Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo. We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. These results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications. Similar to humans, bacteria use an immune system known as the CRISPR-Cas system to protect themselves against invading pathogens such as viruses. CRISPRs are specialized stretches of DNA that guide Cas9 to the right location, while Cas9 proteins act like scissors that can cut foreign DNA.When a virus infects a bacterium, the bacterium steals a piece of DNA from the virus and stores it in its CRISPR region. The bacterium then produces a small RNA template that matches the stolen DNA of the virus and adds a specialized protein to it. When the virus infects the cell again, the protein-RNA complex can recognize the virus and stop the infection.Researchers have successfully adapted this system as a gene-editing tool to target and modify specific DNA sequences in different organisms. Cas9 can target and cut DNA, but until now, it was not clear whether this protein could also efficiently target RNA – the ‘genetic middleman’ between DNA and proteins. RNA is essential to make proteins, and being able to target RNA would allow researchers to answer many important questions about RNA biology.To investigate this further, Strutt et al. used three different subtypes of Cas9 proteins and small RNA sequences in a test tube. The results showed that two of the protein subtypes could target RNA efficiently, and one of which was able to target any RNA sequence. Strutt et al. then used one Cas9 to target specific RNA sequences in bacteria and were able to reduce the amount of protein made from that gene. Moreover, the Cas9 protein helped to protect the bacteria against an RNA virus.This work lays the foundation for using this Cas9 protein as a tool for researchers to study RNA in cells. A next step will be to test if Cas9 can cut RNA in human cells. If this works, it could allow direct targeting of RNA viruses, such as West Nile and Dengue, to stop them from infecting human cells. Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) systems provide immunity against plasmids and bacteriophage by using foreign DNA stored as CRISPR spacer sequences together with Cas nucleases to stop infection . One sucin vivo, Cas9 is exclusively a DNA-targeting enzyme. Nonetheless, multiple studies have harnessed Cas9 for RNA targeting under specific circumstances. For example, the S. pyogenes Cas9 (SpyCas9) can be supplied with a short DNA oligo containing the PAM sequence (a PAMmer) to induce single-stranded RNA (ssRNA) binding and cutting (Francisella novicida (FnoCas9) has been implicated in degradation of a specific mRNA but through a mechanism independent of RNA-based cleavage  and C. jejuni Cas9 (CjeCas9) direct cleavage of RNA in the absence of a PAMmer (F. novicida Cas9 (FnoCas9). While the cleavage efficiencies for both SauCas9 and CjeCas9 are indistinguishable  do not cleave RNA . FurtherSauCas9 targets ssRNA in the absence of a PAMmer, a contrast to SpyCas9 targeting of ssRNA . TestingKd,app) of the SauCas9-sgRNA complex (23 nt targeting region) for the ssRNA target is 1.8 ± 0.09 nM (Given that Cas9 proteins are active with different length guide RNA segments (~20–24 nt) , we test 0.09 nM , which iWe noted that SauCas9-catalyzed ssRNA cleavage is limited to ~30% fraction cleaved , compareInterestingly, the 23-base pair mismatched segment RNA substrates (‘Bubble’ substrates 8 and 9) are targeted more efficiently than their ssRNA counterparts (substrates 1 and 2) . We measAn alternative hypothesis to explain the limited cleavage of ssRNA substrates is that SauCas9 enzyme inactivation occurs over the course of the reaction, even with SauCas9 protein-sgRNA  present in 10-fold excess relative to the ssRNA substrate. To test this, we spiked reactions with fresh SauCas9 protein alone or SauCas9 RNP after reactions reached equilibrium; however, we did not observe an increase in the amount of ssRNA cleavage . We alsoE. coli. A subset of these sgRNAs contained scrambled guide sequences that should not target MS2, providing negative controls. Another sgRNA subset included single-nucleotide mismatches introduced at each position of a target sequence to test for mismatch sensitivity in ssRNA recognition. This plasmid library, comprising 18,114 sgRNAs, was co-transformed into E. coli along with a vector encoding a catalytically active version of SauCas9 and the population of transformants was subjected to infection by bacteriophage MS2 -adjusted p-value<0.05) in the two different MS2 infection conditions . The majStrikingly, mapping enriched guide sequences onto the MS2 genome showed that enriched sgRNAs were clustered at specific regions, which were consistent across both experimental conditions . TogetheE. coli host factor that is necessary for infection.Highly enriched sgRNAs from the screen were confirmed for their ability to confer protection against MS2 phage infection through a soft-agar plaque assay. Reconstitution of SauCas9 with a targeting guide confers approximately a ten-fold protection against the RNA phage . No prota priori, to recognize accessible parts of the MS2 genome. Nevertheless, despite potential noise introduced in this analysis due to guide segments that target inaccessible MS2 regions, we observe an interesting correlation between mismatches in the MS2 screen and in vitro biochemical cleavage assays for the sgRNA with a 23 nt guide segment sequence  for all sgRNAs that contained a mismatch at the same position, and obtained average values for mismatches at each position across the guide. We observed a pronounced gradient of increasing guide stringency with length. On average, short guides were less sensitive to mismatches, while mismatches in longer sgRNAs led to decreased recovery compared to control samples . Previousequence . The firsequence .E. coli chromosome . Cells were grown to an OD600 of 0.6–0.8, induced with 0.4M IPTG, and then incubated overnight at 16˚C with shaking. Proteins were purified using Superflow Ni-NTA affinity resin , followed by a HiTrap HP Heparin column  and gel filtration on a Superdex S200 , as previously described (All proteins were expressed as His-MBP fusions (Addgene vector #29706) in escribed . Cas9 prDNA oligonucleotides were synthesized by IDT . Target RNAs and sgRNAs were transcribed in vitro as previously described . DNA tarCas9 was reconstituted with equimolar sgRNA in 1x cleavage buffer  for 10 min at 37˚C, then immediately placed on ice. Cleavage reactions were conducted with 1 nM target and 10 nM reconstituted Cas9-sgRNA in 1x cleavage buffer unless otherwise noted. Structured RNA substrates were prepared by annealing two separate in vitro transcribed RNAs. The target strand was annealed with 10-fold excess of the non-target strand to ensure that all target is complexed prior to the cleavage reaction. Reactions were incubated at 37˚C for the indicated time and quenched in Heparin-EDTA buffer  at 25˚C for 5 min. Reactions were diluted with 2x Formamide loading buffer and incubated at 95˚C for 5 min prior to separation on a 15% denaturing 7M urea PAGE gel. Gels were dried overnight and exposed to a phosphor imaging screen . Results were visualized on a Typhoon  and quantified in ImageQuantTL . The cleaved fraction of total signal was calculated independently for three separate experiments and were fit with a one-phase exponential decay model in Prism7 .Binding reactions consisted of 750 nM catalytically inactive SauCas9 reconstituted with sgRNA to the final concentrations indicated. Radiolabeled target RNA was added to a final concentration of 1 nM and the reactions were incubated at 37˚C for one hour. Bound probe was separated from unbound using a three-filter system on a vacuum manifold . MembranE. coli cells with a vector containing a tetracycline-inducible wtSauCas9 construct were made electrocompetent and transformed with the MS2-guide plasmid library in triplicate. Approximately 1 × 106 transformants were grown for 30 min at 37˚C with shaking prior to addition of antibiotics and 10 nM anhydrotetracycline (aTc)  for protein induction. After an additional 30 min of growth, cultures were split into three equal pools and treated with none, 3.3 × 106, or 3.3 × 107 MS2 bacteriophage. After 3 hr of infection, cells were plated on LB-agar supplemented with antibiotics and incubated at 37˚C for 16 hr. Plates were scraped with LB and plasmids were isolated using a MidiPrep kit , according to the manufacturer’s protocol. High-throughput sequencing libraries were prepared by PCR amplification of the variable region of the guide plasmid. Dual unique-molecular identifiers (UMIs), included to separate true single-nucleotide mismatches, as well as duplicates, from PCR artifacts  and cloned into a guide expression vector  modifiedrtifacts , were inrtifacts  were cloy method  and plaq2 fold-change values of significantly enriched guides plotted as box and whisker plots in Prism. The secondary structure of the MS2 genome was obtained from  guides for each treatment were partitioned by length and averaged at each position. High-throughput sequencing data accompanying this paper are available through the Sequencing Read Archive under the BioProject accession number PRJNA413805.After applying a low-pass filter, reads were trimmed using cutadapt v. 1.14  and pairned from  and readned from . Log2 foE. coli strain with a GFP reporter gene integrated into the chromosome . Protein expression was induced with 10 nM aTc. GFP and OD600 were measured every ten minutes for at least 18 hr. Curves of GFP expression over time were fit with a logistic growth model in Prism. At 80% of the maximum value, or at least after 16 hr of growth, the GFP signal was normalized by cell density at OD600. To account for effects of guide and protein expression, GFP/OD600 was normalized to a null guide or null protein culture, respectively. As expression of different guides change GFP expression levels, the ratio between normalized RNP and guide values was taken to allow comparison of RNP-based repression across different guides. All experiments were conducted in triplicate and all graphing and quantitative analyses were conducted in Prism. Guide and target sequences can be found in Based on the system outlined previously, SauCas9 was cloned into a tetracycline-inducible vector, while individual guides are under control of a constitutive promoter . Plasmidromosome . Culture 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 favorably evaluated by Gisela Storz (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal his identity: Ailong Ke (Reviewer #2).Thank you for submitting your article "RNA-dependent RNA targeting by CRISPR-Cas9" 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.Your manuscript presents novel Cas9 RNA targeting activity from diverse Type II-A and II-C CRISPR-Cas systems, and show ssRNA targeting and cleavage in a non-canonical PAM- and PAMmer-independent manner. The reviewers universally agree that this is a valuable addition to the literature, and that some of the findings create opportunities for both expansion of the CRISPR toolbox, and for diversification of applications.Unless you are keen on performing additional experiments to address specific items listed below, altering the narrative to present the antiviral activity as a proof of concept, and speculating about the mechanism of action driving the