Drosophila Pumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulated in vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation by DOI:http://dx.doi.org/10.7554/eLife.17096.001 Molecules of DNA contain the instructions needed to make proteins inside cells. Proteins perform many different roles and each needs to be produced at the right time and in the right amounts to enable the cells to survive. The DNA is first copied to make molecules of ribonucleic acid (RNA), which are then used as templates to make the proteins. One way to control protein production is to regulate the RNA molecules. A family of proteins called RNA-binding proteins can recognise and bind to specific RNA molecules and determine whether a RNA molecule is destroyed, used to produce proteins, or stored for later use. In effect, these RNA-binding proteins act as switches that turn protein production on or off.Nanos and Pumilio are two RNA-binding proteins that are found in many organisms, including humans and other animals. Genetic studies in fruit flies show that these two proteins influence development, the nervous system and the behaviour of stem cells by switching off the production of certain proteins. To investigate how Nanos and Pumilio work together to regulate protein production, Weidmann, Qiu et al. used a variety of techniques to study the activity of these proteins in cells taken from fruit fly embryos.The experiments reveal that Nanos acts like a clamp to hold Pumilio close to specific RNAs, which allows Pumilio to switch off the production of the corresponding proteins. The presence of Nanos allows Pumilio to regulate RNAs that it cannot bind to alone. Therefore, the experiments show that by working together with Nanos, Pumilio is able to regulate a wider variety of RNAs than it would otherwise be able to. These findings provide a molecular understanding for why fruit fly mutants that lack Nanos or Pumilio have severe body defects and reduced fertility. The next challenge is to identify the specific RNAs targeted by Nanos and Pumilio in stem cells, the nervous system and during development.DOI:http://dx.doi.org/10.7554/eLife.17096.002 Drosophila melanogaster Nanos (Nos) and Pumilio (Pum), in developmental patterning, fertility, and nervous system development and function. We use this system to explore how two RBPs cooperatively define regulatory networks.Post-transcriptional gene regulatory mechanisms are widespread and mediated by hundreds of RNA-binding proteins (RBPs) that interact dynamically with target mRNAs . Understfem-3 mRNA-binding factor (FBF), or PUF family, of eukaryotic RBPs (hunchback (hb) mRNA, which requires collaboration with Nos  RBP  RBP . Maternaa embryo , whereasa embryo . Where t(hb mRNA . In the hb mRNA by Pum and Nos is dependent on two Nanos Response Elements (NREs) in the 3´UTR  in primordial germ cells and germline stem cells  in the nervous system . The ternary complex structure reveals critical protein-protein and protein-RNA interactions between Nos, Pum and RNA. We also determined a 1.14 Å crystal structure of a binary complex of Pum and a hb PRE2 RNA (5´-UGUACAUA)  , which ehila Pum  and zebrhila Pum , allowedPum with the C-terminal α helix of Nos , the C-terminal region of Pum undergoes notable changes. Loop residues between repeats R7 and R8 rearrange in the ternary complex to promote interaction of F1367x of Nos . In addix of Nos . Since thb NRE2 RNA complex and cooperatively strengthens the binding affinity of Pum for hb RNA. The RNA-binding domain of Pum (PUM-HD) bound hb NRE2 RNA, and addition of equimolar Nos ZC to the binding reaction further retarded the hb NRE2 RNA mobility, indicating formation of a Nos-Pum-RNA ternary complex , we demonstrated that Nos ZC binds stably and tightly to a Pum- complex . Disrupt complex . In addi complex . Nos did its own , even at its own . We appl complex  and incry 3-fold . These dNos to E385Nos) and the non-RNA-binding convex surface of Pum in repeats R7 and R8 . Expression of full-length Nos resulted in 50% repression of WT reporter activity compared to the negative control Halo-tag protein alone  is important for ternary complex formation and repression activity. Substitution of both the −1 and −2 positions of ociation , so we dposition . We thenin alone , similarsequence , mt1. Insequence , consistCyclin B (CycB) NRE contains a PRE that diverges from the consensus with uracils in place of adenines at positions +6 and +8, and it has a different NBS sequence  prevented stable binding of Pum alone  abolished both Pum binding and ternary complex formation . The pros region . Pum binPumilio1 , where bPumilio1 . In cont4 and +5 . Pum binPRE2 RNA . However complex . Thus, N selection, high-throughput sequencing of RNA, and sequence specificity landscapes)    . Pum alodscapes) . Strikindscapes) , 3, consdscapes) , the NosDrosophila embryos and adults (i.e. both Nos-Pum and Pum motifs), such as hb mRNA. Most mRNAs with Pum motifs lack the upstream NBS, suggesting that they may be targeted by Pum alone or with other partners. Finally, we performed gene ontology analyses of target mRNAs bearing Pum or Nos-Pum motifs and observed that significantly enriched terms match the known functions of Nos and Pum, including body pattern formation and germline development, and also suggest new collaborative functions including regulation of cell division, receptor protein signaling, and cell fate determination  or the 1x hb NRE2 ACA mutant that abrogated Pum binding and ternary complex formation. Background repression of the hb NRE2 ACA reporter originating from binding to degenerate motifs in the RnLuc transcript may have limited the sensitivity of the 1x NRE reporters.To evaluate the ability of Nos to enhance repression of mRNA targets with different NREs, we measured repression of reporter mRNAs containing the NREs for which we had examined ternary complex formation . Because protein . We founh 200 ng . Nos alshb NRE2 reporter . As a consequence, Nos appears to expand the Pum target mRNA repertoire beyond those with perfect 5´-UGUAHAUA core PRE consensus sequences by stabilizing Nos-Pum-RNA complexes.To improve the signal-to-noise ratio, we increased the number of NREs in each reporter to three adjacent sites, which dramatically increased cellular repression with lower amounts of Nos expression: 10 ng of transfected Nos expression vector resulted in 79% repression for the 3x reporter  versus 4reporter . Backgrockground . Thus, Nin vivo target RNAs, and expression patterns. Furthermore, as the regulatory targets of individual RBPs are established, it will be crucial to examine overlapping networks to identify cooperative regulation by multiple RBPs. However, our current knowledge of how multiple RBPs collaboratively regulate an mRNA is limited. In this context, the mechanisms of combinatorial regulation by Nos and Pum revealed in this study, which visually capture decades of genetic and biochemical data, provide a foundation to derive general principles for how RBPs co-regulate target mRNAs.Hundreds of proteins that bind mRNAs in cells have now been identified, and large-scale efforts to find their specificities and mRNA targets are underway . To undeNos uses its three functional regions: N, Z, and C  to eliciProtein interactions between Nos and Pum further promote activity of the repression complex and, as revealed by the crystal structures, are mediated by the C-terminal region of Nos and repeats R7 and R8 of Pum. Incorporation of Nos into the Pum-RNA complex induced conformational changes in Pum that added additional RNA contacts. Protein interaction modules may also strengthen regulation by enhancing recruitment of cofactors. The N-terminal region of Nos, not included in our crystal structures, increases repression by the Nos-Pum complex by recruiting the CCR4-NOT deadenylase complex , which snos mutant alleles identified, nosL7, causes a lethal loss of abdominal segments during embryogenesis and encodes a Nos protein with a deletion of residues 376–382 near the C terminus  insertion in the R7-R8 loop region that could augment protein contacts with vertebrate Nos proteins. Interestingly, deletion of these residues allows human Pum1 to interact with Drosophila Nos  adds upstream RNA sequence specificity, which alters mRNA target selection of the primary RBP (Pum) and diversifies the range of target sequences.We found that Nos stabilizes the ternary complex and adds 5´ sequence specificity. Nos specifically recognizes the 5´ sequence only in the context of the ternary complex and also induces a localized conformational change of Pum that adds contacts with the phosphate backbone of nucleotides -1 to -4. Previous reports indicated that the Nos tandem ZFs in isolation exhibit non-specific RNA binding . With ou in vivo  and tern in vivo  to the l in vivo , therebymsl2 RNA by Sex lethal (Sxl) and Upstream of N-Ras (Unr)  , yet thein vivo and explain broadened regulation by Nos and Pum in Drosophila embryos and adults.Nos appears to dramatically alter the repertoire of transcripts bound by Pum. Our analysis of Pum target mRNAs using the SEQRS-derived Nos-Pum motif indicates that the majority of Pum target mRNAs contain the Nos-Pum motif but lack the full canonical PRE Pum recognition sequence. This suggests that, in addition to strengthening Pum repression activity, Nos can alter the identity of mRNAs regulated by Pum. Moreover, enrichment of specific gene ontology terms in the mRNAs bearing the Nos-Pum motif suggest that Nos can alter the biological functions controlled by Pum . These aDrosophila embryo, the Nos protein gradient is highest at the posterior end where hb expression must be repressed for abdomen formation    protein contributes to repression of A motif) . MoreoveA motif)  and the otein 1) . Intriguotein 1) . Thus, totein 1) . Our resIn conclusion, the partnership of Nos and Pum illustrates the profound influence combinatorial control can have on gene expression, demonstrating how location-specific regulation is achieved through the action of RBPs with different spatial distributions, how the addition of a second RBP shifts the RNA recognition motif of the first RBP to modulate target selection, and how regulatory sensitivity can be adjusted by the number and quality of binding sites within a target mRNA and by RBP expression levels. This paradigm emphasizes the importance of understanding how the multitude of RNA-binding factors collaborate to control mRNA stability, translation, processing, and localization.hb 3´UTR were previously described (Drosophila Nanos cDNA (NP_001262723.1) into the XbaI site of pIZ Halo-tag, which contains an N-terminal Halo-tag with a TEV protease cleavage site and a C-terminal V5 epitope. The Nos sequence was amplified from whole fly cDNA and corresponds to isoform Nos-PB, which lacks an alternatively spliced exon encoding a 19 amino acid sequence aa14-VGVANPPSLAQSGKIFQLQ-32 present in the N-terminus of Nos-PA (NP_476658.1). For consistency with the originally identified domain boundaries and mutants, the reported amino acid positions correspond to Nos-PA (e.g. C319Y and C354Y correspond to C300Y and C335Y of Nos-BP). Using the Nos plasmid as a template, the C319Y, C354Y, I376A, M378A, and I382A mutations were generated via QuikChange site-directed mutagenesis . The following mutations and truncations were created using inverse PCR from the Nos plasmid template: F321A, N325A, Y352A, T366A, K368Q, Y369A, NZ (aa1-373), ZC (aa289-401), N (aa1-294), Z (aa289-373), C (aa374-401), Δ376–382 (aa1-375 + 383–401), Δ383–393 (aa1-382 + 394–401), and Δ394–401 (aa1-393).Reporter plasmids pAc5.1 FFluc, pAc5.1 RnLuc escribed , as was Drosophila Pum RNA-binding domain (amino acids 1091–1426) and Nanos ZF domain (amino acids 289–401) were subcloned into the pSMT3 vector with an N-terminal His6-SUMO tag .Bacterial expression and purification of recombinant Pum or Nos for EMSAs was achieved using pFN18K (Promega) with an N-terminal Halo-tag and a TEV cleavage site. The Pum RNA-binding domain sequence, encoding aa1091-1426 of NP_001262403.1, including an N-terminal triple FLAG tag was inserted into pFN18K to create pFN18K Pum plasmid. QC-SDM was used to generate the F1367S Pum mutant and the RNA-binding defective mutR7 (wherein RNA recognition amino acids are mutated: S1342A N1343A E1346A) plasmids. Inverse PCR was used to create Pum Q1337A from the wild type template. The same strategy was applied to generate pFN18K NosZC and the C319Y and C354Y mutant vectors with appended C-terminal V5 epitopes. For crystallographic studies, hb NRE2, 3x CycB NRE, 3x hb NRE2 +3G, and 3x hb NRE2 ACA reporters were generated in an identical fashion using oligos with three repeated NRE elements.Reporters used for Nos enhancement of Pum repression in 32P body-labeled, antisense RNA probe was created by in vitro transcription. The following primers were used to amplify templates for creation of RnLuc RNA probes. The T7 promoter sequence is underlined and gene specific regions are bolded.RNA was purified from 2 million D.mel-2 cells transfected with the plasmids indicated in GGGCGAGGTTAGACGGCCTACCCTRnLuc 3´ forward primer: 5´-GGATCCTAATACGACTCACTATAGGGCGGCCAGCGGCCTTGGRnLuc 3´ reverse primer: 5´-32P 5’ end-labeled DNA oligo with the following sequence.The 7SL RNA was detected on northern blots using a 7SL Probe: 5´-CACCCCTGGCCCGGTTCATCCCTCCTTAGCCAACCTGAATGCCACGG.The radioactive blots were exposed to a storage phosphor screen. The signal on the screen was captured with a Typhoon Trio imager  and subsequently quantified using ImageQuant TL Software . Each RnLuc signal was normalized to the 7SL signal. Statistical analysis of Northern blot data from three replicate cell cultures is reported in The oligos used to create RnLuc 1xNRE reporter plasmids used for Nos enhancement of Pum repression are as follows :hb NRE2 Forward: 5´-TCGACGAAAATTGTACATAAGCChb NRE2 Reverse: 5´-GGCCGGCTTATGTACAATTTTCGhb NRE2 +3G Forward: 5´-TCGACGAAAATTGgACATAAGCChb NRE2 +3G Reverse: 5´-GGCCGGCTTATGTcCAATTTTCGhb NRE2 ACA Forward: 5´-TCGACGAAAATacaACATAAGCChb NRE2 ACA Reverse: 5´-GGCCGGCTTATGTtgtATTTTCGhb NRE1 Forward: 5´-TCGACCAGAATTGTATATATTCGhb NRE1 Reverse: 5´-GGCCCGAATATATACAATTCTGbcd NRE Forward: 5´-TCGAAAGTGATTGTAGATATCTAbcd NRE Reverse: 5´-GGCCTAGATATCTACAATCACTTCycB NRE Forward: 5´-TCGAGACTATTTGTAATTTATATCCycB NRE Reverse: 5´-GGCCGATATAAATTACAAATAGTChb NRE2 -1C Forward: 5´-TCGACGAAAAcTGTACATAAGCChb NRE2 -1C Reverse: 5´-GGCCGGCTTATGTACAgTTTTCGhb NRE2 -2C Forward: 5´-TCGACGAAAcTTGTACATAAGCChb NRE2 -2C Reverse: 5´-GGCCGGCTTATGTACAAgTTTCGSynthetic RNAs (IDT) used in EMSA experiments include the following (with PRE elements underlined and mutations in lowercase bold):hb NRE2 RNA: 5´-Cy5-rUUGUUGUCGAAAAUUGUACAUAAGCC.Cy5- hb NRE2 RNA: 5´-rAAAUUGUACAUAAGCChb NRE2 +3G RNA: 5´-rAAAUUGgACAUAAGCChb NRE2 ACA RNA: 5´-rAAAUacaACAUAAGCChb NRE1 RNA: 5´-rGAAUUGUAUAUAUUCGbcd NRE RNA: 5´-rUGAUUGUAGAUAUCUACycB NRE RNA: 5´-rUAUUUGUAAUUUAUAUChb NRE2 -1C RNA: 5´-rAAAcUGUACAUAAGCChb NRE2 -2C RNA: 5´-rAAcUUGUACAUAAGCC6 cells/mL). For Nos experiments, 10 ng of pIZ Nos expression vector (unless otherwise noted) was balanced with empty pIZ vector for a total mass of 200 ng. For each Nos transfection gradient, pIZ was also used to balance the total mass of transfected expression vector to 200 ng. Transfection conditions for the experiment in hb 3'UTR, and 100 ng of the indicated protein expression plasmid. Total transfected DNA was set at 3 µg per well, balanced by pIZ plasmid. Fugene HD (Promega) transfection reagent was used at a 4:1 ratio (µl Fugene HD: µg of DNA), prepared in 150 µL Sf-900 III SFM media and incubated for 15 min at room temperature prior to application to 2x106 cells in 2 ml of media.D.mel-2 cells were cultured at 28°C in Sf-900 III SFM (Life Technologies) with 50 Units/mL penicillin and 50 µg/mL streptomycin. Transfections were performed as described  using EfLuciferase reporter assays were performed using the Dual-Glo assay (Promega) and a GloMax Discover luminometer. A relative response ratio (RRR) was calculated from RnLuc/FFLuc signals for each sample. Percent repression values were calculated as previously described . The pIZE. coli cells (Promega) in 2xYT media with 25 µg/mL Kanamycin and 2 mM MgSO4 at 37°C to OD600 of 0.7–0.9, at which point protein expression was induced with 0.1% (w/v) rhamnose for 3 hr. Cell pellets were washed with 50 mM Tris-HCl, pH 8.0, 10% [w/v] sucrose and pelleted again. Pellets were suspended in 25 mL of 50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 2 mM MgCl2, 150 mM NaCl, 1 mM DTT, 0.05% (v/v) Igepal CA-630, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml pepstatin, and 10 µg/ml leupeptin. To lyse cells, lysozyme was added to a final concentration of 0.5 mg/mL and cells were incubated at 4°C for 30 min with gentle rocking. MgCl2 was increased to 7 mM and DNase I (Roche) was added to 10 µg/mL followed by incubation for 20 min. Lysates were cleared at 50,000xg for 30 min at 4°C. Halo-tag containing proteins were purified using HaloLink Resin (Promega) at 4°C. Beads were washed 3 times with 50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 2 mM MgCl2, 1 M NaCl, 1 mM DTT, 0.5% [v/v] Igepal CA-630) and 3 times with Elution Buffer . After washing, beads were resuspended in Elution Buffer with 30 U of AcTEV protease (Invitrogen), cleavage proceeded for 24 hr at 4°C, and beads were removed by centrifugation through a micro-spin column (Bio-Rad).Recombinant Pum and Nos for EMSAs were expressed in KRX All RNA-binding reactions were performed in RNA-binding Buffer . Reactions were equilibrated for 3 hr at 4°C. 5% native polyacrylamide TBE mini-PROTEAN gels (Bio-Rad) were pre-run for 3 hr at 50V before loading 5 µl of each sample and then run at 50V for 2–2.25 hr at 4°C.hb NRE2 RNA, reactions contained 1 nM target RNA and the concentrations of PUM-HD and NosZC are as noted in d measurements shown in For EMSAs with fluorescently-labeled d measurements with radioactive RNA oligos, reactions contained 0.3 nM RNA that were labeled at their 5´ ends using T4 Kinase (NEB) with ATP [γ-32P] (Perkin-Elmer). Where indicated for hb NRE2 RNA, the concentration of NosZC was held constant at 400 nM while Pum was titration included concentrations of 0, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 75, 100, 150, and 200 nM. For all other RNAs tested in the presence of Nos, the reactions contained 1 µM NosZC while Pum concentration was titrated from 0, 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 750, and 1000 nM. Gels containing radioactive RNAs were dried onto Whatmann filter paper. The radioactive gels were then exposed to a storage phosphor screen (GE) for 16 hr.For Kd was calculated via nonlinear regression analysis for one site interaction with GraphPad Prism software .EMSAs were imaged with a Typhoon Trio imager  and subsequently quantified using ImageQuant TL software. Fraction bound values from three replicate EMSAs were plotted against titrated protein concentration, and KE. coli strain BL21-CodonPlus (DE3)-RIL (Agilent) at 16°C for 20 hr after induction with 0.2 mM IPTG. The cell pellet was resuspended in lysis buffer containing 20 mM Tris (pH 8.0), 0.5 M NaCl, 20 mM imidazole, 5% (v/v) glycerol, and 0.1% (v/v) β-mercaptoethanol. Both proteins were purified by a similar procedure involving four sequential steps: a Ni-NTA chelating column, Ulp1 overnight incubation to remove the His6-SUMO tag, a heparin column and lastly a Superdex 75 column . The Ni elution buffer contained 20 mM Tris (pH 8.0), 50 mM NaCl, 0.2 M imidazole, and 1 mM DTT. A gradient elution was run on the heparin column with buffer A containing 20 mM Tris (pH 8.0) and 1 mM DTT and buffer B containing additional 1 M NaCl. The Superdex 75 column buffer contains 20 mM Hepes (pH 7.4), 0.15 M NaCl, and 2 mM DTT. For the ternary complex formation, a 16-nt hb NRE2 RNA (5´-AAAUUGUACAUAAGCC) and a 14-nt CycB NRE RNA (5´-UAUUUGUAAUUUAU) were used, separately. Purified Pum and Nos were concentrated and mixed together with RNA in a molar ratio of 1:1:1.1. After overnight incubation at 4°C, the mixture was loaded onto a Superdex 200 10/300 GL column. Peak fractions containing ternary complexes were concentrated to OD280 ~ 7 in a buffer of 20 mM HEPES (pH 7.4), 0.15 M NaCl, and 2 mM DTT for crystallization. For the Pum-RNA binary complex, concentrated Pum (OD280~4.0) was incubated with 8-nt hb PRE2 RNA (5´-UGUACAUA) in a molar ratio of 1:1.2 on ice for 2 hr. The mixture was directly put into crystallization trays. Binary complexes were also formed with 12-nt hb NRE2 RNA (5´-AAAUUGUACAUA), but no crystals were obtained.Pum and Nos proteins (pSMT3 vectors) were overexpressed in hb NRE2 RNA complex were obtained by hanging drop vapor diffusion, mixing 2 μL of sample and 2 μL of reservoir solution  at 20°C. Crystals of Pum-Nos-CycB NRE RNA complex were obtained in the condition of 0.9 M (NH4)2SO4, 0.1 M MES, pH 5.6 by microseeding using the hb complex crystals. Crystals of Pum-hb PRE2 RNA binary complex were obtained in the condition of 15% (w/v) PEG 3350, 0.1 M bis-tris, pH 6.5 and 0.2 M NH4OAc. All crystals were transferred to a cryo-solution containing the reservoir solution with additional 15–20% (v/v) glycerol, and flash frozen in liquid nitrogen. X-ray diffraction data were collected at the SER-CAT Beamline 22-ID at the Advanced Photon Source, Argonne National Laboratories.Crystals of Nos-Pum-522 space group with one complex in an asymmetric unit. The structure of Pum-Nos-hb NRE2 RNA complex was determined by molecular replacement using the structures of Drosophila Pum (PDB: 3H3D) and zebrafish Nanos ZF domain (PDB: 3ALR) as the search model with Phaser. Iterative model building was done with COOT and Phenix  as the search model. Residues 1090–1426 in Pum and RNA bases +1 to +8 of PRE2 are modeled in the binary structure. Data collection and refinement statistics are presented in Crystals of Pum-Western blotting from luciferase assay samples was performed as previously described . Where iProtein extracts of D.mel-2 cells expressing Halo-tag fusions were prepared as previously described . Extractin vivo using the dataset from SEQRS was conducted as described with minor modifications  on the f In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.Drosophila Nanos acts as a molecular clamp that modulates the RNA- binding and repression activities of Pumilio" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by James Manley as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.in vivo tests of the predicted interactions, several predictions have already been born out by previously published results. For example, in vivo studies had implicated sequences upstream of the canonical Pumilio binding sites in hb RNA regulation. Similarly, the new data illustrate the importance of the Nanos C-terminus in 'clamping' Pumilio to RNA, which was so far