Interest in extracellular RNA (exRNA) has intensified as evidence accumulates that these molecules may be useful as indicators of a wide variety of biological conditions. To establish specific exRNA molecules as clinically relevant biomarkers, reproducible recovery from biological samples and reliable measurements of the isolated RNA are paramount. Toward these ends, careful and rigorous comparisons of technical procedures are needed at all steps from sample handling to RNA isolation to RNA measurement protocols. In the investigations described in this methods paper, RT-qPCR was used to examine the apparent recovery of specific endogenous miRNAs and a spiked-in synthetic RNA from blood plasma samples. RNA was isolated using several widely used RNA isolation kits, with or without the addition of glycogen as a carrier. Kits examined included total RNA isolation systems that have been commercially available for several years and commonly adapted for extraction of biofluid RNA, as well as more recently introduced biofluids-specific RNA methods. Our conclusions include the following: some RNA isolation methods appear to be superior to others for the recovery of RNA from biological fluids; addition of a carrier molecule seems to be beneficial for some but not all isolation methods; and quantitative recovery of RNA is observed from increasing volumes of cerebrospinal fluid. A leading goal of modern biomarker studies is to ascertain disease conditions and other biological states by examining molecules in easily obtained biological fluids. For example, the ability to use blood-based markers for diagnosis, location, and staging of cancer, or to predict outcome of several therapeutic options, would have several benefits, from early detection to obviating the need for invasive and expensive biopsies. While some biomarkers may simply be “bystanders” in the disease process, others might be actively involved and thus present targets for novel therapeutics . Animal studies were approved by the Johns Hopkins University Institutional Animal Care and Use Committee and conducted in accordance with the Weatherall Report, the Guide for the Care and Use of Laboratory Animals, and the USDA Animal Welfare Act. Macaques are pair- or group-housed when possible and receive environmental enrichment including manipulanda, foraging, and opportunity to exhibit species-specific behavior. Animals are continually monitored for signs of distress. Ketamine is administered for blood or CSF draws.De-identified plasma was from a healthy donor who provided informed consent in accordance with approval by the Johns Hopkins Institutional Review Board. CSF was from a healthy donor pigtailed macaque (miRbase.org) . A laminar air flow cabinet dedicated to RNA work was used for RNA isolation and reverse transcription preparations. Surfaces  were cleaned thoroughly with RNase-away prior to and after use. Pipettes and other portable objects to be used in the hood were exposed to UV light in a UV crosslinker for 2 min on each side. This was done to avoid introduction of contaminating nucleic acids and, potentially, proteins. We implemented double-gloving, regular glove changes, and wearing of Tyvek sleeves or clean lab coats. Certified RNase-free barrier pipette tips were used. Where possible, all reagents were certified RNase-free. Isolated RNA from all isolation methods was stored at −80°C.g for 5 min in a table-top centrifuge before use. This was done because some column-based clean-up methods introduced a fine white powder into the eluted sample, presumably from the filter materials. Although this material is likely inert, we pelleted it to avoid interference with downstream reactions. It may be preferable to pellet this material and transfer the clarified supernatant to a new RNase-free tube prior to initial freezing.All RT-qPCR reactions were performed with once-thawed RNA as substrate. RNA samples were thawed on ice, mixed gently, and centrifuged at 2500 × g spin for 15 min at room temperature followed by a 2500 × g spin for 15 min at room temperature to remove most remaining platelets. Plasma was obtained from blood collected into ACD anticoagulant. All methods comparison experiments were conducted with aliquots of plasma from a single blood draw from one donor.RNA isolations were performed from platelet-poor primate plasma that had been frozen (−80°C) and thawed once. Platelet-poor plasma was defined as plasma separated from blood cells less than 30 min following blood draw with a 1000 × For all isolations, 5 or 10 pg synthetic cel-miR-39 per isolation was spiked into the respective lysis/denaturant buffer before combining with plasma. We note that synthetic RNA cannot be added directly to plasma or other biological substances because endogenous RNases degrade it immediately  for a total of 18 isolations. Manufacturer’s protocols were followed for the Exiqon and Ambion/Life Technologies kits. For TRIzol LS/column clean-up, 100 μl plasma was diluted with 150 μl nuclease-free water, followed by addition of 750 μl TRIzol LS reagent. Samples were mixed and incubated at room temperature for 10 min. Two-hundred microliters chloroform was added with a glass pipette, followed by vortexing for 1 min and incubation at room temperature for 5 min. Phases were separated by centrifugation at 14,000 × Duplicate isolations from 100 μl plasma were done for each method. Glycogen was added for isolations using both methods; a no-glycogen condition was included only for the Biofluids Kit, for a total of six isolations. The manufacturer’s protocols were followed. miRNAs measured included endogenous miRs-16, -34a, and -126, and spiked-in cel-miR-39. miRNAs were measured by RT-qPCR in triplicate for each isolation. “Fold abundance” was calculated in comparison with the average of results for the two glycogen-added miRCURY Cell and Plant isolations.Triplicate isolations were done for each method from 200 μl plasma. Glycogen was added for all isolations. Manufacturers’ protocols were followed. Triplicate RT-qPCR reactions were performed with material from each isolation to measure endogenous miRs-16 and -150 and exogenous spike-in cel-miR-39. Fold abundance for the Qiagen isolations was calculated against the mean of results for the three Exiqon Biofluids isolations. Separately, for the Qiagen kit, different elution volumes (50 and 100 μl) were compared with the manufacturer-recommended 14 μl.RNA was isolated by the Exiqon Biofluids method with addition of glycogen and spike-in to all isolations. About 50, 100, and 200 μl CSF were used as input volume. Duplicate isolations were performed for each input volume. Higher-abundance miRs-16 and -223 and lower-abundance miR-21 and let-7c were measured in triplicate reactions.Reverse transcription and RT-qPCR steps were performed largely according to the manufacturer’s protocols, with any modifications as described previously. or automatically (CFX96) with checking and manual adjustment by the operator if needed. Mean Cq and standard deviation were calculated for each technical triplicate reaction for each miRNA. Mean Cq and standard deviation (for three isolations) or range (for two isolations) were determined for each type of isolation. Delta Cq values were calculated with reference to the chosen “control” condition for each isolation experiment, as specified above. From these values, fold abundance with respect to the control was calculated .p < 0.02) or by 1.6-fold versus mirVana . TRIzol isolation followed by column clean-up did not perform as well as the other methods in our hands , with large fold abundance differences averaging about six (without glycogen) or >10 (with glycogen). For qPCR Cq results by all methods, standard deviation of the technical triplicates was typically below 0.1 for high-abundance miRNAs. For the low-abundance miRNA miR-34a, high variability was observed.A new, biofluids-specific Exiqon kit became available in December, 2012. According to the manufacturer, this kit allowed better RNA recovery as well as PCR inhibitor removal in comparison with other methods.  The two protocols are similar in terms of time and operator skill requirements. We performed a second isolation experiment to compare the performance of this Biofluids kit (with and without glycogen) with the standard miRCURY kit (with glycogen). For this experiment, duplicate isolations were performed, followed by triplicate miRNA RT-qPCR reactions for each isolation replicate. Both without and with glycogen, the Biofluids kit displayed significantly greater recovery than standard miRCURY with glycogen , with the apparent intent of providing a more concentrated RNA sample.Triplicate isolations were performed followed by triplicate RT-qPCR measurements of endogenous miRNAs and spiked-in cel-miR-39. As with all other experiments performed for this report, we measured miRNAs in 2 μl of purified RNA eluted as per the manufacturers’ protocols. Both kits produced highly consistent results between isolation replicates. Results indicated similar apparent recovery by the two kits Figure , we perfMacaca nemestrina CSF from a single donor. The same amount of spike-in RNA was added to each isolation. In the case of plasma, endogenous miRNA had greater apparent recovery with increasing input volume, but the increase was not proportional from 100 to 200 μl input (Figure To investigate quantitative recovery of RNA using the Exiqon Biofluids kit, we isolated RNA from 50, 100, and 200 μl of plasma and from the same volumes of t Figure . Althougt Figure . InteresOur results indicate that the Exiqon miRCURY Biofluids Kit outperforms other RNA isolation methods we tested, at least for isolation of small RNAs from plasma. This finding, furthermore, should be viewed in light of the relative ease of execution of the Exiqon method when compared with traditional phase-separation-based methods. Ease of use and short processing time are certainly appropriate considerations when results are not compromised.Without glycogen, the standard Exiqon miRCURY slightly underperformed, and with glycogen, it slightly outperformed the Ambion mirVana kit. Both kits outperformed TRIzol LS with column clean-up in our hands. The new Exiqon miRCURY Biofluids kit appeared to be superior for small RNA isolation when compared with the standard miRCURY kit. With miRCURY Biofluids, all miRNAs were apparently recovered at higher quantities in the presence of glycogen, although the difference was not statistically significant at an alpha of 0.05. miRCURY Biofluids also provided better recovery than the Qiagen miRNeasy Serum/Plasma kit in our hands, considering the small elution volume from the Qiagen kit.Although we found that the Qiagen Serum/Plasma kit allowed recovery of most of the on-column RNA with only 14 μl of eluant, it is not clear to us that using smaller elution volumes generally achieves greater RNA concentration. For some protocols and columns, smaller volumes of eluant may simply leave RNA on the column rather than providing greater concentration. An analogy that has been used previously (Eldh et al., Cell article in 2012 after observing that standard TRIzol extraction resulted in apparently poor recovery of low-GC-content miRNAs from low-abundance samples. However, clarity on this point has yet to be achieved.A conclusion from our experiments that we found particularly surprising is that TRIzol LS isolation followed by column clean-up did not perform well in our hands. We are uncertain as to why this method performed so poorly in our case or why the addition of glycogen appeared further to decrease yield, since this method or variations on it have been used with excellent results by others. The variability and low yield we report here is likely due to a separate issue from that reported by Kim et al. , who retWe submit that our results prompt at least three additional recommendations. First, based on our input volume experiments, it would appear that using 200 μl of plasma as input does not necessarily result in quantitatively greater recovery. This echoes previous findings (Kim et al., This study has several limitations, which may be helpful to review as they indicate opportunities for advances in the field. We measured a limited number of endogenous miRNAs in this study. It is possible that recovery may differ from one method to another on a miRNA-by-miRNA basis, similar to the differences reported by Kim et al. , in theiThis research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Qiagen provided a sample of the miRNeasy Serum/Plasma Kit at no cost, as noted in the Acknowledgments, but did not influence the execution or reporting of this study in any way.


