNA abundance provides for insights into the activity of microbial cells and their protein synthesis potential . This isSSUsearch pipeline for metagenomes which aligns shotgun sequence data, and following that extracts reads that mapped to specified regions in the Escherichia coli SSU rRNA gene, which are then used for unsupervised clustering and other analyses as would normally be applied to traditional amplicon data. This improvement holds promise for the use of RNA-Seq in phylogeny and taxonomy studies as it can combine the benefits of wider the breadth of coverage (of RNA-Seq) with the specificity, efficiency and flexibility of a targeted (amplicon) approach.Due to the broad coverage of the transcriptome provided by RNA-Seq, the outcomes are not very amenable to comparative statistical analysis beyond descriptive profiles of taxonomy. Supervised or unsupervised community analyses, for example are not usually possible. To address this shortcoming Solid-associated microorganisms make up the predominant proportion of total rumen microbes . It is eSSUsearch pipeline . After that 0.6 and 0.3 g of the resulting fine powder was weighed into 50 mL tubes and mixed with 6 and 3 mL respectively, of TRIzol reagent. The sample was thawed and incubated at room temperature for 5 min, and RNA was subsequently extracted using the AGPC method . The airRNA concentration and integrity were determined using an Agilent 2100 bio analyzer  and RNA 6000 Nano kit (Agilent Technologies) according to the manufacturer’s instructions. Given that prokaryotes account for the majority of RNA in rumen contents  the prokFirst, a solid-phase sample was manually ground into crude powder in liquid nitrogen, and then 2 g of crude powder was weighed and further ground for 5 min employing the method described above. After grinding, 0.6 g of the resulting fine powder was weighed into 50 mL tubes into which 6, 4.5, or 3 mL of TRIzol reagent were added. The sample was ground and processed to extract RNA, all in replicates. RNA concentration and integrity were estimated using the same methods as described above.Following successful total RNA outcomes from using lower grinding masses and lower TRIzol reagent/mass ratios, twelve solid-phase samples (2 g each), withdrawn from 6 Angus × Hereford cross beef heifers were first ground using the above-mentioned method, and then 0.6 g of fine powder from each of them was weighed into a 50-mL tube and mixed with 3 mL of TRIzol reagent. For each sample, total RNA was extracted in duplicates and the RNA concentration and integrity were estimated using the methods described above. The heifers had been fed a high forage diet comprised mainly of barley straw in a forage-to-concentrate ratio of 70:30 for 102 days.Total RNA (100 ng) was prepared for sequencing using protocols supplied with the Illumina TruSeq RNA Sample Prep Kit-v2, Set A and B , with the exception that steps for the enrichment of mRNA or PolyA selection were excluded. Total metatranscriptomic libraries were validated using the DNA 1000 kit (Agilent Technologies) and were quantified using the KAPA SYBR Fast qPCR kit for Illumina Technologies . Sample libraries were normalized using the qPCR results and 24 samples were multiplexed and paired end (2 × 300bp) sequenced on an Illumina MiSeq using a MiSeq Reagent v3 600 cycle kit .rRNA–HMM  was merged using PEAR , run on rRNA–HMM  tool of rRNA–HMM  was usedE 4.10.2  toolkit,E 4.10.2 . The subR 1.33.1  was usedSSUsearch pipeline of hmmsearch step (of the pipeline) the resulting sequence reads were aligned to the rRNA reference genes template using MOTHUR , corresponding to positions 477–727 in Escherichia coli. From all the samples sequences from this region were extracted and used in subsequent unsupervised clustering analysis. While the SSUsearch) was used as is in this study, the unsupervised analysis pipeline was modified slightly. Apart from the de-replication step where RDP McClust was used all other analysis steps (particularly those covering distance matrix creation and de novo clustering) was done using MOTHUR 1.35.1  shipped with the SSUsearch pipeline, specific HMMs obtained from the Rapid Analysis of Multiple Metagenomes with a Clustering and Annotation Pipeline (RAMMCAP) (hmmsearch component of the (SSUsearch) pipeline - to separate reads into archaea, bacteria and eukaryotic rRNA sequences. The resulting sequence files were aligned to the template (as described earlier). Reads which had a > 50% match to the template and which mapped to the specified 150 bp region were extracted for unsupervised clustering and classification. These downstream analyses, involved de novo OTU picking (in QIIME) with a 97% similarity threshold, taxonomic assignment of OTUs using the SILVA v119 database, construction of phylogenetic tree using FastTree   were useFastTree , alpha aSRP103550 and reads SRR5435167 - SRR5435190.All the sequences in the present study were deposited to the sequence read archive (SRA) of the NCBI under the project accession number Table 1 outlines the results of the comparison. With an initial mass of 3 g, using 0.6 g and 0.3 g isolation masses resulted in average RNA yield of 21.9 and 195.5 μg RNA/g of rumen solid respectively, average RIN values of 7.3 and 6.9 respectively, and average LS/SS  rRNA ratios of 1.95 and 1.68 respectively. 2 g initial grinding mass with 0.6 g and 0.3 g isolation masses resulted in 17.4 and 123.5 μg RNA/g of rumen solids respectively, 1.85 and 1.55 LS/SS rRNA ratios respectively, and RIN values of 5.0 and 6.0 respectively. Means and standard errors are outlined in the table. On both fronts – of LS/SS ratio and RIN number – these results align well with accepted indices of good RNA quality, even when the least amount of ground material was used in the RNA isolation protocol. A combination of 2 g grinding mass and 0.6 g isolation mass was chosen as the benchmark and was used to evaluate the proportion of TRIzol reagent that might be optimum for total RNA isolation.Functional genomics and gene expression studies depend critically on the ability to isolate high quality RNA. An assessment of RNA integrity is therefore an important first step in obtaining any meaningful data for downstream applications and analysis. Two indices of RNA integrity and quality were used for this assessment (1) ratio of the peak areas of the Large Sub Unit (28S/23S) to the Small Sub Unit (18S/16S) rRNAs (LS/SS ratio) and (2) the RNA Integrity Number (RIN). The eukaryotic 28S/18S ratio may reflect unspecific damage to the RNA, including sample mishandling, post-mortem degradation, massive apoptosis or necrosis . RNA of Table 2 and show that yields of 20.0, 34.1, and 36.5 μg RNA/g RS were obtained from 10x, 7.5x, and 5x TRIzol reagent per unit weight of solid material respectively. LS/SS rRNA ratios were 1.85, 1.75, and 1.65 respectively while RIN values obtained were 7.3, 7.25, and 6.55 respectively. As discussed earlier these values are within acceptable limits for good quality RNA, particularly when the computed standard errors are considered. It is worth noting that in this study the average yields observed from using the least amount of TRIzol reagent evaluated  was even higher than that obtained from the amount usually applied. It can be deduced from these results that using 2 g of solid rumen digesta for initial grinding, 0.6 g of ground material for RNA extraction and 3 mL of TRIzol reagent is sufficient for extraction of good quality RNA. This is an improvement on the existing benchmark and will result in significant cost savings for future experiments.The second stage of this study was aimed at a reduction in the amount of TRIzol that can suitably be used in RNA isolation. It compared outcomes  when smaller amounts of TRIzol reagent per unit weight of ground matter were used in the process. Using an initial mass of 2 g of rumen solid-phase digesta sample and 0.6 g of the ground solid sample we extracted total RNA using 10x, 7.5x and 5x TRIzol reagent per unit mass of ground matter. Average RNA yield and quality arising from these treatments are outlined in Figure 1 shows the relative abundances of rRNA transcripts of the microbial phyla represented in the rumen for the cattle sampled. The variation among the animals sampled was clearly seen in these results. The method was also able to produce very reproducible outcomes as the profiles for each of the duplicates for each sample were very similar. Given the reported impact of diet on resulting taxonomic profiles  samples were obtained from each animal, total RNA was extracted in duplicate using the improved protocol outlined and the taxonomic composition was then evaluated. In total 11278779 reads were obtained, averaging 469949.125 ± 62638.8 (mean ± SE) reads per sample. Among these 37.01 ± 0.48% and 62.95 ± 0.46% belonged to Small and Large Subunit rRNA respectively. Among the Small Subunit rRNA (SSU rRNA) reads 1.61 ± 0.05, 60.92 ± 1.29 and 37.46 ± 1.31% were archaea, bacteria and eukaryotic SSU rRNA, respectively. Eukaryotic rRNA reads were further broken down into 10.48 ± 0.43 and 26.97 ± 1.28% Fungi and Protozoa rRNA respectively  only accounted for an average of 0.172% in the rumen samples. The predominant methanogens in the rumen were Methanobrevibacter, Methanomethylophilus and Methanomicrobium, which made up more than 96% of the active archaea population in the rumen of the cattle sampled, with the Methanosphaera making up the remaining. Methanimicrococcus was only present in trace amounts. The bacterial community was very similar across all samples analyzed. At the phylum level the largest active group of bacteria in the 24 samples were classified into the phylum Firmicutes (57% of the bacteria community) followed by Bacteriodetes (17%) Spirochaetes (8%) and Fibrobacter (7.9%). Other bacteria phyla identified in the rumen which accounted for more than 1% of the active bacteria community were Lentisphaerae (2.3%) and Actinobacteria (1.7%). At the family level , Cyllamyces (36.54 ± 0.61%) and Orpinomyces (7.62 ± 0.27) .The two most active protozoan genus found in the rumen samples were Though the relative active proportions of these microorganisms generally agree with proportions reported in previous studies it is important to note that there are differences. These differences in outcome can be explained by the measurement of the active communities using an RNA approach vs. DNA, the use of a robust isolation technique that enables the isolation of representative communities from fiber samples, and varying experimental conditions including: PCR conditions, choice of primers and cloning vector, other host factors including diets and physiological conditions that differ among experiments .Extracting reads mapped to a specific region of the 16S rRNA gene enabled deeper comparison and statistical analysis of the data and an assessment of the structure of the microbial community from genomic material obtained using the improved benchmark. It allowed