Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3′-to-5′ unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our understanding of enteroviruses and the two types of RNA remodeling activities.RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities  ATPase is the most conserved and complex but the least understood. On the basis of sequence analyses, this protein has been predicted as a putative superfamily 3 (SF3) helicase that supposedly plays a pivotal role in enteroviral RNA replication. However, attempts to determine the helicase activity associated with 2CATPase have been unsuccessful. We found that eukaryotically expressed EV71 or CAV16 2CATPase does possess an ATP-dependent RNA helicase activity that 3′→5′ unwinds RNA helices like other SF3 helicases; surprisingly, it also functions as an RNA chaperone that remodels RNA structures in an ATP-independent manner. Moreover, we determined the domain requirements for these two RNA remodeling activities associated with 2CATPase and provide both in vitro and cellular evidence of their potential roles during viral RNA replication. Additionally, our study provides the first evidence that RNA helicase and chaperoning activities can be integrated within one protein, thereby introducing an extended view of RNA remodeling proteins.Enteroviruses contain a large number of closely related human pathogens, including poliovirus, EV71, and coxsackie viruses, and cause ~3 billion infections annually. Among the nonstructural proteins of enteroviruses or picornaviruses, protein 2C For both cells and viruses, the functionality of RNA molecules usually requires correct folding into proper tertiary structures and the association with correct RNA-binding proteins ,2. To maRNA helicases are highly similar to DNA helicases, contain ATPase activity, and utilize the energy of ATP binding and/or hydrolysis to translocate along and unwind RNA duplexes. They are thought to participate in most ATP-dependent remodeling of structured RNAs and are classified into six superfamilies (SFs), designated SF1 to SF6, on the basis of conserved helicase motifs . On the cis-acting elements that play pivotal roles in vRNA replication and translation. These highly structured RNA elements require RNA helicases or chaperones to aid in their proper folding and re-folding. Moreover, during vRNA replication, replicative intermediate double-stranded RNA (dsRNA) must be unwound by virus-encoded or cellular RNA helicases; therefore, vRNA templates can be efficiently re-utilized to synthesize more progeny vRNA strands. Thus far, a variety of RNA viruses, such as flavivirus NS3 , and with an excessive amount (at a ratio of 5:1) of primer RNA, which was a long oligonucleotide and was pre-annealed to the template in the presence or absence of 2CATPase RNA strands from the (-)RNA template by 3Dpol, and the promoting effect of 2CATPase was dose dependent (ATPase was unable to promote (+)RNA synthesis RNA synthesis as expected  cells. RNA replication was measured via quantitative reverse transcription polymerase chain reaction (qRT-PCR) 24 and 48 hours post-transfection, and virus production was detected via immunofluorescent staining of EV71 VP1 in cells. Strikingly, compared to Wt virus, the GK134AA mutation resulted in the loss of EV71 RNA replication -vRNA synthesis and the uridylylation of the protein primer VPg  [http://swissmodel.expasy.org)]. The figure of the modeled EV71 2CATPase 3D structure and the structural alignment with AAV2 Rep40 (PDB ID code 1U0J) were drawn by PyMOL program 1.1  from coordinate file.The 3D structure of EV71 2Cb.org/)] . Five moin vitro transcribed using T7 RNA polymerase . The in vitro transcribed RNA strands were purified by Poly-Gel RNA Extraction Kit  according to the manufacturer's instruction. The two strands were mixed in a proper ratio, and annealed through heating and gradually cooling as previously described . The mixtures were then electrophoresed on 15% native-PAGE gels, followed by scanning with a Typhoon 9200 imager . The RNA strand hybridization assay was performed as previously described [The standard helix destabilizing assay was performed as previously described  with minescribed . The seqNTPase activities were determined via measuring the released inorganic phosphate during NTP hydrolysis using a direct colorimetric assay as previously described . All of in vitro transcription using T7 RNA polymerase, and the 5′-HEX-labeled substrate RNA was synthesized by TaKaRa. Indicated amount of protein was added in 10 μl reaction volumes containing 0.15 nM substrate, 0.3 nM ribozyme, 50 mM HEPES-KOH (pH 7.5), 2.5 mM MgCl2, and 2 mM DTT, 0.01% BSA, 20 U RNasin. The mixtures were incubated at 37°C for 30 min, and then treated by proteinase K  at 37°C for 15 min. The digestion reactions were precipitated with 20 μl isopropanol and 2 μg glycogen , and subjected to by centrifugation at 12 000 g for 15 min. The precipitates were dissolved in formamide and electrophoresed on 15% acrylamide-7 M urea gels, followed by scanning with a Typhoon 9200 imager .The ribozyme RNA was synthesized from the in vitro transcribed by T7 RNA polymerase as described above. The primer RNA strand contains the 1–140 nts of the 5′ end of EV71 (+)RNA, and the (-)-vRNA template contains the sequence complementary with the 1–357 nts of 5′ end of EV71 (+)RNA. The transcribed products were purified and quantified as described above. Then, the primer RNA was pre-annealed to the template. The EV71 3Dpol gene was cloned into the vector pET26b-Ub plasmid, and the protein was purified as the C-terminal His×6 tag fusion protein via a nickel-charged HisTrap HP column  as previously described [pol in 50 mM HEPES-KOH (pH 7.0), 12.5 mM KCl, 5 mM MgCl2, 5 mM DTT, 20 U RNasin (Promega), and 0.25 mM Digoxin (DIG) RNA Labeling Mix (Roche) with total volume of 10 μl at 22°C. The reaction mixtures were supplemented with 10 μl 2×loading buffer , and then denatured at 65°C for 10 min. The samples were electrophoresed on 1.5% agarose-formaldehyde denaturing gels, and transferred onto N+ nylon membranes (Roche). The membranes were incubated with anti-DIG-alkaline phosphatase antibody (Roche), followed by incubation with CDP-Star (Roche) at 37°C for 10 min. The signals were detected by radiography on X-ray film . All oligonucleotides used for in vitro transcription are listed in The RNA primer and template were escribed . The RdRescribed . As prevescribed , the RdRHuman rhabdomyosarcoma (RD) cells were obtained from the American Type Culture Collection (ATCC), and cultured in Dulbecco's modified Eagle's medium  containing 10% fetal bovine serum , 100 units of penicillin and 100 mg/ml of streptomycin. The transfected or infected RD cells were maintained in DMEM containing 2% FBS.Mlu I digestion. 2 μg of each purified linear plasmid was used as the template to in vitro transcribe EV71 RNA using the SP6 RiboMAX Large Scale Production System (Promega) according to the manufacturer's protocols. The RNA transcript was transfected into 80%-90% confluent RD cells using Lipofectamine 2000 (Invitrogen). 24–48 h post-transfection, the total RNA from transfected RD cells was extracted using RNeasy Mini Kit , and stored at -80°C.The wild type and mutant plasmids containing full-length EV71 cDNA were linearized with The amount of viral RNA was quantified by using One Step PrimeScript RT-PCR Kit  according to the manufacturer's protocol as previous described  with EV7RD cells at 90% confluence on glass slides were transfected with indicated EV71 transcript. The cells were fixed at 24 h post-transfection with pre-cooled acetone at -20°C for 30min, followed by washing in PBS for 3 times and incubation with mouse anti-EV71 VP1 monoclonal antibody  for 60 min at 37°C. The cells were then washed in PBS and incubated with Alexa Fluor 488-labeled goat anti-mouse IgG (Invitrogen) for another 45 min. Cells were incubated with DAPI at room temperature for 5 min to stain the nuclei. Finally, the cells were rinsed again with PBS and visualized under a fluorescent microscope .S1 FigATPase was expressed using eukaryotic (baculovirus) system. The purified recombinant proteins were subjected to 10% SDS-PAGE followed by silver staining. M, molecular mass marker.MBP-fusion EV71 2C(TIF)Click here for additional data file.S2 Fig(A) Elution profile of purified MBP-2CATPase from a Superdex 200 increase 10/300 GL column. Protein elution was followed by UV detection at 280 nm. X axis represents the elution volume (in ml). The major elution peak corresponds to a molecular mass of ~600 kDa, which is estimated according to the manufacturer's instruction. (B) SDS-PAGE and silver staining of the eluted protein in the major peak.(TIF)Click here for additional data file.S3 FigATPase (20 pmol). Lanes 1 and 4, reaction mixture without 2CATPase addition; lanes 2 and 5, boiled reaction mixture without 2CATPase. Asterisks indicate the HEX-labeled strand.0.1 pmol RNA helix substrate with 49 (lanes 1–3) or 42 (lanes 4–6) complementary base pairs was reacted with MBP-2C(TIF)Click here for additional data file.S4 Fig(A) The NTPase activity of MBP-2CATPase on each NTP in the absence or presence of 100 pmol ssRNA was measured at 37°C as nanomoles of released inorganic phosphate as indicated. In addition, the ATPase activity of MBP-2CATPase was measured at 22°C as indicated. MBP alone was used as the negative control. Error bars represent SD values from three separate experiments. (B) The unwinding assays of HCV NS3 using the standard helix substrate were performed in the absence or presence of 5 mM ATP or AMP-PNP as indicated. (C) The unwinding assays of MBP-2CATPase using the standard helix substrate were performed at 22°C or 37°C as indicated. Boiled MBP-2CATPase or no protein supplementation was used as negative control. (D) The standard RNA helix (0.1pmol) was reacted with MBP-2CATPase (20 pmol) in the presence of indicated divalent metallic ions (2.5 mM). (E) The standard RNA helix was reacted with 2CATPase (20 pmol) in varying concentrations of Zn2+. (F) The standard RNA helix was reacted with 2CATPase (20 pmol) at the indicated pH. For (B-F), asterisks indicate the HEX-labeled strand.(TIF)Click here for additional data file.S5 FigATPase (20 pmol) in the absence (lane 3) or presence of 5 mM indicated NTP (lanes 4–7). Asterisks indicate the HEX-labeled strand.The standard RNA helix was reacted with 2C(TIF)Click here for additional data file.S6 Fig(D) The standard RNA helix (0.1 pmol) was reacted with MBP-2CATPase wt (lane 4) or ΔSTN mutant (lane 3) in the presence of 5 mM ATP. Asterisks indicate the HEX-labeled strand. (E) Elution profile of purified MBP-2CATPaseΔCTD from a Superdex 200 increase 10/300 GL column. Protein elution was followed by UV detection at 280 nm. X axis represents the elution volume (in ml).The sites of “GK” in helicase/ATPase motif A and “STN” in helicase motif C are indicated as yellow and green, respectively. (TIF)Click here for additional data file.S7 FigThe ATPase activity of indicated proteins was measured at 37°C as nanomoles of released inorganic phosphate. MBP alone was used as the negative control. Error bars represent SD values from three separate experiments.