Using a combination of biochemical and biophysical techniques we have developed 2-dimensional and in some cases 3-dimensional structures for a number of lentivirus packaging signal regions including HIV-1 and FIV. The general features of these regions are of an unusually high level of sequence conservation reflecting the requirement for certain structures to be formed to allow transportation and processing of the viral RNA. There is in addition, data from our work and from that of others that the RNA structure is not fixed and that it changes on interaction with various ligands, including the viral proteins involved in genome RNA encapsidation. The model we are building up is one, in which,


IRES-dependent initiation appears to use different strategies to recruit the translation machinery depending on the RNA organization of the region and the network of RBPs interacting with the element. In this review we discuss recent advances in understanding the implications of RBPs on IRES-dependent translation initiation.RNA-binding proteins (RBPs) are pivotal regulators of all the steps of gene expression. RBPs govern gene regulation at the post-transcriptional level by virtue of their capacity to assemble ribonucleoprotein complexes on certain RNA structural elements, both in normal cells and in response to various environmental stresses. A rapid cellular response to stress conditions is triggered at the step of translation initiation. Two basic mechanisms govern translation initiation in eukaryotic mRNAs, the cap-dependent initiation mechanism that operates in most mRNAs, and the internal ribosome entry site (IRES)-dependent mechanism activated under conditions that compromise the general translation pathway. IRES elements are  Within the cell RNA molecules are associated to RNA-binding proteins (RBPs) forming dynamic ribonucleoprotein particles (RNPs) that affect all steps of RNA metabolism . RBPs ascis-acting regulatory functions in most cases and provide the landing site of many RBPs . O–6. Ocis-. O–6. OcIn response to distinct stresses, cells trigger a differential response that can displace the equilibrium towards cell survival or cell death. Key factors mediating this response are post-translational modification, relocalization, proteolysis or degradation of RBPs. A paradigmatic example of this response is observed in virus-infected cells . Viral eN-ras (Unr), or the Zinc finger protein 9 (ZNF9). Among well characterized RBPs are hnRNPs, a large family of nuclear proteins (hnRNP A1 to hnRNP U) with RNA-binding domains and protein-protein binding motifs [Traditional RBPs consist of modular RNA-binding domains (RBDs), typically RNA recognition motifs (RRM), heterogeneous nuclear ribonucleoprotein K-homology domains (KH), cold-shock domains (CSD), zinc finger domains (ZNF), double-stranded RNA-binding domains (dsRBD), Piwi/Argonaute/Zwile (PAZ) domain, RGG (Arg-Gly-Gly) box, DEAD/DEAH box, Sm domains and Pumilio/FBF (PUF or Pum-HD) domains . Represeg motifs  that shug motifs . HnRNP Kg motifs . Unr is g motifs . Zinc fiGiven the many layers of post-transcriptional control operating in the cell, the number of factors that can be involved in various steps of RNA function is much larger than anticipated. Indeed, the recent development of methodologies aimed at searching for new RBPs has produced a catalogue of factors with RNA binding capacity , such as2.7G(5′)ppp(5′)N structure (termed cap) located at the 5′ end of most mRNAs [Most cellular mRNAs initiate translation by a mechanism that depends on the recognition of the mst mRNAs . In thisst mRNAs . The thist mRNAs . Severalst mRNAs  in eIF3ai and eIF2-GTP) mediates the formation of the 43S complex that is recruited to the mRNA along with eIF1A, eIF1, eIF3, and possibly eIF5. Assembly of a competent 43S complex into mRNA bound to eIF4F is further stabilized by the interaction of eIF4G with PABP, and of eIF4B with eIF4A and PABP. The protein eIF4B harbors an RRM motif and a C-terminal Arginine-rich motif required for RNA-binding [On the other hand, the 40S ribosomal subunit with the ternary complex (TC)  elements. Translation of mRNAs bearing IRES elements, first reported in the genomic RNA of picornaviruses [Cap-dependent translation initiation is inhibited under cellular stress conditions, such as viral infection or apoptosis ,6,39. Praviruses ,41, is tSoon after picornavirus IRES disclosure, other viral RNAs ,43 and ctrans-acting factors (ITAFs) requirement [Although the presence of IRES elements in viral RNAs is well established, data on some cellular elements has been a matter of debate due to the lack of appropriate controls performed to discard the presence of cryptic promoters or alternative splicing transcripts ,66. Indeuirement –69. Morei during internal initiation, driving protein synthesis without the help of eIFs at non-AUG codons [According to the minimal requirement of factors for internal initiation, viral IRES elements can be grouped into two categories. The first category, represented by the intergenic region (IGR) of dicistroviruses, includes those that do not need proteins to assemble the initiation complex. This unique class of IRES elements adopts a compact tertiary structure that functionally substitutes the initiator met-tRNAG codons ,70,71. TG codons –74. In a3.RNA structure plays a fundamental role in viral IRES-dependent translation initiation –77. Stru3.1.C-terminal region of eIF4G in addition to eIF4A, eIF2, and eIF3, but not eIF4E [45  strongly stimulates complex formation in reconstitution assays [Picornavirus IRES elements span from about 280 to 450 nucleotides upstream of the functional start codon . Attendiot eIF4E –91. Furtot eIF4E  which isot eIF4E . Translaot eIF4E . On the ot eIF4E ,96 depenn assays ,97,98.The observation that ITAFs are RBPs previously identified as transcription regulators, splicing factors, RNA transport, RNA stability or translation control proteins  suggestsPTB was the first RBP identified as an ITAF using UV-crosslink assays conducted with radiolabelled IRES transcripts ,100. PTBNonetheless, the large amount of data obtained over the years on the mechanism of action of picornavirus IRES reveals that these regulatory elements are more complex than initially proposed. Proteomic studies based on mass spectrometry analysis of affinity purified RNPs assembled on tagged RNAs with cytoplasmic cell extracts have allowed the identification of RBPs interacting with several picornavirus IRES elements . In suppC-terminal region partially competing out PTB binding [Other examples concern proteins previously reported to perform a role distinct than translation control. A protein recently revealed as a factor regulating translation is Gemin5 , an abun binding , a resul binding .Given their modular organization, RBPs can recognize a large number of targets. This feature raises the possibility that binding of a particular RBP to certain RNAs could facilitate different sorts of regulation depending on the other partners of the complex. One example is Ebp1 (erbB-3-binding protein 1) identified in proteomic analysis bound to domain 3 of the FMDV IRES . Ebp1 co3.2.in vitro [The IRES element of HCV genome differs from picornavirus IRES elements in RNA structure and eIF4G requirement ,123. Thein vitro  despite in vitro . Recent in vitro . MutatioIn addition to eIF3 and the ternary complex, a few RBPs acting as ITAFs are shared between HCV and picornavirus IRES elements  and NS1-associated protein  ,112–114)cis-acting regions identified factors mediating the formation of functional bridges between mRNA regions. This was the case of RNPs assembled on tagged RNAs that contained both the IRES and the 3′ UTR of HCV [Both picornavirus and HCV IRES-dependent translation are synergistically enhanced by the 3′ UTR of the viral genome –129, conR of HCV . One of R of HCV .4.Cellular IRES elements are typically present in mRNAs encoding stress response proteins, such as those needed during nutrient deprivation, temperature shock, hibernation, hypoxia, cell cycle arrest, or apoptosis ,7. HenceMultifunctional RBPs interact with various IRES elements, suggesting a mechanism for the coordinated regulation of translation initiation of a subset of mRNAs bound by shuttling proteins, such as hnRNPs and splicing factors . Nuclear4.1.p53 as well as other factors involved in apoptosis and nutrient deprivation [PTB stimulates translation driven by IRES elements located in mRNAs encoding proteins of the myc family controlling cell growth, the tumor suppressor gene rivation ,136,144.rivation . The unrrivation ,146, a crivation ,147. On rivation ,149.Translation of specific mRNAs in cells with quiescent v-akt murine thymoma viral oncogene homolog 1 (AKT) kinase maintains the levels of proteins involved in cell cycle progression when eIF4E-mediated (cap-dependent) translation is inhibited. This pathway is dependent on SAPK2/p38-mediated activation of IRES-dependent initiation of the cyclin D1 and c-myc mRNAs . Inhibit4.2.IRES located in mRNAs encoding proteins synthesized under apoptosis such as the apoptotic protease activating factor 1 (Apaf-1), and BCL2-associated athanogene (BAG-1), are also responsive to PTB . In partWith the exception of pyrimidine tracts, no distinctive RNA motifs that can be used to predict the binding of RBPs are apparent in cellular IRES elements. Yet, cellular IRES with high AU content, such as XIAP, depend on NF45 , since cAlthough most ITAFs stimulate translation, a few cases where RBPs repress translation have also been reported. IRES-dependent translation of anti-apoptotic proteins XIAP and Bcl-X is repressed by the tumor suppressor programmed cell death 4 (PDCD4), a factor that sequesters eIF4A and, thus, inhibits formation of the 48S translation initiation complex . Phospho4.3.in vivo. In addition, DDX6 knockdown cells show increased secretion of VEGF.As in the case of IRES elements controlling the expression of cell proliferation proteins, stress-dependent relocalization of hnRNP A1 also mediates internal initiation of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF-2) . Howevertrans-acting factor mhnRNP Q [The death-associated protein (DAP5), NF45, G-rich RNA sequence binding factor (GRSF-1), fragile-X mental retardation protein (FMRP), heterogeneous nuclear ribonucleoprotein D-like protein (JKTBP1) and zinc-finger protein ZNF9 are specific for some IRES elements such as DAP5/p97, cIAP1, ODC ,158,164.mhnRNP Q . The ratIn summary, the data available so far indicate that individual mRNAs exploit the activity of multifunctional RBPs to overcome the global repression of protein synthesis.5.ITAFs are RBPs that activate or repress the expression of proteins critical in the cellular response to growth, nutritional, environmental and proliferation signals. It has been proposed that the main role of RBPs is to remodel the mRNA structure in a way that enhances its affinity for components of the translation machinery, or that they substitute some canonical eIFs providing functional bridges between the mRNA and the ribosomal subunits. Elucidating the function of ITAFs demands a deep understanding of their RNA targets and their protein partners with potential modifications. Yet, a subset of IRES elements exhibiting different structural organization interacts with the ribosomal protein RpS25 , consist