translational effect would be acceptable.Summary:S. aureus (Sau) and C. jejuni (Cje) Cas9 can cleave about 30-40% of ssRNA substrates in the absence of a PAMmer  and determined that SauCas9 has roughly 5-fold higher K¬D¬ for ssRNA than dsDNA. Next the authors showed that RNA secondary structure can inhibit cleavage, yet interestingly, introducing mismatches in dsRNA substrates recovered activity and even exceeded the activity towards the ssRNA substrate. SauCas9's ability to cleave RNA also allowed E. coli to defend itself versus MS2 infection and was shown to be useful in knocking down GFP expression in a reporter E. coli line.First, the authors showed that both Essential revisions:The biochemical data provided to establish PAM-independent, RNA-mediated ssRNA targeting is convincing and the narrative articulate. The reliance on guide RNA loading, the presence of active residues (D10 and N580) and divalent metal ions is clear, as well as the impairment by target RNA structural features. The same does not apply to the sections dedicated to defense vs. RNA phages, and to a lesser extent, to the repression of protein translation. In the case of viral RNA defense, while there is admittedly basic proof of concept showing reduction by an order of magnitude, it is unclear how potent and biologically relevant and useful this activity is. The authors may consider testing more guides , and may consider testing the combination of multiple guides concurrently  to show more potent viral resistance. The authors should readily be able to combine guides 23 and 22 to (hopefully) show enhanced resistance , or alter the conclusiveness of their statements. Perhaps two of some of the most potent guides shown on All biochemical experiments were performed on the same pUC RNA target, which ignores the effect that target sequence may have on KD¬, spacer length preference, etc. Additionally, while the pUC RNA is predicted to have secondary structure, there are a number of weak or wobble base pairs. How does RNA sequence change Cas9's kinetic parameters and do RNA targets with stronger secondary structure preclude Cas9 cutting?The authors demonstrated that GFP expression can be reduced by targeting SauCas9 to GFP transcripts. However, the authors didn't provide sufficient evidence to suggest that repression is on the translational level. Cas9 binding may still act on the transcriptional level by interfering with RNAP progression in a Rho-like fashion, or by destabilizing mRNA transcripts, leading to their degradation. Ribosomal profiling should definitively show that Cas9 diminishes expression by blocking ribosome progression.The translational repression effect is presumably due to action on ribosomes, possibly prevention of ribosome loading, elongation inhibition, or reduction of tRNA accessibility. mRNA degradation affects translation but is not the translation mechanism per se. While ribosome profiling may require extra efforts and not fit within the scope of the manuscript, the authors may consider it, or possibly testing the approach on a bi-cistronic system: RBS1-A-RBS2-B, and show whether targeting A only affects A. Of course, revising the text to tone down conclusiveness on translation control is also an option. Essential revisions:The biochemical data provided to establish PAM-independent, RNA-mediated ssRNA targeting is convincing and the narrative articulate. The reliance on guide RNA loading, the presence of active residues (D10 and N580) and divalent metal ions is clear, as well as the impairment by target RNA structural features. The same does not apply to the sections dedicated to defense vs. RNA phages, and to a lesser extent, to the repression of protein translation. In the case of viral RNA defense, while there is admittedly basic proof of concept showing reduction by an order of magnitude, it is unclear how potent and biologically relevant and useful this activity is. The authors may consider testing more guides , and may consider testing the combination of multiple guides concurrently  to show more potent viral resistance. The authors should readily be able to combine guides 23 and 22 to (hopefully) show enhanced resistance , or alter the conclusiveness of their statements. Perhaps two of some of the most potent guides shown on The authors demonstrated that GFP expression can be reduced by targeting SauCas9 to GFP transcripts. However, the authors didn't provide sufficient evidence to suggest that repression is on the translational level. Cas9 binding may still act on the transcriptional level by interfering with RNAP progression in a Rho-like fashion, or by destabilizing mRNA transcripts, leading to their degradation. Ribosomal profiling should definitively show that Cas9 diminishes expression by blocking ribosome progression.The translational repression effect is presumably due to action on ribosomes, possibly prevention of ribosome loading, elongation inhibition, or reduction of tRNA accessibility. mRNA degradation affects translation but is not the translation mechanism per se. While ribosome profiling may require extra efforts and not fit within the scope of the manuscript, the authors may consider it, or possibly testing the approach on a bi-cistronic system: RBS1-A-RBS2-B, and show whether targeting A only affects A. Of course, revising the text to tone down conclusiveness on translation control is also an option.We thank the reviewers for their insights into potential mechanisms of SauCas9-mediated gene repression. In the absence of a PAM, we did not observe binding of SauCas9 to dsDNA in vitro. Thus, the activity of SauCas9 does not seem to be mediated by a CRISPRi-like, PAM-dependent inhibition of transcription. It is possible that SauCas9 blocks the ribosome (either at initiation or during elongation) or, as the reviewers suggest, by otherwise destabilizing the transcript. The text has been revised to include these possibilities and to emphasize the PAM-independent repression of gene expression, more broadly.


Prior to implementing gene expression analyses from blood to a larger cohort study, an evaluation to set up a reliable and reproducible method is mandatory but challenging due to the specific characteristics of the samples as well as their collection methods. In this pilot study we optimized a combination of blood sampling and RNA isolation methods and present reproducible gene expression results from human blood samples.TM blood collection method (Qiagen) was compared with the more recent TempusTM collection and storing system. RNA from blood samples collected by both systems was extracted on columns with the corresponding Norgen and PAX RNA extraction Kits. RNA quantity and quality was compared photometrically, with Ribogreen and by Real-Time PCR analyses of various reference genes  and exemplary of SIGLEC-7.The established PAXgeneTM and TempusTM collection systems resulted in RNA of good quality and quantity for the respective RNA isolation system. No large inter-donor variations could be detected for both systems. However, it was not possible to extract sufficient RNA of good quality with the PAXgeneTM RNA extraction system from samples collected by TempusTM collection tubes. Comparing only the Norgen RNA extraction methods, RNA from blood collected either by the TempusTM or PAXgeneTM collection system delivered sufficient amount and quality of RNA, but the TempusTM collection delivered higher RNA concentration compared to the PAXTM collection system. The established Pre-analytix PAXgeneTM RNA extraction system together with the PAXgeneTM blood collection system showed lowest CT-values, i.e. highest RNA concentration of good quality. Expression levels of all tested genes were stable and reproducible.Combining different sampling methods and extraction kits caused strong variations in gene expression. The use of PAXgeneThis study confirms that it is not possible to mix or change sampling or extraction strategies during the same study because of large variations of RNA yield and expression levels. A reliable analysis of gene expression is still challenging due to the various available blood sampling as well as subsequent RNA isolation methods, which result in robust changes of expression patterns. Nevertheless, gene expression analysis in peripheral blood is an important and obligatory tool in molecular research and diagnostics, especially in large epidemiological studies.As a first major influencing aspect, sample collection and procession has a considerable influence on the data outcome. Consequently, for the pilot study presented here, the bio-sampling standards developed and evaluated during the IDEFICS  study were transferred to this study to ensure reproducibility and comparability of the results . The staTM and (2) the TempusTM collection tubes. The PAXgeneTM collection tubes  are loaded with a patented blend solution, which protects RNA molecules from degradation by RNases and prevents the induction of further gene expression. The TempusTM collection tubes  are preloaded with guanidine and detergent, which stabilizes the RNA by lysing the blood cells and inactivating the RNAses through chaotropic properties.Two major commercially available blood collection tube systems were available and compared in this study, (1) the PAXgeneIFNγ, IL2, IL3, IL4, and IL13 is up-regulated in stimulated vs. control samples, which did not occur when the “PAXgeneTM collection method” was used [Previous analyses agreed that both PAXgene and Tempus systems cannot be combined in gene expression analyses because of large differences in RNA yields and expression levels –6. A welwas used . This reAlso the next stages, i.e. RNA extraction and further quantification steps highly influence the results. In a previous microarray study, that used both collection systems, quantity and quality of RNA from three donors were comparable , but genTM blood collection tubes yield higher amounts of RNA than PAXgeneTM [TM and TempusTM tubes [TempusAXgeneTM . The difTM tubes , 13, howTM tubes .et al. observed in samples from the KORA cohort, that different study designs or reagents (PAXgene™ vs. Tempus™ collection tubes) did not affect reproducibility in a microarray based analysis [In contrast, Schramm analysis .A further parameter, which strongly influences the RNA is the subsequent RNA extraction method. In this study, we compared several on column RNA extraction methods, which were specifically recommended by the manufacturers of the sampling systems. An additional criterion to setup the most appropriate method was the choice of a suitable reference gene for quantitative Real-Time PCR analysis of blood samples. Stable reference gene expression is one, if not the most critical, requirement for accurate and biologically meaningful gene expression normalization. Fluctuations of reference gene expression could lead to false down- or up-regulation of the target gene –16. HereTM collection tubes  and by TempusTM collection tubes  to compare the stability of the isolation (later named series C). All blood collection tubes were gently inverted for 5 min directly after collection and incubated for 2h at room temperature (RT), 48h at 4°C and then stored at -80°C until processing. Samples were thawed on ice and left another 2h at RT before RNA extraction. Ethical approval for the study was obtained by the Ethical Committee of University of Bremen. The participants of the study provided their written informed consent to participate in this study. The ethics committee of University of Bremen approved this consent procedure.All 17 participants are voluntary test persons, of different age, sex and ethnical origin with confirmed good health status, good physical condition, free from medication and no current infectious disease. Two samples from each individual (n = 12) were collected at the same time by PAXgeneTM tubes was commercially available. Therefore, in the first sample set (n = 12), RNA was extracted with a commercial kit from Norgen Biotek , specified for both tested PAX (Norgen Biotek RNA purification Kit II) and TempusTM (Norgen Biotek RNA purification Kit I) collection systems, referred to as Norgen RNA extraction kit throughout the manuscript. A second sample set  and Tempus (n = 4)) was tested with the recommended PAXgeneTM Blood RNA isolation kit, referred to as PAXgene RNA extraction kit. All extractions were performed according to the manufacturers’ protocols as described below.During the test period, only the corresponding RNA extraction kit for PAXgeneTM Blood RNA tube diluent to a final volume of 12 ml. Tubes were vortexed vigorously for 30 s. Mixed samples were centrifuged at 4°C for 30 min at 3,000xg. The dried pellet was re-suspended with 600 μl lysis solution by vortexing. 