comparative interrogation of the dataset and an analysis of the between sample and within sample diversity contained within the data. A total of 104,467 reads  mapped to the specified 250 bp region ranging from 3078 to 9110 reads per sample with a median value of 4146 reads , except that the relative active contribution of the phylum bacteria appears to be more pronounced in the rRNA-Seq output. This could also be the result of differences between the hmmsearch used in the rRNA–HMM program used in RAMMCAP  were made up predominantly of 3 genera, Methanobrevibacter, Candidatus Methanomethylophilus , and Methanomicrobium which together make up 90.6% and account for 44.8, 29.6 and 16.2% of the methanogen community respectively. The least contributors to this population were Methanimicrococcus which made up 0.3%, Methanosphaera which made up 4.3% and an uncultured group of the Methanobacteriaceae family which made up 4.7% of the population. This profile is also very similar to that obtained from an assay of the total rRNA . Additionally this targeted approach revealed deeper differentiation and highlighted (though in trace amounts) the presence of two genera, Methanimicrococcus and an uncultured Methanobacteriaceae, beyond what was possible from the total rRNA assessment. The active bacteria community  with the most predominant subgroups  occurring in similar proportions in both analyses. The active Eukaryota community within the sampled animals is made up primarily of Protozoans  and Fungi  for indices noted in all the animals sampled (and the replicates). Expectedly species richness (estimated by chao1 index), evenness (estimated by Shannon–Wiener index) and the number of observed OTUs were higher in the entire population sampled than was observed for the individual phyla, as the sample space is much wider. Interestingly, while the observed OTUs were markedly different in all the phyla examined, similarly high value for the inverse Simpson’s index  were noted for each of the populations examined. This suggests appropriate diversity, for the microbial populations present in the dataset.It is notable that with this procedure, for 4 of the 5 alpha diversity indices evaluated, higher values were reported for the archaea phylum even though they only make up about 1.4% of the active microbial population. This gives credence to the capability of extracting useful information about microbial populations using this method. Also, even though there were fewer OTUs observed for the Eukaryotic population than was observed for all the other populations examined the eukaryotic sample appeared to have the highest coverage of all with only 10% of the reads occurring from OTUs that appear only once in the sample. On the other hand, the goods coverage index recorded for the entire population was very low compared to that observed for the specific phyla level analysis. This is due to the fact that only a small subset of the entire population is contained within the V4 region and extracting just this region could have negatively impacted the coverage obtained in the sampling.Figure 7) and for the specific phyla  show the capability of this approach to delineate animal level variation with clarity – with the 4 replicates samples for each animal clustering together. These delineations were most visible within the phyla level analyses. The clusters within the eukaryotic and bacterial populations, however, showed tighter clustering than was observed for the archaea population, probably because this phylum (archaea) makes up a very small proportion of the entire microbial community.Between sample (beta) diversity of the communities in this study were evaluated using principal coordinate analysis plots with distances calculated using the Bray-Curtis Dissimilarity index. The plots for the entire population (Comparisons between the results presented in this study and previously published investigations targeting DNA or RNA analysis in the rumen are difficult to make, as the methods of rumen sample preparation are very different. Anaerobic fungal sequences have been virtually absent from previous rumen metagenome and metatranscriptome studies  indicatiThis study set out to explore possibilities of reducing the quantity of materials and reagents that would need to be used to extract total RNA from solid-phase rumen digesta samples, as well as to develop a pipeline of analysis that would elucidate the total active microbial community structure. The purpose was to explore avenues for costs savings in projects involving RNA extraction and to develop a pipeline for total rRNA-Seq analysis. Our method successfully established the possibility of extracting RNA of high quality and integrity from an initial rumen solid digesta mass of as low as 2 g. We also successfully established that a mass of 0.6 g of the ground solid material can be successfully used in the actual RNA extraction procedure and that 5:1 TRIzol reagent/ground mass ratio is suitable for extraction of RNA of good quality rather than the current practice of a 10:1 ratio. This study further demonstrated the possibility of using RNA-Seq in phylogeny and taxonomy studies and in analysis pipelines as would be normally applied to traditional amplicon data by extracting appropriate regions of the total RNA information. This study is the first to elucidate simultaneously the relative active contributions of archaea, bacteria, protozoa and fungi to the rumen community. This method therefore combines the benefits of a breadth of coverage of RNA-Seq with the flexibility and specificity of a targeted amplicon approach, enabling more robust and appropriate statistical analysis of diversity beyond the mere elucidation of taxonomic profiles.All procedures and protocols used in this study were reviewed and approved by the Animal Care Committee at the Lethbridge Research and Development Centre of Agriculture and Agri-Food Canada. Care and management of heifers followed the guidelines of the Canadian Council on Animal Care.CE and RF conceived the research. ZW, XW, and AR conducted the experiments. CE, ZW, and XW analyzed the data. CE, ZW, and RF wrote the paper. All authors approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MP and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Capsid assembly and stability of hepatitis B virus (HBV) core protein (HBc) particles depend on balanced electrostatic interactions between encapsidated nucleic acids and an arginine-rich domain (ARD) of HBc in the capsid interior. Arginine-deficient ARD mutants preferentially encapsidated spliced viral RNA and shorter DNA, which can be fully or partially rescued by reducing the negative charges from acidic residues or serine phosphorylation of HBc, dose-dependently. Similarly, empty capsids without RNA encapsidation can be generated by ARD hyper-phosphorylation in insect, bacteria, and human hepatocytes. De-phosphorylation of empty capsids by phosphatase induced capsid disassembly. Empty capsids can convert into RNA-containing capsids by increasing HBc serine de-phosphorylation. In an HBV replicon system, we observed a reciprocal relationship between viral and non-viral RNA encapsidation, suggesting both non-viral RNA and serine-phosphorylation could serve as a charge balance buffer in maintaining electrostatic homeostasis. In addition, by comparing the biochemistry assay results between a replicon and a non-replicon system, we observed a correlation between HBc de-phosphorylation and viral replication. Balanced electrostatic interactions may be important to other icosahedral particles in nature. Hepatitis B Virus (HBV) is a severe public health problem globally1467910HBV contains four major open reading frames (ORFs), which encode precore/core, surface (envelope), polymerase, and X proteins. The core protein (HBc) contains 183–185 amino acids and can self-assemble into icosahedral capsid particles (capsids)121314151417182021222326in vivo293031323333353637383940Both duck hepatitis B virus (DHBV) core protein and human HBc are known to be phosphorylated baculovirus system, empty capsid particles (empty capsids) were also observedHBV exists in patients as a highly heterogeneous population. The mechanism for the generation of such a high degree of diversity in HBV particles remains to be elucidated. For example, it remains unclear why in the sera of hepatitis B patients, empty virions without any packaged DNA or RNA are highly abundant42436745in vitro this hypothesis by using a capsid disassembly and reassembly system, as well as in vivo by using a charge rebalance approach in a truncated HBc context484243444540Previously, we proposed a charge balance hypothesis which highlights the importance of balanced electrostatic interactions between the positive charge from HBc ARD and the negative charge from encapsidated nucleic acids and HBc itself4048In this study, for the first time, we demonstrated a good correlation between the arginine content of HBc ARD in the full-length HBc context and the size of encapsidated viral RNAs and DNAs. Arginine-deficient mutants encapsidated shorter RNA and DNA than the wild type (WT) HBV. By DNA sequencing, we discovered a number of novel spliced viral RNA species encapsidated in arginine-deficient ARD mutants. These mini-RNA species are not abundant and could not have been found in wild type HBV populations. They become concentrated and enriched only in a capsid environment in severe shortage of positive charge, since they are the fittest match selected by the charge balance principle.E. coli, and human hepatocytes. Furthermore, by using an HBV replicon system, we observed an inverse correlation between the amounts of encapsidated viral and non-viral RNAs. This result suggests that the encapsidated non-viral RNA might function as a charge balance buffer to help maintain an optimum ratio between the positive and negative charges in the capsid interior.By reducing negative charges at acidic residues or serine phosphorylation sites of HBc, we can restore fully or partially the shorter RNA and DNA phenotype of arginine-deficient ARD mutants. Therefore, it appears that negative charge involved in balanced electrostatic interactions in the capsid interior can be donated from three interchangeable or substitutable sources, including encapsidated RNA, acidic residues of HBc, and serine phosphorylation at HBc ARD. Interestingly, using an HBc expression vector for VLP (virus-like particle) production, we demonstrated for the first time that hyper-phosphorylation of HBc is responsible for the formation of empty capsids in insect, In summary, balanced electrostatic interactions appear to be a ground rule in guiding the behaviors of HBV in its life cycle. Balanced electrostatic interaction in the capsid interior is dependent upon (1) positively-charged residues of the core protein; (2) negative charges of acidic or phosphorylated residues; and (3) negative charges of the encapsidated total RNA/DNA contents, controlled by