Toscana virus is an emerging bunyavirus in Mediterranean Europe where it accounts for 80% of pediatric meningitis cases during the summer. The negative-strand ribonucleic acid (RNA) genome of the virus is wrapped around the virally encoded nucleoprotein N to form the ribonucleoprotein complex (RNP). We determined crystal structures of hexameric N alone (apo) and in complex with a nonameric single-stranded RNA. RNA is sequestered in a sequence-independent fashion in a deep groove inside the hexamer. At the junction between two adjacent copies of Ns, RNA binding induced an inter-subunit rotation, which opened the RNA-binding tunnel and created a new assembly interface at the outside of the hexamer. Based on these findings, we suggest a structural model for how binding of RNA to N promotes the formation of helical RNPs, which are a characteristic hallmark of many negative-strand RNA viruses. Bunyaviridae family. Members of this genus also include Rift Valley Fever virus (RVFV), Sandfly Fever Sicilian virus, Severe Fever with Thrombocytopenia Syndrome virus and Uukuniemi virus. These viruses are etiological agents of a wide range of illnesses ranging from self-limiting febrile episodes to hemorrhagic fevers characterized by multi-organ failure and death (Toscana virus is an enveloped negative-strand ribonucleic acid (RNA) virus that belongs to the genus phlebovirus in the nd death . The spond death ,5. The SThe genome of phleboviruses, like other bunyaviruses, is tightly packed with N into ribonucleoprotein complexes (RNPs) . The RNPBunyaviruses replicate in the cytoplasm of host cells. For the initiation of transcription, RdRp employs an endonuclease activity to cleave and ‘snatch’ the caps of host cell mRNA to prime viral transcription. This mechanism has been demonstrated for La Crosse virus and, by sequence comparison, inferred for all bunyaviruses .Bunyaviridae lack a matrix protein that connects the lipid envelope-associated glycoproteins to the RNP. Instead, in Uukuniemi virus, N interacts with the cytoplasmic tails of its cognate glycoproteins. This suggests that the bunyavirus N may, in part, fulfill the role of the missing matrix protein  N and of hexameric N in complex with a single-stranded nonameric RNA. Our structural studies, combined with functional experiments, suggest a molecular mechanism of how RNA binding to N induces the transition from a hexameric ring to a helical RNP assembly.6-fusion, the gene was cloned into pET15b or pET46-ek/LIC vectors (Novagen). RVFV N was expressed from the pET21 vector (Novagen). Plasmids for intracellular reconstitution of RVFV RNPs, pI.18-RVFV_L, pI.18-RVFV_N, pI.18-HA-PKRΔE7 and pHH21-RVFV-vMRen were described previously (A codon-optimized complementary deoxyribonucleic acid (cDNA) of the Toscana virus N gene (GenBank accession code X53794) was commercially synthesized (Genscript) and supplied in the pUC57 vector. For bacterial expression as amino-terminal Hiseviously . The pI.600 of 0.4, followed by a temperature shift to 20°C. The protein was produced for 18 h in the presence of 200-μM isopropyl β-D-1-thiogalactopyranoside. Bacteria were collected by centrifugation and resuspended in lysis buffer containing 20-mM TRIS (pH 8), 10-mM imidazole, 300-mM NaCl and complete ethylenediaminetetraacetic acid-free protease inhibitors, followed by cell disruption in a microfluidizer (Microfluidics). Lysates were cleared by centrifugation at 40 000 g for 30 min at 4°C.The recombinant plasmids were transformed into BL21 DE3 or BL21 DE3 Rosetta cells. Bacteria were cultured in Luria Broth (LB) medium at 37°C until an ODThe supernatant was loaded on a 30-ml gravity flow column filled with 3-ml NiNTA resin pre-equilibrated with lysis buffer. The column was washed with 20-ml lysis buffer, followed by 25-ml washing steps with equilibration buffer  containing increasing concentrations of imidazole . The protein was eluted from the column using 12.5-ml equilibration buffer containing 250-mM imidazole. N was further purified via size exclusion chromatography on a Sephadex 200 10/300 column  equilibrated with TRIS-buffered saline (pH 7.5).Escherichia coli, N bound to the NiNTA column was denatured by incorporating 8-M urea in the washing buffer. Bound RNA was washed off using high salt buffer  and N eluted by 20-mM TRIS, pH 7.5, 250-mM imidazole and 500-mM NaCl.To remove heterogeneously bound RNA from 260/A280 ratio before and after refolding. The refolded N had an A260/A280 ratio of 0.6 while the native RNP complex purified directly from E. coli had a ratio of ∼1.5 indicating about 10% bound nucleic acid.The fraction of bound RNA was determined by the AThe theoretical isoelectric point of N is 9.2 . Cation To verify the integrity of the refolded N, circular dichroism (CD) measurements were performed at room temperature using a protein concentration of 1 mg/ml in phosphate buffered saline, pH 7, in an Aviv 62A spectropolarimeter.260/A280 ratio of the reconstituted RNP complex was 1.1, which corresponds to approximately 5% bound RNA.For reconstitution of a homogenous RNP, the refolded N was incubated at room temperature for 1 h with commercially synthesized nonameric RNA , at a molar ratio of 1:1. A final gel filtration step was added to remove unbound RNA. The stability of the reconstituted complex was monitored by gel filtration and also on a native gel. The Ain vitro-transcribed 8-kb single-stranded RNA comprising a Green Fluorescent Protein (GFP) expression construct  and Topaz system . Various commercially available screens, including PACT, PEG ion, Crystal (Qiagen) and Index Screen (Hampton Research) were used. Initial hits were translated to the hanging drop technique using a drop size of 1 μl + 1 μl at a protein concentration of 18–20 mg/ml. N crystals were grown in 6.5% PEG 3350, 0.1-M sodium malonate, pH 4.5–5.2, while RNP crystals were grown in 11% PEG 3350, 0.1-M sodium malonate, pH 4.5–5.2. Diffraction data of single crystals were collected at BESSY-II BL14.1 and processed using XDS and MOSFLM software .The structure of Toscana virus N was solved by molecular replacement using a homology model of the RVFV N (PDB code 3OUO) calculated by the SWISS-MODEL server as a search model . Both PhBoth models were built with COOT  alternatITC experiments were carried out at 8°C in a VP-ITC or iTC200  using degassed 20-mM TRIS, pH 7.5, 150-mM NaCl. N was diluted and RNA resuspended in this buffer prior to the measurement. A 400-μM solution of single-stranded 6–9-mer RNAs was titrated into a solution containing 30-μM apo N. Binding isotherms were fitted and equilibrium dissociation constants were calculated using the Microcal ORIGIN software.2O buffer  and incubation at 0°C for 10 s and 1000 s. At the indicated time points, the exchange reaction was quenched by adding 12 μl of ice-cold optimal quench solution , immediately frozen on dry ice and stored at –80°C. The non-deuterated control samples and equilibrium-deuterated control samples were also prepared by mixing protein with H2O buffer  and equilibrium-deuterated buffer (0.8% FA in 99.9% D2O). The frozen samples were then thawed at 5°C and passed over an immobilized pepsin column (16-μl bed volume) at a flow rate of 20 μl/min, and the resulting peptides were collected on a C18 trap for desalting and separated by a Magic C18AQ column (Michrom BioResources Inc.) using a linear gradient of acetonitrile from 6.4% to 38.4% over 30 min. Mass spectrometry (MS) analysis was performed using Liquid Chromatography Quadrupole (LCQ) Classic mass spectrometer from Thermo Finnigan, with a capillary temperature of 200°C. Data were acquired in both data-dependent MS/MS mode and MS1 profile mode, and the data analyzed by SEQUEST (Thermo Finnigan Inc.) and DXMS explorer .To obtain optimal fragmentation sequence coverage of Toscana virus N, optimal quench conditions were established as previously described . DeuteriAssays to measure activity of reconstituted RVFV RNPs were essentially performed as described . BrieflyE. coli and purified via affinity chromatography. The resulting protein was associated with E. coli nucleic acids, as apparent by the observed A260/A280 ratio of 1.5. This preparation yielded protein crystals that showed only weak, anisotropic diffraction.N of Toscana virus was expressed in 260/A280 ratio of 0.6 . Correct folding of the protein was confirmed by CD spectroscopy . Analytical gel filtration experiments combined with static light scattering indicated a tendency of the protein to self-assemble even in the absence of RNA  to a control 9-mer RNA corresponding to the reverted 3′ consensus sequence  compared to Toscana virus N in ITC experiments .It was previously reported that Ns of bunyaviruses bind with high affinity to the highly conserved 5′ and 3′ sequences of their genomic RNA segments . In agreTo further analyze the sequence-specificity of N, base pair substitutions were introduced in oligo1. The exchange of guanine 2 (G2) to adenine did not have a significant effect on the binding affinity . Furthermore, the affinity for a 9-mer polyU sequence was only slightly reduced compared to the original 9-mer sequence , indicating a certain degree of plasticity in RNA recognition.in vitro using an 8000-bp single-stranded RNA. By negative-stain electron microscopy, we observed filamentous RNP scaffolds with a diameter of 10–12.5 nm that may have an underlying helical sub-pattern . The interaction of helix α1 with the neighboring monomer included hydrogen bonds and hydrophobic contacts, and a salt bridge between Lys83 and Glu29 of the opposing monomer . A similar oligomerization mode has been reported for RVFV RNPs , pointing to a highly dynamic association of this region in solution.Also in the RNA-bound RNP crystals, oligomerization of N into hexamers was mediated by the amino-terminal α-helical arm, which filled the hydrophobic groove of the core domain at the outside of hexamer Figure . In agreThe orientation of the RNA is evident from the asymmetric distribution of the two guanine (G) bases at positions 2 and 4 which could be discriminated from the remaining pyrimidine bases in the 9-mer