300 μl 96% Ethanol was applied to the samples and mixed. Afterwards, the solutions were transferred to the RNA isolation spin columns. Centrifugation for 1 min at 14,000xg was performed before samples were washed with washing buffer and for the optional DNase 1 digestion, 100 μl RNase-free DNase 1 was applied to the sample, centrifuged for 1 min at 14,000xg and flow through added to the column again. DNA digestion was carried out at RT for 15 min. Thereafter, 400 μl of the washing solution was added and the columns centrifuged for 1 min at 14,000xg and then for 2 min at 16,900xg to dry the column membrane. RNA was eluted by adding 50 μl elution buffer to the samples and incubated for 1 min followed by centrifugation for 2 min at 200xg and 1 min at 14,000xg. The RNA elution step was repeated.Blood samples were transferred to a 50 ml tube and filled with TempusTM tubes were centrifuged for 10 min at 5,000xg. After decanting the supernatant, the pellet was liquefied with 4 ml NPX1 and vortexed until the pellet was dissolved. The tubes were centrifuged again for 10 min at 5,000xg. In the next step, the supernatant was discarded and the pellet was lysed in 600 μl NPX2 by vortexing. Centrifugation was carried out to remove insoluble materials for 1 min at 14,000xg. The supernatants were transferred to new tubes and mixed with 300 μl 96% ethanol by vortexing. The solutions were transferred to the RNA isolation spin columns. Centrifugation for 1 min at 14,000xg was performed before samples were washed with 400 μl NPX3 and centrifuged for 1 min at 14,000xg. Optional DNase 1 digestion was performed as described above. Thereafter, 400 μl of NPX3 was added and columns were centrifuged for 1 min at 14,000xg. Washing with 400 μl NPX3 was repeated and then 2 min of centrifugation at maximum speed was applied to dry the column membrane. The RNA was eluted to the provided storing tubes by addition of 50 μl NPX5 to each sample and incubated for 1 min followed by a centrifugation for 2 min at 200xg and for 1 min at 14,000xg. The RNA elution step was repeated.PAXgeneAn additional DNase1 digestion was performed for series B (Norgen extraction systems). Here 250ng total RNA were digested in three independent experiments with DNase 1 . All the samples were used later for cDNA synthesis in at least technical duplicates (qRT-PCR) .TM tubes did not resolve well in BR1 buffer. Hence, samples were mixed with 300 μl BR2 and 40 μl Proteinase K followed by 10 min incubation at 55°C at a shaker. Samples were pipetted to QIAshredder columns and tubes centrifuged 3 min at 17,000xg. The transferred supernatant of the flow through was mixed with 700 μl 100% Isopropanol. Samples were stored at -80°C until the next step was performed.Tubes were centrifuged for 10 min at 3,000xg. After decanting the supernatant, the dried tubes were refilled with 4 ml RNase free water and pellets re-suspended by vortexing. Thereafter, a next centrifugation step for 10 min at 3,000xg was performed. The pellet was re-suspended in 350 μl BR1 and transferred to a 2 ml tube. The observation was made that samples from TempusSamples were equilibrated to RT, placed in the RNA isolation column and centrifuged for 1 min at 12,000xg. Samples were digested with DNase1 on-column; 350 μl BR3 was used to equilibrate the column conditions before adding 80 μl DNase RDD mix. Following 15 min incubation at RT, the samples were washed with 350 μl BR3. Samples were treated twice with 500 μl BR4 washing buffer and centrifuged 1 min at 12,000xg. A final centrifugation step was performed for 3 min at 17,000xg to dry the membrane. To elute the RNA, 40 μl BR5 were supplied to the membrane, incubated for 1 min and centrifuged 1 min at 12,000xg; the elution step was repeated. For denaturation, the RNA was incubated for 5 min at 65°C in a heating block.® reagent is an ultra sensitive fluorescent nucleic acid stain for quantifying RNA in solution without the absorbance method-based disadvantages such as signals from contaminating proteins or interference of free nucleotides.Integrity and quality of isolated RNA was measured by the classical absorbance measurement at 260 nm by the NanoDrop™ spectrophotometer  and by RiboGreen fluorescence measurements, performed in a volume of 200 μl and fluorescence emission measured at 520 nm against a standard reference curve according to the manufacturer’s instructions. The RiboGreenReverse transcription reactions were performed in a volume of 20 μl containing 500 ng (series A), 250 ng (series B) or 100 ng (series C) of total RNA . First sSIGLEC7 Hs00255574_m1 , PPIA Hs99999904_m1 PCR efficiency 100%, β-Actin Hs99999903_m1 , Tubulin Hs00362387_m1 , all Applied Biosystems, Darmstadt, Germany). Primer efficiencies were tested in dilution series of a single exemplary investigated blood sample. For all amplicons, a cDNA component control and a control without reverse transcriptase were performed. Cycling program: 20 s 95°C and 50 Cycles: Denaturation 1 s 95°C followed by 20 s 60°C annealing and elongation. Results are presented as CT or RQ to a calibrator (ΔCT) [Real-time RT-PCR was performed using hydrolysis probes and the 2x Taqman Universal PCR Mastermix with an ABI Stepone Plus Cycler . Reactions were performed in duplicates in a volume of 10 μl with specific primers and probes (or (ΔCT)  (StepOneor (ΔCT) .2 scale for symmetry improvement. The significance of difference between individual experiments was tested by student’s t-test. P values of p<0.05 was considered to be statistically significant.RT-PCR results were calculated according to the ΔΔCt method and corrected on single gene efficiency to minimize error propagation . The resTM (2.5 ml) and TempusTM (3.0 ml) collection systems (series A + B). RNA of both was isolated either by the Norgen 1 (specific for TempusTM) and 2 (specific for PAX) or the PAX RNA isolation systems. An additional DNase1 digestion for both tubing systems was performed to prevent any possible genomic contamination .The RNA yield was adjusted to the respective kit specified sample and elution volume  and fina260/E280 ratios of Tempus™ 2.1±0.01 vs. PAXgene™ 2.0±0.03, irrespective of the blood collection system. An exception is the RNA from TempusTM collection tubes extracted with the PAXgene RNA extraction kit. Here, five samples from various series showed no RNA and the remaining samples showed a high mean E260/E280 ratio of 2.5±1.26 and were omitted from further analysis.High purity RNA was obtained from both PAXgene and Norgen RNA extraction kits with average ERNA extracted with the Norgen RNA extraction kits from Tempus™ collection tubes showed almost identical concentrations with both analysis methods. In contrast, 2.5-fold higher RNA values were obtained with the photometer from the PAXgene™ collection tubes, compared to Ribogreen . This shTogether with the low RNA quality, RNA extracted with the PAXgene RNA extraction kit from Tempus™ collection tubes showed the lowest yield with almost no detectable RNA. In contrast, the RNA yields from PAXgene RNA extraction kits were improved when compared to the PAXgene collection tubes, but were much lower than those from both Norgen RNA extractions kits .Surprisingly, the average intact RNA yield from PAXgene™ collection tubes was higher with the Norgen RNA isolation kit than with the PAXgene™ kit .In summary, the combination of TempusSIGLEC-7, an adhesion molecule expressed on monocytes [SIGLEC-7 gene expression levels among individuals dependent on their status of health and inflammation were observed in our laboratory before [To further characterize the RNA quality despite the differences in yield, RT-PCR analysis was performed on several reference genes as well as on onocytes  with higonocytes . Differey before . This waPPIA, suggestive of DNA contamination in the RNA, although an on-column-DNA-digestion had been performed within the Norgen kit.Since Norgen RNA extraction delivered the highest RNA yields, this kit was used for further analyses of the same samples of Tempus and PAX collected blood . SamplesT values throughout series A and B, compared to PAX collection tubes . Normalization on 3 reference genes resulted in an upregulation above the set arbitrary threshold of 1  only in subject #2, confirming the upregulation, which also occurred independently in all 3 separate reference gene normalizations.To proof the assay sensitivity, an additional series B was generated, in which a lower amount of 250 ng RNA was digested and subsequently reverse transcribed. DNase1 digestion and RT-PCR was performed in 3 sets of replicates from each sample and ran on different days . No DNA ce genes . These rTM and TempusTM, respectively, were collected from 5 donors and RNA amount and gene expression compared. As expected from the very low RNA yield  in quadruplicate (4 seperate tubes) and subsequent PCR analysis). Data were normalized to PPIA, β-ACTIN, TUBULIN and their combination . Four tubes PAXgeneNA yield , gene exs CT>33; . Therefoon tubes . Stabilibination .SIGLEC-7 gene expression results strongly differed dependent on the normalization to different reference genes . Interestingly, such fluctuation was abrogated when SIGLEC-7 expression was normalized on 3 reference genes. These analyses make the normalization to the pool of reference genes indispensible in order to get reliable expression data.In accordance with the data from Norgen isolation , SIGLEC-ce genes . The low to PPIA . As befoSIGLEC-7 gene expression showed high variations (1) with different sampling systems, (2) with different extraction methods and (3) with normalization on different reference genes. The question remains, which system is the one of choice and results in most stable expression levels.In summary, the exemplary chosen T values of reference genes are obtained from all donors when using the same RNA concentration. As shown in TM collection tubes in combination with Norgen isolation kit delivered higher reproducibility, compared to Pax/Norgen. Nevertheless the coupling of PAX collection tubes with PAXgeneTM isolation kit . The combined PAXgeneTM system presented the highest gene expression as well as most stable results. In contrast, RNA extraction