(TIF)Click here for additional data file.S8 Fig(A) The sequence alignment of the linker region between the HC domain and CTD of 2CATPase proteins from indicated picornaviruses. (B) The sequence alignment of the HC domains of 2CATPase proteins from indicated picornaviruses. EV71, enterovirus 71, genus Enterovirus; CAV16, coxsackie A virus 16, genus Enterovirus; PV, poliovirus, genus Enterovirus; EMCV, encephalomyocarditis virus, genus Cardiovirus; FMDV, foot-and-mouth disease virus, genus Aphthovirus. The alignments were conducted using ClustalX2. The conserved motifs A, B, and C for SF3 helicases are indicated.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Sulfur is an essential nutrient, necessary for synthesis of many metabolites. The uptake of sulfate, primary and secondary assimilation, the biosynthesis, storage, and final utilization of sulfur (S) containing compounds requires a lot of movement between organs, cells, and organelles. Efficient transport systems of S-containing compounds across the internal barriers or the plasma membrane and organellar membranes are therefore required. Here, we review a current state of knowledge of the transport of a range of S-containing metabolites within and between the cells as well as of their long distance transport. An improved understanding of mechanisms and regulation of transport will facilitate successful engineering of the respective pathways, to improve the plant yield, biotic interaction and nutritional properties of crops. Sulfur (S) is as an essential macronutrient for plant growth, development, and response to environmental changes. It is required for the biosynthesis of proteins, co-enzymes, prosthetic groups, vitamins, amino acids like Cys and Met, GSH, and secondary metabolites such as GSL and sulfoflavonoids. Understanding S metabolism in plants is essential for human nutrition as Met is an essential amino acid and as many S containing secondary metabolites and specialized peptides  are important for biotic and abiotic interactions of crop plants and their yield. Sulfur uptake and distribution is tightly controlled in response to developmentally or environmentally induced changes in nutrient demand . AlthougTaken up from the soil, sulfate is incorporated into adenosine-5′-phosphosulfate followed by reduction into sulfite and then sulfide and Cys biosynthesis. In parallel, adenosine-5′-phosphosulfate can be further phosphorylated to 3′-phosphoadenosine-5′-phosphosulfate, which is used for sulfation reactions . Cys is Arabidopsis and about 140 more sulfur metabolites that have not been assigned to the databases to date have been reported recently (2S), and secondary metabolites . Given the large number of metabolites within S-assimilation pathway, and the localization of enzymes and pathways in different compartments, a wide spectrum of plant metabolite transporters has to be expected. Plants have evolved a network of transporters to maintain homeostasis of sulfur and S-derived compounds. Specific intra-and inter-cellular transporters are needed to store the sulfur or to channel it in biochemical processes allowing biosynthesis of important S-containing metabolites.About 300 different metabolites are predicted to incorporate sulfur in recently . These mDespite their importance for sulfur homeostasis, our knowledge of intracellular and intercellular transporters in S assimilation is still limited. In recent years, significant progress has been made in elucidating the functions of some carriers important in S-metabolism in plants. Still, many transport proteins remain unidentified. This review provides an overview of known transport proteins in sulfur metabolism within the cell and plant as a whole and aims understanding of their role in the maintenance of sulfur levels in plants.Stylosanthes hamate, a tropical forage legume. These cDNAs encoded two of high affinity transporters and one a low affinity transporter /sulfate symporters, and sulfate/anion  antiporters . Plants lacking both these transporters are unable to take up sulfate in low concentrations and are strongly affected in growth  and yet to be identified SULTRs localized in various membranes within the plant cell.The SULTR family is best characterized in n growth . The tran growth . On the  mutants . In addi mutants , but mor mutants . The two mutants . Group 4 mutants . The fir mutants . The oth mutants . Interess leaves . Figure Brassica oleracea, poplar, wheat, and Medicago truncatula . Investigations of Cys transport across mitochondrial membrane revealed presence of multiple transport systems with different kinetic properties . This conclusion has been confirmed by showing that cytosolic expression of GSH synthase rescues the seedling lethal gsh2 mutant . In addition, GSH is one of the forms of reduced sulfur subjected to long-distance transport . While the presence of such GSH transporters was long recognized, the molecular nature of such carriers is still far from being determined. The identification of high affinity GSH transporter in yeast . They facilitate transport of GSH and γ-EC, probably as export from the plastids . In addition, traditionally it was thought that Met synthesis in plants requires transport of homocysteine from plastids to the cytoplasm, as the activity of the last enzyme of the biosynthetic pathway, MS, was found in this compartment only . This transporter, similar to yeast mitochondrial SAM carrier, was shown to be additionally localized in mitochondria, and to contains five transmembrane helices . Plant SMM transporters are not known, but expression of a yeast SMM transporter in pea resulted in improved sulfur and nitrogen content of the seeds . The MTOB-transporter was identified as BAT5 or BASS5 following its activation in trans by R2R3 MYB factors regulating GSL biosynthesis . However, the identity of this transporter, which is involved in the export of amino acid, is unknown. It was also not possible to analyze whether keto-acids or amino acids are preferentially exported from the chloroplasts. Theoretically, the export of both types substrates is possible from chloroplasts as the transamination  of keto acids could be catalyzed by both chloroplastidic BCAT3 . The existence of this transporter has been postulated , which is essential for the sulfation of GSLs, flavonols, peptides, and other sulfated compounds in the cytoplasm has been recently identified . Golgi in plants is known to contain the TPST. TPST catalyzes the post-translational Tyr-sulfation of peptides by the transfer of sulfate from PAPS to the phenolic group of tyrosine  phenotype. Several Golgi-localized transporters of PAPS have been described in different organisms like Drosophila , stored there or further metabolized and/or transported . Vacuolar localization of secondary metabolites has been suggested long time ago but it was experimentally demonstrated using water-free environment gradient centrifugation of Arabidopsis leaf tissue only recently  and we consider them below.After completion of biosynthesis, secondary metabolites like GSLs and flavonoids, which are present at high concentrations in the cell, need to be imported into the vacuole , which is maintained by Hnnotated . For comnnotated . In strinnotated . ABC trannotated .S-conjugate pump) in the transport of glutathionylated anthocyanins has been originally reported in maize and petunia  where they can export yet-unidentified but highly toxic compounds to the site of invasion of non-host pathogens of Arabidopsis (Arabidopsis against non-host pathogen (induced defense of Arabidopsis against the grass powdery mildew Blumeria graminis hordei) is a phytochelatin synthase, suggesting possible PEN4-mediated glutathionylation of products to be transported by PDR8/PEN3. Another member of the ABC family in wheat known as LR34, controls durable disease resistance against some of the most devastating fungal pathogens such as leaf rust and stripe rust, potentially by exporting other antifungal metabolites although the mechanism is currently not understood . It could be further demonstrated that anthocyanins are transported to the cell wall or the vacuole by at least two distinct vesicle trafficking pathways  from the production site in ER-associated cytoplasm to the tonoplast  in onoplast . Previou MtMATE2  and it wBrassica napus were shown to move through the phloem to seeds, where the radioactivity was detected . Using Xenopus oocytes as an expression system it could be shown that GTR1 and GTR2 are high affinity plasma membrane-localized GSL-specific proton symporters . The ability of the GTRs to transport GSL may have arisen through neo-functionalization of NRT/PTR family members. The retained ability of both GTR1 and GTR2 to transport nitrate corroborates such a de novo functional specification. Notably, there are more examples of NRT/PTR proteins transporting different types of substrates. Thus, the NRT1.1 which was mainly known as nitrate transporter . A first indication that intercellular or long distance transport may play an important role in the compartmentation of different flavonoids has been obtained in cotyledons and flower buds of Catharanthus roseus -flavonoid conjugates it could be shown that flavonoids can be selectively and unidirectionally transported to specific cell/tissue types and from one organ to another (Flavonoids are produced at high levels in photosynthetically active organs as the expression of key biosynthetic gene encoding for CHS is dependent on light. These secondary metabolites were also found in heterotrophic root tissue where they contribute to many important functions like lateral development, gravitropic response, legume nodulation, the induction of the hyphal branching of arbuscular mycorrhizal fungi, as well as the response to phosphate starvation and the inhibition of polar auxin transport . Althougs roseus . Furtherolecules . Moreove another . It has  another .34S labeling, exploitation of natural variation, and mathematical modeling will be necessary to obtain the comprehensive list of S-metabolite transporters and to understand the role they play in controlling sulfur metabolism and homeostasis in plants. The increased use of these approaches in sulfur research makes hope for a rapid progress in dissection of the network of transporters in sulfur metabolism.Although sulfur-metabolites represent only a small portion of plant metabolites, the pathways of their synthesis are complex and their function and homeostasis require a large number of intra- and intercellular transport steps. Many of these transporters have been characterized, with some of the main gaps having been filled very recently. Thus, the plastidic SULTR has been identified, the GSL transporter found, as well the plastidic PAPS transporter. However, many other transporters still await discovery, above all the transporter responsible for a major flux of sulfate in the cells, the import into vacuole. With better understanding of secondary sulfur metabolism, the need for specific transporters is being recognized, such as the Golgi PAPS transporter, tonoplast GSL, and sulfoflavonoid transporter, and transporters responsible for cellular movement of minor sulfated compounds. Apart from the gene discovery, other questions have to be addressed: the regulation of the transporters of S metabolites, their contribution to the control fluxes of sulfur throughout the plant, both on cellular and whole plant levels, and the role of these transporters in the interactions of plants with other organisms. New tools and approaches, e.g., 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.