RNA Figure . The RNAThe phosphate backbone of the 9-mer RNA is recognized by several positively charged and polar residues. For example, Arg111 and Lys72 formed salt bridges to the phosphates of G4 and U6, respectively. Interestingly, Lys204 and Lys79 formed hydrogen bonds to the phosphate backbone at the 5′ end of the RNA whereas they do not interact with the phosphate backbone at the 3′ RNA end Figure .Assuming an identical conformation of the phosphate backbone, we modeled the nonameric sequence of the 5′ non-coding region  compared to wild-type N , more than the single Y32A mutation, suggesting that these two adjacent residues cooperate in RNA binding. All three single mutants were able to form RNPs in vitro using a synthetic single-stranded RNA as a template, although RNPs formed by the K204A mutant appeared somewhat shorter compared to wild type . Every second N monomer was laterally displaced relative to the neighboring N leading to a staggered assembly of the hexameric ring. In this way, the six-fold symmetry in the N hexamer was broken, resulting in three-fold symmetry of the RNP Figure  and B. C2 at the outside of the hexamer, which did not involve the amino-terminal arm . Since each N binds seven RNA bases, the 6-mer RNA would have enough space to bind to adjacent Ns in the hexameric ring. Our data suggest that RNA binding and the associated structural rearrangements of adjacent subunits prevent binding of a second RNA molecule in the context of a hexameric ring.To further characterize this domain rearrangement, binding of shorter RNA oligomers to N was evaluated. As expected, consecutive shortening of the RNA gradually reduced the affinity for N . Surprisingly, the binding stoichiometries of the shorter RNA oligomers (6–8 mers) did not significantly differ from the 9-mer RNAs  separate upon RNA binding. However, Gln198 does not contact the RNA and, consequently, no inter-subunit rotation is observed. The RNA and DNA sequences used for the crystallization of the RVFV RNP symmetrically fit into one ring at the inside of the RNP. We speculate that, in these cases, a closed ring structure might be more stable than an open helix turn and therefore be favored for crystallization. Similarly, Lys204 and Lys79 do not contact the RNA backbone at the 3′ RNA end of our Toscana virus RNP and, consequently, no major rotation is observed. The 9-mer RNA may be too short at this side and/or the induction of a similar shift as at the 5′ RNA end would induce an open ring and therefore be energetically unfavored. Furthermore, it can be envisaged that the proposed inter-subunit shift is energetically not favoured over the planar architecture, since we do not observe it in the apo N hexamer. Instead, it may be driven by RNA binding. This may also explain why the K204A and K79A mutants still bind with similar affinity compared to wild-type N to our 9-mer RNA—the loss of binding energy due to these mutations may be compensated by a reduced inter-subunit shift. In support of such hypothesis, the It has been known for some time that phlebo- and tenuiviruses are evolutionarily related . Both haCoordinates and structure factors for the Toscana virus N and N–RNA complex have been deposited in the Protein Data Bank under accession codes 4csg and 4csf. GenBank accession code: X53794 and PDB code: 3OUO.Supplementary Data are available at NAR Online.


Cyst nematodes are biotrophs that form specialized feeding structures in the roots of host plants, which consist of a syncytial fusion of hypertrophied cells. The formation of syncytium is accompanied by profound transcriptional changes and active metabolism in infected tissues. The challenge in gene expression studies for syncytium has always been the isolation of pure syncytial material and subsequent extraction of intact RNA. Root fragments containing syncytium had been used for microarray analyses. However, the inclusion of neighbouring cells dilutes the syncytium-specific mRNA population. Micro-sectioning coupled with laser capture microdissection (LCM) offers an opportunity for the isolation of feeding sites from heterogeneous cell populations. But recovery of intact RNA from syncytium dissected by LCM is complicated due to extended steps of fixation, tissue preparation, embedding and sectioning.In the present study, we have optimized the procedure of sample preparation for LCM to isolate high quality of RNA from cyst nematode induced syncytia in Arabidopsis roots which can be used for transcriptomic studies. We investigated the effect of various sucrose concentrations as cryoprotectant on RNA quality and morphology of syncytial sections. We also compared various types of microscopic slides for strong adherence of sections while removing embedding material.The use of optimal sucrose concentrations as cryoprotection plays a key role in RNA stability and morphology of sections. Treatment with higher sucrose concentrations minimizes the risk of RNA degradation, whereas longer incubation times help maintaining the morphology of tissue sections. Our method allows isolating high-quality RNA from nematode feeding sites that is suitable for downstream applications such as microarray experiments. Meloidogyne spp.) and cyst nematodes (Globodera spp. and Heterodera spp.) are sedentary parasites of roots and the primary nematode pathogen of a wide range of crops. Infective stage juveniles (J2s) of cyst nematodes invade the host root and migrate intracellularly until they reach the vascular cylinder. There, these nematodes select an initial syncytial cell (ISC) and become sedentary. Within 24 h of the ISC selection the cells adjacent to the ISC appear hypertrophied and fused together due to local dissolution of the cell walls [Plant-parasitic nematodes (PPNs) are obligate biotrophs that cause significant damage to almost every economically important crop . PPNs arll walls . Three dll walls . The synll walls , 12, 29.Heterodera schachtii and the model plant Arabidopsis thaliana. In recent years a lot of work with this particular pathosystem has shown that the development of the syncytium is accompanied by massive transcriptomic, metabolomic and proteomic changes [Compatible interaction is best studied between cyst nematode  changes , 13, 31. changes  used the changes , 31. In LCM is becoming a popular tool among cell biologists because it allows isolation of RNA, DNA, proteins and metabolites from a heterogeneous cell population. Current LCM techniques are based on two principles. In the “touch-free” approach, cells identified under a microscope are excised by a laser beam and removed from their tissue context by gravity or by catapulting them away with a photonic pulse. Alternatively, cells are fused to a plastic membrane and are subsequently removed mechanically from the remaining tissue (reviewed in Ludwig and Hochholdinger ).Successful LCM requires good morphological preservations of tissues. Furthermore, isolated nucleic acids (RNA or DNA), proteins or metabolites must be of high quality to ensure their utility for downstream applications. Therefore, tissue preparation is the crucial step towards successful RNA isolation of samples generated by LCM. Since LCM requires micro-sections of 2–20 µm, samples must be fixed and embedded prior to sectioning and mounting them on the slides. During this multistep procedure, RNA integrity remains at high risk of degradation due to the presence of RNases. Samples for immuno-histological analyses are generally fixed in paraformaldehyde and embedded in paraffin to ensure well-preserved tissue morphology. In contrast, ethanol:acetic acid (EAA) solution is preferred for fixation in experiments involving RNA extraction because paraformaldehyde cross links with RNA and thus makes its recovery difficult , 18, 30.H. glycines [Nakazono et al.  were theglycines , 18, 19.glycines , 24, 26.H. schachtii. Root segments containing syncytia were cut at 5 days post inoculation (dpi). The infected root segments were subsequently fixed in EAA (3:1) and processed for LCM as described previously [To assess the potential of a previously described giant cell RNA isolation protocol in Arabidopsis for its utility for syncytial samples, we grew Arabidopsis plants in KNOP medium and infected them with J2 nematodes of in EAA 3: and proceviously , 24. HowTo identify the crucial step in which the RNA was degraded, we hand-dissected root segments containing syncytia at 5 dpi and isolated RNA at three different steps Table . The subTo investigate whether RNA degradation might have occurred because of multiple incubations in different sucrose concentrations  during cryoprotection; we modified the tissue preparation procedure by reducing the sucrose incubation steps. For this, root segments containing infected tissues were cut and fixed in EAA solution followed by a direct incubation in 15 % sucrose solution for 3 h (low concentration sucrose-treated samples). These samples were then left overnight in 34 % sucrose solution. Alternatively fixed tissues were directly incubated in 34 % for 5 h (high concentration sucrose-treated samples). Subsequently, tissues from low- and high-concentration sucrose-treated samples were embedded in OCT, cryosectioned and used for RNA isolation. While the RNA from low-concentration sucrose-treated tissues was heavily degraded Fig. a, the seAlthough incubating fixed samples directly in 34 % sucrose for 5 h resulted in improved RNA quality, the tissue morphology was poor  poly-l-lysine-coated glass slides. We mounted 10 µm sections on PET- and PEN-membrane-coated glass slides and washed them with PBS buffer and 70 % ethanol to remove the OCT medium. The complete removal of the OCT medium is necessary for laser cutting. However during washing, all sections were washed away from the slides. We pre-treated the slides with UV light for 30 min to 4 h to enhance adherence, but the majority of the sections was still washed off during the washing step. Next we used Superfrost® positively-charged glass slides, on which tissue retention was greatly improved, while the use of poly-l-lysine-coated glass slides proved robust