The localization of ash mRNA in yeast requires the binding of She2p and the myosin adaptor protein She3p to its localization element, which is highly specific and leads to the assembly of stable transport complexes. ASH1-mRNA transport in yeast to provide a first assessment of where and how localizing mRNAs are specifically recognized and incorporated into mRNPs. By using in vitro–interaction and reconstitution assays, we found that none of the implicated mRNA-binding proteins showed highly specific cargo binding. Instead, we identified the cytoplasmic myosin adapter She3p as additional RNA-binding protein. We further found that only the complex of the RNA-binding proteins She2p and She3p achieves synergistic cargo binding, with an at least 60-fold higher affinity for localizing mRNAs when compared to control RNA. Mutational studies identified a C-terminal RNA-binding fragment of She3p to be important for synergistic RNA binding with She2p. The observed cargo specificity of the ternary complex is considerably higher than previously reported for localizing mRNAs. It suggests that RNA binding for mRNP localization generally exhibits higher selectivity than inferred from previous in vitro data. This conclusion is fully consistent with a large body of in vivo evidence from different organisms. Since the ternary yeast complex only assembles in the cytoplasm, specific mRNA recognition might be limited to the very last steps of mRNP assembly. Remarkably, the mRNA itself triggers the assembly of mature, motor-containing complexes. Our reconstitution of a major portion of the mRNA-transport complex offers new and unexpected insights into the molecular assembly of specific, localization-competent mRNPs and provides an important step forward in our mechanistic understanding of mRNA localization in general.In eukaryotes, hundreds of mRNAs are localized by specialized transport complexes. For localization, transcripts are recognized by RNA-binding proteins and incorporated into motor-containing messenger ribonucleoprotein particles (mRNPs). To date, the molecular assembly of such mRNPs is not well understood and most details on cargo specificity remain unresolved. We used  ASH1-mRNA transport in Saccharomyces cerevisiae to address the question of how localizing mRNAs are recognized and specifically incorporated into mRNPs. Surprisingly, we found that the previously implicated mRNA-binding proteins She2p and Puf6p do not bind to cargo mRNAs with high specificity. Instead, the cytoplasmic motor-adapter protein She3p is responsible for synergistic cargo binding with She2p and for the stable incorporation of specific localizing mRNA into the transport complex. We propose that the specific recognition of localizing mRNAs happens at the very last step of cytoplasmic mRNP maturation. Other organisms might employ similar mechanisms to establish cellular polarity.In eukaryotes, the majority of cells are asymmetric and a way to establish such polarity is directional transport of macromolecules along cytoskeletal filaments. Among the cargoes transported, mRNAs play an essential role, as their localized translation contributes significantly to the generation of asymmetry. To date, hundreds of asymmetrically localized mRNAs in various organisms have been identified. These mRNAs are recognized by RNA-binding proteins and incorporated into large motor-containing messenger ribonucleoprotein particles (mRNPs) whose molecular assembly is poorly understood. In this study, we used the well-characterized process of  In eukaryotes, directional transport and localization of mRNA is widely used to regulate gene expression on a temporal and spatial level. mRNA localization is involved in diverse processes such as inducing cellular asymmetry, guiding key events during embryonic development, and supporting synaptic plasticity Vg1 and VegT RNPs in Xenopus oocytes already showed marked differences between the nuclear and cytoplasmic protein composition of these RNPs A study on the localization of Saccharomyces cerevisiae, ASH1 mRNA is transported as part of large mRNPs from the mother cell to the daughter cell ASH1 mRNA contains four cis-acting regions, termed zip-code elements E1, E2A, E2B, and E3, mediating mRNA incorporation into the mRNPs and its subsequent localization ASH1 mRNA is translated in the daughter cell. The protein product Ash1p acts as a repressor of mating-type switching exclusively in the daughter cell ASH1 mRNA, more than 30 transcripts are localized by this transport complex During mitosis of ASH1 mRNA already in the nucleus at the site of transcription ASH1 mRNA complex binds to the stable cytoplasmic co-complex of the myosin-adapter She3p and the type V myosin motor Myo4p ASH1-E3 RNA element has motile activity She2p is an unusual RNA-binding protein ASH1 mRNA-She2p complex and are required for efficient ASH1-mRNA localization in vivo ASH1-E3, cytoplasmic Khd1p interacts with a region encompassing ASH1-E1 RNA. Both proteins are involved in translational repression during transport In addition to this cytoplasmic core mRNP, the RNA-binding proteins Puf6p and Khd1p associate with the ASH1 mRNA ASH1 mRNP. To date, it is unclear how She2p mediates this function.Among all these factors, She2p is the only RNA-binding protein known to bind to all four zip-code elements of the ASH1-E3 zip code alone can mediate efficient mRNA localization in vivo Since the We found that She2p and Puf6p bind to zip-code elements only with moderately higher affinity than to unrelated RNAs. This raised the question of when and how specificity for mRNP transport is achieved. We further discovered that the myosin adapter She3p is a previously uncharacterized RNA-binding protein. It interacts directly with She2p also in absence of RNA. Addition of zip code containing RNAs results in the formation of highly specific ternary complexes. This specific mRNP has an at least 60-fold higher affinity for zip-code RNAs. Furthermore, incorporation of the RNA increases the interaction between She2p and She3p to a similar extent. Since we find She3p exclusively in the cytoplasm, we propose that highly specific mRNA recognition occurs after nuclear export and is coupled to the assembly of mature, motor-containing transport complexes.ASH1 mRNA (Gene ID: 853650) EAR1 mRNA (Gene ID: 855207) WSC2N) of WSC2 mRNA (Gene ID: 855438) ASH1 mRNA and the weakest binding for the EAR1 zip-code element (Kd = 0.77 µM). No binding was detected to an unstructured Poly-A20 RNA or the MS2-stem-loop RNA Previous studies showed that She2p (Gene ID: 853728) binds to all four zip-code elements of the When repeating RNA filter-binding experiments with HIV-1 TAR RNA (16 mer), a longer HIV-1 TAR RNA (57 mer), and the U1 snRNA hairpin II as controls, we observed Kds ranging from 1.1 µM to 1.6 µM , which binds to the ASH1-E3 RNA in the presence of excess amounts of either specific (ASH1 E3) or unspecific (EAR1) unlabeled competitor RNA. The EAR1 zip-code element was chosen as an unspecific competitor because it lacks a PUF-consensus site ASH1-E3 RNA and recombinant Puf6p was competed for by excess amounts of ASH1-E3 RNA, but only poorly by EAR1 RNA  and lacks predicted Puf6p binding sites To assess the previously suggested direct interaction of Puf6p with She2p in vitro In the cytoplasm, the cargo adapter She3p (Gene ID: 852427) joins the She2p:RNA complex. She3p is thought to connect specifically pre-bound mRNAs to the motor Myo4p (Gene ID: 851204) Since previous data indicated that She3p might be able to join the co-complex of She2p and RNA ASH1 mRNA and colocalizes with ASH1 mRNA at the bud tip, it is considered to be associated with the actively transported ASH1 mRNPs PUF consensus-lacking E3-33-tRNA was used, Puf6p did not form a detectable complex with She2p and She3p  , could b complex . A sligh complex , termed  complex . In cont complex . Interes2, and mass-spectrometric analysis. This recently developed technique ASH1 E3-51 RNA  did not show any defect in vitro , the mutWe also generated mutant versions of She2p and tested them for interaction with She3p and RNA. Two structural regions of She2p show high sequence conservation When performing pull-down experiments with His-tagged She3p, She2p (ΔC) could be co-eluted . In contNext we assessed ternary complex formation with both She2p mutants by size-exclusion chromatography. Whereas She2p (ΔC) still forms stable complexes with She3p and E3-118-tRNA, no ternary complexes assembled with She2p (ΔhE)  and S9C.EAR1 zip-code RNA and observed defects comparable to the ones obtained with ASH1-E3 RNA  mutant showed an about 5-fold reduced complex formation with She3p and the E3 zip-code element . Since t1-E3 RNA .ASH1 zip-code elements than for EAR1 mRNA. These observations are consistent with respective defects in ternary complex formation. Furthermore, they demonstrate that selective reduction of RNA binding, as observed for She2p (ΔC), impairs synergistic RNA binding with She3p.Finally, we analyzed the effects of both mutations on the RNA binding of She2p alone. Since in our hands RNA binding by She2p is too transient for detection by EMSAs , these iDeletion of the helix E of She2p abolishes synergistic complex formation. Because this deletion constitutes a considerable alteration of the overall shape of the protein, we generated more subtle point mutations around this region . Whereasshe2Δ background. Wild-type She2p-Myc efficiently co-precipitated She3p  completely failed to localize to the bud tip  localization by antibody staining in Δshe2 strain with a plasmid expressing She2p (ΔhE) under its endogenous promoter and assessed ASH1-mRNA localization by in situ hybridization. In contrast to wild-type She2p, we did not observe any ASH1-mRNA localization in response to She2p (ΔhE) expression (n>100 cells). Thus, disruption of ternary complex formation as observed in vitro also results in abolished ASH1-mRNA localization in vivo.In addition, we transformed a ASH1-mRNP assembly suggests an early assembly of She2p with ASH1 in the nucleus tsmex67-5. At restrictive temperature, shuttling proteins such as She2p accumulate in the nucleus tsmex67-5 cells revealed wild-type-like localization of She3p at permissive temperature  experiments showed that She2p