There are errors in the author affiliations. The affiliations should appear as shown here:1*‡, Yan Zhang2*‡, Lele Zhao1, Wenjing Zhao1, Chuan He4, Christa F. Honaker3, Zhengxiao Zhai1, Zikui Sun4, Paul B. Siegel3*He Meng* Corresponding authors‡ These authors contributed equally to this work1. School of Agriculture and Biology, Shanghai Jiaotong University; Shanghai Key Laboratory of Veterinary Biotechnology, Shanghai, P. R. China.2. Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, USA.3. Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, Virginia, USA.4. Shanghai Personal Biotechnology Limited Company, Shanghai, P.R. China.menghe@sjtu.edu.cn, Yan Zhang: yzhang@vbi.vt.edu, Paul Siegel: pbsiegel@vt.eduCorrespondence and requests for materials should be addressed to: He Meng:


Lipoptena cervi (Diptera: Hippoboscidae) is a hematophagous ectoparasite of cervids, which is considered to transmit pathogens between animals and occasionally to humans. The principal life stage that is able to parasitize new hosts is a winged ked that just emerged from a pupa. To facilitate efficient transmission of pathogens between hosts, vertical transmission from female deer keds to their offspring is necessary. We investigated vertical transmission of several vector-borne pathogens associated with cervids.Capreolus capreolus and Cervus elaphus, developing larvae dissected from gravid females, and fully developed pupae. The presence of zoonotic pathogens was determined using qPCR or conventional PCR assays performed on DNA lysates. From the PCR-positive lysates, a gene fragment was amplified and sequenced for confirmation of pathogen presence, and/or pathogen species identification.Deer keds from several locations in Hungary were collected between 2009 and 2012. All life stages were represented: winged free-ranging adults, wingless adults collected from Bartonella schoenbuchensis was found in wingless males (2%) and females (2%) obtained from Cervus elaphus, dissected developing larvae (71%), and free-ranging winged males (2%) and females (11%). DNA of Anaplasma phagocytophilum and Rickettsia species was present in L. cervi adults, but not in immature stages. DNA of Candidatus Neoehrlichia mikurensis was absent in any of the life stages of L. cervi.DNA of B. schoenbuchensis is transmitted from wingless adult females to developing larvae, making it very likely that L. cervi is a vector for B. schoenbuchensis. Lipoptena cervi is probably not a vector for A. phagocytophilum, Rickettsia species, and Candidatus N. mikurensis. Lipoptena cervi (deer ked) is an obligate hematophagous ectoparasite of cervids and domesticated animals [ animals ,2, which animals . This sp animals ,5. Withi animals -8.L. cervi is considered a mere nuisance for animals only [L. cervi is also considered a potential vector for a number of zoonotic pathogens, such as Bartonella, Anaplasma, and Rickettsia species [Lipoptena cervi shows a number of interesting life history traits, in which vertical transmission of pathogens from females to offspring seems to be essential to facilitate efficient transmission between vertebrate hosts. The life cycle of L. cervi starts (arbitrarily) with free-ranging winged adult deer keds that search for suitable (cervid) hosts. After landing on a host, they crawl into the fur, shed their wings and thereby become permanently associated with that host. The wingless deer keds frequently take blood meals from that host, which is soon followed by mating. After that, a larva will develop (one at a time) that is retained by the female until the third instar. This third instar larva is deposited on the cervid fur as a white prepupa, which immediately starts to pupate. The fully developed and darkened pupa drops to the ground and remains there until August-September, after which a new generation of winged adult keds can emerge [In general, als only , and clials only ,11. Howe species -14. Lipon emerge ,15.L. cervi are attracted to large moving dark colored objects [L. cervi difficult or impossible [L. mazamae, can be transmitted mechanically from female white-tailed deer to their offspring [When in search for a host, winged adult  objects , fly sho objects . The los objects , makes apossible . Howeverffspring . NeverthL. cervi. Bartonella schoenbuchensis, causing deer ked dermatitis in humans, was isolated from the gut of wingless adult L. cervi [B. schoenbuchensis in wingless adult deer keds was confirmed by others as well [L. cervi life stages. However, DNA of Bartonella species in general, including potential endosymbionts, was detected in L. cervi wingless adults [L. cervi for (zoonotic) Bartonella species.A number of studies show that various zoonotic pathogens are present in a number of the mentioned life stages of L. cervi . Althoug as well ,18, DNA s adults ,18, fulls adults , and freBartonella, Anaplasma species may be transmitted by L. cervi as well. Anaplasma ovis, which causes anaplasmosis primarily in sheep, was detected in a single free-ranging winged adult deer ked. However, none of the additional adult wingless deer keds collected from roe and red deer tested positive for Anaplasma spp. [A. ovis, A. phagocytophilum can cause anaplasmosis both in sheep and cattle, and a zoonotic disease referred to as granulocytic anaplasmosis in humans [L. cervi wingless adults, collected from cervids that tested negative for this pathogen, but not in free-ranging winged adult deer keds [A. phagocytophilum as well [A. phagocytophilum ecotype II was found especially in roe deer, Ixodes ricinus ticks, and deer keds.In addition to sma spp. . In contn humans ,20. Thiseer keds . In anot as well . AdditioL. cervi for Rickettsia species was studied as well. Rickettsiae are non-motile, Gram-negative, highly pleomorphic bacteria, which can be transmitted by competent arthropod vectors, such as ticks, lice, and fleas, and can cause a large number of zoonotic diseases in humans [Rickettsia helvetica and other unidentified Rickettsia species were detected in pools of wingless adult L. cervi, collected from roe deer and red deer, but not in winged free-ranging adult deer keds [R. helvetica is still unclear, this pathogen has been associated with acute perimyocarditis [Finally, vector potential of n humans . Ricketteer keds . Althougcarditis , an unexcarditis , and reccarditis .L. cervi was further explored for four zoonotic pathogens: Candidatus Neoehrlichia mikurensis, A. phagocytophilum, Bartonella spp., and Rickettsia spp. We tested winged free-ranging adults, wingless adults collected from roe deer and red deer, developing larvae dissected from females, and pupae collected from the fur of cervids for the presence of these zoonotic pathogens.In this study, the vector potential of Cervus elaphus hosts, and two Capreolus capreolus hosts . In addition, 94 free-ranging winged adult deer keds  were collected during the same time period from various locations in Hungary. No ethical approval is required for the experimental methods used in this study.Between 2009 and 2012, 345 deer keds and fully developed pupae were collected from different locations in Hungary. A total of 248 wingless adult deer keds , and three developed pupae were collected from one male and ten female In the laboratory, seven wingless adult female deer keds were dissected, and developing larvae were harvested. For all samples, DNA was extracted first by alkaline lysis as described earlier . For a nCandidatus N. mikurensis, A. phagocytophilum, Bartonella spp., and Rickettsia spp. For detection of A. phagocytophilum and Candidatus N. mikurensis DNA, a single multiplex qPCR assay was used, which targets specific regions of genes msp2 (Major Surface Protein 2) for A. phagocytophilum, and groEL (heat shock protein) for Candidatus N. mikurensis. This was followed by conventional PCR and sequencing part of the groEL gene for samples positive for A. phagocytophilum. Detection of A. phagocytophilum and Candidatus N. mikurensis DNA by qPCR, and confirmation by sequencing of positive samples were performed as described earlier [Bartonella spp. a conventional PCR assay was used, which targets a part of the citrate synthase gene (gltA). This was followed by sequencing of positive samples for species identification. Both conventional PCR and sequencing procedures were performed, as described earlier [Extracted DNA was tested for the presence of four zoonotic pathogens:  earlier ,28. For  earlier .Rickettsia species, we used a multiplex qPCR assay, in which two different regions of the gltA gene are targeted. We designed primers and probes to amplify a region of the gltA gene, specific for R. helvetica , wingless adults , and five harvested developing larvae  is able to transmit Bartonella vertically from females to their offspring [ae Table . We dete as well ,12,13,18Anaplasma phagocytophilum DNA was detected in 123 (35%) of deer ked samples, 119 were wingless adults collected from red deer, two were free-ranging winged adults, and two remnants of female adult deer keds after dissection and removal of developing larvae  host, retained their wings somehow, and were able to search for another more suitable host. Other studies reported the presence of A. phagocytophilum in only wingless adults as well [L. cervi is probably not involved in the transmission of A. phagocytophilum. as well ,22, and Rickettsia spp., 29 deer ked samples were found positive for a gltA sequence detected using primers and probes designed by Stenos et al. [Rickettsia helvetica, using primers and probes designed specifically for a gltA region of this species. Four deer ked samples showed positive results based on both gltA regions. Rickettsia DNA was detected in wingless L. cervi males and females, originating from red deer, and in free-ranging winged deer keds. However, qPCR assay results indicated very low levels of Rickettsia DNA present within the samples. Therefore, we were not able to confirm Rickettsia DNA presence by subsequent conventional PCR and sequencing. As for A. phagocytophilum, dissected developing larvae and fully developed pupae showed no positive results for Rickettsia DNA, which indicates that vertical transmission of Rickettsia species by L. cervi females is not very likely. The absence of vertical transmission and the presence of Rickettsia species in winged and wingless deer keds only indicate that L. cervi is probably not involved in the transmission of rickettsiae.For s et al. . In addiCandidatus N. mikurensis DNA in any of the deer ked life stages collected. According to literature, Candidatus N. mikurensis is primarily transmitted by ticks [Candidatus N. mikurensis can be found in several rodent species, which may act as