in holding the tissues during the washing step. Hence, in all subsequent experiments, we used poly-l-lysine-coated glass slides, and the laser capture machine was also calibrated to glass slides.Different types of glass slides with diverse features are available. In this study, four types of slides were tested to obtain a strong tissue adherence: (a) polyethylene teraphthalate (PET)-membrane-coated slides, (b) polyethylene napthalate (PEN)-membrane-coated slides, (c) Superfrost® Arabidopsis ATH1 Genome (Affymetrix UK Ltd). The data analysis showed that amplified cDNA was suitable for microarray experiments (data not shown). Whereas the detailed results from microarray will be presented elsewhere, we analysed the expression of six different genes  in 5-days-old syncytial samples by RT-PCR (see methods for details). These genes were selected based on intensity of expression in syncytium in our microarray data (data not shown). Under optimized PCR conditions, five out of six genes produced a single band of the expected size from LCM-derived syncytial RNA. However, WRKY76 showed an extra band in syncytial samples suggesting unspecific binding of primers , which may result in a rapid loss of water and thus shrunk tissues. Indeed tissue morphology was maintained through longer vacuum infiltration and an overnight incubation in a 34 % sucrose solution see “”. The sl® positively-charged glass slides for giant cells in Arabidopsis roots. However we found that poly-l-lysine-coated glass slides robustly anchored the frozen sections of 5-day-old syncytia.After the morphology of the sections was satisfying, the embedding medium was removed completely by washing which is a perquisite for the laser beam to function properly. The commercially available OCT medium is water soluble and can be removed easily by washing with PBS buffer. However the root sections can also be washed away unless they adhere strongly to the slides. We tried membrane-covered slides (PET and PEN), which permit very fine dissection of target cells, and the targeted area can be catapulted as a single intact piece, but they proved very poor in holding root sections during the washing step. In contrast, Ramsay et al.,  used PENIn conclusion, we have shown that fixation with EAA and a direct infiltration with 34 % sucrose, prior to embedding in OCT medium generates syncytial samples with well-preserved morphology that are suitable to distinguish syncytial cells from uninfected surrounding tissue during LCM. Moreover, high quality RNA was extracted from LCM-processed syncytial samples, which was suitable for downstream applications such as microarrays analysis and RT-PCR. Importantly, we were able to analyse the RNA quality prior to amplification, which has not been shown previously . Our proArabidopsis thaliana plants were grown on KNOP medium and inoculated with 60–70 juveniles of Heterodera schachtii as described previously [10-day-old eviously . SubsequThe fixative solution was replaced with a 34 % sucrose solution prepared in 0.01 M PBS buffer (treated with 0.1 % DEPC), pH 7.4. Safranin-O  was added to visualise the otherwise transparent tissues. Moreover, to facilitate solution penetration, a vacuum was applied for 45 min at 4 °C. Subsequently, samples were incubated in an undulating shaker at 4 °C for 24 h.®, Cat No. 25112. Poly Sciences, Germany) using Cryomold Tissue-Tek® . The embedded tissues were frozen by touching the back of cryomolds to liquid nitrogen. The embedded samples (OCT blocks) were stored at −80 °C until cryosectioning.The tissues were horizontally embedded in a tissue freezing medium, OCT . After collecting 10–15 sections on the cryomicrotome platform, a slide at RT was suddenly placed over the sections to adhere them. The slides with sections were stored in 70 % ethanol, which was already pre-chilled in the cryomicrotome.The OCT blocks containing tissues were left inside a cryomicrotome  pre-cooled to −20 °C for 15 min for acclimatisation before making sections. Longitudinal sections of 10 µm were mounted on poly-® RNA isolation kit  was added to an adhesive cap, incubated at 42 °C for 20 min. The extraction mixture was briefly spun down in a table centrifuge at 8000 rpm and was stored at −80 °C until RNA extraction. The later steps of RNA extraction followed the instructions provided in the kit by the manufacturer. The quality of the isolated RNA was tested with an Agilent 2100 Bioanalyzer using the Agilent RNA 6000 Pico kit . cDNA synthesis was done with NuGEN’s Ovation Pico WTA System  according to the manufacturers’ instructions, starting with 1–50 ng of total RNA. NuGEN’s Encore Biotin Module  was used to fragment 3.95 µg cDNA followed by Biotin-labelling according to the manufacturers’ instructions. The gene expression was analysed through RT-PCR. Each sample contained 6 μL Master Mix, 1 μL template cDNA (1:20 dilution), 0.5 μL of forward and 0.5 μL of reverse primers (10 μM) and water in 20 μL of total reaction volume. Primers are given in Table For OCT removal and dehydration, the slides were treated twice with 70 % ethanol for 30 s each at RT, 95 % ethanol overnight at −20 °C and 99.8 % ethanol for 2 min at RT. After air drying the slides at RT for 2 min, the sections were immediately processed for LCM, and the remaining slides were stored in a box containing silica gel. The LCM captured syncytium cells were collected in RNAse-free adhesive caps  using the following settings for PALM Microbeam laser capture . Energy: 55–57, Focus: 79–80, Cutting Program: “CloseCut+AutoLPC” RNA Extraction, Quality Assessment and Gene expression: 10 µl extraction buffer from the PicoPure


We report the draft genome sequences of marine RNA viruses SF-1, SF-2, and SF-3, which were assembled from metagenomic sequencing of organisms in San Francisco wastewater. These viruses were most closely related to marine RNA virus JP-B and algae viruses. Picornavirales order , capsid, and helicase proteins that have a broad host range, including animals, plants, and insects –3. While–es order , 2. The All three contigs were assembled from a single metagenomic library derived from a wastewater sample taken on 25 January 2010 following a large rainstorm that left >5 inches of rain over the preceding week. Unlike the likely ciliate viruses also discovered in this sample, wastewater samples collected in March 2010 also contained reads to these viruses but at a significantly lower sequence count. This sample was created by 200-fold concentration of 1 liter of wastewater, with particles between the size of 0.22 μm and 300 kDa using Millipore Pellicon XL 300-kDa filters and 0.22-μm spin columns. The viral particle-enriched sample was treated with micrococcal nuclease, nucleic acid was extracted using the Zymo viral DNA/RNA kit, and half of the recovered nucleic acid was treated with DNase. The three contigs were discovered and assembled using PRICE version 1.0 , the GenJN661160, KF412901, and KF478836, respectively.The GenBank accession numbers for marine RNA viruses SF-1, SF-2, and SF-3 are


In addition, RNA-seq datasets from MRI-guided biopsies (n = 92) and glioma stem-like cell cultures (n = 9) were analyzed. Sixty-four DNA-seq datasets from 11 meningiomas and their corresponding blood control samples were also analyzed. Finally, three primary GBM tissue samples were obtained, sequenced using RNA-seq, and analyzed. After in-depth analysis, the most robust virus findings were the detection of papillomavirus (HPV) and hepatitis B reads in the occasional LGG sample . In addition, low numbers of virus reads were detected in several datasets but detailed investigation of these reads suggest that these findings likely represent artifacts or non-pathological infections. For example, all of the sporadic low level HCMV reads were found to map to the immediate early promoter intimating that they likely originated from laboratory expression vector contamination. Despite the detection of low numbers of Epstein-Barr virus reads in some samples, these likely originated from infiltrating B-cells. Finally, human herpesvirus 6 and 7 aligned viral reads were identified in all DNA-seq and a few RNA-seq datasets but detailed analysis demonstrated that these were likely derived from the homologous human telomeric-like repeats. Other low abundance viral reads were detected in some samples but for most viruses, the reads likely represent artifacts or incidental infections. This analysis argues against associations between most known viruses and GBM or mengiomas. Nevertheless, there may be a low percentage association between HPV and/or hepatitis B and LGGs.Next generation sequencing (NGS) can globally interrogate the genetic composition of biological samples in an unbiased yet sensitive manner. The objective of this study was to utilize the capabilities of NGS to investigate the reported association between glioblastoma multiforme (GBM) and human cytomegalovirus (HCMV). A large-scale comprehensive virome assessment was performed on publicly available sequencing datasets from the Cancer Genome Atlas (TCGA), including RNA-seq datasets from primary GBM (The online version of this article (doi:10.1186/s40478-016-0338-z) contains supplementary material, which is available to authorized users. Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. An estimated 77,670 new cases of primary CNS tumors are expected to be diagnosed in the United States in 2016 . In factAlthough an association between human cytomegalovirus (HCMV) and GBM was first observed in 2002 , there iIn an attempt to remedy the high degree of discordance regarding the detection of HCMV in CNS tumors, an HCMV and glioma symposium was convened in Washington, DC on April 17, 2011. At the conclusion of this workshop, a summary paper was published reporting the consensus position in 4 major areas: 1) the existence of HCMV in gliomas, 2) the role of HCMV in gliomas, 3) HCMV as a therapeutic target, and 4) key future investigative directions . Based oNext generation sequencing (NGS) has the ability to globally interrogate the genetic composition of biological samples in an unbiased manner and with relatively high sensitivity. Applying this technology to pathogen discovery has already shown promise, resulting in the discovery of a novel Merkel cell polyomarvirus in Merkel cell carcinoma , for exaAll human specimens were de-identified prior to acquisition. Fresh frozen tissue from 1 GBM sample was obtained from the Louisiana Cancer Research Consortium (LCRC) Biospecimen Core. Additionally, RNA isolated from 2 GBM samples were obtained from BioServe. An RNA-seq dataset from a lymphoblastoid cell line immortalized with EBV (JY) was used as a control for downstream analysis .https://cghub.ucsc.edu) and included RNA-seq datasets  from primary GBM tumors . Non-mapped reads were subjected to a BLAST V2.2.30 search against the Human RefSeq RNA database to identify and remove human reads that fail to be identified through the Novoalignment. Remaining non-human reads were then subjected to a BLAST V2.2.30 search against the NCBI NT database to identify reads corresponding to known exogenous organisms [−6. The results were then fed into MEGAN 4 V4.70.4 for visualization of taxonomic classifications [Metatranscriptome analysis was performed by running single-end or one pair from paired-end sequencing data through our automated RNA-seq exogenous organism analysis software, RNA CoMPASS . Within rganisms . Resultsications . RNA CoMRaw sequence data from RNA-seq and DNA-seq were aligned to a reference containing a human genome  plus a library of 740 virus genomes  . The aliTotal RNA was reverse-transcribed using the SuperScript III First-Strand Synthesis System for RT-PCR . Random hexamers were used with 1 ug total RNA in a 20 μl reaction volume according to manufacturer’s instructions. For the incubation steps (25 °C for 10 min followed by 50 °C for 50 min) a Mastercycler ep  was used. For real-time PCR, 1 μl of the resulting cDNA (diluted to 10 ng/ul) was used in a 10 μl reaction mixture that included 5 μl of 2x iQ SYBR Green Supermix , 1 μl of 10 μM forward and reverse primer mix , and 3 μl of PCR grade water.A list of PCR primer oligos can be found in Additional file Previous studies have used human read subtraction-based approaches to assess the metatranscriptomic profile of primary and recurrent glioma RNA-seq datasets from The Cancer Genome Atlas (TCGA) where a lack of association with HCMV was reported , 26, 27.Analysis of RNA-seq data from a cohort of time course HCMV infected fibroblasts  demonstrAlthough the bulk of viral RNAs are polyadenylated, some viral transcripts are not. To assess whether we were missing viral infections because of a lack of detecting non-polyadenylated viral transcripts, we wanted to test whether sequencing of ribodepleted RNAs (versus polyA selected RNAs as per the TCGA cohort) might yield the detection of viral reads. HCMV has been reported to have a high penetrance in GBM with some studies reporting as high as 90–100 % positivity , 24, 29.No associations were found with any known viruses in the polyA selected RNA sample (00RTS3) Fig.  using eiAnalysis of the ribosomal depleted RNA samples (CAUPRVE and H8CPFRSJ) revealed reads from the murine leukemia virus (MuLV) family  and a low abundance of EBV reads  , RNA-seq datasets from 514 primary and 17 recurrent LGGs from the TCGA were analyzed . Due to To explore the possibility that viruses infecting brain tissue become transcriptionally dormant, resulting in the lack of virus detection in RNA-seq datasets, we subjected the TCGA GBM whole genome sequencing (WGS) datasets to our virome analysis pipeline.Analysis of viruses at the DNA level was relatively unremarkable with the highest read numbers in the primary (TP) GBM WGS datasets being derived from EBV and HAdV-C Fig. : File S5EBV reads were detected in 9 TP GBM WGS datasets for which 6 out of 9 samples analyzed had corresponding blood matched samples  study, a randomized, double-blinded, placebo-controlled trial was recently published showing trends but no significant differences in tumor volumes between the valganciclovir (an anti-CMV drug) and placebo groups at 3 and 6 months . HoweverSeveral recent publications have highlighted the lack of association between viruses, specifically HCMV, and GBM –28, 68.


We describe the initial realization of behavior in the biosphere, which we term behavioral chemistry. If molecules are complex enough to attain a stochastic element to their structural conformation in such as a way as to radically affect their function in a biological (evolvable) setting, then they have the capacity to behave. This circumstance is described here as behavioral chemistry, unique in its definition from the colloquial chemical behavior. This transition between chemical behavior and behavioral chemistry need be explicit when discussing the root cause of behavior, which itself lies squarely at the origins of life and is the foundation of choice. RNA polymers of sufficient length meet the criteria for behavioral chemistry and therefore are capable of making a choice. Behavior is an integral feature of life. Behavior is manifest when a choice is possible, and a living entity responds to its environment in one of multiple possible ways. For relatively simple unicellular life, we tend to think of behavior as a deterministic response. Bacterial cells respond to a metabolite gradient in predictable ways, dictated by the biophysical processes of substrate uptake, second messenger activation, and flagella operation, for example. For relatively complex life such as sentient and conscious humans, we tend to think of behavior as a less predictable function—one that is enmeshed in the concept of free will .vitalism  [Free will has been described as a modern italism  , while Citalism   At this As long ago as ~300 BCE the early atomist Epicurus hypothesized, in an attempt to refute the deterministic nature of the physical world and account for the behavior of choice, that all occurrences are due to small matter (atoms) colliding and interacting in voided space (kenos) and that the atoms are not restricted to straight lines; rather, they exhibit random swerves . EpicuruAs conscious humans, our thoughts on choice and free will range between perceptions that a living entity functions as an absolute mechanism ultimately described by physical laws, and a constant personal awareness of making decisions of our own accord. Without anthropomorphizing molecules, we suggest that even the simplest of life must follow physical laws, but also have the ability to make a “choice” based on their environment and dependent on self-contained information.i.e., substrate uptake or folding pathways, and transitioned from a system in which chemicals had behavior to one in which chemicals could behave.Cashmore recently tackled this problem head on, and came to the conclusion that any atomic “swerve” was unable to lead to more than a perception of free will . In his 2 + 3H2 ∧2NH3. Because of its historical and industrial importance, this is a very well studied reaction that has a deterministic outcome under typical reaction conditions. For example, at elevated temperatures and pressures, the negative enthalpy of this reaction (ΔH = −92.2 kJ/mol) will ensure that about 15% of nitrogen will be converted to ammonia, while the balance of the reactants will remain uncombined.In order to address the ultimate source of “choice” we must differentiate between chemical behavior and behavioral chemistry. Chemical behavior is the essentially deterministic outcome of a chemical reaction among one or a few relatively small molecules. This is a term commonly used by chemists to describe the predicted products of a reaction under study, with the implicit assumption that any deviation from this outcome is simply a failing on our part to understand all the energetic facets of the molecules involved. In other words, chemical behavior is a deterministic process, one that becomes sharper the more we know about a system. A good example is the Haber production of ammonia: NHowever, we would like to introduce the subtly different concept of behavioral chemistry. When the molecules involved are complex enough to attain a stochastic element to their structural conformation in such a way as to radically affect their function in a biological (evolvable) setting, then they have the capacity to behave, and the analysis of such molecular-level behavior would be described as behavioral chemistry. Behavioral chemistry is derived from two processes, either or both of which can be in operation at any given point in time. The first is the expression of quantum mechanical stochasticity “writ medium-sized” in the conformational energetics of macromolecules such as RNA and proteins. Here we mean that atomic swerves do not influence collections of small molecules in any meaningful way, but with the advent of polymeric macromolecules characteristic of life, the stochasticity begins to reassert an influence.At first glance one might claim that the distinction here is arbitrary, because even small molecules can adopt conformational variations in time that can affect their subsequent reactivities; think of the sugar pucker of glucose for example. However, this property does not maintain sufficient complexity to warrant behavior, and does not impact evolution. That said, complexity is not unique to living systems and can be seen in many non-living physical systems as well as in simple mathematical models. Complexity cannot solely determine behavior. A pertinent example of this is the formose reaction, whereby the incubation of the two simple precursors formaldehyde and glycoaldehyde can lead to the spontaneous synthesis of many sugars, including ribose. The formose reaction is both notoriously complex and has the feature of autocatalysis, making it of prebiotic importance . It is iwas the system! In essence, a system requires both a genotype and a phenotype to be able to display behavior. The “self” is now clearly defined  case of RNA during a hypothetical RNA World, it is entirely possible that these variations typically overlapped ... that RNAs arose that had bounded information and were subject