mostly implicated by genetics, for Nanos function in hb regulation and less so in germ line development.The article by Weidman and colleagues addresses a long-lasting question regarding how the RNA binding proteins Pumilio and Nanos regulate their targets. While the structures of Pumilio bound to RNA targets had been identified a while ago, it remained unclear how Nanos may regulate target specificity for a Pumilio-Nanos-NRE tertiary complex. In a beautiful set of experiments connecting in vitro binding and translational repression studies with structural analysis these questions are addressed in the present paper. The analysis is thorough and the data are well controlled and illustrated. The authors are able to identify a new sequence upstream of the Pumilio binding sites important for Nanos contact with RNA in the presence of Pumilio. It also represents one of the few examples of how two RBP cooperate to achieve sequence specificity and how one RBP (nanos) can modulate the sequence-specificity of the other one Pumilio. While this study does not includeThough the structures and binding studies nicely illustrate how cooperativity works in this context, it would have been really worthwhile for authors to use this term early on and would not wait till the middle of their discussion.In addition, the paper would be improved if the interactions foundwere discussed in terms of an evolutionary comparison. How conserved are these contacts? Can one use the structures to predict cooperativity of two RBPs in other organisms? This is not touched upon and would be very valuable.Several issues should be clarified:Much of the analysis is a comparison between the binary Pum-RNA complex and the ternary Nos-Pum-RNA complex. The structure of Pum changes in the region where Nos binds to it and to the RNA element. However, in the binary complex, the RNA doesn't really reach that area. It is shorter and contains only the PRE. Therefore, would Pum assume this altered conformation in the presence of a longer RNA without Nos? Or might it be closer to the conformation it assumes with Nos? This is an important point and bears on the conclusions of this paper.It is very hard to see from the various figures, what is moving in Pum between the binary and ternary complexes. Perhaps a side by side of that area would be easier to follow.The deletions that are discussed on Results section, subsection “Nanos increases the binding affinity of Pumilio for hunchback RNA”, that affect repression involve cutting out a few residues sometimes in the middle of a helix and it's not clear how well the structure would retain its integrity. The extent of repression might correlate with what percentage of folded protein they have in this region. Does this correlate with Pum-RNA binding?The authors show a difference in the RNA structure between the hb and CycB complexes. However, the resolution is fairly low. How confident are they? What do the electron density maps look like in this region?In the discussion the authors mention that the ZFs in Nos are unique compared to other RNA-binding ZFs. In what way?Also in the discussion, the authors compare this cooperative interaction to that between Sxl and Unr. They say that this is also a cooperative interaction and two lines later they say it is additive. Which is it? If Sxl and Unr interaction is additive, then they are distinctly different.These concepts make the whole story here, so it's very important that this is written clearly. The article by Weidman and colleagues addresses a long-lasting question regarding how the RNA binding proteins Pumilio and Nanos regulate their targets.[…].Though the structures and binding studies nicely illustrate how cooperativity works in this context, it would have been really worthwhile for authors to use this term early on and would not wait till the middle of their discussion.Thank you for this suggestion. We have now incorporated the term “cooperativity” into appropriate locations within the Abstract and Results sections.In addition, the paper would be improved if the interactions foundwere discussed in terms of an evolutionary comparison. How conserved are these contacts? Can one use the structures to predict cooperativity of two RBPs in other organisms? This is not touched upon and would be very valuable.We have now addressed these interesting points in the Discussion:“Based on our current understanding of the Pum-Nos interface and phylogeneticcomparisons, the Drosophila Nos-Pum interaction is conserved among Dipteranorthologs, but it is not possible to predict conservation of the interaction in vertebrates.[…]Instead, the Nos and Pum contacts likely coevolved as the number of Nos and Pum homologs increased, perhaps restricting interactions between particular Nos and Pum proteins. Nos and Pum homologs may also interact indirectly, mediated by a bridging partner(s), as was suggested for C. elegans  and mouse .”Several issues should be clarified:Much of the analysis is a comparison between the binary Pum-RNA complex and the ternary Nos-Pum-RNA complex. The structure of Pum changes in the region where Nos binds to it and to the RNA element. However, in the binary complex, the RNA doesn't really reach that area. It is shorter and contains only the PRE. Therefore, would Pum assume this altered conformation in the presence of a longer RNA without Nos? Or might it be closer to the conformation it assumes with Nos? This is an important point and bears on the conclusions of this paper.hb RNA containing 4 additional upstream nucleotides. We therefore cannot rule out the possibility that the unfolding of the C-terminal α helix of Pum observed in the ternary complexes could be induced by a longer RNA as well as by Nos. Indeed the C-terminal region of Pum is involved in crystal packing in the binary complex that could stabilize the terminal α helix. We have edited the Results to clarify this point:We thank the reviewers for their insightful questions about the structure of the C-terminal region of Pum in the binary complex. We conducted crystallization screens, but did not obtain crystals of Pum in complex with a “Comparison of the ternary and binary complexes reveals that the addition of Nos and the upstream nucleotides induces localized conformational changes in Pum that promote Nos-Pum interaction and binding of Pum to RNA upstream of the core PRE site.”And“Since the upstream nucleotides were not present in the binary complex, the change in the structure of the C-terminal region of Pum may be induced by the presence of the upstream RNA and/or Nos. With these conformational changes, Pum and Nos interact with one another and together recognize RNA immediately 5´ of the core PRE motif.”Drosophila Pum, provide some indication that the C-terminal α helix remains formed in binary complexes with longer RNAs. We determined crystal structures of human Pum1 in complex with hb RNAs bearing five additional 5´ nucleotides. In one of the two Pum1:RNA complexes in the asymmetric unit, crystal packing influences the ordering of one additional upstream nucleotide. In the second complex, the upstream nucleotides were disordered and the C-terminal helix was formed as in the binary complex of Pum. The crystal packing in the area does not appear to restrict unfolding of the terminal helix or binding of the upstream RNA in the binary complex. The sequence of the C-terminal region that undergoes rearrangement bears only one substitution in Pum1 (N1421 is a leucine in human Pum1), making our observations in human Pum1 a reasonable model for Drosophila Pum.Although we feel it is not strong enough evidence to include in the manuscript, our previous structural studies of human Pum1, whose RNA-binding domain shares 80% sequence identity with It is very hard to see from the various figures, what is moving in Pum between the binary and ternary complexes. Perhaps a side by side of that area would be easier to follow.hb RNA ternary structure in Thank you for this feedback on the figures illustrating the differences in Pum between the binary and ternary complexes. We now include revised The deletions that are discussed on Results section, subsection “Nanos increases the binding affinity of Pumilio for hunchback RNA”, that affect repression involve cutting out a few residues sometimes in the middle of a helix and it's not clear how well the structure would retain its integrity. The extent of repression might correlate with what percentage of folded protein they have in this region. Does this correlate with Pum-RNA binding?L7nos allele, which disrupts embryonic development. Together, these data and our functional and structural data reveal how that deletion causes loss of function. Unfortunately we were unable to produce recombinant purified Nos Δ376-382 protein to confirm this result but, as noted in the Discussion, Sonoda and Wharton reported the Nos Δ376-382 was unable to form a ternary complex with hb NRE, as assessed by yeast three hybrid assay. We have now clarified these points in the Results:We chose the specific deletions because the Nos Δ376-382 is important in terms of history, biology, and function. This deletion corresponds to the lesion in the “Since deletion of the C-terminal region severely limited Nos repression of the hb 3´UTR reporter in cells , we probWe have now performed additional analyses of the role of the observed Nos-Pum contacts by introducing single amino acid substitutions into the C-terminal region of Nos. As reported in the new “We further examined this region by introducing single amino acid substitutions,including the I376 and M378 residues that contact Pum in the structure . RepressWe also tested mutants in the Nos binding interface of Pum to confirm the importance of the observed Nos-Pum interaction for ternary complex formation. These substitutions blocked incorporation of Nos into the complex, but did not prevent RNA binding by Pum, demonstrating that they did not cause an overall disruption of Pum structure.:“We also found that single amino acid substitutions in Pum disrupt formation of the repression complex. […]Thus, Nos must interact with both repeat R7 and the R7-R8 loop of Pum to form a stable ternary complex.”The authors show a difference in the RNA structure between the hb and CycB complexes. However, the resolution is fairly low. How confident are they? What do the electron density maps look like in this region?hb and CycB ternary complexes, provided as hb and CycB complexes clearly indicates differences in the backbone conformations. The backbone conformation for the CycB RNA appears to change to accommodate the +6U nucleotide that interacts non-canonically with repeat R3 that bears an A-recognition motif. The map-model swaps demonstrate the differences in the two models.We now include comparisons of the electron density maps for the In the discussion the authors mention that the ZFs in Nos are unique compared to other RNA-binding ZFs. In what way?We thank the reviewers for pointing out that we should describe what we did to evaluate the Nos ZFs relative to other RNA-binding ZFs. We elaborate now in the text that no structural homologues were detected using the program DALI (other than the zebrafish Nos). We also considered a manual alignment with the HIV nucleocapsid protein Zn knuckles (ZK), which are also CCHC RNA-binding ZFs. We were pleased to discover that the topology and structures of Nos ZF2 and the HIV ZKs were strikingly similar. We have