splicing of viral RNA or the co-encapsidation of cellular RNA. Our current studies on capsid assembly/disassembly could provide useful information for translational research in anti-virals, VLP-based vaccines, and drug delivery via nanoparticles or liposomes.lower panel of Previously, we characterized an HBc ARD mutant 164 which truncates 19 amino acids from the C-terminus of the full-length HBc183 . This mu25As a DNA virus, HBV replicates via reverse transcriptionin vitro. We noted that WT HBV also encapsidated a highly abundant 2.2 kb spliced RNA . E180 is located near the carboxyl-terminus of HBc with no known structure18To test further the effect of other acidic residues on electrostatic interactions, we performed charge rebalance experiment using E40A and E180A mutations in the context of mutant ARD-III + IV  substitutions which mimicked constitutive serine de-phosphorylation at major phosphorylation sites of mutant ARD-III + IV. Indeed, the short DNA phenotype of mutant ARD-III + IV can be rescued to produce longer DNA species by mutations S155A and S170A . Further373841In addition to ARD-III + IV, we tested the rescue effect of mutation S162A in the context of mutant ARD-III . Individ253750373841In addition to these three major phosphorylation sites at S155, S162, and S170, there are known minor phosphorylation sites at S168, S176, and S178 3031. It 30baculovirus system contained no encapsidated RNAThe charge balance hypothesis predicts that the encapsidated RNA is important for capsid stability by neutralizing the inter-subunit positive charge repulsion from the adjacent arginine residues40baculovirus core protein expression system  into the ARD of HBc .Finally, in addition to insect cells and utations  and 5. Tutations . In thisbaculovirus system, WT and mutant S1A capsids with hyper-phosphorylated HBc were empty with no detectable RNA, while mutants S3A, S5A and S7A contained increasing amounts of packaged RNA. Again, the amount of encapsidated RNA correlated with the degree of ARD de-phosphorylation. In contrast to S-to-A mutants, S-to-D mutant capsids contained only trace amount of encapsidated RNA due to the excessive negative charge in the capsid interior  Important sequence elements required for viral DNA synthesis7332in vitro due to its notorious non-processive nature.In the latter case, we detected some abundant DNA species by PCR assay, yet not as abundant by RT-PCR assay. For example, the full-length 3.2 kb DNA can be identified as a highly abundant species by PCR cloning and sequencing, yet, the full-length 3.2 kb cDNA by RT-PCR can be hardly detected . We attrIt is known that HBc ARD consists of major and minor phosphorylation sites3031In our charge balance hypothesis, we highlights the importance of the stoichiometry between positive and negative charges in the capsid interior in HBV life cycle4048187072777879In conclusion, in addition to HBV, we anticipate that a similar mechanism of balanced electrostatic interactions in the capsid interior could serve as a guiding principle for other non-HBV viruses in nature.ayw genome under a CMV promoter. Site-directed mutagenesis was conducted using the QuickChange XL Site-Directed Mutagenesis Kit . All mutants were confirmed by DNA sequencing. HuH-7 hepatoma cell line was obtained from Dr. M. Lai at Academia Sinica, TaiwanHBc ARD mutants were engineered from the parental replicon plasmid pCHT-9/3091Experimental procedures for the extractions of encapsidated viral RNA and DNA, as well as Southern and Northern blot analyses, are as described previously40488586Experimental procedures for native agarose gel electrophoresis and immunoblot are as described elsewhere18 and 5 μg of core-associated RNAs were used for reverse transcription reaction by SuperScript III RT kit . To amplify HBV ayw cDNA, the PCR reaction was mixed with forward primer 1818 S (5′-GCAACTTTTTCACCTCTGCC-3′), reverse primer 1824AS (5′-AAGTTGCATGGTGCTGGTGCGCA-3′) and 2 unit of Platinum Taq DNA Polymerase High Fidelity . PCR amplification was performed for 35 cycles. Each cycle consists of denaturation at 94 °C for 30 sec, annealing at 55 °C for 30 sec, and elongation at 72 °C for 6 min. Finally, cDNAs were subcloned into the TOPO TA cloning vector  and confirmed by DNA sequencing. The DNA sequences were analyzed by Spidey . Data and statistical analysis were performed with GraphPad Prism Software version 6.0 .For RT-PCR, 55 μM of oligo (dT)Preparations of intracellular and extracellular viral DNAs were as described previously28EcoRI and NotI restriction sites . All mutants were confirmed by DNA sequencing. Spodoptera frugiperda 9 (Sf9) cells were purchased from BCRC baculoviruses containing various HBc mutants were generated with the Bac-to-Bac baculovirus expression system , and amplified in Sf9 insect cells as describedThe full-length WT HBc and various HBc mutants S1A to S7A were cloned into pFastBac1 expression vector at ayw) plasmid pET-Blue-1-HBc183 as a template. All mutants were confirmed by DNA sequencing. WT and various HBc mutants were expressed in E.coli and purified by a 40–70% sucrose gradientHBc mutants S3D, S5D, and S7D were generated with a QuickChange XL Site-Directed Mutagenesis Kit , using the full-length wild-type (For the HBc VLP system (non-replicon), mutants S1A, S3A, S5A, S7A, S3D, S5D and S7D were generated with a QuickChange XL Site-Directed Mutagenesis Kit , using the plasmid pFastBac-CMV-HBc WT as a template. DNA plasmids of WT and various HBc mutants were transfected into HuH-7 cells. Three days post-transfection, intracellular HBc capsid particles were purified using a 25% sucrose cushion and resolved in 1X TBS. For the HBV replicon system used in 2O before incubation overnight at 37 °C with micrococcal nuclease  in the presence of 8 mM CaCl2 and 5 mM dithiothreitol (DTT). De-phosphorylation was conducted by incubation with λ-phosphatase  at 37 °C for 3.5 hr in the presence of 50 mM HEPES, PH 7.5, 0.1 mM EDTA, 10 mM MnCl2 and 5 mM DTT. Complete digestion by micrococcal nuclease or λ-phosphatase was monitored on 1% native agarose gel, using SYBR Green II staining for encapsidated nucleic acids, SYPRO Ruby for capsid proteins5960The protocol of capsid disassembly by micrococcal nuclease treatment was as detailed elsewhere2+-Phos-tag SDS-PAGE 6263HBV capsids were mixed with 5X SDS-loading dye  and boiled for 10 min before loading onto polyacrylamide-bound MnHow to cite this article: Su, P.-Y. et al. HBV maintains electrostatic homeostasis by modulating negative charges from phosphoserine and encapsidated nucleic acids. Sci. Rep.6, 38959; doi: 10.1038/srep38959 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Accurate thermodynamic parameters improve RNA structure predictions and thus accelerate understanding of RNA function and the identification of RNA drug binding sites. Many viral RNA structures, such as internal ribosome entry sites, have internal loops and bulges that are potential drug target sites. Current models used to predict internal loops are biased toward small, symmetric purine loops, and thus poorly predict asymmetric, pyrimidine-rich loops with >6 nucleotides (nt) that occur frequently in viral RNA. This article presents new thermodynamic data for 40 pyrimidine loops, many of which can form UU or protonated CC base pairs. Uracil and protonated cytosine base pairs stabilize asymmetric internal loops. Accurate prediction rules are presented that account for all thermodynamic measurements of RNA asymmetric internal loops. New loop initiation terms for loops with >6 nt are presented that do not follow previous assumptions that increasing asymmetry destabilizes loops. Since the last 2004 update, 126 new loops with asymmetry or sizes greater than 2 × 2 have been measured. These new measurements significantly deepen and diversify the thermodynamic database for RNA. These results will help better predict internal loops that are larger, pyrimidine-rich, and occur within viral structures such as internal ribosome entry sites. Amidst a flood of sequence information, lightening-fast development of RNA therapeutics, whirlwind discoveries of novel RNA gene regulation functions, and the thunderous threat of outbreaks from viral RNA pathogens such as Ebola and Zika, RNA thermodynamics provide a compass to navigate the storm. Accurate thermodynamic measurements of RNA motifs form the core for a wide array of approaches to predicting RNA structure and function from sequence  to produce viral proteins . InternaThe differences between the thermodynamic database and the IRES database may be a contributing factor in the inaccuracies of the current prediction model . This arn × n loops where n is greater than three). Fifty percent of the loops within the thermodynamic database contained only purines. In contrast, only 14% of the loops within the IRES database were purines. Sixty-two percent of the loops were a mixture of purines and pyrimidines in the IRES database. Lastly, the distribution of symmetric and asymmetric loops in both databases was completely opposite. Sixty-five percent of the loops within the thermodynamic database were symmetric, primarily due to the substantial number of 2 × 2 loops. In contrast, 72% of the IRES loops were asymmetric. The differences between the two databases may contribute to the limited accuracy of predictions for viral RNA structures  is more stable than the smallest all-cytidine 2 × 2 loop .As shown in Supplemental Table 1; The loop free energies determined experimentally were then compared to current predicted values (UUU CUGA/3′ACUG UUUU GACU (duplex xix) has the potential for a total of three noncanonical UU pairs and a free energy of 2.2 kcal/mol, whereas 5′UGAC CUC CUGA/3′ACUG CUUC GACU (duplex xx) has the potential for one UU pair and has a free energy of 3.0 kcal/mol.Although the 2004 prediction model  gives a Supplemental Figure 1, four of the five duplexes that have the potential to form UU pairs show peaks in this region. The loop sequence 5′CUUC/3′GCCCCG (duplex xxxv) has no potential UU pairs and thus serves as the control and helps assign imino proton resonances in the stem Watson–Crick pairs. At least seven of the eight expected UU imino peaks were observed in the spectra of the 4 × 4 all-uridine loop (duplex xxviii) , which suggests multiple UU hydrogen-bonded pairs form in this loop. This 4 × 4 all-uridine loop is also among the most thermodynamically stable loops. The imino proton spectra demonstrate the formation of stable hydrogen-bonded UU pairs that can occur adjacent to and also nonadjacent to the closing Watson–Crick pair of the loop. These NMR data support the free energy bonus term for loops with the potential to form UU pairs (see Discussion below).One-dimensional imino proton experiments were then performed to test if UU nucleotides form hydrogen-bonded pairs across asymmetric internal loops. All of the oligonucleotides studied by NMR have identical stems, and thus four GC and two AU imino protons are expected in all of