to kinetic folding traps. Such molecules could in principle respond to environmental conditions, interpret them , and adoOnce behavioral chemistry is possible in a system, the choices that molecules take would be subject to natural selection. We typically imagine the phenotypic state as being the target of selection, but imperfect heritability of phenotypes means that variable conformations can persist over generations, and some of these conformations can have negative fitness consequences. This can be observed empirically in populations of RNA evolving in the test tube . HoweverE. coli have revealed that this bacterial species expresses chemotaxis primarily through the actions of 15 proteins, such as specific chemoreceptors for amino acids. The functions of these proteins serve to bias an otherwise random walk powered by flagellar motion into a directional track toward certain ligands [E. coli genotypes that migrated up a, say, serine concentration gradient when cellular serine stores were low. If this migration were not 100% coincident with a concomitant need to migrate up a, say, galactose concentration gradient, then a molecular choice becomes apparent. The advent of behavior, which is both flexible by definition and robust through genetic underpinnings was key in the establishment of a new phenomenon, evolvability [Ultimately the ability of organisms––now collections of molecules with multiple genes and phenotypic products––to engrain reproducible behaviors would have been crafted by natural selection. The evolution of cellularity grants a more permanent boundary for the self, as discussed above. This second layer of bounding  adds richness to the spectrum of behavior by controlling which elements of the environment the self interacts with. Even single-celled organisms such as bacteria and protists could then express complex behavior, in the form of chemotaxis or phototaxis. For example, years of study on  ligands . Directivability , which iEventually this led to the advent of behavioral ecology, with payoff matrices that could be tied to various behaviors . While mFinally, however, organisms evolved intricate and convoluted means of interacting with their environments and displaying classical behavior, as in a rat choosing paths within a maze . Having (1)There must be at least two options available to the chooser. From a molecular perspective, this would be described in terms of what is allowed energetically e.g., . Perhaps(2)There must be a determination of one option as being optimal for some reason. For intelligent life forms, this generally comes after a cost/benefit analysis of some kind , which r(3)Some action must be taken towards realizing the choice being made. From a physical perspective, the objective determination of a choice being made is reliant upon some observable difference relative to the pre-choice state. In some sense, this is an extension to the organismal level of the molecular memory discussed above for RNA. Unless it can be communicated between intelligent beings in some kind of language, the only evidence of a choice is some subsequent action, which can be observed. Specifically, the choice or the ability to make it must have a fitness consequence.Even at its highest level, behavior reverberates with the basic underlying properties of behavioral chemistry: one genotype can adopt multiple phenotypes, genotypes must sense and respond to their environments, and both the phenotype(s) and the ability to chose among them are subject to natural selection.The question remains whether the type of complex behavior we see in intelligent life is still proximally determined by an underlying behavioral chemistry, or whether the latter has been lost, having been subsumed into the biological operations of the cell long ago in evolutionary history. We essentially lack any understanding of how  the conscious mind interacts with the physical world. The fact that they do interact is most notably apparent in the context of our perceptions of the universe; we experience qualia, or feelings arising from sensations, which cannot be explained by the physical stimulus being sensed. For example, electronic transitions cause the emission of a photon of light, which causes a conformational change in an organic molecule in the eye, which causes an electrical signal in the brain—but we see “blue.” The physical steps leading to the concept of blue would exist without a conscious mind to perceive it, but the representation of photons of specific energies as a color would not. Examples of qualia such as seeing color, smelling smells, and feeling pain are instances where the physical world is causing an effect felt by the conscious mind via sensory organs. etc.) and they are seemingly imperceptible via human’s five senses. We feel that we are moving through time, but we cannot explain or quantify that movement without relying on indirect cues such as watching a second-hand tick or the Sun travel through the sky. Some physical forces are still giving rise to the perceived directionality, but the information collected by our sensory organs serves only as a reference to quantify and conceptualize what we feel.To organisms with the highest level of choice control, perceptions of other aspects of the physical world are not felt quite so directly, such as the feeling of passing through time. Humans understand time in reference to the frequency of a regularly repeating pattern, such as the rotation of the Earth, or Earth’s orbit around the Sun. We feel a sense of directionality in our existence, but we have to use objective visual cues to quantify it. From a physical standpoint, objective metrics that the universe is moving in a constant direction are scarce, (nuclear decay, entropy increasing, the universe expanding, perceiving the universe being actively changed by the choices we are making. Dennett argues that determinism does not imply inevitability, and that furthermore even if the physical reality were deterministic then there indeed are real options to life, not just apparent ones. His premise is that the process of natural selection has operated on an extant physics to create organisms with free will and with responsibilities for their own actions [The interactions discussed above are all causal in that the physics of the environment give rise to the feelings being felt, clearly establishing that the physical world and the conscious mind interact, and that one can affect the other. Yet, scientists are keen to reject the concept of free will for lack of an objective parameter that can be studied empirically . Indeed, actions . Here wePhysical variations in molecules of a certain level of complexity can be trapped into quantum levels, and at this point, a transition from chemical behavior to behavioral chemistry can happen. This is the origin of all forms of behavior, and this transition was more-or-less coincident with the origins of life. The boundaries that define the self allow choice to transcend quantum mechanics and become engrained in chemistry and described better by statistical thermodynamics and Newtonian mechanics. Empirical studies that test the hypothesis that molecules can exhibit measurable properties that would satisfy the constraints of a payoff matrix will shed much light on this issue. Whether behavioral chemistry is still an operative force in the expression and evolution of organismal behavior is uncertain. Moreover, behavioral chemistry may have been an ultimate determinant of free will, but the notion of human perception clouds this issue.


Oncogenic fusion genes underlie the mechanism of several common cancers. Next-generation sequencing based RNA-seq analyses have revealed an increasing number of recurrent fusions in a variety of cancers. However, absence of a publicly available gene-fusion focused RNA-seq data impedes comparative assessment and collaborative development of novel gene fusions detection algorithms. We have generated nine synthetic poly-adenylated RNA transcripts that correspond to previously reported oncogenic gene fusions. These synthetic RNAs were spiked at known molarity over a wide range into total RNA prior to construction of next-generation sequencing mRNA libraries to generate RNA-seq data.a priori knowledge about replicates and molarity of each synthetic fusion transcript, we demonstrate utility of this dataset to compare multiple gene fusion algorithms’ detection ability. In general, more fusions are detected at higher molarity, indicating that our constructs performed as expected. However, systematic detection differences are observed based on molarity or algorithm-specific characteristics. Fusion-sequence specific detection differences indicate that for applications where specific sequences are being investigated, additional constructs may be added to provide quantitative data that is specific for the sequence of interest.Leveraging To our knowledge, this is the first publicly available synthetic RNA-seq data that specifically leverages known cancer gene-fusions. The proposed method of designing multiple gene-fusion constructs over a wide range of molarity allows granular performance analyses of multiple fusion-detection algorithms. The community can leverage and augment this publicly available data to further collaborative development of analytical tools and performance assessment frameworks for gene fusions from next-generation sequencing data.The online version of this article (doi:10.1186/1471-2164-15-824) contains supplementary material, which is available to authorized users. BCR-ABL, PML-RAR, and EML4-ALK fusion products associated with chronic myelogenous leukemia [Oncogenic fusion genes underlie the mechanism of several common cancers and also constitute or encode important diagnostic and therapeutic targets. Fusions may drive oncogenic growth by joining a proliferation-inducing gene to an active promoter, by disrupting the function of tumor suppressor genes, or by creating novel functional products that rewire the biochemical pathways that regulate cellular division . Researcleukemia , 3, acutleukemia , and nonleukemia –9, respeNext-generation sequencing technologies, particularly RNA sequencing (RNA-seq), have revealed an increasing number of recurrent fusions in a variety of cancers, and it is likely that their detection will have growing diagnostic and prognostic utility. As such, validating the laboratory and analysis methods to establish analytical parameters including the limit of detection, linearity, sensitivity, and specificity of fusion detection in tumor RNA specimens is critical for adoption in clinical research settings. For example, does a fusion transcript present at higher molarity (higher transcript abundance) correlate with higher number of fusion-supporting sequencing reads? Are there differences in detection algorithms’ efficacy with respect to specific fusion sequence and independent of abundance? Answering such questions and establishing robust metrics is difficult due to the lack of publicly available RNA-seq data specifically generated to capture gene fusions.We have developed a set of nine synthetic poly-adenylated RNA transcripts that correspond to reported cancer fusion gene sequences , then quantitated using UV absorption. 