associates co-transcriptionally with RNA polymerase II of She3p . It is pshe3 cells. A comparison of the She2p occupancy at the ASH1 locus and three control loci in wild-type cells and in Δshe3 cells showed no significant difference  of localizing mRNAs. The authors concluded that part of the chromatin-associated She2p interacts selectively with localizing mRNAs already during transcription. However, we could not confirm this observation. Although we also detected a reduction of She2p-dependent enrichment after RNase treatment, this effect was observed for all transcripts . Thus, oIn summary, our and previous data suggest the following model: First, She2p binds co-transcriptionally to RNA polymerase II and to nascent transcripts . After tASH1-mRNPs do not have motile activity in vitro On one hand, She2p and She3p function together exclusively in the cytoplasm to select zip-code RNAs. On the other hand, the mRNA cargo itself substantially stabilizes the She2p:She3p interaction. Because this interaction brings together the She2p-dependent pre-mRNP with the cytoplasmic motor complex, we conclude that it is the mRNA cargo itself that triggers joining of all components into the mature transport complex. This interpretation is fully consistent with the observation that RNase-treated Drosophila  Previous publications reported only moderate in vitro selectivity for localizing mRNAs in yeast and Drosophila development, the RNA-binding protein Staufen is involved in the localization of bicoid mRNA. In vitro, Staufen yielded strong binding to specific as well as to control RNAs with extensive secondary structures Drosophila embryos showed that Staufen-containing mRNPs only form and localize efficiently when its native target, the bicoid 3′UTR, is injected Drosophila complexes also require mRNP assembly for specific mRNA binding and localization.An important question arising from this study is whether synergistic binding to RNA cargo is a more general feature in eukaryotes. For instance, during the oocyte-to-embryo transition of In the past, in vitro reconstitution of molecular assemblies has been very successful to provide new insights into biological processes as diverse as transcription and membrane fusion Detailed information on plasmids, oligonucleotides, and yeast strains are found in E. coli BL-21 Star (DE3) cells (Invitrogen) and purified using standard techniques E. coli BL-21 Star (DE3) cells and purified via affinity and size-exclusion chromatography. The GST-tag was cleaved with PreScission protease  during purification, unless stated otherwise. Full-length His-She3p and His-She3p point mutants were co-expressed with She2p using the Bac-to-Bac system (Invitrogen). Recombinant baculovirus was amplified in Sf21 cells and used to infect High Five insect cells. Cells were cultured for 60–70 h. After sonication, His-She3p was purified using HisTrap, HiTrap Q, HiTrap Heparin, and Superose 6 10/300 GL columns . She2p and nucleic acids were removed by extensive washing with 1 M NaCl-containing buffer. At the last step of purification, RNA-binding proteins were assessed by measuring the ratio of A260 to A280 to exclude contaminations by nucleic acids. Myo4p tail was purified as previously described She2p and Puf6p were expressed as GST-fusion in ASH1-E3 RNA was fused to the E. coli initiator tRNA(Met) , and purified by ion exchange chromatography essentially as described RNAs for filter-binding assays and EMSAs were produced by either in vitro transcription or total chemical synthesis . In vitr2, 2 mM DTT, and 30 µg/ml yeast tRNA). Four-fifths of the reaction mixture was applied on the nitrocellulose membrane and radioactivity retained was measured by phosphoimaging. Kd calculation was performed by plotting the fraction of bound RNA versus the protein concentration and applying the Langmuir isotherm. The following serial dilutions were used: wild type–She2p binding to HIV-I TAR  or U1snRNA hairpin II: 0 to 12 µM; She2p-mutant binding to bud-localizing RNAs: 0 to 16 µM; She2p (ΔC)-binding to ASH1-E2A element: 0 to 32 µM; She2p-mutants binding to HIV-I TAR (16 bases), U1snRNA hairpin II, poly(A)20 RNA: 0 to 32 µM; and She2p-F195A-L196A and She2p-Q197A-E198A-I199A to ASH1-E3: 0 to 2 µM. Standard deviations were calculated from at least three independent experiments. To show synergistic RNA-binding effects, serial She2p dilutions  were incubated with 25 nM She3p and 0.5 nM 32P-RNA in Dot-blot buffer (pH 7.9) and treated as described above. For data analysis, the RNA-binding signal of 25 nM She3p alone was subtracted as background. Signal intensities of She2p:She3p:RNA complexes were plotted against the respective She2p concentrations and normalized to the relative signal intensities of the corresponding She2p:RNA complexes.RNA filter-binding assays were essentially performed as described 32P-labeled RNA oligonucleotide in HNMD-buffer (20 mM HEPES (pH 7.8), 200 mM NaCl, 2 mM MgCl2, 2 mM DTT) supplemented with 4% (v/v) glycerol and 30 µg/ml (EMSAs with She2p and She3p) or 100 µg/ml yeast tRNA (EMSAs with She3p or Puf6p only) for 30 min at 20°C. For competition experiments, excess of unlabeled competitor RNA was added to the reaction after 30 min preincubation and incubated for another 15 min at 20°C. Additionally, HNMD-buffer supplemented with 100 µg/ml yeast tRNA was used. Protein:RNA complexes were resolved by PAGE . Gels were scanned with a Storm Scanner and analyzed using the software ImageQuant.In a 20 µl reaction, protein was incubated with 5 nM of 32P-labeled ASH1-E3-51 RNA for 20 min at 20°C in Dot-Blot buffer (pH 7.9). Subsequently, UV cross-linking was performed for 15 min on ice, using a Spectrolinker XL-1500 (Spectroline) with an average intensity of 2,500 µW/cm2. 18 µl of each cross-linked sample were resolved by SDS-PAGE. Gels were scanned and analyzed using the software ImageQuant.In 20 µl reactions, recombinant She2p and She3p at indicated amounts were incubated with 5 nM ASH1-E3-51 RNA, full-length She2p, and His-She3p (334–425)) in 200 µL HNMD-buffer were cross-linked for 10 min at 254 nm. After digestion of the sample with RNase A, RNase T1, and trypsin, cross-linked peptides were enriched on a TiO2 column 100 µg of purified ternary complex  were preincubated for 5 min at 20°C. Then 200 µl samples were loaded on the column in HNMD-buffer (flow rate: 0.5 ml/min). Fractions were analyzed by SDS PAGE (Coomassie blue staining) and 2% agarose gels (in 1× TBE) stained with GelRed DNA Stain (Biotium).ASH1-E3-tRNA-fusion were incubated in HNMD-buffer with 50 µl resin  for 30–60 min at 4°C on a rotating wheel. Binding reactions were pelleted and washed four times with 200–500 µl of HNMD-buffer, followed by a final washing step with 50 µl. For His-tagged She3p, HNMD-buffer for binding and washing was supplemented with 30–50 mM imidazole. Bound proteins were eluted with 50 µl of elution buffer . One-tenth of the input, 1/5 of the final wash, and 1/5 of the elution fraction were analyzed by SDS-PAGE and Coomassie blue staining. RNA content was analyzed with 2% agarose gels (in 1× TBE) stained with GelRed DNA Stain (Biotium).In a volume of 100 µl, 7.5–10 µM of each protein and, if applicable, 5 µM All experiments were performed using a Biacore 3000 system with a CM-5 chip (Biacore) in HNMD-buffer. She3p was covalently bound to the chip surface by standard amine coupling. For measurements with She2p (wt) and She2p (ΔC) concentrations from 0.123 to 30 µM were used. She2p (ΔhE) binding was probed with up to 150 µM protein. All binding signals were below 300 response units. Experiments were performed as duplicates and double referencing was applied. Kds were derived from steady-state measurements, applying the Langmuir isotherm.ASH1 mRNA In situ hybridization was performed using TexasRed-conjugated antisense oligonucleotides against Δshe2 lanes were subtracted from respective Western blot signals. Subsequently, She2p signals from the supernatant fractions were subtracted from input fractions and used to normalize co-immunoprecipitated She3p against the bead-bound fraction of She2p-Myc3.Co-immunoprecipitation of Myc-tagged She2p was performed using monoclonal anti-Myc antibody  coupled to magnetic Protein G beads (Invitrogen) 600∼0.8). To increase the fraction of cells with ASH1 transcription, yeast cultures were treated with nocodazole  for 2 h and washed once with 100 ml YPD as described ChIP experiments were performed as previously described ASH1, a distinct region within the ORF regions of PMA1 and FBA1, the promoter, coding and terminator regions of ADH1, as well as for a heterochromatic control region of chromosome V. The PCR efficiencies were determined prior to ChIP experiments and were in the range of 95%–100%. The sequences and the location of the primers used in this study are shown in The experiments as well as the analyses were performed as previously described Figure S1Control experiments to confirm that Puf6p is an intact protein. (A) In size-exclusion chromatography full-length Puf6p eluted as a single, defined peak. No major elution was observed at the void volume of about 8 ml, confirming that Puf6p does not aggregate. The sharp elution peak of Puf6p further indicates a defined conformation of this protein. (B) Circular dichroism spectroscopy with Puf6p reveals a profile typical for alpha-helical proteins. Since Puf6p contains seven Pumilio-homology domains (0.09 MB PDF)Click here for additional data file.Figure S2ASH1 mRNA variants E3-118, E3-51, and E3-33. Predictions were generated with MC-Fold PUF-consensus sequences UUGU ASH1 mRNA start codon. The zip-code stem loops E3-118 and E3-33 were fused to the anticodon stem of tRNA(Met) Structure predictions of the (0.16 MB PDF)Click here for additional data file.Figure S3ASH1-E3 mRNA core complex, consisting of ASH1-E3 RNA, She2p, She3p, and the interacting Myo4p-tail fragment. Corresponding fractions were analyzed by SDS-PAGE and agarose gel electrophoresis and are shown below the chromatogram.Control experiments for complex assembly with She2p. (A) In pull-down experiments, wild-type She2p did not interact with nickel-sepharose beads. (B) In size-exclusion chromatography experiments, wild-type She2p and She3p eluted as a co-complex in absence of RNA. (C) Size-exclusion chromatography of the reconstituted (0.15 MB PDF)Click here for additional data file.Figure S4ASH1-E3 mRNP. (A) In pull-down experiments with immobilized His-She3p, Puf6p and She2p co-eluted in an ASH1-E3 zip-code-dependent manner . No co-elution of Puf6p was observed when the control RNA ASH1-E3-33 was used (lanes 4–6) or in absence of RNA (lanes 7–9). As observed previously, She3p bound to She2p also in absence of RNA. The small amount of ASH1-E3-33 RNA eluting with She3p from the nickel sepharose beads (lane 6) most likely resulted