from TempusTM collection tubes combined with PAXgeneTM blood isolation kit delivered an extremely low RNA yield and such combination is therefore not advisable.The comparison of various available methods shows that the most consistent results were achieved with the combination of PAXgeneTo assess whether PAXgene™ and Tempus™ peripheral blood RNA collection tubes provide higher quality and RNA yield, we compared their downstream application by RT-PCR; stable RT-PCR results from the same samples would allow evaluation of the RNA quality. Based on such RNA quality and quantity measurements, Tempus™ blood collection tubes combined with Norgen RNA isolation delivered the highest amount of RNA with comparable quality.T values [Our results are consistent with Duale et al  and HantT values , which wTM collection tubes are based on guanidinium hydrochloride as RNA stabilizing agent while PAXgeneTM collection tubes use a chemistry of tetradecyl-trimethylammonium oxalate buffered with tartaric acid [TM blood collection tubes and RNA extraction kits are designed to extract RNA within an alkaline pH range (>8); oppositely to PAXgeneTM blood collection tubes and RNA extraction kit which are performed within an acidic pH range of 3–4. After the first centrifugation step, pellets in acid (PAXgeneTM) and alkaline (TempusTM) pH are generated, respectively. Resuspension of the TempusTM pellet with PAXgeneTM blood RNA extraction kit solution would then increase the pH of the solution affecting binding of the nucleic acid to the column matrix, which has also been optimized for a pH of 3–4 [TM blood RNA extraction kit by their nature. Thus, only RNA from PAXgene™ tube samples could be processed for the downstream RT-PCR analyses. Such PAXgene™ extracted RNA from PAXgene™ (Series C) collection tubes showed higher RNA expression levels by CT values together with improved quality of the Real-Time PCR reaction than series B (Tempus-Norgen). Even with a lower total amount of extracted RNA, the use of the PAXgene extraction kits in the Real-Time PCR resulted in a much better sensitivity of around 3 CT values lower and improved signals with less than half of the initial RNA amount.In contrast, the combination of Tempus™ tubes with PAXgene™ RNA extraction delivered low amounts of RNA. The yield of RNA recovery has been very poor and closed to no RNA was obtained. Such problem becomes clear by looking at the stabilizing agent used in the two kits. Tempusric acid . TempusTH of 3–4 . TherefoTM collection. But only transcripts ≥200 bp can be isolated with the Tempus™ kit [The downstream application of series A and B (Norgen extraction) in Real-Time PCRs showed a more constant signal with the RNA obtained from Tempus™ tubes, compared with those blood samples from the PAXgenepus™ kit , while Rpus™ kit , which cTM RNA isolation kits and the solely on-column DNAse digestion was not sufficient for a complete DNA-contamination removal. Used primers in this study span exon-exon boundaries, except PPIA primers, where the amplicon spans an exon junction and the probe and one of the primers sit within one exon, thus, this assay may be affected from the presence of genomic DNA. Even for primers spanning exon-exon boundaries, DNAse digestion would result in more reliable analyses, because large amounts of remaining DNA possibly disturb the primer annealing during the PCR reaction.DNA traces were detected in all blood samples from PAXgene™ and Tempus™ tubes independent of the use of the Norgen or PAXgeneTM extraction from PAXgeneTM collection delivered lower CT values, i.e. higher gene expression, even when the reaction was performed with a lower starting RNA amount. This suggests that the overall RNA integrity is enhanced using the PAXgeneTM system.The total amount of RNA extracted from the PAXgene™ collection system was higher when isolated with the Norgen instead of the PAXgene™ RNA isolation kit. But as already said above, downstream application of RT-PCR shows that PAXgeneTM blood collection systems. PPIA showed the lowest inter- and intra-assay variability compared to both β-Actin and β-Tubulin. The gene of interest used in the present study (SIGLEC-7) showed high CT values (around 30), which, in particular in blood samples, is associated with higher expression variability. A possible approach is to narrow down the confidence interval with an increase of replicates, but this could often be limited by the availability of samples. To avoid such gene expression uncertainty, we show here that the use of a combination of three not closely related housekeeping genes represents an option of choice to avoid false positives (either up- or downregulated), particularly when genes with known high variability are under scrutiny. Also position of primers and probes, amplicon length as well as RNA’s secondary structures can further affect gene expression levels detected by RT-PCR. In our case β-ACTIN and β-TUBULIN probes bind to the 5’-mRNA region (50–300bp) while PPIA and SIGLEC-7 probes anneal around 1kb from the 5’-end.A previous work demonstrated an approach to minimize systematic errors by the use of multiple reference genes as normalization strategy . Still, T-values/equal amount of RNA from samples collected in the PAXgene™ tubes. Still, the distribution of the plotted CT values was different between the systems. A more linear correlation was obtained with samples generated from PAXgene™ tubes, which confirms the larger variation in gene expression within the samples. In contrast, Tempus tube collection with Norgen RNA extraction (Series A and B) delivered more stable results with the values assembled in a small cloud. Yet, in series C, constant expression levels of the reference genes were measured from the PAXgene system. The smaller the variance the higher was the reliability of the reference gene expression and thus the overall data trustworthiness.In parallel with the higher RNA amount, genes analyzed in series A and B (Norgen extraction) showed generally higher CWhole blood is the easiest obtained and therefore most used human material, which can be used to deduce information about the health state of the donor. With the data determined in this investigation, the important knowledge of “how my RNA was collected”, “how to proceed with the RNA” as well as the importance of the choice of the reference gene are highlighted. In general, both tubing systems deliver RNA in good quality and amount, but random combinations with the RNA extraction systems must be avoided.


Coronaviruses are known to cause a wide range of diseases in humans and animals. Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel coronavirus discovered in 2012 and is responsible for acute respiratory syndrome in humans in the Middle East, Europe, North Africa, and the United States of America. Helicases are motor proteins that catalyze the processive separation of double-stranded nucleic acids into two single-stranded nucleic acids by utilizing the energy derived from ATP hydrolysis. MERS-CoV helicase is one of the most important viral replication enzymes of this coronavirus. Herein, we report the first bacterial expression, enzyme purification, and biochemical characterization of MERS-CoV helicase. The knowledge obtained from this study might be used to identify an inhibitor of MERS-CoV replication, and the helicase might be used as a therapeutic target. mK of ATP for M-nsp13 is inversely proportional to the length of the 5′ loading strand of the partially duplex RNA substrates. Finally, we also showed that the rate of unwinding (ku) of M-nsp13 is directly proportional to the length of the 5′ loading strand of the partially duplex RNA substrate. These results provide insights that enhance our understanding of the biochemical properties of M-nsp13.Middle East respiratory syndrome coronavirus (MERS-CoV) helicase is a superfamily 1 helicase containing seven conserved motifs. We have cloned, expressed, and purified a Strep-fused recombinant MERS-CoV nonstructural protein 13 (M-nsp13) helicase. Characterization of its biochemical properties showed that it unwound DNA and RNA similarly to severe acute respiratory syndrome CoV nsp13 (S-nsp13) helicase. We showed that M-nsp13 unwound in a 5′-to-3′ direction and efficiently unwound the partially duplex RNA substrates with a long loading strand relative to those of the RNA substrates with a short or no loading strand. Moreover, the IMPORTANCE Coronaviruses are known to cause a wide range of diseases in humans and animals. Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel coronavirus discovered in 2012 and is responsible for acute respiratory syndrome in humans in the Middle East, Europe, North Africa, and the United States of America. Helicases are motor proteins that catalyze the processive separation of double-stranded nucleic acids into two single-stranded nucleic acids by utilizing the energy derived from ATP hydrolysis. MERS-CoV helicase is one of the most important viral replication enzymes of this coronavirus. Herein, we report the first bacterial expression, enzyme purification, and biochemical characterization of MERS-CoV helicase. The knowledge obtained from this study might be used to identify an inhibitor of MERS-CoV replication, and the helicase might be used as a therapeutic target. While gastrointestinal symptoms, including diarrhea and queasiness, are also occasionally observed (Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel coronavirus discovered in 2012 and is responsible for acute respiratory syndrome in humans in the Middle East , Europe , North Africa (Tunisia and Egypt), and the United States of America  virus with one of the largest known RNA genomes 30.119 kb) . Followi0.119 kb 6–ATPase as both an RNA helicase and an ATP-independent RNA chaperone which may destabilize RNA duplexes and assist in the formation of more globally stable RNA structures  into two single-stranded nucleic acids (ss) by utilizing the energy derived from ATP hydrolysis –17. Heli–18–ructures . Defectsructures –28.Previously, biochemical characterization of severe acute respiratory syndrome CoV (SARS-CoV) helicase (S-nsp13) demonstrated that S-nsp13 can unwind both double-stranded DNA and RNA in a 5′-to-3′ direction, and it can hydrolyze all deoxyribonucleotide triphosphates (dNTPs) and ribonucleotide triphosphates , 30, Addku) of M-nsp13 is directly proportional to the length of the 5′ loading strand of the partially duplex RNA substrate. While M-nsp13 required a single-strand 5- to 20-nucleotide (nt) 5′ overhang for efficient unwinding of the dsRNA substrate, M-nsp13 was still able to partially unwind (~10% to 30%) a dsRNA substrate with a 0-nt (blunt-ended) to 2-nt 5′ loading strand.Herein, we report the first bacterial expression, enzyme purification, and biochemical characterization of M-nsp13. With double-stranded RNA (dsRNA) as a substrate, M-nsp13 unwound the substrate in a 5′-to-3′ direction, with the helicase requiring more ATP for optimal unwinding of RNA substrates with short 5′ loading strands. We also report that the rate of unwinding , mouse hepatitis virus (MHV) strain A59 (accession number NC_001846), porcine transmissible gastroenteritis virus (TGEV) strain Purdue 46 (accession number AJ271965), and avian infectious bronchitis virus (IBV) strain Beaudette (accession number M95169). The protein alignment shows that all helicases, along with M-nsp13, have the conserved helicase motifs I to VI, with motif I  containing the Lys288 that is recognized as the NTP binding site and the core ATPase site of superfamily 1 to 3 (SF1 to -3) helicases  were incubated with a 5 nM concentration of the partially duplex RNA substrate with a 20-nt overhang  for 30 min. Unless stated otherwise, for this specific experiment, we used the 5′-RNA-20 partially duplex substrate see . As show10.1128/mSphere.00235-16.1Figure S1 Figure S1, PDF file, 0.1 MB.Oligonucleotides and substrates used in this study. The Cy3-labeled strands are marked by asterisks. The green sequences denote the complementary sequences in the two strands. Download Copyright © 2016 Adedeji and Lazarus.2016Adedeji and LazarusCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the Some reports have shown that some helicases have a clear preference for either RNA or DNA , 33–36. 