reservoir hosts [Borrelia burgdorferi s.l. genospecies [Borrelia infections in I. ricinus ticks [Candidatus N. mikurensis as well. Together with our findings, this indicates that although L. cervi and ticks (I. ricinus) can share a common host (cervids), L. cervi is probably not involved in the transmission of Candidatus N. mikurensis.We detected no by ticks ,32, and ir hosts ,33-35. Hospecies ,37, and us ticks ,39. TherBartonella and Rickettsia species, the difference between wingless positive females and males was significant . Free-ranging females also had a higher prevalence for these two bacteria, however not significantly due to low sample size. This is an unexpected difference, since it is reported that when unfed, both male and female deer keds have the same weight, and when blood-fed, males are heavier than females [Rickettsia and Bartonella species (zoonotic or endosymbionts) are able to colonize and/or survive in females more efficiently than in males. Since vertical transmission of pathogens in L. cervi is necessary for efficient transmission between vertebrate hosts, strong selection toward infection of female deer keds can be expected. However, a (molecular) mechanism for this is still unknown.On red deer, we observed larger numbers of positive wingless females in comparison to positive wingless males for all three pathogenic genera. For  females . TherefoWolbachia in another dipteran, Drosophila innubila [Wolbachia endosymbionts [Bartonella and/or Rickettsia spp. infection in female deer ked might lead to more female offspring compared to uninfected females, possibly resulting in the observed asymmetry in the female: male ratio of infected individuals.Another possibility is the involvement of endosymbionts in this phenomenon. In common with other insects, in which all the life stages are dependent solely on blood as the nutrient source, hippoboscids have symbionts. These symbionts are housed in a mycetome on the intestine and are transferred to offspring accompanied to nutrients provided by the mother for her intra-uterine larva . Many mainnubila . Nycteroymbionts  but, unfymbionts . Either Finally, we were not able to test blood or tissue samples of roe or red deer, from which the wingless adult deer keds were collected for presence of pathogens. However, cervids are known reservoir hosts for at least a number of zoonotic pathogens we investigated -49.L. cervi to their offspring. The only deer ked life stage that is able to search actively for new hosts is a winged adult following eclosion from a pupa. Therefore, vertical transmission and detection of pathogens in emerged winged adults is essential to show vector potential of L. cervi.Detection of pathogens in wingless females, developing larvae, and fully developed pupae indicates vertical transmission from female Anaplasma phagocytophilum, or Rickettsia species were not present in harvested developing larvae or fully developed pupae, and only a limited number of winged male and female deer keds were found positive for these pathogens. Therefore, L. cervi is probably not a vector for A. phagocytophilum, and Rickettsia species.Bartonella schoenbuchensis is vertically transmitted from wingless females to developing larvae. It is very likely that L. cervi is a vector for Bartonella, including the zoonotic pathogen B. schoenbuchensis, because we identified this zoonotic pathogen in dissected developing larvae and free ranging winged L. cervi males and females.


C. elegans nervous system. We find that acetylcholine (ACh) is the most broadly used neurotransmitter and we analyze its usage relative to other neurotransmitters within the context of the entire connectome and within specific network motifs embedded in the connectome. We reveal several dynamic aspects of cholinergic neurotransmitter identity, including a sexually dimorphic glutamatergic to cholinergic neurotransmitter switch in a sex-shared interneuron. An expression pattern analysis of ACh-gated anion channels furthermore suggests that ACh may also operate very broadly as an inhibitory neurotransmitter. As a first application of this comprehensive neurotransmitter map, we identify transcriptional regulatory mechanisms that control cholinergic neurotransmitter identity and cholinergic circuit assembly.Nervous system maps are of critical importance for understanding how nervous systems develop and function. We systematically map here all cholinergic neuron types in the male and hermaphrodite DOI:http://dx.doi.org/10.7554/eLife.12432.001 To better understand the nervous system—the most complex of all the body’s organs—scientists have begun to painstakingly map its many features. These maps can then be used as a basis for understanding how the nervous system develops and works.Caenorhabditis elegans. Cells communicate by releasing chemicals called neurotransmitters across the synapses, but it is not fully known which types of neurotransmitters are released across each of the synapses in C. elegans.Researchers have mapped the connections – called synapses – between all the nerve cells in the nervous system of a simple worm called C. elegans.Now, Pereira et al. have mapped all worm nerve cells that use a neurotransmitter called acetylcholine by fluorescently marking proteins that synthesize and transport the neurotransmitter. This map revealed that 52 of the 118 types of nerve cells in the worm use acetylcholine, making it the most widely used neurotransmitter. This information was then combined with the findings of previous work that investigated which nerve cells release some other types of neurotransmitters. The combined data mean that it is now known which neurotransmitter is used for signaling by over 90% of the nerve cells in Using the map, Pereira et al. found that some neurons release different neurotransmitters in the different sexes of the worm. Additionally, the experiments revealed a set of proteins that cause the nerve cells to produce acetylcholine. Some of these proteins affect the fates of connected nerve cells. Overall, this information will allow scientists to more precisely manipulate specific cells or groups of cells in the worm nervous system to investigate how the nervous system develops and is regulated.DOI:http://dx.doi.org/10.7554/eLife.12432.002 Caenorhabditis elegans, both in its hermaphroditic and male form , six classes (26 neurons) are GABAergic  transporter, VAChT (encoded by unc-17) and the ACh-synthesizing choline acetyltransferase ChAT (encoded by cha-1) revealed the cholinergic identity of a number of neurons in the nervous system  and the vesicular ACh transporter ; see ace; four genes in C. elegans; , OH13802 , OH13805 OH13753 .OH13806 , OH13804 , OH13807 OH13803 .OH13808 [otIs354; otEx6380  mutant strain carrying the cho-1 fosmid reporter (otIs544). Worms were fixed using methanol/acetone and stained with a rabbit anti-UNC-17 serum diluted 1/100 (gift from James Rand).Immunofluorescence for UNC-17 was performed as described earlier  using a Immunofluorescence for serotonin was performed using a tube fixation protocol as described earlier . The ant3) and mounted on 5% agarose on glass slides. All images . Values are expressed as mean ± standard deviation (s.d.) or standard error of the mean (sem) as indicated in each figure legend. For results shown in For results shown in unc-3 mutant animals were reconstructed in the anterior region of the ventral cord in order to determine neuron morphology and synaptic circuitry. The reconstructions were made from electron micrographs of serial sections as described in unc-3(e151) allele in trans to a covering deficiency (mnDf5). This strain was generated by crossing unc-3(e151) the strain SP266 mnDp1/ V; mnDf5 X.Wild-type and otIs341 (mgl-1::gfp) transgenic strain was used to identify mutants affecting the identity of the cholinergic RMDD/V motor neurons. A conventional semi-clonal EMS screen identified the ot712 mutation, which was found to be closely linked to the transgene vsIs33V also present in the strain background. ot712 animals are uncoordinated and unc-42 maps on LGV. Complementation tests between ot712 and two alleles of unc-42 (e419 and e270) confirmed that ot712 is an allele of unc-42. Sanger sequencing reveals that ot712 harbors a late nonsense mutation (W181>Stop) in exon 6.The www.wormwiring.org which contains updates to the original wiring diagram (Connectivity data was taken from the latest release of  diagram . Data frhttp://www.weizmann.ac.il/mcb/UriAlon/download/network-motif-software) to find 3-neuron and 4-neuron network motifs. 3-neuron motifs analysis was performed with default parameters. 4-motifs search was performed with the metropolis randomization methodWe used m-finder software  and how many times each NT-combination appeared at each motif-role (approach 2). The number of occurrences of each NT/NT-combination is compared between the real network and the randomized networks by calculating a z-score.We say that a motif-role is significantly associated with a given NT (approach 1), or a NT-combination (approach 2) if the z-score is larger than 2.To assess the risk for false-positives in this approach, we repeated the above calculation while treating 100 randomized networks as real networks, comparing each of them to 100 randomized networks, and choosing events with z-score >2 as significant. For 3-neuron motifs, approach 1, the real network has 5.6 ± 0.7 significant events , while random networks had on average 1.8 ± 1.9 significant events. Hence, the risk for false-positives does not seem high.We obtained the processing depth of each neuron from  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by a Senior Editor, Kang Shen (Reviewing Editor) and two peer reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: Gerald Rubin and Shawn Xu (peer reviewers).Thank you for submitting your work entitled "A cellular and regulatory map of the cholinergic nervous system of C. elegans" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:et al., have written a very interesting and highly informative article which will be extremely beneficial for the C. elegans community. This article sets a broad foundation for cholinergic signaling in the C. elegans nervous system. They use an array of imaging techniques to identify potential sites of release, reception and cholinergic neuron identities, as well as acute observation and discussion of the role of acetylcholine in circuit motifs.Pereira eLife after the following questions are addressed.Both reviewers thought that the manuscript is suitable for publication in Essential revisions:Drosophila has inhibitory nAChRs? In particular, Wormbase lists the closest fly homolog of ACC-4 as the