the NMR spectra. UU imino protons resonate around 10.4–11.3 ppm  and thusAs shown in Symmetric and asymmetric loops show different stabilizing effects at pH 5.5. In general, symmetric loops are more stable than asymmetric loops, and smaller loops are more stable than larger loops at pH 7, which is consistent with previous studies of adenine loops  and cytiUUCUU3′/3′UUUCACG5′ (duplex iv), based on a loop sequence in the Hepatitis A IRES  is less stable than the 4 × 5 all-cytidine loop , however. Thus, the additional stability of the possible U–U pairs may be less than the protonated cytosine pairs at pH 5.5. The different stabilities for IRES loops at different pH conditions may facilitate conformational changes in viral RNA that are triggered by pH changes, such as virus disassembly.Interestingly, a 4 × 5 asymmetric loop, 5′As A IRES , shows a+C+ pairs  is proposed in Equation 016 is prCUCC/3′GCUUCG at pH 7 (duplex xx), add together 3.7 kcal/mol (ΔGloop + ΔGasymmetry), 0.0 kcal/mol , and −0.5 kcal/mol (ΔGUU bonus). Using the new parameters for stabilizing pairs across an internal loop and revised initiation terms for larger sized loops, the prediction of the experimental free energies for asymmetric loops is within 0.3 kcal/mol on average. Sequence-dependent prediction rules for symmetric loops are still under development in collaboration with the Turner laboratory and alumni. Supplemental Table 1. New initiation terms for asymmetric loops are proposed in The 2016 model retains the value of −0.7 kcal/mol for an AU closing base pair. This parameter repeatedly gave the same value within error for all linear regression analyses. The value of −0.7 kcal/mol is consistent with measurements of the free energy of a hydrogen bond and accounts for the difference in the number of hydrogen bonds in AU and GC pairs . The samThe GG bonus varies widely between different loops. In 2 × 4 loops, a GG pair adds −2.0 kcal/mol while a GG pair in a 3 × 4 loop is destabilizing by 1.4 kcal/mol. Guanine nucleotides have many hydrogen bond donors and acceptor sites and are able to form many different types of pairs . GuanineUridine nucleotides have weaker stacking interactions than guanine nucleotides and show less variation in the bonus for UU pairs . The val+C+ pairs could form with two hydrogen bonds. There are several possible conformations for CC and CU pairs that would be disrupted by cytosine protonation  at neutral pH show stacking interactions and a single hydrogen bond between the N3 and amino groups or a bifurcated hydrogen bond from the amino group to the N3 and the carbonyl . This tytonation . Cytidintonation . If the The thermodynamic database for asymmetric internal loops has been curated and expanded. The new initiation terms for loops containing >6 nt greatly improved accuracy in predicting the experimental free energies. The stabilities of large, highly asymmetric internal loops that occur in virus IRES may be more favorable than previously predicted. The bonus terms for GA, GG, UU, and protonated CC pairs vary with the size and asymmetry of the loop. The thermodynamics obtained from optical melting experiments suggests that protonated cytosines and uracil base-pairing at any position in internal loops contribute to loop stability. One-dimensional imino proton NMR spectra validate the formation of UU base pairs, even when the pairs may not stack directly on the closing Watson–Crick pairs. The bonus terms for cytidine loops at low pH will improve predictions for viral RNA that undergo conformational changes in different pH environments. These results will contribute to the ongoing effort to expand and diversify the RNA thermodynamic database and improve RNA structure prediction.The IRES structures studied were obtained from iresite.org , which c2EDTA, and 10 mM sodium cacodylate or 50 mM MES at pH 7.01 or 5.54, respectively. One-dimensional imino proton NMR data were collected with samples at 0.5 mM RNA concentrations in NMR buffer containing 10 mM NaCl, 10 mM potassium phosphate in 90% H2O, and 10% D2O at pH's 6.0 and 4.5 with Watergate solvent suppression on a 500 MHz Varian NMR spectrometer , −11.5 (ΔGstems 5′UGACCUGA/3′ACUGGACU), and −3.3 kcal/mol (ΔGinterrupted nearest neighbor 5′CC/3′GG).For example, in order to calculate the experimental free energy for the 3 × 4 loop 5′Ct and P tests. Loops were grouped by size and asymmetry, and new prediction rules were tested for each loop type.Data from optical melting experiments were fit using the linest function in Excel. The loops at pH 7 from this work were combined with all previous measurements of internal loop stabilities . ThermodSupplemental material is available for this article.


We demonstrated that the UFS was critical for efficient NS5 recruitment and viral RNA synthesis in different flaviviruses. Interestingly, stabilization of the DENV UFS impaired both genome cyclization and vRNA replication. Moreover, the UFS unwound in response to genome cyclization, leading to the decreased affinity of NS5 for the viral 5′ end. Thus, we propose that the UFS is switched by genome cyclization to regulate dynamic RdRp binding for vRNA replication. This study demonstrates that the UFS enables communication between flavivirus genome cyclization and RdRp recruitment, highlighting the presence of switch-like mechanisms among RNA viruses.Viral replicase recruitment and long-range RNA interactions are essential for RNA virus replication, yet the mechanism of their interplay remains elusive. Flaviviruses include numerous important human pathogens, e.g., dengue virus (DENV) and Zika virus (ZIKV). Here, we revealed a highly conserved, conformation-tunable DOI:http://dx.doi.org/10.7554/eLife.17636.001 Flaviviruses include a large family of viruses that are harmful to human health, such as dengue virus, West Nile virus and Zika virus. Understanding the details of the life cycle of these viruses is important for better controlling and treating the diseases that they cause.The genetic information of flaviviruses is stored in single-stranded molecules of RNA. To form new copies of a virus, the RNA must be replicated in a process that involves two critical steps. First, an enzyme called viral RNA polymerase NS5 must be recruited to a specific end of the RNA strand (known as the 5′ end). Then, the ends of the RNA strand bind together to form a circular loop. However, little is known about whether these two processes are linked, or how they are regulated.Using bioinformatics, biochemical and reverse genetics approaches, Liu et al. have now identified a new section of RNA in the 5′ end of the flavivirus RNA, named the 5′-UAR-flanking stem (or UFS for short), which is critical for viral replication. The UFS plays an important role in efficiently recruiting the NS5 viral RNA polymerase to the 5′ end of the flavivirus RNA.After the RNA forms a circle, the UFS unwinds. This makes the NS5 polymerase less likely to bind to the 5′ end of the RNA. Stabilizing the structure of the UFS impairs the ability of the RNA strand to form a circle, and hence reduces the ability of the RNA to replicate. Thus, the UFS links and enables communication between the processes that form the flavivirus RNA into a circle and that recruit the viral RNA polymerase to the RNA.The structural basis of the interaction between the flavivirus RNA 5′ end, including the UFS element, and the viral RNA polymerase now deserves further investigation. It will be also important to explore whether other types of viruses regulate their replication via a similar mechanism.DOI:http://dx.doi.org/10.7554/eLife.17636.002 Cis-acting regulatory elements play essential yet diverse roles in the life cycle of RNA viruses , West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV) and the emerging Zika virus (ZIKV) . Major plication , translalication , viral plication  and hostlication .Hybridization of complementary sequences at the 5′ and 3′ termini, which include the 5′-3′ upstream AUG region (UAR) , downstrmfold RNA folding server . The SHAPE reactivity data were annotated on RNA structure models generated by mparison . The SHAmparison  because mparison , except mparison . This remparison . It was mparison , D3-M13Cmfold online server. It was shown that the overall genome cyclization pattern was only slightly affected by the M1 and M3 mutations -controlled viral translation . To elimutations , and theutations , which nRNAstructure  or with a disrupted cHP structure (cHP-M), were assayed in parallel as controls, which showed non-detected or reduced RdRp activity for de novo initiation , in agreand vRNA in vivo. In fact, the PABP protein, which bridges mRNA ends through poly(A)-PABP-eIF4E-5′ cap interactions, has been shown to bind to the DENV 3′ UTR , it is plausible to consider that the UFS is a general regulatory strategy of viral replication among the flavivirus genus. Although a previous report  has suggBy applying a systemic RNA structure prediction and comparison approach covering different members of the flavivirus genus, the structure of the downstream SLA region in the 5′ end of the flavivirus genome was unambiguously illuminated. The UA-base-pair-rich UFS and the UFS-like element have been identified in MBFVs, two clades of NKVs, and ISFVs, demonstrating that the UFS element is evolutionarily conserved. Interestingly, while the UFS duplex unwinds during genome cyclization in MBFVs and NKVs, neither the conserved mode of genome cyclization nor the unwinding of UFS-like elements during this process was found in ISFVs. The ISFVs have been indicated to be the most divergent outgroup among the genus flavivirus , which lUFS elements usually localize close to, or overlap with, the translation start region, suggesting a possible role for the UFS in viral translation regulation. Moreover, the replication of most positive-strand RNA viruses occurs in virus-induced membrane structures . It is pFunctional long-range interactions are likely to be a general mechanism in positive-strand RNA viruses . Genome E. coli strain BD1528 were kindly provided by Professor Stephen S. Whitehead. All replicon constructs were also manipulated using BD1528. The p4-Dualstop-SP-IRES-Rluc-Rep replicon was generated by introducing a nonsense mutation into the 26th codon of the capsid ORF (CAA to TAA) in the background of p4-cHPstop-SP-IRES-Rluc-Rep  and was cloned into pGEM-T easy  to generate the cloning cassette vector, pBseRI-Dualstop-04. DNA oligos containing the desired mutations of the UFS were annealed and ligated with BseR I-digested pBseRI-Dualstop-04 to acquire Asc I-SnaB I fragments with the desired mutations, which were then subcloned back into p4-Dualstop-SP-IRES-Rluc-Rep.For the convenience of introducing mutations into the UFS region, the Asc I-SnaB I fragment from p4-Dualstop-SP-IRES-Rluc-Rep, which contained the SP6 promoter region and the DENV4 5′ UTR-capsid ORF sequence, was engineered such that the sequence corresponding to the 70–107 nt region of the DENV4 genome