added a figure  to show Also in the discussion, the authors compare this cooperative interaction to that between Sxl and Unr. They say that this is also a cooperative interaction and two lines later they say it is additive. Which is it? If Sxl and Unr interaction is additive, then they are distinctly different.These concepts make the whole story here, so it's very important that this is written clearly.We thank the reviewers for pointing out that we should describe what we did to evaluate the Nos ZFs relative to other RNA-binding ZFs. We elaborate now in the text that no structural homologues were detected using the program DALI (other than the zebrafish Nos). We also considered a manual alignment with the HIV nucleocapsid protein Zn knuckles (ZK), which are also CCHC RNA-binding ZFs. We were pleased to discover that the topology and structures of Nos ZF2 and the HIV ZKs were strikingly similar. We have added a figure  to show “Nos uses its three functional regions: N, Z, and C  to eliciAlso in the discussion, the authors compare this cooperative interaction to that between Sxl and Unr. They say that this is also a cooperative interaction and two lines later they say it is additive. Which is it? If Sxl and Unr interaction is additive, then they are distinctly different.These concepts make the whole story here, so it's very important that this is written clearly.We are sorry for the confusion our abbreviated comparison caused. To clarify, we have now added a paragraph to the Discussion that compares and contrasts cooperative RNA recognition by Sxl and Unr with Nos and Pum, which is quite interesting and instructive of mechanisms of cooperativity:“Cooperative RNA recognition by Nos and Pum is reminiscent of cooperative binding of msl2 RNA by Sex lethal (Sxl) and Upstream of N-Ras (Unr) , yet the mechanisms and effects of cooperative recognition by Nos and Pum display novel distinguishing characteristics. […] As a consequence,Nos and Pum cooperatively bind to RNAs that neither protein stably binds on its own.”


Igh locus undergoes an amazing array of DNA rearrangements and modifications during B cell development. During early stages, the variable region gene is constructed from constituent variable (V), diversity (D), and joining (J) segments (VDJ joining). B cells that successfully express an antibody can be activated, leading to somatic hypermutation (SHM) focused on the variable region, and class switch recombination (CSR), which substitutes downstream constant region genes for the originally used Cμ constant region gene. Many investigators, ourselves included, have sought to understand how these processes specifically target the Igh locus and avoid other loci and potential deleterious consequences of malignant transformation. Our laboratory has concentrated on a complex regulatory region (RR) that is located downstream of Cα, the most 3′ of the Igh constant region genes. The ~40 kb 3′ RR, which is predicted to serve as a downstream major regulator of the Igh locus, contains two distinct segments: an ~28 kb region comprising four enhancers, and an adjacent ~12 kb region containing multiple CTCF and Pax5 binding sites. Analysis of targeted mutations in mice by a number of investigators has concluded that the entire 3′ RR enhancer region is essential for SHM and CSR (but not for VDJ joining) and for high levels of expression of multiple isotypes. The CTCF/Pax5 binding region is a candidate for influencing VDJ joining early in B cell development and serving as a potential insulator of the Igh locus. Components of the 3′ RR are subject to a variety of epigenetic changes during B cell development, i.e., DNAse I hypersensitivity, histone modifications, and DNA methylation, in association with transcription factor binding. I propose that these changes provide a foundation by which regulatory elements in modules of the 3′ RR function by interacting with each other and with target sequences of the Igh locus.The  Igh locus spans ~3 Mb, beginning near the telomere on murine chromosome 12 with the component variable (V), diversity (D), and joining (J) segments of the variable region, followed by the multiple constant region (HC) genes  (enhancers and insulators) has been shown by others to potentially contribute to regulation of variable region formation (VDJ joining) (The Igh locus (Eμ), positioned between the 3′-most HJ segment and the 5′-most HC region, Cμ . (1) Mue 3′ RRs . (3) A ce 3′ RRs . In mouse 3′ RRs . Comparee 3′ RRs . Other se 3′ RRs . (4) Thee 3′ RRs .Other than revealing a conserved palindromic structure, the regions between the 3′ RR enhancers show virtually no homology between rodents and humans . NonetheIn summary, the 3′ RR contains several enhancers located in two structurally distinctive modules – (1) a palindromic region (mouse hs3a–hs1.2–hs3b) and (2) a separate structural unit (hs4). Inter-enhancer regions reveal repetitive, switch-like sequences potentially of functional significance for the Igh locus. Downstream of the enhancer-containing segment of the 3′ RR is additional DNase I hypersensitive sites (hs5–8), which contain CTCF and Pax5 sites and have insulator activity. This hs5–8 region is discussed more fully later.Igh locus were studies of genes of the β-globin locus, which, like the Igh genes, are subject to developmental regulation. Multiple DNase I hypersensitive sites, each with enhancer activity, are located upstream of the β-globin genes. This enhancer-containing region is referred to as the locus control region because endogenous deletions here are found to affect expression of distally situated globin genes. Experimental questions on Igh genes have paralleled experiments carried out in the β-globin locus and in other loci : (1) whIgh locus, including hs1.2 (Electrophoretic mobility shift assays (EMSA) provided a tool to identify proteins with the potential to bind enhancers. Using nuclear extracts, we identified a B cell-specific binding protein with sites throughout the ng hs1.2 , 36. Basng hs1.2 ; and varng hs1.2  , Crip, and mta-1, each had a transcriptional orientation that was opposite to that of all of the V, D, J, and HC elements of the Igh locus. This back-to-back orientation led us to predict that a terminus of the Igh locus might be located in this segment  of a mouse plasmacytoma cell line was shown to have sustained a deletion of the entire 3′ RR , 48. Thiansgenic , 50 and ansgenic ]. Althouansgenic . Now, th of a mou joining . Studies joining . These All death . This reIgh expression  for CSR are situated quite far in a linear distance from the 3′ RR; yet it is implied that they all function together through physical interaction  (3′ CBE). Notably, substitution of hs1.2 by the NeoR gene in a variant of the MPC11 plasma cell line resulted in loss of Igh expression , targeted deletion of which showed their critical role in appropriate regulation of VDJ joining (Presuming that the CTCF/Pax5 region (3′ CBE) of the 3′ RR interacts with other ide ChIP , 60. Mov segment , 61. Themed PAIR . Recent Igh locus  of long-range chromatin interactions has revealed interactions of the 3′ RR with transcribed Igh genes in B cells activated by LPS + IL4 that are not detected in embryonic stem (ES) cells , there are no apparent changes in histone marks in the 3′ RR during switching in cultured cells . This imIgh locus? What is the role of the conserved palindrome? How did the 3′ RR evolve? What are the species-specific aspects of 3′ RR regulation?The 3′ RR enhancer region is critical for GT and CSR, and SHM, and the CTCF/Pax5 binding region could contribute to VDJ joining. We predict that a scaffold formed by modules of the 3′ RR supports physical interactions with target sequences required to accomplish these various activities. With deletions of individual 3′ RR enhancers having little phenotypic consequence, one can ask how many different structural solutions are there to 3′ RR activity? Why are there multiple modules? Do the changes in epigenetic alterations of 3′ RR modules that occur during development indicate specific activities for individual modules? Could the 3′ RR help target DNA repair proteins involved in SHM or CSR? What roles does the 3′ RR share with other cis acting sequences that are critical for SHM, such as those in the light chain loci? What is distinctive about CSR, which is specific to the globin LCR have identified hierarchical regulation by multiple transcription factors  cells. DNA replication initiates ~45 kb downstream of the CTCF/Pax5 module of the 3′ RR between crip1 and Tmem121 (Figure H, JH, DHC, and most proximal V regions (HV genes replicate late in S phase. In pre-B cells, the entire Igh locus replicates early in S phase, indicating the firing of multiple origins that are ordinarily quiescent in non-B cells (Igh replication is under B cell-specific developmental control. In MEL cells, the downstream origin, which is located beyond the limits of the 3′ RR, may be a terminus for Igh locus regulation. It is of interest that changes in Igh DNA replication are associated with changes in nuclear location of the Igh locus (Igh locus is located away from the nuclear periphery, while in MEL and ES cells, and in B and plasma cells, the Igh locus is located at the nuclear periphery. These observations raise the question of whether there are finer demarcations of nuclear subcompartments generally associated with the Igh locus and the 3′ RR? What regulates the movement of the locus from one position to another?Our experiments in collaboration with the Schildkraut laboratory identified an origin of an ~500 kb  regions , 71. All-B cells . These dgh locus  but can gh locus . In pro-Igh locus take place and the role of the 3′ RR are under close scrutiny (Igh genes, which are evident during steps of VDJ joining (The mechanism by which recurrent translocations involving the scrutiny , 76. Epnscrutiny . Furtherscrutiny . The Sko joining . The var joining , 63, shomyc oncogene to which it is juxtaposed as a result of a chromosomal translocation. An inhibitor of BRD4, JQ1, can lead to downregulation of myc expression in multiple myeloma cells. However, myc appears to be suppressed by JQ1 regardless of whether it is associated with Igh sequences through translocation (myc (Recent genome-wide studies have reported “super enhancers” , 80, DNAlocation , potentiion (myc , 63. Is The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Alphavirus vectors present an attractive approach for gene therapy applications due to the rapid and simple recombinant virus particle production and their broad range of mammalian host cell transduction. Mainly three types of alphavirus vectors, namely naked RNA, recombinant particles and DNA/RNA layered vectors, have been subjected to preclinical studies with the goal of achieving prophylactic or therapeutic efficacy, particularly in oncology. In this context, immunization with alphavirus vectors has provided protection against challenges with tumor cells. Moreover, alphavirus intratumoral and systemic delivery has demonstrated substantial tumor regression and significant prolonged survival rates in various animal tumor models. Recent discoveries of the strong association of RNA interference and disease have accelerated gene therapy based approaches, where alphavirus-based gene delivery can play an important role. Togaviridae family [in vivo [Alphaviruses are single-stranded RNA viruses belonging to the e family . The enve family . New proe family ,4. For te family , primarye family  and in v[in vivo . A varie[in vivo  and prop[in vivo . Moreove[in vivo . In thistrans from a helper vector. High-titer replication-deficient particles can be generated from in vitro transcribed RNA from expression and helper vectors co-transfected into mammalian host cells. Direct transfection of layered DNA/RNA expression and helper plasmid vectors (see below) are also able to generate recombinant particles, however, with 10–100 fold lower titers [In vitro transcribed RNA is transfected into mammalian host cells for the generation of recombinant replication-proficient particles. 