GABA receptor Rdl. If evidence for the existence of Ach-gated anion ion channels is limited to C. elegans, then perhaps the wording should be changed to say "the C. elegans genome encodes" to be consistent with the available facts. I note that the two cited references do not contain information to support the broader statement made, as they describe work with C. elegans only.1) It would be helpful to readers to expand on the generality of the statement that "Invertebrate genomes encode not only conventional excitatory Ach-gated cation ion channels, but also inhibitory Ach-gated anion ion channels". For example, is there evidence that the widely studied invertebrate unc-3 is a circuit-associated transcription factor” that unc-3 activity is required to "specify" synaptic connectivity is too strong and misleading. The data just show that unc-3 activity is required for normal synaptic connectivity to occur. That means something very different, at least to me, than "specify". Likewise the statement that unc-3 "defines the function and assembly of an entire circuit" goes way beyond what can be concluded from the results presented. The circuit is indeed abnormal in unc-3 mutants, but to use the word "define" here would require, in my opinion, evidence that in unc-3 mutants the circuit became a different type of functional circuit, not that it is just abnormal. I think one can say that unc-3 is "required", but "define" implies a specific role for unc-3 that has not been demonstrated. Similarly "circuit-associated" is appropriate, but "circuit-defining" is not.2) I think the statement in paragraph seven subsection “acc-4 co-express with at least one other Ach-gated chloride channel in each cell?3) This present study sets a foundation for investigating the role of ACC-4 in cholinergic signaling. ACC-4 however does not elicit strong Ach-gated chloride currents when expressed alone , does this suggest that it is an auxiliary subunit in a heteropentameric receptor? Therefore, does  1) It would be helpful to readers to expand on the generality of the statement that "Invertebrate genomes encode not only conventional excitatory Ach-gated cation ion channels, but also inhibitory Ach-gated anion ion channels". For example, is there evidence that the widely studied invertebrate Drosophila has inhibitory nAChRs? In particular, Wormbase lists the closest fly homolog of ACC-4 as the GABA receptor Rdl. If evidence for the existence of Ach-gated anion ion channels is limited to C. elegans, then perhaps the wording should be changed to say "the C. elegans genome encodes" to be consistent with the available facts. I note that the two cited references do not contain information to support the broader statement made, as they describe work with C. elegans only.Drosophila does have anion-gated LGICs, it’s not clear whether these are ACh channels; in fact, the specific clade of ACC genes in worms is NOT conserved in Drosophila. So, while we can’t rule out that they exist in flies, there is currently no evidence for this. We followed the reviewer’s suggestion and changed the sentence to "the C. elegans genome encodes…"We apologize for this misstatement. While 2) I think the statement in paragraph seven subsection “unc-3 is a circuit-associated transcription factor” that unc-3 activity is required to "specify" synaptic connectivity is too strong and misleading. The data just show that unc-3 activity is required for normal synaptic connectivity to occur. That means something very different, at least to me, than "specify". Likewise the statement that unc-3 "defines the function and assembly of an entire circuit" goes way beyond what can be concluded from the results presented. The circuit is indeed abnormal in unc-3 mutants, but to use the word "define" here would require, in my opinion, evidence that in unc-3 mutants the circuit became a different type of functional circuit, not that it is just abnormal. I think one can say that unc-3 is "required", but "define" implies a specific role for unc-3 that has not been demonstrated. Similarly "circuit-associated" is appropriate, but "circuit-defining" is not.Agree with all, fixed as requested (i.e. eliminated “specify” and “defining”).3) This present study sets a foundation for investigating the role of ACC-4 in cholinergic signaling. ACC-4 however does not elicit strong Ach-gated chloride currents when expressed alone , does this suggest that it is an auxiliary subunit in a heteropentameric receptor? Therefore, does acc-4 co-express with at least one other Ach-gated chloride channel in each cell?C. elegans lab has indeed found such a subunit (an lgc gene). Since this is unpublished data, we have not mentioned this in the manuscript.Yes, that’s correct. Unpublished worked from another


RNA-determined RNPs) are abundant and offer an alternative explanation for the surprising lack of classical RBDs in many RNA-interacting proteins. Moreover, RNAs can act as scaffolds to orchestrate and organise protein networks and directly control their activity, suggesting that nucleic acids might play an important regulatory role in many cellular processes, including metabolism.Post-transcriptional regulation of gene expression plays a critical role in almost all cellular processes. Regulation occurs mostly by RNA-binding proteins (RBPs) that recognise RNA elements and form ribonucleoproteins (RNPs) to control RNA metabolism from synthesis to decay. Recently, the repertoire of RBPs was significantly expanded owing to methodological advances such as RNA interactome capture. The newly identified RNA binders are involved in diverse biological processes and belong to a broad spectrum of protein families, many of them exhibiting enzymatic activities. This suggests the existence of an extensive crosstalk between RNA biology and other, in principle unrelated, cell functions such as intermediary metabolism. Unexpectedly, hundreds of new RBPs do not contain identifiable RNA-binding domains (RBDs), raising the question of how they interact with RNA. Despite the many functions that have been attributed to RNA, our understanding of RNPs is still mostly governed by a rather protein-centric view, leading to the idea that proteins have evolved to bind to and regulate RNA and not vice versa. However, RNPs formed by an RNA-driven interaction mechanism ( RNP code) that determines the fate of the RNA, shaping transcriptome and proteome. RNPs are not static, and their remodelling allows adjustments in gene expression under conditions that require adaptive changes. Dysfunction of RBPs is often linked to disease [RNA assembles with proteins forming dynamic complexes named ribonucleoproteins (RNPs). The sequence of the transcript, its processing and the activity of the available RNA-binding proteins (RBPs) determine the composition of the RNP , 82, est disease , 75, 88,Given the complexity of RNA metabolism, it was estimated that RBPs comprise 3–11 % of the proteome of bacteria, archaea and eukaryotes and they are significantly more conserved across evolution than proteins lacking RNA-binding activity . Most ofA number of studies in the last two decades reported RNA-binding activities in proteins lacking classical RBDs, suggesting that the scope of RBPs was initially underestimated. These newly discovered RNA binders contain protein domains with dual function (e.g. enzymatic and RNA-binding activities) , folds oSaccharomyces cerevisiae. A significant fraction of the yeast proteome (∼4000 proteins) was purified and immobilised on a membrane. The resulting array was subsequently incubated with a mixture of fluorophore-labelled RNAs, and fluorescence retained at each protein spot was used as readout for RNA binding. Using this method, these studies identified 180 [In silico approaches were successfully used to identify proteins harbouring classical RBDs and highly homologous protein domains that may likely bind RNA as well , 40, 65.fied 180  and 42 [fied 180  proteinsMore recently, two independent works described a new method for the unbiased and comprehensive identification of RBPs from living cells, which is referred to as RNA interactome capture , 18. In S. cerevisiae. Surprisingly, many enzymes from classical biochemical pathways, particularly from glycolysis, were found to moonlight as RNA-binding proteins in this unicellular organism. And finally, the interactome of an entire multicellular organism was derived from the worm Caenorhabditis elegans, identifying 594 poly(A) RNA-interacting proteins [Subsequently, this methodology was extended to determine RNA interactomes in different cell lines and species. Kwon et al. added 283 novel RNA-binding proteins from mouse embryonic stem cells to the growing list of RBPs. Sixty-eight of these proteins are highly expressed in undifferentiated cells, suggesting a role in stem cell physiology . Three iproteins .Drosophila melanogaster, Danio rerio, Arabidopsis thaliana) will become available soon, enabling further inter-species comparisons and even deeper insights into RNA biology.Comparison of the datasets revealed a conserved core of eukaryotic RNA-binding proteins, comprising ∼250 RBPs , most ofWith the world of RBPs rapidly expanding and with more and more RBDs being identified, the need arises for a rapid identification of their binding motifs and target RNAs to gain insight into RBP function.Numerous experimental methods have been developed to study protein-RNA interactions. These ribonomic analyses range from the determination of in vitro binding specificities of recombinant RNA-binding proteins (or domains thereof) to the purification and subsequent analysis of native RNPs. Employing a panel of recombinant and purified RBPs in single and competitive binding reactions against a complex pool of RNA ligands in vast excess (RNAcompete) has recently yielded the binding specificities of 207 RBPs from various species. Not surprisingly, it also revealed that two proteins that share a high degree of identity between their RBDs are likely to have similar or even identical RNA sequence specificity . Anotherglobal or transcriptome-wide binding profiles. However, in most cases, studies are limited to individual RNA-binding proteins (or their respective RBDs). Expanding the analyses to other RBPs has been hampered either by the complexity of the experimental procedures, the availability of antibodies or the requirement for recombinant protein production.Common to the aforementioned methods is that they do not only test for binary interactions of one protein (or RBD) with a single RNA species (or short oligonucleotide) but rather produce Structural analyses of protein-RNA complexes have resulted in the identification of interaction