33–2, MgCl2, ZnCl2, or CaCl2. The results showed that M-nsp13 had the best helicase activity with MgCl2, followed by MnCl2, with more than 85% of the substrate unwound. The enzyme showed lesser activity with ZnCl2 and no observable activity in the presence of CaCl2  substrate in the presence of 2 mM MnClof CaCl2 .2 required for M-nsp13 activity, various concentrations of MgCl2 (1 to 6 mM) were incubated with 20 nM M-nsp13 and the 5 nM 5′-RNA-20  substrate for 30 min. The result showed that M-nsp13 had optimal activity with 2 mM MgCl2 . As shown in To determine whether nucleic acid unwinding by M-nsp13 depends on the loading strand length and to determine the minimum length of the 5′ single-stranded overhang required for efficient helicase activity, we designed five partially duplex RNA substrates with single-stranded 5′ ends of various lengths (between 0 and 20 bases) see  but withNTP hydrolysis is known to provide the energy required for translocation of RNA helicases along ssRNA and duplex-RNA during unwinding. To determine whether ATP is required for the unwinding activities of M-nsp13, a standard unwinding assay was conducted with all the RNA substrates described above in the absence of ATP. As shown in ATPase, was shown to possess an ATP-independent RNA chaperone activity NTP requirements of M-nsp13, a standard unwinding assay was conducted as described above, using the 5′-RNA-10  partially duplex RNA substrate in the presence or absence of individual (d)NTPs (2 mM). The results showed that M-nsp13 was able to hydrolyze all the nucleotides for the purpose of nucleic acid unwinding, with slightly more specificity for the NTPs . These rA ⋅ (1 – –kte) + n, where A is the amplitude that corresponds to the maximum fraction of ssRNA that can be generated enzymatically from the substrates, k is the pseudo first-order rate constant of RNA unwinding, t is the reaction time, and n is an additive constant representing the amount of ssDNA present before the reaction started. The results showed that the amplitudes and the rates of RNA unwinding of M-nsp13 were directly proportional to the length of the 5′ loading strand , 5′-RNA-2 (2-nt overhang), 5′-RNA-5 (5-nt overhang), 5′-RNA-10 (10-nt overhang), and 5′-RNA-20 (20-nt overhang) in the presence of 2 mM ATP at different time points (0 to 45 min). The change in the fractions of unwound ssRNA product with time is shown in g strand .Previous reports have shown that SARS-CoV helicase has a 5′-to-3′ directionality and, as such, does not show any activity with a substrate that has a 3′ overhang , 31. To Sequence alignment of M-nsp13 with other helicases suggests that the MERS-CoV helicase belongs to the SF1 family of helicases . HoweverATPase, was shown to possess an ATP-independent RNA chaperone activity  and nsp13 (helicase) physically interact, with nsp12 enhancing the activity of nsp13 , 37, thePrevious studies revealed that SARS-CoV and human coronavirus 229E helicases required at least a 5-nt 5′-end single-strand overhang for efficient unwinding , 38. In While some helicases exhibit a clear preference for either RNA or DNA , 33–36, 33–Comparison of the NTPase and dNTPase activities of M-nsp13 showed moderate variations, as M-nsp13 clearly exhibited better enzymatic activities with the NTPs than with the dNTPs. Nonetheless, the data suggested that the nucleotide-binding site of M-nsp13 has a low specificity among the NTPs. Similar data were also obtained for SARS-CoV nsp13 . Other R39–Other areas for future investigation include establishing the kinetic step size and the natural state of the active protein, .i.e., the monomer of multimers, and characterizing replication complex functions that involve M-nsp13, along with other MERS-CoV nonstructural proteins.In conclusion, we report the first expression, purification, and biochemical characterization of MERS-CoV helicase. We showed that M-nsp13 required more ATP for optimal unwinding of partially duplex RNA substrates with short 5′ loading strands. Our results also revealed that the rate of unwinding of M-nsp13 increased with a partially duplex RNA substrate with a longer 5′ loading strand. These results provide insights that enhance our understanding of the biochemical properties of M-nsp13. As such, M-nsp13 might be used as a target to identify inhibitors of MERS-CoV replication.JX869059.2). The coding region of M-nsp13 was amplified by PCR using 5′-BamHI-nsp13-CGGGATCCTGCTGTAGGCTCTTGTGTTG-3′ as the forward primer and 3′-EagI-nsp13-ATGCGGCCGCTATTGCAGCTTGTAGTTGGTAAAGCTC-3′ as the reverse primer. The PCR amplicons were digested with BamHI and EagI, followed by ligation into expression vector pET52b (Novagen-EMD Millipore), which fused M-nsp13 with a Strep tag at the N terminus. After confirmation of the amplicon nucleotide sequences, the construct was transformed into E. coli BL21 cells. Protein expression and purification were performed as described previously  as described by the manufacturer and expressed and purified as described above for the wild-type enzyme.A600 of 0.9 to 1.0 by the addition of 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). The cells were allowed to grow for 3 more h after induction. The cells were harvested by centrifugation  and stored at −20°C. Frozen cell pellets were resuspended in buffer A . The protein was purified by nickel affinity chromatography with a gradient of 25 mM to 500 mM imidazole in buffer A. Fractions containing pure protein (95%) were pooled and dialyzed against the storage buffer containing 12.5 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 50% glycerol.Expression and purification were as previously described . BrieflyPurified M-nsp13, M-nsp13K288A, and FMDV polymerase were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE), followed by transfer to nitrocellulose membranes . M-nsp13 was detected with anti-M-nsp13 rabbit antiserum as the primary antibody, which was commercially developed upon request (Abcam). A horseradish peroxidase-conjugated swine anti-rabbit immunoglobulin G (Dako) was used as the secondary antibody.Synthetic oligonucleotides were purchased from Integrated DNA Technologies . Sequences of the RNA and DNA substrates are shown in 2, and 2 mM ATP at 30°C for various times. Reactions were quenched by the addition of an equal volume of loading buffer (0.2% SDS and 20% glycerol). Unless otherwise mentioned, reactant concentrations refer to the final concentration in the reaction mixture. The released single-stranded RNA (ssRNA) product and unwound double-stranded RNA (dsRNA) were resolved by 8% nondenaturing PAGE using a running buffer containing 89 mM Tris-borate, pH 8.2, and run for 45 min at 4°C and 110 V. The controls for measuring maximum unwinding were dsRNAs denatured by heat for 5 min at 95°C and loaded immediately on the gel. In this and subsequent assays, the gels were scanned with a Bio-Rad multipurpose imager. When necessary, the band intensities representing ssRNA and dsRNA were quantitated using the Image Lab 5.1 software (Bio-Rad). The fraction of unwound RNA was plotted against time, and the kinetic parameters described in the mechanism were determined by nonlinear regression using GraphPad Prism (GraphPad Inc.).Helicase activity was measured by incubating 20 nM M-nsp13 with a 5 nM concentration of an RNA substrate  in a reaTo obtain the kinetic parameters associated with RNA unwinding by M-nsp13, the fraction of unwound RNA was plotted against time. By nonlinear regression, data were fit to the single-exponential equation in Results using Prism 7.0 (GraphPad Inc.).mK and Vmax of M-nsp13 with regard to ATP hydrolysis, the fraction of unwound RNA was plotted against different ATP concentrations. Data fitting was carried out by nonlinear regression to the Michaelis-Menten equation using Prism 7.0 (GraphPad Inc.).To obtain the kinetic parameters associated with the


Lx animals. Here, we describe long RNA sequencing reads from ribosomal RNA depleted samples from the same animals. This data can be used to investigate splicing-related events.The spontaneously hypertensive rat strain is a frequently used disease model. In a previous study, we measured translational efficiency from this strain and BN-Lx and SHR/Ola rats in biological replicates. Ribosomal RNA was removed and the samples subjected to directional high-throughput RNA-sequencing. Read and alignment statistics indicate high quality of the data. The raw sequencing reads are freely available on the NCBI short read archive and can be used for further research on tissue and strain differences, or analysed together with other published high-throughput data from the same animals.RNA was extracted from rat liver and heart left ventricle from BN- Lx reference strain [The inbred rat strain SHR/Ola (spontaneously hypertensive rat) is frequently used to study cardiovascular and metabolic diseases . In a pre strain , in whichttps://www.ncbi.nlm.nih.gov/bioproject/PRJEB7498.Total RNA was isolated from frozen, pulverized liver and heart left ventricle. Here, we describe the ribosomal RNA-depleted RNA sequencing obtained from these samples. An overview with SRA and BioSample identifiers is given in Table https://www.ncbi.nlm.nih.gov/sra/?term=SRP095829), or accessed through the NCBI BioProject PRJNA314751 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA314751). Per sample, 30–40 million reads were obtained. The adapter sequence GATCGGAAGAGCACACGT was trimmed using Flexbar [The Illumina Ribo-Zero kit (Illumina Cat.-No. MRZH11124) was applied to remove ribosomal RNA and the sequencing libraries were prepared using the Illumina TruSeq Stranded mRNA Library Prep Kit (Illumina Cat.-No. RS-122-2101). The raw sequencing reads can be downloaded from the NCBI SRA (short read archive) under the accession number SRP095829  and a model for hypertensive cardiomyopathy and metabolic syndrome (SHR) [In summary, we provide here a high quality RNA sequencing dataset for two rat strains, with two different tissues from two different animals for each strain. This data can be used to analyze differences between tissues and strains, e.g. variant calling, de novo transcriptome assembly, differential expression and splicing, or circRNA detection. Since the samples originate from the same animals as the ones in the study about translational differences between a healthy rat (BN-me (SHR) , the ribThe data described here is limited to two organs, heart left ventricle and liver.The sequencing depth does not allow for reliable quantification and characterization of very low abundant transcripts.Genomic DNA was not sequenced, and has to be used from other animals of the same strains.