was substituted by the following: 5′- To generate UFS mutants in the p4 infectious clone and p4-GVD, the Asc I-BstE II fragments with desired mutations were generated by overlapping PCR using the corresponding replicon mutants and p4 infectious clone DNA as templates for amplification, cloned into pGEM-T easy, and then subcloned into p4 and p4-GVD, respectively.Site-directed mutagenesis method was employed for the generation of ZIKV fragments containing the desired UFS mutations, the generated Not I-Avr II restriction fragments were then subcloned into the infectious clone of ZIKV strain FSS13025 , which wThe DENV4 mini-genomes were constructed by joining various 5′-300 nt fragments with the 3′ UTR sequence using overlapping PCR techniques. The mutations targeting cHP and 5′CS were also introduced by overlapping PCR.The DENV2 and DENV3 5′ end sequences were acquired by RT-PCR using the DENV2 strain NGC (KM204118) and the DENV3 strain 80–2 (AF317645) RNA as templates respectively, and cloned into pMD-19T-simple . The 5′ end sequence of DENV1 strain WestPac (U88535) was chemically synthesized and cloned into pUC57 by Thermo Fisher Scientific. The SP6 promoter sequence was placed upstream of the DENV1 5′ end sequence. These constructs, together with the JEV infectious clone pAJE70 , which cReplicon and full-length viral RNA were in vitro transcribed as described previously . To gene2. At the indicated time points, the cells in the microplates were lysed with 1× Renilla lysis buffer  and stored at -20°C until use. The Renilla luciferase activity of the collected samples was measured as described previously  and fixed with acetone/methanol (v/v: 3/7) at 4°C. The cover slips were incubated with the anti-dengue envelope protein monoclonal antibody 2A10G6 at 37°C for 1 hr for the cells transfected with DENV4 RNA, and anti-ZIKV envelope protein mAb clone 0302156  was used for the cells transfected with ZIKV RNA. After the incubation with primary antibodies, the cover slips were washed with PBS for three times. AlexaFluor 488-labeled goat anti-mouse IgG  was then added, and after a 1-h incubation, the cover slips were washed as described above. For cell nuclei staining, 4', 6-diamidino-2-phenylindole  was added onto the cover slips and incubated for 5 min. An Olympus BX51 microscope under the control of DP72 software was utilized for image capture.BHK-21 cells electroporated with corresponding vRNA were seeded onto cover slips in 24-well plate-format and cultured at 37°C in 5% CO2, RNA molecules (approximately 9.66 pmol) corresponding to the WT or mutated 5′ end sequence of different flaviviruses were diluted into 12-μl reactions containing 0.5× TE buffer, heated at 95°C for 2 min and snap-cooled on ice immediately. Then 6 μl of 3.3× RNA folding buffer  in the presence of 0.125 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 16°C for 20 hr. The cell pellets were harvested and washed once with Buffer A  and resuspended in Buffer A containing cOmplete EDTA-free protease inhibitor cocktail tablets . After ultra-sonication, the suspension was centrifuged at 30,000 g at 4°C for 20 min. The soluble recombinant NS5Pol protein in the supernatants was then purified using a HisTrap HP column . The fraction containing NS5Pol was eluted with Buffer A containing 300 mM imidazole. The eluted protein was exchanged into 50 mM Tris.Cl (pH 8.0), 300 mM NaCl and 20% glycerol using a HiTrap desalting column  and concentrated to mg/ml grade by ultrafiltration using Amicon Ultra-15 Centrifugal Filter Units . Dithiothreitol (DTT) was added into the purified NS5Pol protein to a final concentration of 1 mM. The final product was dispensed into single-use aliquots and stored at -80°C before use. NS5Pol of JEV SA-14-14-2 strain was cloned, expressed and purified using the same strategy.The coding sequence of the DENV4 NS5 RdRp domain  was cloned into a pET-28a (+) expression vector using Nhe I/Xho I restriction sites. The recombinant NS5Pol carrying an N-terminal His-tag was expressed in 2) was added to the samples, and the RNA was refolded at 37°C for 20 min. The concentration of the renatured RNA was set to 500 nM. The binding reactions contained 50 nM 5′-300 nt RNA, 4 μl of 5× EMSA buffer , 0.05 mg/ml heparin sodium salt, 7.5% glycerol and different amounts of NS5Pol  in a 20 μl volume. Due to the solvent components in RNA and protein preparations, the final reactions contained 64.15 mM Tris.Cl (pH 8.0), 182.5 mM NaCl, 5.5 mM MgCl2, 40 nM EDTA and 375 nM DTT. The reactions were incubated at 30°C for 30 min, and then, 10× gel loading solution  was added, and the mixtures were separated by electrophoresis on 6% native PAGE gels running in 0.5× TBE. The gel apparatus was placed in an ice-water bath to prevent the dissociation of RNA-protein complexes. After running at 60 V for 4 hr, the gels were stained with SYBR Gold nucleic acid gel stain  for 30 min, and gel pictures were captured using a BioSpectrum Imaging System  with a UV transilluminator. EMSAs using 5′-160 nt RNA or mini-genome RNA were performed similarly, with a few exceptions: for the 5′-160 nt RNA-based EMSA, the NS5Pol concentrations were higher ; for the mini-genome-based EMSA, the heparin concentration was set to 0.16 mg/ml, and the electrophoresis was performed with 4.5% PAGE gels at 75 V for 6 hr in an ice-water bath. EMSA assay using JEV NS5Pol and 5′-320 nt RNA was performed with similar conditions for DENV4 5′-300 nt RNA EMSA assay.An RNA-RNA binding EMSA was performed as described previously  with min2) was added, and the RNA templates were folded at 37°C for 20 min. Unless otherwise specified, the 30-μl RdRp reactions contained 450 ng of template RNA (3 μl of 150 ng/μl folded RNA), 55.6 mM Tris.Cl (pH 7.4), 12 mM NaCl, 5.5 mM MgCl2, 2 mM MnCl2, 10 mM DTT, 40 U of recombinant RNase inhibitor (40 U/μl), approximately 5.4 μg NS5Pol (in 1 μl) and 0.5 mM ATP, GTP, and UTP; 0.1 mM CTP; and 0.25 mM biotin-11-CTP . According to previous reports (In vitro RdRp initiation assays were performed using: (I) 5′-160 nt RNA of DENV4, (II) DENV4 mini-genome RNA as templates. The RNA templates were diluted with RNase-free water to a final volume of 8 μl, then heated at 95°C for 2 min and immediately cooled on ice. Two microliters of RNA fold buffer T2 . Briefly, Paired t-test was performed for  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Wenhui Li as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Jeffrey S Kieft (Reviewer #1) and Alexander Khromykh (Reviewer #3).Thank you for submitting your article "Viral RNA switch mediates the dynamic control of flavivirus replicase recruitment by genome cyclization" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:eLife.Liu et al. propose that a previously undetected structural element in the 5' UTR of diverse flaviviruses is important for replication, but that upon genome cyclization this structure is disrupted by 5'-3' interactions. They propose that the RNA acts as a structural switch to coordinate cyclization and RdRP binding. Overall, the reviewers found that the model presented is reasonable but is not adequately supported by the experimental results. If the authors can provide additional data, analysis, and discussion to support this model, the manuscript may become suitable for publication in Essential revisions:1) The authors used many mutants, taking into account the impact on the linear form of the genome. However, the authors must also consider and discuss the impact of these mutations on RNA cyclization and on the internal loop structure (including UFS sequences) in the circular form. The predicted cyclization of DENV4 RNA is similar to that reported for JEV  The data shown in 3) The authors find that NS5 affinity for the RNA is reduced when the UFS structure is disrupted by mutations. Considering that this observation is not caused by changes in SLA, the results do not agree with previous observations reported for DENV2. Specifically, mutations, deletions or complete replacement of UFS by unrelated sequences did not affect NS5 binding nor RNA synthesis in vitro  The authors propose that UFS formation is necessary for NS5 recruitment (linear form of the RNA) and when cyclization takes place, NS5 is released, promoting interaction with the 3' end initiation site of the RNA. This idea is interesting; however, the data presented is not sufficient to support the model. The minigenome WT and mutants bind the polymerase NS5 with low affinity , but not5) 6) The authors indicate that "UFS formation is essential for flavivirus replication" . This RNA structure may function as enhancer of DENV4 replication but is not essential . In addi Summary:Liu et al. propose that a previously undetected structural element in the 5' UTR of diverse flaviviruses is important for replication, but that upon genome cyclization this structure is disrupted by 5'-3' interactions. They propose that the RNA acts as a structural switch to coordinate cyclization and RdRP binding. Overall, the reviewers found that the model presented is reasonable but is not adequately supported by the experimental results. If the authors can provide additional data, analysis, and discussion to support this model, the manuscript may become suitable for publication in eLife.Essential revisions:1) The authors used many mutants, taking into account the impact on the linear form of the genome. However, the authors must also consider and discuss the impact of these mutations on RNA cyclization and on the internal loop structure (including UFS sequences) in the circular form. The predicted cyclization of DENV4 RNA is similar to that reported for JEV . The results of RNA structure prediction by mfold were provided as supplementary file, and the corresponding information has been added in the manuscript. It was shown that the overall genome cyclization pattern was only slightly affected by the M1 and M3 mutations  The data shown in RNAstructure software to generate results of RNA structure predictions by incorporating the constraints of SHAPE data as suggested, and the results of prediction indicated that the secondary structure of SLA was not affected by the DENV4 mutants M1A, M1C, M3A and M3C, thus the changes in NS5 binding and RNA synthesis of UFS mutants were not induced by unwanted disruption of the SLA element. The above results also confirmed that the UFS structure was disrupted/destabilized or reconstituted as designed. These results of prediction were provided as supplementary file (http://www.chem.unc.edu/rna/qushape/). As the M1A/M3A mutants exhibited more highly reactive sites  The authors find that NS5 affinity for the RNA is reduced when the UFS structure is disrupted by mutations. Considering that this observation is not caused by changes in SLA, the results do not agree with previous observations reported for DENV2. Specifically, mutations, deletions or complete replacement of UFS by unrelated sequences did not affect NS5 binding nor RNA synthesis in vitro  The authors propose that UFS formation is necessary for NS5 recruitment (linear form of the RNA) and when cyclization takes place, NS5 is released, promoting interaction with the 3' end initiation site of the RNA. This idea is interesting; however, the data presented is not sufficient to support the model. The minigenome WT and mutants bind the polymerase NS5 with low affinity , but notnot present in these mini-genomes except for the CS-M mini-genome, which cannot circularize since the 5' CS element was mutated. Because of the above reasons, the mini-genomes except CS-M bind the polymerase NS5 with low affinity as shown in Thanks for the suggestions. We have analyzed the results of 5) Thanks for the suggestions. We have re-performed the experiments shown in 6) The authors indicate that "UFS formation is essential for flavivirus replication" . This RNA structure may function as enhancer of DENV4 replication but is not essential . In addiThanks for the suggestions. We have changed the corresponding description as suggested. In order to extend our findings to other flaviviruses, firstly, we performed in vitro EMSA assay using JEV as the model species , which d