surfaces and amino acids of proteins that are directly involved in RNA binding. This has significantly advanced our understanding of the principles that underlie binding specificity and RNP formation , 17. MorBeing able to predict the target RNA motif(s) based on the amino acid sequence of an RBD would greatly advance our understanding of RBPs and facilitate prediction of their cellular function(s), even if the proteins have not yet been extensively studied. However, the molecular basis of sequence-specific recognition of nucleic acids is well understood for only a subset of RNA-binding domains.Pumilio and FBF homology (PUF) proteins employ a repetitive and modular scaffold for sequence-specific binding to RNA, each repeat recognising one nucleobase . BreakinS. cerevisiae that, together with its partner She3p, associates with different cis-acting RNA elements  to mediate subcellular transport and localization of mRNAs [Moreover, RBPs do not necessarily only bind in a sequence-specific manner, recognising a stretch of specific nucleobases. The exon junction complex 4AIII, Y14, Magoh and MLN51) has no apparent sequence specificity and is deposited ∼20 nucleotides upstream of exon-exon junctions by the splicing machinery. Its binding to the RNA is remarkably stable, allowing the EJC to stay associated with the RNA during nuclear export and requiring a dedicated disassembly factor in the cytoplasm , 64. Othof mRNAs .The complexity of protein-RNA interactions is probably best illustrated by the RNA recognition motif. Although being the most abundant and best-studied RBD, the RRM is also one of the most versatile. Despite a shared architecture, RRMs utilise different surfaces for RNA binding and display various different modes of interaction, thus making target prediction extremely difficult , 30. MorDrosophila RNA-binding protein Sex lethal (Sxl) which harbours two RRMs that bind U-rich sequences with high affinity. On the one hand, Sxl can compete with and evict other RNA-binding proteins or complexes such as U2AF, CstF64 and others [Taken together, this further complicates prediction of binding motifs and also highlights that in the cellular context, interactions between RBPs and their targets occur in a complex environment with many different binding partners available. Moreover, association with one ligand often impacts on interactions with other factors. Both mutually exclusive binding and highly synergistic binding have been reported for a number of RBPs. One example is the d others , 37, 44,d others , 44, 48.Synergistic RNA binding of two proteins does not always necessitate a direct interaction between the two RBPs. Neither do antagonising factors always have to compete for binding to the same RNA element to exhibit a mutually exclusive binding behaviour. The RNA itself can relay the information of a binding event to a distal site through structural rearrangements. This can either improve accessibility or impede interaction with other factors. Exemplarily, the 3′ untranslated region (UTR) of vascular endothelial growth factor (VEGF) mRNA shows mutually exclusive and stimulus-dependent binding of two protein complexes, the HILDA (hypoxia-inducible hnRNP L-DRBP76-hnRNP A2/B1) complex and the interferon gamma (IFN-γ)-activated inhibitor of translation complex (GAIT). Binding of the HILDA complex results in a conformational change of the RNA that occludes the binding site of the GAIT complex. This creates a binary, molecular switch to adjust gene expression in response different stimuli , 124.Despite the complexity of the interactions and experimental challenges to characterise RNPs, detailed insights into binding specificities and target RNAs have been obtained for numerous proteins . This habona fide RBD. Several NHL domain-containing proteins were found to co-purify with RNA [In light of this, RNA interactome capture could well prove a treasure trove for RNA biologists. Experimentation can now be extended to proteins that previously had not been implicated in RNA biology but were identified as novel RNA binders by interactome capture, resulting in the discovery of novel RBDs. Exemplarily, interactome capture has contributed to the identification of the NHL domain  as a with RNA , 18, 63.with RNA –71.Furthermore, identification of the RNA targets of newly discovered RBPs will provide insights into their cellular function and further advance our understanding of RNA metabolism. This is exemplified by the fas-activated serine/threonine kinase (FASTK) family. In humans, six FASTK protein family members are present and all of them co-purify with RNA in interactome profiling . FASTK pIn light of this, the newly identified RBPs and the growing number of novel RBDs that have been functionally validated by independent laboratories , 66, 88 Besides the abundance of proteins without a recognisable, classical RBD, large-scale datasets of RNA binders harbour another surprising finding: the identified proteins are enriched in low-complexity regions mainly composed of the amino acids serine (S), proline (P), glycine (G), arginine (R), lysine (K) and tyrosine (Y) . These atails rich in basic amino acids have been reported to play a role in protein-DNA interaction. By projecting into the minor grove of the DNA double strand, they increase local affinity and promote hopping and linear diffusion along the DNA molecule [Interestingly, also ribosomal proteins often contain long extensions enriched in the amino acids G, R and K Fig. . While bmolecule , 113. Anmolecule , was demmolecule . In the molecule .Fig. 2PrIn addition to directly interacting with nucleic acids, IDRs in RNA-binding proteins have been shown to contribute to assembly and formation of RBP ultrastructures such as P bodies, stress granules or nuage . They caComparison of RNA interactome datasets from different organisms has revealed an expansion in the number of short linear motifs in RBPs in higher eukaryotes compared to unicellular yeast, while the number of classical RBDs remains similar . In sum,C. elegans [Surprisingly, interactome capture has also identified a number of proteins as RNA binders which do neither contain an identifiable globular RBD nor a predicted IDR. In human cell lines, more than half of the identified RBPs have no detectable RBD and have not been associated with functions in RNA metabolism before Fig.  8, 13, , 13, 18. elegans , and an  elegans , 81. ThiAlike to proteins, also RNA can fold into intricate three-dimensional structures that serve various functions including catalytic activity, a classical example being the ribosome—a macromolecular and complex RNA machinery. Moreover, it has been demonstrated by systematic evolution of ligands by exponential enrichment (SELEX) that RNA sequences can be derived  to selectively bind various different classes of small molecules . ExampleInsofar, it is surprising that our understanding of RNPs is still mostly governed by a rather protein-centric view and the idea that proteins evolve to bind RNA and not vice versa. Often, it is difficult to differentiate between these two possibilities and there is clear evidence for co-evolution of proteins and RNA that interact to form an RNP. In fact, a continuous and densely populated spectrum exists, with one extreme being protein-determined RNPs (represented by many mRNPs) and the other one being RNA-determined RNPs like ribosomes - the latcanonical RBDs.It is tempting to speculate that in RNA interactome datasets, the abundance of proteins that lack identifiable RBDs might, to some extent, reflect the capture of RNA-determined RNPs. In this case, RNAs have evolved to interact with proteins which hence do not require normal, cap-driven translation [An example of RNA-determined RNPs can be observed in a number of viral RNAs. Complex RNA secondary structures which promote viral translation can be found in the 5′ untranslated region of many different viruses, among them are picornaviruses, hepatitis C virus, herpes simplex virus and others. These so-called internal ribosome entry sites (IRESs) allow non-canonical and cap-independent translation initiation by functionally replacing some  eukaryotic translation initiation factors (eIFs), which are otherwise needed for nslation . Cap-depnslation , 94, andnslation , 109. Thnslation , and strnslation . Moreovenslation . Even thnslation . In sum,nslation , 121.What appears to be a common theme to aptamers and IRES elements is that ligand/protein binding requires (extensive) RNA structure elements. While in rare cases promoting internal translation initiation, extensive RNA structures in 5′ UTRs or even coding regions of mRNAs are otherwise inhibitory to translation. Hence, mRNAs whose major role is to serve as templates for protein synthesis are less likely to contain such intricate structural elements that function in protein binding. However, for non-coding RNAs, these constraints do not apply and one might envision that RNA aptamers that bind proteins might be more common in this class of RNAs. In fact, many of the well-studied non-coding RNAs form extensive secondary structures to associate with proteins. This includes several classes of abundant RNAs such as small nuclear RNAs (snRNAs) involved in splicing , small nThe function of RNA extends well beyond the mere coding for peptides. Exemplarily, RNA is not only the major structural component of ribosomes but also contributes catalytic activity and delivers amino acids for protein synthesis. Moreover, RNAs play integral roles in RNA processing and modification, are involved in sensing of metabolites and act as scaffolds to organise larger complexes. Several classes of small non-coding RNAs provide specificity for proteins, acting as guides. In eukaryotes, microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-associated RNAs (piRNAs) assemble with proteins of the argonaute clade and recruit them to their nucleic acid targets by  sequence complementarity, eliciting gene regulatory pathways . SimilarIn telomerase, the RNA moiety does not only serve as a template for synthesis of telomeric repeats by the telomerase reverse transcriptase (TERT) but also provides a flexible scaffold to assemble the entire RNP, providing binding platforms for the Ku heterodimer, the LSm heteroheptameric protein complex and TERT .Recently, long non-coding RNAs (lncRNAs), a heterogeneous and functionally diverse group of transcripts in eukaryotes, have gained widespread attention. They are polyadenylated, often contain extensive secondary structure and elicit different functions, serving e.g. as scaffolds, decoys and/or guides , 115. ExThe structural role of RNA in the organisation of RNPs, where it e.g. serves as a (flexible) scaffold, is well documented, and its function as guide for proteins, directing them to their sites/sequences of action, is also well established. But can RNAs also regulate the activity of their bound protein partners?Some of the best-documented examples, highlighting the function of RNAs as regulators of protein activity, are found in innate immunity. Toll-like receptors involved in the pattern recognition of pathogens are activated by double-stranded RNA (TLR-3), single-stranded RNA TLR-7/8) and bacterial ribosomal RNA (TLR-13) , 91, 125/8 and baEscherichia coli and Bacillus subtilis. It adopts a rod-shaped secondary structure with a flexible central region, thereby mimicking an open promoter to bind and inhibit the bacterial RNA polymerase [But RNA regulation of protein activity is neither restricted to innate immunity nor eukaryotes. Bacterial 6S RNA is a ∼200-nt-long, non-coding RNA which inhibits transcription of housekeeping genes in lymerase . 