Persicaria minor (kesum) is an important medicinal plant and commonly found in southeast countries; Malaysia, Thailand, Indonesia, and Vietnam. This plant is enriched with a variety of secondary metabolites (SMs), and among these SMs, terpenoids are in high abundance. Terpenoids are comprised of many valuable biomolecules which have well-established role in agriculture and pharmaceutical industry. In P. minor, for the first time, we have generated small RNAs data sets, which can be used as tool in deciphering their roles in terpenoid biosynthesis pathways. Fungal pathogen, Fusarium oxysporum was used as elicitor to trigger SMs biosynthesis in P. minor. Raw reads and small RNA analysis data have already been deposited at GenBank under the accessions; SRX2645684 (Fusarium-treated), SRX2645685 (Fusarium-treated), SRX2645686 (mock-infected), and SRX2645687 (mock-infected). Six weeks old P. minor plants were selected for this experiment. Selected plants were treated with Fusarium along with control . Each treatment had two biological replicates. Plant treatments were carried out as explained in our previous report 2.2Total RNA was isolated using Plant RNA Reagent  using manufacturer's protocol. Purity and concentration of extracted RNA was measured by Nanodrop spectrophotometer (ND-1000) and Qubit respectively. The integrity of RNA was determined by Bioanalyzer analysis (Agilent 2100) using RNA 6000 chip. The only RNA samples with RNA Integrity Number (RIN) over 7 were selected for further analysis.Small RNA libraries preparation were carried out using NEBNext® Small RNA Library Preparation kit according to recommended protocols. First, RNA sample was ligated to 3′ SR adaptor and primer hybridization was carried out to prevent dimer formation. Then, 5′ SR adaptor ligation was carried out followed by cDNA synthesis. PCR amplification was carried out to enrich the library of small RNA. Finally, PCR product was subjected to 6% polyacrylamide (PAGE) gel for size selection.Samples were send to Universiti Malaya, Malaysia for sequencing. Single end reads of 50 base pairs was generated through the Illumina HiSeq 2500™ in Rapid Run mode.2.3https://www.qiagenbioinformatics.com/). Quality cut-off value used was 20. Minimum percent of bases that had the quality was 90. No ambiguous nucleotide were allowed. Trimming of adapter index sequences were carried out and low quality reads were removed to produce clean reads. Reads with length between 18 to 30 nucleotides were used for annotation, while the rest were discarded  http://www.sanger.ac.uk/science/tools/rfam) For annotation of the small RNA, miRNA data set was downloaded from miRBase (version 21) (All the authors have approved the current manuscript for submission. No conflicts of interest involved.


Herein, we determine the net calcification potential and the relative balance of net organic carbon metabolism  and net inorganic carbon metabolism  within 23 coral reef locations across the globe. In light of these results, we consider the suitability of using these two metrics developed from total alkalinity (TA) and dissolved inorganic carbon (DIC) measurements collected on different spatiotemporal scales to monitor coral reef biogeochemistry under anthropogenic change. All reefs in this study were net calcifying for the majority of observations as inferred from alkalinity depletion relative to offshore, although occasional observations of net dissolution occurred at most locations. However, reefs with lower net calcification potential  could shift towards net dissolution sooner than reefs with a higher potential. The percent influence of organic carbon fluxes on total changes in dissolved inorganic carbon (DIC)  ranged from 32% to 88% and reflected inherent biogeochemical differences between reefs. Reefs with the largest relative percentage of NCP experienced the largest variability in seawater pH for a given change in DIC, which is directly related to the reefs ability to elevate or suppress local pH relative to the open ocean. This work highlights the value of measuring coral reef carbonate chemistry when evaluating their susceptibility to ongoing global environmental change and offers a baseline from which to guide future conservation efforts aimed at preserving these valuable ecosystems.Worldwide, coral reef ecosystems are experiencing increasing pressure from a variety of anthropogenic perturbations including ocean warming and acidification, increased sedimentation, eutrophication, and overfishing, which could shift reefs to a condition of net calcium carbonate (CaCO Howev provide , 10. Oceate (Ωa) , 12. Oth systems . Consequ systems .3 dissolution) can drive dramatic changes in the carbonate chemistry of seawater overlying reef ecosystems on these timescales . Fie. Fie3 dis  through time could be critical in guiding conservation and management efforts by contributing to a better understanding of a reef’s ability to deposit CaCO3.A coral reef’s metabolic influence on seawater chemistry is reflected as the rhythmic, diel and seasonal cycles of seawater TA and DIC . Because changes in TA and DIC largely reflect coral reef metabolism , they ca3 to organic carbon fluxes [2 to the atmosphere [Another potential metric of reef biogeochemistry derived from TA and DIC measurements is the slope of TA-DIC regressions, which reflects the balance of NCP and NCC, such that in a system dominated by NCP the slope approaches 0 while in a system dominated by NCC the slope approaches 2. Because of these properties, graphical vector analysis of seawater TA and DIC  quantifies the relative balance of CaCOn fluxes , 32, 33,n fluxes , 35. Themosphere , 23, andmosphere . Consequ2). Based on these studies we evaluate the current net calcification potential and the relative balance of NCP and NCC across a global selection of coral reefs; we qualitatively assess whether TA-DIC slopes can be a useful metric for monitoring changes in reef biogeochemistry due to anthropogenic perturbations; we detail how seawater carbonate chemistry measurements can better inform our understanding of coral reef susceptibility to future ocean acidification; and, we propose that future studies need to take an interdisciplinary approach combining ecological, biogeochemical, and physical measurements to develop a mechanistic understanding of coral reef ecosystems.Herein, we gathered seawater TA and DIC data from 27 temporal or spatial sampling expeditions covering 23 global coral reef locations in all major ocean basins . The stuSurface seawater TA, DIC, temperature, and salinity data were gathered from 23 coral reef locations from previously published and unpublished sources  14, 20,, 36–51. http://www.mbari.org/staff/etp3/regress.htm). The only exception was for data from Eilat Reef in the Red Sea which were grouped according to the year it was collected due to large changes in the offshore TA concentrations between different years [TA-DIC is the slope of the TA-DIC vector.The TA anomaly (ΔTA) was calculated as the difference between reef and offshore TA values , with negative values representing TA depletion (+NCC) and positive values representing TA repletion (-NCC). When offshore TA concentrations could not be obtained from the individual studies, they were estimated from GLODAP gridded data . For TA-nt years . The rel+ scale using the Matlab code equic.m [2) and changes in TA and DIC were predominantly measured over time, or (2) spatially based  if the sampling protocol involved sampling across a large spatial scale (>10km2).For all calculations, TA and DIC data were not normalized to salinity because of a wide range of values between locations, which could potentially introduce artifacts into the data. Regardless, normalizing the data had a minimal impact on both the average ΔTA values and TA-DIC slopes  Figs. To equic.m  at the a-1 (-1) was observed in Western Panama (Pacific), which could be due to high rates of NCC and/or long residence times assuming that no other processes such as upwelling or freshwater input from rain or runoff affected seawater TA [-1 for all locations, with the Cook Island dataset excluded because of substantial groundwater input [3 dissolution at times (mainly at night) as indicated by positive ΔTA values. Therefore, when evaluating ΔTA it is critical to devise a sampling strategy that adequately assesses times when both net CaCO3 production and dissolution are more likely to affect water column TA measurements .Most of the coral reefs in this study showed extensive depletion of TA relative to adjacent open ocean values (negative ΔTA) for the majority of observations Figs  and 3. T-1 . The onlwater TA . On averer input . This is2 concentrations, and low rates of gross calcification [The proportion of positive ΔTA observations at each reef site (indicative of times of net dissolution or negative NCC) ranged from 0 to 67% . Betweenfication , 36, 45.fication , 56 and fication . In factfication .7Be [Based on results from this and previous studies, monitoring changes in ΔTA over time serves as a relatively easy, highly sensitive, and instantaneous indicator of coral reef net calcification under both natural and anthropogenic environmental change. However, it is important to recognize that ΔTA doesn’t provide a quantitative measurement of NCC without knowing seawater residence times and volume. A number of approaches have been used to estimate seawater residence time for different reefs  but2) had significantly higher TA-DIC slopes compared to studies characterizing seawater carbonate chemistry at only one site . Most importantly, the results show that even reefs and/or reef habitats dominated by NCP still undergo substantial net calcification. This is supported by visual and quantitative observations of reefs with low TA-DIC slopes  that have extensive and healthy coral populations and high rates of NCC [in situ benthic communities. However, a recent short-term (24 h) mesocosm experiment with diverse benthic communities from Hawaii illustrated that the TA-DIC slope, TA anomaly, and seawater pH varied predictably according to community composition and relative rates of NCP and NCC [in situ data  would also have a greater depletion of TA due to high rates of NCC. Notably, there was no apparent correlation between average ΔTA or percent occurrences of dissolution and TA-DIC slopes within our dataset . This qus of NCC , 45, 63.s of NCC , 32, 43. and NCC . These ritu data , and indsolution , 64 and solution , 66, makar well below 1 [The local modification of TA and DIC determines how seawater pH changes within that reef. Reef sites with the lowest TA-DIC slopes and the largest absolute change in DIC experienced the greatest variability in seawater pH Figs  and 2. I below 1 . The com3 dissolution, all reefs in this study were net calcifying for the majority of observations. This indicates that, from a chemical perspective, the reefs in this study are currently maintaining positive CaCO3 production critical for the maintenance of reef structure and function . This kS1 Table2 of the TA-DIC slope; (6) slopes of TA-DIC vectors normalized to the average salinity at each site (nTA-nDIC); (7) mean TA anomaly  at each site; (8) percent occurrences of dissolution (%D) at each site; (9) TA of the open ocean endmember used to calculate ΔTA and %D in μmol kg-1; (10) study type, classified as either temporal (T) if the study was conducted mainly at one spot over time or spatial (S) if greater than a 10 km2 area was sampled; (11) total number of samples in each study; (12) length of each study; and (13) citation where the data was taken from.Columns are (1) location of each study; (2) ocean basin the study was conducted in; (3) slope of the TA-DIC vector; (4) percent influence of NCP on changes in DIC; (5) R(XLSX)Click here for additional data file.S1 FigSome locations were combined because there was not enough spatial resolution to show as two distinct points. The colors and symbols indicate whether the reefs are in the Atlantic (green circles), Great Barrier Reef (red squares), Indo-Pacific (blue triangles), and other (grey diamonds) regions.(TIF)Click here for additional data file.S2 FigTo calculate the salinity normalized slope (nTA-nDIC), TA and DIC data were normalized to the average salinity of each site.(TIF)Click here for additional data file.S3 FigFor ΔnTA data were normalized to the average salinity of each reef site.(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.