2.2 ng of each RNA spike were pooled in a PCR plate, and the volume was brought up to 50 μL with RNase free water. A cDNA library was prepared using TruSeq Stranded mRNA LT Sample Prep Kit  and sequenced on an Illumina MiSeq to confirm the sequences of the mRNA transcripts as a final QC step. Fresh aliquots of RNA were taken from storage, washed with 70% ice cold ethanol, resuspended in 1 × TE, and quantitated using RiboGreen (Invitrogen). RNA spikes were mixed together to create a high concentration pool with 40 nM of each spike. This pool was diluted and titrated into to 1 μg aliquots of COLO-829 total RNA (ATCC 1974). cDNA libraries were prepared using the TruSeq Stranded mRNA LT Sample Prep Kit  following the manufacturer’s protocol. The resulting libraries were sequenced on the Illumina HiSeq2500 in Rapid Run mode using paired end reads with 101 cycles in each read.In summary, equimolar amounts of all nine SGFRs were pooled together and this pool was titrated into total RNA from the melanoma cell line COLO-829  at ten dhttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/). We analyzed the data using three fusion detection tools: ChimeraScan [Illumina sequencing data was converted to FASTQ format using Casava pipeline followed by read quality assessment using FASTQC tool  Aligning reads to a reference genome or transcriptome assembly; 2) Identifying discordant read pairs, i.e., pairs for which genomic distance between the two ends’ alignments is significantly different from the expected genomic distance based on library preparation; 3) Extracting split sections of the same read that align to different regions of the genome, thereby, indicating a potential fusion; 4) Algorithm-specific additional steps, such as contig construction, sequence homology search, guided analyses based on exon junction annotation files, etc.TophatFusionPost step) followed by stitching all segments together into full read alignments that are further filtered based on criteria, such as number of fusion-supporting reads. SSH uses 50-bp reads that are aligned by BWA [We emphasize here that our focus is to demonstrate utility of the SGFR constructs for evaluating assay performance and to make them available to the clinical and research communities to further active research in gene-fusion detection methods. To that end, the choice of three representative algorithms and the analysis framework is based on our experience in analyzing such data. Since emphasis is on making RNA-seq gene fusion data publically available, we do not attempt to provide a detailed comparative assessment, pros-cons, or performance characterization of the growing number of gene fusion detection tools discussed elsewhere , 15. Howd by BWA  guided bWe also verified by running a separate parallel sample that the COLO-829 cell line provided a neutral background, i.e., it did not contain any of the nine SGFRs. Therefore, SGFRs in our experiment were not barcoded prior to spiking into the total RNA. However, barcoded SGFRs should be preferred in other cell lines to avoid mixing of spiked-in fusions and potential endogenous fusions.Figure To compare experimental replicates, we calculated the Pearson correlation between number of fusion-supporting reads between replicates Figure  by dividTo observe the effect of changing minimum number of reads required to call a fusion, Figure Figure accepted_hits.bam) generated by THF. We discovered that several fusion-supporting reads were aligned against TMPRSS2 (chr21:42.84-42.9 mb) and ETV1 (chr7:13.93-14.03 mb) loci across various molarities as shown in Additional file TophatFusionPost step was executed. A detailed investigation of actual THF algorithm implementation and specific reasons behind filtering out the fusion is out of scope of this study. However, observations based on additional investigation of unreported fusions highlight the critical importance of tool-specific criteria and parameters that might lead to false negatives or false positives—evidence for fusions from alignment data was processed differently by different tools yielding different results.Notably, some fusions were not detected by one or more tool(s) irrespective of molarity as shown by the points on X-axis in Figure For the sake of completeness, we also note that each detection tool has a large number of input parameters that significantly affect its detection ability. Figure The key contribution of this work is the first publicly available gene fusion RNA-seq data that specifically targets known oncogenic gene fusions that are gaining increasing importance in clinical genomics based on next-generation sequencing. The community can augment this dataset and the proposed analytical framework to further collaborative development of advanced analytical tools for gene fusion detection from RNA-seq data.All sequencing data is available in FASTQ format from the Short Read Archive under accession number SRP043081.Table S2. Fusion read counts across all samples. Table S3. Endogenous fusions. Table S4. Fusion-supporting reads from TMPRSS2-ETV1 Tophat Fusion analysis. (XLSX 31 KB)Additional file 1: Table S1: Fusion sequences.


Cucumber mosaic virus (CMV) 2b and Tombusvirus P19, suppress hairpin RNA (hpRNA)-induced silencing of a β-glucuronidase (GUS) gene in Nicotiana benthamiana. This suppression can be overcome by CMV Y-satellite RNA (Y-Sat) via the Y-Sat-derived small interfering RNAs (siRNAs), which bind to the VSRs and displace the bound hpGUS-derived siRNAs. We also show that microRNA target gene expression in N. tabacum was elevated by CMV infection, presumably due to function of the 2b VSR, but this upregulation of microRNA target genes was reversed in the presence of Y-Sat. These results suggest that satRNA infection minimizes the effect of VSRs on host siRNA and microRNA-directed silencing. Our results suggest that the high abundance of satRNA-derived siRNAs contributes to symptom attenuation by binding helper virus-encoded VSRs, minimizing the capacity of the VSRs to bind host siRNA and miRNA and interfere with their function.Viral satellite RNAs (satRNAs) are small subviral RNAs and depend on the helper virus for replication and spread. satRNAs can attenuate helper virus-induced symptoms, the mechanism of which remains unclear. Here, we show that two virus-encoded suppressors of RNA silencing (VSRs),  Nicotiana species induced by Y satRNA (Y-Sat) of Cucumber mosaic virus (CMV) , which have a 22-nt sequence homologous to CHLI  depend on their associated viruses (helper viruses) for replication, encapsidation, systemic movement and transmission, and are regarded as parasites of their helper viruses. They have small RNA genomes sized from 220 to 1400 nucleotides (nt), which share little or no sequence homology with the genome of helper viruses  processed from double-stranded RNA (dsRNA) or self-complementary hairpin RNA (hpRNA) by Dicer or Dicer-like protein, an RNase III-like endoribonuclease. One strand of each sRNA is loaded onto the RNA-induced silencing complex (RISC) and guides Argonaute (AGO) proteins in the RISC to cleave targeted RNA Zamore, . In planTombusvirus-encoded VSR P19 to suppress hpRNA-induced gene silencing in Nicotiana benthamiana. Both VSRs suppressed hpRNA-induced gene silencing, but in the presence of Y-Sat, this VSR effect was reversed. Using RNA immunoprecipitation, we showed that this reversion is due to the bound hpRNA-derived siRNAs being displaced from the VSRs by Y-Sat-derived siRNAs. We also found that Y-Sat infection minimizes the over-expression of target genes of endogenous miRNAs caused by CMV infection, indicating that Y-Sat inhibited the effect of the 2b VSR on the function of host miRNAs.In this study, we used an Agrobacterium infiltration (agro-infiltration)-based transient expression system to investigate how the satRNA of CMV Y (Y-Sat) affected the ability of CMV-encoded VSR 2b and N. benthamiana and N. tabacum  and 2bm  constructs were also previously described  contains an intron in the coding sequence, which was designed to prevent GUS expression in bacterial cells  or GV3101 .The constructs A. tumefaciens strains containing plant expression constructs were grown overnight at 28°C in Luria-Bertani medium (LB) containing appropriate antibiotics. After centrifugation, Agrobacterium cells were re-suspended in buffer containing 10 mM MgCl2 and 150 μg/mL acetosyringone, with a final optical density at 600 nm (OD600) of 0.7. The bacterial suspensions were incubated at room temperature for ~3 h and then infiltrated into expanded N. benthamiana leaves using flat-pointed syringe . The homogenates were subjected to centrifugation for 5 min at 4°C at 16,000 × g, and the supernatants were collected and kept on ice. Concentration of total protein was determined using the Bradford reagent. MUG reactions were carried out at 37°C and GUS activities were calculated essentially as previously described  assay, agro-infiltrated leaf sections were