3. Layered DNA/RNA vectors: by replacing the SP6 RNA polymerase promoter with a CMV promoter, plasmid DNA can be directly transfected into mammalian cells for recombinant protein expression. Co-transfection with CMV promoter-engineered helper vector allows production of replication-deficient viral particles. The alphavirus vector systems described above permit different approaches, depending on the selected application. In this context, local expression is achieved in vivo by administration of replication-deficient vectors. In case extended spread of expression is requested it is favorable to use replication-proficient virus particles. As far as vaccine development, plasmid DNA vectors have been commonly used [in vitro transcribed RNA for immunization [Due to the potential pathogenicity of alphaviruses in general, vectors for gene delivery and expression have been based on avirulent or attenuated strains. Mainly, Semliki Forest virus (SFV) , Sindbisr titers . 2. Replr titers . In vitrnly used . The chonization .In attempts to enhance the heterologous gene expression levels, reduce the cell cytotoxicity and improve biosafety, a number of modified alphavirus vectors have been engineered. For SFV, the introduction of three point mutations in the p62 precursor sequence resulted in the second-generation helper vector pSFV-helper2, which rendered conditionally infectious particles . InfectiA number of modifications have also been made to both SFV and SIN expression vectors. In attempts to enhance transgene expression, the translation enhancement signal of the capsid protein was introduced into the SFV expression vector, which resulted in 5–10-fold increase in protein expression levels . FurtherE. coli expression system [Other vector designs relate to delivery and encompass generation of virus-like particles (VLPs) and encapsulation of viral particles. In this context, SIN and Ross River virus (RRV) nucleocapsids have been produced in a T7-based n system . Importan system ,31. Moren system . Encapsun system .The broad host range and high level of transient heterologous gene expression are features, which have made alphaviruses attractive for gene therapy applications. Particularly, cancer therapy has been the main area of interest.5-fold reduction in BHK cell transduction was observed. More recently, natural tumor targeting of SIN particles was discovered after systemic delivery in mice implanted with human xenografts [Although the broad host range can be advantageous, it also raises concerns in case of cancer therapy where systemic delivery targets not only cancer cells but also normal tissue. In attempts to improve tumor targeting, IgG binding domains of protein A were engineered into the SIN E2 envelope protein, which enhanced substantially transduction of host cells treated with a monoclonal antibody against surface proteins . At the nografts . This wanografts . A closenografts .9 encapsulated SFV particles; no kidney, liver, bone marrow, neuro- or cardio-toxicity; and the possibility of conducting repeated particle administration without any substantial immune response against liposomes or SFV. Furthermore, SIN replicon RNA-liposome nanoparticles have provided enhanced reporter gene expression and immune responses compared to naked RNA [Another approach to achieve tumor targeting has been the engineering of liposome encapsulated nucleic acids and viral particles. As briefly described in the section on alphavirus vectors, systemic delivery of liposome-encapsulated SFV particles showed enhanced reporter gene expression in tumor tissue due to passive targeting  and alloaked RNA , which mIn the context of cancer therapy, alphavirus vectors have been applied for direct intratumoral administration, initially with reporter genes, followed by application of therapeutic genes. Administration of SFV particles expressing the p40 and p35 subunits of IL-12 showed substantial tumor regression and inhibition of tumor blood vessel formation in a mouse B16 tumor model . Likewis6 particles resulted in strong tumor regression. In another study, oncolytic SFV A7(74)-EGFP vectors also demonstrated significant tumor shrinkage in a mouse model for human osteosarcoma [In attempts to improve vector distribution, replication-proficient oncolytic vectors based on the avirulent SFV strain A7(74) have been engineered leading to lysis of several cancer cell lines . A singlosarcoma . Moreoveosarcoma .+ T-cell and anti-neu IgG responses [Alphavirus vectors have been subjected to cancer vaccine development as naked RNA, recombinant viral particles and plasmid DNA . For exaesponses . A singlesponses .In addition to recombinant particles, layered DNA vectors have been applied for immunization studies. For example, when mice immunized with SIN DNA vectors expressing the murine melanoma cell adhesion molecule (MCAM/MUC18), protection against lethal challenges was obtained . Similarin vitro transcribed RNA replicons for vaccination. In this context, when 1 μg of SFV RNA replicons expressing bacterial β-galactosidase was used for immunization of mice, therapeutic efficacy and complete tumor protection was obtained [An interesting approach has been to apply obtained . The surobtained . HoweverBombyx morii reduced the endogenous BR-C expression and resulted in the failure of larval-pupal transition [Aedes aegypti mosquitoes [The influence of gene expression regulation on gene therapy has become even more important with the discovery of the involvement of RNAi and particularly micro RNA (miRNA) . RNAi waansition . Likewissquitoes .In another approach, tissue-specific miRNA sequences were introduced into an alphavirus vector with the aim of altering tissue tropism for viral replication . IntroduAlphavirus vectors based on naked RNA replicons, layered DNA/RNA vectors, and recombinant viral particles have demonstrated efficient gene delivery and recombinant protein expression in both cell lines and animal models. However, the shortcomings of the application of alphavirus vectors relates to the relatively expensive virus stock production and the highly transient nature of heterologous gene expression. In the context of virus production, packaging cell lines have been engineered ,62, whic


The 35S promoter of cauliflower mosaic virus and that of other plant pararetroviruses gives rise to an RNA, which is both a pre-genome and a polycistronic mRNA. The 600 nucleotide long very structured leader of this RNA is also transcribed separately. The resulting 8S RNA is then converted to a double strand giving rise to a huge set of siRNAs, which suppress silencing. In this Mini-Review I discuss how this versatile stretch of 600 nts constitutes a masterpiece of evolution. Plants respond to virus infections mainly by RNA silencing (RS). RS is generally initiated by recognition of double stranded RNA, usually accumulating as a by product of virus replication. In addition for some cases effector-triggered immunity (ETI) to virus infections was reported  to yield dsRNAs. These are cleaved by dicer-like proteins (DCLs) into 21–24 nt small RNA duplexes (siRNAs).  1 and 3 . The siR 1 and 3 .Cauliflower Mosaic Virus (CaMV), and Rice Tungro Bacilliform Virus (RTBV) allow the extension of the list of viral suppressors to ds viral suppressor RNAs  that mark the starts/ends of minus- and plus-strand DNA synthesis. The minus strand gap is located at the primer (met-tRNA) binding site, the other two at polypurine stretches. .Cauliflower Mosaic Virus encodes seven proteins . Of special interest is the unique transactivator/viroplasmin (TAV). TAV is a multifunctional protein forming viral inclusion bodies and enabling polycistronic translation and virus assembly . The 35S RNA covers the whole genome and is terminally redundant due to a conditional polyadenylation signal, which is passed at the first encounter with the transcription machinery, but recognized at the second .e second . It actse second . Its trae second . Small oBanana Streak Virus (BSV) and RTBV . DCLs 3, 1, and 2 are involved giving rise to species of 24 nt (47%), 21 nt (27%), and 22 nt (14%). Interestingly, the production of 21 nt long siRNAs by RDR6/DCL4/DRB4 is inhibited by TAV in its function as proteinaceous silencing suppressor  of both polarities have been reported, making up half of the total amount of siRNAs in the infected plants. All four plicated . Deep seppressor .Figure 1a), has a cap and ends at a narrow cluster of positions close to the start/end of reverse transcription and lacks a poly-A tail. Interestingly, not only sense 8S RNA (s-8S RNA) was found, but also antisense 8S RNA (as-8S RNA). The as-8S RNA starts roughly where the s-8S RNA ends and ends exactly where the s-8S RNA starts. The as-8S RNA has neither a cap nor a poly-A tail.To learn more about the preferential siRNA production, 8S RNA was isolated and characterized in detail by circularization-reverse-transcription PCR . It starFigure 1d). There must be a time window until the gaps/overhangs of nascent CaMV DNA are removed by repair enzymes and ligase and the supercoil closed. If transcription is initiated before DNA closure, the nascent RNA may fall off at the gap/overhang of the DNA minus strand or near of it as s-8S RNA. A fall-off would explain the lack of polyadenylation. The length of s-8S RNA is thus defined by the distance between start of transcription from the 35S promoter and roughly the primer binding site.How is the poly-A tail-less s-8S RNA produced? Cauliflower mosaic virions are guided to nuclear pores via nuclear localization signals . Due to The mechanism of as-8S RNA production is not yet known. At the relevant antisense positions CaMV DNA contains neither promoter-like sequences nor polyadenylation signals, making ordinary DNA-depending antisense transcription unlikely. Although promoters lacking TATA-boxes exist in plants and other organisms , transcrHepatitis Delta Virus RNAs are replicated by POL II in an α-amanitin-sensitive mode and pospiviroids apparently make use of an RNA-based promoter located on highly structured circular viroid RNA : both RNAs have long stretches of imperfectly matched dsRNA. This suggests that also the ds form of 8S RNA might originate from transcription of 8S RNA by POL II, perhaps using an RNA-based promoter, as in pospiviroids  is involved .One very attractive hypothesis suggests a function for the siRNAs derived from the 8S RNA duplex: they may act as decoys competing with the remaining siRNAs for free AGO proteins. This would also explain the very low amount of siRNAs produced from the CaMV coding region. Experiments using AGO1 antibodies indeed showed that 21 and 22 nt long siRNAs derived from the 8S RNA were bound to AGO1, while those derived from the remaining CaMV region did not The 8S derived siRNAs on the other hand cannot