Here we identified PUF60, a splicing factor and a U2 small nuclear ribonucleoprotein auxiliary factor, as a versatile regulator of transcriptional and post-transcriptional steps in expression of hepatitis B virus (HBV) 3.5 kb, precore plus pregenomic RNA. We demonstrate that PUF60 is involved in: 1) up-regulation of core promoter activity through its interaction with transcription factor TCF7L2, 2) promotion of 3.5 kb RNA degradation and 3) suppression of 3.5 kb RNA splicing. When the 1.24-fold HBV genome was introduced into cells with the PUF60-expression plasmid, the 3.5 kb RNA level was higher at days 1–2 post-transfection but declined thereafter in PUF60-expressing cells compared to viral replication control cells. Deletion analyses showed that the second and first RNA recognition motifs (RRMs) within PUF60 are responsible for core promoter activation and RNA degradation, respectively. Expression of PUF60 mutant deleting the first RRM led to higher HBV production. To our knowledge, this is the first to identify a host factor involved in not only positively regulating viral gene expression but also negative regulation of the same viral life cycle. Functional linkage between transcriptional and post-transcriptional controls during viral replication might be involved in mechanisms for intracellular antiviral defense and viral persistence. Hepadnaviridae family with a partial double-stranded relaxed circular DNA genome. Approximately 240 million people worldwide are chronically infected with HBV. It is estimated that one million deaths occur annually due to HBV-related severe liver diseases such as liver cirrhosis, liver failure and hepatocellular carcinoma1. Although nucleoside analogues and interferons are the major chemotherapies for HBV-positive patients to date, they do not achieve HBV clearance or eliminate the viral genome when in the covalently closed circular (ccc) DNA form, which resides in the nucleus of infected cells. Long-term treatment with these antivirals may also have drawbacks such as development of drug-resistant variants and adverse side effects3. Thus, understanding of molecular mechanisms that determine viral replication, persistence and latency is urgently needed to develop novel treatments to achieve virological cure.Hepatitis B virus (HBV) is a hepatotropic, enveloped virus of the 5). In addition, several forms of spliced RNAs are generated from 3.5 kb RNA. These spliced forms have been observed in sera and livers of hepatitis B patients as well as in cultured cells transfected with the viral genome8. However, their significance and regulatory mechanisms underlying post-transcriptional processing events in the HBV life cycle are essentially unclear.Upon infection, the uncoated viral genome is transported to the nucleus and converted into cccDNA, which serves as the template for synthesis of viral transcripts. Four unspliced viral RNAs, 3.5, 2.4, 2.1 and 0.7 kb, are transcribed from their respective promoters and two enhancer regions (ENI and ENII). The 3.5 kb RNA includes precore and pregenomic RNA species. Precore mRNA encodes precore antigen (HBeAg), and pregenomic RNA directs translation of core antigen (HBcAg) and polymerase. Pregenomic RNA also serves as a reverse transcription template after encapsidation. A variety of liver-enriched and ubiquitous transcription factors target the promoter and enhancer regions to regulate viral transcription and replication  and FUSE-binding protein (FBP), acting as an FBP-interacting repressor (FIR), and is a transcriptional repressor of human c-myc gene10. Here, we found that PUF60 up-regulated core promoter activity through its interaction with transcription factor 7-like 2 (TCF7L2), which is necessary for direct binding with the ENII region. PUF60 also contributed to 3.5 kb RNA degradation and suppression of 3.5 kb RNA splicing.In this study, we aimed to clarify molecular mechanisms controlling transcriptional and post-transcriptional processes during HBV replication, in particular mechanistic coupling between transcriptional regulation and post-transcriptional mRNA processing. During the course of investigating involvement of host cell factors with dual DNA- and RNA-binding capacities in HBV replication in siRNA-mediated gene knockdown and over-expression experiments, we identified PUF60 as a versatile regulator of both transcriptional and post-transcriptional steps in expression of HBV 3.5 kb RNA. PUF60 was first discovered as a poly-U binding, 60-kDa splicing factor that is important for efficient splicing of multiple introns11, were expressed at days 2 and 4 pt in cells  were analyzed by northern blotting. At day 2 post-transcription (pt), the level of 3.5 kb RNA, but not 3.5 kb RNA -derived, 2.2-kb spliced (Sp1) RNA lacking intron nt 2447/489, was higher in PUF60-expressing cells compared to control cells. In contrast, at day 4 pt, both 3.5 kb RNA and Sp1 RNA levels were severely diminished in PUF60-expressing Fig. , probe Slls Fig. . Nuclearlls Fig. , left. Ills Fig. , right. lls Fig. . A dose-lls Fig. . Impact lls Fig. . Increas12 is located at the C-terminus (aa 462–549) of PUF60. To identify the regions in PUF60 responsible for its effects on increased and decreased 3.5 kb RNA expression, PUF60 deletion mutants, pcDNA-F-PUF60-D1, -D2 and -D3, which encode PUF60 lacking one of the motifs  indicated above, respectively, with a FLAG tag were constructed  family and contains two canonical RNA recognition motifs (RRMs) at its N-terminal (aa 129–207) and central (aa 226–304) regions. An additional unusual RRM called the U2AF homology motif (UHM)ted Fig. . The subted Fig. . Wild-tyEffects of over-expression of each PUF60 deletion mutant on 3.5 kb RNA expression at days 2 and 4 pt were tested in cells co-transfected with pUC-HB-Ce Fig. . At day Next, to determine the effect of PUF60 on HBV production, particle-associated HBV DNA in culture supernatants of cells transfected with pUC-HB-Ce with or without pcDNA-F-PUF60 was quantitatively measured Fig. . The res13, to assess the influence of PUF60 on viral infection . As expected, both 3.5 kb RNA and Sp1 RNA levels in infected cells with PUF60 expression were significantly lower than those in control infected cells. At earlier time points, such as day 2 post-infection, it was difficult to assess the influence of PUF60 expression since basal levels of HBV RNAs were quite low in this setting.We next used the HBV infection system with NTCP-expressing HepG2 cells, HepG2-hNTCP-C4 cellsion Fig. . After 1Based on the positive effect of PUF60 on 3.5 kb RNA, in particular at the nuclear level, at the early phase pt Fig. , we nextPUF60 deletion experiments Fig.  identifiTo address the molecular mechanism underlying PUF60 regulation of the core promoter, a series of reporter constructs with partial ENII/BCP deletions were first generated Fig. , left toin vitro synthesized proteins and end-labeled oligonucleotide probes spanning the nt 1689–1726 and nt 1710–1742 regions. Among the proteins tested, TCF7L215, a member of the TCF family of transcription factors that is predicted to bind to nt 1708–1713 (TTCAAAG) from the search program, was found to bind to the nt 1689–1726 sequence  up-regulation of core promoter activity, 2) suppression of pregenome-derived RNA splicing and 3) promotion of pregenome degradation. When the 1.24-fold HBV genome plasmid was introduced into cells with co-transfection of the PUF60-expression vector, the 3.5 kb RNA level increased at an early time point, such as days 1–2 pt, but subsequently decreased compared to control HBV-replicating cells. To our knowledge, this is the first study to demonstrate involvement of a host factor in not only positively regulating gene expression and replication of virus, but also the negative regulation of the same viral life cycle. While why both decreased and increased expression of PUF60 lowered the 3.5 kb RNA at day 4 pt Fig.  is uncle17. PUF60 is classified as a member of the U2AF protein family, where canonical RRMs with distinct features of protein recognition are conserved12. U2AF-related proteins are potentially involved in changes in available splice sites by preventing initial binding of U1 snRNP and U2AF during spliceosome assembly.PUF60 is a splicing factor that associates with splicing factors involved in early spliceosome assembly and plays a role in the recognition of the 3′ splice site during recruitment of small nuclear ribonucleoproteins (snRNPs) to the intron for splicing18. Competition between TIA1 and another RNA-binding factor, HuR, for binding on PDCD4 mRNA is thought to be important for fine-tuning PDCD4 expression in cells. Additionally, TIA1 has been shown to contribute to HBs expression possibly via interaction with a particular HBV RNA sequence, post-transcriptional regulatory element (PRE)19.Although mRNA turnover is critical for gene expression in eukaryotic cells, contribution of decay factors to mRNA degradation machineries remains poorly understood because of their complexity. In particular, evidence for roles of U2AF-related proteins in mRNA degradation is quite limited. T-cell intracellular antigen 1 (TIA1), known to possess U2AF homology motifs, has been shown to contribute to modulation of the mRNA level of programmed cell death 4 (PDCD4) through binding to PDCD4 3′ UTR mRNA20, as seen in retroviruses, alphaviruses and filoviruses23. Cytidine deaminase possibly triggers HBV RNA degradation by tethering the RNA exosome to the viral protein/RNA complex24. Non-stop-mediated RNA quality control is potentially involved in degradation of the viral X mRNA at the RNA exosome complex25. To our knowledge, this study is the first to reveal the role of PUF60 in mRNA degradation, and PUF60-mediated degradation of viral RNAs might be a novel type of antiviral defense mechanism. PUF60 was first identified as a 60-kDa protein that efficiently binds to the poly-U tract. However, no typical or consensus motif for PUF60 binding is observed in HBV RNAs. To determine the HBV RNA degradation mechanism mediated by PUF60, we found direct binding of PUF60 to PRE within HBV RNA but not with its reverse sequence in the in vitro assay  to control antiviral host defense and viral persistence.38, respectively, were gifts from Dr. Mizokami . pUC-HB-Ce, which contains the 1.24-fold HBV genome derived from a consensus