6S RNA lymerase . While tlymerase . Other Rlymerase .In light of RNA being a regulator of protein function, it is intriguing that many of the proteins that are found to associate with RNA are reported to exhibit enzymatic activities. Dozens of enzymes from intermediary metabolism associate with RNA, including almost all glycolytic enzymes , 85, 86.Taken together, RNA is more than a mere bystander molecule that is being regulated by proteins. Various RNAs can play active roles in the organisation of ribonucleoproteins, sometimes even shaping entire subcellular compartments. Moreover, RNAs can control the function and activity of their bound protein partners, adjusting cell physiology to changing cellular requirements. In sum, this has fuelled the hypothesis that RNA may orchestrate enzymatic activities by bringing together all the enzymes that are part of a given metabolic pathway into large protein assemblies, forming metabolons with superior metabolic performance .unconventional RNA-binding proteins that were recently discovered might be a hint that many more examples of functionally active RNAs are waiting to be discovered. Characterisation and functional studies of RNPs that contain novel RNA-binding proteins with enzymatic activities will pave the way for a better understanding of how RNA biology integrates with other, in principle unrelated, cell functions such as intermediary metabolism.Scaffolding, protein binding and regulation of enzymatic activity have, however, only been described for a limited set of RNAs. The large number of


Picornavirales, assembled from metagenomic sequencing of organisms in San Francisco wastewater. This virus likely constitutes a novel genus within the picalivirus family.We report here the draft genome sequence of picalivirus D, a member of picalivirus, a picorna-like superfamily, and likely member of the order  Picornavirales and Caliciviridae, picaliviruses increasingly appear to be members of the Picornavirales order.The picorna-like superfamily is a rapidly expanding taxonomic unit of positive-stranded RNA viruses with conserved RNA-dependent RNA polymerase (RdRp), capsid, and helicase proteins that have a broad host range, including animals, plants, and insects –3. Pical–Picornavirales order, such as Taura syndrome virus, chicken picornavirus 1, Aurantiochytrium single-stranded RNA virus 01, and cricket paralysis virus.While performing weekly metagenomic sequencing of organisms in San Francisco wastewater, we assembled a 9,570-nucleotide contig, which we are naming picalivirus D. This contig aligned by BLASTx to all three members of picalivirus. Picalivirus D consisted of two open reading frames (ORFs) of 7,023 and 2,238 nucleotides containing the nonstructural and structural proteins, respectively. This genome is 604 nucleotides longer than the complete genome of picalivirus A, which was attained by 5′ and 3′ rapid amplification of cDNA ends (RACE) (The viral genome was discovered and assembled using PRICE version 1.0, Geneious version 8.0 Assembler, and SURPI version 1.0 from a total of 15,719,690 paired-end 65-bp reads sequenced on an Illumina GAIIx split between these DNAsed and untreated nucleic acid preparations (The original sample was taken from wastewater on 25 January 2010, after a large rainstorm that left >5 inches of rain over the preceding week. Sample processing was performed on 1 liter of wastewater that was concentrated to <5 ml with particles between the sizes of 0.22 μm and 300 kDa using Millipore Pellicon XL 300-kDa filters and 0.22-μm spin columns. The 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. Other RNA viruses discovered in this sample include ciliovirus, brinovirus, laverivirus, tombunodavirus, and several other marine RNA viruses and phages –9.–KF478837.The GenBank accession number for picalivirus D is


Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs). The streptavidin-binding aptamer S1 sequence was inserted into the 3′ end of dengue virus (DENV) 5′–3′ UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. Several novel host DENV RBPs were subsequently identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which we further implicate in DENV replication. We proposed efficient S1 aptamer-based isolation of viral assembled RNPs from living mammalian cells will be generally applicable to the purification of high- and low-affinity RBPs and RNPs under endogenous conditions. It was well known that host RNA-binding proteins (RBPs) are involved in the viral replication for plus-strand (+) RNA viruses. A limited number of host RBPs affect viral RNA recruitment for replication, help assemble the replicase complexes, control viral RNA synthesis, or affect viral RNA stability . InteresIdentifying RBPs or RNPs could be facilitated by a highly specific RNA affinity tag that allows selective recovery of all proteins associated with the tagged RNA under nondenaturing conditions. However relatively few RNA affinity tags have been identified. Examples include the specific RNA-hairpin structure that binds to bacteriophage MS2 coat protein, nascent “boxB” hairpins from bacteriophages λ and P22, “StreptoTag” which binds to streptomycin ,5, J6f1 in vitro for isolation of RBPs. The “Glutathione RNA Affinity Chromatography” approach is a typical example to identify proteins specifically associated with target RNA [S-transferase (GST). By binding the GST-λ peptide fusion protein to the hybrid RNA including boxB hairpin RNA and then incubating with cell extracts, RNA-protein complexes were assembled on boxB-containing RNAs. Recently, Ward et al. [in vitro-assembled RNA-protein complexes may not represent authentic RNPs which existed under physiological conditions.Based on well-developed RNA affinity tags, many studies utilized cell extracts and synthetic target RNA to assemble the RNA-protein complexes rget RNA ,9. Generd et al.  reportedin vitro approach, several new methods reportedly have been used to identify authentic RNA-interacting proteins from living cells [et al. [et al. [et al. [To avoid potential biochemical artifacts and circumvent limitations of the ng cells ,15,16,17 [et al.  develope [et al.  created et al. [et al. [Formaldehyde and 254 nm UV light cross-linking are commonly used to identify RNA-protein interactions in previously reported strategies . To deteet al.  “froze”  [et al.  also app. This allowed endogenous viral RNP assembly and isolation of RNPs from whole cell extract. By using this method, we identified several novel host proteins binding to DENV RNA. This method, coupled with LC-MS/MS, will allow quick and easy purification of high- and low-affinity RBPs and RNPs under endogenous conditions.In this study, we present a new affinity purification variation based on using the streptavidin-binding RNA aptamer S1 as a tag to isolate DENV-2 (strain Tr1751) RNA-binding proteins in living mammalian cells as demonstration. The S1 RNA aptamer sequence was cloned into the 3′ end of DENV RNA UTR in a plasmid, and after transfection the hybrid RNA was transcribed in living cellsAn overview of the experimental steps of the RNA pull-down assay devised to isolate endogenously-assembled viral RNP complexes from cells is given in , the viral RNA genes were cloned into pHH21 plasmids and fused with S1 RNA affinity tags  in To express the DENV 5′–3′ UTR RNA in 293T cellsity tags B. The moity tags C. To conity tags  and the ity tags D. And acTo investigate whether hybrid RNA with DENV 5′–3′ U and S1 RNA aptamer, assembled in living mammalian cells, was able to bind to streptavidin C1 magnetic beads, we tested the amount of 5′–3′ U RNA after affinity purification with streptavidin . 5 and 58 cells) with a vector control. Five dishes of cells were transfected with either pHH21-5′–3′ U-S1 or pHH21 for 6 h, and incubated with 4-SU containing medium for additional 18 h. After, we cross-linked viral RNA-protein complexes with 365 nm UV light irradiation. After the cells were lysed, the viral RNA-protein complexes were purified from the lysates by binding to streptavidin magnetic beads. The wash conditions of affinity purification were explored by changing the NaCl concentrations in wash buffer  recognized a protein of 24 kDa in whole cell extracts and in the viral RNP (vRNP) sample, but little in the mock sample purified in parallel A. To detIn this study, we devised a novel strategy to isolate endogenously-assembled viral RNA binding proteins (vRNPs) from crude mammalian cell extract by using S1 aptamer-based affinity purification. We expressed S1 aptamer-tagged DENV 5′–3′ UTR RNA in living mammalian cells, allowing endogenous assembly of the tagged RNA into vRNPs, and we purified tagged vRNPs from whole cell extract on Streptavidin magnetic beads. Proteins that were specifically associated with 5′–3′ U-S1 RNA were identified by mass spectrometry, and several newly identified protein partners potentially interact with DENV 5′–3′ UTR RNA. Further study showed that RPS8 interacted with DENV RNA in virus infected cells, and positively participated in DENV replication.in vitro-based methods. Here the utility of S1 aptamer as an RNA tag is demonstrated by isolating proteins that associate with DENV 5′–3′ UTR RNA. Firstly, the S1 aptamer is a short RNA sequence of 60 nt [The expression of the aptamer-tagged RNAs in living mammalian cells may offer several advantages compared to solely of 60 nt , which dof 60 nt ; Secondlof 60 nt . Using tof 60 nt .et al. [in vivo-assembled RNA-protein complexes. Novel RNA-binding proteins associated with a specific target RNA were successfully identified through these methods. However, the limit of these methods is that co-expressing a capture protein such as MS2 and stem-loop