Transcription control is the foundation of gene regulation. Whereas a cell is fully equipped for this task, viruses often depend on the host to supply tools for their transcription program. Over the course of evolution and adaptation, viruses have found diverse ways to optimally exploit cellular host processes such as transcription to their own benefit. Just as cells are increasingly understood to employ nascent RNAs in transcription regulation, recent discoveries are revealing how viruses use nascent RNAs to benefit their own gene expression. In this review, we first outline the two different transcription programs used by viruses, i.e., transcription (DNA-dependent) and RNA-dependent RNA synthesis. Subsequently, we use the distinct stages  to describe the latest insights into nascent RNA-mediated regulation in the context of each relevant stage. All living organisms are fully equipped to synthesize messenger RNA (mRNA) using their endogenously encoded multi-subunit RNA polymerases (RNAPs). However, many viruses form an exception to this dogma and employ a parasitic lifestyle that exploit parts of the host transcription pathways. This co-option allows viruses to dramatically downsize their genomes to the minimum number of genes essential for successful infection. As a result, viruses and their hosts have been involved in an eternal battle of adaptation and counter-adaptation for RNA synthesis since far before the emergence of humans ,2,3. DurRNAs have many attractive properties that can be used by viruses to manipulate gene regulation pathways. First, RNAs, like DNAs, can specifically pair with complementary viral or host sequences using minimal investment of genetic material, thus allowing further minimization of the viral genome. Encoding a DNA-binding protein, on the other hand, consumes significantly more genetic information. A typical DNA-binding domain recognizes ~10 base pairs (bp) or more of DNA and comprises at least ~60 amino acids (aa), thereby requiring 180 bp of encoding DNA . In contFor decades, the principal focus of gene regulation studies has been on protein regulators, but recent discoveries in virology highlight the ever-increasing appreciation that RNAs, including nascent RNAs, also play a major part in gene regulation by viruses. In this review, we will describe several recent discoveries of intriguing viral pathways that manipulate gene expression of the host for their own benefit via nascent RNA. To place these discoveries in context, we will first describe the current understanding of the distinct stages  in the general mechanism of eukaryotic transcription, as used by the host and virus, and present examples of nascent RNA-mediated regulation in the context of each relevant stage.1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7) .,22.21,22Whereas DNA viruses only need to generate mRNA, RNA viruses without a DNA stage have to synthesize both vRNA and mRNA. The vRNA is generated through a replication intermediate, named antigenome which serves as a template for vRNA synthesis. ssRNA viruses evolved sophisticated approaches to generate both RNA species from one template. Using respiratory syncytial virus (RSV) as a model, it was illustrated that RdRp has two RNA synthesis activities which determine nascent RNA fate. RSV is a −ssRNA virus and the major cause of respiratory tract disease in infants and young children, causing between 66,000 and 199,000 deaths per year worldwide . To diffS-adenosyl-l-methionine (AdoMet)-dependent cap (guanine-N7) methyltransferase (MTase) to produce the final cap structure (m7GpppN-). Higher eukaryotes and some viruses include an additional methylation step in which the 2′ OH position of the penultimate ribose is methylated by cap ribose-2′-O-MTase (m7GpppNm-).5′-terminal capping of eukaryotic mRNAs is probably the first co-transcriptional RNA processing event. Formation of the 5′-terminal cap structure  do not have a DNA template and thus cannot rely on the cellular capping enzymes. For this reason, RNA viruses have evolved capping mechanisms that are different from capping during cellular transcription. Most +ssRNA viruses  encode capping enzymes that selectively cap the newly synthesized viral mRNAs ,44,45. FOn the other hand, unsegmented −ssRNA viruses  carry an RdRp termed “L” and a phosphoprotein “P”, which share extensive amino acid sequence similarity among all these viruses . P is anAnother solution to the capping problem of the negative-sense RNA viruses comes from the influenza virus in the form of “cap-snatching” from nascent host pre-mRNAs . To syntAfter entry into the cell, the vRNPs are released and transported into the nucleus. After arrival in the nucleus, the C-terminal domain of the FluPol PA subunit will specifically target and bind the phosphorylated Ser5 of actively transcribing RNAPII CTD  Figure E. SubseqElongation is the repeated addition of a nucleoside monophosphate (NMP) to the 3′ end of the growing RNA chain. Progressive RNA synthesis during the elongation stage can be affected by several hurdles, including transcriptional pause sites, that RNAPII must overcome . These tA promoter-proximal pause will often allow the nascent RNA to fold a structure that can serve as a protein-recruiting scaffold . Additiotrans-acting region  ion TAR; A 76]. A. Atrans-ion TAR; B. Howeveion TAR; B. As a rtrans-activator protein (Tat) [The folding of the TAR hairpin is key to the regulation of HIV-1 transcription  as it isin (Tat)   . P-TEFb in (Tat) . Second,in (Tat) , and thiin (Tat) ,99. Howein (Tat) . Tat wilin (Tat) . As soonin (Tat) .Interestingly, the human T-lymphotropic virus type 1 transcriptional activator Tax also utilizes P-TEFb for viral transcription and displaces P-TEFb from 7SK snRNP through binding CycT1 ,102, sugcis-acting scaffolds to recruit TFs, viruses can also use noncoding RNAs. A recent example described that the Epstein-Barr virus (EBV) uses a virally encoded noncoding RNA named EBV-encoded RNA 2 (EBER2) that acts as a trans-acting guide to promote its own transcription [6 copies; EBER2: ~2.5 × 105) [Instead of using promoter-proximal paused transcripts as cription    and loca5 × 105) . Due to 5 × 105) , but the5 × 105)  found th5 × 105) . EBER2 l5 × 105) . This co5 × 105) ,112. TopThe last stage of transcription is termination, which leads to the release of the RNA transcript and the dissociation of RNAP from the template DNA. Upon encountering sequence-encoded termination signals , factors will be recruited that cleave and modify the RNA 3′ end. Eukaryotic mRNA undergoes multiple types of modifications at its 3′ end throughout its life cycle . For exaMany +ssRNA viruses lack a poly (A) tail at the 3′ end of their genome, but are still efficiently translated. For instance, flaviruses  have a capped RNA genome which contains conserved sequences at the 5′ and 3′ ends, allowing for circularization and efficient translation ,120. OthAdditionally, premature transcription termination events are being uncovered that allow viruses to produce another layer of transcripts encoded by the same genome. By using these sub-genomic transcripts, viruses can create regulatory RNA molecules that undergo a different fate than the main vRNA. This can for instance be miRNA production by retroviruses  or the aViruses can downregulate host transcription by targeted mRNA degradation, to benefit their own gene expression. In this way, the host transcription machinery is deprived of targets and the virus can redirect the machinery to its own viral expression. As a beneficial side-effect to the virus, lowering host transcription will also reduce the effectiveness of the immune system. Therefore, during infection with many human viruses, the accumulation of viral proteins is accompanied by a progressive global reduction in the production of host proteins, a phenomenon that has been termed “host shutoff”. Viral factors that can induce host shutoff include severe acute respiratory syndrome (SARS) coronavirus nsp1 ,129, herLike all influenza viruses, IAV is a segmented negative strand RNA virus. The PA-X protein-encoding gene overlaps with the open reading frame (ORF) for the PA FluPol subunit . RibosomKSHV is a γ-herpesvirus that causes tumors of endothelial cells and B cells. During lytic infection with KSHV, global degradation of host RNA is triggered by a virally encoded protein named shutoff and exonuclease (SOX) ,141,142.Notably, viral mRNA transcription escapes from decay-induced repression, and this escape also relies on XrnI. However, the mechanisms that allow viral mRNAs, unlike cellular mRNAs, to escape from XrnI cleavage are unknown.cis or in trans—as a scaffold to recruit host transcription factors; (ii) use host-capped RNAs as primers for RNA synthesis; (iii) use termination of nascent RNA synthesis to create sub-genomic mRNAs, and (iv) and down-regulate host nascent RNA production for their own benefit.A complex interplay between viruses and the host transcription machinery that is mediated by nascent RNA has recently become increasingly apparent. Here, we explored a few of the myriad pathways by which viruses (i) use their own nascent RNA—in Viral RNA synthesis pathways are promising targets for antiviral development because these mechanisms are essential for virus replication. However, in order to identify additional weak spots in viral infection programs, it is important to understand how viruses regulate their gene expression and to disentangle the complex ways in which these pathways are intertwined with host transcription. Many antivirals display adverse side-effects because the viral target molecule and process was incompletely understood. The recently described new insights into the roles of nascent RNA in viral gene regulation are important steps in the path to new, more effective antivirals with fewer side-effects.