sequence of HBV genotype Ce, was designed in accordance with the most common nucleotide observed among HBV genotype Ce clones  at each position and was artificially synthesized by Eurofins Genomics . DNA fragments of HBV core promoter derived from HBV genotypes B and C were designed in accordance with the most common nucleotide among HBV genotypes Bj and Ce, respectively, via searching the database of HBV sequences, and were then synthesized by Eurofins Genomics. To construct pGL4.74-HBpg-Ce, synthesized fragments corresponding to the nt 900–1817 region of the HBV genome digested by KpnI and HindIII were inserted upstream of the luciferase reporter gene of pGL4.74 . To construct pGL4.74-HBenIIcp-Bj/Ce, synthesized fragments corresponding to the nt 1627–1817 region were amplified by PCR and cloned into pGL4.74 as described above. The sequences of preS1 and preS2/S promoters were obtained from nt 2707–2847 and nt 2937–3204 regions, respectively, from a consensus sequence of genotype Ce. Promoter sequences of human ubiquitin C and human elongation factor 1α subunit were obtained from pUB6 and pEF6 , respectively, and subsequently inserted into pGL4.74. A series of deletion mutants, pGL4.74-HBenIIcp-Bj-D1–D10  supplemented with 10% fetal bovine serum. Cells (1 × 105 cells/well in a 24-well plate) were transiently transfected with 1 µg of plasmid DNA mixed with Lipofectamine LTX (Thermo Fisher Scientific). Synthetic siRNAs were provided by Ambion (Thermo Fisher Scientific) and were transfected into cells using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific).Human hepatoma derived cells [HuH-7, HepG2, HepG38.7-Tet and HepG-hNTCP-C414 was concentrated using Amicon Ultra-15 Centrifugal Filter Devices  and the resulting HBV sample (HBV DNA copies 2 × 108/ml) was used as an inoculum for infection assays. HepG2-hNTCP-C4 cells cultured in a 24-well collagen-coated plate were transfected with pcDNA-F-PUF60 and then inoculated with the HBV sample (50 µl) in DMEM containing 4% polyethylene glycol (PEG) 8000 (Promega) after 12 h of transfection. The cells were washed 3 times with PBS after 24 h of infection and then subjected to RT-qPCR after 96 h of further culture.The culture supernatant of HepG38.7-Tet cells39. To quantify particle-associated HBV DNA, culture supernatants collected from transfected cells were treated with PNE solution  for 1 h on ice. To remove free nucleic acids, the pellets were incubated at 37 °C for 1 h with DNase I  and RNase (TaKaRa). After treatment with proteinase K at 56 °C overnight, HBV DNA was isolated by phenol/chloroform extraction and ethanol precipitation. HBV DNA copies were determined by qPCR with primers 5′-TCCCTCGCCTCGCAGACG-3′ and 5′-GTTTCCCACCTTATGAGTC-3′.Quantification of HBV DNA was carried out as previously described40. Total RNA was extracted from transfected cells with TRI Reagent . After treatment with DNase I and RNase inhibitor, cDNA templates were synthesized and HBV RNAs were quantified by qPCR using the SYBR qPCR Mix kit  with the following primer sets: 5′-TCCCTCGCCTCGCAGACG-3′ and 5′-GTTTCCCACCTTATGAGTC-3′ for unspliced 3.5 kb RNA, and 5′-CCGCGTCGCAGAAGATCT-3′ and 5′-CTGAGGCCCACTCCCATAGG-3′ for spliced RNAs derived from 3.5 kb RNA. 5′- TTCTACAATGAGCTGCGTGTG -3′ and 5′-GGGGTGTTGAAGGTCTCAAA-3′ for β-actin mRNA, 5′-AAGGGTGTTTCGTTCCCTTT-3′ and 5′-TAGTGTTTTCGCCAAGCAAA-3′ for HSPA1B mRNA, and 5′-AGCAGCAGCTCACCAACC-3′ and 5′-CATCGATTGCAAAGGTGAGA-3′ for PUF60 mRNA. For semi-quantitative RT-PCR, cDNA templates were amplified with primers 5′-AGCCTCCAAGCTGTGCCTTGGGTG-3′ and 5′-AACCACTGAACAAATGGCACTAGTAAACTGAGC-3′. Unspliced and spliced forms of 3.5 kb RNA were analyzed by agarose gel electrophoresis. The nucleotide sequences of PCR primers used in the study are listed in Supplementary Table Quantification of unspliced and spliced forms derived from HBV 3.5 kb RNA and host-derived mRNAs were performed as described previously with some modifications2). After washing, the blotted membrane was dried at room temperature. The blot was prehybridized with DIG Easy Hybridization buffer (Roche Diagnostics) in 68 °C and hybridized with an appropriate DIG-labeled RNA probe labeled with DIG-11-UTP at 68 °C overnight using the DIG Northern Starter Kit (Roche Diagnostics). To generate a DIG-labeled RNA probe with specific binding to HBV pregenome and HBs RNA, PCR fragments covering the nt 1998–2447 and nt 3205–488 regions were used as templates for in vitro transcription for the pregenome probe and HBs probe, respectively. RNA was labeled in the T7 promoter transcriptional system with DIG-11-UTP using a labeling mixture from the DIG Northern Starter Kit (Roche Diagnostics). Detection of the DIG-labeled probe on the blot was performed using CDP-Star detection reagent .Total RNA was extracted from cells transfected with HBV plasmids using TRI Reagent. After treatment with DNase I and RNase inhibitor, RNA samples were separated on 1.2% agarose gel with 7% formaldehyde at 60 V for 3 h in 1 × 3-(N-morpholino)propanesulfonic acid (MOPS) buffer . The samples were transferred to a nylon membrane  with 20x SSC transfer buffer for 16 h, and subsequently cross-linked to the membrane by ultraviolet light (120 mJ/cmAt day 2 post-transfection (pt) with pUC-HB-Ce and pcDNA-F-PUF60 or pcDNA-F-PUF60-D1, aliquots of the cells were harvested (designated as 0 h) and the other cells were treated with actinomycin D (5 µg/ml), followed by further culture for 6 or 12 h. At each time point, total RNA was extracted from transfected cells with TRI Reagent and the HBV 3.5 kb RNA level was determined by RT-qPCR using the SYBR qPCR Mix kit (Toyobo).Renilla luciferase reporter which carries either of HBV promoter or host cellular promoter. At 24 or 48 h pt, luciferase activities in cell lysates were measured with the Renilla luciferase reporter assay kit (Promega). Total protein concentrations in cell lysates were measured and used to normalize luciferase activities.Cells were transiently co-transfected with pcDNA-F-PUF60, pcDNA-F-PUF60-D1, -D2, -D3 or empty vector and the in vitro binding between transcriptional factors and HBV DNA sequence of the ENII/BCP region, HNF1α, SRY, TCF7L2, FOXM1, SP1, KLF5 and PUF60 were synthesized in vitro. In brief, cDNAs encoding these seven transcription factors were isolated from MGC clones  and individually inserted into a pEU vector41 to express an N-terminal FLAG-fusion protein. Each transcription factor was synthesized in a wheat cell-free system as previously described42. The 3′-ends of synthesized oligonucleotides (nt 1689–1726) were labeled by DIG-11-dUTP using the DIG gel shift kit (Roche Diagnostics). The labeled oligonucleotide probe was mixed with each synthesized protein, and the gel shift reaction was performed according to the manufacturer’s instructions. The resulting samples were analyzed by native PAGE using a 6% gel. The labeled DNA-protein complexes as well as the probe were blotted to a nylon membrane and detected using CDP-Star detection reagent .To determine 43. Briefly, cell lysates were separated by SDS–PAGE and transferred onto polyvinylidene difluoride membranes. After blocking, membranes were incubated with an antibody against PUF60 , FLAG M2  or GAPDH  or HA  for 1 h. After washing, membranes were incubated with an HRP-conjugated secondary antibody  for 0.5–1 h. Antigen-antibody complexes were detected using the ChemiDoc™ Imaging System . For immunocytochemistry, cells grown on a glass bottom plate were fixed with 4% paraformaldehyde for 15 min and permeabilized in 0.5% Triton X-100 in PBS, followed by blocking with 1% bovine serum albumin (BSA). Immunocytochemistry was performed by incubation with the anti-PUF60 antibody  for 2 h, followed by incubation with Alexa Fluor 488 anti-rabbit IgG (H + L) antibody  for 2 h. Double-stranded DNA was stained with Hoechst 33342 . Subcellular localization of PUF60 was observed under a confocal microscope .Immunoblotting was performed as previously described with slight modification44. Briefly, cells seeded in 100-mm dishes were transfected with siTCF7L2 RNA. After 48 h, the cells were co-transfected with pUC-HB-Ce and the pcDNA-F-PUF60 expression vector or empty vector. After 48 h, ChIP was performed by the Chromatin IP kit . Proteins in the cells were cross-linked with DNA using 1% formaldehyde for 10 min at room temperature. The cross-linking reaction was stopped by the addition of 1 ml of 10x glycine to each dish and incubation for 5 min at room temperature. After washing two times with ice-cold PBS, the cells were scraped into PIC buffer (1 ml PBS and 5 µl 200x protease inhibitor cocktail) and sonicated to shear DNA to lengths between 150 and 900 bp. After 5-fold dilution of the sonicated cell supernatants in 100 µl 1x ChIP buffer and 0.5 µl 200x PIC, immunoprecipitations were carried out overnight at 4 °C with the anti-FLAG M2 antibody. Protein G agarose beads were added and incubated for 2 h at 4 °C with rotation. DNA-protein complexes were eluted from the beads with a buffer containing 1% SDS and 0.1 M NaHCO3. The cross-links were reversed by incubating the eluates with proteinase K solution  overnight at 65 °C. DNA was recovered by phenol/chloroform extraction and ethanol precipitation. ChIPped DNA was analyzed for the presence of HBV gene promoter sequence by qPCR. Viral DNA covering the nt 1589–1828 region was detected by using the SYBR qPCR Mix kit (Toyobo) with the following primer set: 5′-CTTCACCTCTGCACGTCGCATG-3′ and 5′-GTGAAAAAGTTGCATGGTGCTGGTG-3′.ChIP followed by qPCR was performed as previously described with some modification14. Briefly, cells were lysed with lysis buffer (0.5% NP-40 in PBS) and centrifuged at 15,000 rpm for 10 min at 4 °C. The supernatants were incubated with Protein G agarose beads, which were prewashed with lysis buffer, and anti-HA antibody or anti-FLAG antibody for 60 min at 4 °C. The samples were then centrifuged, and the resulting pellets were washed 4 times with lysis buffer and subjected to SDS-PAGE.Immunoprecipitation was performed as previously described with slight modificationCells were suspended with hypotonic buffer , followed by centrifugation at 500 × g for 5 min at 4 °C. The supernatant was collected and termed as the cytoplasmic fraction. The pellet containing the nuclear fraction was re-suspended with disruption buffer .Supplementary Information