homogenized in extraction buffer  following the manufacturer's instructions. Isopropanol precipitation of the total RNA was carried out overnight at −20°C to maximize recovery of the small RNA fraction.Total RNA was isolated from 32P] UTP-labeled RNA probes obtained by in vitro transcription using radioactive UTP from the respective sequences  was separated in formaldehyde-agarose gel  in 0.5× TBE buffer, UV cross-linked, and hybridized overnight at 42°C with [α−32P] UTP labeled, carbonate buffer-fragmented RNA probe  17% polyacrylamide gel. RNA was electro-blotted onto a Hybond-N23 as primer. The synthesized cDNA was amplified using 2b, HcPro or miRNA target gene-specific primers  was amplified for use as loading control.For RT-PCR or qRT-PCR analysis of 2b, HcPro mRNA, or miRNA target genes, total RNA (~1 μg) isolated from infected and uninfected leaves was reverse-transcribed with Superscript III reverse transcriptase (Invitrogen) following the manufacturer's instruction using oligo-dT2O for small RNA northern blot hybridization. For Western blot analysis of the pull-down material, elution step was omitted and washed beads were stored at −20°C for Western blotting.RNA immunoprecipitation was performed as described by Omarov et al.  with minBeads containing the immunoprecipitated P19/RNA complexes were denatured at 95°C for 5 min with addition of Western blot loading buffer . The proteins were separated on a 15% SDS polyacrylamide gel, transferred to Immobilon P membrane (Millipore) and detected by chemiluminescence using 1/2500 anti-P19 antibody  are classified into two subgroups, I and II, based on their serology, symptomatology and sequence homology  and Y-Sat using Illumina deep sequencing technology, and analyzed the relative abundance of Y-Sat and CMV-derived sRNAs. A total of 195,291 and 330,965 reads of 20–24 nt sRNAs  were perfectly mapped to the Y-Sat genome in the two samples S1 and S2, respectively  reporter gene in the leaves was measured. We used the subgroup II CMV strain, Q-CMV, as the helper virus to infect N. benthamiana instead of Fny-CMV. This was because Q-CMV encodes a relatively weak 2b VSR , GUS silencing-inducing hpRNA (hpGUS), strong 2b (2b) VSR encoded by the subgroup I Shan-Dong (SD)-CMV  as a visual marker. A construct expressing a mutant 2b gene (2bm), in which the start codon and three other in-frame AUG codons of the 2b coding sequence were mutated  35S promoter.Cucumoviral 2b proteins are essential for cucumoviruses to cause viral symptoms , Q-CMV-infected (Q-CMV), and Q-CMV+Y-Sat co-infected plants. Northern blotting confirmed that the GUS gene was silenced at the mRNA level  assay in Figure  Figures . However Figures . These rs Figure . The vars Figure , and thes Figure , presumahpGUS transcript  was detected in the s Figure . HoweverhpGUS-induced GUS silencing in mock and Q-CMV-infected N. benthaminana tissues, and the suppression was negated in Q-CMV+Y-Sat-infected tissues -encoded VSR HcPro were analyzed. The HcPro plants rather than wild-type plants were used for two reasons. First, the plants do not develop yellowing symptoms with Q-CMV+Y-Sat infection, presumably because Y-Sat siRNA-directed silencing of the chlorophyll biosynthetic gene CHLI is repressed by HcPro . Northern analysis of the P19-bound sRNAs showed that Y-Sat-siRNAs were present at high levels in the α-P19 RNA-IP from the Q-CMV+Y-Sat-infected tissues  associated with Tomato bushy stunt tombusvirus (TBSV) has shown that abundant siRNAs derived from the DI-RNAs can saturate TBSV-encoded P19 VSR and elevate the levels of free TBSV-derived siRNAs , host sRNA-directed silencing and plant growth are not affected by the high level of Y-Sat siRNAs (Shen et al., satRNA-derived siRNAs have the potential to compete with host plant-encoded sRNAs for endogenous RNA silencing machineries such as DCLs and AGO proteins, which in turn could affect host sRNA-directed gene regulation. Thus, the high abundance of satRNA siRNAs could both reduce symptoms by binding to VSRs and exacerbate symptoms by competing for host silencing machineries. However, we have shown here and previously (Shen et al., In conclusion, our results suggest that satRNAs are likely to attenuate the symptoms via their abundant siRNAs, which bind helper virus-encoded VSRs minimizing their interference with the endogenous siRNA and miRNA pathways as well as the antiviral silencing pathways as proposed in the previous studies. This model could apply to other viral or subviral RNAs that give rise to high levels of siRNAs in infected host cells, for example, DI RNAs. DI RNAs are a truncated form of their associated helper viral genomes and, like satRNAs, also depend on the helper virus for replication and can also attenuate helper virus-induced symptoms (Simon et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ribonucleases (RNases) are important factors for the establishment of virulence in an increasing number of pathogens. These enzymes are responsible for the maturation and degradation of RNA molecules, being key players in RNA metabolism. In this opinion we will focus on exoribonucleases from the RNB family, whose members are known to be involved in the virulence of several microorganisms.Escherichia coli is the prototype of this family, which also includes bacterial RNase R and eukaryotic Rrp44/Dis3 proteins. These proteins present a similar modular organization with cold shock domains (CSD) at the N-terminal region involved in RNA binding, a central RNB domain responsible for catalysis, and a C-terminal S1 domain, also involved in RNA binding , the protein is stabilized , in association with a small RNA-binding protein (SmpB), recognizes stalled ribosomes on defective mRNAs. tmRNA functions both as tRNA and as mRNA, and contains a specialized open reading frame that encodes a peptide tag. tmRNA is loaded onto the defective RNA and the stalled ribosome switches from the translation of the defective mRNA to the translation of the mRNA domain of tmRNA. After translating the tmRNA reading frame, the stalled ribosome is released and the protein carries a C-terminal tag that is recognized and degraded by the cellular proteases .In the foodborne pathogen Taken together, these observations indicate that bacterial strains lacking RNase R exhibit an attenuated virulence phenotype when compared to their parental strains. This is probably connected to the fact that RNase R is a key player in RNA metabolism. Considering the important functions that RNase R has in the establishment of virulence, it can be a potential target to design compounds able to kill specific microorganisms or to reduce their virulence ability. Further studies regarding RNase R function in the control of pathogenesis will certainly help in the comprehension of the RNA-related processes involved in infection.trans-translation, which emphasizes its importance in pathogenesis. We believe that future work will reveal more about the mechanism of action of these proteins and how they affect the infection process. Moreover, recent publications have associated RNB family members with several important diseases (reviewed in Reis et al., Enzymes from the RNB family play crucial roles in the cell, such as RNA processing, turnover, and surveillance. In prokaryotes, several works have highlighted the importance of these proteins, namely RNase R, for the establishment of virulence in some important pathogens. The mechanism by which RNase R is involved in virulence is not yet well-understood. Probably, it has an indirect role by controlling the stability of crucial transcripts that are directly involved in virulence. Moreover, besides its role in RNA metabolism, RNase R has been involved in different cellular processes, such as association with the ribosomes and The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The whole genome RNA-seq data was deposited at GenBank under the accession number PRJNA2284587. The RNA-seq libraries generated 117 million reads of 5.89 gigabases in length. Totals of 21,331, 20,996 and 20,139 expressed transcripts were identified in IP, UIP and AIP, respectively. Furthermore, we identified 757 differentially expressed genes (DEGs) between IP and UIP, and 271 DEGs between AIP and IP. Function enrichment analysis of DEGs found that the highly expressed genes in IP were mainly enriched in energy metabolism, global transcriptional activity and bone development intensity. This study contributes to reveal the genetic mechanism of growth curve inflection point.Animal growth curves play an important role for animal breeders to optimize feeding and management strategies (De Lange et al., 2001  The total RNA was isolated poly (A) mRNA by poly-T oligo attached magnetic beads (Thermo-Fisher). Following purification, the mRNA was fragmented into small pieces using divalent cations under an elevated temperature. Then the cleaved RNA fragments were constructed into the final cDNA library in accordance with the protocol for the Illumina RNA ligation based method . A reverse transcription followed by PCR was used to create cDNA constructs. The average insert size for the single-end libraries was 300 bp (± 50 bp). Then the single end sequencing (50 bp) was performed on an Illumina Hiseq2000 platform. For data analysis, the raw data containing adaptor sequences, reads with low quality sequences and unknown nucleotides N were filtered to obtain clean reads with 50 nt in length. Clean reads were then conducted for quality assessment (data shown in p-value ≤ 0.05 and log2fold-change|log 2 FC∣ ≥ 1. Raw and normalized data were accessible on public database: An enrichment analysis of DEGs found the immune system related genes were in the BIP stage. The energy metabolism rate, global transcriptional activity and bone development intensity were highly expressed in the inflection point period. Superior meat quality was developed in the AIP stage. The raw and normalized data of our study were accessible on public database: GEO submission number GSE69113.For RNA-Seq library preparation, total RNA was extracted from The authors declare no conflicts of interest.