from unspecific RNA binding by She3p , but not in absence of RNA (lanes 4–6). Please note that She3p always migrates as a double band in SDS gels. In a control experiment with immobilized GST protein, no unspecific binding of She2p, She3p, or RNA was observed (lanes 7–9). (C) Control pull-down experiments with immobilized His-tagged She3p confirmed the formation of a ternary complex consisting of She3p, She2p, and ASH1-E3 RNA (lanes 1–3) but did not show an interaction of She3p with Puf6p in the presence of ASH1-E3 RNA (lanes 4–6). No unspecific binding of She2p and ASH1-E3 RNA to the beads was observed (lanes 7–9). In contrast, Puf6p retained a weak affinity to nickel sepharose, which could not be eliminated by using 50 mM Imidazole in binding and washing buffers. Please also note that the band migrating above His-She3p represents a degradation product of Puf6p (compare lane 4 with 7). I, input; W, final wash; E, elution.Reconstitution of a major part of the cytoplasmic see also . (B) Pul(0.42 MB PDF)Click here for additional data file.Figure S5ASH1-E3 (A) or EAR1 zip-code RNA (B) was detected. (C) The minimal She3p concentration required for detection of ASH1-E3 zip code binding in EMSAs was determined to be in the range of 25 nM to 50 nM (position marked by asterisk). (D) This EMSA shows synergistic binding of She2p and She3p on ASH1-E3 RNA in direct comparison to the unaffected Puf6p binding in presence of She2p. A shift to larger molecular weight and thus the formation of a ternary complex was only observed for She2p and She3p. Puf6p alone showed strong but unspecific binding (see ASH1-E3 RNA. (F–I) EMSAs with a constant She3p concentration (25 nM) and varying amounts of She2p show specific ternary complex formation with the ASH1-E1 zip code (F), ASH1-E2A zip code (G), ASH1-E2B zip code (H), and a shortened ASH1-E3-51 zip-code RNA (I). The Kds for these complexes were all estimated to be in a similar range and are comparable to specific complex formation with ASH1-E3 and EAR1 zip codes (see ASH1-E1 (F) and ASH1-E2A (G), resulting in the formation of a subtle band shift. However, RNA:She2p:She3p complexes migrated slightly slower in EMSAs and could thus be distinguished from RNA:She3p complexes.Supporting experiments for studies on synergistic RNA binding by She2p and She3p. (A–B) The She2p:RNA interaction is too transient to be resolved in EMSA experiments. Even at micromolar She2p concentrations, no stable complex with either ding see . (E) Wesodes see . Please (1.11 MB PDF)Click here for additional data file.Figure S6ASH1-E3 (left) and the EAR1 (right) zip codes. Shown are the raw data obtained by dot-blot experiments with radiolabeled zip-code RNA, various She2p concentrations, and 25 nM She3p whenever indicated. The presence of She3p in the reaction significantly enhanced RNA binding, as determined by an increase in signal intensity at lower She2p concentrations. (B) In the corresponding graphs, relative signal intensities are plotted against the She2p concentrations to visualize the larger amount of zip-code RNA bound by the She2p:She3p complex. Because ternary complex formation does not appear to follow simple reaction kinetics, we refrained from calculating Kd values.Control experiments for the observed synergistic complex formation of She2p and She3p on zip code containing RNA. (A) Filter-binding experiments confirm the synergistic binding of She2p and She3p to the (0.19 MB PDF)Click here for additional data file.Figure S7GPLGSMGNSSNNK (G334–K340) of His-She3 (334–425), including the preceding linker region GPLGSM (underlined), cross-linked to an AAU oligonucleotide without any terminal phosphate. [A] represents the adenosine nucleotide, while [A′] stands for the adenine base. All further RNA-related signals are labeled accordingly. The signal at m/z 768.14 indicates that S10 could be cross-linked to U, as it can be calculated as the y4 peptide fragment with an additional U nucleotide and a loss of H2O. The experimental precursor mass 2,163.68 Da equals the sum of calculated peptide and oligonucleotide masses, 1,261.57 Da and 902.17 Da, respectively, within the mass accuracy of the employed Q-ToF instrument. (B) MSMS spectrum of the N-terminal tryptic peptide GPLGSMGNSSNNK (G334–K340) of His-She3 (334–425) cross-linked to a uridine nucleotide. The peptide's N-terminus is carbamylated due to hydrolyzation in urea at elevated temperatures. The sum of the calculated peptide mass  and the mass of a uridine nucleotide (324.04 Da) equals the experimental precursor mass of 1,628.73 Da. (C) MSMS spectrum of the She2 peptide IGSNLLDLEVVQFAIK (I164–K179) cross-linked to a uridine nucleotide. Peptide fragments with neutral loss of ammonia are marked with an asterisk. The experimental precursor mass of 2,081.92 Da equals the sum of calculated peptide and nucleotide mass .UV cross-linking and subsequent mass spectrometric analysis of RNA-bound protein fragments. The peptide sequence and cross-linked nucleotides as well as identified peptide CID fragments are indicated above each MSMS spectrum. (A) MSMS spectrum of the N-terminal tryptic peptide (0.17 MB PDF)Click here for additional data file.Figure S8ASH1-E3 RNA was analyzed by EMSAs. Synergistic RNA binding was not significantly reduced for She3p (S343E S361E) (A) and She3p (R341E) (C) as compared to the wild-type  (F), She3p (S343E S361E) (G), and She3p (L364A V367A) (H) was comparable to wild-type She3p (E). (I) Interactions between His-tagged She3p mutants and wild-type She2p were analyzed by in vitro pull-down with nickel sepharose. She2p binding to She3p (S348E) and She3p (S343E S361E) was comparable to wild-type She3p. In contrast, She3p (L364A V367A) did not interact with She2p.Analysis of She3p point mutants. (A–D) Synergistic binding of His-She3p mutants and wild-type She2p to ild-type . She3p ((0.79 MB PDF)Click here for additional data file.Figure S9EAR1-zip-code RNA and She3p. Deletion of the She2p C-terminus impaired but did not abolish RNA-dependent complex assembly with She3p. In contrast, She2p (ΔhE) failed to assemble co-complexes at concentrations up to 250 nM. (E) She2p (ΔC) and She2p (ΔhE) showed impaired RNA binding. RNA filter-binding experiments demonstrated a significantly reduced affinity of both She2p mutants to the four ASH1 zip codes. Binding of both She2p mutants to the EAR1 zip code and WSC2N was only moderately reduced. However, the She2p (ΔhE) interaction with the WSC2N element was almost abolished. In contrast, binding of both She2p mutants to unrelated stem-loop RNAs was not reduced. This suggests that helix E and the C-terminus of She2p are dispensable for unspecific RNA binding. “% wt-binding” indicates the percentage of RNA-binding affinity relative to wild-type She2p binding. Although wild-type She2p data have been published previously The effect of She2p mutations on RNA binding and complex formation. (A) Elution profiles from preparative size-exclusion chromatography (Superose 12 column) show that wild-type She2p and She2p (ΔhE) eluted at the same volume, whereas She2p (ΔC) showed a retarded elution. Thus, deletion of the C-terminus appears to slightly affect She2p tetramerization without completely disrupting it Click here for additional data file.Figure S10ASH1-E3-zip-code RNA and She3p in EMSAs. (C) In pull-down experiments, the helix E–affecting mutants She2p (Q197A E198A I199A) and She2p (F195A L196A) failed to interact with immobilized His-She3p . Please note that She2p (F195A L196A) migrated slightly slower than the wild type in SDS-PAGE. However, we confirmed the expected mass of both point mutants by mass spectrometry (unpublished data). (D) Control pull-down experiments showed that none of the two She2p mutants bound to nickel-sepharose beads. (E) RNA filter-binding assays demonstrated a comparably mild, 2.5- to 3-fold reduction in binding of the mutants She2p (Q197A E198A I199A) and She2p (F195A L196A) to the ASH1-E3 zip-code RNA. Thus, in these mutants RNA binding seems less severely affected than She3p binding (C). Numbers given in columns “% wt-binding” show the percentage of the RNA-binding affinity relative to wild-type She2p binding, which was determined in a parallel experiment. (F) Circular dichroism spectroscopy with She2p (Q197A E198A I199A), She2p (F195A L196A), and wild-type She2p indicated that none of the mutations impairs protein folding.Analysis of She2p point mutants. (A–B) Mutant proteins She2p (E183A D184A G185A) (A) and She2p (T191A D192A) (B) do not show impaired complex formation with (0.42 MB PDF)Click here for additional data file.Figure S11she3 cells. The lower table shows the same experiment with an additional RNase treatment. For further details, see she3 cells.Supporting information for chromatin immunoprecipitation (ChIP) experiments. (A) Data of ChIP experiments with anti-TAP antibody in TAP-She2p expressing yeast cells. Upper table shows results comparing TAP-She2p expressing wild-type cells with TAP-She2p expressing Δ(0.09 MB PDF)Click here for additional data file.Figure S12Surface representation of the She2p tetramer (0.63 MB PDF)Click here for additional data file.Figure S13S. cerevisiae is boxed in yellow. Branches to species without clear She2p homologs are highlighted in red. Numbers at nodes show bootstrap values. (B) Sequence alignment of She3p homologs from species shown in the phylogenetic tree (A). Among all species, the N-terminal, motor-interacting half of She3p shows higher sequence conservation than the C-terminal half, which interacts with She2p in S. cerevisiae. Species boxed in red lack clear She2p homologs in their genomes. Red bar with the label “crosslink” highlights amino acids of She3p that were contained in a UV cross-linked peptide of She3p (334–425) . Based on this alignment, the phylogenetic tree  and alignment representation were prepared using the CLC Sequence Viewer (www.clcbio.com).Sequence alignment of She3p from different yeast species. (A) Phylogenetic tree with bootstrap analysis. She3p from (0.61 MB PDF)Click here for additional data file.Table S1Plasmids used in this study.(0.10 MB PDF)Click here for additional data file.Table S2Oligonucleotides used in this study.(0.06 MB PDF)Click here for additional data file.Table S3Yeast strains used in this study.(0.07 MB PDF)Click here for additional data file.Table S4Sequences of RNAs used in this study.(0.09 MB PDF)Click here for additional data file.Table S5Primers used for ChIP experiments.(0.06 MB PDF)Click here for additional data file.Text S1Cloning strategy and generation of yeast strains; Circular dichroism spectroscopy; UV cross-linking and enrichment of cross-links for mass spectrometric (MS) analysis; and Nano-LC-ESI-MS analysis of enriched UV cross-links and MS data analysis.(0.13 MB PDF)Click here for additional data file.