tagged target RNA is needed, thus MS2 might affect the RBPs that bind to the RNA sequence besides the stem-loop because the bacteriophage protein MS2 binds tightly to the stem-loop tagged RNA.Recently, the most commonly used strategy in living cells is to insert one or more copies of an RNA-encoded aptamer, which is recognized by a small molecule  or a protein  which is immobilized on chromatography matrix ,32,33. Tet al.  integratet al. ,36. Co-eIn this experiment, we developed an efficient and generalizable method to isolate viral RNA-associated RNPs or RBPs in mammalian cells and overcome current technical limitations for addressing RNA-protein interactions . FurtherThis strategy based on S1 aptamer and PAR-CL to isolate viral RBPs in living mammalian cells was reported for the first time and a scalable method in all steps was described, which can be performed in a single-run MS analysis with high-throughput studies in principle. With the improvement in sample preparation, purification process and data analysis, the high-resolution, quantitative MS may result in increasingly streamlined analysis of crude RNA-protein interactions in a single-step affinity purification at near-physiological conditions. Thus, this strategy has more potential for broad application in studying endogenously assembled RNA-protein complexes. This approach could be used to capture and identify the novel cellular factors associated with various viral RNAs.BsmBI sites, digested with BsmBI, and cloned into the pHH21 vector (kindly gift of Gabriele Neumann) containing the human RNA polymerase I promoter and mouse RNA polymerase I terminator  according to the manufacturer’s protocol. Six hours post-transfection, cells were maintained in 10% DMEM containing 100 µM/L 4-SU for additional 18 h. The photoactivatable nucleotide 4-thiouridine (4-SU) is taken up by cultured cells and then partially incorporated into nascent viral RNAs, and 365 nm UV light irradiation was applied to induce efficient crosslinking. Twenty-four hours post-transfection, cells were irradiated with UV light at 365 nm for 2 min, harvested, and lysed in 3 mL RIPA buffer under RNase-free and protease-free conditions. Total cell lysates were centrifuged for 10 min at 16,000× g, 4 °C. Supernatants were transferred to new tubes and protein was quantified by measuring absorbance at 280 nm.A549, 293T and BHK-21 cells were maintained in DMEM  supplemented with 10% FBS (HyClone), 10 U/mL penicillin, and 10 μg/mL streptomycin  at 37 °C in a 5% CODENV-2 (strain Tr1751) was amplified in C6/36 cells as previously described . For DENThe DENV RNA isolated in the RNA immunoprecipitation assay was detected by RT-PCR according to the protocol. Briefly, 1 μg of RNA was first treated with DNase, and then converted to cDNA using PrimeScript RT Master Mix . The PCR reaction was performed with primers designed against the DENV 5′-UTR-F: 5′-AGTTGTTAGTCTACGTGGAC-3′ and 3′-UTR-R: 5′-AGAACCTGTTGATTCAACAGC-3′. The transcriptional level of pHH21-5′–3′ U-S1 in cells and the binding ability of 5′–3′ U-S1 RNA toward beads were analyzed by RT-qPCR with primers of the DENV 5′–3′ UTR segment , which contain both the segment of 5′ and 3′-UTRs. The DENV RNA level in infected 293T cells was also analyzed by RT-qPCR . The housekeeping gene β-actin  was used as an internal RT-qPCR control. Total RNA was isolated using RNAiso (TaKaRa) and synthesized into cDNA with random hexamers by using PrimeScript™ RT Master Mix  and was then amplified with above primers and SYBR Premix Ex Taq™ II (TaKaRa) for 40 cycles. Data were analyzed using Mx3005P System Software . Relative gene expression refers to the magnitude of the signal generated for the 5′–3′ UTR segment or 5′ UTR of DENV using the β-actin gene as the internal control. The data represent the mean of three independent experiments.® MyOne™ Streptavidin C1 magnetic beads  as follows. In brief, one dish of cell lysate was incubated with 20 μL paramagnetic streptavidin beads (pre-treated according to manufacturer’s protocol) containing 100 μg yeast tRNA (Invitrogen) on a rotation wheel at 4 °C for 60 min. It was then washed four times with modified RIPA buffer  at 4 °C. After washing, RNA conjoined to beads was isolated using RNAiso (TaKaRa) for subsequent RT-qPCR analysis, or RNA-protein complexes were dissociated from beads by boiling for 5 min in 0.1% SDS and then treated with bovine RNase A (TaKaRa) [Twenty-four hours post-transfection, five dishes of cells were irradiated with UV light at 365 nm for 2 min, harvested, and lysed in 3 mL lysis buffer. DENV 5′–3′ U-S1 RNAs and crosslinked proteins were captured with Dynabeads(TaKaRa)  for subsThe samples were analyzed by SDS-PAGE and transferred onto a nitrocellulose membrane. Membranes were incubated with blocking buffer (TBS containing 5% non-fat milk) for 1 h at room temperature and incubated with the primary antibody, a rabbit polyclonal anti-RPS8  diluted 1:500 in blocking buffer overnight at 4 °C. The secondary antibody, IRDye 680RD Goat Anti-Rabbit Secondary Antibody  diluted 1:8000, was incubated with the membranes at room temperature for 1 h. Protein detection was made using the infrared imaging system Odyssey (LI-COR Biosciences).Protein gel bands were excised and in-gel-digested with trypsin, and the tryptic peptides were subjected to LC-MS/MS on an Orbitrap XL instrument  under standard conditions. All sequence and peptide fingerprint data was searched against the Swiss-Prot and NCBI non-redundant databases using MASCOTsoftware .A549 cells were seeded onto eight-well coverslips  and infected with DENV-2 for 24 h. Next the cells were fixed and permeabilized and then incubated with blocking buffer ) for 1 h. Primary antibodies were diluted in blocking buffer  and incubated with cells at 4 °C overnight. Cells were then washed and incubated for 1 h with fluorescently labelled Alexa Fluor 488 goat anti-Rabbit and Alexa Fluor 594 goat anti-mouse antibodies (Invitrogen). Coverslips were mounted in Prolong Gold antifade reagent containing DAPI (Invitrogen) and the cells were visualized under a laser confocal microscope LSM 510 . The images were captured and analyzed by using ZEN software (Zeiss).The interaction of RPS8 with DENV viral RNA during infection was studied by using an RNA immunoprecipitation assay. In brief, the infected cells were harvested and cross-linked with 365 nm UV light. Cells were then disrupted in RIP assay buffer , and the resulting lysates were pre-cleared with protein A-agarose . Immunoprecipitation was carried out using anti-RPS8 polyclonal antibody or normal rabbit IgG (as a negative control). Immune complexes were precipitated with protein A-agarose. Immunoprecipitated viral RNA was isolated using an RNeasy mini kit . Reverse transcript PCR (RT-PCR) was conducted using primers of the DENV 5′-UTR.293T cells, transfected with pOTB7-RPS8 or vector, were infected with DENV for 36 h and culture supernatant was collected. Then, virus yield in the culture supernatant was determined with BHK-21 cells. Briefly, BHK-21 cells were seeded in 6-well plates with 90% confluence. Serial dilutions of virus containing supernatants were added to the cells in a final volume of 1 mL/well. After 2 h of incubation at 37 °C and three time washes, DMEM containing 2% FBS and 1% low-melting-point agarose was added and plates incubated at 37 °C for five days. Plaques were counted after removal of the agarose plug and staining with crystal violet.


Arabidopsis thaliana yields high amounts of good quality RNA in a simple, safe and inexpensive manner.Standard molecular biological methods involve the analysis of gene expression in living organisms under diverse environmental and developmental conditions. One of the most direct approaches to quantify gene expression is the isolation of RNA. Most techniques used to quantify gene expression require the isolation of RNA, usually from a large number of samples. While most published protocols, including those for commercial reagents, are either labour intensive, use hazardous chemicals and/or are costly, a previously published protocol for RNA isolation in We have tested this protocol in tomato and wheat leaves, as well as in Arabidopsis leaves, and compared the resulting RNA to that obtained using a commercial phenol-based reagent. Our results demonstrate that this protocol is applicable to other plant species, including monocots, and offers yield and purity at least comparable to those provided by commercial phenol-based reagents.Here, we show that this previously published RNA isolation protocol can be easily extended to other plant species without further modification. Due to its simplicity and the use of inexpensive reagents, this protocol is accessible and affordable and can be easily implemented to work on different plant species in laboratories worldwide.The online version of this article (doi:10.1186/s13104-015-1119-7) contains supplementary material, which is available to authorized users. The isolation of good quality RNA in sufficient amounts is often a prerequisite for the analysis of gene expression, an approach that is widely used in laboratories worldwide routinely. However, most published protocols are labour intensive and/or use hazardous chemicals, and commercially available reagents or kits usually use toxic chemicals and are costly -3. TheseOñate-Sánchez & Vicente-Carbajosa published an improved RNA isolation protocol for Arabidopsis that is simple, efficient, affordable, and avoids the use of toxic volatiles . This ar260/A230 and A260/A280) were obtained (Table We set out to determine (i) whether the RNA isolation protocol described by Oñate-Sánchez and Vicente-Carbajosa  see Add can be a260/A230 and A260/A280 ratios were above 2.0 for these samples, representing high purity, in all cases, the A260/A230 value in the samples obtained using TRI reagent was generally lower (ranging between 1.40 and 2.06), indicating polysaccharide or, most likely, polyphenol contamination. Of note, it is known that traces of phenol contaminants can strongly inhibit downstream steps in genomic approaches, therefore compromising the results. Additionally, high absorbance at A230 can lead to the overestimation of RNA concentration in the sample.As shown in Table 230.Samples obtained from all three species using either protocol yielded RNA which was good quality Figure . Of noteCitrus reticulata, Citrus limon, Solanum tuberosum, Solanum americanum, Amaranthus viridis and Malva parviflora . Moreover, this protocol yields RNA whose quality is at least comparable to that provided by commercially available phenol-based reagents  [Absorbance curves obtained for the samples shown in Figure a (2008) .