Persons with psychosocial disabilities face disparate access to healthcare and social services worldwide, along with systemic discrimination, structural inequalities, and widespread human rights abuses. Accordingly, many people have looked to international human rights law to help address mental health challenges. On December 13, 2006, the United Nations formally adopted the Convention on the Rights of Persons with Disabilities (CRPD) – the first human rights treaty of the 21st century and the fastest ever negotiated.This study assesses the CRPD’s potential impact on mental health systems and presents a legal and public policy analysis of its implementation in one high-income country: Canada. As part of this analysis, a critical review was undertaken of the CRPD’s implementation in Canadian legislation, public policy, and jurisprudence related to mental health.While the Convention is clearly an important step forward, there remains a divide, even in Canada, between the Convention’s goals and the experiences of Canadians with disabilities. Its implementation is perhaps hindered most by Canada’s reservations to Article 12 of the CRPD on legal capacity for persons with psychosocial disabilities. The overseeing CRPD Committee has stated that Article 12 only permits “supported decision-making” regimes, yet most Canadian jurisdictions maintain their “substitute decision-making” regimes. This means that many Canadians with mental health challenges continue to be denied legal capacity to make decisions related to their healthcare, housing, and finances. But changes are afoot: new legislation has been introduced in different jurisdictions across the country, and recent court decisions have started to push policymakers in this direction.Despite the lack of explicit implementation, the CRPD has helped to facilitate a larger shift in social and cultural paradigms of mental health and disability in Canada. But ratification and passive implementation are not enough. Further efforts are needed to implement the CRPD’s provisions and promote the equal enjoyment of human rights by all Canadian citizens – and presumably for all other people too, from the poorest to the wealthiest countries. Approximately 15 % of the world’s population lives with disabilities. This means there are almost one billion people who face everyday inequality, marginalization, and discrimination through inequitable opportunities, as compared to persons without disabilities . The stoOverall, it is clear that people with psychosocial disabilities worldwide do not have access to the basic mental healthcare, support, and social services that they require. Many are excluded from their community life, face abject levels of intolerance, and are denied the right to marry and have children . Such syThis article assesses the practical impact of the United Nations (UN) Convention on the Rights of Persons with Disabilities (CRPD) on domestic policymaking and court decisions in one high-income country: Canada. As a Western democracy with a long history of human rights protection, Canada theoretically represents a high watermark against which other high-income countries can compare themselves and from which countries with historically fewer human rights protections can learn. It is also a federal country, one in which there are frequent battles over jurisdiction between the federal and provincial governments over their roles and responsibilities. Accordingly, there are challenges with implementing international agreements that are ratified by one level of government and which require changes at another level of government. A case study of Canada is also helpful to reveal the extent to which human rights treaties have any impact on high-income countries. The trend in newer international agreements is to make them relevant for all state parties and to ensure they are not just about dictating poor countries’ policies from afar. But if they have no impact on high-income countries, then this trend may not be taking hold as much as might be hoped.Overall, this study shows that while the CRPD remains conspicuously absent from Canadian legislation, public policy, and jurisprudence, the country’s ratification of the Convention has facilitated an important shift in the social and cultural paradigms surrounding psychosocial disability in Canada. As a result, this new international human rights treaty may be aiding the everyday struggles of persons living with psychosocial disabilities – even in the wealthiest countries – by facilitating larger changes in social norms and expectations around such disabilities.To adequately assess the CRPD’s potential impact on mental health systems in Canada, three steps were taken to collect data. First, an extensive search of all scholarly analyses on the CRPD was conducted. These included reports, commentaries, and articles found on electronic databases . Additionally, a search of all Canadian federal, provincial, and territorial legislation and jurisprudence was conducted for mentions of the UN CRPD. Aggregator databases such as the Canadian Legal Information Institute (CANLII) website were further used to perform this nationwide search.Second, a review of specific Canadian government and UN documents were analyzed to gather insights on Canada’s treaty ratification and implementation process. This included the Canadian government’s 2014 report to the UN on its CRPD obligations. For comparative purposes, the UN CRPD implementation reports of 19 other State Parties were also summarized.Finally, commentaries and shadow reports of both national and local NGOs focused on mental health and disability rights in Canada were consulted to assess the CRPD’s domestic implementation of the treaty. These resources were compared and contrasted with both Canadian government and UN reports on Canada’s CRPD obligations and implementation process.A socio-legal approach was used to directly evaluate the effect of the CRPD’s adoption on psychosocial disability rights in domestic institutions, policies, and judicial decisions . The useHistorically, persons with disabilities have been treated as recipients of welfare, health, and charity programmes, rather than individuals deserving of equal legal rights. Now a prominent domain of international human rights law, disability was long invisible until the early 1970s when it was beginning to be recognized as a fundamental aspect of human rights. At that time, international declarations on disability existed, but were critiqued as non-binding and reflecting a medicalized approach to disability. In 1982, the UN adopted a World Program of Action Concerning Disabled Persons (WPA) that aimed to establish equal rights for persons with disabilities  1 SCR 241, the Court assessed whether the equality rights of a 12-year-old girl with cerebral palsy were violated when her school board placed her in a segregated special education class against her parents’ wishes  3 SCR 624, the Court was asked to determine whether the failure of the British Columbia Medical Services Commission and hospitals to provide sign language interpretation services amounted to a violation of the Canadian Charter of Rights and Freedoms s.15(1) equality rights of persons who are deaf  2 SCR 817, serves as a leading case on how Canada’s international obligations are judicially interpreted [Immigration Act, to determine whether federal immigration authorities should treat the best interests of the Canadian-born children as a primary consideration in their decisions. Justice L’Heureux-Dubé noted that international treaties and conventions do not form part of Canadian law unless implemented by statute, but highlighted that the “values reflected in international human rights law” nevertheless help to inform a “contextual approach to statutory interpretation and judicial review” [Baker demonstrate that Canada’s international obligations can help to inform and develop social, cultural and legal norms on fundamental human rights issues.With respect to international conventions and treaties, ation), 19 2 SCR 8ation), 19 2 SCR 8While some cases speak about disability issues generally, an assessment of the number of court and tribunal decisions that reference the CRPD illustrates the degree of its implementation in Canadian jurisprudence. As of March 2015, the CRPD has been cited in nine Canadian court judgments and three tribunal decisions from those contained in the CanLII database. In seven cases, the Convention is just briefly mentioned, mostly as part of a summary of litigants’ claims to equality rights and access for persons with disabilities. Two cases consider the Convention in more detail, including whether it provides the right to state-funded counsel for persons with disabilities and the relevance of the social model of disability in Canadian society. Two cases  deliberate substantially on the Convention; the first on whether it should influence criminal sentencing of persons with disabilities, and the second on whether it forces governments to distinguish between children and adults with disabilities.Portman v. Government of the Northwest Territories, 2014 CanLII 21552 (NT HRAP), the claimant argued that the Northwest Territories’ Human Rights Act, as well as Article 13 of the CRPD, required the Northwest Territories government to provide her with legal counsel to represent her in her discrimination complaint against the Northwest Territories government and Sun Life Assurance owing to her disability [Human Rights Act relied on by the claimant in her submission does not incorporate the requirements of the Convention in the Act. The Court further ruled that state parties to the CRPD are not required to “afford publicly-funded council to persons with disabilities; instead it requires those states to ensure persons with disabilities have ‘effective access to justice…on an equal basis with others…’” [In sability . Articlethers…’” . The Couthers…’” . After tHinze v. Great Blue Heron Casino, 2011 HRTO 93, the Human Rights Tribunal of Ontario affirmed that Canada’s ratification of the CRPD has contributed to a “paradigm shift” by proposing a social model of disability as a “personal affect deserving of human rights protection” [Quebec (Commission des droits de la personne et des droits de la jeunesse) v. Boisbriand (City), 2000 SCC 27 and Granovsky v. Canada , 2010 FC 587, the Federal Court of Canada assessed the distinction between children and adults with disabilities using the UN Convention on the Rights of the Child and the CRPD for the purpose of adjudicating the applicant’s immigration appeal on humanitarian and compassionate grounds [In  grounds . Counsel grounds .Ultimately, the success of the CRPD and other international laws related to mental health depends on the extent to which they are domestically implemented and followed by its state parties . ImportaIn the Canadian context, disability legislation and policies have not yet fully adopted the social model of disability in how equal rights and accessibility for persons with disabilities are promoted, particularly with its reservations to legal equality under Article 12 of the CRPD.  