To date, the antiviral activities of flavonol kaempferol (Kae) and isoflavonoid daidzin (Dai) against JEV have not been described.New therapeutic tools and molecular targets are needed for treatment of Japanese encephalitis virus (JEV) infections. JEV requires an α-1 translational frameshift to synthesize the NS1' protein required for viral neuroinvasiveness. Several flavonoids have been shown to possess antiviral activity 50) and 50% effective concentration (EC50) against JEV were investigated in BHK21 cells by MTS reduction. Activity against viral genomic RNA and proteins was measured by real-time RT-PCR and western blotting. The frameshift site RNA-binding characterization was also determined by electrospray ionization mass spectrometry, isothermal titration calorimetry and autodocking analysis. EC50 values of Kae and Dai were 12.6 and 25.9 µM against JEV in cells pretreated before infection, whereas in cells infected before treatment, EC50 was 21.5 and 40.4 µM, respectively. Kae exhibited more potent activity against JEV and RNA binding in cells following internalization through direct inhibition of viral replication and protein expression, indicating that its antiviral activity was principally due to direct virucidal effects. The JEV frameshift site RNA (fsRNA) was selected as a target for assaying Kae and Dai. ITC of fsRNA revealed an apparent Kb value for Kae that was nine fold stronger than that for Dai. This binding was confirmed and localized to the RNA using ESI-MS and autodock analysis. Kae could form non-covalent complexes with fsRNA more easily than Dai could.The 50% cytotoxic concentration (CCKae demonstrates more potent antiviral activity against JEV than does Dai. The mode of action of Kae as an anti-JEV agent seems to be related to its ability to inactivate virus by binding with JEV fsRNA. Viral infections are important public health problems worldwide, both in developed and developing countries, due to their morbidity and mortality. Japanese encephalitis virus (JEV) is a leading member of the mosquito-transmitted flavivirus family, and is mainly distributed in China, India and Southeast Asia, where it can cause the central nervous system disease with irreversible neurological damage in humans CCUUUU slippery heptanucleotide motif near the beginning of the NS2A gene, and is stimulated by a downstream RNA pseudoknot structure , membrane (prM/M), and envelope (E) and non-structural proteins . JEV NS1 is involved in viral replication and regulation of the innate immune response. Recent research has identified that NS1' (a larger NS1-related protein), which plays a role in viral neuroinvasiveness, is the product of an α-1 ribosomal frameshift event that occurs at a conserved Y in vitro or in vivo models in vivo or in vitro.Plant-derived flavonoids and dietary isoflavones, a large group of naturally occurring phenylchromones found in fruits, vegetables, tea, soy foods, and herbs, have been shown to possess potential therapeutic benefits in a variety of viral infections using both in vitro conditions Kaempferol, a polyhydric flavonol, is isolated from various plant sources including tea, broccoli, delphinium, witch-hazel, grapefruit, Brussel sprouts, apples, and medicinal herbs. Kaempferol has been reported to exhibit activity against herpes simplex virus, influenza viruses (H1N1 and H9N2) and hepatitis B virus under Comprehensive studies on the nature of interactions between small molecules and DNA/RNA are of fundamental importance for biomedical and pharmaceutical sciences. For example, several classes of mRNA domains, collectively referred to as riboswitches, have been shown to serve as RNA genetic control elements that sense the concentrations of specific metabolites  50 of Kae and Dai against BHK21 cells was 230 and 270 µM, respectively. The maximum non-toxic doses were determined at 180 and 225 µM respectively, and these were used as the highest doses in the antiviral assays. For comparison, 7-hydroxyflavone did not show obvious growth inhibitory effects (data not shown).Cellular toxicity of Kae and Dai were assessed in BHK21 cells using the cell MTS reduction assay. When confluent monolayers were exposed to Kae or Dai at concentrations of 50–400 µM for 72 h at 37°C, both agents exhibited a concentration-dependent inhibition of cell growth compared to controls . The CC550 for Kae was 12.6 µM against JEV and 70% inhibition at 25.7 µM; whereas for Dai, EC50 and 70% inhibition values were 25.9 and 92.4 µM, respectively , red fluorescence was observed in early virus-infected BHK21 cells at 24 h , but notTotal RNA was extracted from each group of cells treated for 48 h. JEV mRNA levels were determined using SYBR Green real-time RT-PCR (relative quantification). Kae and Dai inhibited JEV mRNA transcription of target genes by >14.2% and 27.1%, respectively, at 48 h pre-treatment group, compared with the mock-infected group. In pre-infection group, viral RNA replication was inhibited by 21.6% by Kae and 43.1% by Dai, compared with the mock-infected group . These fThe effect of 25 µM Kae/Dai in each groups on viral E and NS1/NS1' proteins was detected by immunoassay with the Li-COR Odyssey system and quantified using Odyssey infrared imaging software . When coNo changes in E, NS1 and NS1' protein expression was observed in cells treated with 7-hydroxyflavone pre-treatment group .To determine whether Kae and Dai were able to reach the cytosol, and their binding characteristics with JEV RNA, the intrinsic fluorescent properties of flavonoids 5−, 1211.95− and 1216.15−) for the charge state z = 5 and  for the charge state z = 4. The average molecular mass measured from the main peaks was 6048, 6060 and 6072 Da respectively. 5−), 2∶1 (1323.25−), 3∶1 (1380.45−), 4∶1 (1437.85−), 5∶1 (1494.85−), 6∶1 (1552.05−) and 7∶1 (1609.25−) ligand–fsRNA1 complexes for the charge state z = 5 and 1∶1 (1582.44−), 2∶1 (1658.54−), 3∶1(1725.54−), 4∶1 (1701.54−) for the charge state z = 4 complexes were detected in the case of Kae (5− and 1615.04−) and 2∶1 (1375.05− and 1674.74−) ligand–fsRNA1 complexes were observed with Dai (4−) for Kae–fsRNA2 complexes and 1∶1 (1658.54−) for Dai, which suggests that Kae and Dai can both form adducts with fsRNA1 or fsRNA2 in a diverse stoichiometric ratio. These ESI-MS results demonstrated different binding abilities of Kae and Dai. To make semi-quantitative comparisons between Kae and Dai, the affinity of the ligands for fsRNA1 and fsRNA2 was characterized by the concentration of bound ligand for each RNA molecule, as described by Rosu et al. ESI-MS spectra are used to determine the stoichiometry and affinity of flavones for DNA/RNA in the presence of 150 mM monovalent ammonia ions e of Kae , whereaswith Dai . Figure As a control, we selected a 7-hydroxyflavone displaying no anti-JEV activity to test its affinity to fsRNA1 and fsRNA2. Increasing the ratio of 7-hydroxyflavone∶fsRNA1 to 80∶1 allowed the detection of complexes of 2∶1 7-hydroxyflavone∶fsRNA1/fsRNA2 complexes , 7D, but200 method. The fsRNA3 solution was placed at room temperature for 24 h to obtain a stable stem-loop structure before the tests. The thermodynamic parameters for the binding of fsRNA3 obtained at 25°C are summarized in b of 9.02±1.06×106 M−1, a ΔH = −9.5±1.19 kcal mol−1, a ΔS of −0.05 cal mol−1 K−1, and a binding site size of eight nucleotides. The value for Kae with fsRNA3  and a binding site size of 2.7 nucleotides. This entropy was ∼34-fold greater in magnitude than that observed for Dai, suggesting that Kae gave rise to changes in fsRNA3 conformation compared with Dai. In contrast, there were no constant thermodynamic parameters between 7-hydroxyfalvone and RNA (data not shown). Perhaps the use of simpler models is not suitable for 7-hydroxyflavone binding because the thermogram fit values were unacceptable.The interactions of Kae and Dai ligands with fsRNA3 were studied by the ITCh fsRNA3  had an a−1, and elevated by 0.9 kcal mol−1 compared to that of the Kae–R1 complex . This indicated that base G might influence the binding ability between Kae and RNA. The top ranked dock conformation for Kae and Dai with fsRNA is presented in CGG or CCG) when ligands are docked into the fsRNA.The computed binding energy (BE) and corresponding energy difference value (ΔE) for each group are listed in UUU triplet had a BE that was 1 kcal mol−1 lower than that of RNA duplexes that contained UUU triplets with Dai. We obtained similar results for the inclusion complexes of RNA without UUU with Kae and Dai. Visualization of the dockings reveals that the ligands are docked into the minor groove of RNA duplexes. The top ranked dock conformation for Kae and Dai with RNA duplexes and the detailed intermolecular hydrogen bonds are presented in In double stain RNA (dsRNA) docking results, the inclusions U1a, N1a and N1b had the highest probabilities and largest BE. The lowest binding energy and frequencies characterizing inclusion of Dai and Kae into RNA molecules are depicted separately in in vitro models for determining antiviral activity of natural compounds 50 against JEV for Kae and Dai was 12.6 µM  and 25.9 µM (SI = 10.4), respectively. The antiviral activity of Kae and Dai may have been due to inhibition of viral infection or a reduction in viral replication by modulating cellular functions through a number of mechanisms. We next studied viral inactivation in cells that were infected with JEV for 2 h before treatment with Kae or Dai. EC50 for Kae and Dai was 21.5 and 40.4 µM, respectively, suggesting that they inhibited events downstream of viral entry. MTS and IFA results both revealed that Kae had a more effective and higher SI value (15.8) than Dai did, suggesting that Kae is a more potent antiviral agent against JEV, with no toxicity to BHK21 cells. Furthermore, combination of Kae and Dai was more efficient against JEV infection, suggesting a possible synergy in the antiviral properties of flavonoid and isoflavonoid mixtures.Flaviral infections, including JEV, are an important public health problem in mainland China due to the limited therapeutic options and unsatisfactory outcomes. Therefore it is necessary to screen anti-JEV molecules for the development of novel antiviral therapies. Usually, cell culture monolayers infected with different DNA or RNA viruses are the most frequently used To further characterize the effects of Kae/Dai treatment on the cellular response to JEV infection, we investigated their effect on viral genome mRNA and protein expression. The selected NS2A segment, a relatively conserved region in JEV ssRNA coding sequence, was measure by real-time RT-PCR. The Kae and Dai inhibitory effect on JEV mRNA at 48 h suggested that they play critical roles in inhibition of viral genome replication. Meanwhile, the effects of 25 µM Kae/Dai on viral E and NS1/NS1' protein detected by western blot showed that Kae and Dai both inhibited protein expression, when compared to the mock-infected group. Moreover, the inhibitory effect of Kae on JEV protein expression in cells treated before infection was more potent than that in cells treated after infection and in cells treated with Dai before and after infection. This further revealed that Kae was a more potent antiviral agent than Dai. The expression levels of NS1 and NS1' proteins were lower than that of E protein in the mock-infected cells, indicating that the results of protein expression are related to virus strain. The obvious suppression of NS1' expression in cells treated with Kae showed that Kae might inhibit the efficiency of the α-1 ribosomal frameshift of JEV. As a control, 7-hydroxyflavone did not exert any influence on expression of E, NS1 and NS1' proteins.4O, and C2C3 functional groups, but is markedly reduced by the presence of a glycosyl moiety in vivo or in vitro, which may be attributed to the presence of a glycosyl moiety that influences antiviral activity. Greiner et al. The mechanism by which flavonoids and related isoflavones inhibit virus infectivity has