efficiently target the 8S RNA itself or the leader of 35S RNA. The compact structure of these RNA sequences renders them as unfavorable targets for AGO-RISCs. A similar effect is discussed for viroids, the rod-like structures of which are perfect targets for DCLs but very poor ones for AGO/RISCs .Cabbage Leaf Curl (Gemini) Virus (CaLCuV) vector, leading to s-8S like RNA, in this case in a polyadenylated version. Also in this connection large amounts of 8S RNA-derived siRNAs of both polarities were observed, the majority of which was again 24 nt long. The chimeric virus was produced in higher amounts than the empty vector or a CaLCuV vector loaded with a GUS gene, again indicating RNA-based silencing suppression . This resembles the case of human T-cell leukemia virus (HTLV), where a polyadenylation signal is moved over a distance of 290 nts to the facultative polyadenylation site through secondary structure .Like in the case of CaMV 8S RNA, huge amounts of siRNAs are produced also from the RTBV-derived 8S-RNA duplexes . Since RFigure 2).Different results were obtained for BSV-GF . For BSVComparison of the three viruses confirms that 8S RNAs are produced by fall-off at the primer binding site, if properly spaced, and that it does not depend on the compact structure of the leader, which is present in all these three viruses. This compact structure of the leader, however, protects it from AGO-RISC-dependent degradation.The stretch of 600 nts comprising both, the leader of the CaMV pregenomic RNA and the blunt-ended 8S RNA constitutes a masterpiece of evolution. Due to its position it allows unusual fall-off transcription, due to its compact secondary structure it is resistant to AGO-mediated degradation. This secondary structure apparently leads also to an unusual replication mechanism, giving rise to antisense 8S RNA, which hybridizes with its template yielding 8S RNA duplexes as source of huge amounts of decoy siRNAs. The obvious disadvantage of such a structure: inhibition of ribosome scanning and translation is compensated by an ingenious positioning of a small open reading frame, which initiates a shunt mechanism leading the scanning ribosome directly to the start site of translation.Analysis of the siRNA patterns in pararetrovirus-infected plants have led to the discovery of a novel silencing suppression strategy. Although mechanistic details await further experimentation, at least some players of the game are testable. Future science will reveal whether RNA-based silencing suppression is a more widely used strategy and whether host organisms have developed strategies to fight such activity.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, the presence of 8-oxo-dG residues led to a more complete suppression of HCV replication in transfected cells. These modifications did not affect the efficiency of RNase H cleavage of antisense oligonucleotide:RNA duplexes but did alter specificity, triggering the appearance of multiple cleavage products. Moreover, the incorporation of 8-oxo-dG residues increased the stability of antisense oligonucleotides of different configurations in human serum.The inhibitory potency of an antisense oligonucleotide depends critically on its design and the accessibility of its target site. Here, we used an RNA interference-guided approach to select antisense oligonucleotide target sites in the coding region of the highly structured hepatitis C virus (HCV) RNA genome. We modified the conventional design of an antisense oligonucleotide containing locked nucleic acid (LNA) residues at its termini (LNA/DNA gapmer) by inserting 8-oxo-2’-deoxyguanosine (8-oxo-dG) residues into the central DNA region. Obtained compounds, designed with the aim to analyze the effects of 8-oxo-dG modifications on the antisense oligonucleotides, displayed a unique set of properties. Compared to conventional LNA/DNA gapmers, the melting temperatures of the duplexes formed by modified LNA/DNA gapmers and DNA or RNA targets were reduced by approximately 1.6-3.3°C per modification. Comparative transfection studies showed that small interfering RNA was the most potent HCV RNA replication inhibitor (effective concentration 50 (EC The application of complementary DNA or RNA molecules or their derivatives for the modulation biological functions of specific RNA(s) is referred to as antisense technology. Antisense oligonucleotides (ASOs) are the major class of antisense agents used for sequence-specific RNA knockdown , and theThe activity of ASOs depends on many factors, including the efficiency of cell entry, the stability of the complex formed with the targeted RNA and the resistance of the ASO to enzymatic degradation. The low potency of standard RNA and DNA ASOs results from their poor uptake and extremely short intracellular and serum half-lives. Sugar moiety and phosphate backbone modifications have been used to increase the resistance of ASOs to degradation. Some of these modifications also increase the binding efficiency of ASOs to their target sequences  and/or min vivo half-lives, enabling their successful use in clinical trials [The nucleobase moiety represents an alternative option for ASO modification. Several heterocyclic base modifications in ASOs have been described (reviewed in ). Howevel trials .The 8-oxo-2’-deoxyguanosine (8-oxo-dG) residue contains a minimally modified nucleobase, which is naturally occurring and can result from oxidative DNA damage. In the context of ASO, 8-oxo-guanine forms 3- to 4-fold weaker bonds with complementary cytosine (compared to non-modified guanine) , which rin vivo delivery of therapeutic nucleic acids to target tissues and organs represents another important problem that has been only partly solved (reviewed [The reviewed ). In thereviewed  and non-reviewed ,37 modelreviewed ,37, hearreviewed . Clinicareviewed .As ASOs have a clear potential for the treatment of liver-associated pathologies, we chose hepatitis C virus (HCV) RNA as a target for modified ASOs. HCV is a major human pathogen that affects the lives of over one hundred million people. Its positive polarity RNA genome is >9000 nucleotides (nt) long and contains strong RNA secondary structures . Thus faHere, we demonstrate that the incorporation of 8-oxo-dG residues destabilizes not only ASO:DNA but also ASO:RNA duplexes. These modifications also slow down the formation of all-DNA ASO:RNA duplexes, but they have little effect on the formation of duplexes between an LNA/DNA gapmer ASO and its target RNA. Furthermore, the presence of 8-oxo-dG residues does not have a negative impact on the efficiency of RNase H-mediated cleavage of the RNA target. Simultaneously, this modification alters the specificity of RNase H cleavage and increases the stability of ASOs in human serum. RNA interference (RNAi)-guided target site selection was used to identify novel sites in the coding region of the HCV genome that could be efficiently targeted with small interfering RNAs (siRNAs) and ASOs. The incorporation of 8-oxo-dG residues into the DNA region of an LNA/DNA gapmer oligonucleotide that targets such sites led to the development of ASOs that exhibit this novel mechanism of antisense action.Sigma-Aldrich (USA). Validated controls, including non-targeting siRNAs (AM4611 or AM4635) and a combination of siRNAs against firefly luciferase (Luc) [Life Technologies (USA). If not stated otherwise, the non-modified all-DNA oligonucleotides (hereafter designated as “D”), modified all-DNA oligonucleotides (designated as “DM”), LNA/DNA gapmers (designated as “LD”) and LNA/DNA gapmers with modified nucleobases (designated as “LDM”) were synthesized by Exiqon A/S (Denmark). All the obtained oligonucleotides were dissolved in sterile water, aliquoted and stored at -85°C.siRNAs targeting the coding region of the HCV genome were designed using an algorithm that was developed in-house and were obtained from se (Luc)  (AM4629)Shimadzu, Japan) equipped with an SPD-M20A absorbance detector. A Phenomenex Luna 5 μm C18 100A column  was temperature-regulated at 45°C and operated at a flow rate of 1 ml/min. The oligonucleotides were eluted using a methanol gradient in 0.02 M ammonium phosphate buffer, pH 7.0 [4 as the eluent. The fractions were precipitated and washed with acetone. Depending on the downstream application, the oligonucleotide preparations were re-precipitated using ethanol/ammonium acetate (pH 7.0) or ethanol/sodium acetate. The oligonucleotides used in the subsequent analyses contained only trace quantities of impurities.The batches of modified oligonucleotides obtained from different commercial providers were of variable quality. Accordingly, they failed to produce consistent and reproducible results in biological assays. For these reasons, multi-step quality control and purification protocols were developed. The quality of each oligonucleotide batch was independently verified in-house by RP-HPLC analysis using an HPLC LC20-A Prominence system  or Sigma-Aldrich (USA). Complementary oligonucleotides (hereafter designated as “probes”) were labeled in-house with FITC at the 5’-end or purchased with a 5’ FAM label from Exiqon A/S (Denmark). Hybridization was quantified by FRET between the FITC and Cy3 labels or the FAM and TYE563 labels (detected as a decrease in FITC or FAM fluorescence) when they were brought in close proximity due to the formation of a duplex between the probe and its target. The target oligonucleotides were used at concentrations of 25, 50, 100, 200 and 400 nM, and the probe was always used at a concentration of 50 nM. The probe-target interactions were measured in a 384-well optical plate in a volume of 20 μl in buffer containing 150 mM NaCl and 50 mM Tris-HCl (pH 8.0) using an ABI7900HT Real-Time PCR instrument . The temperature was rapidly increased to 95°C, and the complexes were allowed to melt for 10 min. Then, the temperature was decreased to 5°C over 45 min, stabilized for 5 min and increased slowly (over 45 min) to 95°C. The Tm values were calculated from the obtained melting curves.Melting curve data were obtained using FRET as described in detail by You and coworkers . BrieflyExiqon A/S, Denmark) was labeled at the 5´ terminus using γ-33P ATP  and phage T4 polynucleotide kinase  according to the manufacturers’ protocols, purified, and dissolved in buffer containing 10 mM HEPES, pH 7.2, and 20 mM KCl. The labeled RNA was mixed with partially complementary D4676, DM4676, LD4676, LDM4676 or MixLD4676 oligonucleotides .To analyze the spontaneous formation of duplexes between target ssRNA and ASOs, 5 fmol of  LDM4676  in buffein vitro using an mMESSAGE mMACHINE T7 Transcription Kit  according to the manufacturer’s instructions. RNase H assays were performed as described by Kurreck and co-authors [Thermo Scientific, USA), 5 pmol of ASO and 500 ng of FR3131 RNA. The reaction mixture was incubated at 37°C for 0, 1, 5, 10, 20 or 60 min. At each time point, a 10 μl aliquot was collected. The reaction was stopped by adding 10 μl of 2x stop buffer (50 mM EDTA and 1% SDS) and subsequent heating to 95°C for 2 min. The reaction products were separated on a 0.8% TAE agarose gel and detected with ethidium bromide staining.Target RNA (designated as FR3131), consisting of 269 nt from the 5’ end of the HCV genome and the region spanning positions 3081 to 5943, was synthesized -authors . BrieflyGE Healthcare, UK). Quantitative analyses were performed using ImageQuant TL Software .The kinetics of