As SF2 helicase/ATPase family members, ATP hydrolysis is dependent on co-operative RNA and ATP binding. Whereas simultaneous ATP and cognate RNA binding is sufficient to activate RIG-I by releasing autoinhibition of the signaling domains, the physiological role of the ATPase activity of RIG-I and MDA5 remains controversial.The cytoplasmic RIG-like receptors are responsible for the early detection of viruses and other intracellular microbes by activating the innate immune response mediated by type I interferons (IFNs). RIG-I and MDA5 detect virus-specific RNA motifs with short 5′-tri/diphosphorylated, blunt-end double-stranded RNA (dsRNA) and >0.5–2 kb long dsRNA as canonical agonists, respectively. However, A cross-analysis of a rationally designed panel of RNA binding and ATPase mutants and truncated receptors, using type I IFN promoter activation as readout, allows us to refine our understanding of the structure-function relationships of RIG-I and MDA5. RNA activation of RIG-I depends on multiple critical RNA binding sites in its helicase domain as confirmed by functional evidence using novel mutations. We found that RIG-I or MDA5 mutants with low ATP hydrolysis activity exhibit constitutive activity but this was fully reverted when associated with mutations preventing RNA binding to the helicase domain. We propose that the turnover kinetics of the ATPase domain enables the discrimination of self/non-self RNA by both RIG-I and MDA5. Non-cognate, possibly self, RNA binding would lead to fast ATP turnover and RNA disassociation and thus insufficient time for the caspase activation and recruitment domains (CARDs) to promote downstream signaling, whereas tighter cognate RNA binding provides a longer time window for downstream events to be engaged.The exquisite fine-tuning of RIG-I and MDA5 RNA-dependent ATPase activity coupled to CARD release allows a robust IFN response from a minor subset of non-self RNAs within a sea of cellular self RNAs. This avoids the eventuality of deleterious autoimmunity effects as have been recently described to arise from natural gain-of-function alleles of RIG-I and MDA5.The online version of this article (doi:10.1186/s12915-015-0166-9) contains supplementary material, which is available to authorized users. All together our data and previously published work suggest the following model for MDA5 discrimination of self from non-self RNA. A viral dsRNA binds to MDA5 hel Fig. , step 1,5′pppdsRNA is ensured by: (i) strong binding to the CTD; (ii) cis-recognition of the dsRNA moiety by hel locked by cooperative ATP binding; and (iii) reiteration of the cis-binding of the CTD-anchored dsRNA to hel via multiple ATP hydrolysis cycles to sustain enough numbers of activated RIG-I per time unit. In the case of MDA5, the ATPase activity discards any RNA bound to MDA5 hel that does not have a dsRNA structure long enough to allow a timely efficient nucleation of MDA5 into an active oligomeric signalling platform. Interestingly, a potential source of natural cellular (self) dsRNA that RIG-I and/or MDA5 might need to discriminate against, has just been uncovered [We propose that RIG-I and MDA5 use their ATPase motors to kinetically avoid the formation of long-enough lived signal transducing units resulting from the binding of an illegitimate self RNA. In the case of RIG-I, accurate recognition of ncovered . Altogetncovered –45 or enncovered .The cDNA coding for wild-type and variants of human Flag-tagged RIG-I and myc-tagged MDA5 were subcloned into pEF-BOS expression vector and measles virus V protein (Moraten strain) constructs in pCG vector using PCR amplification of cDNA fragments and the InFusion  recombinant technique . MDA5 co5′pppdsRNA was obtained by annealing two T7 transcribed and purified complementary 61-mer-5′ppp-ssRNA (GGUCCUGUCUGUUGUCGGU-CUCGUUUGUUGCGUGUCCGUGUUCGCCUUGGUUCCCCGGUGCC) and (CCAGGACAGACAACAGCCAGA-GCAAACAACGCACAGGCACAAGCGGAACCAAGGGGCCACGG). Both 61-mer-5′ppp-ssRNA contain only three nucleotides to avoid the formation of secondary structure and the production of double-stranded side products RNA often generated by the T7 polymerase [Poly(I:C) and Phi6 dsRNA was purchased from Amersham Biosciences  and Thermo Scientific , respectively. Rabies leader 5′ppp-RNA (GGACGCUUAACAACAAAACCAGAGAAGAAAAAGACA-GCGUCAAUUGCAAACGAAAAAUGUGC) was T7 transcribed and purified by excising the band from a denaturing urea-PAGE . The 61-lymerase .To limit the risk of signals being overshadowed by those possibly coming from endogenous or IFN-induced RIG-I or MDA5, as previously quoted , the ana5′pppdsRNA [The human IFNβ promoter luciferase assay was performed essentially as described , 30. BripppdsRNA . Unless g during 10 minute. The proteins were denatured by the addition of Laemmli 1 x loading buffer before analysis by SDS-PAGE and immunoblotting using anti-Flag , anti-C-Myc (1:50) 9E10, anti-GAPDH  monoclonal antibodies and rabbit polyclonal anti-P/V antiserum .dRIG-I) and chicken MDA5 (chMDA5). 12 bp and 24 bp palindromic duplex RNA (5′-pGGUAGCGCUACC-3′) (12dsRNA) (5′-pGGGACGUCAUGCGCAUGACGUCCC-3′) (24dsRNA) was cy3 labeled using a Mirus kit . The labeled dsRNA was dissolved with the buffer . Reactions (5 μl) contained 1.8 μM labeled dsRNA and varying concentrations of protein. The reaction mixtures were incubated for 10 minutes at room temperature in EMSA buffer . They were then loaded on a 2.5 % low melting temperature agarose gel and run with 0.5 x TBE at room temperature for 15 minutes. The gels were imaged by the fluorescent imager Typhoon Trio (Amersham Biosciences).EMSA was performed to compare the binding affinity between wild-type protein and mutated protein of duck RIG-I . Then 0.5 μM dRIG-I was preincubated with four-fold excess of a 12-mer dsRNA or 8 μM chMDA5 was preincubated with four-fold excess of a 24-mer dsRNA, for 10 minutes at 28° in ATP hydrolysis buffer . The reaction was initiated by adding 2 mM ATP. Reaction aliquots of 10 μl were quenched at time points between 15 seconds and 30 minutes by mixing with 10 μl of quenching buffer . Then, 80 μl of five times diluted Malachite green reagent was added and developed for 60 minutes at room temperature. The absorbance at 622 nm was measured with a plate reader .ATP hydrolysis by


DARIO is one such system that so far has been focussed on animals. Here we introduce an extension of this system to plant short non-coding RNAs (sncRNAs). It includes major modifications to cope with plant-specific sncRNA processing. The current version of plantDARIO covers analyses of mapping files, small RNA-seq quality control, expression analyses of annotated sncRNAs, including the prediction of novel miRNAs and snoRNAs from unknown expressed loci and expression analyses of user-defined loci. At present Arabidopsis thaliana, Beta vulgaris, and Solanum lycopersicum are covered. The web tool links to a plant specific visualization browser to display the read distribution of the analyzed sample. The easy-to-use platform of plantDARIO quantifies RNA expression of annotated sncRNAs from different sncRNA databases together with new sncRNAs, annotated by our group. The plantDARIO website can be accessed at http://plantdario.bioinf.uni-leipzig.de/.High-throughput sequencing techniques have made it possible to assay an organism's entire repertoire of small non-coding RNAs (ncRNAs) in an efficient and cost-effective manner. The moderate size of small RNA-seq datasets makes it feasible to provide free web services to the research community that provide many basic features of a small RNA-seq analysis, including quality control, read normalization, ncRNA quantification, and the prediction of putative novel ncRNAs.  Arabidopsis thaliana  are mostly degraded  leads to a deluge of small RNA-seq datasets. For the analysis of these RNA-seq data, a large array of computational tools has been developed and published. Most tools focus on the prediction and quantification of sncRNA genes, like DARIO a web server for the analysis of small RNA-seq data in animals was introduced  are poorly annotated for ncRNAs and thus a careful and manual annotation of their sncRNAs was essential. A classification of different sncRNAs solely based in their read patterns, as it has been used in DARIO B. vulgaris (RefBeet-1.1)S. lycopersicum (SL2.40)The current version of plantDARIO web service is a list of sequencing read positions mapped to one of the supported reference genomes. Data originating from any sequencing platform and mapped with the user's read alignment tool of choice can be used. However, only data originating from experiments prepared with the small RNA-seq protocol and thus predominantly covering read lengths of about 21–26 nt can be analyzed. Mapped reads can be uploaded in either BAM or bed format. We provide the PERL script map2bed.pl for converting mapped reads to bed format and for merging reads to tags, unique reads. These are represented as coordinate pairs rather than sequences for upload. This reduces the volume of data to be transferred over the internet to a managable amount: 1 GB of SAM formatted mapper output is converted to about 15 MB of compressed bed file that can be uploaded to plantDARIO. User-defined annotations can easily be added to the annotation information stored in plantDARIO's internal database by uploading a list of loci, again in bed format.The user input to the plantDARIO's workflow, which is similar to that of its animal cousin  and the number of mapped reads  is displayed. In addition a link to a genome browser is generated that allows the user to conveniently inspect the expression pattern at each individual locus Figure . This cablockbuster , and infernal  change in expression level is ath-MIR856. This miRNA, which is predominantly expressed in the floral organ  is related to snoR16 and snoR72 families according to a search in Rfam. All these snoRNAs have a primary function in ribosomal RNA processing (Brown et al., Surprisingly, we observe a much larger variability for the processing products of snoRNAs. The extreme case, snoZ102_R77, is a box C/D snoRNA belonging to the SNORD44 clan. Box C/D snoRNA_CD_230 (plantDARIO is such a service that, in contrast to comprehensive analysis environments like GALAXY (Goecks et al., plantDARIO provides the user with a valuable combination of annotation-based, standardized quantitative analysis and a simple facility for guided discoveries of novel small RNA loci.High-throughput sequencing has become the method of choice for the analysis of transcriptome data. For the special case of small RNA-seq data, web services provide a convenient means of conducting standard analyses. In this way the user can avoid the need to install, maintain, and update an array of individual tools. A. thaliana we noticed extreme differences in the levels of small RNAs processed from box C/D snoRNAs. Some of these sdRNAs are known to have a regulatory role in animals, so it might be of possible interest to further characterize small RNA processing from “house-keeping ncRNAs” in plants, and plantDARIO might be a convenient and versatile tool for this purpose.The web service also provides the results in a bed format, which can easily be used for downstream analysis tasks such as the assessment of differential expression. Using publicly available small RNA-seq data for The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.