yet to be fully elucidated. A review of the current literature has indicated that flavonoids affect virus binding to cell membranes, entry into the cell, replication, viral protein translation within the host cell, and formation of certain glycoprotein complexes of the virus envelope Flavonoids are reported to possess the ability to be internalized by platelets and cells. The internalization of Kae observed by confocal microscopy indicated that Kae had high transmembrane permeability in cells. The higher Kae fluorescence observed in infected cells compared to uninfected cells indicates that Kae might bind to JEV RNA. In contrast, no fluorescence was observed in 7-hydroxyflavone- or Dai-treated infected cells or control cells at any time point. In particular, 7-hydroxyflavone was scattered in the extracellular medium, indicating that it has lower transmembrane permeability and viral RNA binding properties than Kae have. Generally, flavonoid activity is attributed to the ability of these compounds to interact with proteins, lipids and polynucleotides through electrostatic and hydrophobic forces, which are necessary for biomolecular interaction within cells and cell–cell interaction, and signal transduction Several analytical methods have been developed to characterize small ligand interactions with DNA/RNA, including fluorescence spectroscopy, nuclear magnetic resonance, and ultraviolet methods In the gas phase, the external medium (vacuum) is widely considered to be hydrophobic. This reinforces the strength of hydrogen bonds compared to hydrophobic and van der Waals interactions However, organic solvents (e.g. DMSO or methanol) strongly influence thermodynamic parameters from ITC because of the exothermic reaction. Hence, we cannot acquire thermodynamic parameters for 7-hydroxyflavone and fsRNA under any experimental conditions. Furthermore, it is not worthwhile exploring in detail the hydrophobic 7-hydroxyflavone.−1, which was elevated by 0.9 kcal mol–1 compared with that of the Kae–fsRNA3 complex. This indicated that dsRNA and the base G in RNA might influence the affinity between Kae and RNA.To further research the two flavonoid compounds binding to the frameshift site in a sequence- and/or structure-specific manner, Kae and Dai were docked into RNA using the AutoDock 4.0 program. From three fsRNA docking results , the lowCCUUUU slippery heptanucleotide that is found ahead of an RNA pseudoknot structure in an α-1 ribosomal frameshift event. The lowest binding energy  and corresponding energy difference value  for each group reflected the ability of RNA to bind ligands Dai and Kae. Compared to the inclusion complexes formed between Kae and dsRNA with UUU triplets, RNA that excluded UUU exhibited the anticipated binding propensity to associate with Kae. We found that Kae binding to RNA without UUU triplets not only had lower BE, but superior probabilities. Correspondingly, another important conclusion can be drawn by comparison with the inclusion complexes of Dai with RNA duplexes without UUU triplets. It may be inferred that the structure of inclusion complexes for Dai binding to RNA molecules without UUU triplets is present in the preferred binding mode as well.To verify the results, the eight dsRNAs sequences selected for the study model were related to a conserved Y UUU triplets and more G bases in the N1 RNA sequence without UUU triplets. Moreover, the specificity remains when initial structures of the selected RNA fragments contain more contiguous U base pairs. Therefore, the eight and nine base pairs are chosen from two RNA fragments, 5′-(UAAAAAAG)2-3′ (U5) and 5′-(GAUUUUUCG)2-3′ (U6), from the second and third RNA sequences given in this work, respectively. For example, the lowest BE of U6b was 0.82 kcal mol−1 lower than that of U6a  even if U5a had a higher frequency of 54% (PU6b = 28%).For instance, the interaction energy for the complex of N1a  decreased markedly compared with that of U1a ), corresponding to the lowest BE of −5.64 kcal mol−1 for N1b and −4.83 kcal mol−1 for U1b. This phenomenon might be ascribed to the specificity of the structures of RNA containing Visualization of the docking into the minor groove of RNA duplexes reveals that Kae and Dai mainly bind dsRNA depending on hydrogen bonds between ligands and RNA, indicating preferential binding for Kae or Dai to a stem-loop structure of the frameshift on JEV RNA. Visualization of the dockings also reveals that the top ranked conformation and binding sites are characterized by containing a special sequence of three base (CGG or AGG) when ligands are docked into the RNA. The inclusion complexes of Kae with RNA are more stable than those with Dai, in agreement with the results of ESI-MS and ITC assay, despite the different measurement principles.Many flavonoids exert their antiviral effects through rings that bind to the viral proteins, or interaction with viral RNA and efficiently blocking viral uncoating. This is the basis of designing new and more efficient antiviral drugs, and their effectiveness depends on the mode and affinity of the binding It is noteworthy that ESI-MS, ITC and docking data for Kae binding to fsRNA are in excellent agreement with the cell studies. The binding characteristics obtained for Kae and the relation to the frameshift structure of RNA suggest that Kae influences frameshift efficiency in virus-infected cells through interfering with a downstream RNA pseudoknot structure. Furthermore, Kae might affect expression of flaviviral NS1', which is involved in viral neuroinvasiveness. Therefore, the programmed translational frameshift site might be the target for Kae as an antiflaviviral drug.In this study, we investigated the ability of Kae and Dai, two natural plant extracts, to suppress JEV replication and E viral protein expression in BHK21 cells. Our results suggest that Kae is more promising than Dai for its anti-JEV effects. In addition, further investigations of the binding properties of structurally different flavonoids to RNA showed that Kae possessed relatively stronger binding affinity for RNA and formed non-covalent complexes more easily. Our results may elucidate new aspects of molecular targets for anti-JEV chemotherapeutics and facilitate our understanding of flavonoid transport in cells and prediction of their antiviral activities by focusing on their structural features. The results are helpful for a better understanding of antiviral mechanisms and have broad applications in dietary prevention of viral infectious diseases.15H10O6 XLogP3,1.9; H-Bond Donor, 4; H-Bond Acceptor, 6, Mr.286.23) and Dai -7- oxychromen-4-one) were purchased from Sigma . For cell experiments, Kae and Dai were both dissolved in DMSO and finally diluted in culture medium. To avoid toxicity or interference from the solvent, the maximum concentration of DMSO in the medium was <0.1%. A downstream fsRNA pseudoknot structure, a stem-loop structure,  of NS1' ribosomal frameshift event were chemically synthesized and purified by HPLC (Integrated Biotech Solutions Co. Ltd.). The two complementary segments of fsRNA3 were also synthesized using the above methods: fsRNA1  and fsRNA2 . fsRNA1, fsRNA2 and fsRNA3 were selected to study interaction with Kae or Dai.Kae (C2. The JEV strain SA 14-14-2 (GenBank accession No. AF315119) used in this study was stored in our Department of State Key Laboratory for Molecular Virology and Genetic Engineering . JEV was propagated in BHK21 cells and stored at −80°C before use.BHK21 cells  were propagated and maintained in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin and 100 µg/ml , streptomycin) at 37°C with 5% CO50 values were defined as the concentration that caused 50% cytotoxicity. Maximum non-toxic dose was determined microscopically by observing morphological changes after 72 h incubation.Cellular toxicity of Kae and Dai were tested according to a previously reported cell viability assay Kae and Dai were tested for their antiviral activity against JEV using the MTS assay with monolayer cultures of BHK21 cells grown in DMEM. Cells were infected with 0.1 MOI JEV in 96-wells plates. After 2 h adsorption at 37°C, plates were washed and the medium replaced with DMEM containing 2% fetal bovine serum and different concentrations of Kae and Dai. After 72 h, 20 µl MTS/PMS was added and the absorbance measured at 490 nm test wavelength and 690 nm reference wavelength using a plate reader. Experiments were also performed by incubating with 5–100 µM Kae or Dai for 2 h, and then infecting with 0.1 MOI JEV. After 72 h incubation at 37°C, absorbance was measured with the MTS method as described above.tv−Acv)/(Acd−Acv)×100%. Atv represents the absorbance of the test compounds with virus-infected cells. Acv and Acd represent the absorbance of the virus and cells control, respectively. The EC50 was calculated by regression analysis.VIR was calculated as  and fixed with 80% acetone for 15 min at room temperature. After washing three times with PBS (pH 7.4), the cells were incubated with JEV E-D3 monoclonal antibody  diluted 1∶400 in PBS (pH 7.4) at 37°C for 1 h. After washing with PBS (pH 7.4), the cells were incubated with the secondary antibody DyLigh594-conjugated goat anti-mouse IgG  diluted 1∶200 in PBS (pH 7.4) at 37°C for 1 h. After washing with PBS (pH 7.4), the cells were stained with 500 nM DAPI solution for 10 min at room temperature. After washing three times with PBS (pH 8.0), the plates were observed by fluorescence microscopy .BHK21 cells were grown on 24-well plates. Cell monolayers were infected with JEV at 0.1 MOI/0.5 ml for 2 h. The solution was removed and replaced with DMEM containing 50 µM Kae or Dai. At 24 h after virus inoculation at 37°C under 5% COIn the follow-up study, 24-well plates were used to test JEV mRNA and protein expression. The experiment was divided into four groups with four replicates for each compound: (1) mock infection group: mock infection for 48 h without compound treatment; (2) pretreatment group: cell monolayers were pretreated with 25 µM Kae or Dai for 2 h, and then infected with 0.1 MOI JEV for 48 h; (3) pre-infection group: cell monolayers were infected with 0.1 MOI JEV for 2 h and then treated with 25 µM Kae or Dai for 48 h; and (4) normal control group: healthy cells without any treatment.5′-CCTCCGTCACCATGCCAGT CTTAG-3′ and reverse, 5′-TTCGCCATGGTCTTTTTCCTCTCG-3′ to amplify a 133-bp length in the region of nt 3976–4108 in JEV strain genome sequence, Tm: 56.8°C. This segment was cloned into pGEM-T Easy vector to construct the standard plasmid, used to obtain a standard curve. In the 48 h pretreatment or pre-infection group, viral RNA was extracted from each well by using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. An RT reaction was carried out using Superscript First-Strand Synthesis System (Invitrogen) in a 20-µl reaction mixture using 1–2 µg total RNA, according to the manufacturer's protocol. Real time RT-PCR was conducted using ABI Prism 7000 Real-time PCR System (Applied Biosystems). Reactions were performed in a 50 µl volume that contained 2 µl cDNA, 1 µl each primer and 25 µl Power SYBR Green PCR Master Mix (Applied Biosystems). The absolute quantity of viral RNA was calculated by using the standard curve, and melting curve analysis was performed to verify the authenticity of the amplification.The following primers were used: forward, −1 each of aprotinin, pepstatin, and leupeptin; 1 mM each of EDTA, phenylmethylsulfonyl fluoride, dithiothreitol, and sodium fluoride; pH 7.4). The lysates were centrifuged at 10,000× g at 4°C for 15 min and the supernatant collected and stored at −80°C until use. The protein content was determined by the Bradford method. Proteins (20 ng) were mixed with electrophoresis buffer, boiled for 5 min, separated on 12% SDS-PAGE, and transferred to nitrocellulose membranes (Amersham). Membranes were blocked with 10% (w/v) non-fat dried milk in PBS with 0.2% (v/v) Tween-20 (PBST) for 1 h, and incubated overnight with primary mouse