ASO:RNA duplex cleavage were analyzed using pre-formed ASO:RNA duplexes. Briefly, 1 fmol of the labeled duplexes was mixed with 0.05 U of RNase H in 1x RNase H buffer, and the reaction was performed at 37°C. Aliquots were collected immediately after mixing the substrate and enzyme (0 time point) or after incubation for 10 s or 0.5, 1, 5, 10 or 20 min. The reactions were stopped by adding an equal volume of 2x stop buffer. The samples were then heated to 95°C for 2 min, cooled rapidly on ice and separated by PAGE in native 15% gels. The gels were dried, exposed to a storage phosphor screen and visualized using a Typhoon Trio scanner  at 37°C. Aliquots were collected immediately after preparation of the mixtures (0 time point) or after incubation for 0.25, 0.5, 1, 2, 4 or 6 h. Next, 2x stop solution was added to each aliquot. The full-length oligonucleotides and their degradation products were separated and analyzed as described above. The average half-lives of each type of ASO were obtained from three independent experiments by fitting the obtained values to an exponential decay function.To analyze the stability of D4676, DM4676, LD4676, and LDM4676  in humanReBlikon GmbH (Germany). The cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with penicillin, streptomycin, 0.5 mg/ml G418, 10% fetal calf serum and 2 mM L-glutamine .Huh-luc/neo-ET cells, which harbor the I389/NS3-3’/LucUbiNeo-ET replicon of HCV genotype 1b (Con1 isolate) , and repin vitro-transcribed RNAs and selection of antibiotic-resistant colonies in the presence of 0.5 mg/ml G418 . The preservation of the introduced mutation was verified as follows. Total RNA was extracted from Huh-luc/neo-ET-3570mut cell line using an RNeasy Mini Kit (Qiagen). Reverse transcription was carried out using a First-Strand cDNA Synthesis kit (Thermo Scientific). HCV-specific cDNA fragment containing the mutation site was PCR-amplified using of primers flanking the mutated region. Obtained PCR products were purified and sequenced using Sanger sequencing.A variant replicon that encodes for NS3 with Thr54Ala mutation, was constructed using site-directed mutagenesis and designated I389/NS3-3’/LucUbiNeo-ET-T54A. The corresponding cell line, designated Huh-luc/neo-ET-3570mut, was obtained by electroporation  of Huh7-Life Technologies, USA). Luc activity was measured 48 h post-transfection (p.t.) using reagents and protocols from Promega (USA). The total protein content in the cell lysates was measured by Bradford micro-assay .For RNAi-guided screening, Huh-luc/neo-ET cells were reverse-transfected with siRNAs (100 nM each) using Lipofectamine RNAiMAX reagent , DOTAP, FuGENE HD  and TurboFect  reagents were used to optimize the transfection of Huh-luc/neo-ET cells. Various amounts of these reagents and forward- or reverse-transfection protocols were used to deliver ASOs . Cytotoxic effects (or lack thereof) were observed using a Nikon Eclipse confocal microscope .Lipofectamine 2000, Lipofectamine RNAiMAX, Lipofectamine LTX  to estimate the effective concentration 50 (EC50) values.Cells were collected and lysed at selected time points. The total protein content in the lysate and Luc activity were measured. For the normalization of HCV replication, which is proportional to Luc activity, the following calculations were performed. First, to enable the comparison of the average Luc activity per living cell, the total protein content of the cells was used to normalize the Luc activity as previously reported . Thus, t8-oxo-dG residues have been reported to destabilize ASO:DNA duplexes ,29,30. HWhen the 8-oxo-dG modification (FITC-Y) was introduced, the Tm values of both the ASO:DNA and ASO:RNA duplexes were reduced by ~1.6–1.8°C compared to those of the duplexes formed by control oligonucleotides. Increasing the number of 8-oxo-dG modifications (FITC-YY) resulted in further reduction of the Tm by ~1.6°C for ASO:DNA and by ~2.5°C for ASO:RNA duplexes. Thus, destabilization resulting from the insertion of 8-oxo-dG modifications occurs not only for ASO:DNA duplexes  but alsoKnown targets for ASOs are located in a 350-nt region at the 5’-terminus of HCV RNA. Clearly, targeting a region that comprises less than four percent of the virus genome is a bottleneck that hinders the development of the most efficient ASOs. RNAi technology was used to search for highly accessible target sites in the HCV coding region. Twenty-eight different siRNAs targeting the HCV genome were designed . Each siThe level of Luc activity in Huh-luc/neo-ET cells is directly proportional to the copy number and replication efficiency of the HCV subgenomic replicon, making it an efficient tool for analyzing the anti-HCV efficiencies of the obtained siRNAs ,56. At aAs the high inhibitory potential of an siRNA indicates the accessibility of the corresponding target sites, it was concluded that RNAi-guided screening enabled the selection of several potential ASO target sites in the HCV coding region. However, an all-DNA ASO based on the sequence of the guide strand of siRNA 4676 was essentially unable to suppress HCV replication. Therefore, HCV-specific 21-mer LNA/DNA gapmer oligonucleotides that contained five LNA monomers at each end and three modified residues in the DNA region were designed Tables .The inhibitory effects of siRNAs and, to lesser extent, ASOs are reduced by point mutations in their target sites. Mutations at distant sites (unless they modify the higher-order structure of the target region) have no such effect. The only mutation in the selected target sites, for which the viability of the mutant replicon has been previously demonstrated, is located in the target site of siRNA 3570 and results in a Thr54→Ala change in NS3 [Huh-luc/neo-ET and Huh-luc/neo-ET-3570mut cells were transfected with different concentrations of siRNA 3570, siRNA 4676, LDM4676, LDM4676inv, LDM3570 and LDM3570inv. A point mutation in HCV RNA that changes the classical A:U base pair in the siRNA guide-strand:target-RNA duplex to the G:U wobble base pair resulted in a marked (10-fold) decrease in the inhibitory efficiency of siRNA 3570 . As expeACEA Biosciences, USA) revealed the same trend. Therefore, as in all previous experiments  characteristic of hepatotoxic LNA/DNA gapmer oligonucleotides , cytotoxnts Figs , the rep50 of 0.13 nM, whereas the EC50 values for LD4676 and LDM4676 were approximately 50-fold higher  or LNA/DNA gapmer ASO (LD4676) .versus LD4676) was between 5 and 10°C . Thus, synthetic ASOs often contain one or more modifications aimed at improving the properties of the compound. The common theme in ASO design is to achieve increased ASO:target duplex stability , which iThe initial idea of using 8-oxo-dG residues was based on theoretical quantum calculations. It was hypothesized that the antisense activity of oligonucleotides containing 8-oxo-dG may be increased due to the ability of corresponding nucleobases to acquire tautomeric forms, which enables enhanced binding to complementary normal nucleobases . HoweverThe efficiency of ASOs critically depends on the physical accessibility of its target site. Very limited information is available about the actual topological structures of mRNA in cells, where they form RNA-protein complexes. In this study, the attempts to target sequences with specific nucleotide compositions or regions lacking predicted secondary structures  were unsuccessful. High-throughput selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) has been used to resolve the secondary structure of the HIV-1 RNA genome . HoweverThe low efficiency of all-DNA ASOs  hinders in vitro-synthesized fragment of HCV RNA  in its backbone. However, should such types of compounds be highly active in vivo, they could contribute to the development of ASO-based HCV treatments. Miravirsen, the first experimental drug of this type, has already been successfully used in clinical trials [Although LDM4676 displayed high activity in different l trials ,75,76. Hl trials ,78, a sul trials ,79, repr50 values similar to their non-modified counterparts but capable of almost completely inhibiting HCV replication in replicon cell lines at higher concentrations.In this study, multiple obstacles that are commonly encountered in the development of new and efficient ASOs were addressed using unconventional and efficient approaches. For the first time, several highly accessible ASO target sequences in the heavily structured coding region of the RNA genome of HCV were revealed. RNAi-based screening represents an efficient and reliable general method for ASO target site selection. This study also provides an important set of findings concerning the use of naturally occurring, minimally modified nucleobases in ASOs. In contrast to nucleobases that contain bulky modifications, the 8-oxo-dG residues reduced the Tm of ASO:RNA duplexes and had no negative effects on RNase H-mediated degradation of RNA strands in ASO:RNA duplexes. Instead, 8-oxo-dG residues facilitated cleavage by RNase H at multiple positions within the target region. Furthermore, the incorporation of 8-oxo-dG residues increased the stability of ASOs in human serum. These effects, possibly combined with other properties of modified nucleobases (such as strong base-pairing of their minor tautomeric forms), outweigh the negative effects on the overall Tm of the ASO. This enabled us to obtain modified LNA/DNA gapmer oligonucleotides with EC


The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3’untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3’ UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3’UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3’UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3’ UTRs. Additionally, the inhibition of packaging  in vitro packaging assay. Moreover, when these same targeted sequences were deleted or mutated in viral genome, viable virus recovery was abolished. Exchanging the terminal sequences between segments failed to recover virus confirming that such changes are deleterious to virus viability. These studies have identified specific regions and sequences key to genome packaging in dsRNA viruses and viability. The specific genome packaging sequences targeted by inhibitory activities of ORNs are bona fide drug target which, as a mechanism common amongst all serotypes, may represent an Achilles’ heel for the development of virus therapeutics.Bluetongue virus (BTV) is an economically important pathogen of ruminants that belongs to a group of viruses whose genome consists of multiple segments of double-stranded RNA. In order for the virus to synthesize viable and infectious progeny, a precise set of the 10 newly replicated BTV segments must be selected for packaging into each new virus particle. How the virus is able to select its own genomic strands from the vast array of cellular RNAs is not clearly understood. One possibility is that that BTV segments harbours an interaction signal that allows them to be sorted and packaged as a set. Correct identification of these signals has basic and applied implications for a possible target of antiviral therapeutics through inhibition of genome sorting and packaging process. Here we showed that a series of short oligonucleotides (ORNs) complementary to multiple sites on the BTV RNA prevented the growth of viable virus in infected cells. ORNs positive for inhibition in virus growth also prevented the genomic RNA to be packaged in an  Reoviridae family) with