NevertheDespite the sparse references to the Convention in federal and provincial/territorial legislation and court decisions, government services and programs are targeting social and structural inequalities faced by Canadians with disabilities, psychosocial and otherwise. Many commentators, however, remain concerned with the absence of independent oversight mechanisms for monitoring CRPD implementation. While the overarching principles of the CPRD may become progressively realized over time, it will take a consistent and determined effort by federal and provincial/territorial legislatures, courts, tribunals, public services, and civil society organizations to ensure that equal participation and accessibility rights are achieved for all Canadians with disabilities.To date, Canada has only submitted one report in 2014 on the domestic implementation of the CRPD since its ratification in 2010. As such, it is yet to be determined whether Canada’s CRPD reports serve as an opportunity to bolster progress on issues stemming from CRPD provisions such as Article 12. Recent criticisms of the Canadian government’s compliance with international treaties, such as the UN Declaration on the Rights of Indigenous Peoples, do not bode well in this regard . Civil sFurthermore, as a dualist, federal state, Canada serves as an excellent case study on the complexities of implementing international human rights treaties. Specifically, Canada’s federal system of provinces and territories, with their independent jurisdiction on decision-making systems and public programs for persons with disabilities, presents obstacles to CRPD implementation. Canada’s dualist system also requires an assessment of international treaties to determine whether current legislation meets international obligations or whether implementing legislation is needed. In cases of the former, as is present with the CRPD, the current legislation deemed to meet Canada’s international obligations is not reinforced through domestic monitoring mechanisms.Though the CRPD had garnered widespread recognition as a progressive human rights treaty on promoting disability rights and equality, challenges remain with actualizing the Convention’s provisions domestically. Even with Canada's progressive treatment of disability rights as an illustration of a wider global shift in human rights law and minority rights, the challenges in implementing the CRPD reflects the difficulties of actualizing international human rights commitments, particularly in advanced, developed countries.Despite the lack of explicit implementation of the CRPD in domestic Canadian law and policy, it seems the Convention has helped to facilitate a larger shift in social and cultural paradigms of mental health and disability. In this regard, Canada is steadily moving forward from its historical legacy of paternalism and discrimination of persons with disabilities, towards inclusive equality . This bo


With the goal of identifying fast, reliable, and broadly applicable RNA isolation methods in European pear fruit for downstream transcriptome analysis, we evaluated several commercially available kit-based RNA isolation methods, plus our modified version of a published cetyl trimethyl ammonium bromide (CTAB)-based method.We confirmed previous work indicating chaotropic agent-based kits produced sufficient, high-quality RNA in freshly harvested, mature ‘Bartlett’ fruit. However, RNA isolation from ‘d’Anjou’ pear peel and especially cortical tissues of fruit stored for 11 months proved challenging to all but our modified CTAB-based method. Generally, more RNA was recovered from peel tissues than cortical tissues. Less toxic dithiothreitol was confirmed to be an acceptable reducing agent as its substitution for 2-mercaptoethanol often yielded high quality RNA. Finally, we present evidence that erroneous signals in the 5S region of Bioanalyzer RNA size plot histograms, that interfered with RNA integrity number calculation, were small RNA fragments that are reduced by simple cleanup procedures, not artifacts as previously reported.The online version of this article (doi:10.1186/s13104-017-2564-2) contains supplementary material, which is available to authorized users. Obtaining sufficient amounts of high quality RNA from plant tissues is a primary hurdle to studying the transcriptomes of plants. Many plant tissues are recalcitrant to RNA extraction for a host of reasons including abundant secondary metabolites and polysaccharides, tough cell walls that may be lignified, high levels of native RNase activity, and widely variable amounts of RNA . FurtherPyrus communis ‘Bartlett’). This study included developing fruit up to harvest maturity that had entered the ethylene climacteric, but did not include older fruit.Climacteric fruits of woody trees, including pear and apple fruit, present challenges to RNA isolation like those mentioned above, but especially due to increases in interfering substances that accumulate during ripening and result in low yields of RNA due to co-precipitation with these substances –8. This P. communis—e.g. ‘d’Anjou’, ‘Bartlett’) have not been reported. To facilitate efforts for functional genomics of tree fruit towards improving postharvest fruit quality [For postharvest fruit physiology, studying fruit pre-harvest through extended storage is routine –15, yet  quality , we presPyrus communis) were obtained from Cashmere, WA on August 31 2015, stored at 33 °F in air for 11 months, and are indicated as “stored”. ‘d’Anjou’ and ‘Bartlett’ pear  were obtained from an experimental orchard located near Orondo, WA on August 4 2016  and were indicated as “fresh”. ‘Gala’ apples were obtained at commercial maturity August 15 2016 from a commercial orchard located near Mattawa, WA.‘d’Anjou’ pears  we added an air drying step to enhance ethanol removal and (5) allowed the elution to incubate at RT for 1 min before centrifugation. Diethylpyrocarbonate (DEPC) treated water was used to make solutions used in this protocol and was prepared as described in Sambrook et al. [See Additional file k et al. .Fruits were washed with mild dish detergent and water, then rinsed with deionized water. Fruit was peeled with a standard vegetable peeler and peel tissues were flash frozen in liquid nitrogen. Immediately following peeling, the pear was cut in half, and roughly 1 cm sub-epidermal cortical tissue from the equatorial region of the fruit was minced finely and flash frozen in liquid nitrogen. Frozen tissues were ground to a fine powder using a clean mortar and pestle chilled with liquid nitrogen. Frozen tissue powder was transferred to a pre-cooled specimen container using a clean, pre-cooled spatula. The specimen containers were immediately stored at −80 °C. To avoid the tedious and inherently inaccurate estimation of small frozen tissue masses we weighed 100 mg of frozen tissue, and thereafter visually estimated an equivalent tissue mass for each preparation in the interest of time.RNA preparations from freshly harvested (“fresh”) d’Anjou peel and cortex using the Macherey–Nagel NucleoSpin Plant RNA Kit and our modified CTAB protocol were pooled and processed with Zymo’s RNA Clean & Concentrator™-5 (cat# R1015) following steps to exclude small RNA fragments (<200 nt) and adhering to the manufactures instructions.http://www.nanodrop.com) to provide sample concentration, A260/280 and A260/230. The Agilent Bioanalyzer (Agilent Technologies—http://www.agilent.com), using the RNA Pico assay (cat#: 5067-1513) and following the manufacturers’ instructions, provided sample concentration, RNA integrity number (RIN), and other detailed quality information.RNA was evaluated with a NanoDrop 2000 spectrophotometer , followed by less recalcitrant tissues (freshly harvested mature pear and apple fruit). We included apple fruit because the Gapper et al.  protocol260/280 values [Ribosomal RNA integrity (frequently estimated by the RNA integrity number (RIN) ) has bee0 values . We ther260/280 ≥1.67 and the best yields across all tissue types ranging from 0.5 to 2 µg from ~100 mg fresh weight tissue , preparations yielding as little as 25 ng of total RNA may be sufficient. This is especially relevant when the target tissues are very small or hard to generate because a brute force approach of processing large amounts of tissue may not be practical or even possible.Importantly, our CTAB based protocol was time equivalent to the kits that were tested, though more equipment intensive . We eliminated intensive preparation of spatulas and mortars and pestles by baking at 200 °C prior to use because native plant RNase activity  renders s et al. , the CTAhttp://www.chem-agilent.com—Publication Number 5988-7650EN), making erroneous non-RNA signals unlikely. To test this hypothesis, we chose 8 samples (4 pairs) to clean-up with Zymo’s RNA Clean & Concentrator-5, which is a kit that includes a step to selectively remove small RNAs (<200 nt). Use of the clean-up process resulted in a substantial reduction in the magnitude of the broad peak seen in our RNA preps in the 5s region , were hampered by what we hypothesized were small fragments of RNA Fig. . Jordon-Based on the results of our comparison, it is clear that our short and reliable modified CTAB protocol would be an excellent choice for European pear fruit at or near harvest maturity through to fruit that had been stored for an extended time. The Macherey–Nagel NucleoSpin Kit and Qiagen RNeasy Plant kit represent the best of the kits we tested—in part because of available alternate lysis buffer chemistry. The kits are less robust than our CTAB method, but with affordable and easy kit based cleanup (e.g. Zymo RNA Clean & Concentrator), the hurdles that low yields and partially degraded RNA pose could be largely overcome. The input amount for all methods tested here was around 100 mg, though when processed with a successful method, the plant tissue yielded 500 ng–2 µg of high quality clean RNA, which is sufficient for a range of state-of-the-art transcriptome analysis methods.Additional file 1. Detailed protocol for our modified CTAB method.Additional file 2. Abbreviated database for all isolations done in this study.Additional file 3. Signal in the 5s region is likely due to small RNA fragments and can interfere with RIN estimation. A, B are replicates of RNA isolations from cortical fruit tissue of freshly harvested d’Anjou pear. C—samples shown in A, B were pooled, and processed with Zymo’s RNA Clean & Concentrator Kit-5 in which we opted to remove RNA fragments <200 nt. The arrows indicate the putative small RNA fragments that are reduced during the cleanup . D—low yield RNA prep with clear 18s and 28s peaks and low signal in the 5s region produces a RIN of 8.5, E—similarly low yielding prep of RNA with clear 18S and 28S peaks but with high signal in the 5S region produces a RIN of 2.8. The portion of intact mRNA in these preps likely does not differ substantially.