anti-JEV E-D3 monoclonal antibody , β-actin monoclonal antibodies  and mouse anti-JEV NS1 monoclonal antibody  diluted in blocking solution at 4°C. Membranes were rinsed three times with PBST, and incubated with the secondary goat anti-mouse IgG conjugated with IRDye700 fluorescein  for 1 h at room temperature. Membranes were rinsed twice with PBST and once with PBS again. Subsequent analysis was performed with a Li-COR Odyssey system and quantified using Odyssey infrared imaging software, with β-actin as a loading control.BHK21 cells were grown on 24-well plates. Cell monolayers were pretreated with 25 µM Kae or Dai or infected with 0.1 MOI JEV before treatment. After 48 h, JEV E, NS1 and NS1' proteins were detected by western blotting. Harvested cells were washed three times with PBS, lysed on ice in lysis buffer . Fluorescence was excited at 405 nm with an argon laser and emitted at 480–500 nm. Three dimensional representations of cells were constructed by the generation of sections in the z-dimension and compiled into z-stacks.ESI-MS was utilized to investigate the binding stoichiometry of small molecules to fsRNA1 (R1) and fsRNA2 (R2). Mass spectra were performed on a Thermo Finnigan LTQ Advantage in the negative ion mode. The heated capillary temperature of the electrospray source was set to 250°C, and the voltage on the heated capillary was 11 V. Full scan mass spectra were recorded in an m/z range from 500 to 2000. The relative intensities of the free and bound RNA in the mass spectra were assumed to be proportional to the relative abundances of these species in solution.200 calorimeter . In the ITC experiment, 8 µM Kae or Dai was loaded into the cell with 100 or 200 µM RNA in the titrating syringe, depending on the binding affinities of the compounds. RNA and flavonoids were dialyzed into the same buffer . The titration experiments were performed at 25°C with an initial 0.4-µl injection for 4 s, followed by 20 2-µl injections of 5 s duration. The spacing between injections was 120 s. The stirring speed during the titration was 500 rpm. Equilibrium association constant (Ka) was obtained by fitting experimental data to a “one set of sites” binding model using the Origin 7 software package . Correction for the enthalpy of RNA dilution was carried out by subtracting a straight-line linear fit from the last three data points of the titration, after the interaction had reached saturation.The binding affinities and enthalpies of the fsRNA3 (R3) and flavonoids relevant to Kae and Dai were measured by an ITC5′-UUUCCUUCUUU UUAUUUUUCUUUCCACCCAUUAAUUCC-3′), as in the control ssRNA. The three dimensional structures of target R1, R2, R3 and R4 were constructed by Accelrys Discovery Studio 2.1. The geometry of the ligand molecules was taken from NCBI database (http://pubchem.ncbi.nlm.nih.gov), which based on X-ray structures. The grid map of 126×64×66 points and a grid-point spacing of 0.558 Å were used during the docking process. During the dock processes, the grid map for R/R3 and R1/R2 were 126×126×126 and 66×126×100 points, and grid-point spacing for R/R4, R1 and R2 were 0.436 Å, 0.525 Å and 0.508 Å, respectively. The initial structures of the selected eight dsRNA fragments were the 20 base pairs shown in UUU triplets assigned as U1–U4, and the others without UUU triplets were assigned as N1–N4, generated using the Nucgen program of the Amber package The two ligand molecules were docked into the RNA using the AutoDock 4.0 program (Scripps Research Institute) to investigate further the interaction of the two ligands with fsRNA. Each docking was performed twice and each operation screened 250 conformations that could have been advantageous for docking, namely each docking could have 500 preferred conformations. The sequences of the selected three fsRNA  are shown in reagents part, in which R1 and R2 were two fragments of R3 (two arms of a stem-loop structure in fsRNA). The single-stranded RNA  contained no G base within its sequence Click here for additional data file.Figure S2ESI-MS full-scan spectra of mixtures of fsRNA1 and Kae at diverse molar ratio of flavonoid∶RNA. (A) Mass spectrum offsRNA1 with Kae at 5∶1 molar ratio of Kae∶RNA. The solution was prepared by mixing equal volumes of 50 µM methanol solution of Kae and 10 µM (150 mM ammonium acetate) solution of fsRNA1; fsRNA1 alone, and 1∶1, 2∶1, 3∶1, 4∶1, 5∶1, 6∶1 and 7∶1 complexes are indicated (B) Mass spectrum offsRNA1 with Kae at 10∶1 molar ratio of Kae∶RNA. The solution was prepared by mixing equal volumes of 100 µM methanol solution of Kae and 10 µM (150 mM ammonium acetate) solution of fsRNA1; 1∶1, 2∶1, 3∶1, 4∶1, 5∶1, 6∶1, 7∶1 and 8∶1 fsRNA1/Kae complexes are indicated.(TIF)Click here for additional data file.


Its sequence and structural features determine its involvement in functional interactions with viral RNA-dependent RNA polymerase and distant RNA domains of the viral genome. This work reports the use of an in vitro selection strategy to select aptamer RNA molecules against the complete HCV-CRE. After six selection cycles, five potential target sites were identified within this domain. Inhibition assays using a sample of representative aptamers showed that the selected RNAs significantly inhibit the replication (>80%) of a subgenomic HCV replicon in Huh-7 cell cultures. These results highlight the potential of aptamer RNA molecules as therapeutic antiviral agents.Hepatitis C virus (HCV) replication is dependent on the existence of several highly conserved functional genomic RNA domains. The  Some 3% of the World’s population is infected with hepatitis C virus (HCV), the major etiological agent of non-A non-B hepatitis ,2. No prcis-acting replication element (CRE) in the 3' end of the NS5B coding region. Recognised as an important partner in viral RNA synthesis [The HCV genome is a 9,600 nt-long positive strand RNA molecule . It encoynthesis ,14, it fynthesis ,14,15,16ynthesis . The speynthesis , to the ynthesis ,19,20 . The T7 promoter region is underlined, the restriction site for EcoR1 is shown in italics, the lower case letters indicate complementary residues, and N30 denotes 30 consecutive A, G, C or T nucleotides, thus providing for different RNAs. A mixture of 3 nmol of each oligonucleotide was heated at 95 °C for 2 min, followed by slow cooling to room temperature. Full, double-stranded DNA was obtained by extension for 1 h at 37 °C with 2.5 U of the Klenow fragment of DNA polymerase  in the presence of 2.5 mM of each dNTP. It was then PCR-amplified (7 cycles) using Taq DNA polymerase  in the presence of the primers 3' PCR-CRE (TCGACTACTCTTTAATTCGCTACCT) and 5' PCR-CRE (GCTATGAATTCTAATACGACTCACTATAGGGATATTATAGTACATAAA). The resulting dsDNA was used as a template for in vitro transcription using the T7 RiboMAXTM transcription kit  to yield the initial RNA population.The template for the synthesis of the RNA population was assembled by the annealing and extension of the 5'aptamerCRE—GCTAT194 RNA fragments. These fragments were obtained by in vitro transcription of the SalI linearised plasmid construct pUC18-T7HCV9181-9371 [EcoRI and SalI restriction sites of the pUC18 vector (Fermentas). HCV-CRE194 was internally biotinylated during its synthesis by adding 188 µM of biotin-16-uridine-5'-triphosphate  to the reaction mix. The amount of biotin-modified nucleotide added was calculated to yield approximately one biotin residue per molecule [2) and stored at 4 °C until use. Prior to the initial selection step, 6.4 nmol of the RNA population (P0) were passed with binding buffer through a pre-equilibrated column lacking the biotinylated target RNA, and incubated for 30 min at 25 °C, to prevent false-positives due to non-specific binding to the column matrix. Aliquots of 4.5 nmol of the resulting RNA molecules were loaded, in 1 mL of binding buffer, onto the target RNA-prepared column and incubated for 30 min at 25 °C. Unbound molecules were discarded by washing three times with 5 mL and once with 10 mL of cacodylate buffer at 25 °C. All RNA molecules bound to the column were eluted by washing with 10 mL of binding buffer heated at 95 °C. They were then concentrated to 10 µL with Centricon YM-10 .The isolation of active RNA molecules for binding to the viral RNA was performed in a sepharose-streptavidin column  loaded with biotinylated viral HCV-CRE181-9371 . The lat181-9371  between molecule . 38 µmolTM III reverse transcriptase  in the presence of 10 pmols of 3' PCR primer and 10 mM of each NTP for 1 h at 55 °C. The reaction was stopped by heating for 15 min at 70 °C. cDNA was then amplified with Certamp mix DNA polymerase (Biotools B&M Labs) in the presence of 25 pmol of the 3' PCR-CRE and 5' PCR-CRE primers. The resulting dsDNA product was used as a template for in vitro transcription using the T7 RiboMAXTM transcription kit (Promega), and the resulting RNA population (P1) either subjected to a new selection round or cloned in the pGEMT-Easy vector (Promega) for sequence analysis. Selection conditions were changed between selection cycles by reducing the target:RNA pool ratio from 5:1 to 1:2 as well as increasing the incubation temperature to 37 °C in round 5, and by reducing the ionic strength from 10 mM MgCl2 to 2 mM MgCl2 in round 7. The new conditions were maintained in the following cycles.The eluted, concentrated active RNA molecules were retrotranscribed using SuperScript−1 G-418 at 37 °C in a 5% CO2 atmosphere. Twenty hours before transfections, ~80,000 cells were seeded onto a 24 well plate in DMEM supplemented with 20% FBS. Cells were transfected using TransFectinTM lipid reagent (Bio-Rad) plus 3 µg of the aptamer RNA molecule, and harvested 20 h post-transfection. Viral HCV RNA was quantified by real time RT-PCR as previously described [HCV replication assays were performed using a human hepatocarcinoma cell line harbouring an HCV subgenomic replicon system  as pescribed . Brieflyescribed . The fluescribed . Quantif18 sequence variants, the experimental constraints limited the number to 1 × 1015. P0 RNA molecules were binding-challenged against the internally biotinylated HCV-CRE194, a 194 nt-long RNA fragment of the HCV 1b genome (The starting RNA population (P0) consisted of 75 nt-long RNA molecules containing a 30 nt-long variable region flanked by fixed sequences that act as primer binding sequences necessary for the amplification of active molecules see . Althougb genome B, fixed b genome . This se194 viral RNA after six rounds of selection (P6) were cloned. Sequence analysis of 43 clones allowed the identification of selected sequence motifs common to several molecules; these were used to classify the selected molecules into five different groups or families . None of the identified consensus sequences were counter selected neither new consensus motifs were selected from P7 to P9. Nevertheless, this analysis was able to identify consensus sequences extended by 1 or 2 nucleotides, and clarified nucleotides whose identity remained ambiguous in the P6 round . Thus, tin vitro selection strategies for identifying RNA molecules as tools for the development of anti-HCV aptamers and to identify potential viral therapeutic targetsTo test the inhibitory activity of the selected molecules on HCV replication, and therefore to evaluate their therapeutic potential, Huh-7 cells that allow the autonomous replication of a subgenomic HCV 1b Con1 replicon were independently transfected with representative aptamers of each selected group, and the number of positive HCV strands quantified by real time RT-PCR at 20 h see . TransfeThis work reports the selection of aptamers, belonging to five different groups, which appear to interact with complementary sequences in the HCV-CRE domain. Representative aptamers showed potent (>80%) inhibition of the replication of HCV replicons, thus demonstrating their potential as antiviral agents. These results confirm the feasibility of using conserved structural RNA genomic domains as targets in new therapeutic strategies.