a double-capsid icosahedral particle and a double-stranded 10-segmented (S1-S10) RNA genome. During virus entry into the cells, the outer capsid of all members of the family including BTV, disassembles from the inner capsid (termed the “core”), which remains intact. The core synthesizes transcripts that are translated into viral proteins, and act as templates for synthesis of genomic dsRNAs pCp (Perkin Elmer) with T4 RNA ligase (Thermo Scientific) in T4 RNA ligase buffer and incubated at 4°C overnight. Unincorporated 32P was removed by exclusion chromatography . Prior to hybridization, unlabelled S10 RNA was denatured at 80°C for 1 min, immediately chilled and then mixed with folding buffer . RNA-ORN hybridization was performed with 0.5pmol of pre-folded S10 RNA annealed with 32P labelled ORNs  in folding buffer in 10 μl final volume [For RNA-ORN hybridization assay, 10pmol of S9 AUG, S9.2, S10 AUG, S10.2, S10.3, S10.5 and Scr ORNs were 3’ end labelled with 10 μCi [l volume . The comIn vitro synthesized VP3 and VP7 were then added to the mixture and further incubated to allow viral core assembly. After eliminating unpackaged RNA by RNase One (Promega) digestion, the assembled particles in the reaction mixture were isolated by a 15% to 65% continuous sucrose gradient followed by fractionation as described previously [in vitro incorporated proteins, the in vitro synthesized viral proteins were radio labelled with 35S-methionine, analysed in 9% SDS-PAGE and detected by autoradiography.The cell-free system for BTV was carried out as described  with som713–718, S10.2725–730, S10728-732 and S10.5743–748, and chimeric S10 and S8) BSR cells were transfected with mutated S10 ssRNA together with the remaining 9 BTV-1 ssRNAs as described previously [To generate the virus with S10 mutants  in 3’UTR are coloured.(TIF)Click here for additional data file.S2 FigThe binding region of S9 ORNs (S9.1 and S9.2) in 3’UTR are highlighted bold.(TIF)Click here for additional data file.S3 FigThe binding region of S1 ORN (S1.3’) in 3’UTR is highlighted bold.(TIF)Click here for additional data file.S4 FigA. Representative examples of BSR cells transfected with different concentration of Scr ORN and stained with crystal violet after 48h showing no sign of cell toxicity. B. For comparison, BSR cells transfected with 2.5uM of Scr were infected with 0.1 MOI of BTV 1 showing non-inhibition of virus replication.(TIF)Click here for additional data file.S5 FigThe binding region of the S10.2 and S10.5 ORNs are coloured. Predicted secondary structure of S10 with deletion corresponding to S10.2 and S10.5 binding regions (ΔS10.2 and ΔS10.5) are shown.(TIF)Click here for additional data file.S6 FigSubstituted regions of the three mutants are coloured. 3’ UTR secondary structure of these mutations predicted with RNAfold are also shown.(TIF)Click here for additional data file.S7 Fig35S-labelled in vitro assembled BTV complexes were fractionated in a continuous sucrose gradient. Fraction #6 from cell-free assembly (CFA) reactions in the absence (+control) or presence of 20 pmol S10.1 and S10.AUG ORNs as indicated were analyzed on 1% denaturing agarose gel.(TIF)Click here for additional data file.S8 Fig35S-labelled protein profile of in vitro translation of assembled ssRNA in the presence or absence of ORN were analyzed on 8% SDS-PAGE gel. Molecular size of each BTV protein and marker are indicated.Fractions 6 and 10 of the complete BTV subcore and transcription complex (TIF)Click here for additional data file.S9 FigSecondary structure of S8 3’UTR from RNAfold.(TIF)Click here for additional data file.S10 Fig32 labelled S9 AUG, S9.2, S10 AUG, S10.2, S10.3, S10.5 and Scr ORNs  were hybridized to 0.5pmol of S9 and S10 RNA in a folding buffer and incubated for 30 min at 30°C. The complex was analysed on 4% native acrylamide gel followed by autoradiography.P(TIF)Click here for additional data file.S11 FigBSR monolayer cells were transfected with mutant S10 together with 9 wild-type ssRNAs (S1-S9) for 3h and overlayed with 1% agarose with DMEM and 1% FCS. At 72hpt the monolayer was fixed with 10% formaldehyde and stained with crystal violet as described in (TIF)Click here for additional data file.


An RNA world has been placed centre stage for explaining the origin of life. Indeed, RNA is the most plausible molecule able to form both a (self)-replicator and to inherit information, necessities for initiating genetics. However, in parallel with self-replication, the proto-organism had to obtain the ability to catalyse supply of its chemical constituents, including the ribonucleotide metabolites required to replicate RNA. Although the possibility of an RNA-catalysed metabolic network has been considered, it is to be questioned whether RNA molecules, at least on their own, possess the required catalytic capacities. An alternative scenario for the origin of metabolism involves chemical reactions that are based on environmental catalysts. Recently, we described a non-enzymatic glycolysis and pentose phosphate pathway-like reactions catalysed by metal ions [mainly Fe(II)] and phosphate, simple inorganic molecules abundantly found in Archaean sediments. While the RNA world can serve to explain the origin of genetics, the origin of the metabolic network might thus date back to constraints of environmental chemistry. Interestingly, considering a metal-catalysed origin of metabolism gives rise to an attractive hypothesis about how the first enzymes could have formed: simple RNA or (poly)peptide molecules could have bound the metal ions, and thus increased their solubility, concentration and accessibility. In a second step, this would have allowed substrate specificity to evolve. We are far from understanding the origin of life, as several of the problems associated with it are yet unsolved ,2. Some The second question, and probably the currently better addressed one, is about how genetics, and with it inheritable evolutionary selection, could have come into place. The most plausible prevailing hypothesis for this event is the so-called RNA world. RNA would have served as a self-replicator, and in this way united the properties of a replicator that is able to inherit information while also possessing the ability to evolve ,8. ExplaThe third equally important, but least understood problem, concerns the origin of metabolism. Metabolism facilitated prototrophy of cells, or at least ecosystems, and in that way rendered them independent of specific geochemical conditions –16. At lin vitro selection experiment, a ribozyme was able to catalyse the aldolase reaction, but only through recruiting a bivalent metal (to be discussed below) which acted as catalyst [What molecules catalysed the reactions in the early form of the metabolic network? The possibility that RNA molecules could have served as catalysts in an early metabolic network is considered ,8: the Rcatalyst . In contcatalyst .Next, the question about the initial trigger is not trivial: to form an RNA nucleotide, not only one, but a series of reactions is necessary. An initial RNA-catalysed reaction system needed, in essence, to provide some sort of function, in order that genetics could select for it and improve catalysis and efficiency. In other words, evolution is selecting for the  product and not for an intermediate step, so an initial RNA-based network could not have come into place one reaction at a time, but only as an already operational entity. This argument renders the origin of metabolism as an RNA-based metabolic reaction system not impossible, but substantially less probable. This problem is amplified by the notion that the least self-sustaining chemical networks lack evolvability, and evolutionary selection cannot change the chemistry in a reaction system, neither are the thermodynamic properties of metabolites subject to genetic selection ,25. ThusOwing to the problems of explaining the origin of metabolism in the RNA world, prebiotic precursors of metabolism were considered as an alternative ,25,26. WPotentially, this scenario predicts that the constitution of metabolism preceded that of genetics in the origin of life . At leasThe discovery of a non-enzymatic glycolysis and PPP does not automatically solve the problem of primordial carbon fixation and does not provide a simple explanation for where the starting materials came from. Perhaps, this problem remains equally strong when considering a post-genetic origin of metabolism ,37, or win vitro selection experiment, they obtained an RNA molecule which bound a bivalent metal, Zn2+. The RNA/Zn2+ complex was fully able to catalyse the aldolase reaction and achieved a substantial rate 4300-fold above the background reactivity [The hypothesis of a metal-catalysed form of early metabolism opens an attractive possibility to give rise to evolution of enzymes. When Fusz et al.  purifiedactivity . Hence, activity ,39.ab initio; only a moderate binding affinity of RNA or a short peptide would suffice to provide the starting point for evolutionary selection. Secondly, iron and other metals not only catalyse or support a single reaction within glycolysis and the PPP, but also catalyse multiple reactions and stabilize their sugar phosphate intermediates [The hypothesis that RNA- and/or peptides bound to metal ions could have functioned as early enzymes is attractive in many ways. First, the origin of such a function does not require the establishment of a complex structure mediates . A simplWhich advantages could the binding of a metal through an RNA or simple peptide provide? First, most of the metal ions only possess average water solubility, thus binding them would increase their accessibility. Secondly, despite iron being abundant in the Archaean sea, other metal ions such as molybdenum or zinc were not as frequent; also iron itself became limiting upon oxidation to the insoluble Fe(III) upon the great oxygenation event –35. The Finally, RNA or peptide binding could increase the specificity of a reaction: metal ions such as Fe(II) act on a broad set of substrates. They are thus in many cases also catalysing unwanted reactions . An examEscherichia coli uses ferrous iron for its catalytic function [E. coli ribulose-5-phosphate epimerase renders this enzyme highly prone to peroxide oxidation, with the result that the bacterial species is sensitive to oxidants [Most modern enzymes of central carbon metabolism are not dependent on metallic co-factors. There are however examples which indicate that this could be the result of a later selection for modern metal-independent enzymes: ribulose-5-phosphate epimerase in function , whereasfunction . These foxidants .The RNA world provides a sophisticated explanation for the origin of genetics, yet has problems in explaining the emergence of metabolism. The recent discovery of non-enzymatic, mainly metal-catalysed, glycolysis and PPP, however, supports a hypothesis that the first form of a metabolic network might have been shaped by environmental molecules that served as its first catalysts. A hybrid theory which considers the RNA world for the origin of genetics and prebiotic non-enzymatic  catalysis for the origin of metabolism could help to solve problems associated with the origin of life. Importantly, this finding gives rise to a hypothesis that enzymes could have come into being as simple RNA and/or peptide molecules that possessed affinity for metal ions frequently found in the Archaean ocean.